Download pdf - Abstracts EASD, 19th Annual Meeting, Oslo, 14–17 September 1983

Diabetologia (1983) 25:135 207 Diabetologia �9 Springer-Verlag 1983

Nineteenth Annual Meeting of the European Association for the Study of Diabetes

Oslo, Norway, 14-17 September 1983

Abstracts

1. Childhood Diabetes in Tokyo and Oslo O. Aagena~s, E. Wannag and T. Kitagawa. Department of Paediatrics, Aker Hospital, Oslo, Norway In Tokyo and Oslo all newly diagnosed Type I (insulin-dependent) diabetes in children under age 15 years from 1 April 1979 to I October 1981, were studied according to a common protocol. Thirty-two children were diagnosed in Oslo (19/100,000/year) and 47 children in To- kyo (< 1/100,000/year). The relative incidence in the age groups 0 5 1�88 6-11 1�88 and 12-14 1�88 did not differ significantly in the two towns. Average age at diagnosis in Tokyo was 8.1 years and in Oslo 8.6 years. Five (12%) of the Tokyo children were diagnosed by routine urine glucose screening at school. This is performed yearly in 20% of the school-children in Tokyo. One of the Oslo children was diagnosed by routine screening. Screening is only performed once in first grade in Oslo. Average insulin dose after 1 year of insulin treatment was similar in Tokyo and Oslo. Forty-five percent of HLA-examined Tokyo children were DR3 or DR4 positive, while that was the case for 100% of the examined Oslo childrenAn spite of the great genetic difference and difference in incidence, the age at diagnosis, clinical picture and clinical course in the first year are surprisingly similar in Tokyo and Oslo.

2. Efflux of Radioactive Ca z+ from Microcarrier-Attached Cloned Rat Insulinoma Cells H. Abrahamsson and P.-O. Berggren. Department of Medical Cell Bi- ology, University of Uppsala, Uppsala, Sweden Calcium metabolism was studied in cloned rat insulinoma cells (RINm5F). Under appropriate conditions these cells aggregate to form islet-like structures which contain a central necrotic zone render- ing such islets unsuitable for analyses. The problem was circumvented by attaching the tumour cells to plastic beads coated with fibronectin. This preparation allowed perifusion studies under standard conditions employed for.isolated islets. Addition of Ca 2+ to a Ca2+-deficient medium resulted in a prompt stimulation of 45Ca effiux from preloaded cells. Removal of Na + from the same type of medium inhibited 45Ca efflux, indicating the participation of a Na+/Ca2+-ex - change in the extrusion of Ca 2+. Furthermore, the RINm5F cells reacted like normal B-cells to the ionophore A-23187. Depolarizing agents, such as high concentrations of K+and tolbutamide, stimulated the 45Ca efflux in a medium containing 2.56 mmol/1 Ca 2+. Never- theless glucose failed to stimulate 45Ca efflux under the same experimental conditions. It is suggested that a primary defect in the RI Nm5 F cells is an inability of glucose to open the voltage-dependent Ca 2+ channels in the plasma membrane.

3. Transmission of Impaired Glucose Tolerance to Subsequent Genera- tions by Female, Not by Male, Youngsters of Experimentally Diabetic Pregnant Rats L. Aerts and F.A. Van Assche. A.Z. St Rafael-Gasthuisberg, K.U. Leuven, Belgium Intravenous injection of 30 mg/kg streptozotocin induces mild diabetes in pregnant rats Oqrstgeneration). Their fetuses (second generation)

show islet hyperplasia and B cell hyperactivity. These modifications of the endocrine pancreas during intra-uterine life have consequences for later adult life, which are not perceptible in basal conditions, but appear in situations stressing B cells, such as an IV glucose load or pregnancy. During pregnancy, these female youngsters of diabetic mothers (mated with control males) have hyperglycaemia and inadequate adaptation of their B cells. Their fetuses (third generation) appear as fetuses of diabetic mothers and, when adult, show disturbed glucose tolerance. In contrast, fetuses of growing control mothers, but with a father who was born to a diabetic mother, appear normal and show normal glucose tolerance in later life. A predisposition for the development of impaired glucose tolerance and gestational diabetes is thus transmitted from one generation to another and is induced in the developing fetus by the hyperglycaemic intra-uterine environment. Whether or not the father was born to a diabetic mother has no influence on the development of the endocrine pancreas of the fetus, thus excluding genetic interference.

4. National Diabetes Programme in Malta: First Epidemiologieal Re- port I. Aganovir, G. Katona, V. Vuksan and Z. Skrabalo. Vuk Vrhovac In- stitute, Krijesnice bb, Zagreb, Yugoslavia The aim of this programme was to provide relevant information on: prevalence of diabetes and impaired glucose tolerance (1GT); characteristics of genetic, anthropometric and biochemical variables; complications related to diabetes and IGT and cardiovascular risk factors. The survey population was a sample of the Maltese population over age 15 years (2149 examinees). Participation rate was 74%. Methods and criteria recommended by WHO were followed throughout the survey (year 1981). Diabetes prevalence was 7.7% (6.1% males; 9% females) and IGT prevalence was 5.6% (5.9% males; 5.4% females). High fasting blood glucose was clearly associated with high body mass index. There was no clear indication that subjects with diabetes and IGT would have more diabetes among their close relatives than non-diabetic subjects. Diabetic and IGT subjects and a control group were invited to participate in a study on diabetic complications. Re- suits: Large vessel disease was found in 25% of non-diabetic, 22% of IGT and 40% of diabetic subjects. Small vessel disease of the eye was found in 35% of male and 29% of female diabetics, 8% IGT males, 6% IGT females and in 4% and 5% of non-diabetic males and females, respectively. Small vessel disease of the kidney was found in 22% of male and 20% of female diabetic subjects.

5. Bacitraein Has Direct Effects on Hepatoeyte Metabolism Indepen- dent of Insulin Action L. Agius and C.Wilding. Department of Clinical Biochemistry and Metabolic Medicine, Royal Victoria Infirmary, Newcastle upon Tyne, UK Bacitracin is a proteolytic inhibitor widely used to inhibit insulin degradation in vitro and in studies investigating the relationship between the intracellular processing of insulin and insulin action. We have examined the metabolic effects of bacitracin, at concentrations currently used in insulin studies, on hepatocytes incubated in the absence of insulin. In hepatocytes isolated from fed rats, bacitracin (0.25-1.0 retool/l) increased the flux through pyruvate dehydrogen-

136 Abstracts

ase, measured with (1-14C) pyruvate by 50-70% (p <0.0005, n =5). This stimulation was independant of the presence of extracellular Ca 2+ and was larger than the stimulation caused by insulin. Bacitra- cin (0.25 to 1.0 mmol/1) decreased the formation of 14CO2 from (2-t4C) pyruvate by 20 to 40% (p < 0.001, n =4) and from (U-~4C) palmitate by 30 to 70% (p < 0.0I, n = 5), indicating a decreased flux through the tricarboxylic acid cycle. It also decreased fatty acid oxidation prior to acetyl-CoA formation (7 to 24%, p < 0.01, n = 5). These changes were associated with a more oxidized mitochondrial redox state but no change in the cellular content of adenine nucleotides. Bacitracin increased the fixation of 14C02 into acid soluble metabolites by 6 to 11% (p < 0.05, n =4). Thus bacitracin has direct metabolic effects. In studies on insulin action in which bacitracin is included, the effects found may be related not only to changes in insulin degradation but also to direct bacitracin effects independent of insulin.

6. Reduction of plasma 1,5-anhydroglucitol in Diabetic Patients and Experimental Diabetes in Rats Y. Akanuma, T. Yamanouchi, S. Mashiko, Y. Iwamoto and H. Akanu- ma. Third Department of Internal Medicine, University of Tokyo, To- kyo, Japan Anhydroglucitol (1,5AG) is a naturally occurring polyol present in human and rat plasma. The level of 1,5AG in non-insulin-dependent and insulin-dependent diabetic patients was 43.9 + 21.9 umol/l (significantly different from normal control subjects: 122.6 + 43.3 gmol/1; p < 0.01). In streptozotocin-induced diabetes in rats, plasma 1,5AG fell to < 1.2 ~mol/l when blood glucose was 27.3 _+_ 3.8 retool/1. Al- though blood glucose was corrected with IV administration of insulin (< 4.4 mmol/1), 1,5AG remained depressed at 4 and 8 h. Four days fasting, glucose administration, liver disease (cirrhosis) and hyperthyroidism exerted no effect on 1,5AG levels. In conclusion, plasma 1,5AG is reduced by deterioration of metabolic control in insulin-dependent diabetes mellitus. Although reduction in 1,5AG occurs rapidly, normalization of this polyol may require appropriate treatment for several weeks. We infer that certain metabolic determinants are present despite normalization of blood glucose. Furthermore, plasma 1,5AG levels may provide a useful marker to indicate the state of metabolic control over an extended time interval, analogous to the measurement of glycosylated haemoglobin.

7. Glucagon is Absorbed Through the Nasal Mucosa and Raises Blood Glucose Levels in Man M. Alberetto, A. E. Pontiroli and G. Pozza. Clinica Medica VIII, Uni- versity of Milan, San Raffaele Scientific Institute, Milan, Italy Previous experience from our group has shown that insulin is absorbed through the nasal mucosa and induces hypoglycaemia in normal and diabetic subjects. The aim of the present study was to evaluate in normal subjects the absorption of glucagon (GLU) through the nasal mucosa. Sodium glycocholate (SGC, 0, 3.3, 5, 10 and 15 mg/mg GLU) was used as a surfactant. GLU (1 rag) was absorbed in the presence of at least 10 mg SGC; with 15 mg SGC/mg GLU peak plasma levels of GLU were equal to IM GLU, although the disappearance of GLU was faster after nasal mucosal than after IM administration. The metabolic effects of nasal mucosal GLU (rise of blood glucose levels and stimulation of insulin release) were slightly lower than IM GLU, and were superimposable when tested repeatedly in the same subjects. No side effects were detected. These data indicate that GLU is absorbed after nasal mucosal administration and exerts its well known metabolic effects. Studies are in progress to improve the effec- tiveness of nasal mucosal GLU and to make this route of administration of value in the management of hypoglycaemia.

8. Platelet Aggregation as a Risk Factor in Diabetes: A Prospective Study P.-H. Althoff 2, J. Krzywanek 1, K. fQberla 3, K. Sch6ffling 2 and K. Bred- dinL Centre of Internal Medicine, ~Department of Angiology and 2Department of Endocrinology, J.W.Goethe University, Frankfurt and 3Institute for Medical Data Processing, Statistics and Biomathe- matics, Munich, FRG The primary aim of the study is to establish whether a constantly enhanced spontaneous platelet aggregation (PAT III) is predictive of the progression of cardiovascular disease. 364 diabetic patients were admitted to the study which was started in 1977 and which will last until 1985. As of November 1982, 52 patients have reached one or more of the defined end points of the study: e.g. death following myocardial infarction, stroke and sudden death (3.3%), myocardial infarction (2.75%), peripheral vascular occlusions (8.8%). An unexpected significant correlation was found between platelet aggregation and fibrino-

gen levels. As expected there was a highly significant correlation between F VIIIc, F VIII RAg and ristocetin cofactor. F VIIIc, F VIII RAg, fibrinogen and ristocetin cofactor were markedly elevated in the diabetic patients. The elevation of these coagulation factors was more pronounced and the incidence of spontaneous platelet aggregation was higher in female than in male diabetic patients. The study will be continued until all patients have reached a 5 year observation period. Preliminary results indicate that a possible risk profile may be helpful in predicting progressive atherosclerotic disease in the individual patient. The most valuable laboratory tests in this respect may be spontaneous platelet aggregation using PAT III, F VIII related antigen and fibrinogen.

9. Allergic Reactions to Human Insulin J. J. Altman, M. Pehuet, G. Slama and C.Tchobroutsky. H6tel-Dieu Hospital, Paris, France Several authors consider one indication for human insulin (HI) to be allergy to animal insulins. An asthmatic man developed acute dyspnoea, swelling at injection site 8 months after starting beef insulin. Porcine insulin (Novo, Nordisk), HI (Novo) caused the same reactions. Desensitization with porcine insulin was successful. A woman with asthma and penicillin allergy, required insulin for gestational diabetes. After short treatment with porcine insulin she developed strong local reactions. Intracutaneous tests resulted in 10 mm weal- and-flare with any porcine insulin, milder reaction with HI. HI therapy was tolerable with only slight local reaction after injection. A woman with penicillin allergy received porcine insulin 10 weeks. She resumed therapy 23 months later (end of pregnancy). She developed swelling of extremities, nausea, dizziness, skin rash. HI giving no improvement, insulin was stopped, patient remained mildly diabetic until delivery. Intracutaneous tests were positive with all insulins at high 1/400 dilutions. Primary allergy to HI is not demonstrated. The hapt- en-carrier theory (allergic response disappearance few months after insulin withdrawal), may not account for reaction 23 months after short insulin treatment. Desensitization with porcine insulin has generally proven effective. In patients with insulin allergy, intermittent insulin treatment, allergic manifestations, HI is unlikely to offer a solution. It may remain of use in beginning insulin in allergic patients.

10. Failure of Glucose to Stimulate 4s-Calcium Uptake into Pancreatic Islets of the Rat Fetus: Relationship with Islet Thiols? H. P.T. Ammon and W. Str61in. Department of Pharmacology, Insti- tute of Pharmaceutical Sciences, University of Tuebingen, Tuebingen, FRG Islets of fetuses do not respond to glucose with insulin secretion and elevation of GSH levels. In islets of adult rats calcium uptake was demonstrated to be related to thiols. Using collagenase isolated islets of adult or fetal rats, uptake of lanthanum non-displaceable 45-calcium was studied in the presence of 3.0 or 16.7 mmol/1 glucose with or without addition of GSH (0A retool/l) over a period of 15 rain. Insu- lin released was measured by radioimmunoassay. In islets of adult rats elevation of glucose from 3.0 to 16.7 mmol/l was associated with a significant elevation of 45-calcium uptake. Addition of GSH significantly increased 45-calcium uptake in the presence of 3.0 and 16.7 mmol/I glucose. GSH augmented the secretion of insulin only in the presence of 16.7 mmol/1 glucose. In islets of fetal rats elevation of the glucose concentration from 3.0 to 16.7 retool/1 was not associated with a significant increase in 45-calcium uptake or insulin secretion and GSH failed to stimulate 45-calcium uptake and insulin secretion at either glucose concentration. In conclusion: our data suggest that the failure of fetal islets to release insulin in response to glucose may be due to the failure of glucose to stimulate calcium uptake, probably by inadequate handling of GSH.

11. Anti-Insulin Antibodies in Type I (Insulin-Dependent) Diabetic Children Before Insulin Treatment: A New Marker for Autoimmune B Cell Damage? C. M. Asplin, J. P. Palmer, P. K. Raghu, P. Clemons, K. Lyen, O. Tatta- ti, B. McKnight and T. Paquette. Departments of Medicine, Biostatis- tics, Paediatrics, Universities of Cincinnati, Pennsylvania, Kansas and Washington, Cincinnati, Ohio, Philadelphia, Pennsylvania, Wichata, Kansas and Seattle, Washington, USA We have employed a sensitive assay for insulin antibodies measuring the 1251 percentage binding of serum obtained from 112 newly diagnosed Type I diabetic children before insulin treatment. 101 acutely ill non-diabetic children and 55 non-diabetic siblings served as controls with percentage binding (mean_+SD) being 1.3+0.3% (range 0.%2.7%) and 1.2 + 0.3% (range 0.8-2.9%), respectively. Considering

Abstracts 137

upper range (2.9%) or mean + 5 SD (2.8%) of controls as maximal normal, 18 of the diabetic children had elevated percentage binding and 36 were above 95th percentile of controls. Mean_+ SD of the 18 definitely elevated diabetic children was 9.2_+ 7.1%. This insulin-binding protein is precipitated by polyethyleneglycol, has a displacement curve that is parallel and over the same concentration range as serum from long-standing Type I diabetic patients and elutes from a sephac- ryl S-300 column at the position of gamma globulin. In conclusion, insulin binding protein(s) are present in a large percentage of newly diagnosed Type I diabetic children and may be a marker of autoimmune B cell damage. Molecular weight and binding characteristics suggest these proteins are anti-insulin antibodies. The ease of this assay makes this possible marker of B cell damage feasible for large scale prospective population studies.

12. Evidence of Asymptomatie Abnormalites of Left Ventricular Func- tion in Diabetes: A Non-invasive Study J. R. Attali, R. N. Sachs, F. Crepin, D. Palsky, S. Lancrenon, P. Tellier, P.Aeberhard, J. Fermanian, M.Vulpillat, J. Lanfranchi and J. Se- baoun. Paris, France 49 diabetic patients (26 insulin-dependent and 23 non-insulin dependent) were compared with 32 control subjects. Absence of ischaemic cardiopathy was confirmed by routine investigations and non-invasive cardiovascular techniques, including an exercise ECG using 12 leads and a thallium 201 scintigraphy. Our results show: (a) a prolonged mean isovolumetric relaxation time (IVRT) as studied by the M mode echocardiography and phonomecanography: diabetics: 0.10_+0.04; controls: 0.05_+0.02s, p<0.001; (b) a reduced mean EF slope: diabetics: 97.48-+37.08ram/s; controls: 125.68+ 34.35 ram/s, p < 0.005 ; (c) a high mean Weissler index (ratio of PEP to LVET): diabetics: 40-+0.08; controls: 33 +0.05, p < 0.01. IVRT and EF slope abnormalities were related to increased myocardial stiffness and impaired LV compliance. In the absence of changes in preload and afterload, the high Weissler index reflected impaired contractility of the myocardium. These abnormalities were related neither to the duration of diabetes nor to the presence or severity of the complications. With the M mode echocardiography, mean diastolic and systolic thickness of the septum is greater in diabetic patients with retinopathy than in control subjects (p <0.005 and p <0.03, respectively); mean diastolic and systolic thickness of the posterior wall is greater in non-insulin dependent diabetic patients than in control subjects (p < 0.001 and p < 0.025). We conclude that there is evidence of LV functional abnormalities specific to diabetes and unrelated to ischaemic cardiopathy and hypertension. Our findings support the hypothesis that they might be due to metabolic disorders and/or myocardial microangiopathy.

13. Effect of Insulin on Erythrocyte Content of ATP and Platelet Acti- vation I.Aursnes, K.Dahl-Jorgensen and K.F. Hanssen, Department of Medicine, Aker Hospital, Oslo, Norway The mechanism behind the abnormal platelet behaviour in insulin-dependent diabetes has not been clarified. A contributing factor could be increased delivery of the platelet activating nucleotide ADP from red cells to platelets. Increase in intra-erythrocytic ATP-concentrations has been found by others after 24 h Biostator treatment. Our aim was to see whether increased plasma levels of free insulin during continuous SC insulin infusion and during multiple daily insulin injections could result in increased erythrocyte content of ATE Blood samples from 25 diabetic patients were obtained on the day before starting and on the day 3 of treatment on which days the free insulin levels were recorded hourly. A correlation (r= 0.46) was found between increase in mean free insulin levels in plasma and ATP concentrations in red cells. With one exception, ATP concentrations rose (on average 13%) in patients with a definite insulin level increase. In another series of experiments, the degree of platelet activation in whole blood upon a standardized stimulus was evaluated in patients using one single injection of NPH insulin daily. Pre-injection blood samples were found to contain platelets which were more reactive than samples obtained 5 h later. The present experiments support the idea that insulin effects on erythrocytes are important in the prevention of thromboembolic complications in diabetes.

14. Quantification of Production and Dispnsal of Ketone Bodies in Splanehnic, Heart, Kidney and Leg Tissues by Means of Simultaneous Arterial-Venous Catheterization and Tracer Techniques A. Avogaro, R. Nosadini, G. Toffolo, C. Cobelli, C. Vigorito, R. Trevi- san, C. Marescotti, E. Duner, L. Sacc~, A. Tiengo and G. Crepaldi. In-

stitute of Medical Pathology, Malattie del Ricambio, Elettrotecnica ed Elettronica, Padova and Clinica Medica I, Naples, Italy We have proposed to quantify ketone body (KB) turnover in normal men from tracer data by using a four compartment model evidencing KB in liver where de novo synthesis is assumed, KB in extrahepatic tissues where utilization takes place and acetoacetate and 3-hydroxybutyrate in blood. Our aim was to confirm the physiological plausibil- ity of this model in four metabolically normal subjects, who underwent diagnostic catheterization. Arterial-venous (A-V) differences of cold acetoacetate and 3-hydroxybutyrate combined with blood measurements can only provide KB net fluxes. In order to quantify sites of production and utilization (p~mol/min), A-V differences were combined with tracer measurements obtained by simultaneous infusion of labelled acetoacetate and 3-hydroxybutyrate. Results: at splanchnic step, production was 271 p~mol/min (range 40-467) and utilization was 44 umol/min (range 16 91). Since acetoacetate CoA transferase is absent in liver, utilization was a measurement of gut KB utilization only. At heart step production was not significantly different from 0 and utilization 6.57 (range 1.85-10.40). At kidney step production was 3.49 (range 1.00-8.57) and utilization 24.67 (range 17.56-35.16). At leg step production was not significantly different from 0 and utilization was 72.7 (range 13-130). We conclude that in normal man in the post- absorptive state: (1) 99% of KB production takes place at the splanchnic step, I% at the kidney step and no production was observed in the leg and heart in agreement with the assumptions of our modeling approach; (2) this technique allows quantitation of gut utilization of KB without portal catheterization.

15. Corticoid-Glucagon Interaction in the Control of Hepatic Protein Synthesis in the Rat in Vivo M.S.Ayuso ~, P. de la Vega ~, C.Alonso ~ and R. Parrilla z. lInstitute G. Marafi6n, C. S. I. C., Madrid and 2University of Extremadura, Ba- dajoz, Spain Adrenal steroids have been shown to exert a permissive effect on the regulatory action of pancreatic hormones on several aspects of hepatic metabolism. Since we have described that glucagon displays a pow- erful regulatory effect on hepatic protein synthesis, we decided to ex- plore whether adequate levels of corticosteroids were also required for glucagon to exert this particular action. Adrenalectomy was found not to induce detectable changes in the rate of hepatic polypeptide chain completion time; under these conditions glucagon had no apparent effect on protein synthesis, but still displayed its characteristic effects of increasing plasma non-esterified fatty acids and increasing the hepatic state of reduction of the NAD system. However the steady-state levels of adenine nucleotides were not changed. The administration of glucagon to adrenalectomized rats treated with corti- colds resulted in 40% inhibition of protein synthesis and this effect was accompannied by a decreased ATP content. These observations seem to indicate that the physiological role of adrenal steroids in controlling hepatic protein synthesis is exerted by modulating the response to pancreatic hormones through changes in the steady-state levels of adenine nucleotides.

16. Production of Monoclonal Antibodies to Islet Cell Surface Antigens Using Hybridization of Spleen Lymphocytes from Non-obese Diabetic Mice S. Baba, K. Yokono, K. Shii, J. Hart, K. Ejiri, K. Ishihara, S. Fujii, T. Kazumi and H.Taniguchi. Kobe University School of Medicine, Kobe, Japan Non-obese diabetic mice established in Japan display a syndrome with dramatic clinical and pathological features similar to insulin-dependent diabetes mellitus in man. Circulating antibodies to the surface of islet cells were demonstrated in some of these mice by the protein A radioligand assay. To produce monoclonal antibodies to islet cell surface antigens, we took the spleens of non-obese diabetic mice, transferred the spleen ceils into heavily irradiated recipient mice, and fused their lymphocytes with FO mouse myeloma cells. The binding of purified antibody from one stable hybridoma to the surface of mouse islet cells and transplantable Syrian golden hamster insulinoma, (In-Ill) , was seven times that of control antibody, whereas absorption of the antibody to mouse spleen lymphocytes or thymocytes resulted in a slight decrease in 12q-protein A binding to In-111 cells. Immuno-enzymatic labelling methods showed that peroxidase binding of In - l l l cells incubated with the antibody was six times greater than that of the cells with control antibody. The monoclonal antibody was visually detected on the surface of these cells. We have developed a novel method for producing monoclonal antibodies to the surface

138 Abstracts

of islet cells for researching the pathogenesis of insulin-dependent diabetes.

17. A 64K Protein Immunoprecipitated from Normal Rat Islets by Anti- bodies from Spontaneously Diabetic BB Rats S. B~ekkeskov, T. Dyrberg and A. Lernmark. Hagedorn Research Lab- oratory, Gentofte, Denmark Islet cell surface antibodies are present in a majority of BB rats which demonstrate morphological and metabolic evidence of the spontaneous diabetes syndrome. In this study, it was tested whether rat islet cell antigens could be immunoprecipitated by sera from diabetic BB rats. Wistar rat islet cell proteins were biosynthetically labelled with 35S methionine. Islets were solubilized in 1% NP40 and the lysate subjected to immunoprecipitation with sera from 10 diabetes susceptible BB rats (33-74 days old), seven of which developed diabetes (74-108 days of age), two had insulitis (day 157) and one impaired glucose tolerance. Control sera were from diabetic non-susceptible BB rats and four normal Wistar rats. Sera from all 10 susceptible BB rats immunoprecipitated a Mr 64000 rat islet cell protein. This component was not detected in lysates of labelled rat spleen lymphocytes. In two animals followed prospectively, immunoprecipitating antibodies were present before diagnosis. In the control group, only serum from one non-susceptible BB rat immunoprecipitated the 64K component. We conclude that autoantibodies in sera from diabetic BB rats recognize a rat islet cell antigen of M~ similar to that previously found to be associated with insulin-dependent diabetes in man.

18. Comparison of Monocomponent Human and Porcine Insulin in the Treatment of Newly Diagnosed Type I (Insulin-Dependent) Diabetic Children with Respect to Immunogenicity, Endogenous Insulin Secre- tion and Metabolic Control H. B~evre, O. Soy• J. Vidnes and K.W. Wefring. Department of Pae- diatrics, University of Bergen, Norway, Department of Paediatrics, The National Hospital, Oslo, Norway and Vestfold Hospital, Tonsberg, Norway The aim of this triple-centre study was to compare monocomponent human and porcine insulins (Novo) in the treatment of newly diagnosed Type 1 diabetic children with respect to immunogenicity, endogenous insulin secretion and metabolic control. Twenty-six patients (17 boys and 9 girls; mean age 8.6 years) were randomly allocated to treatment with human or porcine insulin and were monitored at 3- monthly intervals for 1 year. The two treatment groups were comparable with respect to patient numbers, anthropometric measurements and degree of metabolic derangement at diagnosis. At no time during the first 9 months of treatment was there any statistically significant difference between the porcine and human insulin groups in respect of degree of metabolic control, insulin dose or C-peptide response to a standard meal. Values-+ SD at 9 months were: post-prandial blood glucose (human 15.7 +_ 7.2, porcine 16.2 • 9.2 retool/l); insulin dose (human 16.5 + 5.9 IU/24 h; porcine 16.2 • 7.6 IU/24 h); C-peptide (human 0.15 _+ 0.17 pmol/ml; porcine 0.14_+ 0.09 pmol/ml). In two of the 13 patients on porcine insulin relatively high antibody levels were observed (0.8 mU/ml at 1 year and 2.3 mU/ml at 9 months), but without leading to any statistically significant difference in mean insulin antibody levels between the two treatment groups.

19. Adrenaline is Anti-ketogenic in the Fasted Rat M. Bahnsen and K.G.M.M.Alberti. Department of Clinical Bio- chemistry and Metabolic Medicine, Royal Victoria Infirmary, New- castle upon Tyne, UK Catecholamines stimulate lipolysis and ketogenesis in fed and overnight-fasted man, an important component of the stress response. It is not known, however, whether the major effect of ketogenesis is on the liver or is secondary to increased fatty acid precursor supply. We have therefore used a primed continuous infusion of (3-~4C)-3-hydroxybut - yrate in 48h starved rats to study ketone body production and utilisation. Adrenaline (0.2 gmol/h) was infused for 2h alone, or with somatostatin (10 gg/100 g body weight per h) to remove any influence of glucagon and insulin. Adrenaline infusion, with or without somatostatin, caused a 30-40% fall in blood ketone body concentrations. He- patic ketone body production declined by the same magnitude without any change in metabolic clearance rate. Plasma insulin was unchanged after adrenaline alone but decreased by 50% when somatostatin was infused. Adrenaline -+ somatostatin increased blood alanine (+ 105-170%), lactate (+ 105-170%) and glucose (+ 130-132%), (p < 0.05). Lipolysis was stimulated, reflected by a 70% increase in glycerol concentrations, but plasma non-ester• fatty acids were not altered, suggesting increased re-esterification. Thus in the rat,

adrenaline is anti-ketogenic rather than ketogenic after 48 h starvation. The anti-ketogenic effect could be due to increased intrahepatic oxaloacetate due to increased lactate and pyruvate delivery at a time when delivery of non-ester• fatty acid substrate is unchanged.

20. Recent Data on the Metabolic Action of Gliclazide M.I. Balabolkin, L.I. Levitskaya, T.P. Morozova, L.V. Nodosugova and L.I. Gavriluk. Department of Endocrinology, Moscow Medical Stomatology Institute, Moscow, USSR We have studied 47patients suffering from non-insulin-dependent diabetes (20 patients were treated with glibenclamide and 27 with gliclazide). Before and after treatment for 6-8 weeks, all patients were submitted to oral fructose and glucose tolerance tests. The blood glucose level decreased significantly after treatment with gliclazide (p < 0.05) compared with treatment with glibenclamide. Furthermore, after 6 months of treatment with gliclazide, we found significant decreases in serum immunoreactive insulin, C-peptide and STH levels. The number of insulin receptors on monocytes was decreased before treatment: type I receptors 407• with K affinity (2.17_+ 0.28)x109mol/1; normal 645_+98.6/cell with K affinity (4.41 _+ 0.87) x 109 mol/1; type II receptors 2532 • 276/cell with K affinity (1.61 -+ 0.28) x 108 mol/l ; normal 6 750 + 767/cell with K affinity (1.62-+ 0.53)x 10 a mol/1. After 2 months of treatment with gliclazide, the number of type I receptors was increased to 667 _+ 61.7/cell with K affinity (4.3 + 0.13) x 109 mol/1 and type II receptors to 6799-+ 454/cell with K affinity (2.15 + 0.29) x 108 mol/1.

21. Effect of Fetal Hypoinsulinaemia on Rat Placental and Fetal Liver Glycogen Metabolism V. Barash, A.Gutman and E. Shafrir. Department of Biochemistry, Hadassah University Hospital, Jerusalem, Israel Maternal diabetic hyperglycaemia is associated with placental glycogen accumulation in human and other species despite insulin deficiency. The role of fetal insulin was investigated by producing insulin deficiency through intrafetal injections of streptozotocin (STZ) on days 19.5 and 20.5 of gestation. Control fetuses received citrate buffer only. Upon sacrifice on day 21.5 serum insulin fell from 159_+ 16 to 48 _+ 8 mU/1 in STZ-fetuses, but glucose levels were not affected. Liv- er weight of STZ-treated fetuses decreased from 282 + 10 to 219 • 12 mg, whereas placental weight remained unchanged. In diabetic fetuses liver glycogen decreased from 49.3 _+ 3.7 to 24.2 • 4.8 rag/g, but placental glycogen was steady (1.80 • 0.15 versus 1.76 + 0.10 rag/g). Glycogen distribution between dec• and fetal regions of placenta was 2:1, both in STZ-treated and control fetuses. In maternal STZ- diabetic rats placental glycogen was increased even when the fetuses were also STZ-treated (6.4 • 0.7 versus 5.7 • 0.4 mg/g). Fetal hypoinsulinaemia did not affect placental glycogen synthase activity, whereas fetal hepatic glycogen synthase activity decreased approximately 30%. Fetal hypoinsulinaemia was thus expressed by a reduction in fetal liver weight, glycogen content and glycogen synthase activity, but placental weight, glycogen content + distribution and synthase activity were not affected. These results confirm that placental glycogen metabolism is insulin-independent, but related to maternal hyperglycaemia.

22. Diabetes Control Following Intercurrent Infections A.J.Barnes, B.Haitas, J.C.Moore and R.C.Turner. Diabetes Re- search Laboratories, Radcliffe Infirmary, Oxford, UK Diabetes control is usually aimed at stable conditions, with less attention to brief periods of poor control following intercurrent infections. In a prospective study throughout one winter, the effect of infections was assessed by glycosylation of plasma proteins measured by TBA method. Eight insulin-dependent and eight normal control subjects had an infection, usually influenza; four diabetics had hyperglycaemic symptoms but only two missed work. During stable control, the glycosylated protein in diabetic patients was 0.90__0.06nmol HMF/mg protein per 2 h incubation at 100 ~ (mean -+ SD), and in normal subjects 0.63 + 0.05 nmol HMF. Following infection, glycosylated protein was unaltered in the normal subjects, but increased significantly in diabetic patients at 3, 6 and 13 days, peaking at 6 days at 1.12 + 0.17 nmol HMF (p < 0.01). Eleven diabetic subjects admitted to hospital for marked loss of diabetic control had similar levels of glycosylated protein (1.09 _+ 0.20 nmol HMF) to the out-patients with infection. Glycosylated haemoglobin remained relatively stable throughout the period of infection. Diabetic complications might be particularly promoted by brief periods of poor control, and the marked effect of minor intercurrent infections on protein glycosyla-

Abstracts 139

tion suggest that in the future more attention should probably be paid to diabetic control during brief illnesses.

23. Mechanism of Delayed Removal of Chylomicrons and Very Low Density Lipoproteins Derived from Diabetic Rats H. Bar-On, E. Levy, E. Ziv and E. Shafrir. Departments of Medicine B and Biochemistry, Hadassah University Hospital, Jerusalem, Israel In diabetes, the elevation of plasma triglycerides (TG) in chylomicrons and very low density lipoproteins (VLDL) is attributed to defective removal from circulation. To investigate whether factors other than low tissue lipoprotein lipase activity are responsible, we studied the composition and metabolism of chylomicrons and VLDL obtained from streptozotocin-diabetic rats compared with those from non-diabetic rats. Chylomicrons were collected from intestinal lymph, and VLDL were isolated by zonal-rate ultracentrifugation after in vivo TG labelling with ~4C-palmitate (chylomicrons) or 3H-glyceroI (VLDL). These particles were injected IV into normal recipient rats. Diabetic chylomicrons disappeared from plasma at t,h of 5.5 • "1.1 rain compared with 3.1 • 0.5 min of non-diabetic chylomicrons (p < 0.02). 'Diabetic' VLDL disappeared at 9_+ 1.2min compared with 4.6+ 0.8 min for 'non-diabetic' VLDL (p < 0.005). 'Diabetic' chylomicron remnants, prepared by incubation with lipoprotein lipaSe in vitro, disappeared approximately three times slower than 'non-diabetic' remnants in a liver perfusion system. Apolipoproteins of chylomicrons and VLDL from diabetic rats were depleted of apo-E and apo-A-IV components. In the 'diabetic' apoproteins there was two- to fourfold increase in the peptide-attached carbohydrate residues. Our results indicate that the altered quality of chylomicrons and VLDL produced under diabetic conditions plays a role in their removal, possibly affecting their recognition pattern.

24. Glucose Phosphorytation: Limiting Step in Glucose Metabolism of Adipocytes of Hypophysectomized Rats in the Absence and Presence of Insulin U. Bay and E.R. Froesch. Metabolic Unit, Department of Medicine, University Hospital, Zurich, Switzerland Adipocytes of hypophysectomized rats metabolize 10times less glucose than those of normal rats. Glucose transport (3-0-methylglucose- influx), transport plus phosphorylation (2-deoxyglucose uptake), hexokinase and pyruvate dehydrogenase activity were determined in adipocytes of normal and hypophysectomized rats in order to localize the metabolic block. 3-0-methyl-(U-~4C)-glucose (341xmol/l) influx reaches saturation after 20-80 s. Insulin (200 mU/1) stimulates the influx fivefold in normal cells. Basal influx into hypophysectomized cells is as fast as insulin-stimulated influx into normal cells, but is not further enhanced by insulin. In contrast to 3-0-methylglucose influx, the uptake of 2-deoxy-(U-~4C)-glucose (34txmol/1) is linear from 4-80 s. In normal cells it is stimulated twofold by insulin. The corresponding value in hypophysectomized cells is five times lower and is not changed by insulin. Total hexokinase activity in the homogenate of hypophysectomized cells is decreased to 11% of normal, pyruvate dehydrogenase activity only to 20% of normal. The following conclusions may be drawn: ("1) glucose metabolism in normal cells is regulated by the membrane transport rate, (2) phosphorylation by hexokinase is the limiting step of glucose metabolism in hypophysectomized cells, (3) insulin has no immediate effect on glucose phosphorylation.

25. Peripheral Nerve Function in Type I (Insulin-Dependent) Diabetic Patients with Limited Joint Mobility R.Beacom, B.Sawnhey and L.Kennedy. Royal Victoria Hospital, Belfast, Northern Ireland, UK Limited mobility of joints, mainly small joints in the hands, is a common feature in many patients with long-standing Type I diabetes. Its exact aetiology is unknown. Some studies have suggested an association with neuropathy. We have investigated various modalities of nerve function in patients with Type I diabetes and limited joint mobility (LJM), and in others with long-standing Type I diabetes (> 12 years) but normal joints (NJ). HbA1 levels were similar in the two groups. The biothesiometer threshold for vibration over the ulnar sty- loid was greater in LJM (n = 32) than NJ (n = 33) - 11.04 • 0.99 versus 8.59 _+ 0.62 (mean 4- SEM) respectively, p < 0.025. Vibration threshold over the M P joint of the middle finger was similarly affected ("11.43 4- 1.13 versus 8.87_+0.85, p<0.05). Vibration threshold correlated strongly with duration of diabetes (r = 0.50, p < 0.0025) and the group with LJM had a slightly longer duration of diabetes than NJ group (27.3 _+ 1.8 versus 23.6 _+ 1.4, p < 0.05). Motor conduction velocity in the ulnar nerve was significantly slower in 28 LJM patients than in 19 NJ (50.6 + 1.17 versus 54.3 _+ 1."17 m/s, p < 0.025). Similar trends were

seen in motor and sensory median nerve velocity, but again the LJM group had a slightly longer duration of diabetes. We conclude that although there is a relation between limited joint mobility and nerve dysfunction in diabetes, it is probably not causal but may relate to duration of diabetes.

26. The Poor Evidence for a Direct Feedback Inhibition of Insulin on its Secretion W. Beischer, A. Michael, B. Maier, M. Maas and L. Keller. Department of Internal Medicine I, University of Ulm, FRG The mechanism of insulin feedback inhibition has been studied in vitro. The usual procedures of isolation and incubation of rat islets were followed. The freshly isolated islets were preincubated for 15 min before the final incubation for 120 rain. The medium of preincubation as well as of final incubation varied independently concerning glucose concentration (50, 100 and 300 mg/dl), concentration of added rat insulin (10-400 ng/ml) and binding capacity of added anti-porcine-insulin-serum (10-400 ng/ml). Immunoreactive rat C-peptide and insulin were measured during final incubation. No effect of exogenous insulin was observed at variable glucose concentrations in final incubation following preincubation at 50 mg/dl glucose. No effect of exogenous insulin or of anti-insulin-serum was observed at 300 mg/dl glucose in final incubation following preincubation at 300 mg/dl glucose. An inhibition of the moderately stimulated C-peptide secretion by low concentrations of added insulin was observed in some experiments only; anti-insulin-serum was again without effect at 50 mg/dl glucose in final incubation following preincubation at 300 mg/dl glucose. An unequivocal inhibition of C-peptide secretion by insulin was not obvious from incubation of isolated islets under variable condition. A direct feedback of insulin on its secretion appears unlikely.

27. Insulin Resistance in the Obese Hyperglycaemic (oh~oh) Mouse: Failure of Hyperinsulinaemia to Activate Pyruvate Kinase F. Belfiore, A.M. Rabuazzo, S. Iannello, R. Campione and D. Vasta. Institute of Pathological Medicine, Chair of Pathophysiology of Me- tabolism, University of Catania, Ospedale Garbaldi, Catania, Italy In fed ob/ob mice, as compared to control mice, hepatic pyruvate kinase (PK) activity was enhanced by 35.63% (214.6_+13.6 versus 158.3 • 10.5 nmol-min-1, mg protein-I, mean • SEM, p < 0.01) when measured at saturating (6.6 mmol/1) concentration of the substrate phosphoenolpyruvate (total activity), but the activity recorded at sub- saturating (1.3 retool/l) substrate concentration (active fraction) was unchanged (86.44_6.4 versus 85.7 • 13.6), or even decreased if expressed as percentage of the total activity (40.2 + 2.6 versus 54.1 • 5.1%, p < 0.05). Thus, in ob/ob mice the striking hyperinsulinaemia, although able to increase (by induction) the content of PK, would fail to activate (by dephosphorylation) this enzyme. Considering that the gluconeogenic enzyme fructose-l,6-diphosphatase is increased in these animals (44.7 • 1.9 versus 31.6 _+ 4.00, p < 0.01), a prevalence of gluconeogenesis over glycolysis may be postulated. 48-h-fasting normalized the values of both enzymes in the ob/ob mice. The hepatic concentration of PK effectors (fructose-l,6-P2 and phosphoenolpyruvate) was unchanged in fed ob/ob mice. The in vitro effect of fructose- 1,6-P2 (15 Ixmol/1), which would favour the activation (dephosphorylation) of PK, was preserved. Alteration in cyclic-AMP availability of protein kinase/phosphoprotein phosphatase system may be involved in the insulin resistance of PK.

28. Heart-Rate Variation on Standing-to-Lying: A Proposal for a New Simple Test for the Evaluation of Parasympathetic Versus Sympathetic Cardiovascular Response in Diabetic Autonomic Neuropathy F. Bellavere, C. Cardone, M. Ferri, L. Guarini, D. Fedele. Cattedra di Patotogia Medica, University of Padova, Italy The characteristic pattern of heart-rate response to lying-down consists in a brief R-R shortening over the first five beats after lying (pure parasympathetic withdrawal) followed by heart-rate restoration on beats 20-25. Hence we attempted to standardize the immediate heart- rate response to standing-to-lying (S-L) in 95 male insulin-dependent diabetic patients who underwent a battery of cardiovascular autonomic tests. They were divided, using an especially devised scoring- system, into three groups: (1) 64 subjects without autonomic neuropathy; (2) 18 borderline cases; (3) 13 with autonomic neuropathy. Each subject performed two S-L tests; the results were compared with those of 40 sex- and age-matched control subjects. We evaluated: SLbl = ratio between R-R mean before lying and R-R minimum over first five beats after lying; SLb2=ratio between R-R maximum over beats 20-25 and R-R minimum over first five beats after lying; SLi=SLbl x SLb2. In controls SLbl was 1.2"1_+0.07 (mean+SD),

140 Abstracts

SLb2: 1.47 + 0.18, SLi: 1.75 _+ 0.25. In diabetic patients without auto- nomm neuropathy SLbl was 1.17 +0.1 (p < 0.05), SLb2: 1.48+0.26, SLi: 1.70_+0.3. In autonomic group SLb~ was 1.03+0.1 (p <0.001) SLi: 1.19 _+ 0.09 (p < 0.001). This test, besides providing an evaluation of parasympathetic versus sympathetic effects, suggests the use of SLbl (normal range: 1.07-1.35) and SLi (1.25-2.25) as new diagnostic parameters in autonomic neuropathy.

29. Decreased Noraflrenaline Tilt-Table Response in Type I (Insulin- Dependent) Diabetes - An Early Sign of Diabetic Neuropathy H. Berger, I. Cicmir, A. H0binger, T. Koschinsky and F. A. Giles. Dia- betes Research Institute, Dfisseldorf, FRG The significance of altered catecholamine metabolism in diabetic neuropathy (DN) is still unclear. Therefore, Type I diabetic patients with and without clinical symptoms of peripheral and autonomic DN were studied. Profiles of plasma catecholamines, heart rate and blood pressure were measured in (a) 22 Type 1 diabetic patients without peripheral or autonomic DN (mean age 34years, duration of diabetes 8 years, HbA1 11%), (b) 11 age-matched healthy control subjects, and (c) 10 age-matched Type 1 diabetic patients with autonomic DN (abnormal beat-to-beat variation and pupillary reflex, duration of diabetes 18 years, HbA~ 12%), on a tilt-table (1) after 30 min at rest in supine position, (2) after 10 min tilted up (90~ and (3) after 20 min in supine position again. At rest, noradrenaline levels did not differ between groups a, b and c. After tilting noradrenaline increased in group a from 164 to 291 (pg/Isl; p < 0.005), in group b from 231 to 545 (pg/gl; p < 0.005) and did not change group in c (170 pg/ul). The differences in increment and absolute concentration between groups a and b were significant (p < 0.01) despite similar heart rate and blood pressure changes. In conclusion, a decreased noradrenaline tilt-table response in Type 1 diabetes occurs before other clinical manifestations of peripheral or autonomic DN and may represent the earliest and most sensitive sign of DN.

30. Comparison of Gastroparesis in Diabetic Patients With and With- out Clinical Neuropathy J. F. Bergmann, J. P. Isal, C. Caulin and J. M. Segrestaa. Therapy Clin- ic, Lariboisi6re H6spital, Paris, France Diabetic gastroparesis is known as part of the neurological complications of diabetes. We compared the gastric emptying (GE) of a meal in diabetic patients with and without degenerative complications. 20 diabetic patients (mean age: 57years, range: 24-84years) ingested together with a standard meal (800 cal) 100 undissolvable radio-opaque pellets (volume: 30 mm3). The patients were divided into two groups: group 1 : 10 subjects without clinical evidence of neuropathy; group 2: 10 subjects with neuropathy. The number of pellets in the stomach were counted on X-rays taken 60, 120, 180, 240 and 300 min after the beginning of the meal. For each patient regression curves and the half-life of GE of the pellets were calculated. Results: half-life (mean _+ SEM) and median were respectively: in groups1 and 2: 3.42+0.73h and 2.10h; in groupl: 1.98+0.24h and 1.85h and in group2: 4.86_+1.32h and 3.15h. The five subjects with the higher half-life had clinical neuropathy. There was a statistical difference between groups 1 and 2 (p < 0.05). There was no correlation between GE time and sex-ratio, age, long-standing history of diabetes, type of treatment, retinopathy or nephropathy. We conclude that the GE time is significantly slower in diabetic patients with neuropathy than in patients without neuropathy. These results suggest that the prescrip- tion of oral antidiabetic drugs in patients with clinical neuropathy should take into account the possibility of gastroparesis, which might interfere with the biodisposibility of the drug.

31. Dissociation Between the Short- and Long-term Action of Glucose on the Pancreatic B Cell Content of Intracellular Calcium P. Bergsten. Department of Medical Cell Biology, University of Upp- sala, Uppsala, Sweden

3+ The problem of how glucose affects the content of intracellular (La - non-displaceable) calcium in pancreatic B cells was approached by combining measurements of 4SCa in ob/ob-mouse islets loaded to isotopic equilibrium with determination of total calcium using elec- trothermal atomic absorption spectroscopy. After 24 h of culture in medium containing glucose (5.5 mmol/1), the content of intracellular 45Ca was remarkably resistant to short-term changes of the glucose concentration. There was thus only a 14% increase 30 min after raising the concentration of the sugar from 5.5 to 20 mmol/l and no effects at all on the islet calcium when reducing glucose from 20 to 1 mmol/l. After 7 days of culture at glucose (5.5 retool/I), the amounts of intracellular calcium were higher than after storage at either glucose (1 or

20 mmol/1). The tong-term effect of glucose in suppressing the islet content of intracellular calcium was associated with reduced amounts of immunoreactive insulin as indicated both from histochemical analyses and measurements of the hormone content by radioimmunoassay. The previously unknown effect of glucose to suppress the islet content of intracellnlar calcium may consequently be associated with its mobilization of the secretory granules.

32. Study of Survival Probability in Diabetic Ketoacidosis M. Bergua, M. Rio, I. Levy, I. Halperin, J.M. Oller and D. Figuerola. Secci6 de Diabetis, Hospital Chnic of Barcelona, Departament Esta- distica Facultad de Biologia, Barcelona, Spain In order to estimate the individual survival probability in diabetic ketoacidosis, 107 cases (13 dead and 96 alive), all treated by the same therapeutic regime, were studied. Bad prognostic factors identified by a previous analysis (age, previous diagnosis of diabetes, degree of coma, blood glucose and BUN) were re-analyzed by Cox's linear logistic method:

exp (bo + ~ bj xij) Pi i=1

l + e x p (bo+ ~ bj xlj) j=l

where Pi is survival probability, x represents the different introduced variables, and b, unknown coefficients determined by Engelman's regression program in BMPD (Program PRL). Only age (x0, blood glucose (x2) and previous diagnosis of diabetes (xs) were found to be significant for the regression, (p < 0,04), so that;

L n ~ = 8.826-0.101 (xl)-0.005(x3) + 1.077(x5)

where xa is expressed in years, x2, in mg/100 ml, and xs is equal either to 1 or - 1 , according to whether the disease was previously diagnosed. Considering our results, survival prediction is dependable when probability exeeds 71%. In conclusion, this equation: (1) may allow an accurate prediction of the survival probability of any ketoacidotic patient on admission, (2) offers a retrospective qualitive analysis of any unpredicted death.

33. Distinct Mechanisms for Stimulation of the Phosphatidylinositol Cycle in Rat Islets by Nutrient and Cholinergic Secretagogues L. Best and W.J. Malaisse. Laboratory of Experimental Medicine, University of Brussels, Brussels, Belgium We have demonstrated previously enhanced 32p-labelling of phosphatidic acid and the phosphoinositides in rat islets in response to nutrient (e.g. glucose) and neurutransmitter (e.g. carbachol) secretagogues. Both classes of secretagogue also stimulate labelling of these lipids, at the expense of phosphatidylcholine and phosphatidylethanolamine, using 3H-glycerol as precursor, suggesting that enhanced phosphatidylinositol turnover in islets may be an 'open circle' possibly involving de novo lipid synthesis. In islets pre-labelled with 3H-inositol, both types of secretagogue also stimulated the release of 3H-inositol phosphates, water-soluble products of phosphoinositide hydrolysis, suggesting that breakdown of these lipids also occurred during stimulation. Carbachol was found to induce a small but significant fall in 32p-phosphatidylinositol-4,5-bisphosphate in islets pre-labelled with 32po4, suggesting that breakdown of this polyphospho- inositide could be the initial event in turnover of the phosphatidylinositol cycle stimulated by carbachol. In contrast, no 32p-phosphoinositide breakdown was detected in response to glucose. Treatment of islets with mepacrine, a purported inhibitor of phospholipase C, inhibited carbachol-induced 32p-labelling of phosphatic acid and phosphoinositides, whereas labelling &these lipids in response to glucose was slightly enhanced in the presence of mepacrine. The above observations suggest that nutrient and neurotransmitter secretagogues stimulate turnover of the phosphatidylinositol cycle through distinct mechanisms.

34. Twelve-Month Therapy with Biosynthetic Human Insulin: Results of Two Comparative Double Blind Multicentre Studies with Type I (Insu- lin-dependent) Diabetic Patients Pretreated with Porcine and Bovine In- sulin J. Beyer, R. Haas, F. Enzmann, M. Lauerbach, J. Rademacher, P. Al- thoff, W. Bachmann, K. Brandstetter, U. Cordes, A. Dannehl, G. Diintsch, K. Heun, L. Kerp, N. Lotz, H. Mehnert, H.J. Mitzkat, K. Petersen, C. Rosak, D. Sailer, K. Schlfiter, K. Sch6ffiing, K. E. Schr6der and P. Schumm. Department of Internal Medicine, En- docrinology and Metabolism, University of Mainz, Mainz, FRG

Abstracts 141

In two multicentre double-blind studies 66 Type 1 diabetic subjects pretreated with pork insulin were changed to human insulin or purified pork insulin preparations (regular and NPH-insulin). 65 patients previously pre-treated with beef insulin were transferred in a randomized, double-blind fashion, to human insulin of purified beef insulin of the same preparation (regular and NPH-insulin). All patients were comparable concerning age, mean duration of diabetes, duration of insulin therapy and body weight and all were treated with the new insulin for 12 month. Patients' metabolic control, as demonstrated by fasting and l-h post-prandial blood glucose, HbA~, and daily insulin dosage over 12 months, was nearly unchanged in our four groups compared with the values before changing insulin preparations. In the groups of the human insulin treated diabetic patients, there was only uncertain evidence for a slower blood glucose increase after the first breakfast and in one group for a lower requirement of insulin. No severe hypoglycaemic attacks or skin reactions were reported.

35. Immediate Inhibition by Cortieosterone of 45Ca Efflux from Peri- fused Rat Islets B. Bfllaudel, P. Mathias, B. C. J. Sutter and W.J. Malaisse. Laboratory of Physiology and Endocrinology, University of Bordeaux I, Talence, France, and Laboratory of Experimental Medicine, University of Brussels, Brussels, Belgium Corticosterone inhibits, directly and immediately, glucose-stimulated insulin release. The mode of action of corticosterone (0.6 umol/1) was investigated by characterizing its effect on 45Ca effiux from prelabelled and perifused rat pancreatic islets. Corticosterone decreased 45Ca efflux from islets stimulated by glucose (16.7mmol/1) in a square-wave or steady-state manner. This was associated with a 30% inhibition of insulin release, whether during the initial peak-shaped or later sustained secretory phase. Corticosterone also inhibited 45Ca outflow and insulin release from islets exposed to 2-ketoisocaproate ('10.0mmol/1). In the absence of exogenous nutrient, corticosterone immediately inhibited 45Ca outflow and insulin release in response to the administration of gliclazide (15 ~mol/1) or during exposure of the islets to the combination of gliclazide (60 gmol/1) and the tumour- promoting agent 12-O-tetradecanoylphorbol-13-acetate (0.2 bmol/1). These findings indicate that the inhibition of insulin release by corticosterone coincides with a decreased inflow of Ca 2+ into the islet cells. Since corticosterone impaired the cationic and secretory responses not solely to glucose but also to a non-carbohydrate nutrient and non-nutrient secretagogues, the glucocorticoid could primarily affect cationic fluxes, rather than causing only a specific impairment of glucose metabolism in the islet cells.

36. Subcutaneous Adiposity Influences the Absorption of Radiolabelled Actrapid Insulin A.J. Birtwell, D.R.Owens, S. Luzio, I.R.Jones, T.M. Hayes and A. Volundt University Hospital of Wales, and ~Novo Research Insti- tute, Denmark The absorption of labelled ~25I-Actrapid insulin was studied by simultaneous measurements of the disappearance of radioactivity from the injection site and the appearance of insulin in plasma following SC injection. 6 i.u. insulin was administered into the abdominal wall in nine fasting subjects, 91-115% ideal body weight on two separate occasions. Between-subject variation was significantly greater than within-subject variation for both cumulative plasma insulin expressed as Ts0o/0 AUCo-6h (p < 0.01) and half-time of disappearance, Tso,/o (p < 0.01). The rate of absorption correlated significantly with skinfold thickness Ts00/0 AUC0-6h (p < 0.05) and Ts0~/0 disappearance (p < 0.05). The Tse/0 disappearance (166 + 39 min, mean + SD) and Ts0~/0 AUC0_6h (150_+26min, mean+SD) were not significantly different, being longer than previously reported. There was a linear relationship between appearance of insulin in plasma (AUC0_6h) and disappearance of radioactivity from the injection site (p < 0.01). Subcutaneous adiposity is an additional factor influencing the rate of absorption of ~2q-Actrapid insulin from a subcutaneous depot.

37. Insulin Sensitivity in Starvation: Differential Effects on Splanchnic and Peripheral tissues O. Bj6rkman and L. S. Eriksson. Department of Clinical Physiology and Medicine, Huddinge University Hospital, Stockholm, Sweden Starvation accentuates insulin resistance in obese patients. However, it is not known which tissues are involved in this process (splanchnic and/or peripheral) and whether this applies to healthy subjects. We therefore performed euglycaemic (5mmol/1) or hyperglycaemic (12 mmol/1) glucose clamp studies in non-obese subjects in the post- absorptive state and after 60 h starvation. Net splanchnic glucose bal-

ance was simultaneously measured using hepatic vein catheterization. During both euglycaemic and hyperglycaemic clamps, total glucose disposal was reduced by 50-60% after 60 h of starvation (4.3 _+ 0.4 versus 7.6 _+ 1.1 and 3.1 + 0.2 versus 7.0 _+ 1.3 mg. kg -1. rain -1, respectively). This resistance was totally accounted for by a 40-60% decrease in peripheral glucose uptake. In contrast, net splanchnic glucose uptake was similar before and after starvation both during euglycaemic (0.6+0.2 versus 0 .6+0.1mg-kg- l .min -~) and hyperglycaemic clamps (1.2+0.1 versus 1.0 _+ 0.2 mg. kg ~.min-1). This occurred even though the insulin response in the hyperglycaemic clamp was markedly reduced after starvation (22 versus 55 mU/l). Conclusion: brief starvation in normal man is accompanied by insulin resistance in peripheral tissues while net splanchnic glucose uptake in response to glucose and/or insulin is unaltered. These changes may serve to re- plete primarily the hepatic glycogen stores when glucose becomes available.

38. Left Ventricle Performance in Diabetic Patients During Submaxi- mal Effort Test: Study of Systolic Time Intervals S. Blaga ~, R. Vlaicu ~ , S. Rentrsch ~, I. Corches ~ , M. Motocu 2, AI. Du[u 2, M.L. Rusu 2 and A. H. Suleiman< ~First Medical Clinic and 2Second Medical Clinic, Cluj-Napoca, Romania Thirty-four patients with compensated diabetes mellitus between 16-40 years underwent a submaximal effort test using an ergometric bicycle. The patients in question did not present with signs or symptoms of cardiac disease. During this test we recorded electrocardiograms in D II, phonocardiograms, and carotidograms at rest, at the end of every phase of the effort, and at 1, 5, 10 min after stopping the effort. We calculated pre-ejection (PEP) and ejection periods of the left ventricle (LVEP) and the ratio between them (PEP/LVEP). The effort tests of patients with diabetes were negative. Ten diabetic patients reached submaximal theoretical capacity but with PEP/LVEP alterations. Four diabetic patients with obesity did not reach submaxi- real theoretical capacity. In these four patients the test was stopped because of dyspnoea; all of them had PEP/LVEP alterations. Left ventricle performance alterations in diabetic patients without symptoms of cardiac failure indicate the existence of a cardiomyopathy, the effort test for ischaemic cardiopathy being negative.

39. Purification of a Factor in the Rat from both Hypothalamus and Plasma which Stimulates Insulin Secretion in Vivo and in Vitro E. Bobbioni, B. Jeanrenaud, G. Zahnd and W. Schlegel. Laboratoires de Recherches M6taboliques and Fondation pour Recherches M6di- cales, Geneva, Switzerland Rat hypothalamus has been demonstrated to contain a factor promoting insulin secretion both in vivo and in vitro. This factor has been purified by gel filtrations on Sephadex G-50 and Biogel P2 and by reverse phase HPLC. After each purification step, a single pool of fractions was found to be active in stimulating insulin secretion as assessed by infusing the fractions into isolated, perfused rat pancreas. When plasma from normal rats was purified, an insulin-secretion- promoting activity was found to be linked to the presence of a substance behaving very similarly, both in terms of purification and in the bioassay, to the factor purified from the hypothalamus. These results suggest the presence, in both hypothalamus and plasma of the rat, of a factor which could participate in the control of insulin secretion.

40. Loss of Cholesterol from the Red Cell Membrane: A Possible Cause of Decreased Erythrocyte Deformability in Diabetic Retinopathy W. A. Bode, C. Popp-Snijders, T. Fonk, E. A. van der Veen and J. van der Meet. Department of Internal Medicine, Free University Hospi- tal, Amsterdam, The Netherlands Looking for factors causing decreased erythrocyte deformability found previously, we studied erythrocyte flexibility, ATP-concentration, viscosity and haemoglobin content of erythrocyte ghosts, and cholesterol, phospholipid and protein content of the erythrocyte membrane in a group of 30 Type I (insulin-dependent) diabetic subjects with proliferative retinopathy and 30 age- and sex-matched healthy control subjects. Erythrocyte flexibility (estimated by visco- simetry with washed cells) appeared to be decreased in the patients. The only other significant difference found was a lower cholesterol concentration of the erythrocyte membranes (nmol/mg protein) mean (range) 577 (493-716) versus 618 (469-757) in control subject (p < 0.05). When the same measurements were performed in the 20% youngest and the 20% oldest cells of patients as well as controls, a significant decrease of the cholesterol concentration during cell-aging was detected in the membranes of the patients: 590 (492-745) --~ 566 (463-747) nmol/mg protein, (p < 0.02). In the controls, loss of choles-

142 Abstracts

terol from the membranes could not be demonstrated: 609 (486-717) --~ 618 (462 808) nmol/mg protein (NS). We conclude that the diminished deformability of erythrocytes of diabetic patients with proliferative retinopathy could be due to cholesterol loss from the membranes during circulation of the erythrocyte.

41. Circulating Immune Complexes and Platelet Thromboxane synthesis in Patients with Type 1 (Insulin-Dependent) Diabetes G. D. Bompiani, G. Triolo, G. Davi 1, E. Giardina and A. Strano 1. Istitu- to di Clinica Medica III and qstituto di Clinica Medica I, University of Palermo, Italy Whether circulating immune complexes (CIC) are directly involved in mediating vessel injury in diabetes mellitus is not well established. Platelet abnormalities have been described in diabetes. It is also known that CIC may stimulate the synthesis and release of prostaglandins via Fc receptors for IgG on the platelet surface. This study was designed to investigate the relationship between CIC and the rate of thromboxane (TBX2) synthesis in diabetes. 12 diabetic children with comparable glucose control and without complications and 10 matched controls were studied. CIC were detected by the solid-phase Clq binding assay and by the anti C3 EIA. TXB2 was measured in samples of platelet-rich plasma under thrombin and arachidonic acid (AA) stimulation. CIC were detected in 6/12 patients. Hatelets from diabetic subjects with CIC synthetized greater amounts of TXB2 (154.85_+ 64ng/108 platelets after thrombin; 219.28 + 97.5 ng/10 s platelets after AA) than did platelets from CIC-negative patients (95.1 + 25.54 ng/108 platelets after thrombin; 125.88 • ng/108 platelets after AA; p < 0.05) or controls (107.2 _+ 25.9 ng/108 platelets after thrombin; 128.8 + 34ng/10 ~ platelets after AA; p < 0.05). In view of the known actions of CIC and platelet function, a relationship between these two factors may be suggested in the initiation and progression of microangiopathy in diabetes.

42. Prediabetes in the Spontaneously Diabetic BB Wistar rat: Islet Se- cretion of Insulin and Islet Cell Surface Antibodies A.J. Bone, D. Shirling, M. Gilchrist, F.J. Tames and J. D. Baird. Meta- bolic Unit, University Department of Medicine, Western General Hospital, Edinburgh, Scotland, UK Insulin-dependent diabetes develops spontaneously at 3 4 months of age in 50%-75% of BB rats. Using a multichannel perifusion system, we have studied glucose-stimulated (16.7 retool/l) insulin secretion in islets isolated from BB rats before the development of overt diabetes. HbA1 was used to select two groups of animals for study. The respective mean_+ SEM values for the 'high' and 'low' HbAa groups were 4.8+0.1 and 3.9+0.1% (p<0.0005) for HbA1, 8.6+0.5 and 8A• 0.5 mmol/1 (NS) for random plasma glucose, and 3.3 +and 7.5 • 1.8 mU/l (p < 0.01) for random plasma insulin (IRI). Islets from both groups showed a biphasic secretory response and similar insulin/protein content (378_ 25 and 492_+ 53 lxU IRI/ug islet protein (NS). Is- lets from animals in the 'high' HbAa group showed a low total secretion of insulin (393 + 54 versus 709 • 32 IxU IRI /ug islet protein, p < 0.005). First phase secretion was particularly reduced (52• versus 129+18 IxU IRI/ug islet protein, p <0.01). Islet cell surface antibodies (~2Sl-protein A radiological assay) were detected subsequently in the plasma of 83% of animals in the 'high' and 25% of those in the 'low' HbA1 groups. Results suggest that in this model abnormalities in insulin secretion co-exist with plasma islet cell surface antibodies in the prediabetic period. The nature of this association is not clear at present.

43. Alloxan Effects on Mitochondria: Study of Relationship Between Cycling of Ca 2§ , Net Efflux of Mg 2+, K + and Ca 2+ , Phosphate Trans- port, Oxygen Consumption, Membrane Potential and Volume Changes L. Boquist and L. Nelson. Institute of Pathology, University of Umefi, Ume~, Sweden The aim was to characterize further the recently reported Ca 2+-releasing action of alloxan, using isolated mouse liver mitochondria, dual wave-length spectrophotometry, ion-sensitive electrodes and atomic absorption spectroscopy. When added under non-energized conditions with external inorganic phosphate (Pi) 3 mmol/1 and Ca 2+ 35 amol, alloxan in a minimum concentration of about 10-100 ,umol induced release of Mg 2+, K + and Ca 2 + stimulated state 4 respiration, decreased membrane potential, and swelling. In the absence of external Pi and Ca 2+, and under energized conditions, the corresponding concentration was about "1 mmol/l. The swelling was not inhibited by external Mg 2+ 4 mmol/1 and ATP 50 lxmol/1 to "1 p~mol/1, which prevent membrane-potential-dependent spontaneous efflux of Ca a+. The swelling was more or less completely inhibited by antimycin A, malo-

nate and DNP indicating requirement for coupled respiration; by La 3+, Ni 2+ and ruthenium red emphasizing the importance of cycling of Ca 2+ ; by dibucaine, trifluoroperazine and Sr 2+, possibly indicating activation by Ca 2 + of membrane phospholipase A~; and by mersalyl and N-ethylmaleimide, indicating requirement for transport of Pi. The findings demonstrate that in the presence of Ca 2+, Pi, Mg 2+ and ATP not above the physiological range, even low Concentrations of alloxan induce loss of Mg 2+, K + and Ca2+and probably of adenine nucleotides, and swelling in mitochondria. The loss of Ca 2+ and the swelling agree with data previously obtained using qualitative and quantitative electron microscopy and X-ray microanalysis of islet B cells from alloxan-treated mice.

44. Are Nursing Habits Related to Insulin-Dependent Diabetic Aetiolo- gY? K. Borch-Johnsen, T. Mandrup-Poulsen, M. Christy, B. Zachau Christiansen and J. Nerup. Steno Memorial Hospital, Gentofte, Den- mark Incidence rates of childhood insulin-dependent diabetes vary within and between Scandinavian countries. Incidence curves (incidence against age at onset) are, however, strikingly parallel. Nursing habits vary greatly with socio-economic status, geography and time. Moth- er's milk supplies babies with HLA-DR substance, IgA and lymphoid cells protecting against, for example, viral infections. Breastfed babies have fewer viral infections and young insulin-dependent patients have lower IgA levels than control subjects and virus have been in- criminated in insulin-dependent diabetic aetiology. Thus, nursing habits were investigated in 181 insulin-dependent diabetic children (onset before age 18 years), 94 healthy siblings and a cohort of 6245 children born in 1959-1961. Eleven percent of insulin-dependent children, but only 2% of their healthy siblings, were never breastfed (p = 0.01). Significantly fewer diabetic than control subjects (p < 0.02) were breastfed"-4 months. These differences were most prominent in boys. Thus, insulin-dependent babies were nursed less and for shorther periods of time. In the southern part of Copenhagen County incidence of insulin-dependent diabetes is twice that in the northern, socio-economically more privileged part where breastfeeding was also more in fashion. Interestingly, the mean nursing time was identical in diabetic babies of both regions (2.77 and 2.87 months).

45. Insulin and Insulin-like Growth Factors (I and II) Stimulate Glucose Uptake and Contractility of Isolated Perfused Rat Hearts F. Borner and E.R. Froesch. Metabolic Unit, Department of Medi- cine, University Hospital, Zurich, Switzerland Isometric contraction of intact rat hearts was measured, using the Langendorff technique of recirculating perfusion. The effects of insulin and insulin-like growth factors (IGF) on glucose uptake were compared with those on contractility. Insulin and IGF stimulate glucose uptake in a dose-dependent manner. A positive inotropic effect of insulin and IGF I and II occurred as a threshold phenomenon in a dose range above physiological hormone concentrations. On a molar basis, the hormones were equally effective in stimulating glucose uptake, whereas insulin appeared to be ten times more potent in enhancing contractility. The positive inotropic effect of insulin became detectable at 0.5 mU/ml and was maximal (20% above control) at I rnU/ml. The initial positive inotropic effect rapidly reached a maximum within 1 rain. During continued perfusion only a small further increase in contractility was observed, stabilizing at about 5 rain. Afterwards the contraction amplitude normalized steadily. The inotropic effect of insulin and IGF probably increases the energy requirement of the perfused rat heart. However, stimulation of glucose uptake by insulin and IGF is observed at hormone concentrations below those necessary for an effect on contractile force. Therefore, the effect on contractility does not appear to be a consequence of stimulated glucose uptake.

46. Direct Evidence of Various Immunological Phenomena Associated with the 'Insulitis' Process 1G. F. Bottazzo, 1, 2B. M. Dean, q.M. Mc. Nally and 3E. H. Mackay. 1Immunology Department, Middlesex Hospital; 2Medical Unit, St. Bartholomew's Hospital, London and 3Histopathology Depart- ment, Leicester General Hospital, Leicester, UK Lymphocytic infiltration of pancreatic islets as shown histologically is well established as the hallmark of Type I (insulin-dependent) diabetes. We report here, for the first time, the demonstration of immunoglobulin (IgG) deposition in islet cells and cbaracterisation of the lymphocytic infiltration detected in the pancreas of a newly diagnosed diabetic child. Frozen sections of the pancreas were stained by immunofluorescence using monoclonal antibodies to human im-

Abstracts 143

munoglobulins and various lymphocyte subpopulations. IgG deposition was seen predominantly in islets of the tail portion; the distribution was not uniform in all the cells of the islets but lgG was present around cell membranes and/or within the cytoplasm. Large lymphocytes bearing IgG were seen emanating from vessels and preferential- ly surrounding some islets. Preliminary results indicate that cytotox- • T cells constitute the main bulk of the cellular infiltrate but T-helper cells, and to a lesser extent, killer/natural killer cells were detected also. The great majority of the infiltrating lymphocytes were 'activated', showing full expression of HLA-DR molecules. These studies have demonstrated the penetration of immunoglobulins into living beta cells and have defined the specific nature of the lymphocytes involved in the 'insulitis' process.

47. Chemical Stability of Insulin: Neutral Insulin Solutions J. Brange, L. Langkjzer, S. Havelund and E. Sorensen. Novo Research Institute, Bagsv~erd, Denmark Insulin deteriorates rapidly in acid formulations due to extensive deamidation. The chemical stability is substantially improved when formulated in neutral solution, but significant transformation of insulin still occurs during prolonged storage, resulting from deamidation and covalent dimerization of the insulin molecule. This transformation occurs to the same extent irrespective of the species of the insulin (including human) and strength of preparation, but varies with the formulation of the neutral solution. Formation of insulin dimers and desamido-insulins during 2 years of storage at 4 ~ in the formulation with NaC1 and methylparaben (BP) is 0.13 +- 0.02% and 3.5 • 0.4%, respectively (mean • SEM), and in the formulation with glycerol and phenol or cresol (USP) 0.50 _+ 0.08% and 2.6 ~0.7%, respectively. Af- ter 6 months storage at 25 ~ the comparative figures are 0.31 +- 0.02% and 12,4_+ 0.5% for the BP formulation and for the USP formulation 0.70 • 0.07% and 7.7 +_ 1.3%, respectively. Whereas desamido-insulin generated in acid medium is hydrolyzed in position A-21, the products formed at neutral pH are deamidated in the B-chain partly by hydrolysis of Asn ~3, partly by an a---~/~ aspartyl rearrangement of Asn ~3 with concomitant deamidation. These desamido-insulins possess full biological potency and the immunogenicity in rabbits is not significantly different from MC insulin.

48. The Effect of Triiodothyronine on Glucose Metabolism in Healthy and Diabetic Man R Bratusch-Marrain, S. Gas• W. Waldhfiusl and A. Hofer. I. Medizi- nische Universitfitsklinik, Wien, Austria Experimental hyperthyroidism was induced by triiodothyronine (T3) administration (100 ug/day for 1 week) in healthy and diabetic subjects. Glucose utilization and insulin secretion were determined by means of the hyperglycaemic clamp technique. In seven healthy men, increasing plasma glucose by 7 retool/1 above basal, glucose utilization rose to 14.6-+2.0mg.kg-l.min -a (control: 10.6• -1. min<; 2p<0.02), whereas mean plasma insulin response (53+ 7mU/1) remained unchanged following T3 (serum T3: 2.95+ 0.25 ng/ml). In five non-insulin-dependent diabetics, glucose utilization (control: 4.3 • 0.5 mg. kg -1. min -a) was almost identical after T3 (4.4 • 0.4 mg. kg -1. min-~), as was the plasma insulin response (20 _+ 2 versus 19 _+ 2 mU/l). In the healthy subjects, splanchnic glucose output (SGO) was estimated using the liver vein catheter technique. Basal SGO (101 +_ 11 mg/min) was augmented by 60% when preceded by T3. After oral glucose loading (75 g), SGO was enhanced over 50% during the first 75 rain (2p < 0.02), but was not different from controls thereafter (cumulative SGO: control, 44.2+3.4g/150min; T3, 53.7 • 6.4 g/150 min). Arterial plasma concentrations of glucose and insulin were similar in both groups. Thus, in healthy man exposure to elevated T3 serum concentrations increases insulin sensitivity without affecting the plasma insulin response to hyperglycaemia and augments splanchnic glucose release in the basal state and after glucose ingestion. However, in non-insulin-dependent diabetic patients, exposure to T3 affects neither net glucose consumption nor insulin levels.

49. Non-Antigen Components of Circulating Immune Complexes in Se- ra of Type I (Insulin-dependent) Diabetic Patients F.J. Braun ~, G. Kratzsch t and F.Krapf 2. ~Department of Internal Medicine I, University of Ulm and 2Institute and Clinic for Clinical Immunology, University of Erlangen 2, FRG Circulating immune complexes (CIC) emerging concomitantly with the manifestation of Type I diabetes mellitus presumably participate in the destruction of pancreatic B cells and also appear at the end of the diabetic honeymoon. Identification of non-antigen components of CIC is required for assessment of their pathogenicity. 21 sera were

assayed for the presence of cytoplasmic islet cell antibodies (ICA), and immunoglobulin components (IgG, IgA, IgM) of CIC.CIC were precipitated in serum samples with 2.75% polyethylene glycol 6000. The precipitate was washed, redissolved and incubated with specific antisera. Quantitation was performed by simultaneous measurement in a fully automated laser nephelometric system. CIC of eight sera presented elevated amounts of all components; four of them revealed IgG- and/or IgM-ICA. In one precipitate IgG and IgM, and in another IgA and IgM were elevated. Two sera contained elevated amounts of IgA, isolated elevation of IgM occurred once. IgM-ICA were previously demonstrated in our laboratory, correspondingly IgM is frequently found in CIC. The IgA component may not be directed against B cell antigens. The demonstration of IgM-ICA and IgM-CIC in connection with metabolic derangement supports the al- leged pathogenetic significance of CIC.

50. The Early Identification of Renal Involvement in Diabetes Meilitus G. BrianP, B. Baggio, G. Gambaro, D. Bruttomesso ~, A. Tiengo J, A. Borsatti, G. CrepaldP. Clinica Medica I, ~Patologia Medica e Malattie del Ricambio, University of Padova, Italy The appearance of micro-albuminuria is commonly considered the earliest sign of renal damage in diabetes mellitus. This study describes an attempt to find out some markers of renal involvement more defin- itive than micro-albuminufia. To this end, the urinary excretion of glu- cosaminoglycans, which are very important in determining the barrier properties of glomerular basement membrane, as well as that of two enzymes implicated in their catabolism (fl-galactosidase and N-acetyl- fl-glucosaminidase) have been investigated in 24 patients with a mean HbAlc=9.13_+ 1.78%, with normal renal function and 24h albumin excretion < 25 mg. In comparison with control subjects, diabetic patients showed an increased urinary excretion of glucasaminoglycans (98.14 + 20.00 creatinine versus 43.34+_ 16.61 m. g./g; p < 0.001), N-acetyl-fl-glucosaminidase (52.37• creatmme versus 16.91+_ 4.96 gmol/g; p < 0.001) and/Lgalactosidase (23.44 + 11.46 creatinine versus 9.44+3.39 umol/g; p < 0.001). It is concluded that in insulin- dependent diabetic patients, before the appearance of micro-albuminuria, there are some anomalies in urine chemistry pointing to an abnormal turnover of glomerular basement membrane.

51. Proximal Glomerulo-Tubular Balance in Patients with Insulin-De- pendent Diabetes J.Brochner-Mortensen, M.Stockel, P.Sorensen, A.H. Nielsen and J. Ditzel. Department of Clinical Physiology and Department of Med- icine (Sections of Endocrinology and Nephrology), Aalborg Regional Hospital, Denmark To evaluate the glomerulo-tubular balance for sodium and water in the proximal tubules of diabetic subjects with elevated GFR, the renal plasma clearance of lithium (ClLith.) and GFR were simultaneously determined in 11 Type 1 diabetic patients and in 10 age-matched healthy subjects. C1Lith., which is a measure of the flow into the loop of Henle, did not differ between diabetic and control subjects (22.9 • 4.3 versus 22.8 • 3.7 ml/min per 1.73 m 2, mean + SD), whereas GFR in the diabetic patients was significantly higher than in the control subjects (136 + 10.2 versus 108 _+ 13.6 ml/min per 1.73 m 2, p < 0.001). The regression of absolute proximal reabsorption rate on GFR was identical for the two groups with a regression coefficient which did not differ from 1.00. The results suggest that variations of GFR in man are associated with parallel changes in the proximal tubular reabsorption rate; and indicate that this type of glomerulo-tubular balance is preserved in diabetic patients with elevated GFR.

52. Phospholipid-Sensitive Ca2+-Dependeut Protein Kinase and Phos- phorylation of the Insulin Secretory Granule K.W. Brocklehurst and J.C. Hutton. Department of Clinical Bio- chemistry, University of Cambridge, Addenbrooke's Hospital, Cam- bridge, UK Previous studies on the involvement of Ca 2 +-dependent protein phosphorylation in the stimulus-secretion coupling mechanism of insulin release have shown that insulin secretory granules isolated from a transplantable rat insulinoma will bind cytosolic components in a Ca2+-dependent manner, resulting in the CaZ+-dependent phosphorylation of 10,000, 29,000 and 100,000 Mr proteins. The possible involvement of phospholipid-sensitive Ca2+-dependent protein kinase (PK-C) in mediating this effect was investigated. PK-C activity was detected in the cytosolic fraction of an insulinoma homogenate in an assay which determined the incorporation of radioactivity from [y- ~2PIATP into lysine-rich histone, The enzymic activity bound to intact insulin granules in a Ca2+-dependent fashion and could be subse-

144 Abstracts

quently eluted by lowering the Ca 2+ concentration. PK-C activity and the enzymic activity responsible for the phosphorylation of the 29,000 Mr granule protein migrated identically on sucrose density gradient centrifugation of the cytosolic fraction. Also, in the presence of Ca 2+, phosphatidylserine-liposomes removed both these enzymic activities from the cytosolic fraction. These findings are consistent with a mechanism whereby elevation of cytosolic Ca2+may result in the at- tachment of PK-C to insulin granules and the subsequent phosphorylation of the 29,000 M~ granule membrane protein.

53. Monoclonal BB-Rat Pancreatic Islet Cell Autoantibodies Produced by Rat-Rat Hybridomas C.-H, Brogren, T. Brodin, I.Geriing and B. Mamer. Hagedorn Re- search Laboratory, Gentofte, Denmark and The Wallenberg Labora- tory, University of Lund, Lund, Sweden Various autoantibodies including islet cell surface antibodies, thymo- cyte antibodies, and rat insulinoma cell antibodies are found in serum from diabetic BB-rats using the fluorescence activated cell sorter (FACS) binding assay. The purpose of these studies was to characterize the specificity of these autoantibodies at the monoclonal level. Rat hybridomas were raised by PEG-mediated cell fusion between in vitro lipopolysaccharide stimulated spleen cells from newly diabetic BB- rats and the HAT-sensitive rat myeloma cell-line Y3. Selection for relevant clones was done by an anti-RIN membrane ELISA using an antigen coating of isolated membranes from the RIN-5F or RIN-14B insulinoma cell lines. Monoclonal autoantibodies from 10 established clones were further analysed by FACS binding assays. Two of the clones were found to be stable and to react with the cell surface of RIN-5F and RIN-14B cells, rat II fibroblasts, rat thymocytes, but not rat hepatocytes; they reacted strongly with trypsinized RIN-5F cells and with normal rat islet cells. The two monoclonal autoantibodies were both IgM. Immunofluorescence microscopy on rat thymus and pancreas tissue gave various patterns of non-islet cytoplasmic antibodies indicating that the two clones had different specificities and were reactive to epithelial and endothelial cell structures in addition to their cell surface specificities.

54. Clinical Time-course and Immunochemical Characteristics of Islet Cell Cytoplasmic Antibodies in Childhood Diabetes G. J. Bruining, J. L. Molenaar, C.W. Tuk, H.A. Bruining and J. Linde- man. Sophia Children's Hospital, Rotterdam, The Netherlands 106 patients were analyzed in a cross-sectional and eight patients in a longitudinal study for the presence of islet cell cytoplasmic antibodies (ICA) and complement fixing ICA during childhood diabetes. Con- firming earlier studies, 80% were positive for ICA within 3 months of diagnosis. 75% appeared to have complement fixing antibodies. Com- plement fixation was seen in 50%-75% throughout the first 4 years of clinical diabetes and its titres depended on the IgG-ICA titres. It was necessary to incubate lower titred sera three times to obtain sufficient IgG-ICA on the tissue section for the detection of complement-binding property. Subsequently, 44 IgG-ICA positive patient samples were analyzed for the occurrence of IgG-subclass-antibodies. The higher the ICA titre, the more IgG-subclasses were present. ICA-titres above 16 contained more subclasses, while titres below 16 demonstrated one or two IgG-subclasses. In two children, assayed 28months and 4 months before their disease became manifest, high ICA-titres were found of all IgG-subclasses. Seven patients were studied from diagnosis to 2 years later for IgG-subclasses. If titres changed little the subclass distribution was consistent. In conclusion, detection of complement fixation and IgG-subclasses is dependent on IgG-ICA titres and hence associated with the clinical time-course.

55. Residual B cell function and Enhanced Insulin degradation in Type 2 (Non-insulin-dependent) Diabetic patients with Long-term Dietary Treatment H. Bruneder 1, H.J. Klein 1 and G. SchernthanerL ~Outpatient Clinic for Diabetes, Wiener Gebietskrankenkasse and 2Department of Medi- cine II, University of Vienna, Vienna, Austria To estimate residual B cell function, basal C-peptide and insulin were determined by radioimmunoassay in 99 Type 2 diabetic patients (age 67.5_+ 9.8 years, duration of disease 8.7_+ 5.3 years, body weight 1.15 _+ 0.15 Broca index) after an overnight fast. The patients had been treated exclusively with a relatively low carbohydrate (35%-40%) diet since onset of diabetes. Plasma glucose was 7.6 • 1.5 mmol/1, haemoglobin A1 8.4_+1.4%. Thirty non-diabetic subjects (age 63.5_+ 8.4 years, body weight 1.14 _+ 0.15 Broca index) served as controls. C- peptide was similar in diabetic (0.27 + 0.07 nmol/1) and control subject (0.24 _+ 0.12 nmol/1), but insulin was significantly lower in diabet-

ic patients (8.7 _+ 9.3 mU/1, p < 0.01) than in control subjects (14.2 + 5.6 mU/1). The molar C-peptide/insulin ratio was significantly higher in diabetic patients (4.3_+1.0, p <0.001) than in control subjects (2.3 _+ 2.9). Basal insulin and C-pepfide showed, by analysis of variance, no significant correlation regarding plasma glucose, haemoglobin A1 or duration of diabetes. A significant positive correlation between body weight (Broca index~< 1.05 versus 1.06-1.20 versus> 1.20) and basal insulin (4.5_+2.7 versus 8.0_+5.4 versus 14.1_+ 14.0 mU/1) as well as C-peptide (0.26 _+ 0.05 versus 0.28 _+ 0.09 versus 0.30 _+ 0.07 nmol/1) was observed. In summary, increased B cell function is much more infrequently found in Type 2 diabetic patients with long-term dietary treatment, as previously believed. B cell function shows a positive correlation with body weight, but not with duration of disease and metabolic control (haemoglobin A1, plasma glucose). The higher molar C-peptide/insulin ratio could be caused by an enhanced degradation of insulin.

56. Treatment of Insulin Allergy with Semi-synthetic Human Insulin B. Bruni, M.Campana, M.Carlini, S.Gamba Ansaldi, G.Grassi, M. Spinelli, T. Winkler and G. Regis. Karen Bruni Diabetes Centre and Cattedra Medicina Nucleare, University of Tofino, Italy Monocomponent semi-synthetic human insulin is theoretically indicated in insulin allergy and lipo-atrophy (hypertrophy) persisting when monocomponent (MC) porcine or mixed species insulin is used. Eight insulin-dependent diabetic patients (seven women and one man, 18-64 years, insulin requirement 24-76 U/day) allergic to Mon- otard, Actrapid MC, Lente MC and Rapitard MC (three generalised and local-delayed, three local-immediate and delayed, two local delayed allergy, with associated lipoatrophy in three cases) and two with isolated lipoatrophy to Rapitard MC and lipohypertrophy to Mono- tard were placed on human Monotard and Actrapid. All subjects had high insulin IgG ( > 3 mU/ml : Christiansen) and detectable insulin- specific IgE (> 0.7 U/ml : Falholt). Intracutaneous allergy tests were positive to human, porcine and bovine insulin in six cases; one case was positive to diluting mediums and one to porcine insulin only. Tests were negative in diabetic patients with isolated lipoatrophy and lipohypertrophy. After 6 months, local allergy disappeared in five cases and was reduced in three; systemic allergy disappeared. No lipoatrophy or hypertrophy occurred in new injection areas. In two cases a striking reduction of insulin requirement was noted. However, high levels of IgG antibodies werenot significantly decreased and IgE antibodies were still detectable. Skin positivity to human insulin was unchanged or even enhanced.

57. Action of Glucose and Insulin on Pancreatic Enzyme Release R. Bruzzone, E. R. Trimble, A. Gjinovci and A. E. Renold. Institut de Biochimie Clinique, Geneva, Switzerland Although insulin is known to promote pancreatic amylase biosynthesis, the influences of glucose and insulin on pancreatic exocrine secretion are not well defined. To study their role in enzyme release, we used the perfused rat pancreas with cannulation of the Wirsung duct. The glucose concentration was kept constant at either 5 mmol/l or 15 mmol/1 (the latter to stimulate insulin release) and the cholecysto- kinin analogue (caerulein) was infused successively at 10 -1~, 10-1~ and "] 0 -9 tool/1 (to stimulate enzyme secretion). At 5 mmol/1 glucose, basal secretion of amylase, lipase and chymotrypsinogen was four times higher than at 15 mmol/1 glucose. At 5 mmol/1 glucose also, 10 -a~ mol/l caerulein gave the most potent stimulation of enzyme secretion (approximately ten-fold) with (as expected) 10 -9 mol/l being less potent. The results at 15 mmol/l glucose were markedly different from those at 5 retool/1 in that the stimulatory effects of 10 -1~ and 10 -l~ tool/1 caerulein were significantly reduced. In addition, the profile of the dose response curve was altered with 10 -l~ and 10 -9 mol/1 caerulein being equipotent to stimulate enzyme release. When the amount of insulin secreted at 15 mmol/l glucose was given exogenously during perfusion with 5 mmol/1 glucose, the enzyme response to caerulein was markedly reduced. In conclusion, glucose and insulin inter- act to alter pancreatic enzyme secretion and to decrease the secretory outputs under basal and stimulated conditions.

58. Urine Glucose Estimation, an Unreliable Indicator of Diabetes Con- trol J. Bubeck, W. Beischer and C. Schomann-Ziesenb6ck. Department of Internal Medicine I, University of Ulm, FRG How does the estimation of urine glucose, still a widespread technique of self-monitoring, reflect diabetes control? The test programme included simultaneous estimations and measurements of blood glucose (Haemoglucotest 20-800/Gluco-quant, ACP 5040) and

Abstracts 145

urine glucose (Diabur 5000/Gluco-quant, ACP 5040; double voiding technique) four times daily for at least 10 days. Forty-seven diabetic patients participated, aged 17-76 years. 37 were insulin-treated, 25 had retinopathy, seven had disturbed colour vision, and two had raised serum creatinine. Equivalent correlations between estimation and measurement were reached for blood glucose more frequently than for urine glucose. The renal threshold for glucose was rarely obvious and had to be determined by definition. About half of the patients showed false negative or positive excretion at glucose concentrations > 20% above or below the defined glucose threshold respectively. The defined threshold varied individually between 7.5 and 15.8 mmol/1. Individual blood glucose concentrations differed by up to 9 mmol/1 at the urine glucose level estimated most frequently in each patient. For multiple reasons urine glucose estimates are not a reliable indicator of diabetes control in the majority of diabetic patients.

59. Adaptations of Glucose Metabolism During Lactation in the Rat A.F.Burnol, A.Leturque, P.Ferre and J.Girard. Centre de Re- cherches sur la Nutrition, CNRS, Meudon-Bellevue, France In the post-absorptive state, glucose turnover rate and metabolic clearance rate were increased from 3 days onwards in lactating rats when compared with age-matched non-lactating rats. At peak lactation (12-19 days) glucose turnover (17 umol/min) was 80% higher than in non-lactating rats (9.6 p,mol/min) or in lactating rats 24 h after weaning (11.5 ~mol/min). Basal levels of blood glucose (4.2 mmol/1) and plasma insulin (16 mU/l) were lower in lactating than in non-lactating (5.2 mmol, 1.50 mU/l) and weaned rats (4.8 mmol, 1.72 mU/1 respectively). During an IV glucose tolerance test, glucose disappearance is faster in lactating (9%/rain) than in non-lactating and weaned rats (4%/min). Insulin secretion during the IV glucose tolerance test was similar in lactating and non-lactating rats, but was increased threefold in weaned rats, suggesting an insulin resistant state in non- mammary tissues during lactation. During euglycaemic hyperinsulinaemic (3 500 mU/1) clamp, glucose clearance was increased threefold in non-lactating and lactating rats, and twofold in weaned rats, suggesting that glucose metabolism in mammary gland is stimulated by insulin. This was confirmed in vivo by demonstrating that lipogenesis from 3H20 in mammary gland is stimulated threefold during the clamp, whereas lipogenesis is not stimulated in white adipose tissue.

60. A Prospective Study of Lymphocyte Subsets in Newly Diagnosed Type I (Insulin-Dependent) Diabetic Patients K. Buschard, C. R6pke, J. Mehlsen, S. Madsbad, K. Birch and J. Ry- gaard. Pathological-Anatomical Institute, Kommunehospitalet, Cop- enhagen and Hvidore Hospital, Klampenborg, Denmark T-Lymphocyte subsets in peripheral blood from I 1 newly diagnosed Type I diabetic patients were studied prospectively three times: 1 week, 3 weeks, and 5 months after diagnosis. Lymphocytes were marked with monoclonal OKT antibodies and examined by flow cy- tometry (FACS). No changes were found in percentages of T-lymphocytes (OKT 3). The percentage of helper/inducer T-cells (OKT 4) was 47.3 _+ 2.4 at diagnosis, but decreased at 5 months to 44.1 _+ 2.6 (p < 0.05). The percentage of suppressor/cytotoxic T-cells (OKT 8) was 21.6_+'1.3 at diagnosis hut increased at 3 weeks to 25.9-+2.2% and at 5 months to 24.2_+ 1.1% (p < 0.01). The ratio OKT 4/OKT 8 lymphocytes was 2.28 _+ 0.20 at diagnosis, decreasing to 1.77 + 0.19 at 3 weeks and 1.87 -+ 0.17 at 5 months, (p < 0.001) compared with 1.46 -+ 0.11 in 16 age-matched control subjects. There was no change in the absolute numbers of lymphocytes. There was no correlation between the distribution of subpopulations and glycaemic control. Massive improvement in glycaemic control during a week in other patients with diabetes for H + 4 y e a r s did not change the OKT 4 /OKT 8 ratio (1.81 _+0.31 versus 1.82-+0.23). In conclusion, distribution of T-cell subsets is abnormal at time of diagnosis but changes towards normal within a few weeks. No influence of the glycaemic status could be shown.

61. Secretion and Hepatic Removal of Insulin in Chronic Persistent Hepatitis G. Buzzelli, E. Bonora% C.Coscelli% S.Orioli, U. Butturini ~ and P.Gentilini. Clinica Medica Generale, University Medical Schools, Florence and 1Parma, Italy Hyperinsulinaemia is a consistent finding in chronic active hepatitis. It has been explained by diminished hepatic removal rather than pancreatic hypersecretion of the hormone. The present study was designed to assess insulin secretion and removal in chronic persistent hepatitis (CPH) compared with chronic active hepatitis (CAH). For

this purpose, eight subjects with CPH, eight individuals with CAH, and eight healthy control subjects underwent an oral glucose tolerance test (100 g). Collected blood was used for determination of glucose, insulin and C-peptide concentrations. C-peptide to insulin molar ratios in the fasting state and after a glucose load, as well as relationships between incremental areas of C-peptide and insulin, were used for estimating the hepatic removal of insulin. Subjects with CPH showed glucose and insulin levels comparable to those of the healthy control subjects, whereas individuals with CAH had significantly higher glucose and insulin concentrations. C-peptide responses to oral glucose were similar in the three groups. C-peptide to insulin ratios and relationships did not differ from control subjects in patients with CPH, whereas they were significantly lower in individuals with CAH. These results demonstrate that the diminished hepatic removal of insulin underlying hyperinsulinism of subjects with chronic active hepatitis does not occur in individuals with chronic persistent hepatitis.

62. Molecular Basis for Cationization of Glyeosylalbumin: Importance of Three-Dimensional Structure of Albumin G. Candiano, G. M. Ghiggeri, G. Delfino and C. Queirolo. Haemodi- alysis Service, Hospital of Lavagna, Italy The demonstration of cationic subunits of glycosylalbumin in diabetic sera suggests their implication in the pathogenesis of diabetic nephropathy, as cationic albumins have been shown to be toxic for the glomerulus. The molecular basis for cationization is unclear, in fact, as known, condensation of glucose on amino-groups of albumin would affect a decrease of its isoelectric point. The aim of this study is to explain the anomalous isoelectric point of cationic glycosylalbumin by a derangement of three-dimensional structure of the molecule induced by post-synthetic disulphide breakdown. Albumin was purified from diabetic sera with Blu-Sepharose CL-6B, ultrafiltrated and submitted to chromatography on Concanavalin-A Sepharose. Sul- phydryl groups were determined with 5,5'-(dithiobis(2-nitrobenzoic acid) using cysteine as standard and expressed as nmol per nmol of albumin. Albumin with affinity for lectins had increased contents both of hydroxymethylfuffural, both of sulphydryl groups (0.41 versus 2.52) and a wide range of isoelectric points from 4 to 9. The results show that glycosylation produces breakdowns of albumin disulphide bridges and charged amino-acidic groups buried in disulphide loops get exposed and mediate the increase of albumin isoelectric points.

63. Insulin Resistance in Liver Cirrhosis: an 'in Vivo' Evaluation by Glucose Clamp and 'in Vitro' Study of Insulin Receptor and Glucose Transport M. Cassader, P. Cavallo-Perin, A. Bruno, A.M. Dall'Omo, C. Bozzo, P. Masciola and G. Pagano. Institute of Internal Medicine, University of Turin, Italy To clarify the mechanism of insulin resistance in hepatic cirrhosis, 18 non-diabetic cirrhotic patients (CP) (liver biopsy) and 18 healthy control subjects, age-, sex-matched, were submitted to three euglycaemic clamps (at-100,-1,000 and-10,000 mU/1 steady-state plasma insulin) preceded by 3-3H-glucose infusion (to calculate endogenous glucose production). In five CP and 11 control subjects, 3-0-methyl-glucose transport and % insutin binding were performed on isolated adipocytes. Basal glucose production was similar in CP and controls, but the suppression was incomplete a t -100mU/1 in CP (0.71_+ 0.18 mg- kg -1. min -1, mean _+ S EM); glucose metabolic clearance rate (mg. kg -1. min -l) decreased significantly in CP at -100 mU/1 (2.7 _+ 0.5 versus 8.4-+0.8, p<0.001) and- l ,000mU/1 (4.6-+0.9 versus 12.5+2.1, p<0.00l), but not at -10,000mU/l (13.7_+0.8 versus 14.3_+0.7, NS). Basal 3-0-methyl-glucose transport was significantly lower in CP (0.25 -+ 0.03 versus 0.54 _+ 0.11 pmol/10 s cells, p < 0.001), but the increase at maximal insulin concentration (5 ng/ml) was similar (0.51-+0.07 versus 0.38+0.13, NS); percentage insulin binding was lower in CP (2.6 -+ 0.28 versus 3.93 _+ 0.35%, p < 0.02) without any change in affinity profile (Ke). In conclusion, insulin resistance in cirrhosis is mainly dependent on a receptor defect (rightward shift of the dose-response curve); a post-receptor defect could be excluded (maximum insulin effect in CP both in vivo and in vitro).

64. Effect of Hypothermia on Insulin Secretion: Comparative Study with Hibernating (Hedgehog and Edible Dormouse) and Non-Hibernat- ing Mammals (Rat) C. Castex, R. Hoo-Paris, A.Tahri and B. Ch. J. Sutter. Laboratory of Endocrinology, University of Bordeaux I, Talence, France Hibernating mammals in dormancy can thermoregulate at near-freez- ing temperatures, while non-hibernating mammals cannot survive for

146 Abstracts

long periods at temperatures below 20 ~ Arousal of a mammal from artificial hypothermia requires the reintroduction of external heat but the arousal from natural hibernation depends upon some intrinsic heat-generating mechanisms controlled by hormones. The aim of this study was to compare the thermo-sensitivity of the B cell of two hibernating mammals with that of a non-hibernating mammal the rat. When the pancreases of the hibernating mammals were perfused or perifused with a 16.8 mmol/l glucose medium from 7 ~ to 37 ~ insulin secretion increased at 15 ~ and the first phase of insulin output was higher at 27 ~ than at 37 ~ In the same experimental conditions, the insulin secretion by rat pancreases rose only from 25 ~ of perfusion medium temperature and increased progressively until 37 ~ These results shown that certain biochemical adjustements or a process of acclimatization takes place in the hibernators, i. e. the presence of a particular pool of cold-adapted enzymes or changes in the molecular mechanisms of cell membranes.

65. A Comparison Between Biosynthetic Human and Bovine Insulin in Normal Man by the Glucose Clamp Technique P. Cavallo-Perin, A. Bruno, A. Ozzello, A.M. Dall'Omo and G. Paga- no. Institute of Internal Medicine, University of Turin, Italy To compare biological activity in vivo of biosynthetic human insulin (BHI) and bovine regular insulin, in 18 healthy volunteers, we performed a 3-3H-glucose infusion (1 ~tCi. rain -1- 240 min -1) and an euglycaemic glucose clamp (started at 120 rain) at three different insulin infusion rates for 120 min on three separate occasions: 40 m U / m 2 per min (six subjects), 240 m U / m ~ per min (six subjects) and 1200 m U / m 2 per rain (six subjects). The sequences BHI-bovine were randomized. Glucose metabolic clearance rate values (ml. kg -1. min -~ = DeFronzo M/blood glucose in the same interval) were not significantly different between BHI and bovine: 8.36 + 0.88 versus 8.44 +- 0.81 at 40 m U / m 2 per rain; (mean_+ SEM) 13.38 + 1.53 versus 12.53 + 1.11 at 240 m U / m 2 per min; 15.17 + 0.50 versus 14.32_+ 0.71 at 1200 m U / m 2 per rain. Endogenous glucose production was completely suppressed both with BHI and bovine. Insulin metabolic clearance rate (ml-kg -l. min-l=insulin infusion rate/increase in insulin above basal): 16.01 + 1.52 versus 14.36+0.72 and basal delivery rate (mU.kg -a- min -1 = metabolic clearance rate x basal insulin/1000) : 0.17 _+ 0.03 versus 0.22 __+_ 0.02 were not significantly different between BHI and bovine. These data indicate that BHI and bovine insulin, administered IV at the same doses produce similar biological activity in vivo, both at hepatic level (suppression of glucose production) and at peripheral level (stimulation of glucose utilisation).

66. Influence of Acute Exercise on Platelet Aggregates Ratio in Healthy Subjects and in Type I (Insulin-Dependent) Diabetic Patients F. Cavalot, M. Trovati, G. Tamponi, P. C. Schinco, M. Bazzan, A. Pan- nocchia, Q. Carta, L. Monge, S. Vitali and G. Lenti. Institute of Inter- nal Medicine, University of Turin and Centro Antidiabetico of the S. Giovanni Battista Hospital, Turin, Italy The literature shows that muscular exercise increases fl-thromboglobulin levels in healthy subjects, but not in Type I diabetic patients presenting an already elevated basal fl-thromboglobulin concentration. To investigate the.exercise-induced platelet activation in diabetes mellitus with a different methodology, we measured the platelet aggre ~ gates ratio (PAR) by the Wu and Hoack technique modified according to the Paris Workshop (n. v. 0.76-1.1) in six healthy males subjects (aged 20-30 years) and in 23 Type I diabetic patients, matched for age and sex, without angiopathic complications. Subjects and patients were submitted, 2 h after breakfast, to the following test: 30 min exercise on a treadmill at about 40% VO2max, 30 min rest, 30 min exercise at about 60% VO2max, 30 min rest. PAR was measured before and after these five periods (times A, B, C, D, E). PAR (mean + SD) was: (a) in healthy subjects: 0.87 + 0.08 (time A), 0.80+ 0.13 (time B), 0.82 +- 0.11 (time C), 0.79 + 0.19 (time D), 0.78 _+ 0.18 (time E); (b) in Type I diabetic patients: 0.86 +- 0.12 (time A), 0.77 + 0.12 (time B: p < 0.05 versus time A), 0.88 + 0.09 (time C), 0.75 +- 0.14 (time D: p < 0.005 versus time A and time C), 0.85 +- 0.10 (time E). In conclusion, when diabetic patients do not present a basal platelet hyperactivation, they do not have a blunted aggregatory response to exercise. On the contrary, they show an even greater PAR reduction (i.e. platelet activation) than the control subjects.

67. Ano-rectal Manometry as an Evaluating Test for Impaired Ano-ree- tal Function in Diabetes F. Caviezel, A. Bossi, A. Baresi, A. Salvini and G. Pozza. Department of Clinical Medicine, Ospedale S. Raffaele and Continence Centre, University of Milan, Milan, Italy

Diarrhoea and/or rectal incontinence may represent a sign of autonomic neuropathy in diabetes. The present investigation was performed to study ano-rectal (A-R) function and reactivity to appropriate stimuli in 13 diabetic patients with or without autonomic neuropathy (five non-insulin-dependent, eight insulin-dependent, mean age: 44.2 years, mean duration of diabetes: 11.3 years). Twenty- five healthy subjects (mean age: 43.5 years) were studied as controls. All subjects underwent an A - R manometry by means of special open- ended-tip cathers connected to a six-channel polygraph. A rectal latex balloon was inflated with 30 or 60 ml of air to induce a stimulus which in normal conditions is able to relax the internal sphincter and con- tract the external one (A-R inhibitory reflex). Six diabetic patients had symptoms and signs of autonomic neuropathy. Marked abnormalities of A - R function were found in all diabetic patients (but one) with autonomic neuropathy (i. e. no response even to maximum stimulus or contraction of both sphincters), while all non-neuropathic patients presented a normal pattern of A - R manometry. A relationship between abnormal response to rectal stimulus and the presence of an autonomic neuropathy involving pelvic parasympathetic section or regional intramural plexuses may be suspected.

68. Insulin Resistance Versus Impaired Insulin Secretion in Type 2 (Non- Insulin-Dependent) Diabetes: Predominance of the Role of the Pan- creas E. Cerasi, R. Nesher, Y. Litvin and L. Della Casa. Department of En- docrinology and Metabolism, Hebrew University Hadassah Medical Center, Jerusalem, Israel, and Department of Medicine, University of Modena Medical School, Modena, Italy The role of insulin secretion versus insulin resistance for glucose tolerance was evaluated in 17 control subjects, seven lean Type 2 diabetic, 12 normoglycaemic obese, and 14 obese-diabetic patients matched for weight with the normoglycaemic obese and for hyperglycaemia with lean-diabetic patients. Pancreatic responsiveness was determined by measuring endogenous insulin response to IV glucose boluses (0.1, 0.3 and 0.9 g/kg at 75 rain intervals). Peripheral sensitivity to exogenous insulin was determined by measuring changes in K-glucose (0.3 g/kg IV glucose) in response to 25, 50 and 100 mU/kg insulin (75 min intervals) under somatostatin blockade. Obese subjects showed increased Vrnax of the glucose-endogenous insulin dose-response curve, while all the diabetic patients revealed minimal responsiveness. The dose-response curve of K-glucose versus exogenous insulin showed a marked shift to the right of control in the normoglycaemic obese and obese diabetic patients, the latter two being similar. The response in lean- diabetic patients was slightly lower than the control subjects only at the lowest insulin dose, the curves being otherwise superimposable. Thus, the pancreatic response to glucose separated subjects as diabetic/non-diabetic (without considering obesity), while the peripheral response to insulin separated them as obese/non-obese (without considering diabetes). Thus the principal defect in Type 2 diabetes is islet deficiency, insulin resistance playing a secondary role by aggravating the metabolic consequence of insulin deficiency.

69. Near-Normal Control of Glycaemia Does Not Retard Progression of Mild Diabetic Retinopathy The Kroc Study Group: London, Canada; London, UK; Chicago, IL; Rochester, MN; New Haven, CT, USA We sought to determine in a prospective multicentre study whether improvement of control of glycaemia affects progression of background retinopathy (BR). Patients with mild BR and absence of C- peptide were randomly assigned to pump (n =35) or conventional therapy (n = 34). Glycaemia and retinopathy were assessed throughout the subsequent 8-10 months. Age, duration of diabetes, insulin dosage, glycaemic control and degree of retinopathy were similar in the two groups. The mean diurnal glycaemia in both groups were similar at onset and did not change in the conventionally treated group. Mean diurnal glycaemia 10.3 +_0.5 mmol/l with glycosylated haemoglobin 11.0 + 0.3 % fell to 6.4 + 0.3 mmol/1 and 8.7 _+ 0.3 % respectively during insulin pump therapy. BR was assessed by fundus photography (Wisconsin Reading Centre, USA) and microaneurysm counts on fluorescein angiograms. Progression of BR occurred by both criteria particularly in those patients with relatively mild BR. The rate of progression of BR was at least as rapid in the pump-treated as in the conventionally-treated group. In conclusion, (1) substantial improvement of glycaemic control for 8-10 months does not prevent progression of BR even when BR is mild, and (2) these results suggest that a primary prevention trial may be required to determine if intensive treatment affects diabetic retinopathy.

Abstracts 147

70. Comparison of Highly Concentrated Versus Diluted Insulin for Continuous Subcutaneous Insulin Infusion E. Chantelau, A. Rajab, G.E. Sonnenberg, V. JSrgens, H.J. Ciippers and M. Berger. Medical Department E, University of Dtisseldorf, FRG Continuous subcutaneous insulin infusion (CSII) therapy with Mill Hill Infusers HM 1001 is usually performed utilizing regular insulin diluted in saline to 15 U/ml ; newer insulin pumps require highly concentrated insulin. Pharmacokinetics of identical doses of regular insulin in strengths of 15 U/ml (concentration 1), 40 U/ml (concentration 2) and 100 U/ml (concentration 3) were investigated in six healthy normal weight men. In randomized order, 10 U of monocomponent porcine regular insulin were administered SC via Butterfly cannula into the abdominal wall, using a switched-off Mill Hill Infuser HM 1001. Insulin was delivered as bolus by turning the manual injection knob (boost dose/half-turn 53 .ul). Blood glucose and serum insulin were measured at intervals of 10 to 15 rain during 3 h with the following results (mean):serum insulin maximum 35.5 mU/l (concentration 1) versus 25.5 mU/l (concentration 3) NS; serum insulin increment above base-line level (area under the curve) 3 069 (concentration 1) versus 2 023 mU-1-1.180 min -1 (concentration 3), p < 0.05 ; blood glucose minimum 2.2 (concentration 1) versus 2.6 mmol/l (concentration 3) NS; time of blood glucose minimum post injection 73 rain (concentration 5) versus 93 min (concentration 3), p < 0.05. The differences between concentrations 2 and 1 or concentration 3 were not statistically significant. In conclusion, regular insulin at strength 15 U/ml appears to be more efficient for bolus injection during CSII than regular insulin at strength 100 U/ml.

71. Insulin Secretion and Insulin Hepatic Extraction in Obese Subjects with Normal Glucose Tolerance V. Cheli, P. Buzzo, P. L. Melga, E. Ansaldi, P. Odetti, G. Falzetti and R. Prando. Department of Internal Medicine, Metabolic Diseases Ser- vice, University of Genoa, Italy It is well known that C-peptide concentration in peripheral blood pro- vides a more accurate assessment of insulin secretion. We have performed C-peptide and insulin determinations after an oral glucose load (100 g) in obese subjects with normal glucose tolerance to evaluate whether the hyperinsulinaemia is due to an increased pancreatic secretion or to a decreased hepatic extraction. Obese subjects were grouped into normo-responders (Ob-I) and high-responders (Ob-II) according to their insulin incremental areas. Only incremental areas 0-30 were considered since glucose incremental areas 0-30 were superimposable in different groups. A significant relationship between glucose and C-peptide incremental areas 0-30 was found in normal subjects (r= 0.83 ; p < 0.001), in Ob-I (r= 0.76; p < 0.001), but not in OB-II (r=0A7). According to the 95% confidence limits for a single observation around the regression line of normal subjects, we observed that only seven subjects out of 12 Ob-II were high-responders. The molar ratio between C-peptide and insulin incremental areas 0 30 was reduced in Ob-I (2.49 + 0.21 ; p < 0.02) and especially so in Ob-II (1.84 _+ 0.09; p < 0.001) compared with normal subjects (3.72 + 0.33). In conclusion, peripheral hyperinsulinaemia after glucose in obese subjects is mainly due to an impaired insulin hepatic extraction and partly to an early increased insulin secretion.

72. Interactions of Glucose and Insulin on Glucagon and Pancreatic Polypeptide Secretion: Studies in Normal and Diabetic Dogs D. Chenon, E.R.Trimble% G. Ribes and M.M. Loubati~res-Mariani. Facult6 de M6dicine, Laboratoire de Pharmacologic et de Pharmaco- dynamic, ERA 786 du CNRS, Montpellier, France, and JInstitut de Biochimie Clinique, Universit6 de Gen~ve, Switzerland It has been reported that both plasma glucagon and pancreatic polypeptide (PP) concentrations are elevated in diabetes. The present study was undertaken to test whether hyperglycaemia per se or insulin lack is responsible for these abnormalities. Experiments were performed on 18 h fasted non-diabetic and alloxan-diabetic dogs. In conscious, non-diabetic dogs glucose (0.2 g/kg, IV) decreased peripheral plasma glucagon and PP and increased insulin concentrations. Simi- lar results were obtained from the pancreaticoduodenal vein of anaesthetized non-diabetic dogs, where somatostatin levels were also raised after glucose. Insulin (0.2 U/kg, IV) given to conscious, non- diabetic dogs caused hypoglycaemia and stimulated PP secretion only after 20 min, when blood glucose levels fell below 3 retool/1. Diabetic dogs treated by daily injections of insulin had fasting blood glucose levels of "17-8 retool/1. Insulin injection (0.2 U/kg, IV) caused an immediate reduction of the initially high levels of plasma glucagon and PE These effects occurred before any significant change in blood glu-

cose, which progressively fell to 8-9 mmol/1 at 60 min. The results suggest that secretion of PP and glucagon can be suppressed in vivo by glucose only in the presence of sufficient amounts of insulin.

73. Early or Incipient Diabetic Nephropathy and Effect of Anti-hypertensive treatment C.K.Christensen. Second University Clinic of Internal Medicine, Kommunehospitalet, Aarhus, Denmark Kidney function was studied in eight male juvenile diabetic patients with incipient diabetic nephropathy (D3; no clinical proteinuria but albumin excretion> 15 ~tg/min), 10control subjects, eight diabetic patients with normal albumin excretion (D2) and in 10 diabetic patients with overt nephropathy (D4). GFR in D3 patients was at the same level as in D2 and elevated compared with control subjects. RPF was significantly lowered compared to controls but similar to D2. In D3 patients mean blood pressure and renal vascular resistance were elevated compared to controls and D2. Seven D3 patients were followed for 5.9 years. Diastolic blood pressure increased significantly (86+9 to 92+12mmHg; mean + SD; 2p=2.8%). GFR was unchanged (142 _+ 16 to 139 +_ 18; NS.) but RPF decreased from 544 _+ 51 to 489 + 29 (2p=2.9%). Albumin excretion increased by a mean of 25~g.min-1 year 1 in seven D4 albumin excretion increased 2300 ~tg. min 1.year-% in both groups correlated to blood pressure. Seven D3 patients were followed for a mean period of 7 months during anti-hypertensive treatment with metoprolol. We found a significantly positive correlation between change in blood pressure and change in albumin excretion (r= 0.89, 2p= 0.67%). Anti-hypertensive treatment may prove useful in preserving the kidney function in patients with incipient diabetic nephropathy.

74. Improvement of Autonomic Nerve Functions After One Year of Con- tinuous Subcutaneous Insulin Infusion I. Cicmir, S. Kashiwagi, H. Berger, T. Koschinsky and F. A. Gries, Dia- betes Research Institute, D%seldorf, FRG The long-term effect of improved metabolic control on autonomic nerve functions has been studied in 19 Type I (insulin-dependent) diabetic patients (10 males, 9 females, mean age: 30 years, duration of diabetes: 13 years, HbA1 9.7%) during 1 year of insulin pump therapy (HbA1 after 1 year: 8.2%;p < 0.05). The coefficient of variation of the beat-to-beat variation at rest (parasympathetic) has been quantified with the Neurocard-analyzer, and the speed of mydriasis (sympathetic) with the infrared pupillometer. The mean coefficient of variation and speed of mydriasis did not change until the third month. Thereaf- ter, the coefficent of variation increased steadily from 2.0% to 2.7% (p< 0.005) and the speed of mydriasis from 0.73 to 1.13 mm/s (p< 0.005) during 1 year of insulin pump therapy. The significant improvement occurred in all patients, those with both autonomic nerve functions still in the normal range (n = 9), as well as those with impaired autonomic nerve functions (n= 10). In conclusion, a stable long-term improvement of metabolic control in Type 1 diabetic patients can improve even severely impaired parasympathetic and sympathetic nerve functions of the heart by 25% and of the eye by 55% within 1 year.

75. Impairment of Insulin Action After Acute Cortisol Excess in Man D.Clerc, H.Wick and U. Keller. Division of Endocrinology and Metabolism, University Hospital, Basel, Switzerland Stress or cortisol therapy results in acute elevation of plasma cortisol. To assess the impact of acute cortisol excess in insulin sensitivity, glucose production and peripheral clearance (3-3H-glucose infusion), plasma non-esterified fatty acid (NEFA), amino acid and C-peptide levels were determined during a euglycaemic insulin clamp. Nine normal subjects were studied 7 h after 80 mg hydrocortisone and after placebo. Plasma cortisol levels peaked at 2 h (2.8 ~.mol/1) but had declined by 7 h. After hydrocortisone, modest hyperglycaemia and elevated levels of branched chain amino acids, alanine, insulin and C- peptide ensued. During the clamp (insulin infusion of 20 m U / m 2 per min for 120 min, plasma insulin of - 5 3 mU/1), glucose production was less suppressed after hydrocortisone than after placebo (to 0.4 + 0.1 versus 0.13_+ 0.1 mg. kg -1.rain -1 =p < 0.05), glucose clearance increased less (to 2.4+0.2versus 3.3 +0.3 ml .kg- l -min 1, p<0.01) and plasma C-peptide decreased less after hydrocortisone than after placebo (0.42+0.08 versus 0A5+_0.02nmol/l, p< 0.001). Plasma NEFA and amino acids were similarly lowered during insulin after hydrocortisone and placebo. Thus, acute elevation of plasma cortisol results after 7 h in (1) insulin resistance of peripheral clearance and hepatic production of glucose, (2) diminished insulin-induced inhibition of insulin secretion, and (3) unimpaired insulin action on plasma NEFA and branched-chain amino acids.

148 Abstracts

76. Prolonged Hypoglycaemic Clamp Technique to Evaluate Intrave- nous and Subcutaneous Kinetics of Porcine and Human Insulin in Nor- mal Subjects and Type I (Insulin-Dependent) Diabetic Patients C. Cobelli, A. Mari, S. Del Prato, E. Duner, A. Frigato, R. Fabris, A. Tiengo, G. Crepaldi, A. Berger and L. Heding. University of Pado- va, Italy and Novo, Denmark Porcine and human insulin kinetics were investigated during prolonged hypoglycaemic clamp (2.78 retool/l) in order to inhibit endogenous insulin secretion and to achieve stable insulin and glucose plateaux so that insulin clearance (PCR) and bioavailability are com- puted rigorously on the same day. Five normals and seven newly diagnosed Type 1 diabetic patients were studied during 12 h clamp (2 h stabilization, 2 h IV load, 8 h SC load) with constant IV insulin infusion (0.5 mU.min -1 .kg -1 and 0.75 U IV and 0.15 U/kg by SC injection. In normal subjects, PCR was also evaluated at euglycaemia. En- dogenous insulin secretion was completely suppressed (C-peptide nearly undetectable). Absorption after SC injection was brisker and higher in diabetic patients for both insulins, and generally spiky. Re- sults: (1) a two compartmental model was necessary to describe the IV data; (2) PCR (ml/min, mean + SD) was 1142_+ 180, 1304 + 516 in normal subjects and 1274_+433, 1315 +476 in diabetic patients for porcine and human insulin respectively ; (3) bioavailability was higher in diabetic patients (87 +_ 32, 90 _+ 25 for porcine and human insulin) than in normal subjects (56_+ 14, 63_+ 39); (4) hypoglycaemia increases PCR (1327_+ 250 euglycaemic, 1512+ 116 hypoglycaemic, p < 0.05); (5) glucose requirements normalized to glucose and insulin concentrations (mg. min-lml -~ per rag.1-1, mU 1) were higher during SC for human insulin (6.2-+ 2.3 in normal subjects, 8.3 -+ 6.0 in diabetic patients) than for porcine insulin (3.8 -+4.5 in normal subjects; 4.2 _+ 3.1 in diabetic patients, p < 0.05 normal and diabetics pooled.

77. The Dawn Phenomenon in Type I (Insulin-Dependent) Diabetic Pat- ients Treated by Continuous Subcutaneous Insulin Infusion A. Consoli, F. Capani, F. Della Loggia 1, M. Massi-Benedetti, E. Vita- colonna and S. Sensi. Istituto di Clinica Medica, Universitfi di Chieti and Istituto di Patologia Medica, Universitfi di Perugia, Italy The optimization of basalinsulin infusion during continuous SC insulin infusion (CSII) treatment is usually based on blood glucose values in the morning before breakfast. This approach has been criticized because of the occurrence of the dawn phenomenon, i.e. the increase of blood glucose in the early morning. Hence the possibility occurs that, when a near-normal glycaemic value in the morning is obtained, asymptomatic hypoglycaemia may occur during the night. In order to verify the existence and if so, to quantify this phenomenon, 14 Type 1 diabetic patients treated by CSII were admitted to hospital for one night. Blood glucose, free insulin, serum cortisol and plasma non-esterified fatty acids (NEFA) were measured at 24.00, 02.00, 04.00, 06.00 and 08.00 h, without interruption of sleep. Mean_+ SEM blood glucose was: 24.00h: 3.95+_0.41; 02.00h: 4.92_+0.39; 04.00h: 5.45+ 0.43; 06.00 h: 5.91 -+0.44; 08.00 h: 5.46+0.41 mmol/1. The mean value at midnight was significantly lower (paired t test) than the morning values (24.00 versus 04.00 h : p < 0.02; 24.00 versus 06.00 h : p < 0.005; 24.00 versus 08.00 h: .p< 0.005). It is concluded that the dawn phenomenon occurs, its magnitude is small (A 24.00/06.00 h: 2.03 retool/l) and we suggest that blood glucose values before breakfast should be maintained at levels not lower than 5.5 mmol/l.

78. Dissociation and Degradation of Specifically Bound Insulin: Effect of a Sulphonylurea in Vitro R. Cordera, G. Chimini, M. Bagnasco and R. Gherzi. Department of Internal Medicine, University of Genoa, Italy Discordant data were produced regarding the ability of sulphonylureas to increase insulin binding. This effect might be mediated by inhibition of transglutaminase, an enzyme involved in protein internalization. We tested this hypothesis by evaluating the effects of glipizide on degradation and dissociation after insulin binding to cultured human lymphocytes (MOLT4 line). We also compared its effects with those produced by bacitracin, known to inhibit the membrane mediated degradation of specifically bound hormone. All experiments were performed in the presence of 4% bovine serum albumen to decrease non-receptor mediated degradation. After binding equilibrium was reached (120 min at 24 ~ cells (107 cells/ml) were warmed in a shak- ing bath at 37 ~ and dissociation and degradation monitored every 5 min for 45 rain. The same procedure was followed after an overnight pre-incubation with glipizide (1 Ixg/ml) or bacitracin (300 llmol/1). Degradation (TCA solubility) and dissociation (expressed as percentage decrease of equilibrium binding) were markedly and similarly affected by both bacitracin and glipizide. The rates of both processes

were approximately halved by these treatments. The similarity between the effects exerted by these two drugs allowed the hypothesis of a glipizide-induced inhibition of surface enzymes responsible for hormone degradation.

79. Insulin and C-Peptide Responses to Oral Glucose Tolerance Tests in Idiopathic Reactive Hypoglycaemia C. Coscelli, E. Bonora, G. Speroni and U. Butturini. Istituto di Clinica Medica Generale, Parma University Medical School, Parma, Italy The occurrence of idiopathic reactive hypoglycaemia during an oral glucose tolerance test is a relatively frequent condition. In this study insulin and C-peptide responses to oral glucose as well as the C-peptide to insulin molar ratios were evaluated in 10 non-diabetic subjects with reactive hypoglycaemia (plasma glucose nadir < 2.8 mmol/1 on one or more occasions during an oral glucose tolerance test), and in 10 sex-, age-, and weight-matched control subjects. Insulin levels and areas (1.12 _+ 0.17 versus 0.92 + 0.10 nmol. 1-1.240 rain -1) did not differ significantly in the two groups, whereas C-peptide concentrations and areas (4.22_+ 0.61 versus 2.53 _+ 0.41 nmol. 1-1. 240 min -~) were significantly higher in the group with reactive hypoglycaemia (p < 0.05). C-peptide to insulin molar ratios after glucose load as well as relationships between incremental areas of the two peptides were higher in subjects with reactive hypoglycaemia than in the control subjects. Our results indicate that B cell response to oral glucose as well as insulin uptake by the liver in subjects with reactive hypoglycaemia are greater than in control subjects. As uptake and biological effect of insulin are known to be strictly related phenomena, and because of the major role played by the liver in glucose homeostasis, we suggest that idiopathic reactive hypoglycaemia might be due partially to an increased insulin secretion together with an enhanced biological effect of the hormone on the liver.

80. Hepatic Responsiveness to Insulin in Normal and Diabetic Rats C. J. Crace, J. R. Dryburgh and H. Keen. National Institute for Medi- cal Research, Mill Hill, London, UK Intraportal insulin infusion ( < 10 mU/kg) to normal conscious, fasted rats for 1 h stimulated 14C-glucose incorporation into hepatic lipid from 0.059 + 0.002% of a subcutaneously injected dose up to 0.105 + 0.11%. Higher portal doses, and all doses infused peripherally (0-1000 mU/kg) decreased hepatic lipid radioactivity and increased lipid radioactivity in peripheral muscle. In acutely diabetic rats no significant change in hepatic lipid radioactivity was observed after infusion of insulin by either route despite the expected fall in plasma glucose. Daily SC injection of 4-6 U Ultratard plus Actrapid insulin for periods > 2 weeks to streptozotocin-diabetic rats resulted in fasting plasma glucose levels ranging from 1.75 to 27.5 mmol/1. In this insulin-maintained group hepatic lipid radioactivity after 1 h insulin infusion was variable. However, the ratios of hepatic lipid radioactivity: circulating plasma radioactivity approached normal values in individuals with plasma glucose < 11 retool/1 but not in hyperglycaemic animals. We conclude (1) that peripherally administered insulin tends to exert a 'peripheral steal effect', which diverts circulating glucose from the liver to other tissues, and (2) that basal circulating insulin levels are required for maintenance or restoration of the normal hepatic responsiveness to insulin.

81. Improvement of Diabetic Peripheral Neuropathy with Ganglioside Treatment: A Multicentre, Randomized, Double-blind Trial G. Crepaldi, D. Fedele, P. Negrin, G. Pozza, G.Comi, G. Pagano, W. Troni, F. Frigato, C. Mezzina, F. Grigoletto and D. Massari. Catted- ra di Patologia Medica, University of Padova, Italy Positive effects on metabolic peripheral neuropathies have been reported with gangliosides. A double-blind, randomized, cross-over, multicentre trial versus placebo, with a wash-out period, was performed on insulin-dependent diabetic patients with impairment of nerve conduction velocity in at least two nerves. Nerve conduction velocity and glycosylated haemoglobin were evaluated at 0, 6, 10, 16weeks, while daily plasma glucose profile and glucosuria were monitored at 0, 3, 6, i0, 13 and 16 weeks. 140 patients were divided in- to two groups according to the absence (group A = 97) or the presence (group B = 43) of neurological symptoms, and then randomly assigned to the drug-placebo or placebo-drug sequence. The trial consisted of the IM administration of gangliosides (20 rag/day) or placebo for 6 weeks and in a 4 week wash-out period. No significant variation of metabolic parameters was observed during the trial. A significant main effect on nerve conduction velocity was observed in group A for motor peroneal nerve (p < 0.03) and in group B for sensory median nerve (p<0.06) and motor ulnar nerve (p<0.002). In

Abstracts 149

group B paresthesias also improved significantly. This trial confirms the positive effect of gangliosides on diabetic peripheral neuropathy.

82. Long-term Strict Control in Type I (Insulin-Dependent) Diabetes Mellitus: Effect on Late Diabetic Complications K. Dahl-Jorgensen, K.F. Hanssen, E. Smeland, O. Brinckmann-Han- sen, O. Aagenaes and Aker Diabetes Group, Aker Hospital, Oslo, Nor- way We studied 45 Type 1 diabetic patients without C-peptide response (duration 7 22years) without proliferative retinopathy (median 4 microaneurysms). After 2 months' run-in period, they were randomly assigned to; group 1:15 receiving CSII; group 2:15 receiving multiple SC injections via Butterfly, cannula 5-6 times daily; group 3:15 receiving twice daily rapid long-acting insulin. All groups improved blood glucose control in the run-in period (.p < 0.00l) as they started home blood glucose monitoring. After change of treatment groups 1 and 2 improved further (< 0.01) but group 3 was unchanged. After 6 months HbAlc was 6.8% in group 2, 6.7% in group 1 and 7.6% in group 3 (p < 0.05); mean blood glucose was 5.5 mmol/l in group 1 and 7.2 mmol/l in group 3 (p< 0.01). Glomerular filtration rate was supranormal and decreased in groups 1 and 2. Retinopathy was evaluated blind by 3-monthly fluorescein angiography and progressed in groups 1 and 3. Transient cotton-wool exudates developed in some subjects from groups 1 and 2. One patient in group I had transient proliferative retinopathy which regressed spontaneously without laser treatment. We conclude that patients in group 3 with home blood glucose monitoring, improve glycaemic control, but less so than groups 1 and 2. Retinal response to near normoglycaemia is complex. A transient deterioration may be seen, perhaps associated with the rapid normalisation of blood glucose during CSII.

83. Increased Glomerular Permeability to Albumin in Type 2 (Non-Insu- lin-Dependent) Diabetic Patients Before and After Exercise E. M. Damsgaard, J. R. Nielsen and C. E. Mogensen. Institute of Clini- cal Genetics, Odense University; Department of Medicine, Fredericia Hospital; Department of Clinical Physiology, Odense University Hospital and Second University Clinic of Internal Medicine, Kom- munehospitalet, Ptrhus, Denmark In the epidemiological survey of diabetes mellitus in Fredericia of subjects aged 60-74 years, the excretion of albumin and fl-2-microglobulin before and after exercise was studied. Seventy-six newly-diagnosed diabetic patients, 54 diet-treated, 72 tablet-treated, and 30 diabetic patients treated with insulin were compared with a sex- and age-matched control group of 285 non-diabetics. All subjects performed a multistage exercise test to exhaustion. The basal excretion of albumin (median (range) (gg/min) was control: subjects: 7.5 (1.7-3120.0), newly diagnosed patients: 14.3 (2.4-256.3), diet-treated patients: 14.5 (1.5-731.1), tablet-treated patients: 15 (2-6399.8) and insulin-treated patients: 31.6 (1.9-8120.0). The excretion of albumin after exercise was: control subjects: 16.3 (1.3 2992.5), newly diagnosed patients: 34.1 (1.7-586.3), diet-treated patients: 28.6 (2.4-1356.3), tablet-treated patients: 33.4 (2.6-6965.0) and insulin- treated patients: 24 (3.4-5 548.6). All the diabetic groups, including the newly diagnosed group had significantly higher excretion of albumin both before and after exercise than did the control group (p < 0.05). All the groups except the insulin-treated subjects showed a significant increase in albumin during exercise, fl-2-microglobulin did not increase in any of the groups. This indicates an increased glomerular permeability in Type 2 diabetes by the time of diagnosis.

84. Metabolic Effects of Nasally Administered Somatostatin Analogue L-363, 586 in Normal Subjects C. J. Davies, S. Williams, D. R. Owens and T. M. Hayes. Department of Medicine, Welsh National School of Medicine, Cardiff, UK Intravenous somatostatin enhances glucose tolerance in Type 1 (insulin-dependent) diabetes ; its therapeutic use, however, being limited by its short halfqife and the mode of administration. We have examined the effects of a longer acting analogue of somatostatin (L-363, 5 8 6 -

MSD) on carbohydrate metabolism in 10 normal subjects, following a standard meal (700 Kcal, incorporating 5 g xylose to assess absorption). Post-prandial glucose was significantly higher than control values during IV infusions of L-363, 586 at doses > 1.25 ~g/h, (mean_+ SEM at 2h: placebo 4.46_+0.26mmol/1; L-363,586 7.75+0.24 mmol/1; p< 0.001) with a significant decrease in plasma insulin response with infusions> l0 gg/h (at 1 h: placebo 43_+ 5, L-363, 586 6+1.5mU/1; p<0.01). Following nasal administration (200gg, 15 min before the meal) post-prandial plasma glucose was significantly elevated (at 1 h: placebo 4.85 + 0.39; nasal L-363, 586 8 + 1.2 mU/1;

p<0.02). The meal-induced rise in plasma glucagon was similarly suppressed by both IV and nasal L-363, 586. L-363,586 is similar in action to somatostatin, the nasal route offering a practical route of administration.

85. Comparison of the Pharmacokinetics of Tritiated Insulins Labelled at Various Positions of the Molecule J. G. Davies, D. Carmignac, C. Bradshaw and R. E. Offord. Departe- ment Biochimie M6dicale, Centre M~dicale Universitaire, Geneva, Switzerland Previous studies have shown that semi-synthesis can produce an au- thentic tritiated tracer, [Phem-3H] insulin, and extensive pharmaco- kinetic studies have been conducted with this analogue. Chromato- graphic and electrophoretic systems have been used to analyse the tritiated species found in the circulation after injection of this tracer. However, as this derivative is specifically labelled at phenylalanine B1, there are a few sites on the molecule where modification of the amino-acid sequence would be difficult to detect by these methods. Therefore, two new tracers have been produced by semi-synthesis, [GlyA1-3H] insulin and [Ala133~ insulin. Thus the metabolism of the amino-terminal region of the A-chain and the carboxy-terminal region of the B-chain can be observed more easily. These new tracers, as well as [Phe m 3HI insulin, have been injected SC into rats. The appearance of tritium in the plasma was followed. Also, the plasma components were separated by gel filtration on Sephadex G50. The changes as a function of time in the gel-filtration profiles of the various [3H] insulins differed from one to another. However, the differences relate mainly to the recycling of label from the hormone once degraded and there is, as yet, no further evidence fo r major differences in the pharmacokinetics of these tracer.

86. Evaluation of New Automatic Recording System of Diabetic Patient Information - Diadata J. L. Day, J. Metcalfe and H. Alban-Davies. The Ipswich Hospital, Ips- wich, UK Interpretation of diabetic patient data is prone to error. Diary presentation may be poor, rarely determines event-time relationships, and depends on recall and preconceptions. Interpretation is time consuming and subject to bias. Diadata enables entry of information (blood tests, injections, meals, reactions and activity) into portable slaves (time automatically recorded). Data are rapidly extracted, analysed and presented in total or graphical form. Mean + SD glucose overall or in different time blocks, specific events (glucose above or below pre-set limits, hypoglycaemic reactions), trends and time relationships are identified and summarised. In 19 insulin-dependent diabetic patients information provided by Diadata was compared with diary records, patient and doctor questionnaires and the influence of Diadata analysis on clinical assessment examined. Diadata and diaries provided identical mean overall and time-block glucose values. Diadata identified far greater variation in meal and injection times and showed significantly more hypoglycaemic reactions than patient or doctor questionnaires or diaries (p < 0.0001, < 0.0001, < 0.005). Event identification revealed a mean of 11.8 high, 9.4 low blood glucose and four reactions per patient. 70% of 'events' were explicable by data analysis. Diadata is easily operated, can provide useful accurate information and significantly improve clinical decisions.

87. Does Continuous Subcutaneous Insulin Infusion Prolong the Remis- sion Phase of Type 1 (Insulin-Dependent) Diabetic Children? C.E. de Beaufort, G.J. Bruining, R. S. R. Aarsen, N.C. den Boer and W. F. A. Grose. Sophia Children's Hospital, Rotterdam, The Nether- lands After diagnosis 30 Type 1 diabetic children were treated either by continuous SC insulin infusion (CSII) or by conventional insulin administration. Semi-synthetic human insulin was used in both groups. The children were studied prospectively, after randomization and after written informed consent was obtained. 17 referred children have participated for up to 18 months; seven on CSII (ages 5 15 years) and 10 conventionally treated (ages 3 13 years). The proposed duration of study for each child is 2 years. At monthly intervals home-blood@u- cose-(profiles), HbA~, intermediary metabolites and endogenous insulin secretion are measured. Glucagon-stimulated C-peptide reserve (measured every 2-6 months) correlated with 24 h urinary C-peptide secretion (r=0.8). Urinary C-peptide secretion expressed per gram creatinine correlated with 24h secretion (r=0.9). After 4 and 8 months of treatment, HbA1 and C-peptide secretion were significantly better in the CSII treated children (c~=0.05, Mann Whitney, CSII-treated children secreted 17.4_+2.0nmol/24h C-peptide after

150 Abstracts

8 months, conventionally treated children 4.5 + 2.8 nmol/24 h. HbA1 the values were 8.7 + 0.9% respectively 11.4_+ 0.5%). Insulin-dosages were 0.4 versus 0.6 U.weight-Lday -1. So far, CSII has resulted in much better preservation of endogenous insulin reserve.

88. The Contribution of Adrenaline to the Recovery from Hypoglycae- mia in Type I (Insulin-Dependent) Diabetes Mellitus: Dependency on A-Cell Function and Mediation Through fl-2-Adrenoceptors P.De Feo, G.Perriello, G. Bolli, S.De Cosmo, R Compagnucci, G. Angeletti, F. Santeusanio and R Brunetti. Istituto Patologia Medi- ca, Perugia, Italy, and Endocrine Research Unit, Mayo Clinic, Ro- chester, Minnesota, USA To assess the counter-regulatory contribution of adrenaline during hypoglycaemia in patients with Type I diabetes and to determine whether it is mediated through/~1 or/~2-adrenoceptors, insulin was infused for 1 h (30 mU. m -2- rain -1) in seven normal volunteers, in five Type 1 diabetic patients (duration < 1 year) with normal and in seven Type 1 diabetic patients (duration 8.5 + 1 years) with blunted glucagon responses to hypoglycaemia, alone or together with propranolol (non- selective beta-blocker) or metoprolol (beta<selective blocker) (3 mg bolus + 100 lxg/min for 150 rain). Post-nadir plasma glucose recovery of Type I diabetic patients with normal glucagon response was identical to that of non-diabetics (30+-0.4versus 33-+0.5[xmo1.1 -~. rain- 1, NS) and was unaffected by propranolol or metoprolol. In the diabetic patients with blunted glucagon response, plasma glucose recovery was slower (21 +-0.8 gmol . l -a .min -1, p<0.001 versus non- diabetics and diabetic patients of shorter duration) despite larger secretion of adrenaline, cortisol and growth hormone, and was further impaired by propranolol (I 4_+ 0.6 ~tmol. 1 1. rain-1, versus saline p < 0.001, whereas metoprolol had no effect (22+-0.9 Ixmol.1-1. rain -1, NS). In conclusion, adrenaline is not essential for plasma glucose recovery from hypoglycaemia in Type I diabetic patients who have normal glucagon responses, but becomes critical in long-term Type 1 diabetic patients with blunted glucagon responses. In contrast to propranolol, metoprolol does not interfere with plasma glucose recovery from hypoglycaemia in long-term Type 1 diabetes, indicating that the beta2-adrenoceptors mediate the action of adrenaline.

89. Lipids, Free Radical Attack and 'Control' in Insulin Dependent Dia- betes A. P. Delamothe, J. Lunec 1, A. C. Dickson, D. N. W. Griffith, T. L. Dor- mandy 1 and D.J. Betteridge. Department of Medicine, University College London Medical School, and 1Department of Chemical Pa- thology, Whittington Hospital, London, UK It has recently been recognised that free radicals may be generated in biological systems with potentially pathological results. In this study, lipid peroxides (products of free radical reactions) were estimated in 12 diabetic and nine healthy subjects. Peroxides were measured in chloroform-methanol (2:1, v:v) extracts of plasma and were represented as 'diene conjugation' at 240 nm in total plasma and individual lipoprotein subfractions. Results are given in arbitrary absorption units. Lipid peroxides were elevated in diabetic compared with healthy subjects (0.54_+ 18 versus 0.37 +- 0.09, mean+- SD, p< 0.05) and were found predominantly in the VLDL and LDL fractions in all subjects. When peroxides were calculated per mmol of cholesterol and triglyceride for whole plasma and each of the lipid subfractions, there was no difference between the diabetic and healthy subjects, suggesting that the increase in peroxides in the diabetic subjects was due to their increased amount of peroxidisable substrates. Six poorly controlled diabetic subjects were retested after 2 weeks of continuous subcutaneous insulin infusion and their peroxides fell in VLDL and LDL in proportion to their reduced lipid levels. These finding may be of importance in the aetiology of tissue damage in diabetes.

90. Binding of Hypoglycaemic Sulphonylureas to an Artificial Phospho- lipid Bilayer M. Deleers and W.J. Malaisse. Laboratory of Experimental Medicine, University of Brussels, Brussels, Belgium Prior radioisotopic studies have suggested that hypoglycaemic sulphonylureas bind to the plasma membrane of the pancreatic B cell, which could be equipped with sulphonylurea receptors. In the present

3 3 study, we observed that H-glibenclamide and H-gliquidone bind to multilamellar liposomes formed of egg yolk phosphatidylcholine, steady-state values being reached within 15 min incubation at 37 ~ At a high concentration of sulphonylurea (0.1 mmol/l), the total binding corresponded to a sulphonylurea/phospholipid molar ratio close to 0.0"1-0.02. In this artificial model, both specific and non-specific components of the binding phenomenon could be characterized by

the same criteria as those used in studies performed with natural membranes. The relative ability of distinct sulphonylureas (gliquidone, gliclazide, tolbutamide, chlorpropamide) to inhibit the specific binding of 3H-glibenclamide (or 3H-gliquidone) paralleled their relative potency as insulin secretagogues in the same manner as observed in intact islets. It is proposed that the insertion of hypoglycaemic sulphonylureas into the phospholipid domain of the B cell membrane may represent a primary event in the mechanism by which these agents stimulate insulin release. Moreover, the ability of distinct sulphonylureas to penetrate into such a domain may play a r61e in condi- tioning their biological potency.

91. Normalization of Intermediary Metabolites by Insulin + Glucagon Replacement in Pancreatectomized Diabetic Patients S. Del Prato, S. Vigili de Kreutzenberg, R. Trevisan, E. Duner, A. Vale- rio, U.Baccaglini, C.Tremolada and A.Tiengo. Patologia Medica, Malattie del Ricambio and Clinica Chirurgica II, University of Pado- va, Italy We have demonstrated recently that insulin does not correct the metabolic features of pancreatectomized diabetic patients (TPx). The metabolic effect of insulin (0.15 mU. kg - 1. min-1) and insulin + glucagon (2 ng. kg-1. min 1) infusion (10.00 14.00 h) was studied in four TPx patients. Intermediary metabolites, glucose turnover and recycling (~H and a4c-glucose injection) were evaluated in the post-absorptive steady-state. The effect of a meal challenge was also investigated. During insulin infusion free-insulin was 20 _+ 5 mU/1, glucagon (30 K antibody) 110 + 15 pg/ml and glucose 2.83 + 0.83 mmol/l (mean + SEM). Lactate was 1.126+0.172, pyruvate 0.071 +0.012 and alanine 0.306+0.050 mmol/l. ~H-glucose turnover was 0.113 +0.017 mmol. kg ~.min -1 and recycling 0.002+0.034mmol.kg-lmin-L During insulin + glucagon replacement, free-insulin was 19 + 4 mU/1, but glucagon (335 • 64 pg/ml; p < 0.05) and glucose (8.94_+ 3.38 retool/l; p < 0.05) rose. Lactate (0.649 _+ 0.053 mmol/1; p < 0.05), pyruvate (0.035 + 0.004 mmol/l; p < 0.05) and alanine (0.150_+ 0.025 mmol/l ; p<0.05) almost halved. 3H-glucose turnover was higher (0.245+_ 0.030 mmol.kg 1. min-1; p < 0.01) and glucose increased (0.073 + 0.037 mmol. kg -~. min -1 ; p < 0.05). Glycerol and ketone bodies were similar in both treatments. Meal consumption resulted in an increase of glucose with both treatments (zenith = 10.00 +- 2.61 and 19.78 +- 4.33 mmol/1; p< 0.05) and in a slight increase of gluconeogenic metabolites. Glycerol was more rapidly inhibited by meal during insulin replacement (30min after meal 0.082+-0.004 versus 0.110+ 0.008 mmol/l ; p < 0.05). In conclusion: glucagon deficiency per se is responsible for TPx metabolic features. Glucagon replacement restores normal values of gluconeogenic precursors.

92. The Role of Ca 2+ ions in the Activation of Fat-cell Pyruvate Dehy- drogenase by Insulin R. M. Denton, J.G. McCormack and S.E. Marshall. Department of Biochemistry, University of Bristol Medical School, Bristol, UK Insulin activates pyruvate dehydrogenase (PDH) in fat tissue by causing the dephosphorylation of the ~-subunits. Persistence of the activation during preparation and subsequent incubation of intact mitochondria greatly aids investigation of the mechanism and has allowed the demonstration that activation of PDH-phosphatase is involved rather than inhibition of PDH-kinase. Since PDH-phosphatase is activated by Ca 2+, it has been speculated that insulin action may be brought about by increased intramitochondrial Ca 2+. This view was supported by the finding that increasing calcium in fat-cell incubation media activated PDH. However, subsequent evidence was strongly against a role for Ca 2+ as a mediator of this insulin action. Findings included: (1) Ruthenium red (an inhibitor of Ca2+-transfer into mitochondria) blocked the effect on fat tissue PDH activity of high Ca 2+ in the medium, but not that of insulin; (2), activation of PDH in mitochondria from tissue previously exposed to high Ca 2+ was lost on incubation of isolated mitochondria in the presence of Na + (which allows Ca2+-efflux) while the effect of insulin persisted; (3), NAD-iso- citrate dehydrogenase and oxoglutarate dehydrogenase are activated within the same Ca2+-concentration range as PDH-phosphatase, but no evidence of activation of these enzymes was found in mitochondria from insulin-treated tissue.

93. Infectious Morbidity Following Caesarean Section: An Increased Occurence in Diabetic Pregnancies M. P. Diamond, S.L. Salyer, W. Vaughn, F.H. Boehm, and S.S. Ent- man. Departments of Obstetrics and Gynaecology and Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee, USA

Abstracts 151

We reviewed the infectious morbidity of all Type 1 (insulin-dependent) diabetic patients delivered by Caesarian section (CS) between 1977 and 1981. Of 83patients, 55 (group1) had no risk factor for post-CS morbidity; 24 (group 2) were at risk for infection on the basis of ruptured membranes or labour. Comparison was made with 287 consecutive non-diabetic patients delivered by CS; there were 34 (group 3) without risk factors and 235 (group 4) with risk factors. All groups had similar demographics and gestational age. Four diabetic and 18 non-diabetic subjects were excluded because of pre-operative antibiotic therapy. Frequency of patients with post-partum endome- tritis and/or wound infection was: group 1 :5 /55 (9.1%), group2: 6/24 (25%), group 3:1/34 (2.9%) and group 4:12/235 (6.0%) (g2 analysis of group 2 versus group 4; p < 0.01). A trend was seen when comparing groups 1 and 3. The infections among diabetic patients were independent of White's classification and obesity. For low-risk patients, infections were independent of diabetic control; high-risk patients with poor control were greatly at risk. It is generally considered that perioperative prophylactic antibiotics are not cost-effective in low risk CS. However, the high incidence of infection among both low risk (9%) and high risk (25%) diabetic subjects suggests that perioperative prophylactic antibiotics are indicated for all diabetic patients undergoing CS.

94. Platelet and Coagulation Factors Related to Soluble Immune Com- plexes or Insulin Anti-Insulin Complexes in Type I (Insulin-Dependent) Diabetic Subjects With or Without Proliferative Retinopathy U. Di Mario, D.Q. Borsey, M. Franco, C.V. Prowse, L.J.P. Duncan and D. Andreani. Department of Endocrinology University of Rome, Italy and Diabetic Department, Royal Infirmary, Edinburgh, UK Soluble complexes are thought to be capable of triggering platelet adhesiveness and aggregation. The possible relationship between soluble immune complexes (Ag/Ab) or insulin anti-insulin complexes (Ins/Ab) and the levels of platelet factor 4, B thromboglobulin, factor- VIII related antigen, fibrinogen, fibrinopeptideA and antithrombin 3, in diabetic subjects with and without proliferative retinopathy, was investigated. 20 Type I diabetic patients with proliferative retinopathy (group A), 20 patients without any sign of microangiopathy (group B) and 20 healthy subjects (group C) were individually matched for age, sex and where applicable, duration of diabetes. C3 binding Ag/Ab were detected by conglutinin and the class of immunoglobulins involved was analyzed. Total, free and bound insulin antibodies were assayed. A significant correlation was found between the presence of class G Ag/Ab and the levels of platelet factor 4 and factor VIII related antigen and between Ins/Ab and antithrombin 3 in group A patients. In Ag/Ab positive group A subjects, a significantly greater number of the platelet and coagulation factors were elevated. This study points out that Ag/Ab and Ins/Ab are among those factors possibly inducing platelet and coagulation abnormalities and thus contributing to aggravate the pathological events or retinopathy.

95. Affinity Labelling of Lysosome and Granule Specific Proteinases in a Transplantable Rat Insnlinoma K. Docherty and J. C. Hutton. Department of Clinical Biochemistry, University of Cambridge, UK Studies on the conversion of proinsulin to insulin in rat pancreatic islets have indicated that a cysteine proteinase with a slightly acidic pH optimum may be involved. This processing occurs at pairs of basic amino acids. To identify such proteinases, purified granule and lysosome/granule fractions from a transplantable rat insulinoma were affinity labelled with ~25I.-peptide chloromethyl ketones and subjected to SDS polyacrylamide gel electrophoresis. The peptide analogue 125I- Tyr-Ala-Norleu-Arg-CH2C1 with a single basic amino acid, labelled peptides of Mr 25,000, 31,500 and 39,000. 12SI-Tyr-Ala-Lys-Arg- CH2C1, a peptide analogue containing a pair of basic amino acids, labelled the 31,500 and 39,000 Mr peptides to a similar extent, but expressed a much lower affinity for the 25,000 Mr peptide. Antibody precipitation and inhibitor studies suggested that these peptides were cysteine proteinases, and that the 31,500 Mr peptide was cathepsin B. Parallel enzymatic marker studies indicated that the 25,000 Mr peptide was of lysosomal origin, and that the 31,500 and 39,000 Mr peptides were distributed in both lysosomes and granules. The latter peptides are therefore candidate enzymes for proinsulin processing.

96. The Hypotensive Effect of a Low Sodium, Low Fat and High Fibre Diet in the Hypertensive Diabetic Patient P. M. Dodson, P.J. Pacy, P. Bal, R.F. Fletcher and K.G. Taylor. De- partment of Diabetes and Endocrinology, Dudley Road Hospital, Birmingham, West Midlands, UK

We have previously reported the hypotensive effect of a high cereal fibre, low fat and low sodium (40-50 mmol/day) dietary regime in essential hypertensive patients. We now report the effects of this regime over a 3 month period on 50diabetic hypertensive patients (age: 55.8 + 8.1 years, 22 males and 28 females, ideal body weight: 133.2 + 22%) with regard to blood pressure and glycaemic control compared with a matched control group (n = 25). At 3 months, there was a significant fall in blood pressure (mean+SEM: systolic, 179.4+19.7 to 164.2+21.5mmHg; p<0.001: diastolic 97.7_+9.4 to 88.2+ 12.0 mmHg; p < 0.001), accompanied by a significant fall in 24 h urinary sodium excretion (197.2 +_ 73 to 136.1 + 67.1 mmol; p< 0.001), urinary sodium/potassium ratio (2.88 + 1.1 to 1.9 + 0.8; p < 0.001), weight (76.9 + 14.0 to 73.9 _+ 13.3 kg; p < 0.001), and glycosylated haemoglobin (11.6 + 3.1 to 9.5 +_ 2.9% ; p < 0.001). There was a significant reduction in the treated group at 3 months of diastolic blood pressure (p < 0.01) and urinary sodium/potassium ratio (p < 0.001) compared with the 3 month control values. We conclude that this dietary regimen has a significant hypotensive effect with improvement in diabetic control.

97. Thyrotropin-Releasing Hormone in Islets of Langerhans; Increased Production by Growth Hormone, Decreased Release by Somatostatin L. O. Dolva, J. H. Nielsen and K. F. Hanssen. Department of Medicine B, Aker Hospital, Oslo, Norway and Hagedorn Research Laboratory, Gentofte, Denmark Thyrotropin-releasing hormone (TRH) is synthesized and released from isolated pancreatic rat islets in organ culture. Recently growth hormone immunoreactivity and TRH immunoreactivity have been found in the same hypothalamic cell in the rat hypothalamus. Islets were isolated from 6 day-old rats by the collagenase method and cultivated for (a) 14 days or (b) 1 day in RPMI 1640 medium, containing (1) 1 lxg growth hormone/ml or (2) 0.5 ~g somatostatin/ml. TRH immunoreactivity was measured in medium and islet extracts. Following GH, the TRH immunoreactivity in the medium increased from 19.0 + 1.7 to 35.3 + 1.3 fmol. islet - I . day -1 the first day and remained significantly higher with GH throughout the study. The TRH in the islets increased by approximately 100%. Somatostatin decreased the TRH in the medium from 12.8 _+ 1.4 to 7.5 + 1.0 fmol. islet -1- day 1, while the TRH in the islet slightly, but significantly increased. Conclu- sion: TRH in both islets and medium increased following GH, indicating increased synthesis. Following somatostatin, TRH in the medium decreased while it increased in the islets, indicating inhibition of the TRH release.

98. Internalized Insulin Inhibits Intracellular Proteolysis in Isolated Rat Hepatocytes B. Draznin. University of Colorado Health Sciences Center, Denver, USA We have recently suggested the necessity of insulin internalization for its inhibitory effect on intracellular proteolysis (IP). In this study we attempted to investigate the relationship between the rate of insulin internalization and its ability to inhibit IP. We used acidification techniques to separate surface bound 125I-insulin (Sur) from internalized ligand (In). The In/Sur ratio plotted as a function of time permits the calculation of the rate of insulin internalization (K~-). Ke was 0.049 per rain at low insulin concentration (0.4ng/ml) and increased to 0.131 per rain at an insulin concentration of 3.2ng/ml. Insulin in a dose-dependent manner increased the rate of 14C-glucose incorporation into glycogen and inhibited the rate of degradation of intracellular proteins prelabelled in vivo with 14C-valine. When insulin internalization was blocked by phosphoarsineoxide (10 .6 mol/1), the amount of surface bound ligand and its effect on glucose incorporation into glycogen were unaffected, whereas insulin's effect on IP was markedly diminished. There was a direct and significant correlation between IQ- and insulin-induced inhibition of IR We conclude that (1) insulin internalization is essential for insulin to inhibit intracellular proteolysis and (2) inhibition of intracellular proteolysis by insulin is proportional to the rate of insulin internalization.

99. Antibody Responses following Change-over to Mixed Species (Por- cine/Bovine) Insulins M. I. Drury, D. Cregan, R. Drury, A.B. Kurtz, S.R. Bloom and L.S.Gerlin. Mater Misericordiae Hospital, Dublin, The Middlesex Hospital and Hammersmith Hospital, London, and Novo Laborato- ries Limited, Basingstoke Twenty-seven insulin-requiring diabetic patients, who had been treated with non-purified insulins for 4 years, were transferred to a regimen of highly-purified mixed species (porcine/bovine) insulins. They

152 Abstracts

were seen at 3-monthly intervals for 2 years, during which the binding of labelled porcine and bovine insulins by serum was monitored, as was insulin dose, glycosylated haemoglobin and mean fasting blood glucose. Insulin binding fell from a mean + 1 SD of 13.3 + 13.7% (bovine) and 8.1 _+ 8.4% (porcine) at the beginning of the study to 6.8 + 8.0% (bovine) and 2.3 ___ 1.9% (porcine) 2 years after the change to mixed species insulins. This reached statistical significance 60 weeks after the transfer. Antibodies to the insulin contaminant pancreatic polypeptide were significantly reduced 4 weeks after transfer to the mixed species insulins. They fell from 64+27% at week 0 to 14.8+ 7.2% at week 108. This indicates the relatively high immunogenicity of highly-purified mixtures of beef and pork insulins. In addition, the faster reduction of antibodies to pancreatic polypeptide suggests that this immunogenic impurity has been removed from the preparations and that the continuing insulin antibody production was caused by the intrinsic immunogenicity of the insulin.

100. Impaired Regulation of Plasma Renin Concentration in Type t (In- sulin-Dependent) Diabetic Patients with Microvascular Disease P. L. Drury and H.J. Bodansky. Departments of Endocrinology and Diabetes, St Bartholomew's Hospital, London, UK The aetiology and importance of hypertension in the pathogenesis of diabetic microvascular complications are unknown. We measured blood pressure and plasma renin concentration (PRC) in 31 patients with Type 1 diabetes and proliferative retinopathy, and in 17 patients free of complications (controls); groups were matched for age, sex and duration of disease. Plasma samples were taken between 0800-0900 h after 1 h recumbency. Patients took their usual carbohydrate-restricted diet with free sodium intake, but no drugs except insulin. Supine blood pressure (phases I/V) was recorded by mercury sphygmomanometer taking the mean of five readings. PRC was measured, in terms of MRC renin, using excess nephrectomised sheep substrate; intra- and inter-assay variations were 9.1% (n=10) and 16.3% (n= 8). Non-parametric statistics were used, and logarithmical- ly transformed PRC for linear regression. Systolic and diastolic pres- sures were higher in patients with retinopathy than the controls subjects (median: 134/77 versus 120/70 mmHg, p < 0.025 and p < 0.05, respectively). Median PRC was higher in the group with retinopathy (21.3 versus 15.4 mU/1, p < 0.025). Among controls PRC was inversely related to systolic pressure (r= - 0.43, p < 0.05) and to age (r= - 0.42, p < 0.05), but not in those with retinopathy (r= + 0.08 and -0.12 respectively, p > 0.1). These findings demonstrate altered regulation of PRC in diabetic patients with retinopathy; disordered control of renal renin secretion may contribute to the hypertension associated with microvascular disease.

101. A Comparison Between the Efficacy of Intravenous and Intraperi- toneal Insulin in Maintaining Short-term Basal and Prandial Glycae- mia in Insulin-Dependent Diabetes M. Ducasse, J. Jimenez-Gonzales and K.G.M.M. Alberti. Depart- ment of Clinical Biochemistry and Metabolic Medicine, University of Newcastle upon Tyne, UK Insulin therapy is given into the peripheral circulation and may be metabolically less effective than intraportal insulin. The peritoneum affords a possible route for intraportal insulin delivery. We have compared effects of intraperitoneal versus peripheral intravenous insulin infusion in five C-peptide negative insulin-dependent diabetic patients. Glycaemic control was achieved overnight using a Biostator with insulin given IV on one occasion and IP on the other. A standard breakfast (450 kcal) was then given. On the IV day, glycaemic control was maintained by Biostator after a 3 unit square wave pulse of insulin. On the IP day, the identical insulin delivery profile was used but started 12 rain earlier. Basal normoglycaemia was achieved by both routes with identical insulin requirements. During the meal glucose remained below 8.5mmol/1 and returned to preprandial levels (5.5-7 retool/l) within 3 h with IV insulin. With IP infusion, however, peak glucose was above 10.4 mmol/1 in all cases and was at least 2 mmol/1 above basal at 3 h. Thus basal glycaemic control is similar with both routes but under the conditions used prandial glycaemic control was poorer with IP insulin, suggesting delayed absorption and/or poor portal entry of IP insulin.

102. Aldose reductase activity in pancreatic islets S. P. Dufrane, W.J. Malaisse and A. Sener. Laboratory of Experimen- tal Medicine, University of Brussels, Brussels, Belgium Sorbitol is rapidly formed when pancreatic islets are exposed to D- glucose. We have now characterized aldose reductase activity in rat islet homogenates, using a new fluorometric micromethod based on the

measurement of NADP + formed from NADPH (30 ~tmol/1). The reaction velocity averaged 0.75 + 0.12 pmol/min per islet in the presence of 10 mmol/1 D-glucose, and 2.59 _+ 0.41 pmol/min per islet in the presence of 0.1 mmol/1 D-glyceraldehyde. The apparent Km for D-glucose was close to 16.3 mmol/1 and that for D-glyceraldehyde close to 0.04 mmol/l. In the presence of D-galactose, the velocity averaged 101 _+ 6% of that found at the same concentration (10 mmol/1) of D-glucose. Both sorbinil and, to a lesser extent, 3-3-tetramethylene- glutaric acid (0.1 mmol/1) decreased the reaction velocity, whether in the presence of D-glucose or D-glyceraldehyde. The reaction velocity was higher with the a-anomer than the fl-anomer of D-glucose, the fl/a ratio in velocity averaging 0.61 _+ 0.18 and 0.70_+ 0.08 with the islet homogenate and semi-purified calf lens aldose reductase, respectively. These results demonstrate the presence of an a-stereospecific aldose reductase activity in pancreatic islets and warrant further investigation on the participation of the sorbitol pathway in the physiology and pathophysiology of pancreatic islets.

103. Phospholipid-Dependent Protein Phosphorylation and Glucose-induced Phospholipid Turnover in Neonatal Rat Islets M. E. Dunlop and R.G. Larkins. University of Melbourne, Depart- ment of Medicine, Repatriation General Hospital, Heidelberg, Victor- ia, Australia A protein kinase activated by phospholipid, diacylglycerol and calcium (protein kinase C) has been demonstrated in rat pancreatic islets. This study examined the physiological relevance of this kinase to insulin release by determining whether it has an endogenous substrate and whether glucose induces the changes in phospholipid turnover necessary for activation. Neonatal rat islets were cultured in RPMI 1640 (glucose,32 11.1 retool/I)... Homogenates were prepared and incubated with P-ATP. Addmon of phosphatidylserine, diolein and Ca 2+ enhanced (by 37%) phosphorylation of a protein of apparent MW40K. Cyclic AMP and Ca2+/calmodulin had no effect on the phosphorylation of this protein, but enhanced phosphorylation of another endogenous protein (approximately 55 K). To determine whether glucose may provoke phospholipid turnover necessary to provide diacylglycerol for protein kinase C activation, cultured islets were in-

37 cubated with -P to steady state in RPMI 1640 (glucose, 11.1 retool/l) and then challenged with glucose (1.67 mmol/l) or (16.7 mmol/1) for 15 rain. Glucose (16.7 retool/l) caused a threefold increase in insulin release and increased ~2p incorporation into phosphatidic acid (by

0 32 38 V0) with a concomitant increase in P-phosphatidylinositol, indicating activation of the phosphatidylinositol cycle. It is concluded that islet protein kinase C causes phosphorylation of an endogenous protein and that glucose induces the acute changes in phospholipid turnover necessary to activate this process.

104. Study of the HbAlc Levels at Monthly Intervals in Gestational Dia- betes: Their Relationship with Insulinaemia, C-Peptide and Weight of the Newborn - Comparison of Two Therapeutic Regimes S. Duran 1, I. FernandeW, M.C. Gonzalez ~, N. Gonzalez ~, E. Roldan ~ and C. Bedoya. ~Endocrinologia Medica and 2Catedra de Obstetricia, University Hospital of Seville, Seville, Spain We have studied HbAlo levels (thiobarbituric acid test) at monthly intervals in 30 normal pregnant women and in 104 gestational diabetic patients (GDP). The GDP were divided into two groups: 51 were treated with diet alone (1700-2100 Kcal/day) and 45 with the same diet plus a low dose insulin regime. Eight GDP were studied in the last week of their pregnancy without previous treatment. In the non- treated GDP up to the ninth month we found a significant increase in the I-IbAlc levels (8.5 _+ 0.35% versus 7.5 + 0.15%, p < 0.001). In the two GDP treated groups we found no significant differences from normal pregnancy in monthly HbAlc levels. A significant linear relationship was found between the first HbAlc value and the glycaemic value 2 h after on oral glucose tolerance test in GDP (r: 0.266, p < 0.05). We also found a linear relationship between the mean of HbAlc levels during the last trimester of pregnancy and the newborn weight corrected for gestational age (r: 0.355, p < 0.01). We found no relationship between HbAlo and insulinaemia or C-peptide levels on cord blood of GDR The fetal deaths (6%) and hypoglycaemia (2%) were present only in the GDP treated with diet alone.

105. Insulin Resistance in Isolated Cardiocytes of Genetically Obese Zueker Rats: Evidence for a Post-receptor Defect J. Eckel, A. Stocks, A. Wirdeier, L. Herberg and H. Reinauer. Diabetes Research Institute, Dtisseldorf, FRG Muscle tissue accounts for the major proportion of glucose utilization in the intact animal. The role of cardiac muscle in the abnormal glu-

Abstracts 153

cose handling observed in obesity is presently unclear. Cardiac myocytes isolated from obese and lean Zucker rats have now been used to characterize the defective sites related to the obesity syndrome. No difference in viability and ATP/ADP content has been observed between myocytes from lean and obese rats. Insulin binding under steady-state conditions (60 rain at 37 ~ was found to be 0.46 and 0.59 fmol/3.6 x 10 4 cells in lean and obese animals, respectively. No significant difference in insulin-receptor affinity and receptor number could be detected between the two groups. Insulin action was determined by measuring the effect of the hormone on the initial rates of 3-0-methylglueose transport. Basal transport activity was unaltered in obese rats when compared to the lean controls. However, at all insulin concentrations studied (2.9 x l0 11 to 10-7mol/1), a decreased responsiveness (30-50%) was observed in cardiocytes from obese rats. In conclusion, our data show that insulin resistance in cardiac muscle of obese Zucker rats does not involve the receptor itself, but appears to be located at a post-receptor site.

106. A Novel Peptide HI (PHI) - Localization and Effects in the Rat Is- lets S. Efendi6, J. Fahrenkrug, T. H6kfelt, J. Lundberg, E. Sandberg and J. Szec6wka. Departments of Endocrinology and Histology, Karolins- ka Institute, Stockholm, Sweden and Department of Clinical Chemis- try, Fredriksborgs Central Hospital, Hillered, Denmark PHI is a newly isolated gastrointestinal polypeptide showing structural homology to vasoactive intestinal polypeptide (VIP). In this study we investigated localization of PHI in rat pancreas and characterized its effects on the release of insulin, glucagon and somatostatin from the isolated perfused rat pancreas. Pure, natural PHI was kind gift from Professor V. Mutt and Dr. K.Tatemoto. Somatostatin and PHI antibodies were raised in our laboratories. Histochemical studies revealed PHI immunoreactive nerves in association with the pancreatic islets. In the absence of glucose, PHI (1 nmol/1) stimulated glucagon release, whereas in the presence of 4.4 retool/1 glucose it enhanced secretion of insulin. In the next series of experiments, the interaction of PHI with glucose-induced insulin and somatostatin release was studied. In the presence of 6.7 mmol/1 glucose, the polypeptide enhanced secretion of both insulin and somatostatin during the entire 60 min perfusion period. In contrast, in the presence of 16.7 retool/1 glucose, PHI exerted a clear-cut stimulatory effect on the early (1-7 min), but not on the late (7 60 rain) phase of insulin release and had no influence on somatostatin secretion. We conclude that: (I) PHI immunoreactivity is present in the nerves in the islets. (2) PHI exerted glucose- dependent, stimulatory effects on the pancreatic A, B and D cells.

107. Minimal Improvement of B Cell Function after Continuous Subcu- taneous Insulin Infusion D. W. Erkelens and A.J. Spijker. Department of Internal Medicine, Utrecht, The Netherlands Presumably, B cell function is irreversibly lost in Type 1 (insulin-dependent) diabetes. To test whether C-peptide reactivity could return after improved control, glucagon tests were performed before and at least 3 months after institution of continuous subcutaneous insulin infusion (CSII) in 11 diabetic patients (median duration of diabetes 19 years). C-peptide was measured by radioimmunoassay (Novo-kit, detection limit 0.01 nmot/l) at - 4, 0, + 4 and + 6 min after 1 mg glucagon IV. For calculation, the values below the detection limit were assumed to be 0.01 nmol/l . Pairwise differences were tested with the Wilcoxon test. In eight patients, basal 0.014 nmol/1 (mean of - 4 and 0 min values) and stimulated 0.022 (mean of + 4 and + 6 min values) C-peptide increased during CSII to basal 0.029 (p < 0.02) and stimulated 0.045 (p< 0.02). These eight patients were not different from three patients who showed no improvement regarding the duration of diabetes, daily insulin dose or type of insulin used. The data suggest that in a number of patients with long-standing Type 1 diabetes, CSII is accompanied by reappearance of measurable C-peptide and reactivity to glucagon. It remains to be evaluated whether this is due to de- repression of B cell function or reappearance of functioning B cells.

108. Intracellular Muscle and Aorta Metabolism in Pigs with High Lev- els of Insulin Antibodies K. Falholt, K.G.M.M. Alberti and L. G. Heding. Novo Research In- stitute, Denmark and University of Newcastle upon Tyne, UK The peripheral hyperinsulinaemia usually found in conventionally treated insulin-dependent diabetes have deleterious metabolic effects. We have used a hyperinsulinaemic model to examine this hypothesis in two key peripheral tissues, aorta and muscle. Seven pigs were immunized with crystalline insulin. Subsequently they shared an insulin-

binding capacity of 13.2 + 4 mU/1 and the immunized pigs had impaired glucose tolerance. Immunized pigs showed marked elevation of muscle and aorta triglycerides compared with control pigs ( n= 15) but similar non-esterified-fatty acids. Several enzymes were found to be affected. Thus glyceraldehyde-3-phosphate dehydrogenase activity was decreased in muscle (289 + 72 versus 417 + 40 l~U/g, p < 0.01) and aorta (14.7 _+ 1.4 versus 22.8 _+ 4.0 IU/g, p < 0.001) in immunized pigs whilst 3-hydroxyacyl-CoA-dehydrogenase, glucose-6-phosphate dehydrogenase and malic enzyme activities were all increased significantly (by < 50% to 300%) in both muscle and aorta. At the same time hexokinase and pyruvate kinase activities were increased in muscle alone. Thus in the presence of increased peripheral total insulin levels in animals with a normal pancreas, profound metabolic changes were noted. There was a general increase in peripheral lipogenic activity with pronounced effects on vascular tissue. These findings have potentially important implications with regard to macrovascular disease in diabetes.

109. Influence of Maternal Metabolic Control and Insulin Antibodies on Neonatal Complications in Infants of Diabetics Mothers F. Fallucca 1, P. Gargiulo ~, F. Troili 1, R. Fiore ~, O. Zuccarini ~, G. Gerli- ni z, A. PachP, M. IavicolP. ~I Cattedra di Endocrinologia, :Istituto di Puericultura and 3Cattedra di Puericultura Prenatale, - University of Rome, Italy A relationship between poor maternal metabolic control (MMC) and neonatal complications (NC) has been clearly established in diabetic pregnancy. Insulin antibodies (IAb) could induce some NC, as we and others have reported. The respective and related roles of MMC and of IAb have not been studied. Our aim was to investigate the prevalence of NC, correlated with IAb, in infants of diabetic mothers (IDMs) in the same conditions of MMC. For this purpose we assayed IAb and C-peptide (CPR) in cord blood of 68 IDMs. MMC was divided into two groups - poor and good control - on the basis of three- monthly fasting blood glucose levels and the presence or absence of acetonuria and hypoglycaemia. NC occurred more frequently in IDMs born of mothers with a poor MMC. Cord blood-CPR levels were significantly higher in IDMs born of mothers with a poor MMC than in those with a good MMC (585 + 96 versus 353 + 63 pmol/1), while no differences were observed in IAb levels. On the other hand, at the same level of MMC, the presence of IAb in cord blood was associated with more elevated cord blood-CPR levels and with a significantly higher prevalence of some NC. These data suggest that both the metabolic and immunological factors, present in diabetic pregnancy, have an influence on the genesis of fetal hyperinsulinaemia and NC in IDMs.

110. Ovarian-Adrenal Interactions with Pancreatic B Cell After Oestra- diol Treatment in the Rat: Ultrastructure and Insulin Secretion A. Faure, L.Aerts, B.Ch.J. Sutter, and F.A.Van Assche. Laboratoire d'Endocrinologie, University of Bordeaux I, Talence, France Previous results demonstrated the importance of the adrenal gland on the oestradiol enhancing effect on insulinaemia. To study this further B cell ultrastructure and islet insulin secretion were compared in ovariectomized rats after 14 days of treatment with 17-fl-oestradiol (1 rag/ day) or oil vehicle as control, and in four groups of adrenalectomized- ovariectomized animals receiving oil, oestradiol, corticosterone (0.4 rag/day), or corticosterone + oestradiol. Morphometric analysis of B cells did not show any difference in total granule number per test area between all the groups studied. The percentage of light granules inside the total granule population was not affected by ovariectomy but decreased after adrenalectomy. Oestradiol treatment increased this percentage only in the presence of the adrenals or corticosterone. Insulin secretion, expressed as the area under the glucose-dependent insulin curve, was not affected by ovariectomy but decreased after adrenalectomy and was reversed by corticosterone supplementation. Oestradiol treatment enhanced insulin secretion only in the presence of the adrenal glands or corticosterone. A good correlation was found between the enhancement of percentage of light granules and oestradiol-induced insulin hyperseeretion. Our data suggest that corticosterone is necessary for the oestradiol-stimulating effect on B cell function.

111. Obesity and Arterial Hypertension in Diabetic Patients Without Nephropathy T. Fekete, L. Levi and A. Teszler. Second Medical Clinic, Cluj-Napo- ca, and Policlinic, Baia-Mare, Romania In order to assess the relative role of obesity in connection with other factors in causing arterial hypertension (AHT) in diabetic subjects

154 Abstracts

without nephropathy, 829 ambulatory diabetic patients without proteinuria (598 Type2 and 231 Type 1) and 856 age-matched control subjects were examined. AHT (>160/100mmHg) was present in 330 diabetic patients (40%) versus 108 (13.6%) control subjects (p< 0.001). Obesity was recorded in 514diabetic patients (62%) versus 310 control subjects (36%) (p< 0.001). Diabetic patients with obesity had AHT in 232 cases (70%); diabetics without obesity had AHT only in 98 cases (30%) (p< 0.001). AHT was more frequently encountered in women (209 = 48%) than men (121 = 30% p < 0.001). This was due to the higher frequency of obesity in women (286=35%) than men (228 = 28% p < 0.01). Type 1 diabetes taken separately showed obesity less frequently and also less AHT, albeit more often than controls. The prevalence of AHT increased with age, and duration of diabetes. Both these effects were significantly enhanced by the presence of obesity; probably due also to the higher prevalence of atherosclerosis (obese diabetics with atherosclerosis = 285 of 514 (56%) versus non- obese diabetics with atherosclerosis: 84 of 314 (27%,p< 0.001). Thus, obesity, besides being an important causal factor of AHT in diabetic patients without nephropathy, also enhances the effect of other related factors (age, sex, duration of diabetes).

112. Autonomic Neuropathy in Diabetes Mellitus: A Combined Evalua- tion of Five Cardiovascular Tests by Discriminant Analysis M. Fernfindez Castafier, A. Sorribas, F. Fernfindez Fernfindez, E. Ni- colau, R. Casamayor, E. Esmatjes, D. Figuerola and T. Micalo. Dia- betic Department of Clinic Hospital and Statistics Division, Universi- ty of Barcelona, Spain Five tests of autonomic function: deep breathing ratio (B), Valsalva ratio (V), standing ratio (S), blood pressure response to standing (SP) and sustained handgrip (H), were evaluated in 115 diabetic patients (age range: 12-49years, duration of disease 1-43 years) and 56 normal subjects. Cardiovascular autonomic neuropathy (CAN) was first assessed by conventional analysis. Two groups were defined, CAN + (at least one abnormal or two borderline tests, n = 44, all diabetic patients) and C A N - (n= 127). The quantitative values of the tests were then assessed by discriminant analysis. Standardised canonical discriminant function was F=-0 .80158 B' -0.19597 V' -0.22274 S' -0.24431 SP'. Canonical correlation was 0.7580109. F values (mean and ranges) were -0.64591, -3.9377, 1.0922 ( C A N - ) and 2.06333, 1.1895, 3.6026 (CAN+). Six subjects (three C A N - , three C A N + ) were reclassified in the opposite group. F ranged from -3.5700 to 0.7808 in control subjects, from 1.0857 to 3.6026 in diabetic patients with symptomatic autonomic neuropathy, and from -3.9377 to 3.2202 in asymptomatic diabetics. We concluded that (a) deep breathing ratio is the best discriminant test for CAN screening, (b) combined assessment of deep breathing, Valsalva, standing and blood pressure response to standing ratios by discriminant analysis provided information not forthcoming from conventional analysis and (c) assessment of sustained handgrip is unhelpful in diagnosis of cardiovascular autonomic neuropathy.

113. The Response to Glucose Ingestion in Type 2 (Non-Insulin-Depen- dent) Diabetes: A Double Tracer Study E. Ferrannini, L. D. Katz, D. Simonson, S. Bevilacqua, G. A. Reichard, Jr, E.J. Barrett and R. A. DeFronzo. C. N. R. Institute of Clinical Phys- iology and Second Medical Clinic, University of Pisa, Pisa, Italy, Yale University School of Medicine, New Haven and the Lankenau Hos- pital, Philadelphia, USA Following glucose ingestion, endogenous glucose production (EGP) is restrained, thus contributing to normal glucose tolerance. To examine whether this response is maintained in diabetes, glucose kinetics after oral glucose administration were studied in 12 Type 2-diabetic and 11 healthy control subjects by labelling the endogenous glucose pool with 3H-glucose and the oral load with 14C-glucose. Basally, EGP was similar in diabetic and normal subjects (0.014_+ 0.001 vs 0.013 + 0.003 mmol. min -1 kg-1) though the former had higher plasma glucose (10.5 + 1.0 versus 5.1 + 0.1 retool/l, p < 0.001) and insulin (23 + 4 versus 13 + 2 mU/1, p < 0.05) concentrations. Following glucose ingestion, oral glucose appeared in the peripheral circulation in similar time-course and amounts (N75% of the load over 3�89 h) in the two groups. EGP was promptly inhibited in both diabetic and normal subjects, and was still suppressed at 3�89 (0.007+0.002 and 0.005+ 0.001 mmol- min -1- kg -~, respectively). In diabetic patients, average inhibition of EGP during absorption was virtually the same as in normal subjects (54_+ 5% and 53 _+ 5%). This, however, occurred in the face of greater hyperglycaemia (16.6 + 1.3 versus 7.7 + 0.3 mmol/l, p < 0.001) and comparable insulinaemia (46 + 10 versus 42 + 5 mU/1). Furthermore, EGP was positively correlated with glycaemia both basally (r=0.92, p<0.001) and after glucose (r=0.63, p<0.05), Tissue

glucose removal was markedly reduced in diabetics (3.5+ 0.3 mmol/kg over 3�89 h versus 5.2 + 0.3 of controls, p < 0.001). We conclude that in Type 2 diabetes after oral glucose (a) EGP is inhibited to normal absolute values, (b) the liver is resistant to the restraining action of hyperglycaemia on EGP, and (c) tissue uptake is impaired.

114. Management of Animal Insulin Allergy with Human Insulin P. Fireman, A. Chamovitz, M. Ackerman, R. Levine, D. Greene and J. Galloway. University of Pittsburgh, Pittsburgh, PA and Lilly Re- search Laboratories, Indianapolis, IN USA Human insulin (HI) has been suggested as an alternative for diabetic patients with animal insulin allergy. Although HI cross-reacts anti- genically with pork insulin and beef insulin, HI appears less immunogenic. Prospective studies (> 1 year) show that 6% HI-treated diabetic subjects developed anti-insulin IgE antibodies compared with 17% pork and 19% beef insulin-treated subjects. To date, HI has been studied in nine insulin-dependent patients whose allergy to animal insulins was classified as follows: IgE systemic (four), IgE local (two), non-IgE systemic (one) and non-IgE local (two). After desensitization with HI, three out of four patients with IgE systemic allergy improved, with a subsequent decrease in IgE antibody in one patient. Of the two patients with IgE local allergy, one did not improve after HI desensitization, but then tolerated HI by constant infusion pump: the other improved when changed to HI. Two of the three patients with non-IgE systemic or local reactions (? pseudo-allergy) continued to have adverse reactions when changed to HI and did not improve after desensitization. Thus, HI appears to be an appropriate alternative to animal insulins in certain diabetic patients with IgE systemic or local allergic reactions to animal insulins.

115. Effects of Cationic Modification on Intracellular Uptake and Su- perficial Binding of 45Ca by Decapsnlated ob/ob Mouse Pancreatic Is- lets P. R. Flatt, P. Rorsman and S.K. Swanston-Flatt. Department of Bio- chemistry, University of Surrey, Guildford, Surrey UK, and Depart- ment of Medical Cell Biology, University of Uppsala, Sweden Decapsulated ob/ob mouse islets were used to examine the effects of mono- and divalent cations on 45Ca uptake and binding as discrimi- nated by La3+-wash technique. At 3 mmol/1 glucose (1.28 mmol/1 Ca2+), intracellular 45Ca uptake was increased 1.9-4.5-fold by omission of 125 retool/1 Na +, 1.5-fold by omission of 1.2 retool/1 Mg 2+ and 2.3-fold by reduction o fH + (pH 8.5). Omission of 5.9 mmol/1 K + was without effect. 30.9 mmol/1 K + increased uptake 4.7 6.8-fold, whereas addition of H + (pH 6.3) or 12 mmol/1 Mg 2+ decreased uptake by 1.8 and 1.8-2.0-fold, respectively. In control incubations,

4s 20 mmol/1 glucose increased intracellular Ca uptake fivefold. Stim-

uptake was decreased 3.I-fold by Mn 2+ and increased 4.7 and 3.I- fold by Zn 2+ and Ba 2+. Mn 2+ also decreased uptake 7.2-fold at 20 mmol/1 glucose, as did Ni 2+, Co 2+, Cd 2+ (1.4 5.8-fold) but not

2 + 7 + 45 Zn or Ba . Superficial Ca binding was increased 7.4. and 10.3- fold by Zn 2+ and Cd 2+. However, binding was not affected by other divalent cations, by 20 mmol/1 glucose in their absence or presence, or by the major ionic modifications made with and without adjustments using choline chloride or sucrose.

116. The Dawn Phenomenon: Is It a Major Clinical Problem? A. J. Francis, P.D. Home, N. Mann, W. G. Reeves and S. Walford. De- partment of Clinical Biochemistry and Metabolic Medicine. Royal Victoria Infirmary, Newcastle upon Tyne and University Hospital, Nottingham, UK The 'dawn phenomenon', comprising a rise in blood glucose starting before breakfast and refractory to conventional twice daily insulin therapy, has been recently described. The incidence of this phenomenon, and whether it occurs in a clearly definable separate group of diabetic subjects, is unknown. 1100, seven point, laboratory measured blood glucose profiles from 95 unselected patients on twice daily short- and intermediate-acting insulins have been analysed. Mean blood glucose levels before (10.1 +0.3 mmol/1) and after (2.1 +0.3) breakfast were significantly higher (p < 0.001) than at lunch (7.3 + 0.2 and 9.1 +_ 0.2 mmol/1) and at dinner (8.4 _+ 0.2 and 9.8 + 0.2 mmol/l). Blood glucose distribution was unimodal at all time points - not sup- porting the existence of a separate 'escape' group. Seventy-eight percent of patients had their highest glucose levels before or after breakfast. When compared to those with satisfactory fasting blood glucose, patients with fasting hyperglycaemia had similar blood glucose pro-

Abstracts 155

files from lunch-time onward. There was no correlation between the evening intermediate acting insulin dose and fasting blood glucose. Blood glucose profiles were independent of insulin species (pork, beef). Insulin binding antibodies did not protect against fasting hyperglycaemia. Thus pre- and post-breakfast hyperglycaemia is a major problem with twice daily insulin therapy, and is independent of insulin species and insulin antibodies.

117. 1,25-Dihydroxy-Vitamin D3 Increases 4SCalcium2+ Uptake by Is- lets from ob/ob Mice in Vitro B.J. Frankel, J. Sehlin and I.-B.T~iljedal. Department of Histology and Cell Biology, University of Umefi, UmeS, Sweden It is generally accepted that 1,25-dihydroxycholecalciferol [l,25(OH)2D3] is necessary for normal calcium metabolism by stimulating calcium uptake in the intestine. The steroid also has a direct effect on the islet B cell. Hand-dissected islets from UmeS-ob/ob mice were cultured 7days at +37~ in tissue culture medium RPMI 1640+10% fetal calf se rum+Hepes+ant ib io t ics+l l . lmmol /1 added glucose + 1,25(OH)2D3 (1 nmol/1 or 1 ~mol/l) or ethanol vehicle (0.0001% or 0.1%) preincubated 30 min with glucose (3 mmol/1) _+ 1,25(OH)2D3, and then incubated 1 or 2h with 3 or 20mmol/1 ~lucose_+ 1,25(OH)zD3 for the measurement of insulin release or

Ca 2+ uptake. 1,25(OH)zD3 (] nmol/1) significantly increased 45Ca 2+ uptake in the presence of glucose (3 mmol/l), but did not increase the uptake stimulated by glucose (20 mmol/l), possibly because all islets had been exposed to sufficient vitamin D3 during culture with normal fetal calf serum. A greater concentration of the vitamin (1 gmol/1) gave similar results. Islets 'cultured' for only 3 h showed similar tend- encies. These data could have implications for B cell function under conditions of vitamin D-deficiency.

117. Phenformin-Induced Changes of the Insulin Binding Parameters in Type 2 (Non-Insulin-Dependent) Diabetes are Dependent on the Dura- tion of Treatment P. Fratino, G. Bellomo, F. Travaglino, F. Mirabelli and R L. Nicotera. Clinica Medica II, University of Pavia, Pavia, Italy The present study was performed in order to clarify the effects of duration of phenformin treatment on the insulin binding parameters in monocytes derived from non-obese patients with Type 2 diabetes. Phenformin was given orally at a dose of 50 + 50 mg daily and the insulin binding parameters were investigated before and after treatment. Before treatment insulin binding was 1.91 +0.31%, sites/cell were 5 530 _+ 1050, K~/I 0 s tool was 1.25 -+ 0.28. Three, 7 and 30 days after treatment insulin binding were 4.21_+0.80%, 3.90+2.12%, 6.03_+1.78%; sites/cell 6010_+920, 18100_+11370, 22115_+8095; K~_ 2.26 _+ 0.45, 0.46 _+ 0.28, 0.55 + 0.09, respectively. The results demonstrate that the after short-term treatment (3 days) this effect was achieved by an increase of the K~t (p < 0.001) without changes of the number of sites/cell. After longer treatment (7 and 30 days), the effect was due to a great increase of the number of sites/cell (p < 0.001) associated to a decreased Ka (p< 0.001). In conclusion, phenformin increases the insulin binding to monocytes of diabetic patients by two different mechanisms, depending on the duration of treatment.

119. A Comparison of the Pharmacokinetics and Biological Potency of Human Insulin Zinc Suspension (Recombinant DNA) with Highly- Purified Porcine Insulin in Normal Man B. M. Frier, F.N. Sullivan, F.S. Mair, I.M. Koch and J.B. Scotton. Gartnavel General Hospital/Western Infirmary, Glasgow, and Lilly Research Centre, Surrey, UK A single dose cross-over study was performed in 10 fasting, non-diabetic men to compare the 24 h profiles of blood glucose, plasma insulin and C-peptide following a single SC injection of either human insulin zinc suspension (human crystalline) or highly-purified porcine insulin, (HPPI Novo Monotard), in standard dosage (0.2 U/kg). Both insulins produced moderate hypoglycaemia within 3 h which persisted for 24 h after administration. The rate of fall of blood glucose was similar from 0-3 h but was significantly lower after HPPI at 4 h (p < 0.05). Mean plasma insulin values after HPPI were higher than after human crystalline insulin with significant differences between 3-6 h (p < 0.02). Mean plasma C-peptide was significantly lower following HPPI between 1-9 h after injection (p < 0.05). We conclude that the duration of hypoglycaemic action of human crystalline insulin is equivalent to HPPI over 24 h. The rise of plasma insulin after SC administration, and the concomitant suppression of endogenous insulin secretion, were significantly less after human crystalline insulin than after HPPI, suggesting that the biological potency of human crystalline insulin is lower in normal subjects.

120. International Trends in Diabetes Mortality J.H.Fuller and S.P.Edmeades. The Middlesex Hospital Medical School, London, UK Using statistics published by WHO, mortality rates for diabetes as the underlying cause of death have been compared for most European countries and the USA. For 1976 the highest age-standardised mortality rates per 100,000 population occurred for Malta (males 73.6; females 179.5) and Belgium (males 20.3 ; females 45.4) while the lowest rates were for Northern Ireland (males 6.3 ; females 10.4) and Norway (males 5.7; females 7.7). Trends in mortality over the period 1968-1979 have been analysed using the Spearman rank correlation coefficient. Significant downward trends occurred for females only in Belgium, Austria, England and Wales, and Scotland, and for both sexes in Northern Ireland and the USA. Diabetic mortality rates increased significantly over this period for both sexes in Spain, Poland and Bulgaria. There are several possible reasons for international differences in diabetic mortality rates including variations in death certi- fication practice, diagnostic criteria and disease prevalence, but the continued monitoring of such mortality statistics is important for the evaluation of new preventive and therapeutic practices in the care of the diabetic patient.

121. Insulin Deficiency and the Dawn Phenomenon in Insulin-Treated Patients E. A. M. Gale, C.S. Yajnik, T.S. Chadwick, R.F. Smith and T.D.R. Hockaday. Radcliffe Infirmary, Oxford, UK Relative insulin deficiency in the latter part of the night is a common problem with conventional insulin therapy. We examined the effect of partial or total insulin withdrawal at 06.00 h upon the rate of rise of plasma glucose, plateau levels achieved, and upon other metabolites. Six male insulin-dependent diabetic patients (aged 19-37 with duration of diabetes > 10 years), low insulin antibody levels and minimal C-peptide responses, took part. They were studied on four occasions, each preceded by 36 h of soluble insulin therapy and an overnight IV infusion of insulin. At 06.00 h insulin was either stopped or continued at 25%, 50% or 100% of the basal infusion rate. The rate of rise and plateau levels of glucose were inversely related to the infusion rate. Even so, the rate of rise of glucose was near-maximal with the 50% reduction. In contrast, ketones rose only with the 25% infusion. This simple technique allows detailed analysis of the rate and amplitude of metabolic changes during controlled insulin deficiency. Relative insulin lack may be sufficient to explain the rapid 'dawn rise' of glucose commonly seen in diabetic patients on conventional therapy, but fasting ketone levels are a better index of the degree of insulin deficiency.

122. Factors Which Affect Insulin Dose: Do Insulin Antibodies Matter? J. Galloway, S. E. Fineberg, N. S. Fineberg, and J. Rathbun. Lilly Labo- ratory for Clinical Research and Indiana University School of Medi- cine, Indianapolis, Indiana, USA Previous studies have failed to demonstrate simple relationships between insulin requirements and insulin antibodies. Variables other than antibodies which might affect insulin dose include body weight, degree of adiposity, endogenous insulin secretion, and intensity of glycaemic control. We examined these variables in patients complet- ing the first year of an open-label trial of human insulin of recombinant DNA origin ([rDNA]; n= 115) and a double-blind trial which includes purified pork insulin (n = 140). Multiple regression analysis revealed that weight, concentrations of total glycosylated haemoglobin and bound insulin were positively correlated with dose, whereas C-peptide concentrations were negatively correlated. Weight was the most significant variable for both treatment groups (p < 0.001), whereas C-peptide, glycosylated haemoglobin, and bound insulin levels varied in importance. Patients who had good glycaemic control (total glycosylated haemoglobin < 10%; normal 6.0-8.8%) had the lowest insulin doses (p < 0.01) but did not differ with regard to bound insulin. Levels of insulin antibodies resulting from treatment with purified pork or human insulins (rDNA) contributed only weakly to insulin dose requirements. Any inferences regarding insulin dose must take into account body weight.

123. Improvement of Peripheral and Autonomic Neuropathy During One Year of Treatment with Continuous Subcutaneous Insulin Infusion in Type I (Insulin-Dependent) Diabetes S. Gambardella, A. Napoli, V. Spallone, A. M. Verrastro, F. Jacoangeli, C. GeraldinF and G. Menzinger 2. I Cattedra Endocrinologia and qstituto Neurologia*, University of Rome and 2Catt. Med. Costit., Uni- versity of Naples, Italy

156 Abstracts

In eight Type 1 diabetic patients (mean age 39 years, mean duration of diabetes 15 years) maximum motor conduction velocity (MCV) of median and peroneal nerves, sensory conduction velocity (SCV) of median nerve and beat to beat variation of heart rate during deep breathing (mean rate difference: MRD) were studied during poor diabetic control and after 1, 3, 6 and 12 months of improved metabolic control maintained by CSII (mean daily blood glucose -104.1 • 8.3 versus 175.5 -+ 21.7 mg/dl, p < 0.05 and HbA~ : 6.8 • 0.3 % versus 9.3 • 0.5%, p< 0.01). Our data, analyzed by the paired t-test, show long lasting significant improvement both in MCV (mn=basal: 48.5-+ 1.7 m/s: 1 month: 53.4 -+ 2, p < 0.001 : 3 months: 56.3 _+ 1.5, p < 0.001 ; 6 months: 54.7+2, p<0.01; 12months: 51.7+3m/s, NS) and SCV (basal: 52.6 -+ 1.4; I month: 56.3 • 1.9, p < 0.001 ; 3 months: 57.8 -+ 1.6, p < 0.001 ; 6 months: 57.2 • 1.6, p < 0.05; 12 months: 55.5 • 3.9, NS). Five out of the eight cases had abnormal MRD scores (10, lower limit of normal range). In these patients a progressive increase in MRD was observed (basal: 4.64+ 1.6; 1 month: 6.36+ 1.4; 3 months: 5.99+0.8; 6 months: 6.8 • 1.5; 12 months: 7.74 • 0.8) reaching significance at 1 and 6 months (p < 0.01 and 0.05).

124. Effects of Cortisol and Adrenaline Deficiency and Replacement in Glucoregulation in Adrenalectomized Dogs C. Gauthier, H. L A. Lickley, K. El Tayeb and M. Vranic. Departments of Physiology, Surgery and Medicine, University of Toronto and Women's College Hospital, Toronto, Canada To delineate cortisol-adrenaline interactions, we studied effects of adrenalectomy with or without hormonal replacement in five dogs. Basal plasma insulin (IRI) was 50% normal (8.3 • 7 mU/1), while glucagon (IRG, 168 • 14 pg/ml), glucose turnover (3.6 • 0.2 mg. kg -1. rain -~) and concentration (5.14 + 0.4 mmol/1) were normal. Thus, hypoinsulinaemia compensated for adrenal insufficiency. Cortisol infusion (2.5 !xg. kg -1. rain -1) restored normal values. Adrenaline remained undetectable. Peripheral IRI was unchanged, portal IRI oscillations transiently abolished, and IRG decreased to 126+ 45 pg/ml (p < 0.05). Glucose production and clearance (MCR) fell to 2 .6+6mg-kg -1-min -1 and 2.9_+0.3 ml-kg -I .rain -1, thus glucose concentration remained unchanged. Decreased glucose production reflects the fall in IRG, whereas decreased MCR could be an acute effect of cortisol replacement. During basal adrenaline replacement (0.02 ug. kg -1- min -1) followed by an infusion (0.1 Ixg. kg -1. min -1) to provide levels consistent with stress, adrenaline was 110+ 14 and 541 _+ 50 pg/ml. Cortisol remained undetectable. In contrast to normal dogs, adrenaline did not cause transient insulin release. Relative suppression occurred during the higher infusion rate. Glucagon increased by 40% during adrenaline infusion. During basal adrenaline replacement glycaemia was unchanged but adrenaline (0.1 gg-kg -1. rain -1) caused hyperglycaemia (7.1 -+ 3 mmol/1) comparable to that in normal dogs. However, this was due solely to a 40% decrease in MCR. Lack of adrenaline effect on glucose production must reflect cortisol deficiency, whereas the exaggerated MCR decrease could reflect the effect of adrenaline in the absence of an insulin spike.

125. Glucagon, Somatostatin and Insulin Secretion from the Isolated Pancreas of Normal and Streptozotocin-diabetie Rats During Perfusion with Tolbutamide, Meglitinide, Pirogliride and HB 7t0, a Benzamidine K. Geisen and H. Ritzel. Pharma Research, Hoechst AG, Frankfurt, FRG These four structurally distinct insulin-releasers lower blood glucose in normal laboratory animals equipotently. In concentrations of 300 gmol/1, they were compared with each other for their effect on insulin, somatostatin and glucagon release in the isolated perfused pancreas. The hormone release data given are average percentage changes (t20-t40) in comparison to preperfusion phase (t0-t19). In the normal pancreas, all four compounds, in the presence of 3.3 mmol/1 glucose and 0.05 mmol/1 IBMX, released insulin (+ 697%) and somatostatin ( + 1660%) and suppressed basal glucagon secretion ( - 62%). In the pancreas of the diabetic rat, 6 weeks after administration of 60 mg/kg streptozotocin, all four compounds, in the presence of 3.3 or 25.5 mmol/1 glucose and 0.05 retool/1 IBMX, induced weak insulin release (+37%, +165% respectively) and increased elevated spontaneous somatostatin release (+42%, + 83% respectively). Glucagon secretion (+16%, +11% respectively) was not inhibited. Without IBMX, all compounds caused glucagon release (+222%, +129% respectively) in the streptozotocin rat pancreas in the presence of 5.6 and 25.5 mmol/1 perfusate glucose concentrations. In conclusion, all compounds stimulate secretion of A, B and D cells: Glucagon suppression in the normal pancreas is caused by a paracrinal interaction predominantly with insulin. Stimulation of glucagon secretion in the

isolated pancreas is confirmed by our findings that tolbutamide also releases glucagon in the isolated perfused rat stomach.

126. Direct Effect of Insulin on Liver Ketogenic Capacity in Man P. P. G. Gerber, U. Keller and W. Stauffacher. Department of Medicine and Research, University Hospital, Basel, Switzerland During insulin deficiency lipolysis is enhanced, and increased levels of non-esterified fatty acids (NEFA) stimulate hepatic ketogenesis. In addition to this NEFA-mediated ketogenic effect, studies in vitro suggested a direct effect of insulin on hepatic ketogenic capacity. To assess whether insulin can directly affect hepatic ketogenesis in man, total ketone body production rates were determined in three groups of five to seven subjects fasted overnight, using a primed continuous infusion of 3-14-acetoacetate. Insulin secretion was suppressed by infusion of somatostatin, and insulin was infused during 170 rain to yield plasma levels of approximately 10mU/1 (groups I and 2) or of approximately 100 mU/l (group 3). In each group glucose was kept at equal levels using the glucose clamp technique and a variable glucose infusion (20%). In groups 2 and 3 NEFA levels were elevated and maintained constant at 1.8 mmol/1 by combined infusion of intralipid (0.8 ml/min) and heparin (40 U.kg - l . h 1). During intralipid infusion total ketone body production was 9.1 ___ 1.0,umol.kg -1-min -I (group 2) and 0.7 ___ 0.2 umol. kg -1. min -1 in group 1 receiving no intralipid. During hyperinsulinaemia of 100 mU/1 (group 3) the intralipid-induced increase in ketone body production was reduced to 3 76 • 0.81 umol- kg - I . min-l(p < 0.01). Thus, ketogenic effects of elevated NEFA levels were partly inhibited by insulin. The results demonstrate that insulin may alter the ketogenic capacity of the liver in m a n .

127. Mechanisms for Impaired Glucose Counter-Regulation and Post- hypoglycaemic Hyperglycaemia in Type I (Insulin-Dependent) Diabetes J.Gerich, G.Bolli, P.Cryer, and E.Tsalikian. Endocrine Research Unit, Mayo Clinic and Medical School, Rochester, Minnesota and Washington University, St. Louis, Missouri, USA To assess glucose counter-regulation after SC insulin in Type I diabetes, glucose production and utilization, and plasma glucose, free insulin and counter-regulatory hormones were determined for 12 h after SC insulin injection (0.15 U/kg) in 20 Type 1 diabetic patients without clinical neuropathy maintained euglycaemic (5 mmol/1) overnight (Biostator) and in 11 normal volunteers. Nine patients had only exaggerated (nadir 2.3 +0.2 versus 3.4+0.1 retool/1 in normal subjects p < 0.001) and prolonged (8.6 ___ 0.5 versus 5.3 • 0.6 h < 4 retool/1 in normal subjects, p < 0.01) hypoglycaemia; 11 patients had similar hypoglycaemia followed by rebound hyperglycaemia (16_+1 versus 4.2 + 0.1 retool/1 in normal subjects at 12 h, p < 0.001). The exaggerated hypoglycaemia resulted from prolonged suppression of glucose production accompanied by paradoxically suppressed glucose utilization, increased plasma free insulin, absent glucagon responses and relatively impaired adrenaline, growth hormone and cortisol responses. Rebound hyperglycaemia resulted from late accelerated glucose production as plasma-free insulin decreased below baseline and could be abolished by early infusion of insulin plus prevention of counter-regulatory hormone responses (euglycaemic clamp), but not by infusion of insulin alone. Conclusions: prolonged hypoglycaemia after SC insulin in Type 1 diabetic patients results from impaired counter-regulatory hormone secretion and decreased insulin clearance with reversal primarily via decreased glucose utilization rather than increased glucose production (2). Increased counter-regulatory hormone secretion, albeit subnormal, is essential for post-hypoglycaemic hyperglycaemia but insulinopenia is not.

128. Urinary Excretion of Glycosylalbumin with Abnormal Isoelectric Points in Diabetic Nephropathy G. M. Ghiggeri, G. Candiano, G. Delfino and C. Queirolo. Hemodial- ysis Service, Hospital of Lavagna, Italy To evaluate diabetic nephropathy in the light of current knowledges on membranes selectivity towards charged macromolecules, isoelectric points and affinity for lectins of urinary albumin have been evaluated in ten normal subjects and 28 Type I (insulin-dependent) diabetic patients classified as: (A) normal (Albur < 10 ~tg/min), (B) functional nephropathy (10 < Alb~r < 100 ~tg/min), (C) organic nephropathy (Albor> 100 gg/min). Albumin was purified by Blu-Sepharose CL- 6B. Ultrathin isoelectric focussing was performed in silanized glass plate and stained with photochemical ultrasensitive silver stain. Car- bohydrates were evaluated with affinity chromatography on Concan- avalin-A Sepharose. Normal subjects and group A showed bands between 4 and 4.7 isoelectric points with a main peak at 4 with affinity

Abstracts 157

for lectins. Group B showed a main peack at 4.6 isoelectric points. Group C showed a main peack at 4.6 isoelectric points and some cationic bands with affinity for lectins. These results show that anionic glycosylalbumin is the main urinary albumin in normal subjects and in diabetic patients without nephropathy and account for a physiological mechanism of albumin excretion mediated by carbohydrate content. They further support the idea for a tubular origin of increased albuminuria in diabetic functional nephropathy.

129. A Role for Endogenous Prostaglandin E in the Biphasic Pattern of Insulin Release in Man D. Giugliano, E Di Pinto, F. Saccomanno, R. Torella, N. Passariello, N. Frascolla and F. D'Onofrio. Institute of Medical Pathology, I Fac- ulty of Medicine, University of Naples, Italy The inhibitory effect of prostaglandin E upon insulin secretion in man is well established. In the present study, we examined the possibility that endogenous prostaglandin E may account for the biphasic pattern of insulin secretion in man. Normal non-obese and non-diabetic volunteers were studied. Each subject served as his own control and received, on two separate days, a square-wave glucose stimulation (0.5 g/kg as bolus followed by a 20 rag-kg -1- rain -~ glucose infusion) previously determined to give maximal insulin release. Infusion of lysine acetylsalicylate (LAS, 72 rag/rain) caused a twofold increase of total insulin secretion (4 787 _+ 840 versus 2142 _+ 502 mU. 1-4. min-~, p < 0.01) and converted the biphasic insulin response to a multiphasic pattern. To test the possibility that LAS worked via the inhibition of endogenous prostaglandin E synthesis, prostaglandin E~ (0.2 ~Lg. kg 1.rain 1) or prostaglandin E2 (10 ug/min) were infused in addition to LAS. Both prostaglandins re-established the biphasic pattern of insulin release and also decreased insulin to control values. Infu- sions of either prostaglandin E1 or prostaglandin E2 in the absence of LAS reset insulin secretion to a lower level without altering the kinetics of release. On the basis of these results, we hypothesize that endogenous prostaglandin E released in response to glucose exerts an inhibitory effect on insulin secretion that becomes biphasic in appearance.

130. Insulin Secretion and Glucokinase Activity in Ventral and Dorsal Parts of the Rat Pancreas R.Goberna, J.M.L6pez Martinez, R.Ramirez, F.J.Bedoya and J. Jim~nez. Department of Biochemistry. Faculty of Medicine, Seville, Spain It has been established that the endocrine cell composition of islets is different in differents parts of the pancreas. We have studied the secretory response of ventral and dorsal parts of the pancreas towards some secretagogues by developing a technique for selective perfusion of dorsal and ventral areas of the rat pancreas and by using islets isolated from both parts. In addition, we have studied the activity of glucokinase in islet extracts from both parts. The insulin secretory response of perfused ventral pancreas was 50% lower than in dorsal pancreas despite the fact that insulin and protein contents were similar. This was confirmed by measuring the insulin secretion of islets isolated from both parts. In contrast, there was no difference in the glucokinase activity (3.55 + 0.32, in ventral islets (n = 7) versus 3.84 + 0.01 in dorsal islets (n = 6) pmol of D [U14C] glucose, min-a.islet-4). The results suggest that the differences in insulin secretion may indeed be due to paracrine factors rather than intrinsic B cell differences.

131. Glucose Disposal, Insulin Secretion, and Insulin Resistance in Obesity and Diabetes Mellitus A.Golay, R.DeFronzo, D.Thorin, M. Dusmet, E.Jequier, and J.P. Felber. University of Lausanne and CHUV, Lausanne, Switzerland and Yale University, New Haven, USA Using the euglycaemic insulin clamp (approximately 100 mU/1) with indirect calorimetry, we characterized the metabolic disturbances in the following groups: (a) 17healthy control subjects, (b) six obese non-diabetic subjects, (c) eight non-obese Type2 (non-insulin-dependent) diabetic patients, (d) five obese hyperinsulinaemic diabetic patients, (e) six obese hypoinsulinaemic Type 2 diabetic patients. In- sulin secretory status was determined with a 4-h oral glucose tolerance test. During the insulin clamp, total body glucose uptake was similarly reduced (p < 0.01) in obese non-diabetic subjects (4.1 _+ 0.3 mg-kg -1. rain 1) and non-obese diabetic patients (4.4_+ 0.5), compared with control patients (7.2_+ 0.5). In the obese hyperinsulinaemic (2.7 +

1 1 0.4 mg. kg- �9 min- ) and hypoinsulinaemic (3.2 _+ 0.2) patients, total glucose uptake was further reduced versus obese non-diabetic and non-obese diabetic subjects (/2 < 0.02). Glucose oxidation

(1.3-1.7 mg-kg -1. min -1) was decreased similarly in all experimental groups versus the control subjects (2.7+0.2, p<0.001). However, quantitatively the decrease in non-oxidative glucose uptake was two- to threefold greater in all groups compared with controls (p< 0.01). Hepatic glucose production suppressed normally (> 80%-90%) in all groups except obese hypoinsulinaemic diabetic patients (50% _+ 5%, p< 0.05). Basal plasma non-esterified fatty acids were elevated by 50%-100% versus control levels (p < 0.01) and was strongly correlated (r > 0.80) to the observed defects in total glucose uptake, glucose oxidation, and non-oxidative glucose disposal. We conclude that the same metabolic disturbances contribute to the insulin resistance in obesity and diabetes mellitus and may partially be explained by elevated non-esterified fatty acid levels.

132. Mechanism of Calcium Effect on Insulin-stimulation of Hexose Transport J. Goldman and V. Fisher. Henry Ford Hospital, Detroit, Michigan, USA Calcium (Ca) ions are required for optimal insulin action. To study the mechanism of this requirement, the dependence of basal and insulin-stimulated 2-deoxy-glucose transport on hexose concentration was investigated in isolated rat adipocytes, with and without Ca (2.5 mmol/1) at 37 ~ (n= 6). Carrier-mediated hexose transport was assessed by subtracting from total uptake the diffusional component measured in the presence of cytochalasin b (50 p.mol/1). Km and Vmax values were calculated from Lineweaver-Burk plots. Without added Ca + +, Km values were 10.0 and 12.5 mmol/1 for basal and insulin- stimulated transport, respectively, while the corresponding Vmax values were 2.4 and 3.3nmol.min-l.105 cells -~. In the presence of Ca ++, Km values were 12.5 and 11.1 mmol/1 for basal and insulin- stimulated transport, respectively, and the corresponding Vm= values were 4.2 and 8.3 nmol. rain -1.10Scells -1. In conclusion, the hexose transport Km is affected by neither Ca + + nor insulin, while Vma~ is increased from basal values by insulin in the absence of Ca + +. Vmax is further enhanced by Ca ++ for both basal and insulin-stimulated transport. These results are consistent with a Ca + + mechanism for the potentiation of basal and insulin-stimulated hexose transport involving increased numbers of transporter units and/or enhanced effec- tiveness of hexose translocation.

133. Sorbitol as a Marker of Diabetic Control in Pregnancy J. Goldman, B. Greenblatt and M. Karp. Hasharon (Golda) Medical Center, Petah-Tikva, Israel Sorbitol is an intermediate in the polyol pathway whose activity is increased when ambient glucose rises above physiological levels. Accu- mulation of sorbitol has been reported to be one of the factors responsible for pathogenesis of diabetes associated complications. Sorbitol was measured enzymatically in the erythrocytes of 20 insulin-dependent diabetic pregnant women, and in a control group of 12 healthy pregnant women. No correlation was found between gestational week and sorbitol levels. It was also three times higher in the erythrocytes of the diabetic group. A significant correlation was found between sorbitol levels of the diabetic group and percentage glycosylated haemoglobin. Furthermore, the correlation between mean erythrocyte sorbitol levels during pregnancy and fetal weight was significantly correlated, while none was established between fetal weight and glycosylated haemoglobin. Mean sorbitol levels in homogenates of diabetic placentas and those of the control group were found to be significantly different. Consequently, while erythrocyte sorbitol levels are an excellent monitor of diabetic pregnancy, they seem of little prognostic value for outcome of pregnancy.

134. Significance of Transglutaminase-Mediated Crosslinking of Pro- teins in Insulin Release R. Gomis, P. Lebrun, A. Herchuelz, F. Malaisse-Lagae, A. Sener and W. J. Malaisse. Laboratories of Experimental Medicine and Pharma- cology, University of Brussels, Brussels, Belgium Transglutaminase is an ubiquitous enzyme catalyzing crosslinking of proteins and participating in various processes, such as blood coagulation, post-ejaculatory clotting and receptor-mediated endocytosis. Islet homogenates display Ca 2+-dependent transglutaminase activity,

3 14 catalyzing the incorporation of [2,5- H] histamine, [1,4- C] P-tosyl- 14+ putrescine and [ C] p-tosyl-methylamine into N,N-dimethylcasein.

Methylamine, p-tosylglycine and monodansylcadaverine inhibited enzyme activity. Methylamine rapidly accumulated in intact islets and was incorporated in endogenous proteins. The oxidation of glucose and endogenous fatty acids or glutamate was unaffected by methylamine (2 retool/l). Methylamine caused a rapid, reversible and dose-

158 Abstracts

related (1-10 retool/l) inhibition of insulin release evoked by D-glucose, 2-ketoisocaproate, L-leucine, L-lysine, L-arginine and gliclazide. However, insulin release evoked by phorbol 12-myristate 13-acetate or the association of Ba 2+ and theophylline was less sensitive to methylamine than that evoked by other secretagogues. The inhibitory effect of methylamine was decreased after pre-incubation with glucose and inversely related to the extracellular Ca 2+ concentration. Methylamine increased 86Rb outflow from prelabelled islets and decreased nutrient-stimulated 45Ca uptake, whilst failing to affect 45Ca uptake or efflux evoked by an increase in intracellular K + concentration. Thus, transglutaminase represents a novel target responsive to changes in cytosolic Ca 2+ concentration, and participates both in the control of K + conductance and exocytotic process in the B cell.

135. Decreased Myocardial Calcium Uptake and Beta-Adrenergie De- sensitization in Streptozotocin Diabetic Rats O. Gotzsche. University Institute of Pathology and ~ University Clinic of Internal Medicine, Kommunehospitalet, Arhus, Denmark Myocardial catecholamine sensitivity was investigated in the short- term (8 days) in streptozotocin diabetic rat. Myocardial calcium up-

-45 take ( Ca labelling followed by cold washout) was measured in the isolated perfused heart (a. m. Langendorff) in response to isoproterenol (ISO) (10 -4 mol/1). In vivo and in vitro production of cyclic AMP after ISO stimulation was measured in control (n = 8) and diabetic rats (n=8). Basal myocardial calcium uptake (nmol.g protein -a. rain-l; mean+SEM): control: 86.1_+5.1; diabetic: 90.6_+4.3 (NS); after ISO: control: 118.6 _+ 3.7; diabetic: 95.4 _+ 5.3 (2 p < 0.001). The in vivo cyclic AMP response 60 rain after a SC injection of ISO (30 rag/ kg) was diminished in diabetic rats (control: 5.13_+0.47; diabetic: 2.95 _+ 0.18 pmol/mg protein, 2 p < 0.001) as was adenylate cyclase activity (pmol cyclic AMP.rag protein-l .min -I) when expressed as ISO induced increase: control: 50.0_+ 8.7%; diabetic: 1.2_+ 5.7%, (2 p< 0.00l). The adenylate cyclase activity after stimulation via the nucleotide binding protein with GTP and NaF was intact in diabetic rats. An abnormal coupling from beta-receptor to the catalytic unit of adenylate cyclase seems to exist. As the permeable cyclic AMP analogue, db-cyclic AMP lead to an increased calcium uptake only in control hearts (43%), the coexistence of a decreased myocardial calcium uptake and a beta-adrenergic desensitization in the myocardium in experimental diabetes is suggested.

136. The Influence of Insulin Binding Antibodies on Bovine and Biosyn- thetic Human Insulin Action in Type I (Insulin-Dependent) Diabetes R. S. Gray, P. Cowan, U. Di Mario, B.F. Clarke and L.J.P. Duncan. Diabetic Outpatient Department, Royal Infirmary, Edinburgh, Scot- land, UK Sixteen Type 1 diabetic and eight age and weight matched non-diabetic subjects were infused IV with bovine and biosynthetic human insulin (BHI) on separate days at 1 mU. kg -1. rain -1. Antibody binding of bovine and BHI measured 22.3 + 3.4 and 20.0-+ 4.0% (mean + SEM; NS) in the diabetic subjects. Blood glucose concentration was clamped for 2 h at 3.9 retool/1 and insulin-mediated glucose metabolism was determined during the second hour. The clamp was maintained and glucose metabolism determined having discontinued the insulin infusion. Glucose metabolism values were similar during bovine and BHI infusions in non-diabetic (361.1-+33.3 and 383.3_+ 33.3 gmol. kg -1.rain -1) and were greater (p < 0.01 and < 0.01) than those of diabetic subjects (228.9 _+ 23.3 and 207.2_+ 19.4 lxmol, kg 1. min-1). Having discontinued the insulin infusion, glucose metabolism declined at a similar rate following bovine and BHI infusions in control (4.86 _+ 0.72 and 3.85 _+ 0.44 lxmol/kg per min 2) and diabetic subjects (3 32_+ 0 70 and 4.50_+ 0.77 .umol/kg per rain2). When the results of bovine and BHI infusions in diabetic subjects were combined, insulin antibody binding correlated negatively with glucose metabolism during the insulin infusion (r= -0.37, p< 0.05) and with rate of decline of glucose metabolism having discontinued the insulin infusion (r= - 0.62, p < 0.001). Conclusions: (1) IV infused bovine and BHI have similar biological activities; (2) Type 1 diabetic subjects are insulin resistant; (3) antibody binding of insulin contributes to this insulin resistance; (4) antibody binding of insulin profoundly influences the rate of insulin deactivation.

137. Prostaglandin E in Rat Islets of Langerhans and its Relationship to Insulin Secretion in Vitro I. C. Green, D. Perrin and S. Bogdanovich. Department of Biochem- istry, The University of Sussex, Brighton, UK Rat islets of Langerhans synthesize several species of prostaglandin. The relationship between islet prostaglandin production and insulin

secretio n was investigated, since prostaglandin E1 and E2 (PGE) have been implicated in the impaired acute insulin response in non-insulin dependent diabetes. Collagenase-isolated rat islets were incubated for 15-60rain in groups of 20-30, in medium containing 6mmol/ l glucose _+ additions. Insulin secreted into the medium was measured; prostaglandins in islet tissue were extracted and radioimmunoassayed for total PGE. Sodium salicylate, a prostaglandin synthesis inhibitor, caused a time-dependent fall in islet PGE content from 15.9 _+_ 1.9 to 6.5 + 1.0 pg PGE-islet -~. h-1, and significantly stimulated insulin release. An opioid peptide, dynorphin-(1-13), 6x10-9mol/1), and chlorpropamide (70 gmol/1), decreased islet PGE from 5.45 + 0.65 to 2.20 _+ 0.25 and 2.94_+ 0.26 pg/ug islet protein per 20 min respectively, while significantly stimulating insulin secretion. Exogenous PGEI and PGE2 (10-9mol/1) actually stimulated insulin secretion, but when used at a higher concentration (10 -5 mol/1) both depressed control, and abolished dynorphin-stimulated relase. We conclude that there is a negative correlation between islet PGE content and insulin secretion. Sodium salicylate and chlorpropamide, which are known to restore the acute insulin response, lowered islet PGE levels. The action of opioid peptides may be linked to lowering prostaglandin E production in islets.

138. Immune Intervention Trial in Newly Diagnosed Type I (Insulin-De- pendent) Diabetes B. Greulich, T. Lander, E. Standl, H. Kolb, K.-D. Gerbitz, D. Kuschak and F. A. Gries. Dtisseldorf and Munich, FRG There is strong evidence that the immune system plays a role in islet cell destruction leading to human Type 1 diabetes. The immunomod- ulatory drug inosiplex has been shown to diminish islet B cell destruction in mice with low dose streptozotocin diabetes. The clinical trial was designed to evaluate the possible benefical effect of inosiplex in newly diagnosed Type 1 diabetic patients. Eleven patients (mean age 18 + 2 years) with recent onset Type 1 diabetes received, in addition to insulin, inosiplex (50 mg/kg body weight daily) for two periods of 4 weeks, the first dose being given within 5 days of diagnosis. These patients and a control group (eight patients) were seen regularly for metabolic control and evaluation of immune status during a follow- up period of 6 months to date. Results: daily mean insulin requirement at 6 months was indentical in the inosiplex and control groups (0.43 _+ 0.1 IU/kg body weight), fasting C-peptide levels were 0.26 _+ 0.05 mmol/l in inosiplex-treated patients versus 0.22 + 0.08 nmol/l in control subjects. HbAa values were 8.2 + 0.3 versus 8.6_+ 0.8%. The development of islet cell antibody titres did not differ in both groups, mean insulin antibody titres were indentical also. No side effects of inosiplex treatment, apart from one transient hyperuricaemia, were seen. It is concluded that so far the above treatment schedule has no clinically important effect on the endogenous insulin reserve.

139. Impaired Cerebrovaseular Response to Hypercapnia in Diabetic Patients D. N. W. Griffith, S. Saimbi, k C.A.Watson and D.J. Betteridge. De- partment of Medicine, University College London School of Medi- cine, The Rayne Institute, University Street, London, UK The normal response of cerebral blood flow is to increase when arterial CO2 tension rises. This response has been widely used as one test on the functional integrity of the cerebral vasculature. We have examined this CO2 reactivity in a diabetic population, using intravenous 133Xe- non to measure cerebral blood flow. Readings were first performed with subjects breathing room air, and subsequently 5% CO2. In a control group of 10 normal subjects (four women, six men; age range 23-68 years; mean age 48.3 years) cerebral blood flow increased normally. However, in the 17 diabetic patients studied (seven women, ten men; age range 18-65 years; mean age 39.4years; 16 insulin dependent, 1 insulin independent) no increase in flow was found in five, and a further three patients showed an impaired response. This abnormal reactivity did not correlate with the presence or absence of diabetic complications, age or duration of diabetes, some of the patients exhibiting this finding being young, with a short duration of diabetes and no clinical evidence of complications. The cause of this abnormality is not understood, but has been found also in other stroke- prone people (hypertensive patients) and could constitute early evidence of potential cerebrovascular complications.

140. Insulin Receptor Kinase of Human Erythrocytes as a Diagnostic Tool F. Grigorescu, J. S. Flier and C. R. Kahn. Joslin Diabetes Center, Beth Israel Hospital and Harvard Medical School, Boston, Massachussets, USA

Abstracts 159

Recently we have shown that the fl-subunit of the insulin receptor of human erythrocytes undergoes an insulin dependent tyrosine-specific phosphorylation consistent with the receptor being a tyrosine protein kinase. For clinical studies we have developed a method to measure the binding and kinase properties of erythrocyte insulin receptor which requires only 100 ml of blood. A standard assay consists of incubation of solubilized receptor with [32p-?']-ATP in the presence of Mn 2+ and various concentrations of insulin, immunoprecipitation with receptor antibodies and identification of phosphorylated receptor by gel electrophoresis and autoradiography. In controls (n = 8) insulin stimulated the phosphate incorporation two- to fivefold in a dose response manner. When the dose response curve for phosphorylation is correlated with the fractional occupancy of the receptor, non- linear coupling is observed. Using this method we also studied a patient with variant of type A syndrome of insulin resistance and a defect in insulin binding on erythrocytes, monocytes and fibroblasts. Prel- iminary results indicate a decrease in autophosphorylation of the insulin receptor in erythrocytes, suggesting an abnormality in both binding and kinase properties of the receptor.

141. Rise in Plasma Somatostatin Levels in Response to Glucose is Im- paired in Non-Insulin-Dependent Diabetic Patients V. Grill, M. Gutniak, A. Roovete and S. Efendi6. Department of En- docrinology, Karolinska Hospital, Stockholm, Sweden In diabetic animals with impaired metabolic control, glucose fails to stimulate somatostatin secretion from pancreas and gut. To investigate whether glucose regulation of somatostatin secretion is impaired in diabetic man, we compared somatostatin responses to 1 g/kg of glucose perorally in controls (fasting blood glucose < 5.2 mmol/1 and IV glucose tolerance > 1.0), in subjects with decreased glucose tolerance (blood glucose 5.2-7.0 mmol/l and IV glucose tolerance < 1.0) as well as in patients with diet-treated non-insulin-dependent diabetes mellitus (fasting blood glucose ~> 7.0 mmol/l). Somatostatin-like immunoreactivity (SLI) in plasma was concentrated and extracted on silica glass before radioimmunoassay as previously described. Fasting levels of SLI did not differ significantly between groups. In control subjects glucose elicited a 45% sustained rise in SLI (incremental effect 285 _+ 75 pg. ml ~. 90 rain ~, n= 6, p < 0.02). A similar response was seen in subjects with decreased IV glucose tolerance (n= 6, p < 0.05). Conversely no effect was seen 0-120 min after glucose ingestion in diabetic patients (n= 6). Using identical protocols except for glucose omission, no effect on SLI was seen in any group. We conclude that in man peroral glucose stimulates somatostatin release into the systemic circulation and that this effect is lost in overt non-insulin-dependent diabetes.

142. Monoclonal Antibodies to the Insulin Secretory Granule Mem- brane K. A. Grimaldi, J.C. Hutton and K. Siddle. Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK Hybridoma technology has proved useful in the production of antibodies of defined specificities to antigenic components which are not readily purified and available only in small amounts. We have raised monoelonal antibodies to secretory granule membranes isolated from a rat insulinoma. Spleen cells from immunized Balb/c mice were fused with NSO or NSI myeloma cells. Hybridoma supernatants were screened with granule membranes immobilized in poly-L-lysine coated wells of flexible microtitre plates using 125I-labelled sheep anti- mouse immunoglobulin to detect binding. 196 positive cell lines were generated of which 60 were stable after 3 months of continuous sub- culture. Subcellular and tissue specificity is being assessed and results so far indicate that no antibodies bind to membranes prepared from tumour mitochondrial and lysosomal fractions or erythrocyte ghosts. Western blotting of SDS-polyacrylamide gels of insulin secretory granule proteins using the hybridoma supernatants showed positive reaction to proteins of molecular weights in the range of 50,000- 60,000 M r (5 cell lines) and 100,000-120,000 Mr (14 cell lines). The availability of large amounts of antibody specific to the molecular components of the secretory apparatus will be valuable in the study of the mechanism of stimulus-secretion coupling of insulin release.

t43. Insulin Secretion and Insulin Sensitivity in Relation to the Duration of Type 2 (Non-Insulin-Dependent) Diabetes L. Groop. Fourth Department of Medicine, University of Helsinki, Helsinki, Finland Although not proven, it has generally been suggested that a progressive deterioration of B cell function may explain the increasing rate of

secondary drug failure with time in Type 2 diabetes. To study this problem, we investigated 288 patients with onset of non-ketotic diabetes between the ages of 35 and 70 years with respect to B cell function (C-peptide response to IV glucagon) and insulin sensitivity (insulin tolerance test). The patients were divided into four subgroups with different duration of their diabetes; (a) < 2 years (n = 60); (b) 2-4years (n=50); (c) 5-10years (n=55); and (d)> 10years (n=63). Diabetic control and mean relative body weight were similar in the four groups. The C-peptide concentrations did not differ significantly between the four groups, indicating unaltered B cell function in relation to time in these patients. In contrast, the insulin sensitivity (expressed as glucose disposal in response to IV insulin, Kirr) was decreased in groups b, c and d compared with group a (p < 0.01). The data suggest that progressive deterioration of B cell function with time is not a main feature of Type 2 diabetes, whereas insulin sensitivity decreases relatively soon in the course of the disease.

144. Sex-Associated Differences in Insulin Binding and Action on Glu- cose Transport and Metabolism in Rat Adipocytes M.Guerre-Millo, A.Leturque, M.Lavau and J.Girard. U. 177 INSERM, Institut Biom6dical des Cordeliers, Paris, France The few studies of insulin action on glucose metabolism in adipocytes from adult female rats have reported a low responsiveness to the hormone. In this study, insulin binding and action were compared in ovarian and epididymal adipocytes from 90-100 day-old female and age-matched male rats. Ovarian and epididymal adipocytes were similar in size (0.178 versus 0.154 ~g lipids/cell). The insulin dose-re-

14 sponse curves of 2-deoxyglucose transport and [U- C]-glucose incorporation into CO2 and lipids were shifted to the left in ovarian adipocytes compared with epididymal adipocytes, indicating increased insulin sensitivity in females. The maximal stimulation of glucose transport ( x 4) and metabolism ( x 3) by insulin was similar in both sexes. These data were consistent with insulin binding-curves: ovarian adipocytes bound significantly more insulin than epididymal at low insulin concentrations (< 13 mU/1) due to an increased affinity for insulin of specific receptors. No differences were found in the number of insulin receptors per cell between females and males. In contrast to previous studies, our data demonstrate that adipocytes from female rats are highly responsive to insulin in vitro.

145. Relationships Between Aerobic Capacity, Glyeaemic Control and Serum Lipoproteins and the Effects of Long-term Regular Physical Training in Female Type I (Insulin-Dependent) Diabetic Patients R. Gunnarsson, H. Wallberg-Henriksson, S. R6ssner and J. Wahren. Huddinge Hospital and Karolinska Hospital, Stockholm, Sweden The Framingham Study showed that insulin-treated diabetic women have a high relative mortality from coronary heart disease. Diabetic patient are often found to have serum lipid abnormalities that could contribute to premature atherosclerosis. Regular physical training in normal subjects is generally considered to beneficially influence lipoprotein levels. We have examined the relationships between maximal oxygen uptake (VO2max), HbA1 and serum lipoproteins in 18 female Type 1 diabetic patients. Lipoproteins were determined by preparative ultracentrifugation. Before the training period, there was no correlation between VO2max and HBA~ (r= -0.23; NS). A positive correlation was found between VO2max and HDL and HDL2 cholesterol (r= 0.54 and 0.61; p < 0.05) while LDL cholesterol correlated negatively with VO2max (r= -0.59; p< 0.05). HDL and HDI< cholesterol correlated negatively with HbA1 (r=-0.48 and -0.52; p< 0.05). Half the patients were randomized to a 5 month training program with 20 rain bicycling daily. Their VO2max increased with 8% (p < 0.05), but there were no changes in serum lipoprotein concentrations or in glycaemic control assessed by home monitored blood glucose and monthly determinations of HbAv In summary, the pre- training correlations between VO2max, HbA1 and lipoprotein levels suggested that a high aerobic capacity and good diabetic control fa- vourably influence serum lipids in Type 1 diabetes, but the study did not demonstrate any effect of long-term regular training on either lipid status or glycaemic control.

146. Somatostatin Response to Mixed Meal in Healthy and Diabetic Subjects and in Pancreatectomized Patients M. Gutniak ~, V. GrilP, K. L. Wiechel 2, L. Thulin 3 and S. Efendi61 . ~De- partment of Endocrinology, Karolinska Hospital, 2Department of Surgery, SOdersjukhuset, and 3Department of Surgery, Huddinge Hospital, Stockholm, Sweden The aim of the study was to evaluate somatostatin response to mixed meal and its modulation by improvement of metabolic control of dia-

160 Abstracts

betes mellitus. Somatostatin in plasma was measured after extraction on silica glass before, during and 120min after breakfast (1258 kJ, 26% carbohydrates, 23% protein, 51% fat). Subjects: (group A) six healthy volunteers - fasting blood glucose (FBG) 4.2 _+ 0.2 mmol/1, (group B) six subjects with decreased glucose tolerance - FBG 6.4 + 0.5 mmol/l, (group C) four Type 2 (non-insulin-dependent) diabetic patient - FBG 10.2 _+ 0.9 retool/l, (group D) four Type I (insulin-dependent) diabetic patients - FBG 12.5 + 1.1 retool/l, (group E) four pancreatectomized patients - fasting blood 12.4_+3.2mmol/1. Groups D and E were studied firstly, after withdrawal of insulin treatment for 12 h and secondly, when optimal metabolic control was achieved by Biostator. Basal somatostatin levels in groups A-E were (pg/ml): 15.5_+5 (group A), 21.1 _+5 (group B), 18.0+3 (group C), 21.7 _+ 5 (group D), 36.8 + 16 (group E). Thus significantly higher levels were found in group E. Mixed meal significantly stimulated somatostatin response in groups A - D but not in E (pg-m1-1.120 rain-a): 1293-+186 (groupA), 1819_+272 (groupB), 1919+227 (groupC), 1478 -+ 290 (group D), 114 +_ 39 (group E). In Biostator experiments the response was increased in groupD (2220+417pg.ml -~. 120rain -1) and normalized in groupE (1497_+284 pg.ml -a. 120 min-1). In conclusions, (1) mixed meal stimulates somatostatin release in healthy subjects, Type 1 and Type 2 diabetic patients but not in pancreatectomized patients, (2) short-term normalization of blood glucose enhances this response, (3) gastrointestinal somatostatin contributes greatly to somatostatin activity appearing in plasma after meal.

147. Gastroparesis Diabeticorum: The Role of Surgery and the Histo- logical Abnormalities of the Vagus Nerve R.J.C.Guy, A.K.Sharma, P.K.Thomas and P.J.Watkins. Diabetic Department, King's College Hospital, London, and the Department of Neurological Science, The Royal Free Hospital, London, UK Gastroparesis causing intractable vomiting is an exceptionally rare feature of diabetic autonomic neuropathy. We describe two cases in whom all conservative measures failed. The results of surgery and, for the first time, detailed measurements on the abdominal vagus are reported. The patients, aged 28 and 29 years, had diabetes for 12 and 16 years. Both were overwhelmed with problems from an autonomic neuropathy with severely abnormal autonomic function tests (heart rate variability < 3 per min). X-ray screening showed no effective gastric peristalsis. A gastroenterostomy in the first case was complicated by reflux gastritis requiring further surgery and failed. In the second a gastrojejunostomy was successful. These observations confirm that even surgery is not always successful. Electron microscopic studies of the vagus from the second case showed a severe reduction in the density of unmyelinated axons (49,000/mm 2, normal: 209,000 _+ 66/ram 2, mean + SD). The surviving axons tended to be of small calibre suggesting either preferential loss of axons of large fibre size or diffuse involvement with the presence of small regenerating axons. The basal lamina surrounding the Schwann cell was thickened to 293.5 + 12 nm (mean + SEM; normal: 72.5 + 11 nm; p < 0.01). These gross abnormalities support the view that diabetic gastroparesis is related to vagal denervation.

148. Six-year Analysis of a Prospective Natural History Study of Diet Treated Type 2 (Non-Insulin-Dependent) Diabetes D. R. Hadden, A. L. T. Blair, A. B. Atkinson, L. Kennedy, E. A. Wilson, J. D. Merrett and J. A. Weaver. Diabetic Clinic, Royal Victoria Hospi- tal, Belfast, Northern Ireland, UK Prospective data on the Belfast Diet Study are now available up to 6years. 172 symptomatic newly-diagnosed diabetic patients aged 40 69 years were managed by energy-restricted diet (approximately 1500 kcal, 42% CHO, 20% protein, 38% fat, 13 g fibre content). Life table analysis showed a 5-year survival of 89.2% among the study group, which compares with a 91.9% survival for an age/sex matched cohort constructed from the tables of the Registrar General for the whole population of Northern Ireland. There were 23 deaths in the study group. Significant risk factors (p < 0.05) by analysis of variance for mortality were: greater age at onset (55.7years for survivors, 61.0years for deaths); higher fasting plasma glucose at onset (11.1 mmol/1 for survivors, 12.6 mmol/1 for deaths); and lower fasting plasma insulin at onset (12.0 mU/1 for survivors, 9.6 mU/1 for deaths). Weight, percentage of ideal weight, fasting plasma triglyceride or cholesterol at onset did not predict survival. After 6 months of dietary management, the values for fasting glucose and insulin no longer predicted survival.

149. Organ-Specific Autoantibodies in Type I (Insulin-Dependent) Dia- betic and Non-Diabetic Families B. H~iggl61 n, G. Holmgren ~, A. Huen 3, A. Lernmark 2, P. Mackay 2, A.Rabinovitch and A.Rubensteint 1University of Umefi, Umegt, Sweden, ZHagedorn Research Laboratory, Gentofte, Denmark, and 3University of Chicago, Chicago, USA Islet cell surface antibodies (ICSA), complement-dependent antibody-mediated cytotoxicity (C'AMC), and thyrogastric antibodies (TMA, TGA, and PC) were analysed in 30 Type 1 diabetic families (n = 124) with 1-2 Type 1 diabetic probands and in 30 healthy control families (n= 106). None of the Type 1 diabetic relatives and the control individuals had a diagnosed disease. The control families had no history of diabetes. The Type I diabetic probands (age: 5-23 years, duration of diabetes: 2-19 years) had ICSA (20%) and C'AMC (10%) while these antibodies did not differ in control of diabetic families, be it parents or siblings. The diabetic patients (p= 0.02) and their parents (p < 0.005) had an increased prevalence of thyrogastric autoantibodies compared to controls. The prevalence of autoantibodies among one or both parents to a Type 1 diabetic child was 43% compared to 17% in control families (p = 0.05). Autoantibodies in diabetic patients and their relatives were associated to the HLA-DR3 (p < 0.02) while autoantibodies in the healthy controls occurred without association to HLA. In conclusion: (1) ICSA and C'AMC are primarily found in Type 1 diabetes; (2) relatives to Type I diabetic children have autoantibodies associated to HLA-DR3; and (3) Type 1 diabetic parents, more often than HLA-DR matched control parents, have organ-specific autoantibodies.

150. Effect of Pregnancy on B cell Replication and Insulin Content in Normoglycaemic Rats with a Reduced B cell Mass B. Ziegler, S. Noack, J. M. Diaz-Alonso and H.J. Hahn. Central Insti- tute of Diabetes 'Gerhardt Katsch', Karlsburg, GDR The present investigation was undertaken to clarify whether the effect of pregnancy on pancreatic B cells is preserved in normoglycaemic animals with a reduced pancreatic B cell mass induced by a single IV injection of streptozotocin (STZ; 30 mg/kg). Such STZ-treated animals were characterized by a reduction of pancreatic insulin content (up to 50% on extraction) and of B cell mass (to 65% measured by im- munohistochemical morphometry) and they do not restore their pancreatic insulin content despite normoglycaemia over a period of 50 days. During pregnancy, 20% of these animals developed a progressively increasing hyperglycaemia which was paralleled by a marked decrease of pancreatic insulin content. The other STZ-treated rats with a compensated glucose metabolism during pregnancy were characterized by an increase in their pancreatic insulin content, in B cell mass, and in islet DNA synthesis in comparison to non-pregnant STZ-treated rats. Our results show that residual B ceils in normoglycaemic STZ-treated rats respond to pregnancy-associated factors with an increased replication to compensate for the enhanced insulin demand.

151. Abnormal Vascular and Endothelial Response to Ischaemia and Venous Stasis in Diabetic Microangiopathy B. Haitas, A. J. Barnes, S. C. Williams, M. E. C. Shogry, J. C. Moore and R.C. Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Oxford, UK Vascular responses in insulin-dependent diabetic patients with and without retinopathy have been studied. After 10min, forearm ischaemia produced by a sphygmomanometer cuff at 200 mmHg, the hyperaemic response was measured with a transcutaneous oxygen monitor. Eleven diabetic patients with proliferative retinopathy had a diminished response at 60 s (9 _+ 3 mmHg) compared with 11 matched diabetic patients without retinopathy (15 + 4 mmHg, p < 0.005) and 13 normal subjects (14 + 4 mmHg, p < 0.005). Patients with microangiopathy had an impaired vibration sensory threshold (p < 0.01) compared to patients without retinopathy. The vibration sensory threshold correlated significantly with the hyperaemic response (r = -0.54, p < 0.05). Abnormal vascular reactivity may be a consequence of microangiopathy or abnormal neural control. After 10 min forearm venous stasis with the cuff at 80 mmHg, diabetic patients with proliferative retinopathy had diminished fibrinolytic activity (median 0.25 tL/ ml) compared with normal subjects (0.64bt/ml; p<0.005) and diabetics without retinopathy (0.72 g/ml; p < 0.01). After venous stasis, plasma fibronectin rose significantly in diabetic patients with retinopathy (317_+ 96 to 399_+ 74 lxg/ml; p< 0.002), and without retinopathy (301 + 77 to 364 + 66 gg/ml; p < 0.05), but not in normal subjects. These abnormal vascular and endothelial responses could

Abstracts 161

promote and perpetuate diabetic microangiopathy, and contribute to the inexorable progression of advanced nephropathy and retinopathy despite excellent glucose control.

152. Modified Proinsulin is Degraded Rapidly within Pancreatic B Cells P. A. Halban, M. Amherdt, L. Orci and A. E. Renold. Institute of Clini- cal Biochemistry, University of Geneva, Geneva, Switzerland Modified cytosolic proteins are known to be degraded more rapidly than native products. We wished to determine whether the same applies to a modified protein within the potentially protective environment of secretory granules. Rat islets were labelled (30 min, [3H]leucine) in the presence, or absence (controls), of canavanine and thialysine (arginine and lysine analogues), followed by a 24 h chase without analogues. Labelled products were assayed by immunoprecipitation followed by gel chromatography to separate proinsulin from insulin. Incorporation of analogues into proinsulin inhibited its conversion to insulin during the chase, but the labelled modified proinsulin was released from islets at the same rate as native labelled proinsulin and insulin from controls, suggesting sequestration of such material into secretory granules. Despite this, 36% of prelabelled modified proinsulin was degraded within islet cells during the chase, compared with only 7% for native products in control islets. The modified proinsulin, if not released, is thus recognized as such and dis- posed of rapidly by B cells. To account for this rapid destruction, we envisage: (1) secretory granules containing modified proinsulin have altered properties 'sensed' by lysosomes, resulting in their rapid destruction, or (2) modified proinsulin, unable to be packed as native insulin, is more sensitive to proteolysis.

153. Do Obese Subjects Have Selective Insulin Resistance? P. J. Hale, J. Crase, R. M. Baddeley and M. Nattrass. General Hospital, Steelhouse Lane, Birmingham, UK Obese subjects have impaired peripheral glucose disposal compared with normal subjects. It is unclear whether this insulin resistance is found for other effects, such as inhibition of hepatic glucose production or lipolysis. Four normal subjects (percentage ideal body weight 94-112%) and six non-diabetic, grossly obese subjects (percentage ideal body weight 207-265%) were studied during a stepped, low-dose insulin infusion. During four consecutive I h periods saline, actrapid insulin 0.005 U. kg -a. h-~, actrapid 0.01 U. kg -~. h- l , and actrapid 0.05 U.kg -1 .h -1 was infused and blood samples obtained for intermediary metabolite measurement. In normal subjects, blood glucose fell by 0.2, 0.5, and 1.7 retool/1 during the three insulin infusions compared with 0.2, 0.5 and 1.3 mmol/l in obese subjects (NS). Total ketone bodies fell throughout by 0.01 retool. 1 -~. b -1" in normal subjects and by 0.02, 0.01 and 0.05 mmol/1 in obese subjects. Blood lactate in normal subjects fell by 0.05 mmol/1 with the lowest infusion rate, then rose by 0.02 and 0.039 mmol. 1-1- h ~. The latter rise was associated with a stimulation of the metabolic clearance rate for glucose (3H-glucose infusion method). In obese subjects lactate fell by 0.03, 0.03 mmol-1-1, h -~ and then rose by 0.06 mmol/1. The results suggest that insulin resistance may be confined to glucose disposal in obese subjects.

154. Metabolism of Phospholipid Classes in Isolated Mouse Pancreatic Islets: Effects of Starvation A. Hallberg. Department of Medical Cell Biology, Uppsala Universi- ty, Uppsala, Sweden The capacity for membrane biosynthesis in islets starved in vivo or in vitro was studied by estimating the incorporation of D-(UJ4C) glucose into different islet phospholipid classes separated by thin-layer chromatography. In islets isolated from non-starved mice the uptake of isotope into phosphatidylserine (PS), phosphatidylinosotol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine plus phos- phatidylglycerol (PE + PG) increased in an almost linear fashion with increasing glucose concentrations. In lysophosphatidylcholine, phosphatidic acid and sphingomyeline, however, no significant incorporation could be detected. Starvation for 60 h in vivo decreased the incorporation rates into PS, PI, PC and PE+ PG both at low (2.8 mmol/l) and high (16.7 retool/l) glucose concentrations. In cultured islets the ~4C-glucose incorporation into phospholipids was generally higher than in freshly isolated islets. Only the incorporation into PC, which was decreased, was affected by culturing the islets at a low glucose concentration (starvation in vitro). The hydrolysis of PI associated with stimulated secretion was studied in cultured islets pre-labelled with 3H-glycerol. Preliminary results suggest that a 'PI-response' to 10 4 mol/l carbamylcholine is present also in islets exposed to starva-

tion in vitro. It is concluded that islets starved in vivo or in vitro still have capacity for biosynthesis of membrane phospholipids from exogenous glucose, despite the marked impairment of glucose-stimulated insulin release.

155. Effects of Noradrenaline and Insulin on Lipolysis and Fatty Acid Re-Esterification in Isolated Human Adipocytes V. A. Hammond and D. G. Johnston. Department of Medicine, Royal Victoria Infirmary, Newcastle-upon-Tyne, UK Substrate cycling between triglyceride and non-esterified fatty acid (NEFA) has been demonstrated previously in rat adipose tissue but information is lacking for man. We have therefore studied lipolysis and fatty acid re-esterification in isolated human adipocytes from SC tissue obtained during elective abdominal surgery. Lipolysis was assessed as glycerol release and NEFA re-esterification by the non-isotopic balance technique, utilizing cell and medium glycerol and NEFA concentrations. Basal and stimulated (10 -6 tool/1 noradrenaline) lipolysis were linear for 4 h at 37 ~ and were similar at medium glucose concentrations of 5 and 20 mmol/1. Basal lipolysis was increased 43%-330% by noradrenaline alone and decreased 20%-60% by insulin alone (80 pmol/1) at 4 h. Insulin inhibited noradrenaline- stimulated lipolysis 11-33 % irrespective of the glucose concentration. In the basal state 14% 50% of NEFA released were re-esterified as reflected by the NEFA: glycerol ratios. The proportion of NEFA re- esterified was decreased by noradrenaline alone to 0%-14%. This decrease was partially reversed by insulin. A substrate cycle between triglyceride and fatty acid thus occurs in man, enabling control of NEFA mobilisation through alterations in lipolysis or in fatty acid re-esterification. Insulin and noradrenaline may act through both mechanisms.

156. Modification in Control of the Enteroinsular Axis by a High-Fat Diet S. M. Hampton, J. A. Tredger, P. Kwasowski, L.M. Morgan, J. Wright, R. Cramb, M. Dunn and V. Marks. Department of Biochemistry (Divi- sion of Clinical Biochemistry and Nutrition), University of Surrey and St. Luke's Hospital, Guildford, Surrey, UK Eight healthy volunteers (normal dietary intake < 130 g fat/day) ate a low-fat diet (30 g fat/day) for 9 days and after reverting to their usual diet for 15 days consumed a high-fat diet (150 g fat/day) for a further 9 days. Oral fat tests (100 g) with or without IV insulin (0.2 U/kg) were carried out on days 7 and 9 of each diet. Insulin significantly inhibited GIP secretion in response to fat during the low-fat regimen (area under plasma GIP curve 3 979 + 547 pg. m1-1- h 1 after fat plus insulin versus 5490+599pg.ml -~ .h q after fat alone p <0.01) but not during the high-fat regimen (5 652 +_ 962 versus 4 503 _+ 654 pg. m l i h-~ p = 125). Five of the volunteers were maintained on a high-fat diet for 30 days and subsequently ate a low-fat diet for a further 9 days and the tests were repeated. Insulin now failed to inhibit fat-stimulated GIP secretion. We conclude that (1) 9 day high-fat diets reduce feedback control of GIP secretion by insulin and (2) ingestion of long-term, high-fat diets by healthy subjects reduced feedback control of fat- stimulated GIP release by insulin regardless of their diet immediately preceding the test.

157. Measurement of 'Free' Insulin: The Influence of Temperature, Storage, and Duration of Incubation on the Desired Result I. Hanning and P. D. Home. Department of Clinical Biochemistry and Metabolic Medicine, Royal Victoria Infirmary, Newcastle-upon- Tyne, UK Plasma insulin concentrations of insulin-treated diabetic subjects must be measured after extraction of insulin antibodies, usually with polyethylene-glycol. Commonly, frozen plasma is incubated at 37 ~ to restore the presumed equilibrium between free and antibody- bound insulin, but the method has not been validated. In six C-peptide negative diabetic subjects, measured free insulin levels were independent of the temperature of centrifugation (1 min) when extracted immediately thereafter (polyethylene-glycol 30% w/w), but drifted down (68 +_ 6%, p < 0.02) or up (117 _+ 5%, p < 0.05) if extraction was delayed by 30 rain incubation at 0 or 37 ~ respectively. This was independent of whether insulin levels were rising or falling at the time of venepuncture, suggesting that equilibrium between free and bound insulin is indeed maintained in vivo. Extracts stored at - 2 0 ~ were unchanged by incubation at 0 or 37 ~ after thawing. In contrast, measured free insulin levels in samples stored as plasma were significantly higher after incubation at 0 ~ for 2 h (140 _+ 16%, p < 0.05) with a wide scatter (105%-214% (range)). Samples incubated at 37 ~ for 2 h before extraction of insulin antibodies gave even more erratic results

162 Abstracts

(74%-257%). Thus, extraction of insulin antibodies from plasma at the bedside is mandatory in the accurate estimation of free insulin concentrations in insulin-treated diabetic subjects.

158. Binding Affinity of Monoiodoinsulin Tracers of Human (Pancre- atic or Semi-Synthetic) and Porcine Insulin Origin B. Hansen, S. Linde, and O. SgnneL Hagedorn Research Laboratory, Gentofte, and ~University of Arhus, Arhus, Denmark Iodine substitution in specific tyrosine residues in porcine insulin has an influence on the binding affinity to isolated rat adipocytes, rat hepatocytes, and cultured human lymphocytes of the IM-9 line. The aim of the study was to elucidate whether a change in one amino acid residue in the C-terminal part of the B-chain and/or the way of preparation had any additional effect on binding affinity. Porcine insulin, pancreatic human insulin, and semi-synthetic human insulin (derived from porcine insuIin) were iodinated with 125I in urea-containing buffer using the lactoperoxidase method. The four monoiodinated insulin isomers (substituted in Tyr A14, A19, B16, or B26) were separated and isolated using disc-electrophoresis and ion-exchange chromatography. The binding affinities to the cell types mentioned were determined. All binding affinities were calculated relative to A14 monoiodoinsulin, known to have the same binding affinity and biological activity in adipocytes as native insulin (porcine). In general, A19 monoiodoinsulins had lower binding affinity, B26 monoiodoinsulins higher (adipocytes, IM-9) or equal (hepatocytes) binding affinity and B16 monoiodoinsulins intermediate binding affinity relative to A14 monoiodoinsulins of the same origin. No difference in binding affinities in the three cell systems could be demonstrated when comparing the same specific tracers derived from different species.

159. Phosphorylation and Dephosphorylation of Insulin Receptor Kinase from Hepatoma Cells H. U. Hfiring, M. F. White, M. Kasuga and C. R. Kahn. E. P. Joslin Re- search Laboratory, Harvard Medical School, Boston, Massachusetts, USA Insulin stimulates phosphorylation of the insulin receptor at serine and tyrosine residues of the/3 subunit within 1 min in intact Fao-hepatoma cells. Addition of anti-insulin antibody causes both insulin dissociation and dephosphorylation of the receptor with an identical time course, suggesting the presence of active receptor phosphatases in the intact cell. With the partially purified insulin receptor obtained from solubilized cells by lectin affinity chromatography and [?" 32p ATP], the fl subunit was phosphorylated only at tyrosine residues. In- sulin stimulated the Vm~x of autophosphorylation 10- to 15-fold with no change in Kin. About 2 molecules of phosphate were incorporated per insulin binding site. Tryptic peptide mapping by HPLC resolved at least five insulin-stimulated phosphopeptides. Two peptides were phosphorylated maximally within 20 s, whereas the other sites did not reach steady-state for several rain. Dephosphorylation of receptors labelled with [32p] occurred after addition of unlabeled ATP to the reaction mixture, suggesting a phosphatase was retained with the partially-purified receptor. After further purification of the receptor with receptor antibody, insulin-stimulated phosphorylation was retained, whereas dephosphorylation no longer occurred. These results confirm the kinase nature of the insulin receptor and indicate that phosphatase is a separate protein.

160. Relationship of Diabetic Control to Left Ventrieular Ejection Frac- tion During Cold Stimulation Tests in Type I (Insulin-Dependent) Dia- betes A. D. B. Harrower, R. Railton and P. Newman. Medical Unit, Monks- land District General Hospital, Airdrie, Lanarkshire, Scotland We have reported previously a high incidence of abnormal cold stimulation tests in Type 1 diabetic patients with no clinical evidence of heart disease. While we have shown that this may reflect impaired myocardial peffusion, any possible effect from age, duration of diabetes, cardiac autonomic neuropathy or diabetic control must be excluded. Thirty-one Type 1 diabetics aged from 25 to 29 years and with duration of diabetes from one month to 37 years were studied. None had any symptoms or signs of cardio-respiratory disease. The change in left ventricular ejection fraction during cold stimulation was measured by nuclear angiography. Cold stimulation was produced by im- mersion &both hands to the wrists in ice-cold water for 2 min. Plasma glucose and glycosylated haemoglobin (HbA1 c) levels were measured and cardiac autonomic function was assessed by the heart rate change on standing (30/15 ratio) and during the Valsalva manoeuvre (Valsal- va ratio). Fifteen patients (48.4%) had abnormal cold stimulation tests. There was no correlation between the change in left ventricular

ejection fraction during cold stimulation and age, duration of diabetes, plasma glucose levels, 30/15 ratio or Valsalva ratio. However, there was a significant negative correlation with HbA1 levels (r= 0.42, p= 0.02). Metabolic derangement may produce abnormalities of left ventricular function indicating that long-term control is important in the aetiology of heart disease in diabetes.

161. Pregnancy in Insulin-Dependent Diabetic Patients: The Effects of Changing Policy T. M. Hayes, J. Birtwell, J. Peters, P. Morris, J. Murphy and J. F. Pear- son. University Hospital of Wales, Cardiff, UK In 1978 the obstetric policy for insulin-dependent diabetic patients in Cardiff was changed to allow them to stay out of hospital until admitted in spontaneous labour at term instead of elective induction at about 37 weeks. This study reports the results of 35 pregnancies in insulin-dependent patients between 1972-1978 and 45 pregnancies in insulin-dependent patients between 1979-1982. There was no difference between the groups in the quality of diabetic control as judged by mean blood glucose at each trimester. There was a reduction in the incidence of the respiratory distress syndrome from 16.2% in 1972-1978 to 2.4% in 1979-1982. One neonatal death but two still- births occurred in the first group and no neonatal deaths but two still- births (one an anencephalic) in the second group. The incidence of congenital abnormalities and the Apgor score was similar in both groups, but neonatal hypoglycaemia was less frequent in the second group. Birth weights were higher in the second group, 73% being over the 50th centile for gestational age compared to 47% in the first. There was no difference in maternal weight gain or the changes in insulin dosage. A reconsideration of the policy for the delivery of pregnant insulin-dependent patients appears to be justified.

162. Decreased Whole Body Proteolysis by Leucine Infusion in Man M. Haymond, P.Tessari, E.Tsalikian, F. Schwenk and S.Nissen. Mayo Clinic, Rochester, Minnesota, USA Leucine decreases proteolysis and stimulates protein synthesis in rat tissues. To determine whether a leucine infusion affects protein metabolism in vivo, normal subjects were studied using [2H3] leucine and [14C] a-ketoisocaproate (the a-ketoacid of leucine) to estimate total leucine carbon flux and leucine Oxidation. Following baseline sampling, subjects were infused with saline or leucine at 0.16 or 0.261xmol.kg-~.min -1. Plasma leucine increased by 9, 19, and 26 ~xmol/1, respectively. Leucine flux decreased (p < 0.05) during saline but did not change during the leucine infusions. Leucine appearance from protein (flux minus leucine infusion) decreased (p < 0.05) during the leucine infusion ( - 0.19_+ 0.03 and - 0.27 _+ 0.05 ~mol. kg -1- min -1) when compared to saline ( - 0.11 + 0.04). Leucine oxidation decreased during saline but increased (p < 0.05) during teucine infusions, whereas Ieucine going to protein did not change. Plasma glucagon, insulin and C-peptide remained constant. In summary, infusions of leucine decreased proteolysis and increased plasma leucine and leucine oxidation, independent of hormonal changes, but did not affect estimates of protein synthesis. In conclusion, infusion of leucine at rates approaching 10-15% of leucine flux decreases proteolysis. These data suggest that, in man, proteolysis may be regulated by a feedback mechanism involving plasma leucine itself.

163. Effect of Continuous Ambulatory Peritoneal Dialysis on the Insu- lin Resistance of Uraemia A. Heaton, D.G. Johnston, L.A. Ashworth, M.K. Ward and D. N. S. Kerr. Departments of Medicine and Clinical Biochemistry and Meta- bolic Medicine, Royal Victoria Infirmary, Newcastle-upon-Tyne, UK Patients in endstage renal failure have marked carbohydrate intolerance due to peripheral insulin resistance. Continuous ambulatory peritoneal dialysis (CAPD) involves considerable glucose absorption from the dialysis fluid. We have therefore examined the effects of CAPD on glucose and insulin metabolism. Six uraemic patients on standard diet were studied for 24 h before starting CAPD, and compared with 13 matched control subjects. Mean blood glucose was 6.6+ 1.0mmol/l in uraemic and 5.2_+0.5 in control subjects (p< 0.01); serum insulin was 36.3 _+ 14.5 and 15.4_+ 6.0 mU/l, respectively (1o<0.01). The gluconeogenic precursors lactate, pyruvate and alanine, and ketone bodies, were also significantly raised. Using a three- step hyperinsnlinaemic euglycaemic clamp, glucose disposal ('M') (~xg. kg 1. min-1) was found to be lower than in control subjects: 2.0 at 0.02 units, kg 4. h - 1 (control 3.2), 4.7 at 0.032 units, kg - 1. h - ~ (control 6.5) and 7.3 at 0.05 uni ts .kg- l .h -1 (control 7.9), confirming marked insulin resistance. After 3 months' CAPD, repeat 24 h profiles showed decreased mean blood glucose (6.0+_0.8 mmol/l ; p<0.05)

Abstracts 163

and insulin (31.4_+ 13.4 mU/1) despite peritoneal absorption of 120 g glucose/day. Ketone body levels fell. Thus the insulin resistance of uraemia is confirmed, and CAPD restores metabolic profiles towards normal, probably by improving insulin sensitivity.

164. Effects of glucose and other secretagogues on the cytosolic ratios of free N A D H / N A D + and free N A D P H / N A D P + in mouse pancreatic islets C.J.Hedeskov, K.Capito and P.Thams. Department of Biochem- istry A, Panum Institute, University of Copenhagen, Denmark In this study we have used measurements of intracellular concentrations of lactate, pyruvate and malate to compute the two ratios free N A D H / N A D + and free NADPH/NADP + in the cytosol of mice pancreatic islets after incubation with different secretagogues. With glucose (3mmol/1) the ratio free N A D H / N A D + was 0.00094+_ 0.00009 and the ratio free NADPH/NADP + was 26.8 _+4.4. Glucose (20 retool/l) did not raise the cytosolic ratio of free N A D H / N A D +, which was increased to 0.0021_+0.00035 by (10mmol/1 leucine+ 10 mmol/1 glutamine; p< 0.001). The cytosolic ratio of free N A D P H / N A D P + was enhanced 115% by glucose (20 mmol/l ; p < 0.005) and 131% by (10 mmol/l leucine + 10 mmol/1 glutamine; p < 0.001). Fructose (20retool/I), glucose (3mmol/l)+fructose (20 mmol/1) and leucine (10 mmol/1) had no effect on either of the two cytosolic redox couples. We conclude that an increase in the cytosolic ratio of free NADPH/NADP + (but not of free N A D H / N A D +) may be involved in coupling B-cell glucose metabolism to insulin secretion and that an increase in cytosotic N A D P H / N A D P + ratios is not a prerequisite for stimulation of insulin secretion by leucine or by glucose (3 mmol/l) + fructose (20 mmol/1).

165. Comparison of the Insulin Suppression Test and the Euglycaemic Clamp for Constructing Insulin Dose Response Curves R.J. Heine, T. Fonk, H. J.G. Bilo, C.P. de Vries, E.A.v.d. Veen, J. v.d. Meet. Department of Internal Medicine, Free University Hospital, Amsterdam, The Netherlands In a previous study, we showed a highly significant correlation between the insulin suppression test (IST) and the euglycaemic clamp for assessing insulin sensitivity in vivo at one insulin level. To differen- tiate between impaired insulin sensitivity and/or responsiveness, insulin dose response curves are necessary. We constructed these curves both with the euglycaemic clamp technique and the IST in eight normal subjects and eight Type 2 (non-insulin-dependent) diabetic patients. In the IST and euglycaemic clamp, insulin was infused sequentially at rates of 50, 150 and 500mU.kg-a .h -1 and in the IST concomitantly glucose at rates of 6, 8 and 10 mg. kg -1-min 1. In the euglycaemic clamp, fasting glucose level -0 .3 mmol/1 was maintained for three periods of 120 min. The metabolic clearance rate of glucose was determined for each insulin rate in both tests.

Insulin infusion rate (mU.kg I. h-a)

Metabolic clearance rate (ml. kg 1.rain-l)

50 150 500

Normal subjects IST 10.8 _+ 1.0 17.5 + 0.2 18.7 + 2.2 clamp 11.4+0.5 16.5_+0.8 19.8_+2.4

Type 2 diabetes IST 3.6 + 0.5 8.5 + 1.0 10.7 +- t .5 clamp 4.0+0.8 7.9+0.5 9.2_+0.6

Results expressed as mean _+ SEM.

The metabolic clearance rate derived from both tests strongly correlated for the three insulin infusion rates: r=0.96, 0.93 and 0.93, respectively. The I ST is simple to perform and the results of this study justify its use for examining insulin dose response curves.

166. Physiological Changes in Portal Blood Concentrations of Pancre- atic Hormones in Freely moving Rats A. He]man, D.Castaing, J. Morin, N.Pfister-Lemaire and R.Assan. Diabetes Department, H6pital Bichat, Paris, and Hepatic Surgery De- partment, H6pital R Brousse, France In order to study the kinetics of pancreatic hormone secretions in freely moving rats, we set up a technique for portal vein cannulation, via the left branch of the portal vein, and established its physiological validation. Catheter patency allowed portal blood sampling in 80% of

rats on post-operative day 8 and in 30% on day 15. Rat growth, brom- sulphthalein clearance and serum amino-transferase level were normal from day 7 on. Blood flows measured with radiolabelled microspheres were normal (pancreas: 0 .91+0.12ml.min- l .g -1 of tissue, controls 1.39 + 0.33 ml. rain -1. g 1 ; liver 4.93 + 0.48 ml/min per 100g body weight, controls 4.02+_1.23 ml/min per 100g body weight). In freely moving, 16 h-fasted Sprague-Dawley rats, basal portal concentrations were: glucose: 6.27 -+ 0.22 retool/l, insulin (IRI): 67 ___ 20 m U/ l and glucagon: 480 + 14 pg/ml. These values were stable for a 1 h observation period. A clear-cut portoperipheral concentration gradient was shown for IRI and glucagon. IV glucose induced a biphasic IRI release in portal blood, significant from the first minute, maximal at minute 15 (308 + 74 mU/l), and a significant reduction of portal glucagon level, minimal at minute 15 (227 _+ 28 pg/ml). IV arginine induced mild hyperglycaemia (7.75 + 0.55 mmol/l) and a biphasic release of IRI (peak: 543+57mU/ml) and glucagon (peak: 1044-+ 190 pg/ml). During a meal test, portal concentrations of both hormones increased rapidly, before any change in blood glucose level: IRI rose from minute 3 (peak at 20 min: 333 +- 36 mU/1), and glucagon as early as the first min, immediately reaching its maximal value: 800 _+ 125 pg/ml.

167. Reappraisal of the Role of Cyclic AMP in Stimulus-Secretion Coupling in Mouse B Cells J.C.Henquin and H.P.Meissner. Unit~ de Diab~te et Croissance, University of Louvain, Brussels, Belgium, and I Physiologisches [nsti- tut und Medizinische Klinik, University of Saarland, Homburg/Saar, FRG Two stimulators of adenylate cyclase (glucagon and forskolin) were used to study the effects of endogenous cyclic AMP on insulin release, ionic fluxes and membrane potential in mouse B cells. At 3 mmol/l glucose, glucagon (1 l.tmol/1) and forskolin (5 Ixmol/1) had no effect on membrane potential or insulin release. At 7 mmol/1 glucose, they triggered electrical activity (slow waves of depolarization with spikes) in silent cells, and stimulated release. They reversibly potentiated release and electrical activity induced by 10-15 mmol/1 glucose: the slow waves with spikes lengthened and their frequency increased because of a shortening of the silent intervals. The effects of forskolin on electrical activity were abolished by cobalt and those on insulin release were inhibited by cobalt or calcium omission. At 7-10retool/1 glucose, forskolin stimulated 45Ca2+ influx (5 rain uptake) in islet cells and, in the presence or absence of extracellular calcium, increased 86Rb+ effiux rate (K permeability) in preloaded islets. All these effects of glucagon and forskolin were mimicked by dibutyryl cyclic AMR These data suggest that the potentiation of insulin release by cyclic AMP not only involves mobilization of intracellular calcium but also facilitation of Ca 2+ influx in B cells, possibly through a direct influence of the nucleotide on the gating of calcium channels.

168. Effect of Amino-oxyacetate upon Ionic Movements Induced by Nu- trient Secretagogues in Islet Cells A. Herchuelz, P. Lebrun and W. J. Malaisse. Laboratories of Pharma- cology and Experimental Medicine, University of Brussels, School of Medicine, Brussels, Belgium Amino-oxyacetate, an inhibitor of transamination reactions, was recently reported to prevent the induction, by glucose or 2 ketoisocaproate, of a more reduced state in cytosolic redox couple of rat pancreatic islets. The cationic effects of amino-oxyacetate were examined in islets prelabelled with either 86Rb or 45Ca. Amino-oxyacetate augmented the outflow of 86Rb from the islets, whether in the absence or presence of an exogenous nutrient, and slightly impaired the capacity of nutrients especially 2-ketoisocaproate, to decrease 86Rb outflow. Amino-oxyacetate abolished the nutrient-induced rise in 45Ca efflux, which normally results from the stimulation by glucose or 2-ketoisocaproate of *~ influx into the islet cells. Amino-oxyacetate, how-

45 ever, failed to suppress the early inhibitory effect of nutrients on Ca efflux. The alteration by amino-oxyacetate of the cationic response to nutrient secretagogues coincided with a suppression of insulin release, whereas the cationic response to a non-nutrient stimulation by a high concentration of extracellular K § was unaffected by amino-oxyacetate. These findings suggest that the induction of a more reduced cytosolic redox state represents an essential link between metabolic events and both the decrease in K + conductance and stimulation of Ca 2+ inflow in the process of nutrient-induced insulin release.

164 Abstracts

:169. Reversal of A, B, and D Cell Insensitivity to Glucose in Alloxan Diabetic Dogs by a Glucose Controlled Insulin Infusion System K. Hermansen, O. Schmitz and H. Orskov. First and Second Universi- ty Clinics of Internal Medicine, Institute of Experimental Clinical Re- search, University of Aarhus, Denmark Insulin deficient diabetes is associated with islet cell insensitivity to glucose. The present study was designed to determine whether this abnormality could be counteracted either by increasing the intra-islet insulin level or by normalizing the diabetic state by a glucose controlled insulin infusion system (GCIIS; Biostator). Using the isolated, perfused pancreas of dogs with severe untreated alloxan diabetes of 3-5days duration, we found that 5mmol/1 arginine (n=4) and 5 mmol/1 calcium (n = 4) stimulated A, B, and D cell secretion, whereas an increment in glucose from 1.3 to 11 mmol/l (n=4) had no effect on islet hormone secretion. In the pancreas from untreated alloxan diabetic dogs, the acute infusion of large amounts of insulin (25 m U / ml) simultaneously with an elevation of perfusate glucose from 1.3 to 11 mmol/l failed to restore glucose sensitivity of the islet cells. In contrast, the treatment of alloxan diabetic dogs (n = 3) by a GCIIS for 24 h revived the glucagon, insulin, and somatostatin responsiveness to glucose (1.3 to 11 retool/l) of the subsequently perfused pancreas. It is concluded that the insensitivity to glucose of islet cells in insulin deficient diabetes is not ascribed to an intra-islet insulin deficiency per se but rather to an abnormal metabolic state secondary to insulin deficiency.

170. Role of the Availability of Gluconeogenetic Substrates in Fasting Hypoglycaemia at Late Gestation in the Rat E. Herrera and A. Zorzano. Departamento de Investigaci6n, Centro Ram6n y Cajal, Madrid, Spain Fasting hypoglycaemia increases in the rat at late gestation. To determine the role of endogenous substrates involved, 24-h starved 21-day pregnant rats (P) were compared with virgin controls. Basal circulating amino acids and glycerol levels were respectively decreased and augmented in P. Animals were injected IV with 0.2 or 1 mmol of either (U54C)-L-alanine or (uJac)-glycerol. Tail blood was collected at 2 and 5 min and animals were decapitated at 10 rain. With 0.2 mmol of glycerol but not with alanine, ~4C-glucose production was enhanced in P, whereas it was enhanced with 1 mmol of both substrates. Fetal blood ~4-C-glucose remained below but changed in parallel to maternal values. After 90 min incubation of liver and kidney cortex slices from other rats in the presence of 1 or 5 retool/1 (UJ4C)-L-alanine or (U-14C)-glycerol, 14C-glucose formation always increased in proportion to the substrate concentration in medium and values were higher in liver slices from P than from controls with both concentrations of (U-14C)-L-alanine and in kidney cortex slices with the two concentrations of each substrate. Glucose synthesis in P appears limited by the reduced availability of certain substrates. This factor, rather than the fetal glucose draining, is the main element responsible for intense maternal hypoglycaemia during fasting.

17:1. Absorption on Infused Insulin: Kinetics and Relation to Subcutane- ous Blood Flow E Hildebrandt, K.Birch, S.Levin Nielsen and L.Sestoft. Hvidore Hospital, Klampenborg, Denmark Nineteen Type 1 (insulin-dependent) diabetic patients were given a constant infusion of 125I labelled Actrapid insulin (1.12 i. u./h) into the abdominal subcutaneous tissue. In 12patients SC blood flow was measured simultaneously at a symmetrical contralateral location by means of the mXenon technique. In seven patients a constant insulin infusion of 2.24 i.u./h was given in the other side for 7 h. Measure- ments of the size of the infused depot and of SC blood flow were done for the first 7 h. After 24 h, SC blood flow was measured in all patients, and after 26 h infusion was either continued while SC blood flow was changed by local cooling or heating over the sites of insulin infusion and SC blood flow measurements (n = 7) - or the infusion was stopped without (n = 7) or with (n = 7) removal of the needle. On the first day, a depot was built up, which reached steady state after > 6 h. Next morning the steady-state depot was 5.15 i.u. (mean). A dou- bling of infusion rate doubled the depot size. After stopping the infusion the depot disappeared according to first order kinetics. The size of the steady-state depot was inversely correlated with SC blood flow, and the insulin absorption rate from the depot was positively correlated with SC blood flow in a curvilinear way.

172. Retinopathy is Associated with Higher Glycaemia in Maturity-On- set Type Diabetes R. M. Hillson, J. Howard-Williams, A. Bron, P. Awdry, J. I. Mann and T. D. R. Hockaday. Radcliffe Infirmary and Oxford Eye Hospital, Ox- ford, UK In 1982, 149 diabetic patients diagnosed 7.3 +- 1.0 years earlier attent- ed for an ophthalmological review. Diagnosis was by glucose disappearance rate constant < 1 after IV glucose (20 g/m2). None required insulin initially, but in 1982, 22 were on insulin, 72 on oral hypoglycaemic agents and 55 on diet alone. Excluding three patients with retinopathy at diagnosis and ten not then examined did not alter conclusions. Retinal photography showed background retinopathy (/> 1 microaneurysm +_ other changes) in 55 patients (37%). None had proliferative retinopathy. Compared with patients without retinopathy those with retinopathy had greater fasting plasma glucose at diagnosis (13.2 _+ 4.0 versus 10.9 +_ 3.8 mmol/1, p < 0.001) and during treatment (mean of 1, 3 and 5year values 9.0_+2.9 versus 7.5+2.3mmol/1, p=0.002). In 1982, the retinopathy patients had greater haemoglobin Ale (8.9_+ 1.8 versus 7.7_+ 2.0%, p < 0.001) and random glucose (10.8 _+ 4.8 versus 8.5 ___ 4.2 mmol/l, p = 0.007). Half those with fasting glucose >~12.0 mmol/l at diagnosis had retinopathy 7years later compared with a quarter below this. The percentage of patients with retinopathy at 7 years increased with mean fasting glucose during treatment rising from 18% at < 6.0 mmol/1 to 88% at >~14 mmol/1. Body mass index was greater in those with retinopathy (30.0+_ 5.7 kg/m 2) than in those without (27.9 +_ 4.9 kg/m 2, p< 0.05) in 1982 but not initially.

173. Haemodynamie Changes During Insulin-Induced Hypoglyeaemia J. Hilsted, F. Bonde-Petersen, M.-B. Norgaard, M. Greniman, N. J. Christensen, H.-H. Parring and M. Suzuki. August Krogh Insti- tute and Kommunehospitalet, Copenhagen, Denmark Haemodynamic variables were measured before (1), during (2) and after (3) insulin-induced hypoglycaemia in seven healthy males (age 22_+ 1 years, mean_+ SEM). Blood glucose levels were 4.2_+ 0.2 retool/1 (1), 1.9 +- 0.1 (2) and 4.3 +- 0.2 (3). Plasma volume (~25I-albumin method) decreased significantly from 3107+163ml (1) to 2887 +- 136 (2) and 3072 +- 158 (3). Heart rate, cardiac output and cardiac stroke volume all increased significantly (heart rate: 63 + 3 beats/ rain (1), 82_+4 (2) and 63 +_4 (3), cardiac output 5.8+0.3 l/rain (1), 8.5 +_ 0.8 (2) and 5.5 +__0.4 (3), cardiac stroke volume 94_+ 11 ml (1), 119 _+ 14 (2) and 87 + 12 (3)). Mean blood pressure decreased significantly: 95_+3mmHg (1), 90+-2 (2) and 94_+3 (3). No significant change was found in hepato-splanchnic vascular resistance, whereas lower-extremity vascular resistance decreased significantly: 3.2+- 0.5 mmHg. ml- 1.1000 g- 1 (1), 2.3 +- 0.2 (2) and 3.5 +_ 0.4 (3). Plasma catecholamines increased significantly (noradrenaline 0.21+_0.03 ng/ml (1), 0.43+0.10 (2) and 0.32+-0.08 (3), adrenaline 0.04+ 0.01 ng/ml (1), 0.76_+0.10 (2) and 0.08_+0.01 (3)). Thus, insulin-induced hypoglycaemia, a frequent complication of insulin therapy, has profound but transient effects on the circulation. Haemoconcentra- tion and increased cardiac output are likely to be risk factors in patients with arteriosclerotic cardiovascular disease.

174. Impaired Insulin Receptor Binding and Postreceptor Defects of Adipocytes from Normal and Diabetic Pregnant Women E. Hjollund, O. Pedersen, T. Espersen, J. Klebe and N. Schwartz Sorensen. Department of Medicine, County Hospital, Department of Obstetrics and Gynecology, Kommunehospital, Arhus, Denmark Adipocytes, monocytes and erythrocytes from eight non-diabetic pregnant women (NP), eight pregnant women with insulin-dependent diabetes (DP) undergoing Caesarian section and 10 normal non-pregnant women (N) undergoing gynaecological surgery were studied. ~z5 Compared with N, ( I)-insulin receptor binding at tracer concentration to isolated adipocytes from NP and DP was reduced by 45% (p < 0.001) and 30% (p < 0.02). No differences were found in insulin receptor binding to pure monocytes or erythrocytes. In non-insulin- stimulated adipocytes from NP and DP, glucose transport rates (lipogenesis at tracer glucose concentration, where glucose transport is rate-limiting) were similar to N, whereas maximally insulin-stimulated adipocytes exhibited significantly lower transport rates (p < 0.05, p < 0.05). The insulin concentration giving half-maximal stimulation of glucose transport (EDs0) was significantly higher in both pregnant groups (p < 0.05, p < 0.05) Moreover, when compared with normal non-pregnant women, glucose metabolism rates (lipogenesis at higher glucose concentration) in insulin-stimulated adipocytes were lower in NP and DP (p < 0.05, p < 0.01). In DP, non-insulin-stimulated glucose metabolism rates also were lower than N (p < 0.05). In conclusion, in

Abstracts 165

late normal and diabetic pregnancy, insulin receptor binding to adipocytes is reduced. This is accompanied by the predicted consequence, decreased sensitivity to insulin of adipocyte glucose transport. Maximally-insulin-stimulated glucose transport and glucose metabolism rates are decreased in adipocytes from pregnant diabetic and non-diabetic women; this is suggested to be caused by postreceptor defects.

175. Intravenous Glucose Tolerance and the Accompanying Insulin Re- sponse Correlate with Plasma Concentrations of Group-II Elements in Non-Insulin-Treated Diabetic Patients T. D. R. Hockaday , R.F. Smith, N. Ward, H. Dhar and C.S. Yajnik. Radcliffe Infirmary, Oxford, and Imperial College Reactor Centre, Silwood Park, Ascot, UK Mather and Pilkington reported an inverse correlation between plasma magnesium and glucose in diabetes. While we confirmed such a trend among 28 insulin-dependent patients ( r ~ = - 0.33, p < 0.09), it was absent among 49 non-insulin-treated diabetic patients fasted overnight. However, for 69 of these diabetic patients undergoing an IV glucose tolerance test (20 g /m 2 body surface) the K~ rate factor correlated with initial Mg (r~ = 0.3'1, p < 0.0'1) and more strongly with the Mg:Ca (calcium) ratio (r~=0.37). However, it was only this latter association that held for the 41 non-insulin diabetic patients alone (r~=0.37, p<0.02). Elemental losses from previous glycosuria are probably irrelevant, as the relationship was stronger in the upper half ( > 0.70) of the I ~ range ( I ~ versus Mg, r~ = 0.58, p < 0.005). Associa- tions between plasma concentrations of Group-II elements (analysed by neutron activation) and insulin secretion could contribute to these findings. Thus, initial Mg correlated with the insulin secretory area (as percent basal) from 5-30 min, and the 90 min Mg with the 30-90 min insulin values (p< 0.025 and < 0.01, respectively). Plasma barium at 60 min correlated inversely with the 30-90 min absolute insulin secretory area (p< 0.01). The Mg :Ca ratio at 60 min correlated with the 0-90 absolute insulin area (p < 0.005).

176. Human Ultralente Insulin R. R. Holman, J. Steemson, P. Darling and R. C. Turner. Diabetes Re- search Laboratories, Radcliffe Infirmary, Oxford, UK The greater solubility of human insulin, and its reported faster action, has raised doubts that it would be possible to produce a sufficiently long acting human formulation to provide a basal supply for diabetic patients. Conventional highly purified beef ultralente insulin and semi-synthetic, human ultralente insulin have been compared in a double blind crossover study. After 6 weeks on each therapy, overnight glucose profiles in nine Type 1 and nine Type 2 diabetic patients showed that human ultralente insulin was as effective as beef ultralente insulin in controlling basal plasma glucose levels (04.00-07.00 h, median 5.7 mmol/l on human and 6.3 mmol/l on beef ultralente insulin, respectively). There was no greater pre-breakfast plasma glucose rise on human insulin, after a morning injection 20 h earlier, and no difference between patients receiving adequate or insufficient ultralente insulin. Three patients had their insulin dose reduced whilst on human insulin because of hypoglycaemic episodes. Insulin antibody binding was reduced on human insulin (p < 0.02) suggesting it is less antigenic than beef insulin. No significant changes were observed in weight, glycosylated haemoglobin or LDL levels. Once daily human ultralente insulin appears to be a suitable formulation for the basal insulin requirement of diabetic patients.

177. GRP-Nerves in the Control of Pancreatic Endocrine Secretion J. J. Holst, S. Knuhtsen, S. L. Jensen, O. V. Nielsen and T. W. Schwartz. Institute of Medical Physiology C, the Panum Institute and Depart- ments of Surgery C and Clinical Chemistry CL, Rigshospitalet, Uni- versity of Copenhagen, Denmark GRP (gastrin releasing peptide), a 27-aminoacid peptide with extensive sequence homology with the frog-skin peptide bombesin, was recently isolated from porcine tissue. Having produced antibodies against this peptide and using a subsequently developed specific radioimmunoassay, we now provide the following evidence for the participation of GRP-producing nerves in the control of the endocrine pancreas. (1) A peptide indistinguishable from GRP is present in porcine and human pancreatic extracts (3-30 pmol/g). (2) By immuno- histochemistry this peptide has been localized exclusively to nerve fibres, some of which contact the pancreatic islets. (3) GRP added to the arterial line of the isolated perfused porcine pancreas in concen-

11 8 trations from '10- to '10- tool/1 (a) inhibited somatostatin output (92-84% of basal), (b) did not affect glucagon output, (c) stimulated pancreatic polypeptide output (106 15'1% of basal), and (d) stimulat-

ed insulin output (160-250% of basal), the latter in concentrations as low as 10 -11 mol/1. (4) GRP ist released from the isolated perfused porcine pancreas during stimulation of the parasympathetic nerve supply, the amount released rising from undetectable to 0.8+ 0.2 pmol/min (n = 6). (5) Vagal stimulation strongly increased insulin secretion from the isolated perfused pancreas; but during perfusion with a potent GRP-antiserum, this insulin response was markedly reduced. Our results suggest that GRP may be a physiologically important transmitter peptide in the vagal control of pancreatic islet secretion.

178. The Advanced Diabetic Glomerulopathy - Is There a Distribution Pattern Within the Kidney? A. Horlyck, H.J.G. Gundersen and R. Osterby. University Institute of Pathology and the Second University Clinic of Internal Medicine, Kommunehospitalet, Aarhus, Denmark In advanced diabetic glomerulopathy every glomerulus is affected, but to a varying degree. In the normal kidney structural and functional differences are known between superficial and deep nephrons. This is a study of the relationship between glomerular position and glomerulopathy. Autopsy material from 12 long-term, Type 1 (insulin-dependent) diabetic patients was used. On slides presenting the whole depth of cortex three zones of equal width were drawn. Two parameters were estimated in each zone separately: The PAS-positive material as percentage of the glomerular tuft, and the relative number of totally occluded glomeruli. Further, the distribution of occluded glomeruli was tested for a non-random pattern. The results showed that the volume fraction of PAS-positive material, as well as the frequency of occluded glomeruli were similar in all zones. The occluded glomeruli tended to lie in rows perpendicular to the kidney surface, a tendency which could not be explained by the general tendency for glomeruli to show such an arrangement. We conclude that (1) it is unlikely that combined structural and functional differences that do exist between deep and superficial glomeruli play a major role in the development of diabetic glomerulopathy, (2) kidney topology influences the process of glomerular occlusion.

179. Constant Infusion of Glucose with Model Assessment as a Measure of Insulin Resistance in Man J. P. Hosker, D.R. Matthews, D. F. Treacher, M. Burnett, E.G. Bown, A. Smith and R. C. Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Oxford, UK We have developed a new method, constant infusion of glucose with model assessment (CIGMA), for assessing insulin resistance in normal subjects or non-insulin-dependent diabetic patients. CIGMA was compared with other measures of resistance: (1) euglycaemic hyperinsulinaemic clamp, (2) fasting plasma insulin, (3) percentage ideal body weight. CIGMA consists of a continuous infusion of glucose (5 mg/kg ideal body weight per rain) for '1 h, measuring glucose and insulin levels achieved. These levels are interpreted, using a computer model of glucose-insulin homeostasis, to give values for each subject's resistance. 12 normal and '1'1 non-insulin-dependent diabetic subjects were studied. The inverse of the steady-state glucose infusion rate during the clamp was used to provide an independent measure for resistance. There was a significant correlation between resistance measured by CIGMA and the clamp (Rs = 0.88, p < 0.0'1) and between resistance measured by CIGMA and both fasting plasma insulin (Rs = 0.84, p < 0.01) and percentage ideal body weight (Rs = 0.53, p < 0.0'1). Median resistance for normal subjects was '1.35 by CIGMA and 1.43 by clamp, and for diabetic patients was 3.5 by CIGMA and 3.69 by clamp (a normal weight resistance defined as '1). CIGMA appears to be a simpler, less labour-intensive way to measure insulin resistance than euglycaemic clamping.

180. Commercial Antisera and Insulin-Like Stimulatory Activity of Adipocyte Lipogenesis S. P. Houlder, M. A. Khokher and P. Dandona. Metabolic Unit, Royal Free Hospital, London, UK We have recently described an insulin-like stimulatory effect of human immunoglobulin G on adipocyte lipogenesis; this effect of IgG has been shown to be mediated by the Ec fragment of the IgG molecule. During the course of our experiments involving the neutralisa- tion of the biological activity of IgG molecule and its fragments, we observed that the commercially obtained antisera exerted their own stimulatory effect upon the adipocyte lipogenesis system. We hence assayed several antisera from commercial sources on the adipocyte lipogenesis system by standard techniques. These antisera were directed against insulin, IgG, Fab and Ec fragments, IgM and IgA. All

166 Abstracts

antisera produced 60% 80% above basal stimulation of adipocyte lipogenesis in dilutions of up to 1 : 100. This order of stimulation is similar to that produced by 2.5 to 5 mU/1 of insulin. These data show clearly that care must be exercised in assessing biologically active insulin and non-suppressible insulin-like activity in serum samples since anti-insulin antisera used in such assays are bound to contribute to insulin-like activity of their own.

181. 'Stress' Hyperglycaemia During Acute Myocardial Infarction - A Myth? D. J. Husband, D. G. Julian and K. G. M. M. Alberti. Royal Victoria In- firmary and Freeman Hospital, Newcastle upon Tyne, UK Hyperglycaemia occurring at the time of admission with suspected acute myocardial infarction is generally held to represent 'stress' hyperglycaemia. Twenty-six patients, not previously known to be diabetic, had blood glucose values >~ 10 mmol/l on admission to a Coronary Care Unit. Of these, 16 survived for 2 months at which time a 75 g oral glucose tolerance test showed diabetes in ten (63%) and impaired glucose tolerance in one (WHO criteria). All those with abnormal glucose tolerance at 2 months had an elevated glycosylated haemoglobin (> 7.5%) on admission indicating pre-existing diabetes. All those with a Hb& level over 8% had abnormal glucose tolerance. Nine of the ten, who died or did not have an oral glucose tolerance test, also showed an elevated HbAI. We conclude that an admission blood glucose >~ l0 mmol/1 in patients with severe chest pain is more likely to indicate previously undiagnosed diabetes than 'stress' hyperglycaemia, that there is no evidence that myocardial infarction precipitates diabetes and that the glycosylated haemoglobin level can be used to distinguish stress hyperglycaemia from that due to diabetes.

182. Biological and Immunogenic Activity of Human Monocomponent Insulin: Two years Follow-Up M. Iavicoli ~, G.A. Coronel, A.M. Dawud, P. Arduini, P. Pietravalle, k Valle and A. Maldonato. Prima Cattedra die Endocrinologia, Uni- versitfi degli Studi di Roma 'La Sapienza '1, Policlinico Umberto I, Rome, Italy Biological and immunogenic activity of semi-synthetic monocomponent human insulins in 68 patients receiving insulin for the first time were examined: 15 treated with human insulin (group 1), 28 with porcine monocomponent (group 2) and 25 with conventional insulins (group 3). Ten long-standing diabetic patients treated for I year with porcine insulin and then switched to human insulin for the second year were included. Glycosylated haemoglobin (HbA~c) insulin antibodies, circulating immunecomplexes by two different methods were assayed at the beginning of the study and after 1, 2, 3, 4, 6, 12 months in all the subjects and also after 13, 14, 15, 18 and 24 months in the 10 long-standing diabetics patients who switched to human insulin. At the end of follow up, insulin requirement (U- kg-1- day-~) was 0.44 in group I, 0.52 in group 2 and 0.62 in group 3. Insulin antibodies were significantly lower in group 1 than in group 2 (p < 0.05) and in group 3 (p<0.01). hnmunecomplexes prevalence was similar in the three groups. In the long-standing diabetic patients insulin requirement and HbA~c maintained similar levels during the 2-year follow up. Insulin antibodies did not change during the first year and showed a significant reduction after 2 years (p < 0.05). No difference was found during the 2-year observation period in the immunecomplexes prevalence. In conclusion, human insulin has similar biological activity and lower immunogenic activity than porcine insulin.

183. Cyclic-AMP Involvement in Gap Junction Formation between Pan- creatic B Cells P.A. in't Veld, D. G. Pipeleers 1 and W. Gepts. Department of Patholo- gy and 1Department of Metabolism, Vrije Universiteit Brussel, Brus- sels, Belgium Both morphological and functional studies support the idea that the functional integrity of the endocrine pancreas depends on intercellu- lar communication within the islets of Langerhans. As in other tissues one mode of communication could occur via gap junctions. Gap junctions have been described between islet cells and their presence can be influenced by factors affecting insulin release. The present study, was undertaken to identify agents involved in the formation of gap junctions between pancreatic B cells. Gap junctional structures were quantified on freeze fracture replicas of the central part of islets cultured for 20 h under various conditions. When compared to values measured in whole rat pancreas, islets cultured at glucose (5.6 mmol/1) exhibited a threefold decrease in the number of gap junctional particles per unit of membrane area. The reduction was not observed after culture at glucose (11.2 retool/l) or after addition of di-

butyryl-cyclic AMP (1 mmot/l); an 80% increase was noted with theophylline (1 mmol/1). These results provide one possible explanation for the twofold lower glucose-induced insulin release from islets cultured at glucose (5.6 retool/l), compared with freshly isolated islets. It is concluded that cyclic AMP might regulate gap junction formation between B cells and thus contribute to the functional status of the insulin release apparatus in vivo.

184. Abnormal Cephalic Insulin Secretion as Well as Oral Glucose Tol- erance in Genetically Obese (fa/fa) Rats E.Ionescu and B.Jeanrenaud. Laboratoires de Recherches Mrtabo- liques, Geneva, Switzerland The aims of the present study were to study reflex insulin secretion and oral glucose tolerance in lean and genetically obese fa/fa rats. Freely moving rats previously implanted with chronic IV catheters were used. Experiments were done at 13-14weeks of age. In obese rats, the percentage increase in reflex insulin secretion elicited by sac- charin (0.15%) was only half that seen in lean controls (p < 0.01). No reflex insulin secretion was observed in lean or obese rats following water ingestion. Spontaneous ingestion of 1.8 ml of a glucose solution (0.7 g/ml) within 90 s had the following effects: (a) reflex insulin secretion (occurring before any change in glycaemia) of obese rats was only half that of controls; (b) the subsequent glucose-induced insulin secretion was similar in both groups; and (c) post-absorptive glycaemia was significantly higher in obese than in lean rats, an abnormality that persisted for 40 min following the meal. These data suggest that obese fa/fa rats have defective cephalic insulin secretion that may be due to abnormal afferent or efferent signals to the central nervous system. These abnormalities may be responsible for an abnormal pattern of insulin output following a meal, thereby contributing to glucose intolerance.

185. Characteristics of Severe Diabetic Ketoacidoses Occurring Before and After 60 Years of Age C. Ionescu-T/rgoviste, I. Mincu, C. Dumitrescu, A. Danciu, N. Miha- lache and D. Cheta. Clinic of Nutrition and Metabolic Diseases, Bu- charest, Romania Between 1974 and 1980, 459 cases of severe diabetic ketoacidosis (pH ~< 7.20, total CO2~ < 10 retool/l) were admitted to our Intensive Care Unit. 416 (270 male, 176 female) were aged 60 years (41 _+ 7) and 43 (22 male, 21 female) above the age of 60 years (67 + 7). The precipitating causes were fairly similar, with infections foremost (68%). Pre- senting biochemical parameters, before and after 60 years showed differences in: blood glucose: 32.7 + 12 versus 34.8 + 8.1 mmol/l : plasma sodium 137 _+ 14 versus 143 + 16 mmol/l; plasma potassium 4.2 _+ 0.9 versus 4.9 _+ 0.7 mmol/1; hydrogen ion concentration 95 _+ 17 versus 73 _+ 12 nmol/1; (p < 0.01); plasma osmolarity 327 + 98 versus 379 +_ 96 mOsm/1. The difference was more significant (p < 0.01) in frequency of hypernatraemic hyperosmolarity (initial sodium > t 47 retool/l), 7.4% of 202 cases before age 60years versus 24.1% of 29 cases after age 60 years. The fluid amounts administered IV within the first 24 h was greater before (7516+_2121ml; p<0.01) than after 60years (5 276_+ 1132 ml); insulin requirements were greater after 60 years (236__+_ 78 U/24 h; p < 0.01) than before the age of 60 years (182 + 59 U/24 h). The mortality rate in the course of severe diabetic ketoacidoses was significantly lower before 60years (1.3%, p<0.01) as against 6.9% after age 60years. The excess mortality in the latter group was due to unavoidable causes (cerebral vascular accident, myocardial infarction, chronic renal failure or severe infections).

186. Freshly Isolated Islets: Possible Specific Effect of Collagenase to Inhibit Somatostatin Action E. Ipp, R. Oron, R. Nesher and E. Cerasi. Hebrew University-Hadas- sah Medical Centre, Jerusalem, Israel The potency of somatostatin to inhibit glucose-induced insulin release is markedly reduced in freshly isolated islets. Dose-response experiments demonstrated that the ID50 of synthetic somatostatin-14 was 375 nmol/1 (n = 5) in fresh, collagenase-digested islets - three or- ders of magnitude greater than observed in physiological preparations, e.g. isolated perfused pancreas. However, after overnight incubation at 37~ in culture medium (DMEM H-16), sensitivity increased markedly, i.e. ID50 of somatostatin was 22 nmol/1 (n=4). These islets are functional as determined by their response to glucose, 16.5 mmol/1 (% A =440% versus % A =300 in freshly isolated islets, n = 6). The mechanism for this effect is being studied by examining somatostatin-receptor interactions within the islets in freshly isolated and cultured islets. To determine the specificity of the diminished responsiveness of freshly isolated islets to somatostatin, the inhibitory

Abstracts 167

effect upon insulin secretion of another hormone, also acting at the plasma membrane of the B cell, was examined. Adrenaline-induced inhibition of insulin secretion was 51% at 27.3 mmol/1 (n= 3), a concentration which is within the pathophysiological range. It is generally thought that the somatostatin impotence observed in freshly isolated islets is due to collagenase 'injury' of the plasma membrane of the B cell, which recovers after prolonged culture. If so, this may be a selective effect by which collagenase influences somatostatin action.

187. Long-Term Safety of Insulin Infusion: The Intravenous or Intra- peritoneal Route for Pump Treatment K. Irsigler, H. Kritz, G. Hagmiiller, C. Najemnik and S. Leodolter. Metabolic Unit and L. Boltzmann Research Institute for Metabolic Diseases and Nutrition, City Hospital Vienna-Lainz, Vienna, Austria Pump treatment by the intravenous or intraperitoneal route has achieved metabolic control superior to that by the subcutaneous route. Data from 52patients years with externally-worn pumps, 16patient years with implants and 117 catheters in 87 patients were evaluated concerning risks and safety in long-term performance. Ketoacidotic episodes per 10 patient years: intravenous external devices 1.7, intraperitoneal external 2.4, implants none. Severe hypoglycaemic episodes per l0 patient years: external intravenous 0.5, external intraperitoneal 1.5, intravenous implant none, intraperitoneal implant 0.8. With intravenous external and (intraperitoneal external) devices, respectively, there were one (four) skin infections, five (12) breaks, four (three) slippages, six (one) obstructions. With implants none of these occurred. No thrombosis or embolisation from intravenous catheters was detected. Two intraperitoneal polyethylene and two intraperitoneal silicon catheters showed tissue growth and wors- ening of control. Catheter patency was restored at endoscopy. Five catheters were found free-floating after more than 300 days of good function. In conclusion, frequent catheter problems seen with externally-worn pumps are minimized with implanted pumps. The long- term problem with intraperitoneal catheters is tissue growth, and is independent of catheter material and insulin preparation. Variable length and number of outlets need evaluation.

188. Forearm Oxygen Uptake Before and After Oral Glucose Loading R. A. Jackson, J.B. Hamling, B.M. Sire, P.M. Blix, A.H. Rubenstein and J. D. N. Nabarro. The Middlesex Hospital, London, UK and De- partment of Medicine, University of Chicago, USA The relationship between energy metabolism and glucose disposal remains obscure. Using the forearm technique, we have studied peripheral oxygen consumption by superficial (skin and adipose tissue) and deep (muscle) forearm tissues before and after oral glucose loading in normal men, aged 19 32years. Basally, mean-+SEM oxygen saturation of arterialised, superficial and deep venous blood was 94.9 -+ 0.7%, 65.6 _+ 2.6% and 45.0 _+ 2.4% respectively (p < 0.001). Oxy- gen consumption by superficial and deep forearm tissues was 0.80

1 0 and 6.83 ~tmol 100ml forearm- rain- , respectively. Only 17V0 of the former and 25% of the latter was accounted for by concurrent glucose uptake, namely 0.06 and 0.29 umol- 100 ml forearm-I-rain-l , respectively. Net lactate production was observed by superficial (0.076 ~tmol. 100 ml forearm -1- min -~) but not deep tissues. Neither peripheral oxygen utilisation nor lactate production were increased by glucose loading despite 9.4-fold and 4.5-fold elevations in concurrent glucose uptake by deep and superficial tissues respectively. In conclusion, (1) arterialised, superficial and deep venous blood differs strikingly in composition, (2) glucose uptake accounts for < 25% of basal peripheral oxygen consumption suggesting that the latter is mainly dependent on lipid metabolism, (3) the failure of glucose loading to increase peripheral oxygen utilisation or lactate production despite marked elevations in glucose uptake, suggests that the fate of the glucose taken up is storage.

189. Stroke Related Factors Among Normotensive Diabetic Patients D. Janeczko, A. Czy~yk, J. Niewiadomska and J. Kopczyflski. Depart- ments of Gastroenterology and Metabolic Diseases, and of Epide- miology, Medical Academy, Warsaw, Poland The purpose of the study was to elucidate which factors, excluding hypertension, are related to cerebrovascular events, a relatively frequent concomitant of diabetes. The study group consisted of 476 diabetic patients (231 males and 245 females; participants of the WHO Multinational Study of Vascular Disease in Diabetes; aged 35-56 years) with diabetes of 2-32 years duration. The prevalence of stroke as definied by WHO criteria was 4.3% (3.9% in males, 4.5% in females). None of the stroke patients had hypertension at the time of the study, nor any reported hypertension in the past. For each stroke

patient three diabetic patients free of the cerebrovascular event were selected, matched for the sex and age. The following variables were tested for association: the obesity index (Quetelet), systolic and diastolic blood pressure, fasting glycaemia, total serum cholesterol and B-lipoprotein cholesterol, serum triglycerides, effort angina by Rose's criteria, ECG findings (Minnesota code) and leg vascular disease. Stroke patients had significantly longer duration of diabetes (14.4_+ 10.3 years, mean_+SD) compared with the control subjects (5.3_+ 4.8 years; p < 0.001) and poorly controlled diabetes: fasting glycaemia was 12.3 _+ 7.9 and 8.7_+ 4.4 mmol/l for the stroke and control group respectively. The results are suggestive of the hypothesis that long- lasting marked hyperglycaemia is a risk factor for stroke in diabetic patients.

190. Plasma Fibronectin and Factor VIII Related Antigen in Diabetic Patients with Atherosclerusis: The Sehwabing Study H. U. Janka, G.Waldmann, and H. Mehnert. Schwabing City Hospi- tal, Munich, FRG Raised levels of plasma fibronectin (FN) and factor VIII related antigen (F VIIIR: Ag) have been interpreted as a sign of endothelial injury. However, no data are reported in diabetic patients with atherosclerosis. Therefore, 675 unselected diabetic outpatients (aged 40-79 years) participating in the Schwabing Study on Macroangiopa- thy were screened for FN and FVIIIR: Ag measured by immunoelec- trophoresis (Laurell technique using specific antisera). In addition, all patients were classified according to peripheral vascular disease, coronary heart disease and carotid artery stenosis. FVIIIR: Ag was significantly increased in age adjusted diabetic patients with atherosclerosis (234.6+5.9%; mean-+SEM) compared to patients without detectable large vessel disease (186.3 +4.5%; p< 0.001). Differences of FN did not reach significance (107.1 -+4.0 versus 96.4_+3.2%). A multivariable analysis of variance revealed that FVIII: Ag was associated with atherosclerosis independent of blood glucose, HbAI, duration of diabetes, and kidney function. These results indicate that considerable endothelial damage is present in diabetic patients with atherosclerosis. F VIII: Ag, but not FN, may prove to be a valuable indicator of endothelial integrity of the large vessels.

191. Dissociation Between Pancreatic Islet Blood Flow and Insulin Re- lease in the Rat L.Jansson. Department of Medical Cell Biology, University of Upp- sala, Uppsala, Sweden The blood flow to the pancreatic islets was estimated in fed or fasted (72 h) rats with the aid of non-radioactive microspheres. The pancreatic blood flow (PBF) in fed animals was 0.55 _+ 0.04 ml/min x g and in the starved animals 0 .30+0.04ml/minxg (p<0.001), and the corresponding islet blood flow values (IBF) 82.0-+ 12.4 and 50.5-+ 9.7 ul/min x pancreas (p< 0.05). Serum insulin concentrations were lower (p < 0.001) in the starved animals. IP injection of 2 m130% solution of D-glucose increased IBF in both fed (p < 0.05) and starved animals (p < 0.01) to approximately the same level. The glucose injection caused a marked increase of serum insulin levels in the fed animals (p< 0.001) but no change in the starved group. A similar injection of mannoheptulose, a known inhibitor of islet insulin release and glucose metabolism, significantly stimulated IBF in both groups (p< 0.01) while circulating insulin levels were decreased (p < 0.01) and serum glucose increased (p < 0.01). It is concluded that changes in IBF do not reflect alterations in rates of insulin release. The positive correlation between IBF and the concentration of serum glucose irrespective of the rate of insulin release, indicates that glucose regulation of IBF is mediated through mechanisms other than the glucose metabolism of the islet B cell.

192. Glycohaemoglobins and Glycosylated Plasma Proteins During Im- proved Metabolic Control of Diabetic Patients Gy. Jermendy, J. Soegaard, P. Aa. Svendsen and J. Nerup. Steno Me- morial Hospital, Gentofte, Denmark Glycohaemoglobins and glycosylated plasma proteins (GPP) were measured in 18 diabetic patients (13 newly diagnosed and five known diabetics, aged 42.7_+3.4years; mean -+SEM) over a period of 2 weeks during which glycaemic control was rapidly improved. Ion exchange chromatography was used for HbA~+b as well as for HbAac while thiobarbituric acid assay was applied for ketoamine-bound hexose values and for GPR The mean blood glucose concentration (13.5-+0.7mmol/1) as well as the mean values of stable HbAla+b (2.35 _+ 0.08%), of stable HbAI~ (11.9 + 0.4%), of labile HbA~ (1.37 _+ 0.13%), of ketoamine-bound hexose (32.25 -+ 1.03 pmol HMF/mmol Hb), and of GPP (1.89_+0.07 ~mol HMF/g protein) were markedly

168 Abstracts

elevated at patients' admission. During improved control, the mean decreases of values as a percentage of the initial values after 1 and 2 weeks were, respectively: mean blood glucose 31% and 39%, stable HBAlc 3% and 9%, labile HbAI~ 49% and 46%, ketoamine-bound hexose 8% and 13%, GPP 22% and 35%. No significant change in mean values of stable HbAla+b could be observed during that time. Stable HbAaa+b, stable HbAlo and ketoamine-bound hexose values were poor indicators of the changes seen in glycaemic control, while GPP and especially labile HbAlc reflected more accurately the rapidly improved hyperglycaemia of the diabetic patients.

193. Evaluation of Methods of Studying the Chlorpropamide-Alcohol Flush in Type 2 (Non-Insulin-Dependent) Diabetes P.Jerntorp and L.-O.Alm6r, Department of Medicine, University of Lurid, Maim6 General Hospital, Malta6, Sweden Alcohol flushing after chlorpropamide exposure (CPAF) has been claimed to be associated with Type 2 diabetes and relative freedom from late diabetic complications. Since the facial flush is often dis- crete, objective methods to measure the CPAF reaction, and to separate it from the simple alcohol flush, are needed. We studied 164 Type 2 diabetics. Standard CPAF-tests were performed. Alcohol tests without chlorpropamide premedication were performed in patients positive to the earlier CPAF-test. The reation (flush/no flush) was judged by the same examiner. The increase in plasma acetaldehyde concentration (A P) (gas chromatography) and facial skin temperature (A T) were measured, and the malar thermal circulation index (A MTCI) (after Wilkin) was calculated. In 85 of the patients (52%) a flush reaction was noted during the CPAF-test. Using the following definitions of a positive test, all methods had high sensitivity and specificity: A T > I . 0 ~ (91%; 87%), AMTCI_->I.5 (88%; 94%) and /x p > 4 .umol/1 (86% ; 85%, respectively). A facial flush during the alcohol test was seen in 19/50 (38%) of the CPAF-test positive patients. In no alcohol test did A P exceed 4 gmol/1. Thus, irrespective of the reaction during the alcohol test, a flush reaction with A P > 4 kmol/1 during the CPAF-test indicated a flush related to inhibition of alcohol metabolism by chtorpropamide. Using these methods CPAF could be studied objectively: 52% were flushers (47% CPAF-positive and 5% alcohol flushers without CPAF).

194. Incipient Nephropathy in Type I (Insulin-Dependent) Diabetic Pat- ients K.Johansen, E.Mathiesen, B.Oxenboll, P.Aa.Svendsen and T. Deckert. Steno Memorial Hospital, Gentofte, Denmark The aim of the study was to single out in patients with Type 1 diabetes mellitus those patients who will later develop diabetic nephropathy as defined by persistent proteinuria. 71 diabetic patients with albustix- negative urine were followed for 6 years. Additionally, 227 patients were studied cross-sectionally. All patients were albustix-negative, < 50 years old, diabetes started before the age of 40 years, blood pressure was normal and urinary albumin excretion rate (UalbV) was < 200 ug/min. The best prognostic parameter was UalbV. During the follow-up period, 10 patients (14%) increased UalbV to > 200 gg/min and five of these developed diabetic nephropathy. Since all seven patients with UalbV > 70 Ixg/min at the beginning of the follow-up developed diabetic nephropathy or UalbV > 200 .ag/min, we designate them incipient patients with nephropathy. The cross-sectional study showed that patients with incipient nephropathy had increased blood pressure and normal serum creatinine. GFR and stable HbAac were higher than normal and retinopathy more prevalent in patients with elevated UalbV compared to normoalbuminuric diabetic subjects. In conclusion, incipient diabetic nephropathy is characterized by UalbV >70txg/min, slightly elevated blood pressure, and supernormal GFR.

195. Platelet Aggregation in Diabetic and Non-Diabetic Subjects: Role of Glucose, Lipids and Prostaglandin Precursor Fatty Acids D. B. Jones, T. M. E. Davis, E. Bown, R. D. Carter and J. I. Mann. Dia- betes Research Laboratories, Radcliffe Infirmary, Oxford, UK ADP induced platelet aggregation was measured in 15 insulin-dependent diabetic patients, 15 non-insulin-dependent diabetic patients and 15 non-diabetic control subjects and correlated against blood glucose, glycosylated haemoglobin, serum insulin, plasma lipids and platelet phospholipid fatty acids. In the insulin-dependent diabetic patients, the highest correlations were seen with blood glucose (Rs = 0.69, p < 0.01) and glycosylated haemoglobin (Rs=0.31). In the non-insulin dependent diabetic patients, the highest correlations occurred with total cholesterol (Rs = 0.62, p < 0.05), the ratio of the combined percentages of arachidonic (C20:4c06) and linoleic (C18:20)6) acids to the

combined percentages of eicosapentaenoic (C20:5~o3) and linolenic (C18:303) acids (Rs = 0.46, p < 0.05), the percentage of eicosapentaenoic acid (Rs = 0.45) and blood glucose (Rs = 0.45). In the non-diabetic control subjects, the highest correlations occurred with very low density lipoprotein cholesterol (R = 0.48, p < 0.05) high density lipoprotein cholesterol (R= -0.39) and with total cholesterol (R= 0.35). The results suggest that platelet aggregation may be primarily determined by glycaemic control in insulin-dependent diabetic patients but also by the level of thromboxane analogue precursor fatty acids in non-insulin dependent diabetic subjects. Platelet aggregation in normal subjects may be determined by plasma lipid levels.

196. The Relationship Between Carbohydrate Tolerance and Differen- tial Hepatic Extraction of Insulin in Response to Serial Glucose and Mean Tolerance Tests in Normal Subjects I.R.Jones, D.R.Owens, S. Luzio, C.J.Davies, A.J.Birtwell and 1". M. Hayes. Department of Medicine, Welsh National School of Medicine, Cardiff, UK Five normal fasting males (aged 21-29 years, within 10% of their ideal body weight) were studied for two 24-h periods involving serial 4 hourly glucose (oral glucose tolerance test, 75 g, GTT) or standard meal tolerance tests (500 Kcal, 60% CHO) of equivalent carbohydrate content. For each 4-h cycle, plasma glucose, insulin (IRI), C-peptide and glucose-dependent insulinotropic polypeptide (GIP) were measured at half-hourly intervals. The glucose tolerance was significantly better following the meal tolerance test than after the oral GTT for each 4-h cycle throughout the day (p < 0.001). The B-cell secretory response as indicated by C-peptide levels was similar for the two carbohydrate loads. However, the peak peripheral plasma insulin responses to the meals were significantly greater than those following the repeated oral G T r (p< 0.001). The basal IRI: C-peptide ratio was 0.1, but following the oral GTI" this ratio doubled and in response to the meal tolerance test increased up to five times the basal value. The liver extracts approximately half of the insulin but only a negligible amount of C-peptide. Following the meal tolerance test, hepatic insulin extraction is reduced, with a consequent increase in the peripheral insulin concentration and significantly better glucose tolerance in response to this more physiological stimulus.

197. Electronic Whole Blood Platelet Aggregation Measurement in Diabetes R. J. Jones, A. P. Delamothe, S.J. Machin 1 and D.J. Betteridge. Depart- meat of Medicine, University College London School of Medicine, The Rayne Institute, London and ~Department of Haematology, Mid- dlesex Hospital,London, UK Abnormal platelet function may be involved in the pathogenesis of vascular disease in diabetes. A novel method for observing platelet aggregation by measuring the.accretion of platelets to two electrodes in whole blood by a change in impedance between the electrodes has been reported recently. This electronic method is more physiological since the platelets do not need to be separated from red and white cells which have been shown to produce factors which alter platelet aggregation. Whole blood platelet aggregation was assessed in 12 insulin-dependent diabetic subjects (nine female, three male, aged 36_+ 4.2years, mean_+SEM) and 12 age, sex matched healthy subjects (35.6+3.1 years). Statistically significant increased platelet aggregation in whole blood was observed in the diabetic subjects following the addition of the agonists: collagen (I Ixg/1) and arachidonic acid (1 retool/l) (p < 0.01 ; p < 0.05, Wilcoxon's signed rank sum test). Dia- betic subjects showed a decreased sensitivity to the anti-aggregatory prostaglandin epoprostenol (5.4_+ 1.9 versus 2.7 +_ 0.7 ng/ml, mean + SEM). These changes may be important in the pathogenesis of diabetic vascular disease.

198. An Insulin Gene Polymorphism that Relates to Diabetic Hypertriglyceridaemia N.I.Jowett, L.G.Williams, G.A.Hitman and D.J.Galton. Medical Professorial Unit, St. Bartholomew's Hospital, London, UK Diabetes and hypertriglyceridaemia are associated with altered levels of plasma insulin, which may in turn be affected by insulin gene tran- scription. A polymorphic region 5' to the human insulin gene has been identified, which may affect gene expression. We have studied 51 hypertriglyceridaemic patients and 33 control subjects (normolip- idaemic and non-diabetic) using leucocyte DNA digested with en- dmmclease Bgll. This was Southern blotted and hybridised with a 32p-labelled human insulin gene probe. Autoradiography showed small (S); 0-600 bp or large (L); 1600-2200 bp, DNA inserts and patients were genotyped as LL, LS or SS. A standard 75 g oral glucose

Abstracts 169

tolerance test was performed on 42 of the lipaemic patients, who were grouped for either a normal or abnormal response (2 h blood glucose level < or> 8 retool/l). The genotype distribution for the lipaemic group was LL: 6, LS: 30, SS: 15 compared to controls LL: 2, LS: 16, SS: 15 (p< 0.01). Within the lipaemic group those with an abnormal glucose tolerance showed genotypes LL: 5, LS: 9, SS: 7 compared to normal glucose tolerance LL: 0, LS: 14, SS: 7 (p< 0.05). The long in- sert appears to segregate with hypertriglyceridaemia, and within this group homozygosity (LL) associates with abnormal glucose tolerance.

199. Processing of the Insulin-Receptor Complex in Isolated Hepato- cytes S. M. Juul, R. H. Jones, J. L. Baker and P. H. Srnksen. St Thomas' Hos- pital, London, UK Processing of the insulin-receptor complex has been studied in isolated hepatocytes using an 12SI-labelled photoreactive insulin analogue - Nmg-((4-azido-2-nitrophenyl)-acetyl)insulin - ('photoprobe'). Cells were incubated with the 'photoprobe' in the dark at 37 ~ in the absence and presence of excess cold insulin. Aliquots of the incubation medium were taken at timed intervals and irradiated with ultraviolet light. Non-covalently bound label was removed, the cells homoge- nized and centrifuged in Percoll density gradients to obtain subcellular fractions. At all time intervals, specifically-bound radioactivity was found at a density of 1.03-1.05 g/ml and corresponded to the position of plasma membrane marker enzymes. The radioactivity appeared as two peaks, the second, of slightly greater density, increased, relative to the first, with time from 0.5-10 rain. Over the same time interval, the specific activity (cpm/gg protein) increased threefold. A third small peak was noticeable from 2.5 min at a density of 1.055 g/ml. No radioactivity was found in the lysosomal region. The results demonstrate that the 'photoprobe', covalently bound to its receptor, was associated with the plasma membrane/low density vesicle subcellular fractions. No binding occurred with fractions of greater density during a 10-min incubation. It therefore appears that insulin degradation is initiated early during internalisation.

200. Teaching of Diabetic Patients is an Important Part of Treatment of Diabetes Mellitus: Results after 3 Years G. Kacerovsky-Bielesz, H. Hohenecker, H. Schindler and B. Willms. I. Medizinische Abteilung, Hanusch Krankenhaus, Wien, Austria and Fachklinik ffir Diabetes und Stoffwechselerkrankungen, Bad Lauter- berg, FRG The effective teaching of diabetic patients is an important prerequisite for successful treatment, which requires a lot of personnel and much time. Our solution has been a teaching programme including eight videofilms, books and questionnaires. The doctors and nurses rein- force this basic instruction with individual explanations and diet counselling. Three years later, we evaluated its success for a group of 300 Type 1 and Type 2 diabetic patients. Average blood glucose fell from 14.3 to 8.8 mmol/l, the average HbA~ fell from 12.5 to 8.7% in the Type 1 diabetic patients, annual hospitalisation dropped from 10 days to 1 day. The ability to recall the instructions was related to the good metabolic balance in this young group. In the Type 2 group, with its wide age range and various treatments, an improvement of metabolism was also observed depending on age and education. Almost all patients reported an improved life style (less frequent hypoglycaemia, better efficiency). Not one case of coma was recorded. In conclusion, with a multi-media instruction programme, all age groups up to 70 years with varying levels of schooling proved teachable, making it possible to control long-term metabolic balance and thereby reducing hospitalisation and preventing long-term complications.

201. The Interaction of Insulin with Vascular Smooth Muscle N. Kaiser, A.Tur-Sinai and E.Cerasi. Department of Endocrinology and Metabolism, Hebrew University-Hadassah Medical Center, Je- rusalem, Israel Vascular smooth muscle cells are involved in diabetic macrovascular disease. Proliferation of smooth muscle cells following endothelial injury is induced by various serum factors including insulin. In order to clarify the role of hyperinsulinaemia in macrovascular disease, we studied the interaction of insulin with cultures of smooth muscle cells from the bovine aortic arch. These cells exhibited specific binding of 125I-insulifi which was reversible, and dependent on pH. Insulin binding was accompanied by internalization and degradation of the hormone. Pre-exposure of cell cultures to insulin for 18 h resulted in dose-dependent down-regulation of the binding. Sparse, serum- starved smooth muscle cells were very sensitive to the mitogenic ac-

tion of insulin: significant increase in 3H-thymidine incorporation occurring already at 1 ng/ml of insulin. The maximal stimulatory effect observed at 1000 ng/ml insulin was approximately 50% of the effect of 10% serum. In view of the high sensitivity of smooth muscle cells to the mitogenic effect of insulin, and the saturation of the effect at very high concentrations, it is suggested that the growth promoting action of insulin in this system is mediated both by direct interaction with the specific insulin receptors, and crossreaction with the IGF-1 receptors.

202. The Relationship of the Control of Diabetes to the Development of Coronary Heart Disease: a S-Year Follow-up Study B. Karamanos, A. Kofinis, Ch. Hadjigeorgiou, P. Christacopoulos and Z.Komninos. Diabetic Center, Hippokration Hospital, Athens, Greece One hundred diabetic patients were followed for 5 years in order to investigate the relationship between the control of diabetes and the development of coronary heart disease (CHD). Twenty-two of these patients had CHD from the beginning, 59 remained without CHD (group A) and 19 developed CHD during the follow-up period (group B). The control of diabetes was assessed bimonthly by the determination of post-prandial blood glucose, pre-and post-prandial urinary glucose and glycosylated haemoglobin. The mean values of the parameters measured in group A compared with B were: age: 60 + 2 versus 70_+ 2 years; p < 0.01 ; percentage ideal body weight: 112.0_+1.8 versus 110.7_+3.7%, p>0.05; duration: 6.7_+8 versus 7.8_+1.2years, p>0.05; blood glucose: 8.87_+0.27 versus 8.69+ 0.42 retool/l, p> 0.05; urinary glucose pre-prandial: 6.33 + 1.1 versus 3.55+1.1mmol/1, p>0.05; post-prandial: 7.10+1.1 versus 5.1_+ 1.1mmol/l, p>0.05; glycosylated haemoglobin: 7:72_+0.1 versus 7.58 + 0.4%, p > 0.05; cholesterol: 6.21 -+ 0.19 versus 5.87 + 0.35retool/l, p>0.05; high density lipoprotein cholesterol: 1.14+ 0.06 versus 1.13-+0.12mmol/1, p>0.05; blood pressure maxima 138.6 _+ 2.4 versus 146.1 _+ 3.6 mmHg, p > 0.05; minima 82.6 _+ 0.8 versus 83.4_+1.1 mmHg, p > 0.05. All hypertensive patients were under treatment. The prevalence of smoking was not different between the two groups (35 versus 27%, p > 0.05), while the prevalence of hypertension (treated or not) was significantly higher in group B than A (58 versus 27%, p < 0.05). Conclusion: the development of CHD in diabetes is related to age and presence of hypertension (treated or not), while it is not related to the control of diabetes or smoking.

203. Effects of Dietary Protein on Blood Glucose and Serum Insulin in Type 2 (Non-Insulin-Dependent) Diabetic Patients S.-G. Karlander, E. Andersrn, K. Kindstedt and K. Hellstrrm. Depart- ment of Medicine, St Erik's Hospital, Stockholm, Sweden Amino acids may stimulate insulin secretion in Type 2 diabetic patients with an impaired insulin response to glucose. We therefore want- ed to investigate whether protein-rich foodstuffs may help to improve glucose control in these patients. Protein-poor and protein-rich test meals were given one week apart to fasting Type 2 diabetic patients (26 on diet alone and 12 on diet+glibenclamide), aged 39-75 years. Both fish and lean meat served with mashed potato (24-30 g protein) doubled the insulin response to potato alone (5 g protein). The blood glucose response was moderately, but significantly, reduced, while serum glucagon levels were unchanged by protein. The stimulating effect of protein on insulin secretion remained when fat was also added. However, complete breakfasts, containing 20 and 37 g protein, respectively (13 and 25% of calories), did not differ in responses of blood glucose and serum insulin. We conclude that the metabolic effects of protein-rich food-stuffs seen in experimental test meals are not reproduced in complete meals, possibly due to a lesser variation in protein content. Therefore, it does not appear that an increased proportion of protein in the diabetic diet will be of value in improving glucose control in Type 2 diabetic patients.

204. C-Peptide-like Substance in Serum of Patients with Acute Pan- creatitis T. Kasperska-Czy~ykowa, L. G. Heding, B. Tronier and A. Czy~yk. De- partment of Gastroenterology and Metabolic Diseases, Medical Academy in Warsaw, Poland, and Novo Research Institute, Bags- vaerd, Denmark The functional capacity of the B cells of the pancreatic islets was evaluated in five cases of acute pancreatitis (two men and two women, of whom one was examined twice after two successive attacks). This assessment revealed that in the fasting state the serum level of C-peptide exceeded the normal value by about five times on the first 2 days after attack when this peptide was determined with Byk-Mallinckrodt kits,

170 Abstracts

but it was within the normal range when M 1221 C-peptide-antibodies ad modum Heding were used. The data presented below displays this difference:

Serum C-peptide (nmol/1) (mean _+ SEM)

Days after (t) (2) (3) (5) (10) attack

Byk-Mallinck- 2.77• 2.97• 1.94• 1.94• 1.36• rodt kits M1221 0.36• 0.36_+0.12 0.34+0.15 0.43• 0.64• antibodies

Serum insulin (7.2-31.2 mU/l) and serum proinsulin, determined ad modum Heding (0.017-0.036 nmol/1), were normal. These results suggest that in acute pancreatitis a C-peptide-like substance, which does react with some C-peptide-antibodies but is not identical with 'true' C-peptide nor with proinsulin, is released into the blood. The nature and origin of this substance, as well as the diagnostic value of its determination, remain to be elucidated.

205. Subcutaneous Insulin Infusion with a Closed-loop Glycaemie Con- trol System R. Kawamori, M. Shichiri, N. Asakawa, N. Iwama and H. Abe. Osaka University Medical School, Osaka, Japan It is obligatory to infuse insulin SC for the purpose of long-term glycaemic control by a wearable artificial endocrine pancreas with a needle-type glucose sensor. In order to develop an optimal algorithm for SC insulin infusion, desired plasma insulin concentration, IRI(t), is set as IRI(t)=a G(t)+b G'(t) + c, where G(t) and G'(t) are blood glucose concentration and the rate of change in blood glucose concentration respectively, and a, b and c denote the coefficients of proportional- and derivative-action, and the constant for basal insulin supplementation, respectively. Secondly, the pharmacokinetics of SC injected insulin were analysed to establish IRI(t) by using a two-compartment process. To obtain physiological plasma insulin profiles against oral glucose challenges, a simulation study revealed that insulin should be infused SC in a waveform in rapid response to glycaemic rise, resulting in much weight on b, the coefficient for derivative- action (a=0.5, b=79, c = - 3 3 ) . In pancreatectomized dogs, almost identical glycaemic control and IRI profile as those seen with IV insulin infusion by artificial endocrine pancreas (a= 1, b=4, c = - 72), were obtained with this SC infusion algorithm. In summary, both a physiological glycaemia-related plasma insulin profile and glycaemic control were obtained in pancreatectomized dogs with a newly-developed closed-loop SC insulin infusion algorithm.

206. Haemoglobin A1 as a Routine Test at the Diabetic Clinic - Com- parison with Plasma Glucose Measurements L. Kennedy, E. Byrne, G.Savage and J.D.Merrett. Royal Victoria Hospital and Queen's University, Belfast, Northern Ireland Most published studies of HbA1 as a measure of glycaemia have been conducted in relatively "controlled" conditions (e. g. research wards), whereas the test's main clinical application lies in the outpatient clinic. In December 1980 we introduced HbA1 measured by agar gel elec- troendosmosis (Coming) after incubation of red cells in saline, as a routine test at our clinic. 302 insulin-treated patients had an initial value (mean+SEM) of 10.72_+0.13% and all were reviewed at least twice during the following year. Levels after 2-4 months (n = 182) and 5-7 months (n= 197) were significantly reduced to 9.9_+0.16% and 10.13 + 0.16% respectively (p< 0.001 when compared with initial values on a paired basis). After 9-12 months HbAa was no different from initial level - 10.74_+0.14% versus 10.74_+0.15% (n=251). In contrast, at all these reviews 2.5 h post-prandial plasma glucose was unchanged, compared with values at the initial visit. A change of HbA1 of > 1.5% (either increase or decrease) from initial visit to subsequent reviews was arbitrarily designated "clinically significant", as was a change of > 3 mmol/1 in plasma glucose. In paired observations (i, e. increase, decrease or no change), HbA1 and glucose had approximately 40% concordance only. HbA~ measurement yields clinical information not available from plasma glucose measurement in the outpatient clinic. However its routine availability does not seem to have made a sustained impact on improving control.

207. Effect of Immunoglobulin G on 3-0-Methylglueose Uptake and In- sulin Binding by Rat Adipocytes M. A. Khokher and P. Dandona. Metabolic Unit, Royal Free Hospi- tal, London, UK

Following the demonstration that human IgG and its Fc fragment cause stimulation of adipocyte lipogenesis, we have now examined whether IgG exerts this effect through the insulin receptor and whether it stimulates transport of glucose across the adipocyte membrane. Specific binding of insulin to adipocytes and 3-0-methylglucose uptake by these cells was measured by standard techniques following collagenase digestion of rat epididymal fat pads. A concentration of 400 rag/1 human IgG produced a consistent stimulation of 3-0-methylglucose uptake (348% above basal) by adipocytes after 1 min incubation at 37 ~ This amount of uptake was greater than that produced by 500 mU/1 insulin. IgG concentrations in the range of 400 mg/l to 1 g/ l were unable to alter either the binding of 125I-insulin to adipocytes or to interfere with the displacement of ~25I-insutin by cold insulin. These data provide the first evidence that human IgG stimulates glucose transport and that this stimulation is independent of the insulin receptor.

208. Immunotherapy of Experimental Insulin-Dependent Diabetes in Mice: Effect of Antibodies to Immune Response Gene Products U.Kiesel and H.Kolb. Diabetes Research Institute, University of Dtisseldorf, Diisseldorf, FRG Low-dose streptozotocin-induced diabetes in mice serves as a model of insulin-dependent diabetes. Suppression of the development of diabetes (hyperglycaemia) could be achieved by administration of antibodies in vivo to immune response (Ir)-gene products. Male C3H mice were treated on five consecutive days with 40 mg streptozotocin/kg bodyweight. The experimental group received 2 h before the first streptozotocin injection 20-40 Ixl of alloantisera or monoclonal antibodies IV. The development of hyperglycaemia could be substantially suppressed by administration of two monoclonal antibodies to I-A ~ gene products (p < 0.05) and, surprisingly, by an alloantiserum to I-J determinants (p< 0.001). The suppressive effect of antibodies I-J k gene products could be reproduced in mice of strain B10.BR (also carrying the I-J k allele) but, as a control, not in strain B10 (carrying the

b I-J allele). These results suggest that I-A and I-J positive T lymphocytes mediate cellular autoimmunity to pancreatic islets. Such lymphocytes can be used as targets for immunotherapeutic approaches.

209. A 6-Year Follow-up Study of Impaired Glucose Tolerance in the Micronesian Population of Nauru H. King and P. Zimmet. WHO Collaborating Centre for the Epidem- iology of Diabetes Mellitus, Royal Southern Memorial Hospital, Mel- bourne, Australia A longitudinal study of 266 randomly selected non-diabetic Nauruans - 215 normal subjects and 51 with impaired glucose tolerance (IGT) - has permitted the prognosis of IGT to be studied in this Micronesian population. Nauruans are known to suffer from a very high prevalence of diabetes. The subjects were first examined in 1975-1976, and a follow-up examination was performed in 1982. Of the IGT subjects, 26% developed diabetes during the study period (4% per annum) compared with 7% of normal subjects (1% per annum). After controlling for the effects of both age and obesity, the risk of subsequent diabetes for IGT subjects remained significantly higher than for normals (odds ratio: 3.6, p < 0.01). However, of those with IGT initially, 39% were normoglycaemic at follow-up. In IGT subjects, of nine physiological, biochemical and environmental factors examined, only baseline plasma glucose concentration was consistent in predicting progression to diabetes, when the data were examined by both univariate mad multivariate methods. These findings are in accord with recent findings from longitudinal studies in other populations, and support the recognition of this category of glucose intolerance.

210. Cyclic-AMP Induced Insulin Resistance at Receptor and Post-receptor Level in Isolated Rat Adipocytes D. Kirsch, W. Kemmler and H. U. H~iring. Diabetes Research Group, Munich, FRG Preincubation (30 rain) of rat fat cells with catecholamines (isoprena- l ine 10 -7 to ]0-Stool/1 or noradrenaline 10 9 t o 10-6mol/1) decreases insulin sensitivity of glucose transport by 90 and 50%, respectively, and reduces high affinity binding of insulin. As catecholamine- stimulated lipolysis is mediated by cyclic-AMP, we investigated whether this insulin resistance is cyclic-AMP-dependent as well. Ep- ididymal rat fat cells were excised and isolated by the collagenase method; glucose transport was measured with 3-0-methylJ4C-glucose and insulin binding was determined with A14-~25I-insulin. Pre-incubation (30min) with dibutyryl-cyclic AMP or 8-bromo-cyclic AMP

4 3 0 (10- to 10- tool/l) led to a 15-30Y0 inhibition of high affinity binding of insulin and a 30-50% inhibition of glucose transport. IBMX

Abstracts 171

(10 4 to 10 -3 tool/l) reduced insulin binding by 25% and glucose transport by 70%. However, whereas the catecholamine effect on glucose transport is mainly due to the decrease in insulin binding, an additional post-receptor resistance seems to be induced by the cyclic- AMP analogues and IBMX. In conclusion, an increase of cyclic- AMP induces insulin resistance at receptor and post-receptor level.

211. Long-Term Timolol Treatment After Myocardial Infarction in Dia- betes J. K. Kjekshus and T.Gundersen. Baerum Hospital, Sandvika and Rogaland Hospital, Stavanger, Norway The effect of long-term treatment with the non-selective beta-receptor antagonist timolol on mortality and non-fatal reinfarction was studied in diabetic patients surviving an acute myocardial infarction. The analysis was based on 99 diabetic patients in the Norwegian timolol multicentre study. All patients were randomized to timoIol 10rag twice a day or matching placebo. The follow-up period was 12-33 months (mean 17 months). The mortality among diabetic patients in the placebo group was twice that of non-diabetic subjects, 30.5% and 13.5% respectively. Non-fatal reinfarctions occurred more often in diabetic (21.7%) than in non-diabetic subjects (13.5%). Analy- sis of all randomized diabetic patients showed 14 deaths in the placebo group and 6 deaths in the timolol group, a reduction of 62.8% (p < 0.01). Non-fatal reinfarctions were 10 in the placebo and two in the timolol group, a reduction of 82.7% (p< 0.01). Inclusion rate, side effects and withdrawals were comparable in diabetic and non-diabetic patients. Although long-term treatment with beta-receptor antagonists may induce slight carbohydrate intolerance, it has been shown for the first time that excessive rates of cardiac mortality and morbidity among diabetic patients may effectively be prevented by administration of timolol.

212. Immunocytochemical Morphometry of the Endocrine Pancreas in Obese and Non-obese Type 2 (Non-Insulin-Dependent) Diabetic Pat- ients G. K16ppel, C. R. Drenck, K. Habich, G. Bommer and Ph. U. Heitz. In- stitutes of Pathology, Universities of Hamburg and Basel, FRG and Switzerland To acquire further insight into the pathogenesis of Type 2 diabetes, the volume density and the absolute mass of pancreatic endocrine cells were studied in obese and non-obese Type 2 diabetic patients and compared with respective controls. Insulin (B), glucagon (A), somatostatin (D) and pancreatic polypeptide (PP) cells were immunocyto- chemically stained and quantitated by point counting. The PP lobe, accounting for about 10% of the total pancreatic volume, was not considered. The pancreas of obese non-diabetic (n = 4) and obese diabetic patients (n = 7) showed a significantly higher parenchymal volume (mean: 80 and 40ccm) than that of normal weight non-diabetics (n = 7) and diabetics (n = 8) (mean: 40 and 30 ccm). Volume density of B celis and total endocrine cells was significantly reduced in lean diabetics compared with controls (0.40 versus 0.69; 0.66 versus 1.02). In obese diabetic patients the same values were only slightly below those of obese non-diabetics (0.56 versus 0.51 ; 0.75 versus 0.73). The most significant changes were found when the absolute B cell masses were calculated. The mean values of obese and normal weight controls (0.4 and 0.28 ccm) contrasted with those of obese and lean diabetics (0.2 and 0.1 ccm). It is concluded that the different B cell masses of obese and non-obese subjects with and without diabetes reflect, at least in part, the heterogenous insulin secretory capacity of these patients.

213. An Immunogenetic Concept of Insulin-Dependent Diabetes in BB Rats: The Mutant RT1 u~ Haplotype of the Major Histoeompafibility Complex I. K16ting and O. Stark. Central Institute of Diabetes, Karlsberg, GDR and Institute of Biology, Charles University, Prague, Czechoslovakia Diabetes in BB rats is associated with the RTI ~ haplotype of the major histocompatibility complex which might be due to a mutation within this complex genetic region. Since such a mutation results in a change of immune reactions determined by major histocompatibility complex coded genes, we have studied the graft-versus-host reaction (GVHR, popliteal lymph-node-weight-assay) in adult non-diabetic BB rats, in their parental Wistar rat strain, and in their interstrain F1 and first backcross hybrids as well as in LEW rats of congenic and recombinant strains. A strong GVHR was ascertained in the majority of interactions with BB rat recipients. On the contrary, only a weak GVHR was shown in most cases with BB donors &the lymphoid cells transplanted. The lowered GVHR of BB rat lymphoid cells might

correspond to our additional observations of a reduced immune response to skin allografts and against mycoplasma infections as well as to the low immune response to pig insulin. Taken together, these findings lead to an immunogenetic concept of diabetes in BB rats which might serve as a model of insulin-dependent diabetes in man.

214. Experience of the Initiation of Continuous Subcutaneous Insulin Infusion Treatment on an Outpatient Basis G. Knight, A.J.M. Boulton, J. Drury and J. D. Ward. Royal Hallam- shire Hospital, Sheffield, UK Previous reports of the use of continuous subcutaneous insulin infusion (CSII) have described in-patient commencement of the treatment. As part of a feasibility study of the use of CSII in our diabetic clinic, 86 subjects started the therapy as outpatients. Twenty-seven (32.6%) discontinued within 1 year, 23 doing so in the first 3 months. Principal reasons were excessive pump size (eights subjects) and dis- comfort (five). One patient developed ketoacidosis 3 days after starting CSII due to lack of understanding of the treatment, despite education in the use of the treatment. Two subjects developed needle insertion site abscesses within I week of CSII. No other subjects encountered serious clinical problems during the initial period. After 9 months those subjects using CSII and those using an intensified injection regimen had significantly improved glycosylated haemoglobin leveis (CSII: mean HbAI: 55.9 mmol H MF/mol Hb prestudy; 46.0 at 9 months, p< 0.001; intensified injection regime: 55.4prestudy; 50.5 at 9 months, p< 0.001 ; reference range 29-39 mmol-HMF/mol Hb). There is no significant difference in drop out rates between the two groups. We conclude that for most subjects CSII may be initiated safely and practically as outpatients.

215. Effects of Selective Adrenergic Blocking Drugs on the in Vivo Ad- renergic Effects on Plasma Levels of Giucagon, Insulin and Glucose in Rabbits J. Knudtzon. Pediatric Research Institute, Rikshospitalet, Oslo, Nor- way In fasted rabbits, the plasma levels of glucagon are mainly increased by a-receptor stimulation, whereas in man /?-receptor stimulation seems to be of most importance in releasing glucagon from the endocrine pancreas. The aim of the present study was to investigate which subtypes of c~- and/?-receptors are involved in the regulation of the plasma levels of glucagon, insulin and glucose in fasted rabbits, a2-receptor blockade (yohimbine) significantly blocked the adrenaline-induced increases in the plasma levels of glucagon and glucose and the adrenaline-induced decreases in the plasma levels of insulin, whereas al-receptor blockade (prazosin) was inactive. The slight isoproterenol-induced hyperglucagonaemia was not affected by/71- or fl2-receptor blockade. The isoproterenol-induced hyperinsulinaemia was inhibited at a lower dose by/?2-receptor blockade (ICI 118.551) than ill-receptor blockade (metaprolol). The isoproterenol-induced hyperglycaemia was blocked only by fl2-receptor blockade. It is concluded that in fasted rabbits: (1) plasma levels of glucagon are mainly increased by az-receptor stimulation; (2) plasma levels of insulin are decreased by az-receptor stimulation and increased more efficiently by flz-receptor stimulation than by ill-receptor stimulation; and (3) plasma levels of glucose are increased by a2- and/?2-receptor stimulation.

216. Incidence and Management of Severe Hypoglycaemia in 402 Type 1 (Insulin-Dependent) Diabetic Patients: Follow-Up 18 Months after an Intensified Teaching and Treatment Programme J. Koch, I. Mtihlhauser, V. Jrrgens, A. Kunz, W. Graninger, G. Schern- thaner and M. Berger Departments of Medicine, Universities of Dfisseldorf, FRG and Vienna, Austria At the two hospitals, an identically structured 5-day-in-patient, groupteaching and treatment programme was carried out with particular emphasis on self-management by the patient in order to achieve nearnormoglycaemia. All 402 Type I diabetic patients (age 30+ 10years, duration of diabetes 11 +8 years) who participated in the programme between January 1980 and June 1982, were included in the study. In a representative subgroup of 88 patients, significant improvement of metabolic control was documented (fall of mean HbA~o from 11.1% to 8.8% at 12months and 9.2% at 21 months after the teaching programme). 18 months after the programme, patients were asked about the incidence and management of severe hypoglycaemia requiring glucagon injection by relatives, assistance by a physician or emergency hospitalisation. Answers were obtained from 388 patients (97%): 56 had experienced 107 severe hypoglycaemic episodes, i.e. 10% of the patients experienced at least one severe hypoglycaemic ep-

172 Abstracts

isode per year. In 39% of the 107 hypoglyeaemic episodes, glucagon was injected (effective in all but one case), in 21% a physician was called and 42% of patients with severe hypoglycaemia led to emergency hospitalisation. Our successful attempt for continuous improvement on HbA~ by a 5-day diabetes teaching and treatment programme was not associated with an unduly high risk of severe hypoglycaemia, when compared with respective data from the literature. Glucagon injection by relatives should be encouraged in teaching programmes.

217. Evolution of Retinopathy in Relation to Control in Type 2 (Non-In- sulin-Dependent) Diabetic Patients A. Kofinis, B. Karamanos, K. Petrou, J. Roussos and Z. Komninos. Diabetic Center, Hippokration Hospital, Athens, Greece In order to investigate the relationship between the control of diabetes and retinopathy in Type 2 diabetic patients, we followed 90 patients for 5 years. The patients were examined bimonthly and the control was assessed by measurement of post-prandial blood glucose and glycosylated haemoglobin; cholesterol and high density lipoprotein cholesterol (HDL) were also measured. The status of the retina was assessed by fundus photography and in selected patients by fluo- roangiography. At the end of the 5-year period, 11 patients had catarract and were excluded from further evaluation, 41 patients had no retinopathy (group A) while 38 had retinopathy (group B). Group A compared to B had mean: age 63.8_+2.3 versus 60.6_+2.4years p > 0.05 ~ duration 12.1 _+ 0.8 versus 14.7 _+ 1.1 years p > 0.05 ; glucose 8.51 _+ 0.34 versus 9.21 _+ 0.28 mmol/1 p > 0.05 ; glycosylated haemoglobin 7.69+0.5% versus 7.68_+0.05%, p>0.05; cholesterol 6.32+ 0.21 versus 6.08+0.25mmol/1 p>0.05; HDL 1.14-+0.06 versus 1.07 _+ 0.07 mmol/l, p> 0.05. Twenty-two patients from group B had retinopathy from the beginning while 16 developed it during the follow-up period (sub-group Ba). Comparing sub-group Ba with group A, the following were found: age 61.3-+3.3 versus 63.8• p> 0.05; duration 12.3 + 1.4 versus 12.1 -+ 0.8 years; p> 0.05; glucose 9.13 -+ 0.46 versus 8.51 -+ 0.34 mmol/1, p > 0.05; glycosylated haemoglobin 7.58 + 0.3% versus 7.69 -+ 0.05%, p > 0.05; cholesterol 6.17 -+ 0.4 versus 6.08-+0.25 mmol/l, p > 0.05; HDL 1.16+0.06 versus 1.14+ 0.06 mmol/l, p > 0.05. Conclusions: In Type 2 diabetes, age, duration, degree of control and lipid profile are not related either to the presence or the development of retinopathy.

218. Continuous Subcutaneous Insulin Infusion Combined with Physical Training Normalizes Insulin Sensitivity in Type I (Insulin-Dependent) Diabetes V. A. Koivisto, H. Yki-J~rvinen and R.A. DeFronzo. Third Depart- ment of Medicine, Helsinki University, Helsinki, Finland, and De- partment of Medicine, Yale University, New Haven, USA Type 1 diabetes is characterized by insulin resistance. We examined the effect of continuous subcutaneous insulin infusion therapy, alone or combined with physical training, on insulin sensitivity and glycaemic control in 9 Type 1 diabetic patients (age 27 +_ 1 years, mean + SEM). Insulin sensitivity (euglycaemic insulin clamp at 1 mU/1) and maximal aerobic power (VO~max) were determined before and after 6 weeks' pump therapy and after an additional 6 weeks' infusion therapy combined with physical training (ergometer exercise I h four times a week). Three patients already pump-treated were studied only before and after training. During pump therapy, HbA1 decreased from 10.6_+ 0.6% by 20% (p < 0.005). Insulin requirements fell from 49 _+ 5 U/day by 30% (p < 0.005). The rate of glucose metabolism pre- pump (4.2 _+ 0.5 rag- kg-i . rain-1) rose by 40% (p < 0.05). During combined pump therapy and training, VOzmax rose by 9% (p < 0.02) and insulin requirements fell by a further 9% (p < 0.05), whereas HbA1 and body weight (102_+2% of ideal) remained unchanged. Glucose metabolism rose by 40% to 7.4 _+ 0.9 mg- kg-1. min-1 (p < 0.01 ), comparable to that in matched healthy control subjects (7.1 +0.7 mg-kg 1. min -1, n = 7). In conclusion, (1) pump therapy improves insulin sensitivity and decreases insulin requirements in the face of near normoglycaemia and (2) pump therapy with physical training can normalize insulin sensitivity.

219. Autoimmune T-lymphocytes with specificity for pancreatic islets H. Kolb, A. Ben~Nun, I. R. Cohen, I. Barberena and U. Kiesel. Diabe- tes Research Institute, University of DUsseldorf, Diisseldorf, FRG, and The Weizmann Institute of Science, Rehovot, Israel It is thought that in insulin-dependent diabetes T-lymphocytes mediate a cellular immune response towards islets. We now present direct evidence - in an animal model - for autoimmune T-lymphocytes with

specificity for islets antigens. Mice of strain C57BL/6 were immunized with islet homogenates. Lymphocytes from draining lymph nodes show a specific proliferative response when challenged in vitro with mouse or rat islet homogenate (stimulation index 15-20) but do not respond to homogenate from liver or spleen (stimulation index < 1.3). The relevant islet antigen can be solubilized from islet cells by ultrasonication, but it is different from islet hormones. Autoimmune T-lymphocytes have been propagated in vitro for several weeks by the addition of interleukin 2. Islet antigen specific lymphoblasts could be restimulated by another challenge with islet antigens in the presence of syngeneic macrophages. Autoimmune T-lymphocyte lines will allow a closer analysis of cellular immunity in insulin-dependent diabetes.

220. Evanescent Effect of Glucagon on Hepatic Glucose Production in Vitro M. Komjati, H.Vierhapper, P.Bratusch-Marrain and W.Waldh~iusl. I. Medizinische Universit~itsklinik, Wien, Austria To elucidate in vitro the transience of glucagon induced hepatic glucose generation, the effect of glucagon on hepatic glucose (HPG) and cyclic AMP production was evaluated in perfused isolated rat liver (5 mmol/1 glucose Krebs-Ringer buffer, 20% human erythrocytes) in a non-recirculating system (n= 7). After a pre-equilibration period of 60 rain, glucagon was added to the infusate in stepwise increasing concentrations at 0, 60 and 100 min to give a final concentration of 2.5 x 10 -11,10 -9 and 5 x 10 -8 mol/1, respectively. Adding glucagon to the infusate (2.5 x 10-11 mol/1) caused cyclic AMP production [basal,. 12.2 + 2.5 pmol/(min x 100 g body weight); mean + SEM] to rise rapidly in hepatic effluent, and to plateau at 23.5 _+ 1.5, whereas HGP [basal, 3.7 _+ 1.6 l.tmol/(min x 100 g body weight)] increased only transiently to a maximum of 15.3 __ 3.1. The enhanced cyclic AMP production during the consecutive glucagon infusions was accompanied again by a transient rise in HGP during the second, but not during the last glucagon infusion. When 3-isobutyl-l-methylxanthine, a potent phosphodiesterase inhibitor, was added to the perfusion medium (500 txmol/l), cyclic AMP response to 2.5 x 10 -n mol/1 glucagon was enhanced [247 _+ 124 pmol/(min x 100 g body weight)], as was HGP (+21%, p< 0.05). Thus, the transient effect of glucagon on HGP as observed in vivo may not depend on glucagon induced insulin secretion and hyerglycaemia, but represents an evanescent action of glucagon generated cyclic AMP on glycogen breakdown.

221. Lipolytic Effect of Glucagon on Perifused Isolated Human Fat Cells L. Kor/myi. Medical Department III, Amtssygehus, Aarhus, Den- mark, and First Department of Medicine, Semmelweis University, Budapest, Hungary Although glucagon has no or negligable lipolytic effect on incubation with human fat cells, recent data in vivo suggest its importance in peripheral lipolysis. To prevent the rapid degradation of glucagon observed during incubation isolated human fat cells (2 ml) were perifused with gassed Hanks-Hepes buffer at 37 ~ at a rate of 60 ml/h. The glycerol concentration of collected perifusate fractions (2 ml) was measured. The degradation was investigated by TCA precipitation of 125I-glucagon. Basal glycerol production was significantly stimulated by 10 -I~ mol/l of glucagon and it was dose-related up to 10 -6 mol/l of glucose (from 3.7 + 2.5 to 6.3 + 3.1 and 8.9 + 3.2 nmol. 109cells- 1. rain-l). The glycerol production was inversely correlated with degradation of glucagon (r= 0.93) and the cells from older patients had higher degradating activities. Neither trasylol nor bacitracin prevented the degradation while the latter had an anti-lipolytic effect. In conclusion, we suggest: (a) glucagon has a lipolytie effect on human fat ceils but high degradation of the hormone makes it difficult to investigate, (b) glucagon could mobilize huge amounts of fuel from periferi- al fat cells in spite of the low sensitivity and high degradating capacity of the fat tissue.

222. Diabetic Growth Peptides of Very Low Molecular Weight for Hu- man Vascular Cells in Serum from Type2 (Non-Insulin-Dependent) Diabetic Patients T. Koschinsky, C. Btinting, R. Rtitter and F.A. Giles. Diabetes Re- search Institute, Diisseldorf, FRG Dialyzable serum growth factors for human arterial smooth muscle cells and fibroblasts occur in Type 2 diabetic patients with poor control (HbA~:10%-13%) but not with good metabolic control and are more potent than insulin, insulin-like growth factors I and II or growth hormone. The nature of these growth factors has been studied

Abstracts 173

further. These growth factors are characterized by: (1) complete recovery, (a) in the dialyzate of Spectrapor6 membrane (molecular weight cut off: 1000 daltons) and (b) in the filtrate of Amicon UM05 (500 daltons); (2) linear dependence of growth stimulation over a 10 x concentration range but only in combination with dialyzed serum proteins (> 12,000 daltons); (3) reduction of the growth stimulating activity to control levels by pretreatment with two different pro- teases: serva pronaseE (streptomyces griseus; EC3.4.24.4), calbio- chem protease (Subtilisin carlsberg; EC3.4.4.16); (4) separation of two fractions with different growth stimulating activity, enriched 6000 x compared with the original diabetic serum, after serum dialysis as in (1 a) and subsequent chromatography on Biogel P2. In conclusion, serum from poorly controlled Type 2 diabetic patients contains growth peptides of very low molecular weight for human vascular cells, which might easily penetrate the arterial wall and could contribute to the increased angiopathic risk in diabetes.

223. Normalization of Gastric Inhibitory Polypeptide (GIP) Secretion After a Meal During the First Year of Insulin Treatment in Type 1 (Insu- lin-Dependent) Diabetes T. Krarup, S.Madsbad and A.J.Moody. Hvidore Hospital, Klamen- borg and Novo Research Institute, Bagsv~erd, Denmark We have described previously diminished secretion of GIP after a meal in the first 3 months after the diagnosis of Type I diabetes. The present study describes the relation between plasma-GIP and B cell function in the first 18 months after starting insulin treatment. Fifteen Type l diabetic patients (age < 40 years) were examined 7 and 14 days and 3, 6, 9, 12, and 18 months after the onset of the disease. After an overnight fast, blood glucose, plasma C-peptide, and GIP were measured before and over 3 h after a standard meal. Twenty-one normal subjects were studied for comparison. The integrated GIP response was significantly lower than that of the normal subjects (63 _+ 4pmol . l - l .m in 1, mean_+ SEM) after 7 and 14days and 3 months (46_+ 4, 48_+ 3, 45 _+ 5 pmol. 1-1. min-1). Thereafter it increased to near normal levels after 6, 9, 12, and 18 months (55 _+ 5, 57 _+ 5, 60 _+ 5, 58 -+ 4 pmol. 1 1. min-1). GIP was neither correlated with the endogenous insulin secretion, which increased in the first 3 months after onset and thereafter decreased, nor to blood glucose. In conclusion, newly diagnosed Type 1 diabetic subjects have diminished GIP secretion after a meal in the first 3 months after diagnosis. Thereafter, the response becomes normal. The increase in GIP is accompanied by a decrease in B cell function. No mutual relationship between GIP and insulin secretion seems to exist in newly diagnosed Type 1 diabetes.

224. Production of C-Terminal Specific Glucagon-Antibodies After Synthesis of a Defined Antigen U. Krause, J. Fessler, D. Saunders and J. Beyer. Department of Endo- crinology, University of Mainz and Deutsches Wollforschungsinstitut, Aachen, FRG Heterogeneity of glucagon immunoreactive peptides in plasma often impedes the interpretation of measured values. Peptides of different sources with opposite regulation of their secretion are measured. The use of antisera produced with chemically defined antigens may overcome some of these problems. We therefore tried to raise C-terminal specific antisera to glucagon using a new antigen exposing only C- terminal fragments of glucagon. The 19-29-glucagon fragment was prepared by digestion of glucagon with TPCK-trypsin. N-terminal 19- alanine of the fragment was reacted with cysteic acid. HO3S-Cys-19-29-glucagon reacts with succinylated albumin to give: BSA (Succinyl)• Two rabbits were immunized every 3 or 4 weeks with an equivalent of 1 mg glucagon emulsified with complete Freund's adjuvant. Both rabbits produced antibodies. After the fourth injection the titres rose up to 1:3 200. Our results provide evidence for the usefulness of the new antigen in the production of C-terminal specific glucagon antibodies. The specific coupling of the fragment prevents the formation of antibodies reacting with the mid-regional portion of the glucagon molecule.

225. Basal Insulin Infusion with Implanted Pumps in Type I (Insulin- Dependent) Diabetes: First Results on Neuropathy and Retinopathy H. Kritz, C. Najemnik, and K. Irsigler. Metabolic Unit and L. Boltz- mann Research Institute for Metabolic Diseases and Nutrition, City Hospital Vienna-Lainz, Vienna, Austria In 31 unstable Type 1 diabetic patients with poor metabolic control under intensified conventional therapy, a basal rate insulin infusion pump (Infusaid) was implanted using the IP catheter route. Where necessary, additional SC injections were administered. Long-term performance (for 4,681 patient days), complications, feasibility, influ-

ence on metabolic control and on neuropathy and retinopathy were evaluated 6 months after implantation and the following results were obtained: Metabolic data - mean _+ SEM blood glucose improved significantly from 11.6 _+ 1.5 to 7.4 _+ 1.5 mmol/1, glycosylated haemoglobin A1 from 11.5% to 8.5% (intensified conventional therapy) (p < 0.005). Neurological data - proportion of subjects showing improvement in motor nerve conduction velocity (MNCV) 77%, sensory nerve conduction velocity 64%, autonomic dysfunction 42%; proportions showing deterioration: MNCV 6%, sensory nerve conduction 6%, autonomic dysfunction 3%. Ophthalmological data - improvement on ophthalmoscopy in 26%, angiography 26%, vitreous fluorophotometry 38%. Deterioration on ophthalmoscopy in 22%, angiography 22%, vitreous fluorophotometry 29%. Complications: three pumps had to he exchanged under local anaesthesia. Two severe hypoglycaemic episodes, but no ketotic derangement occurred. In conclusion, good metabolic control was achieved in all patients, neuropathy was improved. Functional improvement of retinopathy, no severe deterioration. Basal rate insulin infusion is a feasible way to improve metabolic control and is well accepted by patients who value improved life style and absence of hypoglycaemia.

226. Temporal Relationship Between Free Insulin and Glucose Utiliza- tion in Insulin Infusion Studies V. Kruse, Aa. Volund, W. Waldh~iusl 1, P. Bratusch-Marrain 1, I. Jensen and R Nowotny 1. Novo Research Institute, Bagsvaerd, Denmark and 11. Medizinische Universitfitsklinik, Wien, Austria The relationship between free IRI and glucose utilization was studied in five normal, five insulin-dependent diabetic subjects with low and five with high levels of insulin-binding antibodies during and up to 4 h after a square wave infusion of semi-synthetic human insulin. In- sulin was infused for 1 h; dose 4 U/70 kg and 20 U/70 kg on separate days. Euglycaemia was maintained by glucose infusion. Significantly less glucose was needed by both groups of diabetic subjects than by the control group at 4U/70kg (p<0.01) and at 20U/70kg (p< 0.001). By means of a graphical analysis of the accumulated amount of infused glucose and the corresponding area under the curve for free IRI, the infused glucose was partitioned into an early need and a late need for glucose. The early need reflects the amount of glucose infused while free IRI is elevated, whereas the late need occurs after free IRI has returned to basal level. The early as well as the late need for glucose were reduced in both groups of diabetic subjects compared with the control group (p= 0.05 to p < 0.001) indicating insulin resistance in the diabetic subjects. The reported increase in apparent insulin sensitivity during prolonged insulin infusion (2-8 h) may be explained by the late occurring need for glucose.

227. Influence of Intensified Pre- and Post-conceptional Metabolic Control on Early Fetal Growth Delay in Diabetic Pregnancy C. Ktihl, B. Moller-Jensen, J. F. Pedersen, and L. Molsted-Pedersen. Hvidore Hospital, Klamenborg, Ultrasound Laboratory, Glostrup County Hospital, and Diabetes Centre, Department of Obstetrics and Gynaecology Y, Rigshospitalet, Copenhagen, Denmark In spite of intensive metabolic control during diabetic pregnancy, congenital malformations still occur three to four times more frequently in infants of diabetic mothers than in infants of non-diabetic mothers. We found recently that fetal growth, as estimated by ultrasound, is often considerably delayed in early diabetic pregnancy and that these 'delayed' fetuses have a significantly increased risk of developing malformations. The aim of the present study was to investigate whether strict metabolic control, before and after conception, might normalize early fetal growth in diabetic pregnancy. Until now, seven diabetic women (White classes B-F) have conceived after a preceding period of strict metabolic control obtained by insulin pump treatment (CSII) for at least 2 months. Seven conventionally treated diabetic women (White classes B-D) served as controls. At the conception, the mean_+SEM HbA~c level of the CSII-group was 7.2_+0.9%, and 6-8 weeks thereafter it was 6.9 + 0.9% and 6.5 _+ 0.9% in the CSII- and control groups, respectively. Fetal growth was delayed in three of seven fetuses in the CSII-group but only in one of seven fetuses in the control group. These preliminary results indicate that strict metabolic control around the time of conception will not necessarily normalize the early fetal growth in diabetic pregnancy.

228. Pharmacological and Biochemical Evaluation of 2-(fl-Methyl- cinnamyhydrazono)-Propionate (MCHP), A New Compound with Hypoglycaemic Activity H. F. Kfihnle, I. V. Deaciuc and F. H. Schmidt. Boehringer Mannheim GmbH, Medizinische Forschung, Mannheim, FRG

174 Abstracts

Given IP or per os (5-10 mg/kg) to fasted guinea pigs, MCHP produced a statistically significant decrease in glycaemia and ketonae- mia. Rats were found to be five times less sensitive to MCHR In the perfused guinea-pig liver, MCHP (1-10 ~xmol/1) produced a strong inhibition of gluconeogenesis from lactate, pyruvate, alanine and other precursors. Ketogenesis from oleate but not from octanoate was also strongly inhibited. Likewise, in isolated guinea-pig liver mitochondria ketogenesis from palmitoyl-camitine and, to a lesser extent, from palmitoyl-CoA+carnitine, but not from octanoate, was depressed by MCHR The same effects were seen on the oxygen uptake in the presence of the above mentioned substrates. MCHP had no effects on the oxygen uptake by isolated skeletal or heart muscle mitochondria in the presence of long chain fatty acid derivatives. Prelimi- nary results suggest that MCHP inhibits the transfer of long-chain fatty acids across the mitochondrial inner membrane. The results of toxicity and mutagenicity studies did not show any effects which would exclude the use of the compound in human experiments.

229. Prevalence of Coronary Heart Disease Among Maturity Onset Diabetics and Non-Diabetics in East and West Finland. An Interim Re- port M. Laakso, K. Py6rNfi, A. Aro, T. R6nnemaa, P. Puukaka and V. Kallio. Department of Medicine, University Central Hospital of Kuopio, Kuopio, and Rehabilitation Research Centre of the Social Insurance Institution, Turku, Finland Coronary heart disease (CHD) is more frequent in East Finland than in West Finland. This is in part explained by higher levels of CHD risk factors in the population living in East Finland. In January 1982 a collaborative study was started aiming to compare the prevalence of CHD and the levels of CHD risk factors among maturity onset diabetic patients and non-diabetic control subjects living in East and West Finland. About 600diabetic and 600non-diabetic subjects, males and females aged 45-64 years, will be examined in both areas. Preliminary results with respect to the prevalence of symptomatic CHD in diabetic and in control subjects living in East and West Fin- land are as follows: previous myocardial infarction, male diabetics: East 29%, West 8%; female diabetics: East 14%, West 6%; male con- trois: East 6%, West 4%; female controls: East 3%, West 1%; angina pectoris, male diabetics: East 46%, West 10%; female diabetics: East 46%, West 18%; male control subjects: East 20%, West 9%; female control subjects: East 20%, West 8%. These results show that the excess of CHD prevalence in East Finland becomes evident in diabetic and non-diabetic subjects of both sexes, with an additional excess in male diabetic patients.

230. A Method for the Mass Isolation of Islets from the Canine Pan- creas P. Lacy, E.Finke, H.Gebel and D.Scharp. Washington University School of Medicine, St. Louis, Missouri, USA The canine pancreas contains wide bands of collagen which interfere with islet isolation by the collagenase technique, since it entraps pancreatic tissue during digestion. We have found that partially-digested collagen can be retained by using strips of Velcro in the digestion tubes. The spiny portion of Velcro holds the gelatinous collagen and permits separation of intact islets from the parenchyma. The major steps in the procedure are as follows: the canine pancreas is continuously infused through the pancreatic ducts with collagenase (1 mg/ ml) for 30 rain at 37 ~ large pieces (2-3 cm) of pancreas are placed on Velcro strips, incubated with collagenase (3 mg/ml) for I rain, centrifuged, the pellet is saved and the procedure repeated six times; islets are isolated from the digest by centrifugation on a Ficoll gradient. The yield is approximately 40-60000 islets per single dog pancreas. Previously, we had shown that destruction of passenger lymphoid cells in donor rat or mouse islets prevented rejection of islets. Using these procedures, initial studies indicate prolongation of islet allograft survival following incubation of canine islet aggregates in 95% O2 and fluorescent antibody studies with a monoclonal Ia antibody have shown that canine islet cells are Ia negative similar to the mouse.

231. Humoral and Cellular Immunological Events in Type 1 (Insulin- Dependent) Diabetes with Recent Onset: Immune Modulatory Effects of Inosiplex T. Lander, E. Standl, H. Mehnert, B. Bertrams, B. Greulich, H. Kolb, F. A. Gries, E. R Rieber, G. Riethmiiller and E. D. Albert. Institute for Immunology and Diabetes Research Unit, Diisseldorf and Munich, FRG Immune modulatory effects of Inosiplex have been shown to be bene- ficial in an animal model of insulin-dependent diabetes as represen-

ted by the streptozotocin-induced autoimmune diabetes in mice. In this study 20 Type 1 diabetic patients with recent onset (duration of symptoms < 4 weeks, aged from 13 to 34 years) have been followed over 5-10 months. Measurements of islet cell antibodies (ICA) and distinct lymphocyte subpopulations by means of monolonal antibody technique (OKT 4/8, T 811) were performed every 4 weeks. Inosiplex 50 mg/kg per day (n = 11) or 100 mg/kg per day (n = 9) was administered orally for 2 months after clinical manifestation. Thirteen diabetic patients were persistently ICA-positive and seven were negative. No correlation was observed between the occurrence of ICA and quantities of lymphocyte subsets. Whereas a low dose of Inosiplex seemed to modulate neither absolute numbers nor ratios of helper or supressor cells, patients given 100 mg/kg per day showed a significant depression of helper cells (suppressor 0.67+0.05 versus helper 0.50 + 0.04, p < 0.05). A significant decrease of suppressor cells in patients with recent onset of diabetes as described by others could not be confirmed by our data.

232. Hazards of Asymptomatic Hyperglycaemia: A 17-year follow-up of the Study of Men Born in 1913 B. Larsson, L.-O.Ohlson, H. Eriksson, K. Sv~irdsudd and L.Welin. Department of Medicine I, Sahlgrenska Hospital, G6teborg, Sweden This report examines the relationship between fasting blood glucose baseline levels and subsequent 17-year coronary heart disease (CHD) and CVL morbidity, and total mortality. For a prospective study of risk factors for CHD, 975 men aged 50 years were recruited from a general Swedish urban population in 1963. Of 855 men (85%) who agreed to participate, those who showed no signs of CHD or did not have known diabetes mellitus were selected and observed for 17 years. One hundred and eight men developed CHD (hard criteria), 42 CVL and 147 died. When quintiles or deciles of fasting blood glucose distribution were considered, no trend of 17 year CHD morbidity rates was apparent. However for individuals with fasting blood glucose levels above the 95th centile (5.8 mmol/1), the risk for CHD was doubled. A similar non-linear trend was also found for both the association between baseline fasting blood glucose and CVL morbidity and total mortality.

233. Pancreatic Polypeptide Secretion in Diabetic Patients with Idi- opathic Haemochromatosis V. Lassmann 1, P. Passa, E Vague% B. Vialettes ~ and J. Canivet. Depart- ments of Diabetology, Marseille 1 and Paris, France It has long been stated that glucose intolerance in patients with idiopathic haemochromatosis (IH) is due to islet dysfunction resulting from the pancreatic iron overload. But some data on glucagon secretion in IH are at variance with this theory. In order to investigate endocrine pancreatic function resulting from iron overload, plasma pancreatic polypeptide (PP) secretion induced by a protein-rich meal was studied in 30 Type 1 (insulin-dependent) diabetic patients without autonomic neuropathy, ten with IH, ten with chronic pancreatitis (CP) and ten with idiopathic diabetes. Body weight, sex, duration of diabetes and control were identical in the three groups. Age was lower in idiopathic diabetic patients (30 + 3 versus 46+2 and 51 +3 years). The sera were all negative for anti-PP antibodies. Fasting plasma PP levels were significantly higher in Type 1 diabetic patients with IH and idiopathic diabetic patients (36 + 9 and 30.3 _+ 4 pmol/l) than in diabetic patients with CP (14.8_+4.7 pmol/1). Following a meal, in the first two groups, PP levels rose to a peak obtained at 30 rain (139.3 _+ 23.6 and 100.6 _+ 36 pmol/l, respectively) and lasted for 4 h. In diabetic patients with CP, no rise was observed. These findings show that PP secretion is comparable in diabetic patients with IH and in idiopathic diabetes. Considering these results with similar ones concerning glucagon secretion, we conclude that only the B cell is functionally in- jured by iron overload in haemochromatosis.

234. The Effect of Near-Normal Blood Glucose Levels upon Retinopa- thy: Two- year Follow-up T.Lauritzen, K.Frost-Larsen, H.-W.Larsen, T.Deckert and Steno Study Group, Gentofte, Denmark Thirty patients with Type 1 (insulin-dependent) diabetes and background retinopathy were randomized to either continuous SC insulin infusion (CSII) or conventional treatment (CT). Patients had a mean + SEM of 175 _+ 6 blood samples during the first year and 91 _+ 8 during the second year. Patients were seen in the out-patient clinic at monthly intervals during the first year and every third month during the second year. Fundus photography, fluorescein angiography and measurements of oscillatory potential, macular recovery time, and posterior vitreous fluorophotometry were performed at 0, 6, 12, and

Abstracts 175

24 months. The individual mean blood glucose during the first year ranged from 7.1-14.1 mmol/1 (mean 9.9) for conventional treatment and 5.4-7.8mmol/1 (mean 6.2) for CSII (p<0.01). No significant changes were seen in mean blood glucose or in haemoglobin Alc between the first and second year in either treatment groups. Seven patients (four CSII, three conventional treatment) developed proliferative retinopathy. Three occurred during the first year, four during the second year. There was no significant difference between mean blood glucose, frequency of blood glucose < 2.5 mmol/1, or duration of diabetes between those patients who developed proliferative retinopathy and those who did not. Retinal function deteriorated significantly from 0 to 12 and from 12 to 24months with conventional treatment, whereas a significant improvement was seen from 0 to 12 months with CSII with no further significant improvement during the second year. In conclusion, near-normal blood glucose levels can be maintained by CSII with less intensive assessment of control. Two years of near-normal blood glucose levels cannot prevent development of proliferative retinopathy in patients with established retinopathy.

235. Hyper-Responsiveness of Glucose Metabolism to Insulin in-the Adipocytes of Young Hyperinsulinaemic Obese Zucker Rats M. Lavau, M. Guerre-Millo and C. Guichard. U 177 INSERM, Insti- tut Biomedical des Cordeliers, Paris, France Hyperinsulinaemia and insulin resistance are characteristics of the adult obese Zucker rat. In this study we investigated the responsiveness of adipocyte glucose transport (2-deoxyglucose) and metabolism (u-a4c-glucose----~CO2, lipids and lactate) to insulin in the early phase of hyperinsulinaemia which develops in obese pups after 17 days of age. Inguinal adipocytes of 30-day-old obese and lean (Fa/fa) rats were used. Cell size was 0.186 gg lipids/cell in obese versus 0.040 in lean rats. Basal glucose transport and metabolism were x 3 higher in obese than in lean rats. Under maximal insulin stimulation, transport was x 8 in lean and x 20 in obese and metabolism was x 3 in lean and x 10 in obese rats. These increases were achieved at 30 mU/ l in lean and 100 mU/1 in obese rats, i. e. in the range of their respective insulinaemia. The dose-response curves were shifted to the right in obese rats. However, insulin was much more efficient in obese than in lean rats even at 8 mU/ l which produced an increment of total glucose metabolism of 1 pmol/cell x 2 h in obese versus 0.2 in lean rats. Soon after the emergence of hyperinsulinaemia, adipocytes of obese rats exhibit a markedly increased responsiveness to insulin. This is likely to be of major importance in the development of the obese syndrome.

236. Insulin and Glucagon Release by the Perfused Ventral and Dorsal Parts of the Rat Pancreas V. Leclercq-Meyer, J. Marchand and W.J. Malaisse. Laboratory of Experimental Medicine, University of Brussels, Brussels, Belgium The ventral and dorsal parts of the pancreas differ by their embryology, vascularization, exocrine drainage, relative richness in distinct endocrine cell types and hydrolase content. Insulin and glucagon release was studied separately in each part perfused through the coeliac (l.4 ml/min) or superior mesenteric artery (0.6 ml/min). Relative to tissue weight, the insulin content was comparable in both parts, whereas the glucagon content was five times higher in the dorsal than ventral part. When expressed as percentage of hormonal content, the basal insulin release at low glucose concentrations was not vastly different in the two parts. Such was also the case for insulin release evoked by glucagon in the presence of 7 mmol/ l glucose. However, the more marked release of insulin provoked either by arginine at three different glucose concentrations, or by glucose (8.3 retool/l) in the absence or presence of carbamylcholine was higher, relative to hormonal content, in the dorsal than ventral part. In contrast, glucagon release (relative to content) was either the same or higher in the ventral compared with the dorsal part. These results suggest that B cells in the ventral part are poorly reactive to glucose, and that this defect is not corrected by such stimuli as arginine or carbamylcholine.

237. Measurable Subclinical Hand Joint Limitation in Diabetic Pat- ients M. Letanoux ~, G.Slama ~, N.Thibult 2 and G.Tchobroutsky 1, 2. 1De_ partment of Diabetes, H6tel-Dieu, Paris and 2U21 INSERM, Ville- juif, France Cheiroarthropathy may be more frequent in diabetic patients prone to late complications. We aimed to detect a measurable joint limitation at a subclinical stage. Maximal passive joint amplitude (flexion and /or extension) was measured in the pre-eminent hand, using a goniometer, for the wrist, the third and fifth fingers. We compared 50 diabetic patients (including 32 Type l) to 118 non-diabetic subjects

similar in age, sex, hobby and professional activity repartition. Intra- subject reproducibility of a measure was within 6%. Joint mobility was not influenced by age (except for the wrist), sex, body mass index, social class or alcohol consumption. Diabetic patients had significant limitation of wrist flexion (74 _+ 10 versus 80 + 10; degrees; mean _+ SD; p < 0.001), metacarpophalangeal flexion of the fifth finger (92 + 6 versus 94 + 7; p < 0.06), fifth finger extension (43 _+ 19 versus 56 + 19; p < 0.001) and third finger extension (25 + 12 versus 33 + 14; p < 0.01). Wrist and fifth finger extensions correlated to plasma creatinine levels ( r= - 0.38, p < 0.001 ; and r = - 0.27, p < 0.06 respectively). Fifth finger extension was more limited in patients with retinopathy than without (48 + 21 versus 35 + 14; p < 0.06). Wrist extension correlated to diabetes duration. We conclude that extension of the fifth finger is the best measurable parameter for quantifying subclinical changes in joint mobility. Moreover, it correlates with the presence of retinopathy and plasma creatinine levels.

238. Nature of Resistance to Autoimmune Diabetes in Biobreeding/ Worcester Control Rats A.A.Like, E.J.Weringer, A.Holdash, P.McGill and A.A.Rossini. University of Massachusetts Medical School, Worcester, Massachu- setts, USA The Biobreeding/Worcester (BB/W) colony contains one control and nine diabetic-prone family lines in the 14-15 generation of inbreed- ing. 40-55% of susceptible rats have evidence of autoimmune diabetes. Control (W-line) animals have been without diabetes for nine generations. Koevary et al reported the adoptive transfer of diabetes to susceptible BB/W rats with diabetic spleen cells activated in-vitro with T-lymphocyte mitogen concanavalin-A. Wistar-Furth rats were resistant to adoptive transfer of diabetes. We utilized this technique to determine whether resistance to diabetes in W-line rats is expressed at the level of the lymphoid system. Spleen cells from acutely diabetic rats were cultured for 3 days with concanavalin-A. 35-50 x 106 cells were injected IV into untreated W-line rats, and W-line animals after immune-modulation by: splenectomy, three intraperitoneal injections (1.5ml) of anti-lymphocyte serum, splenectomy+anti-lymphocyte serum, 150mg/kg cyclophosphamide or partial (>50%) neonatal thymectomy. Results: diabetes was induced in 0/8 untreated, 0/10 anti-lymphocyte serum, 1/14 splenectomized, 5/24 splenectomized+anti-lymphocyte serum, 15/26 cyclophosphamide and 2/3 partially neonatal thymectomized W-line animals. Since the immunological manipulations could not be expected to modify the antigenic nature of the B ceils, we conclude that W-line resistance to diabetes resides in the lymphoid system and is overwhelmed by measures altering the cellular components of immune surveillance.

239. Effects of Aprotinin on Insulin Absorption and Subcutaneous Blood Flow in Insulin-treated Diabetic Patients B. Linde and R. Gunnarsson. Huddinge Hospital, Stockholm, Sweden The protease inhibitor aprotinin (Apro) added to a SC insulin injection in healthy subjects enhances the early phase of insulin absorption. To study whether this is coupled to an increase in SC blood flow 10 insulin-treated diabetic patients on two occasions received 10 U of 12SI-Actrapid together with 10,000 KIU of Apro or its diluent. The insulin was injected SC in the thigh, while in the opposite leg the flow indicator ~ Xe was given with the same additive. The elimination

1)5 133 rates of - I and Xe and the plasma concentrations of glucose and free insulin were followed for 4 h . With Apro plasma insulin rose more rapidly during the first 30 45 rain and the rate constant for 125I tended to be higher. However, during the late phase (2-4 h), the elimi-

1)5 nation of - I was lower with Apro (p < 0.05) but free insulin was unaltered. The elimination of 133Xe was higher following Apro (p < 0.05) but the rate of plasma glucose reduction was similar. The findings suggest that Apro enhances the early elimination of subcutaneously

1)5 injected insulin by an increase in SC blood flow. The lower - I-elimination following Apro during the late phase indicates some local degradation of SC-injected insulin which can be inhibited by Apro.

240. Glucose Increases Intraeellular pH of Pancreatic Islet Cells from ob/ob mice P. Lindstr~m and J. Sehlin. Department of Histology and Cell Biolo- gy, Ume~, Sweden The transmembrane distribution of 14C-labelled, 5,5-dimethyloxazoli- dine-2,4-dione (DMO) in islets isolated ob/ob-mice was used as a probe for mean intracellular pH. D-glucose (20 mmol/1) increased the uptake of DMO corresponding to a rise in intracellular pH by about 0.15 units. The effect of glucose was concentration-dependent with a apparent Km of about 4mmol / l and a maximal effect at about

176 Abstracts

10 mmol/1 glucose. In a bicarbonate-buffered and oxygenated medium the effect of glucose was inhibited by mannoheptulose (20 mmol/ l), suggesting that it may be associated with glucose metabolism. The effect of glucose was not affected by theophylline (2 mmol/l), but potentiated by L-5-hydroxytryptophan (4 mmol/l); it appeared rapidly (7 min), persisted for at least 30 rain, and it was obtained both with HCO3-/COz-buffered media and with Hepes-media with or without oxygenation. It has been suggested previously that reduction in pHi is an important link in the B cell stimulus-secretion coupling. The present data suggest that glucose increases mean intracellular pH of islet cells. This effect may be coupled to glucose metabolism but is probably not the result of insulin release.

241. Hormonal Responses to Insulin-Induced Hypoglycaemia After Optimized Glycaemic Control in Type I (Insulin-Dependent) Diabetes P. E. Lins, U. Adamson, M. Kollind, B. Hamberger and S. Efendi& De- partment of Medicine, Danderyd Hospital and Departments of Sur- gery and Endocrinology, Karolinska Hospital, Stockholm, Sweden Severe hypoglycaemia has been reported to occur less frequently during continuous subcutaneous insulin infusion (CSII) than during conventional therapy. In contrast, mild and particularly asymptomatic hypoglycaemia is often recorded during CSII, most probably reflecting less precipitate falls in blood glucose on such treatment. Alterna- tively the responses to hypoglycaemia might change after a period of near-normoglycaemia, making the patient less aware of hypoglycaemia. The aim of this study was to investigate whether CSII altered the hormonal component of this response. We studied eight Type 1 diabetic patients, age 29-48 years, duration 3 24 years, without signs of autonomic neuropathy. During a mean CSII period of 14weeks, HbA~c improved from 11.6 _+ 0.5% to 9.3 + 0.4%. Hypoglycaemia was induced before and at the end of this period. SC insulin administration was discontinued 24 h before the hypoglycaemia, control being maintained by an IV infusion of insulin. After an overnight fast, insulin (2.4 U/h) was given IV until steady-state glucose levels were obtained. Glucose, insulin, growth hormone, glucagon, cortisol, adrenaline, noradrenaline, pulse rate and blood pressure were measured every 15 rain. Steady-state glucose levels of 2.3 mmol/1 were obtained in both experiments, and the hormonal responses to hypoglycaemia were the same. We conclude that the hormonal responses to hypogly: caemia do not change following a period of optimized glycaemic control by CSII.

242. Skeletal Lesions and Medial Arterial Calcification of the Feet in Diabetes and in Patients with Familial Amyloidosis and Polyneuropathy F. Lithner, S.-O. Hietala and L. Steen. Department of Medicine, Uni- versity Hospital, Ume~, Sweden Skeletal osteolytic destruction (diabetic osteopathy) and medial arterial calcification in the feet of diabetic patients may be due to neuropathy. We have performed radiographical examinations of the feet of diabetic patients with (A) and without (B) gangrene, non-diabetic patients (C) and patients with familial amyloidosis (D). The patients with familial amyloidosis had severe distal neuropathy but normal oral glucose tolerance test curves. Radiographical findings were classified without knowledge of the clinical findings.

Group AGr0up B Group C Group D (n=162)(n=59) (n=30) (n=45)

Mean age (years) 67 64 65 60 Mean duration of diabetes 13 14 - - (years) Osteopathy (%) 30 0 0 9 Medial art. calcification (%) 61 51 10 42

Osteopathy was more common in diabetic patients with gangrene than in the other groups of patients. Medial calcifications were more common among diabetic patients with gangrene compared with groups C and D. There were no differences between the groups B and D, but both of these groups had more calcification than group C. In conclusion, this study suggests that diabetic osteopathy is not caused by peripheral neuropathy per se. The skeletal and skin lesions in diabetic patients are probably equivalent lesions, localized to different tissues in the feet. In accordance with the hypothesis of neuropathy as the cause of medial calcification, the patients with familial amyloidosis had medial calcification. However, this does not exclude other ae- tiological factors in diabetic patients.

243. Is High Blood Pressure a Risk Factor for Diabetic Retinopathy? P. Lombrail 1, E.Eschwege 2 and Ph. PassaL ~Service de Nutrition-En- docrinologie, Saint Louis Hospital, Paris and 2INSERM U. 21, Ville- juif, France The aim of this study was to investigate the r61e of hypertension in the prevalence of diabetic retinopathy. The 786 diabetic patients of this cross-sectional study were systematically recruited over a 1 year period. There were 323 Type 1 (insulin-dependent) diabetic and 463 Type 2 (non-insulin-dependent) diabetic patients without macropro- teinuria; their mean age was 52 + 12 years, the known duration of the disease was 10 +_ 8 years (mean • SD). Two hundred and ten were hypertensive (supine blood pressure > 160/90 mmHg on day3 in hospital, or patients already on antihypertensive treatment); 55 were Type 1 diabetic and 155 were Type 2 diabetic patients. In all patients fluorescein angiography of the retina was performed and retinopathy was considered to be present when at least two microaneurysms in the posterior pole were observed. In Type I diabetic patients the prevalence of the retinopathy was 69% in the hypertensive versus 47% in the non-hypertensive subjects (NS). In Type 2 diabetic patients, it was 39% in the hypertensive versus 25% in the non-hypertensive subjects (p < 0.01). Results were similar when known duration of diabetes was taken into account. These data differ from those of the WHO Multi- national Study but are in keeping with previous reports.

244. Role of the Kidney in the Removal of (12sI)-Glicentin from Rat Plasma J. M, L6pez-Novoa, F.J. Garrote, G. Calvo and I. Valverde. Fundaci6n Jim6nez Diaz. Independent University of Madrid, Madrid, Spain The disappearance from plasma of (125I)-glicentin (prepared by U. D. Larsen, Novo, Copenhagen) after a single IV injection was studied in nine control, nine binephrectomized (BNX) and 14 bilater- ally ureteral-ligated (BUL) rats. Seven blood samples were obtained at intervals during 60 rain after injection and total and TCA-precipitable radioactivity was measured. Values are referred to as percentages of the TCA- recipitable counts in 5-min plasma (mean +_ SEM).

~ 125 60-rain plasma, ( I)-glicentin and INa were chromatographed on Sephadex G-100 and eluates counted for radioactivity. Plasma gel-filtration revealed a peak of radioactivity in the elution volume of (125I)- glicentin, and another in that of 125I-Na; their relative proportions were similar to the respective TCA-precipitable and TCA-soluble fractions. No TCA-precipitable radioactivity was recovered in urine of control rats. Disappearance of (12sI)-glicentin from plasma was slower in BNX and BUL than in controls. 10-rain values were 70.2 + 1.7%, 67.1 + 1.0% and 48.5 + 1.8%, falling at 60 min to 26.2 + 1.0%, 19.5 + 1.1% and 9.6 + 0.7%, respectively. On a semi-logarithmic plot, control rats showed a straightline from 20-60rain (t~/~=27.5+ 2.0 rain), whereas BNX and BUL failed to reach a straight line. These results suggest that the kidney plays a major role in the removal of glicentin from the circulation, and glomerular filtration and renal catabolism seem to be involved, while peritubular uptake could be of minor importance.

245. Ca 2+- and Phospholipid-Dependent Protein Kinase in B Cells J. M. Lord and S.J.H. Ashcroft. Nuffield Department of Clinical Bio- chemistry, John Radcliffe Hospital, Oxford, UK Ca 2+ and phospholipid-dependent protein kinase (Ca-PL-PrK) may play a role in stimulus-secretion coupling. We have investigated the presence and properties of Ca-PL-PrK in collagenase-isolated rat islets of Langerhans and in a cloned hamster B-cell-line. Islets or B cells were sonicated and centrifuged at 20,000 g for 20 min. The supernatant was used directly for the study of endogenous protein phosphorylation. Purification of Ca-PL-PrK was achieved by DEAE-cellulose chromatography of the supernatant. Ca-PL-PrK was assayed by measuring the incorporation of radioactivity from {~/YP}ATP into protein in the presence of calcium, phosphatidylserine and diacylglycerol. Histone H1 was used as an exogenous substrate for Ca-PL-PrK in supernatants and column eluates. Endogenous proteins phosphorylated in a calcium and phosphalipid-dependent manner (in the absence of added histone) were resolved by SDS-gel electrophoresis and autoradiography. Extracts of both islets and B cells were shown to contain Ca-PL-PrK activity which was stable for several days at 4~ Phos- pholipid-dependent phosphorylation of histone H1 was maximal at 10 Ixmol/1 calcium. The major endogenous B cell substrates for B cell Ca-PL-PrK had mol. wts. of 11, 14.5, 19, 31 and 40 kilodaltons. It is concluded that B cells contain Ca-PL-PrK and endogenous substrates for this kinase, consistent with a role for Ca-PL-PrK in insulin secretion.

Abstracts 177

246. Evidence for Adenosine Receptors on Pancreatic A Cells M. M. Loubati~res-Mariani, J. Chapal and M. Roye. Faculty of Mede- cine, Laboratoire de Pharmacologie et de Pharmacodynamie, ERA 786 du CNRS, Montpellier, France Previous results from this laboratory gave evidence for the presence of purinergic receptors for ATP (P2 purinoceptors according to Burn- stock's classification) on B cells. The aim of the present work was to investigate whether the glucagon secretory cells had also purinergic receptors. The experiments were performed on isolated rat pancreas perfused with a medium containing 2.8 mmol/1 glucose. Adenosine and ATP (1.65-165 ~,tmol/1) induced an immediate and transient glucagon secretion in a peak form. The effect was dose-dependent. 2- chloroadenosine (1.65 ~tmol/1), which is an adenosine analogue resistant to tissue uptake and not a substrate for adenosine deaminase, was more potent than adenosine in stimulating glucagon secretion. In contrast, a,fl-methylene ATP, a phosphate-modified ATP analogue, resistant to hydrolysis to AMP and adenosine, was not effective. An antagonist of adenosine receptors, theophylline (50 umol/I) suppressed the effect of adenosine (1.65 gmol/1), ATP (1.65 umol/1) and 2 chloroadenosine (1.65 ]xmol/1). These results support the hypothesis of a purinergic receptor for adenosine (or Pl purinoceptor) on the glucagon secretory cells.

247. The Confidential United Kingdom Enquiry into the Outcome of Babies of Diabetic Mothers (1979-1980) C. Lowy, R. W. Beard* and J. U. Goldschmidt. Unit of Endocrinology and Diabetes, St. Thomas' Hospital, and *St. Mary's Hospital, Lon- don, UK 1034pregnancies, 773established diabetic patients (EDM) and 261 diabetic subjects diagnosed in pregnancy (GDM) were reported from 186 hospitals. Half the EDMs had diabetes in excess of 9 years and 86% were insulin-dependent. 43% of the EDMs and 23% of GDMs were delivered by elective Caesarean section, tSeterm delivery ( < 36 weeks) was reported in 10% of the EDMs and 22% of GDMs. The perinatal mortality was 11.5 and 55.6 per 1000 for GDMs and EDMs, respectively. The EDM perinatal mortality was related to the third trimester blood glucose, no measurement 7/20 (35%), > 6 mmol/1 19/272 (7%), < 6 mmol/l 8/283 (2.8%). The EDM malformation rate was 7.1% and was increased in the 441 women with no reported blood glucose in the first trimester. The malformation types reported for the UK population were compared with those of the enquiry; cardiac: 15 observed 0.2 expected, anomalies of the spine: 8 to 0.09 expected, hypoplasia of the lung: 5 to 0.09 expected, reduction deformities of the lower limbs: 4 to 0.09 expected. This first prospective national study probably captured half of the EDMs delivered in 1979-1980. Congenital malformations and perinatal mortality were associated with metabolic control in the first and third trimester, respectively.

248. Plasma and Platelet fl-Thromboglobulin and Platelet Thrombox- ane B2 Synthesis in Insulin-Dependent Diabetics: Influence of Glycae- mic Control J. Lubetzki, P.J. Guillausseau, E. Dupuy, J. Maclouf, C. Mear and J. R Caen. Departments of Diabetology and Angio-Haematology, La- riboisiere Hospital, Paris, France In 12 recent onset insulin-dependent diabetic patients and 12 sex- and age-matched control subjects, plasma/?-thromboglobulin r total flTG platelet content (triton), flTG platelet release induced by thrombin (0.025, 0.1, 0.25 U/ml), calcium-ionophore A 23187 (0.0125, 0.025, 0.05 gmol/I) and thrombin-induced thromboxane B2 synthesis were studied. The diabetic patients were studied before and after 15 days of intensive conventional therapy as inpatients. Plasma flTG was enhanced in the diabetic patients (39.6+4.1 versus 28.5-+2.3 ng/ml, p<0.025) without any change after control (45.8-+5.8 ng/ml). Total flTG platelet content was the same in diabetic and control subjects (66.5 _+ 3.8 versus 61 + 2.3 lag/109 platelets) and unchanged after control (64.8 -+ 3.8). Platelet flTG release was slightly increased in the diabetic patients after low thrombin concentration (10.2 + 2 versus 5.5 _+ 1 gg/109 platelets), without any difference with the two highest thrombin concentrations and after glycaemic improvement. With the highest ionophore concentration, an increased flTG release was observed in diabetics (24.8_+1.8 versus 15.4_+1.7.ag/109 platelets, p< 0.025) with improvement after control (18.1 _+ 1 ; p < 0.025). Similar normalization was noted with the lowest ionophore concentration. As for flTG release, an enhanced thromboxane B2 synthesis was observed in the diabetic patients with the only low thrombin concentration (7.2_+1.8 versus 1.6_+0.2ng/109 platelets, p<0.05) and unchanged after control (9.6 _+ 3.7).

249. Plasma HDL Cholesterol and Liver Lipid and Protein Concentra- tions in Type 2 (Non-Insulin-Dependent) Diabetes Mellitns P.V.Luoma, M.J.Savolainen, E.A.Sotaniemi and R.O.Pelkonen. Clinical Research Unit, Departments of Internal Medicine, Medical Biochemistry and Pharmacology, University of Oulu, Oulu, Finland Plasma HDL cholesterol in diabetes may vary from low to high. Earli- er, we observed a positive relationship of plasma HDL cholesterol level to liver phospholipid and protein concentration. To clarify whether this is true also in diabetes, we investigated 12 subjects with a diagnostic liver biopsy who had Type 2 diabetes. Low liver phospholipid concentration was associated with a low plasma HDL cholesterol level and low HDL cholesterol/total cholesterol ratio, and with reduced hepatic microsomal enzyme activity that was assessed by liver cytochrome P-450. The HDL cholesterol/total cholesterol ratio was directly proportional to hepatic phospholipids and cytochrome P-450. The results suggest that in diabetes also a change in hepatic microsomal function may influence the plasma cholesterol distribution profile. Variation in plasma HDL cholesterol level in diabetes may reflect variation in liver phospholipid concentration.

250. Glycosylated Fibrinogen in Normal and Diabetic Subjects A. Liitjens, E.A. van der Veen and J. van der Meer. Laboratory of Clinical Chemistry, Andreas Ziekenhnis and Department of Internal Medicine, Free University Hospital, Amsterdam, The Netherlands Glucose is non-enzymatically bound to various proteins, Altered structure of the proteins may influence their function. Few data exist on fibrinogen glycosylation in diabetes. We studied fibrinogen glycosylation in badly controlled diabetic patients (n = 8, mean glucose at the time of sampling 17.4mmol/1; mean HbA1 14.3%). Fibrinogen was isolated from citrated plasma by serial glycine precipitation. A fibrin clot was formed by the addition of thrombin, and this clot was extensively washed in saline. Using this method < 0.1% of other plasma proteins was found in the clot. Subsequently the clot was hydrolysed by heating with oxalic acid; the amount of 5-hydroxymethylfurfural formed was estimated by the 2-thiobarbituricacid method. In eight normal subjects (mean glucose level 4.0 mmol/1, mean HbA1 6.2%) 5.0+ 0.2 mol glucose per tool fibrin was present. In the eight diabetic subjects significantly more glucose was bound to the fibrin molecule: 7.0 + 0.4 tool per mol. As no lysine is present in fibrinopep- tides A and B, we may extrapolate these data to fibrinogen. The effects of the increased glycosylation on fibrinogen function and metabolism are presently under investigation.

251. Disaturated Phosphatidylcholine Release and Lung Deflation Sta- bility of Newborn Rabbits from Control and Chemically Diabetic Preg- nancies R. J. MacFadyen. Department of Physiology and Pharmacology, Uni- versity of Strathclyde, Glasgow, Scotland The influence of maternal chemical diabetes on neonatal lung function was examined by measurement of pressure-volume lung deflation curves and phosphatidylcholine (PC) release into lung lavage in premature newborns (day 28.5) of control and chemically diabetic rabbits. Rabbits were treated with saline (control) or alloxan (65 rag/ kg, IV) on day 3 of gestation and chemical diabetes defined as fasting eugylcaemia, impaired glucose tolerance and non-fasting hyperglycaemia in relation to control. Newborns from both groups showed increases in lung wash total PC content which were significantly greater in newborns of chemically diabetic pregnancies at 8 h (p= 0.037). Lung wash disaturated PC content increased with time after delivery in newborns of both groups to similar levels (control 8 h: 80_+29 versus chemical diabetes: 77.9 + 21 gg phosphorus/g). When expressed as a percentage of total lung disaturated PC content, lung wash disaturated PC was significantly less than control at 2, 4 and 10 h and the relative disaturation of lung wash PC was significantly reduced 2, 6, 8 and 10 h after delivery in chemical diabetic pregnancies. Newborn rabbits from chemically diabetic pregnancies showed significantly reduced lung deflation stability during the neonatal period compared with controls.

252. Effect of Two Periods of Intensified Insulin Treatment on B cell Function in Type I (Insulin-Dependent) Diabetic Patients: A 1.5-year Prospective Study S. Madsbad and T. K~arnp. Hvidore Hospital, Klampenborg, Den- mark At time of diagnosis, 15 consecutive Type I diabetic patients were allocated to conventional treatment (group A, n=9, mean blood glucose 11.7 _+ 0.5 mmol/1) or treatment with multiple insulin injections

178 Abstracts

for 10 days after diagnosis (group B, n = 6, mean blood glucose 6.3 + 0.7mmol/1) and for 7days after 15months (mean blood glucose 6.5 _+0.5 mmol/1). For the remaining time group B was treated conventionally as group A. Islet B cell function was calculated by C-peptide measurements on days 7 and 14 and every third month using a test meal. After 14 days, B cell function was maximal in group B and 61% greater than in group A (p < 0.05), where maximal B cell function was seen after 3 months. No difference in B cell function was found between the two groups after 3 months. Subsequently B cell function declined in both groups until after 15 months, when strict glycaemic control induced a B cell improvement (mean 33%, n = 6) in five of the six patients in group B. After 18 months B cell function in group B had again declined in all six patients and was similar to that of group A. Conclusion: short periods of strict glycaemic control induce functional improvement in B cell function but do not influence long-term outcome of B cell function.

253. Cold Test Effects on Clotting Changes in Type I (Insulin-Depen- dent) Diabetes M. Maiello, U. Armani, D. Boeri, R. Bonadonna, A. Piana and A. Sac- carello. Scientific Institute of Internal Medicine, University of Genoa, Italy Many studies have reported platelet hyperactivity in diabetes, but nearly all were performed in vitro. The aim of this study was to test such platelet behaviour after an 'in vivo' pro-aggregating stimulus. Af- ter an overnight fast, two blood samples were collected in eight Type 1 diabetic and eight normal subjects, before and 15 rain after cold pressure test fl-thromboglobulin (fl-TG), fibrinopeptideA (FPA) and maximal width of ADP-induced-platelet aggregation were measured. Before the cold test diabetic and normal subjects showed significant differences both in fl-TG (2 p < 0.001) and maximal width (2 p < 0.025) levels; after the test significant differences were found in/3-TG (2 p < 0.001), FPA (2p < 0.025) and maximal width (2p < 0.02) levels. Dur- ing the test, normal subjects showed a significant decrease of fl-TG (2 p < 0.05) and FPA (2 p < 0.01); a higher and significant decrease of /3-TG (2 p < 0.005) was found in diabetic patients who showed also, unlike the normal subjects, a significant increase in FPA levels (2p < 0.05). Opposite FPA behaviour in normal and diabetic subjects suggests that cold test induced stimulation, inefficient in normal subjects, activates clotting mechanisms in diabetes. Such an activation could be related to the greater decrease of fl-TG shown by diabetic patients through the hypotized inhibitory action of/3-TG on endothelial prostacyclin synthesis.

254. Biosynthesis of Enteroglucagon in the Gut V. Maier, A. Greischel, A. Schnabel and E.F. Pfeiffer. Department of Internal Medicine I, University of Ulna, FRG A family of glucagonoids reacting with antibodies raised against the N-terminal part of the glucagon molecule has been found in the gut of many animal species, including man. The chemical structure and biological significance remain to be established. In addition enteroglucagon (GLI) seems to be synthezised and released from other organs. A completely isolated segment (approximately 15 cm) of the rat jeju- num was perfused via the mesenteric artery with Krebs-Ringer buffer, plus human albumin 1%. The perfusate was collected via portal vein. In this system, after infusion of sucrose into the lumen GLI is released in a biphasic pattern. To establish whether or not GLI is synthezised in the gut, biosynthesis of GLI was examined by incorporation of tritiated tryptophane in a recirculating experiment over 2 h. GLI was purified by affinity chromatography, using antibodies against glucagon coupled to Sepharose-4-B. Afterwards GLI was separated into two components.of molecular weights 9 000 and 3 500 Daltons by gel filtration. In addition, isoelectric focussing resulted in the highest specific radioactivity in the material of 9~176176 Thus in conclusion, (1) Biosynthesis of GLI occurs in the isolated organ, and (2) heterogene- ous forms of GLI are released.

255. Metabolic and Secretory Interaction of L-Asparagine and L-Leu- cine in Pancreatic Islets F. Malaisse-Lagae, A. Sener, M. Welsh, C. HellerstrOm and W.J. Ma- laisse. Laboratory of Experimental Medicine, University of Brussels, Brussels, Belgium and Department of Medical Cell Biology, Universi- ty of Uppsala, Uppsala, Sweden L-asparagine augments insulin secretion provoked by 2-ketoisocaproate or L-leucine, but not that evoked by D-glucose. The metabolic response of rat islets to L-asparagine and L-leucine was investigated. L-asparagine failed to modify the sparing action of L-leucine on

14 14CO2 output from islets prelabelled with [U- C] palmitate and its en-

hancing effect on 14CO2 production by islets prelabelled with L-[U- 14C]-glutamine. L-asparagine augmented the oxidation of L-[1-a4c] or L-[U-14C] leucine, but conversely L-leucine inhibited the oxidation of L-[UJ4C] asparagine. Studies performed with L-[U-14C] aspartate, in the presence of unlabelled L-asparagine, indicated that the latter inhibition was due to impaired deamidation of L-asparagine. In islets first cultured for 18-42 h in Parker's medium, the increment in 02 uptake evoked by the combination of L-asparagine and L-leucine was not significantly higher than that evoked by L-leucine alone. L-leucine augmented the malate/pyruvate production ratio, which was further enhanced by L-asparagine. Thus the augmentation by L-asparagine of L-leucine-stimulated insulin release may be due as much to an intracellular redistribution of reducing equivalents as to an increase in their generation rate. The present study emphasizes, therefore, the essential role of the cytosolic redox state as a coupling factor in the process of nutrient-induced insulin release.

256. Diabetes Education: Effects of a Systematic Course on Patients' Knowledge, Behaviour and Control A. Maldonato, V. Cascella, F. Marani, A. Cama, C. Blasi, P. L. Maestri- pieri, C. Gnessi and F. Barbetti. 1st Department of Endocrinology, La Sapienza University, Rome, Italy To evaluate the long-term effects of a systematic 18 h course about diabetes, we called in September 1982 all the 215 patients who started attending our clinic regularly during 1981, 62 of whom followed the course (C) and 153 who did not (NOC). Our call was answered by 42 C (25 Type 1 (insulin-dependent), 17 Type 2 (non insulin-dependent)) and 57 NOC (11 Type 1 and 46 Type2 patients). A list of 25 multiple-choice questions was presented to them to evaluate their knowledge (11 questions), behaviour (nine questions) and control (five questions, two about hypoglycaemia). The percentage of haemoglobin A1 was also measured to evaluate control. Among the Type 1 diabetic patients, knowledge and behaviour obtained significantly higher scores (p< 0.001 and 0.005), whereas control scored significantly worse (p < 0.05) in the C than in the NOC patients. Among the Type 2 patients, differences in scores went in the same direction as for the Type I diabetic patients, but no statistical significance was reached. Haemoglobin A1 was similar for C and NOC patients irrespective of treatment (Type I / C 10.5_+ 0.48%; Type 1/NOC 10.4_+ 0.88% ; Type 2/C 9.0 _+ 0.64%; Type 2 /NOC 8.78 _+ 0.24%). In conclusion, our course appeared to improve patients' knowledge and behaviour showing the influence of education on motivation. Surprisingly no improvement in control could be shown.

257. High Density Lipoprotein Cholesterol Levels and Basal B cell Se- cretion in Type 2 (Non-Insulin-Dependent) Diabetes V. Manicardi, E. Bonora and C. Coscelli. Central Medical Clinic, Par- ma University Medical School, Parma, Italy Studies on high density lipoprotein cholesterol (HDL-C) levels in non-insulin-dependent diabetes have given contradictory results. The importance of insulin in lipoprotein metabolism is well known. C- pepfde is considered as a reliable indicator of endogenous insulin production more than insulin itself. The aim of this study was to investigate HDL-C levels and their relationship with fasting C-Peptide in 20 non-obese Type 2, 38 obese Type 2 diabetic patients and 20 non- obese, healthy control subjects, matched for age and sex. Mean_+ SEM HDL-C levels resulted 48.7 _+ 2.5 mg/dl in controls; 38.4 _+ 3.2 in non-obese (p<0.05 versus controls) and 38.1_+2.2 in obese Type2 patients (p < 0.01 versus controls; NS versus non-obese Type 2 patients). Fasting C-peptide was 1.1 _+ 0.11 ng/ml in controls; 1.54 _+ 0.18 in non-obese Type 2 patients (p < 0.05 versus controls); 2.44 _+ 0.36 in obese Type 2 diabetic patients (p < 0,01 versus controls). A significant inverse relationship between HDL-C and fasting C-Peptide was found in non-obese (r= - 0.525,p < 0.05) and in obese Type 2 diabetic patients (r=-0.330, p< 0.05). No correlation occurred in controls (r= -0.012, NS). In conclusion, these results give evidence for an inverse relationship between HDL-C and insulin secretion in Type 2 diabetes, suggesting that endogenous hyperinsulinaemia might play a role in the control of plasma HDL-C concentration.

258. Dietary Fibre and Poorly Controlled Diabetes J. I. Mann, D. B. Jones, S. Lousley, R. D. Carter, R. Jelfs and P. Slaugh- ter. Radcliffe Infirmary, Oxford, UK Published data relating to the benefit of high fibre diets for diabetes are based on investigations in relatively well controlled patients. We have studied patients whose random clinic glucose levels remained persistently > 12 retool/l, despite maximal doses of oral hypoglycaemic agents. Fifteen consecutively eligible patients were recruited from

Abstracts 179

the Oxford Diabetic Clinic. Twelve were being considered for conversion to insulin. A 24h metabolic profile was performed after which patients were randomised either to continue on a reinforced low-carbohydrate regimen or to a high-carbohydrate high fibre alternative (55% energy from carbohydrate, 60 g dietary fibre daily). A further profile was performed after 6 weeks on the diets and a final 24 h study after the dietary periods had been reversed. Four patients found high fibre unacceptable and did not complete the study, the remainder experienced no problems. Mean _+ SD haemoglobin Alc values initially, after the reinforced low carbohydrate period and after high fibre were 10.6 _+ 2.1%, 10.3 _+ 2.2%, 8.5 _+ 1.9%. Levels on high fibre were significantly lower than at other times (p < 0.01). Total and LDL cholesterol and total triglycerides were significantly lower on high fibre. Thus the majority of poorly controlled diabetic patients on maximal oral therapy can achieve substantial improvement by dietary modification without conversion to insulin.

259. Prospective Analysis of Islet Cell Antibody and C-Peptide Levels During the First 24 Months of Insulin-Dependent Diabetes B. Marner, T. Agner, C. Binder, A,. Lernmark and J. Nerup. Hagedorn Research Laboratory and Steno Memorial Hospital, Gentofte, Den- mark Islet cell cytoplasmic antibodies (ICA) were determined in 60 (21 female, 39 male, 11-69 years old) insulin-dependent diabetic patients followed prospectively for 24 months. ICA was titrated by indirect immunofluorescence on sections of a human pancreas showing high reproducibility in repeated tests. The prevalence of ICA at diagnosis was 62%; four (7%) additional patients, negative at diagnosis, were later found to develop ICA, while the remainder were ICA-negative. The prevalence of ICA decreased progressively, being 57% after 3 months, 43% after 12 months, and 24% after 24 months. The average ICA titres decreased from 1:9 at diagnosis to 1:6, 1:3, and 1 :% respectively. The patients remaining ICA positive after 24 months of insulin-dependent diabetes had an average titre of 1:9 compared with 1 : 81 at diagnosis. The drop in ICA titre paralleled a decrease in fasting C-peptide from 0.24pmol/1 at 3months to 0A9pmol/l at 12 months and 0.14 pmol/1 at 24 months, but this progressive decline did not differ from that in the ICA-negative patients. It is concluded that insulin-dependent diabetic patients with ICA persistent for 2 years represent individuals with high ICA titres at onset and that fasting C-peptide decreases with increasing duration of diabetes, independent of the presence of ICA.

260. Atherosclerosis and Diabetes Mellitus in Sand Rats (Psammomys Obesus) G. Marquie 1, S. Mahtout 1, P. Hadjiisky 2, P. Gendre 3, J. DuhaulP, and M. Boulanger 4. ILaboratoire de Nutrition, Universit~ des Sciences, Al- ger, Algeria, 2H6pital Boucicaut, Paris, 3Centre Microscopic Electro- nique, Bordeaux, and qnstitut Recherches Servier, Suresnes, France In sand rats fed on laboratory chow, a few animals developed diabetes leading to ketoacidosis and death; most animals developed obesity before the onset of diabetes (non-insulin dependent then insulin- dependent diabetes). Animals fed only on fresh plants did not develop diabetes. Light microscopic, histochemical and ultrastructural studies were made in the aorta in these animals. Some enzyme activities linked to the pentose pathway and the tricarboxylic cycle were higher in aortas of diabetic animals. In contrast, cholinesterase activities, which induced an aortic lipolysis, were reduced. Insulin-dependent animals showed a diffuse aortic lipoi'dosis with foamy cells. Morphological and ultrastructural changes in the wall of the aortas were observed: alteration of conjunctive tissue with disorganization of elastic tissue and development of collagen tissue which induced a late fibrosis. Advanced degenerative changes of smooth muscle cells promoted a real cellular necrosis. The progressively increasing severity of these arterial lesions was particularly observed in the later phase of the disease in insulin-dependent animals. The sand rat seems to be a valuable model for investigating the pathogenesis of diabetic angi- olopathy.

261. Adrenergic and Cholinergic Modulations of Rat Gastric and Pan- creatic Somatostatin-Like-Immunoreactivity Release in Vivo M. Man'e, S. Hadjeri, L. Rene-Corail and R. Assan. Diabetes Depart- ment, H6pital Bichat, Paris, France To study whether adrenergic and cholinergic systems influence portal somatostatin-like-immunoreactivity (SLI) release from stomach and pancreas in vivo, 12 h-fasting rats were pancreatectomized or gastrec- tomized and then subjected to an acute hypovolaemia, in the presence or absence of propranolol, phentolamine, or atropine. The initial por-

tal SLI concentration was 1.44 _+ 0.24 ng/ml ; it rose up to 3.27_+ 0.29ng/ml after pancreatectomy (n=14; p<0.001). These values were further enhanced by hypovolaemia. After propranolol injection, gastric SLI declined to 1.81 +0.48 ng/ml (n=6; p< 0.05); it did not rise during hypovolaemia. Phentolamine or atropine administration did not modify SLI values. By contrast, gastrectomy was followed by a significant decline of portal SLI concentrations (from 1.24_+ 0.12 ng/ml to 0.77 _+ 0.08 ng/ml; n = 18; p < 0.005). Pancreatic SLI did not rise significantly during hypovolaemia. Propranolol or atropine injections did not alter SLI concentrations. Conversely, SLI rose from 0.85 -+ 30.14 to 2.11 _+ 0.40 ng/ml after phentolamine injection (n = 6; p < 0.05). In conclusion, these results suggest that: (l) in the rat, the contribution of the stomach to portal SLI concentrations is predomi- nant compared to that of the pancreas; (2) in these experimental conditions, gastric SLI is stimulated by beta-adrenergic mechanisms; (3) pancreatic SLI release may be unmasked by an alpha-adrenergic blockade.

262. Use of Intraperitoneal Insulin in Brittle Diabetes S. M. Marshall, D. J. Husband, S. Walford, P. D. Wright and K. G. M. M. Alberti. Royal Victoria Infirmary and Freeman Hospital, Newcastle upon Tyne, UK We describe the use of continuous intraperitoneal insulin infusion in six 'brittle' diabetic patients. All were female, aged 15-30 years, had had multiple previous admissions with ketoacidosis, were resistant to SC insulin and could no longer be managed with IM or IV insulin. A tunnelled catheter was inserted into the peritoneum under general anaesthetic. Silicon-rubber-covered polyethylene catheters (Siemens) were used initially, but six catheters in three patients failed after 2�89 weeks (mean 7�89 weeks). We then used a Broviac Silastic catheter with an integral Dacron cuff. One failed because of dislodgement, but six continued to function after 4-12 weeks. Highly purified acidic porcine insulin (Hoechst) was delivered via a Siemens Promedos pump. Twenty-four-h blood glucose profiles were performed on IV and IP insulin. Mean blood glucose did not differ significantly (5.9 versus 7.3 mmol/1) but mean daily insulin requirements were lower via the IP than the IV route (54.6 versus 112.8 U/day; p < 0.01). Indi- vidual patients have been treated for 4-34weeks (mean 15weeks); three have returned to a normal lifestyle. We conclude that although there are technical difficulties, continuous IP insulin is a useful and safe therapeutic option in severe, 'brittle' diabetes.

263. Improved Metabolic Control During Continuous Subcutaneous In- sulin Infusion with Variable Basal Infusion Rate in Type I (Insulin-De- pendent) Diabetic Patients M. Massi Benedetti, M.A. Antonella, P. G. Fabietti, A. Bueti, G. Cala- brese, G. Bellomo, F. Santeusanio and P. Brunetti. Istituto di Patologia Medica, University Perugia, Italy The aim of the study was to improve metabolic control during continuous insulin infusion (CSII) avoiding the dawn phenomenon without risking nocturnal hypoglycaemia. A group of eight Type I diabetic patients on dual rate CSII for 12-24 months was switched to an infusion regime characterized by an automatically variable basal rate which provided a higher nocturnal infusion from 03.00 until 06.00 h. The daily insulin dose was similar during the two infusion regimens (fixed basal rate (FBR): 38.0-+2.9 U/day; variable basal rate (VBR: 41.8_+3.SU/day). The basal rate during FBR was 0.84-+ 0.07 U/h, while during VBR it was 0.62_+ 0.05 U/h throughout the day and the night until 03.00h; from 03.00h to 06.00h it was increased to 1.33 _+ 0.12 U/h. During VRB fasting blood glucose (4.9 _+ 0.6 mmol/l) and mean blood glucose (5.8_+0.4 mmol/l) were lower than during FRB (respectively 9.5 _+ 0.8 and 8.7 _+ 1.3 mmol/1; p < 0.05 for both). A significant difference was observed in fasting 3-betahy- droxybutyrate levels (FBR: 0.381_+0.06mmol/1; VBR: 0.118_+ 0.02 mmol/l ; p < 0.05). The mean plasma free insulin dose was lower during VBR (20.6_+ 2.2 mU/1) than during FBR (28.5_+ 3.3 mU/1; p < 0.05). In conclusion, the different distribution of insulin infusion allowed for better metabolic control and reducing the peripher hyper- insulinization, especially during the early phases of the night, minimized the risks of nocturnal hypoglycaemia.

264. Discrimination of Glucose and Mannose Anomers by Glueokinase from Liver and Transplantable Insulinoma F. M. Matschinsky, M. D. Meglasson and M. A. Schinco. Diabetes Re- search Center, University of Pennsylvania, Philadelphia, Pennsylva- nia, USA Glucokinase (GK) is claimed to be the glucose sensor determining the B cell glycolytic rate. Stimulation of insulin secretion by glucose or

180 Abstracts

mannose anomers occurs with c~-D-glucose more effectively than with its fl-anomer at concentrations < 16 mmol/l and with ~z-D-mannose more effectively than with/%D-mannose at 27 retool/1 or less. GK may be the site of discrimination of hexose anomers by the B cell. This was tested with GK partially purified from rat liver or insulinoma by ion exchange chromatography. Hexose phosphorylation by GK was measured for 2 or 3 rain at 25 ~ using pure hexose anomers. GK from either tissue had higher affinity for c~-D-glucose than/3-D-glucose (Kin: 4.0 versus 10.4 mmol/1). Similar discrimination of mannose anomers was observed, c~-D-mannose having higher affinity (Km: 8.1 versus 16.9 mmol/1). Cooperativity was observed with both anomers of glucose and mannose. Maximum phosphorylation rates were slightly higher (10%-20%) for /~-hexose anomers. Comparison of reaction rates for hexose anomers with GK shows preferential phosphorylation of a-anomers at glucose concentrations < 15-20 mmol/1 and mannose < 30-60 mmol/1. These results support the glucokinase- glucose sensor hypothesis by showing discrimination of hexose anomers by GK paralleling the anomer potencies as insulin stimulants.

265. Assessment of Insulin and C-Peptide Half-Life in Man by Soma- tostatin Suppression and Demonstration that Insulin Delivery in the Basal State is an 'on-off' phenomenon D. R. Matthews, M.A. Burnett and R.C.Turner. Diabetes Research Laboratories, Oxford, UK The half-lives of insulin and C-peptide in vivo have been assessed by a method which examines the decline of endogenously produced insulin and C-peptide levels. Venous blood samples were taken each minute from seven normal subjects: IV glucose (0.1 g/kg) was given over 1 rain, followed by a bolus of 250 l.tg of somatostatin-14 (Serono) and an infusion of a further 250 !-tg over 30 min. Plasma samples were analysed for C-peptide, glucose and insulin. Thus glucose stimulated insulin and C-peptide which were then suppressed by somatostatin. The half-life of endogenously produced hormone was calculated from log-transformed data. Insulin half-life was initially short (2.0 • 0.12 min over the first 3 min • SEM), and over the subsequent 10 min was 5.5 _+ 0.5 rain. C-peptide declined according to a simple exponen- tial relationship (half-life 11.7 • 0.9 min), and thus it is unlikely that the complex decline of insulin is a mixing phenomenon. Basal insulin delivery from the pancreas was calculated from the plasma values using these estimates of the endogenous half-life. The 14-min plasma oscillations of both insulin and C-peptide in normal subjects were de- convoluted to show that insulin production was turned off almost completely for a few minutes at the nadir of each cycle, and that there was a selective hepatic extraction of insulin at the peaks.

266. Plasma C-Peptide, Cortisoi and Growth Hormone Responses to Hypoglycaemia, Induced by Human Insulin Compared with Porcine In- sulin C. Mazzi. Department of Endocrinology, St. Anthony Hospital, Gal- larate, Italy The present study was designed to measure the hormonal responses to hypoglycaemia induced by human monocomponent insulin (Ac- trapid, Novo) compared with porcine insulin (Actrapid, Novo). With a time interval of one week, insulin tolerance tests (0.2 U/kg body weight) were performed on seven healthy women (aged 18-37 years) in the early follicular phase, who had a body mass index of 19-24. In venous blood samples, at -15 , 0, + 15, 30, 60, 90, 120, 180, 240 rain, insulin, C-peptide, cortisol and growth hormone levels were determined by radioimmunoassay, and glucose by GOD-POD colorimetric method. The sensivity for human and porcine insulin was identical; and the two insulins showed the same plasma insulin peak values. The hypoglycaemic effect and the growth hormone increase also behaved similarly with both insulins. The secretion of endogenous insulin, represented by C-peptide levels, was less inhibited by the exogenous human insulin (p < 0.05). Following the human insulin, the cortisol increase was less pronounced in comparison with porcine insulin (human insulin: from 560.3_+52.4 to 794.9+66.2nmol/1, p<0.05; porcine insulin: from 554.8 • 46.9 to 858.4 • 52.4 nmol/1, p < 0.01). These results suggest that the hormonal responses to hypoglycaemia depend in part on the molecular structure of insulin.

267. Teaching Diet 'Live' over Lunch or with a Videotape Leads to Im- proved Knowledge, Compliance and Glycaemic Control in Type 1 (Insu- lin-Dependent) Diabetes D. K. McCulloch, R. Mitchell, J. Ambler and R. B. Tattersall. Queen's Medical Centre, University Hospital, Nottingham, UK Dietary non-compliance is a major cause of poor control in Type 1 diabetes. It is common to blame the patient but it is equally possible

that teaching methods are at fault. We compared three teaching methods: group A - standard dietary instruction from a dietician, group B - practical lunch-time demonstrations and group C - a purpose-designed videotape. During a 3 month run-in period 1 HbA1 fell from 12.9 + 2.0 to 11.7 + 1.7% in 40 poorly controlled adult Type I diabetic patients. They were then randomised to groups A, B or C and seen 3- monthly by a doctor and dietician. Knowledge was assessed by questionnaires, compliance by 7 day food records and glycaemic control by serial HbAI measurements. Over 6 months follow-up, there was no significant improvement in those given standard instruction by a dietician. In the lunch-time demonstration group, HbA1 fell to 10.6 • 2.1% (p< 0.025) and in the videotape group to 9.6 + 2.3% (p< 0.001). Im- provement in control was paralleled by increased knowledge and compliance. This study shows that new and interesting educational methods can have a major influence on knowledge, compliance and metabolic control in Type 1 diabetes.

268. Independence of Glyeogenic Effects of Insulin and of a Glucose load in cultured Rat Fetal Hepatocytes P. Menuelle and C. Plus. Laboratoire Interactions Cellulaires, Univer- sity of Paris 7, UER Odontologie, Paris, France Primary cultures of 18 day-old rat fetal hepatocytes, grown for 2 days in NCTC 109 medium, 10% fetal calf serum, cortisol (10 umol/l), glucose (5.5 mmol/1) were used to measure glycogenesis stimulation after addition of insulin (10 nmol/1) or glucose (18 mmol/1). Maximal 14C-

14 stimulation of glycogen labelling from C-glucose occurred after 3 h in the presence of insulin (370%) and 1 h in the presence of the glucose load (380%). These stimulatory effects were found additive and unaffected by cycloheximide (10 p~mol/1). The contribution of glucose medium to glycogen formation, determined after continuous labelling with 14C-glucose for the first 48 h of culture (65%), was increased after a 4-h-exposure to insulin and glucose load (75 and 80%, respectively). In hepatocytes desensitized to insulin after pre-exposure to the hormone, insulin was unable to stimulate glycogenesis, whereas the effect of the glucose load was maintained. A similar situation was obtained in the presence of chloroquine (70 p.mol/1), which modifies the fate of the insulin-receptor complex in the cell. The newly formed glycogen during 4 h in the presence of insulin or glucose load was mobilized (by 85%) by glucagon (10 nmol/1) or adrenaline (10 nmol/1), which are known to act on the same hepatocyte population. Therefore cultured fetal hepatocytes respond to insulin and a load of glucose by utilizing distinct metabolic pathways.

269. Effect of One Month Prednisone Treatment in Newly Diagnosed Type I (Insulin-Dependent) Diabetic Children F. Meschi, L. Beccaria, A. Flores d'Arcais, C. Pellini and G. Chiumel- lo. Endocrine Unit, Paediatric Department, University of Milan, Italy 26 diabetic children were randomly divided into two groups after diagnosis with parents' informed consent. Group 1 was treated with oral prednisone (60mg/m 2 daily for 14days and 30 and 15mg/m 2 for 7 days). Group 2 was a matched control group. Blood samples and clinical data were obtained after 15, 30, 60, 90, 180, 270, 365 days. Re- sults are expressed as mean + SD. Insulin dose (U. kg-1. day-l) was higher in group 1 (1.47 • 0.16) than in group 2 (0.74 • 0.16) during the first 30 days of treatment (p < 0.001). The greatest number of subjects with insulin requirement < 0.5 U. kg -1. day -a, C-peptide concentrations of > 0.3 nmol/1 and minimal glycosuria was observed after 2 months in both groups (four in group 1 and two in group 2). The lowest mean stable HbA~ level was reached in both groups after 6 months (group 1 : 6.5 _+ 0.76%; group 2:7.05 + 0.75%). Mean fasting and post-breakfast C-peptide concentrations (nmol/1) increased in both groups within the first 6 months (group 1 : 0.79 +_ 0.40 and 1.04 + 0.43; group 2:0.35 _+ 0.17 and 0.49 + 0.27). In conclusion, the role of prednisone therapy remains undecided in newly diagnosed Type 1 diabetic children.

270. Endogenous Platelet Thromboxane Az Production in Diabetic Pat- ients With and Without Peripheral Vascular Disease D. P. Mikhailidis, M.A. Barradas, J. Y. Jeremy, J. Mohiuddin, L. Gra- cey and P. Dandona. Metabolic Unit, Royal Free Hospital, London, UK Platelet aggregation and thromboxane A2/B2 (TXB2) production were investigated in insulin-dependent (n = 12) and non-insulin-dependent diabetics (n = 28), non-diabetic patients with peripheral vascular disease (PVD; n= 8), and 12 healthy control subjects. Platelet aggregation and TXB2 production in the diabetic patients as a group were not significantly different from those in the control group. However, diabetic patients with severe PVD had hyperaggregable platelets and

Abstracts 181

generated markedly m o r e T X B 2 than diabetic patients with no PVD (with or without microangiopathy) or control subjects (p < 0.01). Pat- ients with PVD but no diabetes also had platelet hyperaggregability and produced markedly greater amounts of TXB2 than control and diabetic subjects without PVD (p<0.01). Platelet aggregation and TXB2 production by patients with PVD and diabetes were slightly, but not significantly greater, than in patients with PVD alone. We conclude that macrovascular disease is associated with platelet hyperaggregability and enhanced thromboxane production, and that in the absence of macrovascular disease diabetes is not associated with either of these features despite the presence of microvascular disease. Platelet hyperaggregability in diabetes is probably related to the concomitant presence of macrovascular disease but not microangiopathy.

271. Testing Histocompatibility Antigens (Loci A and B) in a Group of Type I (Insulin-Dependent) Diabetic Patients in Bucharest I. Mincu, D. Cheta, C.I. Truia, M. Truia, C. Ionescu-T/rgovi~te and D. Ilinca. Clinic of Nutrition and Metabolic Diseases, Oncologic In- stitute and Center of Haematology, Bucharest, Romania For a preliminary estimation of the prevalence and significance of HLA antigens, tests were carried out on the A and B loci in an unselected group of 107 patients with Type 1 diabetes in Bucharest. Mono- specific antisera furnished by NIH, Bethesda were used. For HLA-A the following were obtained: A2 (20.3% of the total specificities); A1 (18.4%); A3 (14.0%); AI0 (11.1%); A28 (10.1%). Provisional estimation for the healthy population likewise indicated HLA-A2 as being more frequent followed by A30/31, A1, A9, A3. For HLA-B: B7 (38.2%); B5, B12 and B14 (14.0% each); B8 (11.1%). In the healthy subjects the order was B12, B35, B5, B8 the same as B18, then B7 (which does not exceed 11%). The most frequently encountered hap- lotypes in the diabetic patients were: A2/B7 (8.4% of the total haplo- types); A3/B7 (6.9%); A1B7 (6.6%); A10/B7 (3.8%); A9/B7 and A11/B7 (3.6% each). An unexpectedly high frequency of the HLA-B7 antigen was found in this group of diabetic patients, contrasting with its assumed 'protector' role in the Caucasoid population. The frequency of antigen HLA-A3 and haplotype HLA-A3/B7 infringes their listing in the 'resistance axis' to diabetes.

272. Circadian and Circamnual Variations of Insulin Requirements in Type I (Insulin-Dependent) Diabetic Patients on Portable Pumps J. Mirouze and J. L- Selam. Metabolic and Endocrine Department, St. Eloi Hospital, Montpellier, France The study was done on 20 Type 1 diabetic patients chronically treated for 1-2.5 years by continous peritoneal insulin infusion via portable pumps. 20%, 40% and 40% of the daily carbohydrates (180+ ]0g) were given at breakfast, lunch and supper. The patients were instruct- ed to adapt by themselves both basal and prandial rates 15 min before the three meals, and basal rate again at bed-time according to 6 + 1 capillary glucose testings/day. Prandial doses were higher (p < 0.01) for breakfast than for lunch and supper: 160+40, 90+25, 92+ 20 mUI /g of carbohydrate respectively (mean_+ SD). Basal rate was higher during afternoon and evening than during morning and night: 1.2+0.5, 1.2_+0.6, 1.0+0.5 and 1.0_+0.6 U/h, respectively. These results confirm our previous findings using the artificial pancreas in short-term protocols. Monthly evaluation of the total daily doses showed the highest values between June and September, and the lowest between December and April: 43 _+ 6 and 38 _+ 4 U/24 h, respectively (p < 0.01).

273. Inhibition of Insulin Degradation by Insulin-like Growth Factors R.Misbin, E.Almira, T.Mehl, T.Merimee and W.Duckworth. De- partments of Medicine, University of Florida, Gainesville, Florida and Indianapolis, Indiana, USA We studied a patient who responded to small doses of IV insulin but was resistant to large doses of SC and IM insulin. His serum had a normal level of insulin-like growth factors I (IGF) (18 versus 24_+ 2 nmol/1, mean _+ SEM in 25 other diabetic patients) and a low level of IGF II (22 versus 94 _+ 5 nmol/1). His serum contained a heat-labile factor that degraded 12sI-insulin in vitro to products that were soluble in 5% trichloroacetic acid. Addition of insulin (50 nmol/1), IGF I, and IGF II (gift from Drs J. Zapf, E. R. Froesch and R. E. Humbel, Zurich) to serum inhibited 12sI-insulin degradation by 39%, 28% and 85%, respectively. Further studies were performed using insulin protease isolated from rat skeletal muscle. IGF II was a more potent inhibitor of 125I-insulin degradation by insulin protease than was insulin itself. At a concentration of 50 nmol/1, 125I-insulin degradation was inhibited by 35%, 13%, and 51% by insulin, IGF I, and IGF II, respectively. In-

125 125 sulin protease also degraded I-IGF I and I-IGF II but less rapid-

ly than it degraded ~25I-insulin. Conclusions: (1) IGF II is a potent inhibitor of insulin degradation, (2) IGF I and IGF II can be degraded by insulin protease, (3) low serum levels of IGF II may be associated with insulin resistance in some diabetic patients.

274. Microalbuminuria in Maturity Onset, Primarily Type 2 (Non-Insu- lin-Dependent) Diabetes, Predicts Clinical Proteinuria and Early Mor- tality E.E.Mogensen. Second University Clinic of Internal Medicine, Kommunehospitalet, Aarhus, Denmark This study was conducted to clarify whether an increased urinary albumin concentration (UAC) of 30-140 ug/ml, ('microalbuminuria') predicts proteinuria and early mortality in primarily Type 2 diabetic subjects. Patients, aged 50-75 years with age at diagnosis > 45 years, were included. These patients (n = 76) were compared with lower concentration groups: < 15 ~g/ml (n = 75) and 15-30 ug/ml (n = 53); and higher: > 140 ug/ml (n = 28). During 1973, all morning urines of diabetic subjects attending the clinic were examined by radioimmunoassay and re-examined 9.5 years later. The four groups of UAC were compared for development of proteinuria and for mortality by 1983. Data for age, sex, diabetes duration, treatment (81% on oral agent and/or diet), plasma glucose, blood pressure, height and weight were analysed. The 30-140 gg/ml group exhibited a very marked increase with respect to development of proteinuria (17 of 76 compared with 7 of 128 in the < 30 llg/ml groups, p< 0.01) and in mortality: in the < 15 llg/ml group 33 died versus 24 expected, + 38% mortality compared with the control population; the 16-29 ~g/ml group: 30/17, + 76%; the 30-140 ~g/ml group: 59/24, + 146%, and the > 140 llg/ml group: 20/9, + 122%, mortality in all groups significantly and increasingly elevated. Thus microalbuminuria predicts proteinuria and early mortality in Type 2 diabetes.

275. Plasma fl-Thromboglobulin Responses to Insulin-Induced Hypo- glycaemia in Type I (Insulin-Dependen 0 Diabetes L. H. Monnier, J. L. Richard, C. Colette, A. Orsetti and J. Mirouze. De- partment of Metabolism, Saint-Eloi Hospital, Montpellier, France The effect of insulin-induced hypoglycaemia was examined in 10 Type 1 diabetic subjects. After an overnight blood glucose normalization each patient received at 09:00 h (time 0) an additional IV bolus of 3 U regular insulin. Blood glucose was continuously recorded up to 180rain, and plasma samples were drawn at regular intervals for fl- thromboglobulin (fl-TG) determinations. The patients were divided into two groups according to whether blood glucose decrements from baseline were greater (group 1, n = 6) or lower (group 2, n = 4) than 1.78 mmol/1 at nadir. The time courses of fl-TG decrements or increments from baseline were compared in groups ] and 2. In group 1, fl- TG increased during the insulin stress while a decrease was observed in group 2. Significant differences between the two groups were observed at 30 min (+ 87.6_+ 41.8 versus - 32.0_+ 7.1 ng/ml, p < 0.05) and 60 min (+ 36.0 _+ 15.7 versus - 62.5 _+ 32.1 ng/ml, p < 0.02). In group1 the fl-TG response remained elevated at 120 and 180rain (p < 0.1 compared with group 2). It appears, therefore, that transient hypoglycaemia can result in sustained platelet aggregation in vivo provided that the blood glucose fall from baseline exceeds a threshold approximately equal to ].78 mmol/l.

276. Monolyers of Rat Endocrine Pancreas Embedded in Collagen Gels Reorganize into Three-Dimensional Islet-Like Structures R. Montesano, P. Mouron, M. Amherdt and L. Orci. Institute of His- tology and Embryology, University of Geneva Medical School, Ge- neva, Switzerland Islets of Langerhans are complex micro-organs whose constituent cells show a characteristic topographical distribution probably crucial to the coordinated function of the islet. One in vitro model of the endocrine pancreas, monolayer culture, allows the study of islet cell physiology, but not the assessment of the role of the topography in situ, because of the two-dimensional organization of these cultures. To find conditions permitting islet cells to express the characteristic association pattern observed in situ, we have grown pancreatic endocrine cells in a three-dimensional collagen gel matrix. Islet cells disso- ciated from neonatal rat pancreas are first allowed to spread on the surface of hydrated collagen gels, then covered with a second layer of gelling collagen. Within a few days, the flattened endocrine cell dus- ters reorganize into rounded three-dimensional structures which appear in sections perpendicular to the bottom of culture dishes as smooth-contoured multilayered aggregates of well-granulated endocrine cells. As in islets in situ, non-B cells (identified by electron microscopy and immunofluorescence) have a preferential distribution at

182 Abstracts

the periphery of the B cell aggregates. We therefore propose to use this new culture system to investigate the role of this characteristic pattern in islet cell function.

277. Lymphocyte Transfusions Prevent Diabetes in Biobreeding/ Worcester Rats J. P. Mordes, B.A. Woda, A.A. Like, D. Faustman and A.A. Rossini. University of Massachusetts, Worcester, MA and Washington Univer- sity, St. Louis, MO, USA The Biobreeding/Worcester (BB/W) rat is an animal model of autoimmune diabetes. We recently reported that transfusions of whole blood from the non-diabetic W-subline of BB/W rats completely prevent diabetes in diabetes-prone recipients. We now report three experiments designed to determine which blood component is responsible for this protection, In all experiments, diabetes-prone BB/W rats 35 days of age were randomized to receive 5 weekly IV injections. (1) Animals were injected with either saline or red blood cells, white blood cells, or plasma from W-line donors. Diabetes occurred in 7/23 red cell, 1/27 white cell, 10/25 plasma and 13/28 saline recipients. At 120days of age, peripheral blood was obtained from non-diabetic rats. FACS analysis of OX 19 tagged leucocytes revealed 39% T-lymphocytes in white cell recipients (n = 15) versus 9% in saline recipients (n = 9). Responsiveness to concanavalin A was also increased in the white cell group. (2) Diabetes-prone rats received either W-line blood treated with anti-T-lymphocyte antibody plus complement, untreated blood, or saline. Diabetes occurred in 8/20, 1/20, and 13/19 rats in each group respectively. (3) Finally, 1/19 rats transfused with W-line spleen cells and 11/18 recipients of diabetes-prone spleen cells developed diabetes. We conclude that transfusions of W-line T-lymphocytes prevent diabetes in the BB/W rat.

278. The Measurement and Validation of Non-steady C-peptide Secre- tion Rates in the Dog T. Morishima, K. Polonsky, H.Tager and J. Radziuk. University of Western Ontario, London, Canada and University of Chicago, Chica- go, USA If C-peptide (CP) kinetics are linear and the first-pass hepatic extraction is negligible, the non-steady secretion rate of CP can be calculated from its peripheral levels and a predetermined decay curve by de- convolution. This theory was tested as follows: five overnight-fasted dogs were injected with 25 nmol of dog CP and the plasma decay curve obtained over 2 h. Dog CP was then infused at variable rates (sinusoidal etc.) for 2-3 h. Plasma CP determined by radioimmunoassay ranged from 0.2 to 8.5 pmol/ml. The basal CP secretion rate was 17.8 + 2.5 pmol/min (near basal insulin secretion). During the variable infusions, the calculated (y) and infusion (x) rates were compared by linear regression analysis (y = a + bx). a and b were not significantly different from 0 (p>0.5) and 1 (p>0.2) respectively. 95% confidence limits for b were (0.970 <f l< 1.088). In separate studies (n=7) hepatic fluxes of CP were determined from portal, arterial and hepatic venous CP levels and the hepatic blood flow (using an indocyanine green infusion): (1) under basal conditions, (2) with somatostatin and low-dose (3 pmol. kg-1. min-1) CP infusion and (3) with somatostatin and high dose (15 pmol.kg -1 .rain -1) CP infusion. Arterial CP levels were: (1) 0.237+0.039, (2) 0.275+0.02 and (3) 1.45+ 0.14pmol/ml and the fractional hepatic extraction was: (1) 4.3+ 4.5%, (2) - 5 +4.4% and (3) 2.1 +2.4%. Conclusions: (a) hepatic extraction of CP in dogs is near zero, and (b) CP secretion rates can be accurately determined from plasma levels and the response to an injection and can be used in the estimation of insulin secretion.

279. Glycosylation of Human Haemoglobin A: Kinetics of the Forma- tion and Dissociation of Haemoglobin Ale H. B. Mortensen, A.Volund and C.Christophersen. Paediatric De- partment, Glostrup Hospital, Novo Research Institute and Marine Chemistry Section, Department of General and Organic Chemistry, H. C. Orsted Institute, University of Copenhagen, Denmark The stability of the ketoamine adduct, termed HbAac, was investigated using an isoelectric focussing method. Prolonged saline incubation of purified HbAlo followed by renewed isoelectric focussing revealed that the HbAI~ concentration decreased while a corresponding increase in the haemoglobin A (HbA) concentration occurred. The emergence of HbA on saline incubation indicates that the non-enzymatic glycosylation of HbA to HbAlc is a reversible process. This finding was further substantiated by kinetic studies on the formation and breakdown of the ketoamine adduct during incubations of red cells in glucose and saline. It appeared that the rate constant for the formation k2 was 14.2 x 10-6/s at 37 ~ while the rate constant for the disso-

ciation k-2 was 1.7 x 10-6/s . These data were included in a biokinetic model which also took into account the removal of HbAlo by decay of erythrocytes, and the results were compared with an irreversible model for HbAac formation. The steady-state relation between mean blood glucose and HbAI~ was similar to the corresponding relation based on the irreversible formation of HbAlc. However, contrary to the irreversible model, the steady-state HbAac concentration was reached after 3 to 4 weeks after a change in blood glucose level. This finding indicates that, in assessing continous glycaemic control in diabetic patients, HbAlo should be measured at least once every 3-4 weeks.

280. Evaluation of a Structured Diabetes Teaching and Treatment Pro- gramme in Two University Hospitals: Significant Improvement of Dia- betes Care in 88 Unselected Type I (Insulin-Dependent) Diabetic Pat- ients for up to 21 Months I. Mfihlhauser, V. J6rgens, V. Scholz, L. Hornke, A. Kunz, W. Granin- get, G. Schernthaner and M. Berger. Departments of Medicine, Uni- versities of Dtisseldorf, FRG and Vienna, Austria In the two hospitals, an identical diabetes teaching and treatment programme (in-patient, Monday-to-Friday, group teaching) was set up. All patients (age 28 + 10 years, duration of diabetes 9 _+ 7 years, undetectable C-peptide in 70% of patients) referred to the two hospitals during a certain period were re-investigated 12 months later and again (for assessment of metabolic control only) 21 months after the teaching and treatment programme. Initially HbAa~ was 11.1%, after 12 months 8.8% and after 21 months 9.2% (p < 0.001). Hospitalisation was reduced from a median of 10 to 0-1 days per patient per year (p < 0.001). At 12months' follow-up, 36% of the patients had normal HbAlc values (groupA) and 25% had an HbAao~>10% (groupB). There were no differences between groups A and B with regard to severe hypoglycaemia, intelligence, diabetes-related knowledge or duration of diabetes. Group A had better compliance rates to some objec- tives and higher C-peptide levels than group B. The data indicate that the diabetic teaching and treatment programme resulted in a substantial long-term improvement of metabolic control and a striking reduction of hospitalisation. The study substantiates the feasibility of ap- plying this teaching and treatment programme on a large scale to community hospitals in order to improve the quality of diabetes care and to decrease health care costs.

281. Sulphonylureas Do Not Improve Impaired Insulin Sensitivity in Type I (Insulin-Dependent) Diabetes R. Mfiller, U. Keller and W. Berger. Division of Endocrinology and Metabolism, University Hospital, Basel, Switzerland Sulphonylureas have been demonstrated to enhance insulin sensitivity in vitro and in vivo. Since insulin resistance has been documented recently in Type 1 diabetes, we assessed insulin sensitivity in six Type l diabetic patients without endogenous insulin secretion and treated with portable insulin pumps using the euglycaemic insulin clamp technique. They were studied twice: once before, once after 8 days of glibomuride (2 • 50 mg four times a day) therapy. During insulin infusion (20 m U / m 2 per rain for 120 rain), glucose production (3-3H-glucose infusion) decreased similarly by 80% and 75% after glibornuride and control therapy, respectively. Glucose clearance failed to increase during insulin in both studies. Insulin sensitivity was impaired in the diabetic patients since in six normal control subjects, the metabolic clearance of glucose increased during the clamp by 113% (p< 0.01 versus diabetics). Suppression of glucose production by insulin was similar in normal subjects (by 85%) and in diabetic patients. Plasma non-esterified fatty acids were similarly lowered during insulin in both studies in diabetic and normal subjects. Metabolic control and insulin requirements were not influenced by glibornuride. The results demonstrate that insulin resistance of peripheral glucose uptake is present in Type 1 diabetic patients but uninfluenced by glibornuride therapy. Extrapancreatic sulphonylurea effects appear to be absent in diabetic patients without endogenous insulin secretion.

282. Improvement of Diabetic Neuropathy in Type l (Insulin-Depen- dent) Patients with Continuous Insulin Infusion Therapy C. Najemnik, H. Kritz and K. Irsigler. Metabolic Unit and k Boltz- mann Research Institute for Metabolic Diseases and Nutrition, City Hospital Vienna-Lainz, Vienna, Austria We sought to evaluate the effect of intraperitoneal or intravenous insulin infusion therapy over 12 months on the neurological features of diabetic neuropathy. Patients were divided into two groups. Group A: 20 Type I diabetic patients, age 34+ 13 years, duration of diabetes 16+_ 8 years, used continuous intraperitoneal or intravenous insulin infusion. Group B: 20 Type 1 diabetic patients, matched for sex, age,

Abstracts 183

duration of diabetes, symptoms and neurophysiological parameters, used conventional SC injection therapy. Patients of both groups had moderate to severe peripheral and/or autonomic neuropathy. Motor and sensory nerve conduction velocity symptom score and tests for autonomic dysfunction were evaluated. Motor nerve conduction velocity measured in the median, peroneal and tibial nerve, and sensory nerve conduction velocity measured in the median and sural nerve, improved significantly (p< 0.005) in group A but were unchanged in group B. A significant improvement (p_-< 0.05) was also seen in the autonomic heart function tests (Valsalva, 30:15 ratio) in group A, but there was no change in group B. Glycosylated haemoglobin improved from 12.5% to 8.6% in groupA (p<0.005) but did not change in group B. Thus, improved metabolic control over 12 months can positively influence not only peripheral but also autonomic diabetic neuropathy.

283. Biphasic Insulin Release is the Expression of Time-Dependent In- hibition and Potentiation of the Secretion Rate R. Nesher, L. Waldman and E. Cerasi. Department of Endocrinology and Metabolism, Hebrew University-Hadassah Medical Center, Je- rusalem, Israel Repeated stimulation of the perfused rat pancreas with arginine (5.0 mmo!/1) or with glucose (8.3 mmol/1) resulted in a time-dependent inhibition (TDI) of insulin release, the secretion rates being 30% and 75% of the respective controls. Twenty-rain priming with glucose, but not with arginine, potentiated subsequent insulin release approximately twofold. Thirty-min stimulation of the pancreas with arginine revealed monophasic dynamics of insulin release, while similar stimulation with glucose lead to biphasic dynamics with a 10-min period of nadir in the secretion profile. Synergistic interaction between glucose and arginine during a 10 rain stimulation resulted in the abolition of the expression, but not the generation, of time-dependent inhibition signal for insulin release. Introduction of a glucose stimulus during the refractory period of arginine-generated insulin secretion, or introduction of arginine stimulus during the nadir period of glucose-activated insulin release, abolished the nadir-TDI. We suggest that the biphasic dynamics of insulin secretion during a glucose stimulus is the result of a fast-generated TDI signal, followed by a slow-accumulat- ing signal for time-dependent potentiation of insulin release. Arginine stimulus generates TDI exclusively, exhibiting acute first phase.

284. Biosynthesis of Thyrotropin-Releasing Hormone Immunoreactivity in Isolated Pancreatic Islets in Organ Culture J. H. Nielsen, B. Welinder, L. O. Dolva and K. F. Hanssen. Hagedorn Research Laboratory, Gentofte, Denmark, and Hormone Laboratory and Medical Department B, Aker Hospital, Oslo, Norway In new-born rats thyrotropin-releasing hormone immunoreactivity (TRH-IR) is present in higher concentrations in the pancreatic islets than in the hypothalamus. Although TRH-IR was found in isolated islets kept in culture, it is not known whether TRH biosynthesis occurs in islet cells. In the present study, new-born rat islets were incubated with 3H-histidine for 3, 6, or 20 h, extracted with 90% methanol and subjected to reversed-phase high-performance liquid chromatography (HPLC) for 60 min on a Hamilton PRP-1 resin eluted with ammonium bicarbonate 0.1 mol/1 (pH 7.0) containing a linear acetonitrile gradient from 2.5 to 17.5%. A radioactivity peak with the same retention time as synthetic TRH was recovered from islet extracts and from immunoprecipitates of extracts treated with TRH- antiserum and protein A-Sepharose. Labelling ofislets for 20 h resulted in incorporation into the TRH-like peak similar to labelling for 3 h followed by a 17 h chase period, while little incorporation was seen after 3 and 6 h labelling. These results demonstrate biosynthesis of TRH in islet cells and it is suggested that the islet TRH biosynthesis pro- ceeds via a precursor by a slow conversion process.

285. Stimulation of Human Adipose Tissue Lipoprotein Lipase by Insu- lin: Relation of Response to Basal Activity E. A. Nikkil~i, H. Yki-J~irvinen, M.-R. Taskinen and V.A. Koivisto. Third Department of Medicine, Helsinki University Hospital, Helsin- ki, Finland We have measured the heparin-releasable activity of lipoprotein lipase (LPL) in human adipose tissue and the serum lipoprotein levels before and after a 5-h euglycaemic (insulin infused at rates 1 mU and 4.5 mU- kg -1. rain -1) and a hyperglycaemic clamp. The subjects were 10 healthy, normal-weight volunteers with normal serum lipid levels. Mean steady-state plasma glucose concentrations were 4.7+0.1, 5.0 _+ 0.1 and 8.8 _ 0.3 mmol/1 and corresponding plasma insulin levels 101,592 and 148 mU/1 during low- and high-insulin euglycaemic

and hyperglycaemic clamps, respectively. The corresponding rates of glucose metabolism averaged 9.9_+ 0.6, 15.8 + 1.5 and 17.9 + 1.3 mg. kg -1. rain -1, respectively. Compared with a control experiment using saline infusion, the LPL activity increased by an average of 20%, 35% and 50% (p < 0.05) during the low-insulin euglycaemic, hyperglycaemic and high-insulin euglycaemic clamps, respectively. The change in LPL was inversely related to basal LPL activity in euglycaemic low- insulin ( r= -0 .84 , p<0.01), euglycaemic high-insulin ( r= -0 .84 , p<0.05) and in hyperglycaemic ( r= -0 .83 , p<0.01) clamps. The VLDL triglyceride decreased during all three clamps by an average of 50% (p< 0.01), but there was no significant correlation between the rise of LPL and the fall of VLDL triglyceride. LDL cholesterol decreased by 5% (/7<0.05) and by 10% (p<0.05) during high-insulin euglycaemic and hyperglycaemic clamps, respectively. HDL cholesterol did not change. We conclude that basal LPL determines the response of LPL to insulin and therefore insulin stimulates the LPL only when it is primarily low.

286. Different Effects of HB699 and Glibenclamide on Islet Function L. Norlund and J. Sehlin. Department of Histology and Cell Biology, University of Ume~, Ume~, Sweden The anti-diabetic agent glibenclamide acts mainly by stimulating insulin release from the pancreatic B cells, although the precise mechanism of action is still unclear. A new drug, HB 699, an acyl-amino-alkyl benzoic acid derivative, which corresponds to one major part of the glibenclamide molecule, may provide us with means to correlate specific cellular effects of glibenclamide with certain domains of the glibenclamide molecule. Basal insulin release (glucose 3 retool/l) was stimulated by HB 699 (25-200 ~mol/1), whereas secretion induced by glucose (20 retool/l) was not affected. HB 699 (20-200 ~tmol/1) induced dose-dependent 45CaZ+uptake at glucose (3 retool/l), but at 200 mmol/1 it did not affect the osmotic resistance of isolated B cells. Glibenclamide-induced insulin release showed a complex dose-response relationship; one component saturated at 0.1 gmol/1 and the other was detectable above 10 ~tmol/1. Maximal stimulation of 45Ca 2+ uptake by glibenclamide was obtained even at 1 ~tmol/1. Glibencla- mide (1-200 l.tmol/1) increased the osmotic resistance of B cells. It is suggested that the action of glibenclamide on insulin release can be resolved into a 'high-affinity' component correlated with 45Ca2+ uptake and increased osmotic resistance in B cells, and a 'low-affinity' component which may be related to 45CaZ+uptake but not to change in the osmotic resistance. The latter component may be due to acyl- amino-alkyl benzoic acid residue.

287. Influence of Fixation and Staining Techniques on the Ultrastruc- ture of the Insulin Secretory Granule R. Norlund, L. Norlund and I.-B.TNjedal. Department of Histology and Cell Biology, University of UmeS, Umegt, Sweden The ultrastructure of pancreatic B-cell granules was examined after fixation of isolated mouse islets in different fixatives. Islets fixed in glutaraldehyde with or without post fixing in osmium tetroxide, or in osmium tetroxide alone, exhibited the classical picture of electron- dense core plus lucid halo. After fixation in potassium permanganate, with or without post-fixation in osmium, the secretory granules were often completely filled with a fairly homogenous material and the vesicle membrane outline was distinct. The peri-granular halo of glutaraldehyde- and osmium-fixed B cell granules remained unstained in cells exposed to the osmophilic amine, 5,6-dihydroxytryptamine. When glutaraldehyde-fixed B cells were stained according to Ram- bourg to visualize reducing groups, possibly carbohydrates, two distinct classes of secretory granules were observed: one darkly stained and one without noticeable precipitation of silver; the peri-granular halo remained unstained. Islets from both obese mice (non-inbred Umegt ob/ob) and their lean littermates showed these two types, although the proportions of Rambourg-positive and -negative granules may differ between the mice depending on the functional state of the B cells. After fixation in permanganate the Rambourg staining was lost.

288. Left Ventricular Function in Type I (Insulin-Dependent) Diabetes Mellitus: Evidence for the Presence of a Diabetic Cardiomyopathy? R. J. Northcote, C. M. Kesson and D. Ballantyne. Department of Clin- ical Medicine, Victoria Infirmary, Glasgow, UK This study was undertaken to determine left ventricular function using gated equilibrium bloodpool angiography in Type 1 diabetes. The study group comprised ten male patients with Type 1 diabetes of at least 10 years' standing. Prior to inclusion in this study, coronary artery disease was excluded as far as possible, each subject being

184 Abstracts

asymptomatic and normal on clinical examination with respect to cardiovascular disease. In addition, each subject had a normal standard 12-lead electrocardiogram, exercise electrocardiogram and a normal chest X-ray. Each patient had no coronary artery disease risk factors identified. Gated equilibrium bloodpool angiography was undertaken at rest and during isometric handgrip exercise. The results were compared with a group of healthy, age-matched male control subjects. Global ejection fraction in the diabetic group (59+4.2%, mean + SD) was reduced compared with control subjects (68 + 6.3%) at rest, and fell further during exercise. There is, therefore, evidence of impaired left ventricular function in Type 1 diabetes which may be due to a cardiomyopathy rather than coronary artery disease.

289. Effect of Metformin on Glucose Disposal and Production, Inter- mediate Metabolites and Insulin in Type2 (Non-Insulin-Dependent) Diabetic Patients during Dose Response Insulin Glucose Clamp R. Nosadini, S.Del Prato, R.Trevisan, S.Vigili De Kreutzenberg, M. Muggeo, A. Valerio, P. Tessari, A. Tiengo and G. Crepaldi. Patolo- gia Medica e Malattie del Ricambio, Padova, Italy To investigate the effect of metformin on glucose metabolism, 11 Type 2 diabetic patients were studied before and after 4 weeks of taking metformin (500rag thrice daily). The insulin-glucose disposal dose-response curve was studied with insulin glucose clamp at insulin infusion rates of 21, 73, 760mU.m-2.min -1. Glucose disposal (mmol.m-2.min -1 was 5.1+1.1; 14.0+1.7 and 22.4+1.7 before metformin and 7.5_+ 1.1, 16.2+ 2.4 and 26.8_+ 2.5 after metformin (mean _+ SEM; p < 0.05). Basal lactate concentration was 0.64 + 0.04 and 0.88_ 0.07 mmol/l (p< 0.05); alanine was 0.24 + 0.02 and 0.25 +__ 0.05 and lactate/pyruvate ratio 13.8 __+_ 2.3 and 16.6 + 2.0 (p < 0.01) before and after metformin, respectively. During insulin glucose clamp a 34% and 31% increase of lactate along with a 16% and 13% decrease of alanine was observed before and after metformin. Basal glucose production (isotopic technique) was 14% lower after metformin but not during insulin glucose clamp. Insulin binding on erythrocytes and monocytes was not changed by mefformin. We conclude that: (1) metformin ameliorates insulin sensitivity, improving maximal glucose disposal but not insulin binding, thus with a postreceptor mechanism; (2) lactate rather than alanine is the final product of glycolysis in presence of high insulin and euglycaemic levels, suggesting a reduced availability of NH2 because of inhibited proteolysis; (3) metformin inhibits hepatic gluconeogenesis and increases blood lactate with low insulin but does not stimulate peripheral lactate production with high insulin levels.

290. Evidence for Metabolic Regulation of Pancreatic Glucagon Secre- tion by L-Glutamine C-G. 0stenson. Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden To investigate the effects of L-glutamine on glucagon secretion and on glucose and glutamine metabolism, isolated pancreatic islets from normal and streptozotocin-treated guinea-pigs (A cell rich islets) were incubated in the presence of glucose (5.5mmol/1)+L-glutamine (10retool/l). L-glutamine significantly enhanced glucagon release from 297+54 to 528 _+ 53 pg- ,ug D N A - I . h -1 in normal islets and from 553 _+ 31 to 806 _+ 50 pg- ttg DNA -1. h -1 in A cell rich islets. Si- multaneously, glucose oxidation was significantly suppressed by L- glutamine in both types of islets. Islet content of ATP was reduced by L-glutamine to about 60% (p < 0.02) in A cell rich islets, but not significantly changed in normal islets. Glutamine oxidation, at 5.5 mmol/1 glucose, was considerably higher in A cell rich islets (1377+ 86 pmol- ~tg DNA -1. h -1) than in normal islets (610+93 pmol.l-tg DNA -1. h-~). Addition of porcine insulin (25 mU/ml) counteracted these effects by L-glutamine, i.e. suppressed glucagon release, hut increased glucose oxidation and ATP content of the A ceil rich islets. In conclusion, the present findings demonstrate that L-glutamine stimulates glucagon release and is readily metabolized within A cells. Fur- thermore, the regulation of glucagon secretion by L-glutamine appears to be reciprocally related to factors affecting glucose metabolism and ATP levels in the A cell.

291. Insulin Binding and Effects in Hyperthyroidism and Hypothyroid- ism J. Ostman, P. Arner, J. Bolinder and A. Wennlund. Huddinge Hospital, Stockholm, Sweden The aim of the investigation was to examine mechanisms behind changes in insulin requirement in diabetic patients developing either hyperthyroidism or hypothyroidism. Subcutaneous adipose tissue was removed from six hyperthyroid and six hypothyroid patients and

eight healthy control subjects, all matched for age, body weight and fat cell size. Segments of adipose tissue were incubated for measurements of glycerol release and 14CO2-production from glucose-U-14C. The mean EDs0 for the antilipolytic effect of insulin was 10 mU/1 in hyperthyroid patients, 2.5 mU/l in hypothyroid patients and 50 mU/1 in the control subjects. The lipolytic responsiveness to insulin did not differ between the three study groups. Maximum insulin-induced glucose oxidation was elevated by 180% in hyperthyroid patients and decreased by 60% in the hypothyroid subjects. Insulin binding to isolated fat cells was decreased by 40% (p < 0.05) in hyperthyroid patients and increased by 70% (p < 0.01) in hypothyroid subjects. These differences were largely due to differences in the receptor number. In hyperthyroidism, the increased insulin demand seems due to a decrease in the insulin receptor number. In hypothyroidism postreceptor defects may lie behind the impaired effect of insulin on glucose oxidation, since insulin binding and the sensitivity of the antilipolytic effect of insulin were increased.

292. Distensibility of Foot Veins in Diabetic Patients K. Ostrowski and A.Czy~yk. Department of Gastroenterology and Metabolic Diseases, Medical Academy in Warsaw, Poland The aim of this study was to assess changes in the volume of foot veins in relation to changes of effective venous pressure (EVP) in the range from 0 to 30 mmHg. Water plethysmography modified after the method of Wood was used for this purpose. Measurements were carried out in 40 diabetic patients, aged 16-40 years, with duration of disease for 2 to 9 years, and in 20 healthy control subjects, aged 18-40 years. In the diabetic group background retinopathy was found in only four cases, and two patients had clinically manifest polyneuropathy. The resting (control) venous volume rise following EVP rise to 30 mmHg was in diabetic and healthy subjects 2.6 _+ 0.5 and 2.8 +_ 0.6 ml/100 ml, respectively (mean +_ SD). Thermal stimulus (cooling) caused only a reduction of venous volume increment in healthy subjects, while in the diabetic group this increment in response to increased EVP was identical to that under control conditions. Similarly, only in healthy subjects a rapid IV infusion of phentolamine (0.2 mg/kg body weight) increased the increment of venous volume. On the other hand, a rapid IV infusion of papaverine (0.6 mg/kg body weight) produced an increase in venous volume increment of a similar order in both groups of probands. These results point to an impairment of the innervation and nervous reflexes of pedal veins in a relatively early stage of diabetes, before clinical manifestation of other signs of angiopathy and neuropathy.

293. The Significance of Hyperglyeaemia Following Acute Myocardial Infarction G. Oswald, S. Corcoran, M. Casbum-Budd, and J. S. Yudkin. Diabetic Department, Whittington Hospital, London, UK The admission blood glucose in 100 patients surviving documented acute myocardial infarction (AMI) was compared retrospectively with the admission blood glucose in subgroups of patients dying from cardiogenic shock or dysrhythmia following AMI. None of these patients was previously known to be diabetic. Admission blood glucose was significantly higher in patients dying from cardiogenic shock than in survivors (2p = 0.001) or in those dying from dysrhythmia (2p = 0.01). The extent of infarction was estimated using a QRS scoring system in 40 patients who had had standardised electrocardiograms recorded at regular intervals following AMI. No relationship was demonstrated between admission blood glucose and the estimated infarct size (r = 0.14, NS, Spearman rank correlation). Prospective study in 37 patients following AMI, none of whom was known to be diabetic, revealed a highly significant relationship between admission blood glucose and HbAic, determined by an iso-electric focussing technique (r = 0.501, p < 0.01). We suggest that hyperglycaemia following AMI reflects pre-existing glucose intolerance or undiagnosed diabetes. A disproportionate number of patients dying from cardiogenic shock following AMI are probably undiagnosed diabetic subjects.

294. HLA-D Region Genomic DNA Sequences in a Genetic Analysis of Type I (Insulin-Dependent) Diabetes Mellitus D. Owerbach, A. Lernmark, G. Holmgren, and B. H~iggl6f. Hagedorn Research Laboratory Gentofte, Denmark and University of Umegt, UmeS, Sweden Restriction fragment length polymorphism was analysed in 104 HLA-DR typed individuals from 22 families, using a HLA-D region class II antigen/3-chain eDNA clone, pDR-/3-1, as a hybridization probe. Twelve of the families had offspring with Type 1 diabetes, while the members of the 10 control families were all healthy and

Abstracts 185

without any family history of Type 1 diabetes. DNA was isolated from peripheral blood leucocytes, digested with restriction endonucleases and analysed by the Southern transfer technique. PstI, BamHI, and EcoRI digestion showed extensive fragment length polymorphism with 5-9 variable fragments per enzyme. The variable sequences were linked to HLA-DR. A BamHI 3.2 kilobases (kb) fragment was decreased in frequency among the Type 1 diabetic children (1/14) compared with either the healthy siblings (10/22, p < 0.05) or the control children (12/24, p < 0.02). A BamHI 5.8 kb fragment was increased among Type 1 diabetic children (10/14) compared with control children (8/24, p < 0.05). These results were largely due to an association with HLA-D2 and DR4, respectively. A 3.7 kb BamHI sequence was present in 50% (3/6) of parental HLA-DR4 containing chromosomes from control families, but was absent on the HLA-DR4 chromosomes (0/13) from diabetic individuals. These results suggest that the HLA-D region r-chain probe detects HLA-DR4 subtypes, only some of which are associated with Type 1 diabetes.

295. The Insulin Sensitivity Index Calculated from the Intravenous Glu- cose Tolerance Test Accurately Measures Insulin Sensitivity when Com- pared with the Glucose Clamp Method G. Pacini, D.T. Finegood, C. Cobelli and R.N. Bergman. LADSEB- CNR, Padova, Italy and USC, Los Angeles, California, USA We proposed the insulin sensitivity index (SI) as a measure of insulin resistance of peripheral tissues. S~ is defined as the increase in fractional glucose disappearance per unit increase in plasma insulin and can be determined from an intravenous glucose tolerance test (IVGTI') using a minimal model of glucose disappearance. Here we validate in eight normal dogs, Sj calculated from IVGTT, using the glucose clamp technique as an independent reference method. IVGTT: glucose was injected (0.3 g/kg) and 43 blood samples were taken over 3 h. K~ was 2.9 _+ 0.2/min (mean _+ SD). SI ranged from 1.3 to 6.1 x l0-4/min per mU/l . Euglycaemic clamps: Euglycaemia (Gb=5.2_+0A mmol/l) was maintained despite insulin infusions. Level I: insulin infusion = 8 mU/min (0-150 min), plasma insulin was elevated from 15 _+ 4 to 44 _+ 14 mU/ l (p < 0.01), glucose infusion was 108 _+15 mg/min. Level 2: insulin infusion = 40mU/min (151-300 rain), plasma insulin = 177 _+ 37 mU/1, glucose infusion = 357_+43 rag/rain. S~ ranged from 0.02 to 8.6 x 10-4/rain per mU/1. Validation: the two SI values (from clamp studies and IVGTY) were compared in each animal. The two independent measures were highly correlated (r= 0.93, p < 0.001) and the slope of their relationship was not different from 1.0 (slope = 0.85, p>0.2, NS). Conclusions: Sl from IVGTF measures insulin sensitivity accurately when compared with glucose clamp, and therefore constitutes a non-invasive, low-cost tool for quantifying insulin resistance in a routine clinical setting.

296. Study of Insulin Sensitivity in Newly Discovered Type 2 (Non-Insu- lin-Dependent) Diabetic Subjects by Tritiated Glucose Infusion, Hyper- glycaemic Glucose Clamp and Insulin Binding to Monocytes G. Pagano, A. Lombardi, E. Pisu, C. Bozzo, D. DeBenedictis and P. Masciola. Institute of Internal Medicine, University of Turin, Italy A group of 30 newly discovered Type 2 diabetic patients (WHO criteria), mean age 48_+5years, - 4 +20% ideal body weight, fasting blood glucose 110-155 mg/dl, was studied in order to evaluate: basal and sustained hyperglycaemia, endogenous glucose production (eGP) (by 3H-glucose infusion = rag. kg -1. rain-l), early (0 10 rain) and late (10-120 rain) mean plasma insulin levels during hyperglycaemic clamp (glycaemia 100 mg/dl over fasting value x 2 h), metabolic clearance rate of glucose (M-coefficient of DeFronzo • 100/mean glycaemia = ml. kg-1. min 1) and insulin binding to isolated monocytes (% of specific binding, R0 and Ke). Eight well matched healthy subjects were studied as controls. The mean results were: (1) endogenous glucose production similar in diabetic and control subjects (2.76_+ 0.94 versus 3.19 + 0.75 mg. kg -1- rain -1) but incomplete suppression was seen during hyperglycaemia in 10 diabetics; (2) similar mean plasma insulin (early 44_+6 versus 38+4, late 34_+3 versus 34_+2); (3) significant decrease of metabolic clearance of glucose (1.55 -4- 0.1 versus 3.01 -+ 0.36 ml- kg-1. min-1, p 0.001); (4) significant decrease of insulin binding (2.57 -+ 0.17 versus 3.93 _+ 0.35%, p 0.001). These results indicate that hyperglycaemia in Type 2 diabetes is mainly dependent on reduced glucose utilization and decreased insulin binding. Individual data show a wide variation in insulin response during clamp in diabetic patients, but an almost constant decrease of peripheral glucose utilization and insulin binding, suggesting that insulin resistance is the main pathogenetic mechanism in Type 2 diabetes.

297. Early and Aggressive Antihypertensive Treatment Reduces the Rate of Decline in Kidney Function in Diabetic Nephropathy H.-H. Parving, A. R. Andersen, U. M. Smidt and P. Aa. Svendsen. Ste- no Memorial Hospital, Gentofte, Department of Clinical Physiology, Bispebjerg Hospital, and Department of Medicine F, Herlev Hospi- tal, Copenhagen, Denmark The effect of early and aggressive antihypertensive treatment upon kidney function in diabetic nephropathy was studied prospectively in 10 Type I (insulin-dependent) diabetic patients with a mean age of 29 years. Renal function was assessed by the glomerular filtration rate (GFR, single bolus SlCr-EDTA technique) and the urinary albumin excretion rate (radial immunodiffusion). During the mean pretreatment period of 29 months (range 23-38 months) GFR decreased from 105 to 81 ml/min per 1.73 m 2 (p< 0.01), urinary albumin excretion increased from 607 to 1158 ug/min (p< 0.01) and arterial blood pressure rose from 130/88 to 155/103 mmHg (p< 0.01). GFR decreased by a mean of 0.91 ml/min per month (range 0.44 to 1.46 ml/min per month). Antihypertensive treatment was initiated with metoprolol, hydralazine and fruosemide or thiazide. During the 39 month (range 28-48 month) treatment period arterial blood pressure decreased from 144/97 mmHg (mean pretreatment) to 128/84 mmHg (p < 0.01), urinary albumin excretion diminished from 977 (mean pretreatment) to 433 ~g/min (p < 0.01), and GFR declined from 80 to 62 ml/min per 1.73 m z. The rate of decline in GFR decreased from 0.91 ml/min per month before treatment to 0.39 ml/min per month (range 0.08 to 0.68 ml/min per month) during treatment (p < 0.01). Our prospective study indicates that effective antihypertensive treatment reduces the decline in kidney function in young Type I diabetic patients with diabetic nephropathy.

298. Comparison of Monocomponent Porcine and Semi-synthetic Hu- man Insulin with Regard to Glucose Control, Insulin Requirement and anti-Insulin Antibodies P. N. Paus, H. H. Bassoe, K. Dahl-Jorgensen, T. Gjemdal, J. Jervell and O. Myking. Rikshospitalet, Oslo, Haukeland Sykehus, Bergen and Sentralsykehuset, Fredrikstad, Norway Preliminary results from a long-term double-blind investigation of the effect of transferring insulin-requiring diabetic patients from highly purified porcine to semi-synthetic human insulin (SHI) are presented. 106 patients entered the study. After a run-in period of 3 months, they were randomly assigned to two different groups. One group (n= 55, mean age 30years (range 15-61 years), mean duration of diabetes 15.4 years (range 2-43 years)) was given porcine short- and intermediate acting insulins (Novo). The other (n = 51, mean age 32years (range 18-53years), mean duration of diabetes 13years (range 2 30 years)) was transferred to the corresponding SHI. During the first 6 months, four patients were excluded because of pregnancy. Two subjects from each group withdrew because of hypoglycaemia. After 6 months, there were no significant differences (p> 0.05) in glucose control as judged by HbA1 (porcine 9.85% versus SHI 10.65%), in insulin requirement (porcine 42.7 IU/day versus SH142.9 IU/day) or in anti-insulin antibodies (in lab 4.19 versus 4.19; ratio of bound/free insulin porcine 0.88 versus SHI 0.73). In conclusion, the transfer from porcine insulin to SHI can be made without difficulty. After 6 months no differences in glucose control, insulin requirements or insulin antibodies were found.

299. Continuous Subcutaneous Insulin Infusion for 6 months Aggra- vates Non-Insulin-Mediated Glucose Metabolism and Postreceptor De- fects in Insulin Action of Adipoeytes from Type I (Insulin-Dependent) Diabetic Patients O. Pedersen, E. Hjollund, H. Beck-Nielsen, B. Rickelsen and N.S. Sorensen. Medical Department 3, County Hospital, Aarhus, Den- mark Twelve ketosis-prone Type I diabetic patients (mean age 28 years, mean duration of diabetes 11 years, mean daily insulin dosage 43 units) were treated with continuous SC insulin infusion (CSII) for 6 months. These patients were compared with age- and sex-matched groups of conventionally treated Type 1 patients and normal subjects. Compared with healthy subjects both groups of Type I patients had a 35% reduction in insulin binding to adipocytes (p< 0.01) accompanied by decreased insulin sensitivity of glucose transport and antilipo- lysis (p < 0.001) and decreased basal and maximally insulin stimulated glucose metabolism of adipocytes (p < 0.01). Insulin binding to monocytes and erythrocytes from Type I diabetic patients was similar to that of normal subjects. After CSII the mean diurnal plasma glucose concentration decreased from 9.8 + 1.9 to 6.8 _+ 1.4 mmol/l (p < 0.01) whereas the mean diurnal plasma insulin concentration was un-

186 Abs~acts

changed. Insulin binding to monocytes was 50% reduced (t7< 0.01) but to adipocytes and erythrocytes was unaltered. Compared with the pre-CSII-period both basal and maximal glucose metabolism of adipocytes was further decreased (p< 0.02). In conclusion, CSII for 6months aggravates the glucose metabolism of adipocytes from Type 1 diabetic patients. It is suggested that the improved glycaemic control of these patients during CSII may be due to a more appropriate control of insulin on hepatic glucose release, whereas the basal and insulin-stimulated glucose utilization of peripheral tissue are even more impaired.

300. Endogenous Versus Meal-Related Contributions to Total Rates of Post-prandial Glucose Production in Diabetes and the Effects of Con- tinuous Subcutaneous Insulin Infusion G. B. Pehling, P. Tessari, J. E. Gerich, M. W. Haymond, R. A. Rizza and F. J. Service. Mayo Clinic, Rochester, Minnesota, USA To determine whether post-prandial hyperglycaemia observed in conventionally treated diabetes is due to impaired suppression of endogenous glucose production or abnormal disposition of meal-related glucose, and whether 2 weeks of near-normoglycaemia from continuous subcutaneous insulin infusion (CSII) restores glucose disposal rates to normal, total rates of glucose turnover (2-3H glucose), meal-related glucose ([6,6-2H2] glucose added to the standard meal) and endogenous glucose production (total minus meal-related) rates were determined in seven lean Type 1 (insulin-dependent) diabetic patients and

f ive lean normal subjects. The diabetic patients were studied when poorly controlled and again under CSII. The elevated basal and peak plasma glucose from poor control (13 _+ 1.8, 23.7 _+ 1.1 mmol/1) were decreased by CSII to levels (5.2_+ 0.3, 10.7 _+ 0.6 mmol/1) approximat- ing the normal subjects (5.2 +0.1, 7.7-+ 0.7 retool/l). Total and endogenous glucose production in poor control (172_+29, 101_+ 26mmol.kg-1.480min 4) were decreased by CSII (88_+5, 26_+ 4mmol .kg- l -480min -1) to those of the normal subjects (95+2, 35 _+ 6 mmol. kg - 1.480 rain- 1). Meal-related glucose production did not differ between poor control, CSII and normal subjects (71_+4, 62 _+ 2, 61 _+ 5 mmol-kg-~. 480 rain-1 respectively). Conclusion: post- prandial hyperglycaemia in uncontrolled diabetes is primarily due to excessive endogenous glucose production which can be restored to normal by CSII.

301. Correlation between C-Peptide and Islet Cell Antibodies in Newly Diagnosed Type I (Insulin-Dependent) Diabetic Patients: A Follow-up Study M. Peig, R. Pujol-Borrell', I. Levy, R. Casamitjana, D. Figuerola and J. M.McNally 1. Servicio de Diabetes, Hospital Clinico Provincial, Barcelona, Spain and 1Department of Immunology, Middlesex Hos- pital Medical School, London, UK Cross-sectional studies have failed to show any relation between the presence of circulating islet cell antibodies (ICA) and beta cell function in Type 1 diabetic patients. However sera containing complement fixing islet cell antibodies (CF-ICA) have been shown to inhibit ro- dent islet function 'in vitro'. In order to study possible correlation between ICA and insulin reserve, an IV glucagon-C peptide test (samples taken at 0, 2, 4, 6, 8, 10 and 12 rain) was performed at diagnosis and at 3 and 6 month follow-up periods in a group of 34 Type 1 diabetic patients (age 17.1 + 6.0 years). C-peptide responses derived from the area under the time curve for C-peptide ratios (pmol x 102/mmol blood glucose) at onset, 3 and 6 months were 17.1 _+ 14.8, 44.5 _+ 27.1 and 27.4 + 21.5 in 23 patients persistently positive for ICA-IgG and 33.9 _+ 14.1, 48.0 + 31.3 and 70.3 + 31.9, respectively, in 11 ICA-negative patients. ICA-positive patients showed a significantly lower beta cell residual function at diagnosis and at 6months (p<0.01) compared with ICA-negative diabetic patients. CF-ICA-positive patients behaved in a similar manner to the ICA-positive group as a whole. These results indicate that ICA is a marker of a more rapid loss of beta cell function occurring in newly diagnosed diabetic patients.

302. Determination of 'Tingling' and Pain Threshold in Lower Limbs by Electrical Stimulation in Diabetic Patients With and Without Clinical Neuropathy R. Perciun, C.Ionescu-Tirgoviste, V.Vl~sceanu, V.Pop, M.Perciun, I. Privantu, and I. Mincu. Clinic of Nutrition and Metabolic Diseases, Bucharest, Romania We have determined the 'tingling' and pain threshold of an electrical stimulus (alternative asymetrical spike wave, 20 Hz), applied at left shank level through two acupuncture needles introduced at 30 cm distance between each other, and at approximately 1 cm depth. The mean current intensity value for the two sensibility thresholds was ex-

pressed in a A + SD. The study group included 98 diabetic patients (59male, 39female; mean age 56 +16 years) from which 66 were Type1 (insulin-dependent) and 32 Type2 (non-insulin-dependent); 60 cases (40 Type 1 and 20 Type 2) with and 38 cases (26 Type I and 12 Type 2) without clinical signs of peripheral neuropathy. The data have been compared with those obtained in 26 age-matched normal subjects. The results showed: (1) a large recorded current intensity value dispersion in the diabetic and normal groups; (2) the values were greater in the diabetic patients than in control subjects both for 'tingling' (79+39 versus 176 _+ 92 ~tA, p0.01) and for pain threshold (517 _+ 156 versus 556_+211 ~tA, p < 0.01); (3) parallel to a longer duration diabetes ( < 10 years, 10-20 years and > 20 years) we recorded an increase of both 'tingling' (141 _+ 102,183 _+ 112 and 211 + 132 ~tA) and pain threshold (398 _+ 208, 551 _+ 241 and 979-+ 386 ~tA respectively); (4) a significant statistical difference was recorded between diabetic patients with and without clinical signs of neuropathy (p < 0.01), both for 'tingling' (295 -+ 98 versus 116 -+ 72 ~tA) and pain threshold (882 _+ 219 versus 381 _+ 186 ~tA).

303. Effect of Sulphonylureas on 'In Vivo' Sensitivity to Insulin in Type I (Insulin-Dependent) Diabetes A.Pernet, F.Kuntschen and E.R.Trimble. Institute of Clinical Bio- chemistry, University of Geneva, Switzerland In a previous study, we showed that Type 1 diabetic patients were insulin-resistant at physiological insulin concentrations. The present study was undertaken to assess whether sulphonylureas could modify the sensitivity to insulin in Type 1 diabetes. Six male Type I diabetic subjects (age 34_+ 3 years, 108 _+ 4% ideal body weight, mean + SEM) without residual insulin secretion and without clinical insulin resistance (total insulin dose 43 _+ 5 U/day) were studied twice by the euglycaemic clamp technique; one of the two experiments was preceded for 15 days by glibenclamide treatment (3 x 5 rag/day). Insulin was infused for two periods of 2 h sequentially at 0.5 and 1.0 mU.kg -1. min -~. During both periods, the plateau levels of plasma free insulin were identical with or without glibenclamide. Glucose concentrations were maintained during the experiment at 4.9 + 0.03 mmol/1 without sulphonylurea and at 4.9 + 0.03 mmol/l with sulphonylurea. Glucose disposal rate was greater with glibenclamide: 2.7 _+ 0.45 versus 2.03 _+ 0.27 mg. kg -~ -rain -~ without glibenclamide during the first insulin plateau (NS), and 6.44 + 0.45 versus 5.59 + 0.59 mg. kg -1.min 1, respectively, during the second insulin plateau (p < 0.05). The sum of glucose infused over the last 30 min of the two periods was significantly higher with glibenclamide: 9.39 _+ 0.76 versus 7.82_+ 0.59 mg. kg -~. min -1 (p < 0.001). Thus, a short-term treatment with sulphonylureas improves 'in vivo' sensitivity to insulin in Type 1 diabetes at physiological insulin concentrations.

304. The Role of Portal Flow Diversion in Insulin Resistance of Cirrhotic Patients A. Pezzarossa, S.Contini, A. Marni, E. Capocasale, E. Bonora and U. Butturini. Department of Medicine and Departement of Surgery, University of Parma, Niguarda Hospital, Milan, Italy Impairment of carbohydrate metabolism and hyperinsulinaemia are common findings in liver cirrhosis. Resistance of peripheral tissues to insulin and reduced degradation of the hormone by the liver are accepted explanations for these metabolic features. The liver is the major target organ for insulin action and removal. To investigate the relative roles in insulin resistance of liver cell damage and diversion of portal blood flow to the liver we performed oral and IV glucose tolerance tests in ten cirrhotic patients a week before and 2 weeks after portocaval shunting. Insulin, C-peptide and blood glucose levels were evaluated. After portocaval shunting, these was a significant elevation of blood glucose between 0 and 180 min, a significant increase in insulin between 60 and 120 min and a significant decrease of C-peptide between 30 and 90 min during the oral glucose tolerance test. There were no significant variations of blood glucose and insulin during IV glucose tolerance tests. Unlike orally administered glucose, IV glucose is not taken up to a significant extent by the liver. These results confirm that the diversion of portal blood flow (spontaneously or sur- gically determined) is a more important factor in the impaired carbohydrate homeostasis in cirrhotic patients than the liver cell damage itself.

305. Protection Mechanisms of Pancreatic B Cells against Cytotoxic Agents D. Pipeleers and M.van De Winkel. Department of Metabolism and Endocrinology, Vrije Universiteit, Brussels, Belgium

Abstracts 187

Type 1 (insulin-dependent) diabetes develops after destruction of > 90% pancreatic B cells. To examine the mechanisms leading to B cell specific damage, single B cells - purified by fluorescence-activated cell sorting were briefly exposed to diabetogenic agents and then investigated during culture at glucose (5.5 mmol/1). More than 90% B cells were killed after addition of alloxan, streptozotocin or islet-cell surface antibodies isolated from Type 1 diabetic patients and supple- mented with complement; purified islet non-B cells were unaffected. Pretreatment of B cells with glucose (20 mmol/1) protected against alloxan-, but not against streptozotocin-cytotoxicity; this protection was attributed to the glucose-induced increase in cellular NAD(P)H; similarly, a lower alloxan toxicity was measured in B cells that were selected for their high NAD(P)H content. When B cells were exposed to alloxan, streptozotocin or complement, and then cultured with glucose (20 mmol/1) and/or nicotinamide, a 50-100% reduction in cytotoxicity was observed. These results indicate that the B cell toxic effect of alloxan, streptozotocin and diabetic islet-cell surface antibodies plus complement can be modified at the target site. It is suggested that the B cells can be protected by decreasing their sensitivity to cytotoxic agents or by stimulating their defence mechanisms.

306. Influence of Exercise and Alcohol on the Need for Insulin and Glu- cose Homeostasis under Hybrid Insulin Infusion K. Piwernetz, G. Fr6hlich, G. Kleppmeiel; R. Renner and K. D. Hepp. Stfidt. Krankenhaus Mtinchen-Oberf6hring, Munich, FRG It is well-known that exercise and alcohol ingestion can alter glucose homeostasis and consequently the need for insulin. To quantify these effects seven Type 1 (insulin-dependent) diabetic patients were treated with a hybrid (open-loop combined with closed-loop) insulin infusion (modified Siemens Promedos E 1). The infusion was started at the beginning of the meal with a rate of 4 U/h and was controlled simultaneously by a GCIIS (proportional-differential-algorithm). In a randomized sequence the patients received a standard meal (1425 kJ) containing 36 g carbohydrate with and without a preceding ingestion of 30 g pure alcohol (cognac 40 vol%) or in another experiment with and without a following exercise (50W, duration 3 min, rest 2 min; nine times repeated). Exercise reduced the meal-related need for insulin by 42% (p< 0.01), alcohol by 25% (p< 0.05 both procedures compared to their respective control period). Mean blood glucose was markedly lower with alcohol than without. Therefore diabetic patients treated with continuous insulin infusion should be well informed of these important influences on glucose concentration. Otherwise they run the risk of hypoglycaemia during post-prandial periods of hyperinsulinaemia. Moreover, the findings demonstrate in- herent problems for outpatients with usual muscular activity who are treated with insulin doses determined by GCIIS under bedside conditions.

307. The Fast Acetylator Phenotype in Diabetes Mellitus: Abnormal Prevalence and Association with Blood ABO Groups and with the Chlorpropamide-AIcohol-Flush A. E. Pontiroli, A. De Pasqua, A. Mosca, L. Bonisolli and G. Pozza. Clinica Medica VIII and Chimica Biologica III, University of Milan, Istituto Scientifico San Raffaele, Milan, Italy The acetylator phenotype and blood ABO groups were determined in 55 normal subjects and in 156 diabetic patients (6"1 with Type "1 and 95 with Type 2 diabetes). The prevalence of fast acetylators was significantly higher in Type "1 diabetes than in control subjects (52.5 versus 29."1%, p< 0.02). In Type 2 diabetes, the prevalence was 38.9% (NS versus controls and versus Type I diabetes). In Type 2 diabetes, but not in controls or in Type 1 diabetes, a significant association was found between the fast acetylator phenotype and the B blood group, since the B group was present in 24.3% of fast acetylators and in 5A% of slow acetylators (p<0.02). A small additional group of diabetic patients ("18 with Type 1 diabetes and 32 with Type 2 diabetes) was tested for the acetylator phenotype and for the occurrence of chlorpropamide alcohol flush (CPAF, 40 ml Vermouth after chlorpropamide, 250 mg twice a day for 2 days). CPAF was more frequent in fast than in slow acetylators (6"1."1 versus 25.8%, p< 0.05).

308. Red Cell Sorbitol Determined by Capillary Gas Chromatography in Type t (Insulin-Dependent) Diabetic Patients Before and After Sorbi- nil Treatment C. Popp-Snijders, M.Z. Lomecky-Janousek, J.A.Schouten and E. A. van der Veen. Departments of Endocrinology and Internal Med- icine, Free University Hospital, Amsterdam, The Netherlands Intracellular accumulation of sorbitol in lens and nerve tissue of diabetic patients is thought to contribute to diabetic-associated complica-

tions, such as cataract and neuropathy. To study the effect of aldose reductase inhibitors on intracellular sorbitol contents, erythrocytes can be used as a model for the less accessible tissues. Results of red cell sorbitol estimations, however, vary considerably, probably due to lack of specificity of the method used. We developed a specific (confirmed by mass spectrometry) and precise method (interassay coefficient of variation = 4%) to measure red cell sorbitol, using fused silica capillary gas chromatography. Subsequently we studied the effect of the aldose reductase inhibitor sorbinil (CP-45,634) on the sorbitol content in erythrocytes of 15 Type 1 diabetic patients. Following a 4- week placebo period, a 4-week treatment with 200 mg sorbinil once daily decreased the mean red blood cell sorbitol content from 13.6 + 5.1 to 3.3 + 1.2 nmol/ml (mean + SD). The mean red cell sorbitol content in 19 healthy subjects was 5.8 _+ 1.6 nmol/ml. The decrease of the red cell sorbitol content in diabetic patients after the sorbinil treatment was not the result of better diabetic control, as indicated by unchanged percentages of glycosylated haemoglobins.

309. Non-Insulin-Dependent Diabetes in the Adult Rat as the Spontane- ous Evolution of Neonatal Diabetes: Insulin and Glucagon Secretion In Vitro B. Portha, M. H. Giroix, M. Kergoat and L. Picon. Laboratory of Phys- iology, University of Paris 7, Paris, France Non-insulin-dependent diabetes was produced in adult rats following a neonatal streptozotocin injection. Rats with non-insulin-dependent diabetes exhibited chronic low insulin response to glucose in vivo and slightly elevated basal plasma glucose values (< 11 mmol/1). Insulin and glucagon secretions were evaluated by isolated pancreas perfusion. Insulin response to glucose (5.5-22 mmol/1) was lacking. Even in the presence of theophylline, the B cells remained insensitive to glucose. In contrast, glyceraldehyde elicited an insulin release as high as that in the controls, thus indicating that the B cell dysfunction in non-insulin-dependent diabetic rats involves a block in glucose metabolism in the early steps of glycolysis. In the absence of glucose the B cells of non-insulin-dependent diabetic rats exhibited hypersensitivity to arginine and leucine. Glucagon release was normal in the basal state; in response to arginine and acetylcholine it was lower than in controls; it was suppressed by glucose as efficiently in diabetics as in controls. Thus, in this model of non-insulin-dependent diabetes, extreme glucose insensitivity of the B cells contrasts sharply with normal or occasionally higher response to other stimuli.

310. First Year Follow-up Study of Lymphocyte Subsets in Susceptible Relatives of Type I (Insulin-Dependent) Diabetic Families P. Pozzilli, M. Sensi, K.M. Spencer, O.Zuccarini, L. A1-Sakkaf and D. Doniach. Departments of Diabetes and Immunogenetics and Im- munology, St. Barholomew's Hospital and Middlesex Hospital, Lon- don, UK and Cart. Endocrinologia, University of Rome, Italy A prospective study of lymphocyte subsets in genetically and immu- nologically susceptible first degree relatives of Type 1 diabetic probands was started in order to search for early cell mediated markers leading to beta cell destruction. Subjects: 10 complement fixing islet cell antibody (CF-ICA) +, 12 conventional ICA(IgG-ICA) § and 29 1CA-, all of whom had at least one HLA haplotype in common with the diabetic proband. Lymphocytes were collected every 4 months and phenotyped with a panel consisting of the monoclonal antibodies: UCHT1 (total T cells), Leu 3a (helper cells), UCHT4 (suppressor/cytotoxic cells), H25 (K/NK cells), 41=2, TAC (activated T cells) and DA2 (Ia cells). Minimal variations of total T cells, helper T cells and K / N K cells were observed. On the other hand, the suppressor/cytotoxic T cell subset showed a significant decrease with time in both CF-ICA + (30_+7% versus 19+9%) and IgG-ICA + subjects (33+4% versus 25• but not in the ICA- subjects. Two CF- ICA § subjects showed repeatedly activated T cells. These preliminary results suggest that modifications of lymphocyte subsets may provide further indication of the immunological abnormalities preceding onset of overt diabetes.

311. Regulation of Ca z+ Transport by Rat Insulinoma Microsomes M. Prentki, D.Janjic and C.B.Wo]lheim. Institute of Clinical Bio- chemistry, University of Geneva, Geneva, Switzerland In an attempt to define which of the intracellular organelles participate in the regulation of cytosolic Ca 2§ during secretagogue-induced insulin release, Ca 2+ handling by rat insulinoma microsomes was investigated. The regulation of extramicrosomal [Ca2+]o concentration maintained by suspensions of microsomes was studied using Ca 2 +-selective mini-electrodes. Microsomal Ca2-uptake was MgATP-dependent, indicating Ca 2+ ATPase activity. No evidence for a Na+/Ca 2+

188 Abstracts

counter-transport was obtained. Within 5 min, microsomes lowered medium [Ca2+]0 to about 0.2 ~xmol/l. They had a high Ca 2+ sequester- ing activity as judged by their capacity to take up and retain several se- quential Ca 2+ pulses. The accumulated Ca 2+ could be released either by the Ca 2+ ionophore A23187 or by transforming ATP into ADP by the addition of glucose plus hexokinase. Cyclic AMP, 3-isobutyl-l- methylxanthine and Na + were unable to release Ca 2+. ADP (I mmol/1) but not AMP induced a reversible net efflux of Ca 2+ from the microsomes, raising [Ca2+]0 from 0.2 to 0.4 gmol/l. It is concluded that insulinoma microsomes are able to maintain extramicrosomal [Ca2+]0 in the low submicromolar range and might thus contribute to the regulation of cytosolic Ca 2 + and insulin release.

312. Does Insulin Pump Treatment Impair Glucose Counter-regulation ? M. Press, W. Tamborlane, D. Simonson and R. Sherwin. Yale Univer- sity, New Haven, USA Hypoglycaemia is a major risk of continuous subcutaneous insulin infusion (CSII). To determine whether CSII impairs glucose counter- regulation, two studies were performed. First, the hormonal response to hypoglycaemia was measured in six diabetic patients before and after 4-8 months of CSII. On each occasion, patients received IV insulin (J mU. kg -~. rain-l). Plasma glucose was clamped at 5 mmol/1 for 2 h, then allowed to fall to 2.8 mmol/l where it was clamped for another hour. Despite an identical hypoglycaemic stimulus, reduced plasma levels of both adrenaline (0.7 versus 1.7 nmol/1, p < 0.05) and cortisol (0.3 versus 0.6 umol/l, p < 0.05) were observed after CSII. Sec- ondly, physiological amounts of exogenous cortisol and adrenaline were infused into six CSII patients and six conventionally-treated control subjects. In CSII patients the increase in plasma glucose in response to adrenaline was reduced (3.6 versus 6.8 mmol/1, p < 0.05, after 4 h) while the hyperglycaemic effect of cortisol (4.7 retool/1 after 6 h in controls) was totally abolished. These diminished glycaemic responses were mediated by corresponding reductions in hormone

3 -stimulated hepatic glucose production ( H-glucose turnover). Thus, CSII reduces not only the secretion of adrenaline and cortisol in response to hypoglycaemia, but also the hepatic response to these hormones. Impaired counter-regulation may be a potentially serious side-effect of CSII.

313. Oestradiol Modulates Insulin-stimulated Gluco-regulatory En- zymes in Mouse Soleus Muscle J. A. Puah and C. J. Bailey. Department of Biological Sciences, Univer- sity of Aston in Birmingham, Birmingham, UK Oestradiol improves the hypoglycaemic action of insulin in ovariectomised female mice. This is associated with increased glucose uptake and phosphorylation, glycogen formation and glucose oxidation in soleus muscles. Increased insulin receptor binding may account in part for these effects, as reported previously. This study examines postreceptor sites at which oestradiol modulates insulin action through alterations in the activities of rate-limiting glueo-regulatory enzymes. Maximum activities of hexokinase (EC 2.7.1.1), glycogen synthase (EC 2.4.1.11) and 2-oxoglutarate dehydrogenase (EC 1.2.4.2) were determined in the absence and presence of insulin (750 mU/1), using soleus muscle homogenates from ovariectomised mice treated orally for 10 weeks with either placebo or a replacement dose of oestradiol- 17fl (5 gg. kg- 1. day- ~). Oestradiol significantly increased the activities of hexokinase type II and the active (I) form of glycogen synthase by 47% and 34% respectively in the absence of insulin, and 51% and 56% respectively in the presence of insulin. 2-oxoglutarate dehydrogenase activity was increased (98%) by oestradiol only in the presence of insulin. The results indicated that oestradiol improves the hypoglycaemic action of insulin in female mice partly by enhancing the activities of enzymes which regulate glucose phosphorylation, glycogenesis and glucose oxidation in skeletal muscle.

314. The Effect of Additional Insulin on Intravenous Glucose Tolerance and the Forearm Uptake of Glucose Compared to that following Oral Glucose J. Radziuk. University of Western Ontario, London, Canada We have shown previously that the forearm glucose uptake and insulin levels are increased following oral glucose administration, compared to the IV case. To test the hypothesis that insulin is the cause of this improvement in glucose tolerance, the following studies were performed: six normal overnight-fasted subjects were given on three separate occasions (1) a 100 g glucose drink, (2) 90 g IV glucose infused at the absorption rate of glucose and (3) the same IV infusion with additional insulin infused peripherally during the first 2 h, attempting to match plasma insulin concentrations following oral glucose. Arterial-

ized hand vein glucose and insulin concentrations and deep vein glucose levels were determined. Forearm blood flow was measured by capacitance plethysmography. Arterial glucose rose to peaks of 159 _+ 7, 209 +_ 5 and 149 + 8 mg/dl for the three cases. Glucose and insulin concentrations were not significantly different at any point for the oral and IV+ insulin cases (p> 0.2) for the 180 rain monitored, but were both higher than following the IV case during the first hour. This situation was reflected in the forearm glucose uptakes: (1) 25.8 _+ 1.7, (2) 15.5 _+ 2.0 and (3) 27.4 + 1.8 mg- dl-1. h-1. Conclusions: by infusion of peripheral insulin alone we can match both the insulin levels and the glucose tolerance seen after oral glucose. That this improvement is at least partially peripheral can be seen from the 70% increase in forearm glucose uptake compared to IV glucose infusion.

315. Persistent Neonatal Hypoglycaemia: Deficiency of D Cells or Hy- peractivity of B Cells? J. Rahier, S. Falkmer, K. Falt, H. Muntefering, K. Becker and W. Gepts. Departments of Pathology, Universities of Louvain, Belgium, Lund, Sweden, Mainz, FRG and Brussels, Belgium The pathogenesis of the syndrome of neonatal persistent hypoglycaemia remains poorly understood. After immunoperoxidase staining, B, A, D and pancreatic polypeptide (PP) cells were quantitated morphometrically in the pancreases of 15 hypoglycaemic infants with hyperinsulinism and of 23 age-matched controls. The size of B-cell nuclei was also measured as an index of functional activity. Nesidio- blastosis was observed not only in hypoglycaemic infants but also in controls, and thus is not pathognomonic for the disease. An adenoma essentially containing B cells was present in three cases. The volume density of all cell types and the size of B-cell nuclei were normal out- side the adenoma. In 12 cases no localized lesion was found. The volume density of B cells was not significantly increased; that of A cells was unchanged and that of PP cells was markedly augmented. The average volume density of D cells was decreased by 30%, but was within the normal range in certain cases. B cells with a very large nucleus were present in all hypoglycaemic infants and the average nuclear ra- dius of B cells was increased by nearly 12%. The data suggest that this syndrome could result from the hyperfunctioning of a certain population of B cells, rather than from marked changes in the mass of B or D ceils.

316. Synergism between HLA-DR3 and DR4 Homozygosity and Level of Hyperglycaemia as Risk Factors for Proliferative Diabetic Retinopa- thy L. I. Rand, A. S. Krolewski, J. H. Warram and J. S. Soeldner. Joslin Dia- betes Center, Boston, Massachusetts, USA Severity of diabetes and HLA-DR phenotype were examined as risk factors for proliferative diabetic retinopathy (PDR). Cases of PDR (n = 101) and controls without retinopathy (n = 93) were all insulin-dependent, matched for duration of diabetes (mean 25 years) and followed at Joslin Clinic since diagnosis. The proportion of visits with post-prandial glycaemia >- 11.1 mmol/l was used as an index of diabetes severity. Patients with an index >-- 50% had a higher risk of PDR than those with an index < 50% (odds ratio =4.6,p< 0.001). Apparent homozygotes for HLA-DR (4/0, 3/0) also had increased risk of PDR relative to heterozygotes (odds ratios = 4.6, 2.2). The odds ratio for all homozygotes was 2.8 (p<0.02). This association could not be explained by a relationship between HLA-DR phenotype and severity, whether measured by this index, HbAlc, glucagon or growth hormone response to IV arginine. Among those below the median in our severity index, there was little relationship between HLA-DR phenotype and PDR; while in those above the median, ie. severe diabetes, homozygotes were in great excess among cases and deficient among controls (odds ratio = 9.6, p < 0.005). In summary, HLA-DR homozygosity seems to magnify an association between PDR and severity of hyperglycaemia.

317. Different Endogenous Insulin and Glucagon Secretion Inhibition by Human Insulin (Recombinant DNA) in Man S. Raptis, F. Enzmann, D. Hadjidakis, A. Raptis, J. Hadjioannou, E.Diamantopoulos j and N.Thalassinos. Second Department of In- terual Medicine-Propaedeutic and 1Department of Clinical Thera- peutics, Athens, Greece The action of human insulin (BHI) and purified porcine insulin (PPI) on the secretion of C-peptide and glucagon was investigated and compared after the latter was stimulated by a protein meal in 11 healthy volunteers. Both insulins were administered as a constant infusion for 180 min at 20 mU/min. Significant inhibition of C-peptide (from 1.3 + 0.3 to 0.4 _+ 0.2 ng/ml) and of glucagon (from 120 + 42 to

Abstracts 189

44 + 14 pg/ml) by BHI occurred under basal conditions and without any change of blood glucose. The stimulated secretion was also inhibited but with greater significance (p < 0.005) and for a longer period by BHI than by PPI. The surface areas of C-peptide were 120+ 21 ng. min- 1. m1-1 under B HI and 225 + 70 ng. rain- 1. ml-1 under PPI, while those of glucagon were 12 000 + 2114 pg. min - 1. ml- 1 under BHI and 20400 + 3 212 pg. min 1. ml-1 under PPI. It is thus demonstrated that both A and B cells of human pancreas recognize BHI as homologous insulin compared with porcine insulin. It is concluded that a more physiological action and better diabetic control can be achieved by human insulin compared with purified porcine insulin.

318. Site-Specific lodination of Insulin and Measurement of Insulin Antibodies M.J. Rathbun, S.E.Fineberg and B.H. Frank. Indiana University School of Medicine and Lilly Laboratories for Clinical Research, In- dianapolis, Indiana, USA Variability in iodination sites may lead to differences in detection and quantitation of insulin antibodies. Monoiodinated insulins usually consist of mixtures of A14, A19, B16 and B26 labels. Monoiodinated insulins were purified by high pressure liquid chromatography and identified by sequence analysis. Eight human antisera were studied with pork insulin iodinated at each site and titrated against non-radioactive pork insulin (0-1.9 x 10-6mol/1). Median characteristics were for A14, AIg, B16 and B26 respectively: (%) 44.9, 34.8, 48.3 and 33.5; non-displaceable binding (%) 6.9, 3.7, 7.3 and 4.6; N1 ( x 10 -6 tool/l) 1.7, 2.5, 2.0 and 2.0; N2 ( x 10 6 tool/l) 40.6, 33.2, 44.9 and 21.5. The probability that characteristics differed among labels were: < 0.001, 0.001, 0.02 and 0.04, respectively. When displacement curves were compared, initial binding, non-displaceable binding (at 1.9x 10-6mol/1) and high affinity binding sites were B16>A14> B26 > A19. Individual antisera differed greatly. Iodination site significantly affects antibody measurements. Inconsistency in labelling and purification will lead to increased variability and error in detection and quantitation of insulin antibodies.

319. A Novel Bio-Artificial Pancreas with Rapid Glucose-lnsulin Kinet- ics G. Reach, M.Y.Jaffrin and J.F. Desjeux. INSERM U83, Paris and CNRS ERA 999, Compi~gne, France To date, insulin release from hybrid pancreatic devices in response to glucose is delayed by a lag which is not compatible with closed-loop insulin delivery. We devised a novel bio-artifical pancreas according to a model of glucose transmission achieved by ultrafiltration across the membrane. In this system, islets are placed between two fiat poly- acrylonitrile membranes (AN69S, Rhone-Poulenc). Blood circulates successively above the first, and below the second membrane in the reverse direction, resulting in a U-loop surrounding the islets. Theo- retical analysis indicates the presence of a counter-current flux, i. e. ultrafiltration across the first, and reabsorption of fluid across the second membrane. The system was perfused in vitro (4 ml/min) with Krebs buffer, the glucose concentration of which was increased from 2.8 to 20 retool/1. Insulin release in the effluent of the device increased significantly within 3 rain and reached a maximal level after 10 rain, which was fourfold higher than the insulin concentration detectable simultaneously at the turning point of the U-loop. This indicates that, as predicted, insulin was mostly dragged by the counter-current. Fi- nally, insulin release returned to basal levels 20 rain after the end of the stimulation. Conclusion: this 'ultrafiltration chamber' has appropriate glucose-insulin kinetics and is therefore a potentially implant- able closed-loop insulin delivery system.

320. Platelet Behaviour in Diabetic Ketoacidosis, Paradoxical Insensi- tivity to Adenosine Diphosphate J. P. D. Reckless, R.R. Campbell, C.M. Stirling, D. Mundy and K.J. Foster. Royal United Hospital, Bath, UK Diabetic ketoacidosis may be complicated by thromboembolism, but possible altered platelet behaviour has been little studied therein. Ten ketoacidotic patients (pH 7.0), aged 15-65 years, were studied during conventional infusion treatment with human insulin, on admission, daily for I week and at 2 weeks. Plasma platelet-specific proteins, fl- thromboglobulin (~FG) and platelet factor 4 (PF4), were measured by radioimmunoassay. Platelet aggregation induced by adenosine diphosphate (ADP sensitivity), and its half-inhibition by prostacyclin (PGI IDs0 ) were measured in an aggregometer. Platelet data was log- transformed before analysis. (mean, (+ SD, - S D ) ) flFG: 90.2 ng/ml (153.4, 53.1) and PF4 (75.3 ng/ml (105.3, 53.8)) were initially elevated, falling to nadir at day 3 (41.5 ng/ml (52.9, 32.5), p < 0.01 ; 15.1 ng/ml

(24, 9.5), p < 0.01, respectively). ADP sensitivity was abnormally reduced (4.94nmol/1 (10.5, 3.0)) increasing on day 1 (2.4nmol/l (2.9, 2.0), p < 0.05) and day 2 (1.49 nmol/1 (1.64, 1.35), p < 0.001), with little change thereafter. PGI2 IDs0 was initially reduced (12.8 nmol/1 (21.5, 7.6)), sensitivity increasing by day 1 (3.8 nmol/1 (7.55, 1.9), p< 0.01) with little subsequent change. In-vivo measures of platelet aggregation (fiTG, PF4) and prostacyclin sensitivity improve with treatment, but paradoxically ADP sensitivity increases from initial low levels with possible thrombogenic significance. This could reflect pH, granule depletion, insulin lack or phospholipid change.

321. Apo A-I Gene Polymorphism in Diabetic and Non-Diabetic Hypertriglyceridaemia A. Rees, C.C. Shoulders 1, J. Stocks, F.E. Baralle I and D.J.Galton. Medical Professorial Unit, St. Bartholomew's Hospital, London and ~Dunn School of Pathology, University of Oxford, UK Hypertriglyceridaemia is commonly associated with diabetes mellitus. Recently a DNA point mutation has been identified flanking the 3'-end of the apo A-I gene that appears to segregate with hypertriglyceridaemia. It's prevalence has been studied in diabetic and non-diabetic hypertriglyceridaemic subjects (matched for sex and plasma triglycerides) to see if it discriminates between the two. Leucocyte DNA was digested with Sstl restriction endonuclease, and Southern blotted with a cloned 32p-apo A-I gene probe. Gene fragments were identified by autoradiography. Thirty-six normolipaemic, non-diabetic control subjects were genotyped, and no polymorphic allele was identified. Of 10 hypertriglyceridaemic diabetic patients analysed, five displayed this polymorphic allele (p< 0.001 compared to controls). Further- more, seven out of 18 non-diabetic hypertriglyceridaemic patients displayed the polymorphic allele (p < 0.001 versus controls). Thus, whilst the frequency of the DNA polymorphism flanking the Y-end of the A-I gene is significantly increased in diabetic lipaemic subjects compared to controls, it does not appear to distinguish between diabetic and non-diabetic hypertrigtyceridaemia.

322. Continuous Intraperitoneal Insulin Treatment in Type I (Insulin- Dependent) Diabetes: Comparison Between Square Wave Infusion and Bolus Application R. Renner, K. Piwernetz and K.D. Hepp. Stfidt. Krankenhaus Mfinchen-Oberfrhring, Mtinchen, FRG The optimal meal-related insulin infusion pattern during IP infusion is still unknown. After a period of excellent control five Type I diabetic patients were therefore treated at lunch on two successive days by a 60-min square wave infusion (SWI) (Promedos E 1 pump, Siemens) and by a bolus (CPI model 9100, Lilly) in a randomised fashion. Each procedure delivered 11% of a patient's daily insulin requirement. Glu- cose and insulin curves (free insulin) were compared with those from a group of six healthy volunteers who also received a test meal (1900 k J) with 48 g carbohydrate in the form of rice. Meal and insulin were started simultaneously. Peak insulin values at 35 mU/1 were gained after 30min with bolus, and after 60min with SWI. Fifteen min after commencement of SWI, insulin was still at zero level (p < 0.002 versus bolus treated group). Peak concentrations from healthy volunteers were not exceeded by either treatment. Glucose values under bolus treatment decreased during the first 30 min (p < 0.001 versus SWI, p < 0.01 versus control subjects). Peak glucose was obtained after 60 rain with bolus and SWI and after 45 min in case of healthy subjects. Difference between peak and initial glucose concentration was 16mg/dl for bolus, 54mg/dl for SWI (p<0.05), and 15mg/dl for healthy volunteers. Thus neither bolus nor SWI treatment renders physiological conditions. A mixture of both might optimize diabetic control.

323. Beta-Thromboglobulin, Platelet Factor 4, Platelet Retention, Aggregation and Circulating Aggregates in Diabetid Patients J. Reynders, J.J. Rodzynek, P. Leautaud and A. Delcourt. Department of Internal Medicine, H6pital d'Ixelles, Brussels, Belgium In a prospective study, beta-thromboglobulin, platelet factor 4, platelet retention, platelet aggregation and circulating p]atelet aggregates were measured in 58 healthy volunteers (control group) and in 72 consecutive ambulatory diabetic patients. The average values of beta- thromboglobulin, platelet factor4 and platelet retention were significantly higher in the diabetic group (p<0.01), while no statistical difference was observed for circulating platelet aggregates and platelet aggregation. A high correlation was found between beta-thromboglobulin and platelet factor 4 values (r= 0.84, p < 0.05). Beta-thromboglobulin, platelet factor4, platelet retention, platelet aggregation and circulating platelet aggregates values were respectively abnormal

190 Abstracts

in 52.2%, 59.7%, 20.8%, 11.I% and 19.4% of the diabetic patients and in 1.7%, 0%, 0%, 1.7% and 0% of the control patients. Beta-thromboglobulin and platelet factor 4 appear to be the most frequent abnormal parameters. In our series, these abnormalities were not related to the presence of micro- or macroangiopathy nor with the degree of blood glucose control (mean HbAlc) or with the type of treatment. The significance of altered platelet function in diabetes mellitus remains to be investigated.

324. Effect of Mouse Fibroblast Interferon on Insulin Secretion, Total Protein and Proinsulin Biosynthesis in Mouse Pancreatic Islets C. J. Rhodes and K. W. Taylor. Department of Biochemistry, The Lon- don Hospital Medical College, London, UK In response to a viral infection, interferon may be produced by many host cells, to inhibit protein synthesis in an effort to halt viral replication. We investigated the effect of incubating mouse fibroblast interferon (500 units/ml) for varying time periods between 1-50 h, at a glucose concentration of 2 retool/l, in isolated islets from fed DBA/2 adult male mice. Islets were then assayed for insulin secretion, total protein and proinsulin biosynthesis in response to glucose concentrations of 2 and 20 retool/1. In islets incubated with interferon from 4 h onwards, there was an observed reduction of 20-30% in total protein biosynthesis at both glucose concentrations in comparison with control islets. A similar effect was observed for proinsulin biosynthesis at glucose (2 mmol/1). However, using glucose (20 mmol/1), interferon produced a selective inhibition of proinsulin biosynthesis (60-70%), statistically significant after 7 h incubation (p < 0.02). This effect might be one of the factors inhibiting proinsulin biosynthesis when islet B cells are infected with some viruses. By contrast, interferon had no effect on insulin secretion at glucose (2 or 20 mmol/1).

325. Extrapancreatic Action of the Sulphonylurea Gliquidone: Post-Re- ceptor Effect on Insulin-Stimulated Glycogen Synthesis in Rat Hepato- cytes in Primary Culture F.Rinninger and W.Kemmler. Institute for Diabetes Research, Munich, FRG The mechanism of the extrapancreatic action of the sulphonylurea drugs is still not well understood. We have therefore studied in vitro the effect of sulphonylurea on insulin binding and insulin action in the liver. Rat hepatocytes were maintained in primary culture for 48 h; during the second 24 h of culture, the cells were incubated with or without the sulphonylurea drug gliquidone (5.0 gg/ml). After culture, 125I-insulin binding (2 h, 20 ~ and glycogen synthesis rate from 14C glucose (2 h, 37 ~ were measured. Gliquidone had no effect on insulin binding or basal rate of glycogen synthesis. Insulin (10-10,000 m U/l) stimulated glycogen synthesis rate in a dose-dependent manner with a maximal effect of 250% above the basal rate (insulin 1000 m U / 1). Gliquidone increased glycogen synthesis rate at all insulin concentrations tested significantly, reaching, for instance, 250 to 320% above basal level at insulin concentration 1000 mU/1. Half-maximal stimulation was reached in both groups at insulin about 80 mU/1. Thus, responsiveness was increased but sensitivity was unchanged. These results permit the conclusion, that (a) sulphonylureas have an extrapancreatic effect which (b) is mediated by a post-receptor mechanism.

326. Is Physical Activity Related to Glucose Tolerance? A. Rivellese, G.Annuzzi, O.Vaccaro and G.Riccardi. Department of Internal Medicine, 2nd Medical School, University of Naples, Italy The aim of this study was to evaluate whether a relationship exists between impaired glucose tolerance (IGT) and physical activity. 65 subjects with IGT (according to EASD criteria), age range 40-59 years, and 125 normal subjects, age sex and weight matched were selected from the participants in a healthy examination survey. They were retested by oral glucose tolerance test 2 and 4 months apart. Immediate- ly after, they were asked to fulfil a questionnaire on daily physical activity during work and leisure time and then to exercise on a cycloer- gometer for 20 rain at a work load of 75 W for males and 60 W for females. No difference in physical activity at work was recorded between the groups; conversely the proportion of individuals who were physically active during leisure time was significantly lower among IGT subjects (19.7% versus 8.1%, p<0.05). Similarly IGT subjects showed a reduced working capacity, as demonstrated by the increased heart rate at the end of standard exercise period (113.4+ 15.8 versus 105.7 + 17.7 beats/min, p < 0.005; mean-+ SD). The difference was even more striking between individuals with IGT on two occasions and those with two normal oral glucose tolerance test results (114.8 _+ 15.4 versus 104.6 + 17.8 beats/min, p < 0.001). This supports

the hypothesis that low physical activity is an independent risk factor for IGT.

327. Diabetic Mortality and Morbidity Statistics in the Evaluation of Diabetic Care N. Robinson, S. P. Edmeades and J. H. Fuller. The Middlesex Hospital Medical School, London, UK Emphasis is currently being placed on methods designed to improve the metabolic control of diabetic patients; such as providing diabetic community nurses and the use of home blood glucose monitoring. As these methods are expensive, their impact on measures of diabetic mortality and morbidity should be evaluated. For England and Wales (1968-1980) mortality rates for diabetes as the underlying cause of death have been examined. Rates for females declined steadily for all age groups (p < 0.05) but remained relatively constant for males. Dia- betic mortality rates showed considerable regional variation, being higher in the North compared with the South-East. Hospital discharges for diabetes as a percentage of discharges for all causes were considered as a measure of morbidity for England and Wales (1968-1978) and there was a significant increase for both sexes, particularly males with highest percentages in the North and West. A similar analysis was carried out for 32 health districts in North Lon- don for 1975-1981 during which time nine districts introduced diabetic community nurses. Using 3-year moving averages a marked fall in the percentage of diabetic discharges after 1978 was apparent in districts implementing this service.

328. Atropine Lowers the Elevated Insulin Levels of Genetically Pre- Obese Rats in Response to Glucose or Arginine F. Rohner-Jeanrenaud, S.C. Woods and B.Jeanrenaud. Laboratoires de Recherches M6taboliques, Geneva, Switzerland The aim of the study was to determine the aetiology of obesity in genetically obese (fa/fa) rats. Normal and pre-obese rats were tested at 17 days of age. All animals were then weaned. Those which became obese were retrospectively designated 'pre-obese'. For the tests, glucose (300 mg/kg) or arginine (600 mg/kg) was injected IV with or without atropine (5 mg/kg). Plasma insulin (IRI) levels were measured 2.5 and 10 rain later. Areas under the response curves for glucose-induced IRI output were (ng/10 min): lean (n = 8): 26.6 _ 3.8; pre-obese (n= 7): 39.7 + 3 (2p < 0.02). Pre-obese rats given atropine (n = 9) had a response (30.4 + 3) statistically identical to that in lean rats (in which atropine had no effect). Response curve areas for arginine-induced IRI output were: lean (n = 21): 67 + 9; pre-obese (n = 5): 99_+11 (2p<0.05). Atropine lowered the values in pre-obese rats (n = 6) to 58 _+ 8; ie, statistically the same as in lean rats given atropine. We are currently investigating the effect of vagotomy at age 17 days on the subsequent development of obesity. We conclude that substrate-induced insulin oversecretion in pre-obese rats is cholinergical- ly mediated. As it occurs early, it may cause overstimulation of lipogenic pathways and contribute to obesity.

329. Evidence for a Disturbed Metabolism of Glucose in the Clonal B Cell Line RINm5F R Rorsman and E. Grapengiesser. Department of Medical Cell Biolo- gy, University of Uppsala, Uppsala, Sweden In normal pancreatic B cells the metabolism of glucose seems to be a prerequisite for the recognition of the sugar as a secretory stimulus. Glucose metabolism is therefore a primary target for investigation when elucidating why RINm5F cells do not respond appropriately with a stimulated insulin release upon exposure to the saccharide. In the present study, the utilization and oxidation of glucose were measured as the production of 3H20 and 14C02 respectively. The RINm5F cells exhibited typical tumour cell features such as a high glycolytic flux with an oxidation-to-utilization ratio as low as 0.10-0.15 (normally -0.50). Maximal oxidative degradation was attained already at 1 mmol/l. This concentration was also equivalent to the Km for the glucose utilization, indicating a substantial left-hand shift of the dose- response curve compared with the normal B cell. Other abnormal characteristics were decreased glucose oxidation at concentrations of

tahne d S~ggar baeyOe~ldr4e~tl~Ul, xa2ditohe Tfi~ldi~ligsttuhr~te~mmiSe~ia~nlofCaZa~

associated with both a relatively low cellular content of cytochromes as revealed by dual wavelength spectrophotometry, and ultrastructural evidence of atypical mitochondria.

330. Disturbed Vascular Permeability in Streptozotocin-Diabetie Rat Hearts P. R~sen and R. R6sen. Diabetesforschungsinstitut, Dtisseldorf, and Pharmakologisches Institut der Universit~it, Cologne, FRG

Abstracts 191

Since endothelial diffusion is assumed to play a crucial role in regulat- ing the overall supply of the heart with energy providing substrates ('nutritional flow') and hormones, local myocardial perfusion was examined in hearts of streptozotocin (STZ)-diabetic rats (60rag STZ, 4 weeks) characterized by hyperglycaemia and glycosuria, but without ketoacidosis. Regional epicardial perfusion was estimated in Langen= dorff perfused hearts on the left ventricle from fluorescence indicator elution kinetics after 100 ~1 bolus injections (FITC-Dextrane-3, MG 3000, 100 Ixl/100 ms, microscope fluorimeter). The elution profile is characterized by two pseudo-first order kinetics: a fast vascular (tl~) and a slow component (TJ~) indicating interstitial exchange. The vascular component accelerated with coronary flow (5-10 ml/min) and was not different in hearts from control (n = 9) and diabetic (n = 7) animals: t�89 ranging from 3.78 +_ 0.22 to 2.03 + 0.09 s. However, the rate of interstitial exchange was significantly prolonged in diabetic rat hearts (T�89 + 0.07 compared with 0.79_+ 0.05 min in control hearts), although coronary vascular resistance was not different in both groups. Concomitantly, the amount of exchanged fluorescence indicator was significantly diminished in diabetes. It is tempting to speculate that the reduction of endothelial diffusion could be an important factor for impaired myocardial energy metabolism and cardiac performance and indicates microangiopathic alterations in STZ- diabetic hearts.

331. Some Effects of Synthetic Fragments of Human Growth Hormone on Glucose Metabolism C. G. Rudman% N. Stebbing ], L.G. Frigeri 2 and U.J. Lewis 2. IAMGen Inc., Thousand Oaks, California and 2Whittier Institute for Diabetes, LaJolla, California, USA A synthetic peptide of 15 amino acid residues (deletion peptide) from the amino terminal region of human growth hormone has been tested for effects on glucose metabolism in several animal models. The peptide and a number of analogues have been shown to potentiate the action of insulin on plasma glucose clearance in both normal and glucose intolerant rodents. This effect was dose-dependent with regard to both insulin and the peptides. The deletion peptide has potentiating activity in the db/db mouse, ob/ob mouse, the Y-Y mouse and the normal and streptozocin-treated rat, where the peptide, insulin and glucose were administered IP. In studies with dogs, peptides and glucose were administered via indwelling cannulae and in primates glucose was given orally and peptides by IM injection. Of the various peptide analogues, one has been shown to be about 25 times more potent than the complete deletion peptide with regard to the hypoglycaemic potentiation of insulin. This phenomenon appears to be indirect via a lowering of serum non-esterified fatty acids and recent studies indicate that deletion peptide also enhances glucose uptake in muscle, as measured by 14C glucose incorporation.

332. Long-Term Effect of Continuous Subcutaneous Insulin Infusion Therapy on the Autonomic Diabetic Neuropathy of the Cardiovascular System G. Sachse, J. Neuzner and K. Federlin. III. Med. Klinik und Polikli- nik, Giel3en, FRG Recently we have described the effect of short-term treatment with continuous subcutaneous insulin infusion (CSII) therapy on the autonomic diabetic neuropathy of the cardiovascular system. To examine whether the improvement of cardiovascular reflex tests would persist over a longer period, the following study was performed: eight Type 1 (insulin-dependent) diabetics (six of them with an improvement after short-term CSII therapy, two of them with no effect of short-term therapy on the autonomic diabetic neuropathy of the cardiovascular system) underwent CSII therapy for a period of 6-12months (mean: 8.2 months). Before treatment, all patients showed pathological cardiovascular reflex tests but no clinical signs of autonomic neuropathy. Cardiovascular reflex tests were performed every 4 weeks. In six of the patients, we observed a normalization of cardiovascular reflex tests throughout the treatment period, in the two patients with no effect after short-term treatment, no improvement could be observed during long-term treatment either. The results suggest that short-term normalization of blood glucose levels might influence autonomic diabetic neuropathy of the cardiovascular system possibly based on metabolic disturbances, but even long-term CSII treatment seems to be ineffective in reversing those pathological changes of the autonomous nerve system based on established microvascular lesions. Cardiovascular reflex tests should therefore be performed in diabetic patients before clinical signs of autonomic neuropathy become apparent.

333. Effect of Two Years of Strict Metabolic Control on Kidney Func- tion in LongTerm Type I (Insulin-Dependent) Diabetic Patients J. Sandahl Christiansen, T. Lauritzen, H.-H. Parving, T. Deckert, and the Steno Study Group. Steno Memorial Hospital, Gentofte, Den- mark The effect of 2 years of strict metabolic control on glomerular filtration rate (GFR) and urinary albumin excretion rate (UalbV) was investigated in 29 long-term Type 1 diabetic patients during either unchanged conventional treatment (UCT, n = 13) or continuous subcutaneous insulin infusion (CSII, n=16). All patients had albustix negative urine, normal serum creatinine and arterial blood pressure. Near-normalization of blood glucose and HbAlc was achieved in the CSII group, while the same two variables remained unchanged and elevated in the UCT group (p < 0.01). GFR decreased from 132 _+ 19 to 111 _+ 16 ml/min per 1.73 m z (mean_+ SD) during the 2 years of CSII treatment (p < 0.01) and was normal after 2 years of treatment in 12 of 13 patients with initial low UalbV-< 70 jxg/min). GFR remained nearly unchanged in the UCT-group during the 2-year observation period. UalbV remained unchanged during CSII treatment in 12 out of 13 patients with initially UalbV -< 70 .ug/min. One of the three hyper- albuminuric patients (> 70 ~tg/min) in the CSII group and all five hy- peralbuminuric patients in the UCT group progressed to persistent proteinuria (UalbV > 200 ~tg/min). In the remaining eight UCT-patients, UalbV kept low and nearly constant during the 2 years. Our 2- year study demonstrates that GFR can be reduced and frequently also normalized during prolonged strict metabolic control in long-term Type l diabetic patients. Urinary albumin excretion did not decrease during CSII-treatment.

334. Protection by a Hydroxyl Radical Scavenger, Dimethyl Urea, Against Hyperglycaemia, but not against Insulitis, in the Multiple Low Dose Streptozotocin Model of Diabetes S. Sandler. Department of Medical Cell Biology, University of Uppsa- la, Uppsala, Sweden Injection of five consecutive sub-diabetic streptozotocin (STZ)-doses (40 mg/kg body weight, IP) to C57BL/KsJ mice gradually leads to increasing hyperglycaemia and a Concomitantly developing pancreatic insulitis. Furthermore, dimethylurea (DMU) has previously been found partially to protect against diabetogenic injections of STZ. In the present investigation, C57BL/KsJ mice were given saline or DMU (4 g/kg body weight, IP) 30 min before the daily administration of STZ. The serum glucose was followed and the animals were sacrificed at different time points. DMU almost totally prevented the development of hyperglycaemia, whereas the saline-treated group gradually became diabetic, the serum glucose concentrations after 2.5 months being 11.8 _+ 0.5 and 26.2 _+ 1.6 mmol/1, respectively. Two weeks after the first STZ injection, six out of ten DMU-treated and 12 out of 15 saline-treated mice had insulitis and similar proportions of insulitis in the two experimental groups were also found after 3 weeks. At 4 and 10weeks after the STZ treatment there were no or scarcely any cases of insulitis observed. It is suggested that DMU protects against B-cytotoxic effects induced by the repeated injections of low doses of STZ by free radical scavenging. A greater number of B cells may then survive and the animals thus became less prone to develop hyperglycaemia during the episode of pancreatic insulitis.

335. Evaluation of a Blood Glucose Photometer for the Diabetic Patient with Impaired Vision N. Saurbrey, K. Birch, S. Arnold-Larsen and C. Ktihl. Hvidore Hospi- tal, Klampenborg, Denmark To make it possible for the blind diabetic to achieve better glycaemic control, the Hypocount II B system for blood glucose monitoring has been modified to communicate results by buzzer tones. Capillary blood is placed on the test strip using a special guide. Results are expressed by one to six buzzer tones indicating blood glucose values between 0 and 18 mmol/l. The aim of the present study was to evaluate the meter with respect to accuracy and to seek patient opinion on its practical use. Trained staff using the meter performed 138 blood glucose measurements. Simultaneously obtained blood samples were analysed by a laboratory hexokinase method. The coefficient of correlation was r= 0.98 (p < 0.05). After thorough instruction and practice, five blind Type 1 diabetic patients performed 89 blood glucose measurements as in-patients. Comparison of blood glucose results measured by patients and laboratory, respectively, yielded a coefficient of correlation of r=0.85 (p<0.05). All patients found blood glucose monitoring of great value. Three of the blind subjects found the in- strument easy to operate. The remaining two had difficulties with one or more steps in the prescribed procedure. In conclusion, the Hypo-

192 Abstracts

count 1I B, Audio model, for blind diabetic patients is accurate but difficult to operate for some patients.

336. Effect of Insulin on Somatostatin Release: Studies with Freshly Isolated and Precultured Rat Pancreatic Islets G. Sch~ifer, A. E. Heyer and H. Schatz. III Medical Department, Uni- versity of Giessen, FRG To study further the assumed inhibiting effect of insulin on pancreatic D cell function, we measured somatostatin release from isolated rat islets. Methods: (1) collagenase-isolated rat islets were per]fused for 30 min at 8.3 mmol/1 glucose (a) with or without 25 U/1 rat insulin; (b) with anti-insulin serum or normal guinea-pig serum (both at 10 ~1/ ml). (2) Following precultivation for 48 h at 8.3 retool/1 glucose, islets were (a) incubated statically for I h at 2.7 or 16.7 mmol/1 glucose or (b) per]fused for 30 rain at 8.3 mmol/1 glucose, in both cases with or without added rat insulin (25 U/l). Results: (]a) Exogenous insulin did not cause a change in the pattern of somatostatin release, compared with controls, whereas 0b ) in the presence of anti-insulin serum, significantly lower somatostatin values were measured. (2a) Ad- dition of insulin resulted in a reduced somtostatin content of the incubation medium after ] h at 16.7 retool/1 glucose (2.8 _+ 0.4 versus 5.2_+ 0.7 pg/islet; n = 7, p < 0.01). (2b) Per]fusion experiments are still under investigation. Conclusions: inhibition by exogenous insulin of somatostatin release appearing in precultured islets, in contrast to fresh islets, may indicate a restoration of altered D cell insulin receptors. The reduction of somatostatin secretion by anti-insulin serum might be a consequence of diminished feed-back inhibition of insulin secretion, or also of an insulin-like action of anti-idiotypic antibodies possibly present in our anti-insulin serum.

337. Biological Activity In Vivo and Pharmacokinetics of Biosynthetic Human Proinsulin in Comparison to Human Insulin H. Schatz, S. Ammermann, F. Enzmann I and K. Federlin. III Medical Department, University of Giessen, and JFa. Lilly, Bad Homburg, FRG The extent and duration of hypoglycaemia after IV biosynthetic human proinsulin (hPI) were determined in comparison to equimolar amounts of human insulin ( ~ 1 IU/kg body weight) in six rabbits with implanted central-venous catheters. Human C-peptide and glucose were injected as a control, all in randomized order on different days. Determinations were made in serum (0-240 min) of glucose, immunoreactivity for insulin and human C-peptide. Results: Serum glucose decreased maximally by about 35% and 60% following hPI and human insulin, respectively. After hPI, the duration of hypoglycaemia was not prolonged in any case, normoglycaemia was restored even earlier than after insulin. The immunoreactivity curve for insulin after IV insulin was linear on a logarithmic plot (first order), in contrast to that after hPI. After 90-120min, a very slight increase was found again for human C-peptide immunoreactivity. However no discontinuity was apparent for the blood glucose curve. Conclusions: hPI ex- hibits a relatively strong bioactivity in vivo compared with insulin, in contrast to several in-vitro results. No prolonged hypoglycaemic action was apparent. It cannot be excluded, however, that cleavage of exogenous hPI to insulin in vivo might contribute to some extent to the hypoglycaemic action of hPI.

338. A 2-Hour Nocturnal Interruption of Continuous Subcutaneous In- sulin Infusion Induces a Delayed and Sustained Metabolic Deteriora- tion in C-peptide Negative Type I (Insulin-Dependent) Diabetic Patients A.Scheen, M.Castillo, B.Jandrain, G.Krzentowski, E Henrivaux, A. Luyckx and P. Lefrbvre. Division of Diabetes, University of Lirge, Belgium We investigated the metabolic consequences of a 2-h nocturnal interruption of a continuous subcutaneous insulin infusion (CSII) in eight C-peptide negative Type I diabetic patients. The changes in blood glucose, plasma free insulin, glucagon, non-ester]fled fatty acids (NE- FA) and 3-hydroxybutyrate concentrations were compared during two randomized tests carried out either during the normal functioning of a Mill Hill pump from 22.00h to 08.00 h (1.04_+0.07 U insulin/h, maintaining adequate metabolic control) or during the same conditions but with a deliberate arrest of the pump between 23.00 h and 01.00 h. Taking the 23.00 h value as reference, interruption of the CSII resulted in: (1) a rapid (significant after ] h) and sustained (-17_+ 5 mU/ l at 03.00 h) decrease in plasma free insulin; (2) a delayed (significant after 4 h) and linear rise in blood glucose ( + 3.7 _+ 1.1 mmol/l at 05.00 h); (3) an early (significant after I h) and prolonged rise in plasma NEFA ( + 328 _+ 142 gmol/1 at 03.00 h); (4) a delayed (significant after 3 h) and sustained increase in plasma 3-hydroxybutyrate

(+332_+78 Ixmol/1 at 03.00h); (5) no significant changes in plasma glucagon. Thus, 2-h nocturnal interruption of a CSII at rest is sufficient to induce a significant, delayed and sustained, metabolic deterioration in C-peptide negative patients despite a good baseline blood glucose control.

339. Immunogenicity of Human Monoeomponent Insulin in Man: Long-Term Follow-Up of 77 Newly Treated Type 1 (Insulin-Dependent) Diabetic Patients G. Schernthaner, M. Borkenstein, E.Schober, and M. Fink. Depart- ment of Medicine II, Department of Paediatrics, University of Vienna and Department of Paediatrics, University of Graz, Austria The immunogenicity of human monocomponent (MC) insulin (Ac- t rapid/Monotard) was studied in the long-term follow-up of 77 newly treated Type ] diabetic patients. Sixty-two diabetic patients treated with the respective porcine MC insulins served as control subjects. IgG-insulin antibodies were measured by radioimmunoelectrophore- sis. In addition HLA-DR antigens were also determined in all patients. Results: (1) IgG-insulin antibodies ( > 0.05 units/litre) were not found in any of the diabetic patients before insulin treatment. After 3, 6, 9 and ]2 months, insulin antibodies were found significantly less often in the human insulin-treated patients than in the porcine group (3 months: 7% versus 21%, p < 0.0]; 6 months: 14% versus 29%, p < 0.01; 9months: 27% versus 44%, p<0.05; ]2months: 31% versus 48%, p < 0.05); (2) Insulin antibody formation against human MC insulin is not controlled by genes of the HLA complex, whereas antibody production against porcine MC insulin is mainly found in DR4- positive Type I diabetic patients. In summary, human MC-insulin is immunogenic in man, but incidence and titres of insulin antibodies are significantly lower in human-insulin than in porcine-insulin treated diabetic patients. The IgG-insulin antibody production against human insulin did not correlate with the age of the patients at onset or their immunogenetic background (HLA-DR antigens).

340. Onion Leaf Sheet Model for Insulin Release Y. Scholler, V. De Maertelaer and W. J. Malaisse. Laboratory of Exper- imental Medicine and Institute of Interdisciplinary Research in Hu- man and Nuclear Biology, University of Brussels, Brussels, Belgium A mathematical model was recently proposed for the interaction between Ca 2+, calmodulin and cyclic AMP in insulin release. Although this model provided a fair picture of functional events under steady- state conditions, it was found inadequate to simulate rapid changes in cationic fluxes and insulin release. Two new features are now introduced in this model. Firstly, the cytosolic domain was fragmented into a cortical layer and a central core, to account for geographical heterogeneity in cytosolic Ca 2+ concentration. The vacuolar system was distributed evenly among the cortical layer and central core. The relative volume of the central core was fixed in each model between extreme values of 80%-99%. The Ca 2+ outflow from the central core, relative to its Ca 2+ content, was fixed between extreme values of 0.00]-0.060/min. Secondly, the effect of glucose to increase Ca 2+ in-

2+ flow into and decrease Ca outflow from the B cell was assumed to be discontinuous, a 2-rain silent period separating the first 2 min from the later period of stimulation. With these two features, the normal dynamics of insulin release could be adequately simulated. Our work emphasizes, therefore, the importance of cytosolic heterogeneity and signal discontinuity in the time course of insulin secretion.

341. Regulation of Cyclic AMP Formation in Pancreatic B Cells F. Schuit and D. Pipeleers. Department of Metabolism and Endocri- nology, Vrije Universiteit, Brussels, Belgium We reported previously that single B cells release 30-fold less insulin in response to glucose than intact islets and that this poor secretory ability can be partly attributed to low cellular cyclic AMP levels. Studies were undertaken to assess whether the higher cyclic AMP levels in isolated islets are maintained by structural cell-coupling or by locally released hormones. Cyclic AMP accumulation was therefore measured in various B cell preparations after 10 min incubation with isobutylmethylxanthine. Both at low and high glucose, intact islets accumulated over fourfold more cyclic AMP/~tg DNA than single or reaggregated B cells. This difference disappeared in the presence of

8 10- tool/1 glucagon. Furthermore, cyclic AMP formation in single B cells was stimulated at lower hormone concentrations (]0 -9 mol/1) than in intact islets (10 -7 mol/1). Glucagon-induced cyclic AMP accumulation in purified B cells was inhibited by somatostatin (10 7 to 10-1~ but unaltered by pancreatic polypeptide (up to 10 -6 tool/l). In all conditions, the magnitude of glucose-induced insulin release correlated with the degree of cyclic AMP accumulation;

Abstracts 193

this was also the case after addition of adrenaline. It is concluded that locally released glucagon and somatostatin might determine the rate of cyclic AMP formation in pancreatic B cells and thus contribute to the insulin secretory response in vivo.

342. Analysis of the Insulin Response Using Photoreaetive Insulin Ana- logues A. Schttttter, D. Saunders, C. Diaconescu, G. Klotz and D. Branden- burg. Deutsches Wollforschungsinstitut, Aachen, FRG Covalent binding of photoreactive insulins to isolated rat adipocytes produces prolonged stimulation of lipogenesis. Cells incubated with B2 (2-Nitro, 4-azidophenylacetyt = NAPA) des Phem-insulin (B2), B29 (NAPA) insulin (B29), or A1 (NAP)-insulin gave, after ultraviolet irradiation and washing, 80%, 55%, and 0% respectively of the maximal stimulation. The response from B2 decreases linearly with time to about 30% after 3 h, suggesting a half-life of about 80 min for the biologically effective covalent complex at 37 ~ Inactivation of the covalent complex may be connected with internalization processes as it could be influenced by inhibition of endocytosis by lowering the temperature to 20~ as well as the lysosomotropic agent chloroquine. The relation of receptor occupancy to response was studied for the covalent B2- and B29-complexes using iodinated analogues. These in- sert into receptors with efficiencies of 21% and 13% respectively. The response is proportional to the covalent occupancy, but the molar potencies of the covalent compared to non-covalent systems are reduced to 26% for B2, and 50% for B29 complexes. The ratio of occupancy to response is constant between pH 7-8 for non-covalent complexes, but decreases threefold for B2-complexes. This indicates that the covalent complexes are dynamic not static aggregates.

343. Cardiovascular Responses to Tyramine and Noradrenaline in Dia- betic Autonomic Neuropathy. I. N. Scobie, P.T. Rogers, P.M. Brown, C. Fitzpatrick and P.H. S6nksen. Department of Medicine, StThomas' Hospital Medical School, London, UK Denervation hypersensitivity has not been documented in diabetic autonomic neuropathy. Cardiovascular responses to intravenously infused tyramine (an indirectly acting sympathomimetic; 1-71*g. kg-~. min-1) and noradrenaline (0.01-0.04/zg- k g - 1 min-~) were measured in group 1 (five normal subjects), group2 (five insulin-dependent diabetic patients) and group 3 (five insulin-dependent diabetic patients with autonomic neuropathy). Groups were matched for age. No significant change in heart rate was seen in any group. Rest- ing heart rate was higher in group 3 (p < 0.01). During tyramine infusion, no change in systolic blood pressure occurred in groups l or 2, while mean -+ SD systolic blood pressure rose from 142 ___ 23 to 151 -+ 13 mmHg (p< 0.001) in group 3. Diastolic blood pressure did not alter. During noradrenaline infusion, systolic blood pressure increased in group l (109_+9 to 118_+t9mmHg; p<0.01) and in group3 (141 _+ 24 to 173_+25 mmHg; p< 0.001), being significantly greater in group 3 than groups 1 and 2 (p < 0.001). Diastolic blood pressure rose significantly only in group I (83 +_ 7 to 89 -+ 11 mmHg; p < 0.05). Thus super-sensitivity to noradrenaline occurs in diabetic autonomic neuropathy indicating post-denervation hypersensitivity. Tyramine hypersensitivity also occurred indicating that denervation is not complete and suggesting biochemical dysfunction at a pre-synaptic level.

344. Regulation of Insulin Release from Pancreatic Islets R.S. Scott, D. Hopcroft, D. Mason, D. Howarth and C. Mockett. Christchurch Clinical School, Christchurch, New Zealand Insulin release may be subject to regulatory mechanisms from within the islet through localised hormone influences and structural connec- tions between cells. These mechanisms were studied by comparing insulin responses from intact rat islets to those observed with dispersed islet cell populations and re-aggregated 'pseudo-islets'. Basal insulin release from the perifused dispersed islet cells (0.246 _+ 0.015 pmol. ug DNA 1-min-~) was approximately fivefold greater than obseiwed with whole islets (0.054_+ 0.005 pmol �9 DNA -1. min -1) or with reaggregated islets (0.070 _+ 0.009 pmol. gg DNA-1- min- 1). Following glucose stimulation, incremental first phase responses were not significantly different from those of intact islets. Second phase insulin responses were, by comparison, attenuated. Re-aggregated islets responded to glucose with exaggerated insulin release. Peak insulin levels during the first phase were 1.316+0.248pmol.lzg DNA -1. min -~ compared to intact islets (0.471+0.051) and dispersed cells (0.539_+0.104pmol.gg DNA -1-min-1). Peak insulin levels during second phase release were correspondingly higher for the re-aggregated islets. These results indicate that disruption of islet integrity alters

the B cell responsiveness to a nutrient stimulus. The functional changes observed after islet cell re-aggregation give strong support to the importance of intra-islet modulating factors for insulin release.

345. Diabetic Retinopathy is Associated with Raised Concentrations of Plasma Fibronectin G.SeghierP, L.A. De Giorgio ~, A.Gironi 2, L.AlviggP, U.Bartoli ~, L.Giuntini ~ and G.Venturino J. Outpatient Clinic for Diabetes ~ and Department of Clinical Chemistry-', Spedali Riuniti, Pistoia, Italy Plasma fibronectin (PF) is a glycoprotein released by the endothelial cells. A factor that induces fibronectin production by these ceils is vascular injury. To study whether vascular damage of diabetic microangiopathy could modify PF levels, we investigated the correlation between PF concentrations, assessed by a turbidimetric assay, and the presence of retinopathy in 36 insulin-treated patients (group A) and in 55 diabetic patients on sulphonylureas (group B), suffering from diabetes for at least 5 years and with identical HbA1 levels. Both groups were compared with control subjects (n = 51), strictly matched for sex and age. Retinopathy (background, except for two proliferative in group A) was diagnosed by examination of the fundi after pupil dila- tation. Mean_ SD PF levels of control subjects (323.5 _+ 58.9 rag/l) did not differ either from that of groupA (349.3_+82.9mg/1) or group B (351.6_+84.1mg/1). Group A patients with retinopathy (n=23) had higher PF levels when compared to patients without retinopathy (n = 13), (371.3 _+ 92.5 mg/1 versus 310.5 _+ 42.1 mg/l; p < 0.05) and control subjects (p<0.01). Similarly group B patients with retinopathy (n = 21) had PF levels significantly increased than those without retinopathy (n = 34), (408.4_+ 79.5 mg/l versus 316.6 -+ 66.6 rag/1; p< 0.001) and control subjects (p< 0.001). In conclusion, our data, although needing further investigation, suggest a relationship between diabetic microangiopathy and raised PF levels.

346. Pharmaeokinetics of Insulin Infused Intraperitoneally Via Por- table Pumps. Regional Variations and Comparison with Subcutaneous Infusion J.L.SeIam, M.Raymond, A.Orsetti and J.Mirouze. Metabolic and Endocrine Department, St-Eloi Hospital, Montpellier, France Venous plasma free insulin was measured repeatedly during 4 h following a standardized breakfast in 28 C-peptide-negative chronically pumped Type 1 (insulin-dependent) diabetic patients and five normal subjects. In the former group, insulin was given as a 1 U /h basal infusion and a 1 h meal-dose of 6 U via a peritoneal (IP) catheter lying in the low (n = 10), or in the mid-abdomen (n = 10), or SC (n = 8). The re- suits of the IP patients were correlated with haemoglobin A1, and M- value. Fasting free insulin of IP patients was found in the normal range, lower than those of SC patients (14.7_+0.5 versus 18.0_+ 1.6 mU/1, p < 0.01). Bolus-induced peak was in the normal range in both groups although earlier in IP than SC patients (70_+ 6 versus 139 _+ 23 min, p < 0.001) Values tended to basal values by the end of the test in IP patients while remained elevated in SC patients (17.6 _+ 1.2 versus 28.8 _+ 4.1 mU/1 at 240 min, p < 0.001). Results of the mid- and low-peritoneum subgroups differed only for peak values (31.5 _+ 2.9 versus 25.0_+ 1.6 mU/1, respectively, p < 0.05), and did not correlate with haemoglobin A1, and M-values. Thus, IP insulin infusion produces a more physiological plasma profile than SC infusion, with a further slight improvement if the catheter is high-placed in the abdomen.

347. Long-Term Effects of Glipizide on Endocrine Pancreas and Insulin Receptors in Type 2 Non-lnsulin-Dependent Diabetes M. Serrano Rios, A. Ordofiez, M. S. Sfinchez, M. T. Villalba, S. de la Vi- fia, R.Nash and M.E.Carb6. Centro Especial Ram6n y Cajal, Madrid, Spain Long-term effect of glipizide on endocrine pancreas and insulin receptors were studied in Type 2 diabetic patients. Diets were individually adjusted. Groups A and B were treated with 5-15 mg/day of glipizide and were classified previously according to fasting glycaemia ; A (9.36 _+ 0.26 retool/l, n = 28); B (13.85 _+ 0.44 mmol/l, n = 7). Group C was managed only on diet (8.11+0.32 retool/l, n=9). Glycaemia, IRI, C-peptide and glucagon were determined before and after breakfast testing (75 g carbohydrates), initially and at 2, 4 and 6 months after admission, Insulin binding to erythrocytes (only groups A and C) and HbA1 were measured in parallel. Fasting glycaemia was significantly lowered after 2 months (A, p < 0.0001 ; B, p < 0.025) with no further changes; HbA1 diminished after 6 months (A, p < 0.0001 ; B, p < 0.005). Boths parameters remained unaltered in group C throughout the study. No change in B cell function was apparent at any time in any of the three groups. Glucagon levels did not change in groups A

194 Abstracts

and C and diminshed significantly in group B at 6 months (p < 0.005). In group A (not in group C), insulin receptor affinity increased after 6 months (p<0.01) without changes in receptor numbers in either group. Long-term glipizide therapeutic benefits occurred without significant changes in insulin response. We suggest that the major effect of glipizide takes place at the extrapancreatic (receptor) levels.

348. Randomized Clinical Trial of the Effect of Glucose Control from Continuous Subcutaneous Insulin Infusion on Peripheral Nerve Func- tion in Type I (Insulin-Dependent) Diabetes F.J.Service, P.J. Dyck, J.R.Daube, P.C.O'Brien, L.D. Hall, R.E. Westland and R.A. Rizza. Mayo Clinic, Rochester, Minnesota, USA Twelve Type 1 diabetic patients with mild abnormalities of nerve function were randomly assigned to continuation of conventional therapy (three men, three women) or to continuous subcutaneous insulin infusion (CSII) (three men, three women). There were no significant differences between the treatment groups at entry for nerve function: neurological symptom score, neurological disability score, computer assisted sensation examination and measurements of amplitudes, la- tencies and conduction velocities of motor and sensory fibres of six limb nerves; nor for mean blood glucose (seven capillary specimens/ 24 h; 12.4 versus 11.9 retool/l) and glycosylated haemoglobin (]0.6 versus 11.0%), respectively. During the study, both mean blood glucose (7/24 h, monthly; 4.4 versus 10.2 mmol/1, p= 0.004) and glycosylated haemoglobin (every 1 2months; 8.3 versus 10.5%, p=0.002) were improved in CSII in contrast to conventional therapy. After 8 months, there was significantly better function in the 34 nerve conduction parameters in CSII (p= 0.03). However, no differences were observed for the other measures of nerve function. Conclusion: near- normalization of glycaemia for 8 months in Type 1 diabetic subjects appears to improve peripheral nerve conduction.

349. Glycaemic Control in Diabetic Patients by a Wearable Artificial Endocrine Pancreas with a Needle-Type Glucose Sensor M. Shichiri, R. Kawamori, Y. Goriya, Y.Yamasaki, N. Hakui and H. Abe. Osaka University Medical School, Osaka, Japan We have shown that physiological glycaemic control can be established in pancreatectomized dogs with a wearable artificial endocrine pancreas incorporating a needle-type glucose sensor. The applicabili- ty of this device to human diabetic patients was examined. Needle- type glucose sensors were inserted into the SC tissue of the forearm or abdomen of normal and diabetic volunteers and the output values were compared with blood glucose concentrations monitored by the bedside-type artificial endocrine pancreas. The measured glucose concentrations in SC tissue (y) were highly correlated with blood glucose concentrations (x) [y=0.81 x+7.1, r=0.85, in forearm, y=0.78 x+ 11.3, r=0.91, in abdomen]. No time delay in measurement was recognized. Insulin delivery IV and SC based on sensors' outputs by the wearable artificial endocrine pancreas could regulate the glycaemic excursions perfectly in insulin-dependent patients. Scanning electron-microscopic examinations revealed only a minimal deposition of blood cells and fibrin to the surface of the sensor after 2-3 days continuous SC sensing. Serum anti-glucose oxidase antibody determined by radioimmunoassay was not detected in any of the subjects studied. These results indicate the feasibility of long-term glycaemic control with this system.

350. Lipolytic Effect of Somatostatin in Human Adipocytes M. A. Sim6n, A. Tortes, P. Mayor, A. Santos and C. Calle. Department of Biochemistry and II Department of Surgery, Faculty of Medicine, University Complutense, Madrid, Spain We reported previously a lipolytic effect of somatostatin (ST) in isolated rat adipocytes. To gain further insight, we examined the lipolytic action of ST in isolated human adipocytes. In normal patients, SC adipose tissue was obtained from the right upper quadrant of the abdomen during cholelithiasis surgery (C) (6) or from the femoral region during the repair of inguinofemoral hernias (H) (3). In patients with abnormal fat stores, the adipocytes were extracted from three lipomas La (1), L2 (1), L3 (1) and from one Madelung's lipomatosis (M) (1). To measure glycerol, the cells (].5-2.5 x 105 cells/ml) were incubated for 1 h at 37 ~ in Krebs-Hepes buffer (pH 7.4) with BSA fatty acid-free (1%), bacitracine (2.3 mmol/1) with or without different ST concentrations (0.8 x 10 -9 to 3.48 X 10 7 mol/1). The basal glycerol productions (nmol-105cells-1. h- l) were (C: 44 + 21), (H: 279 -+ 99), (L1 : 92.8), ( L 2 : 37.0), (L3: 65.8) and (M: 67.3), statistically significant site differences in glycerol values were observed between the two groups of normal patients. The presence of ST (1.74 x 10 -7 mol/l) augmented basal lipolysis in all groups having different average percent age increases (C:

93.7+21; !07%) (H: 569-+128; 104%), (LI: 197.7; 105%), (L2: 69.4; 87%), (L3: 84.8; 28%) and (M: 279.2; 315%). in conclusion, it would seem that ST greatly enhances lipolysis, as much in normal as in pathological human adipocytes.

351. Effect of Beta- and AIpha-Adrenergic Blockade on Glucose-In- duced Thermogenesis in Man D. Simonson, A. Golay, J.P. Felber, D.Thorin, E. Jequier, and R.A. DeFronzo. Yale University, New Haven, CT, USA and University of Lausanna and CHUV, Lausanne, Switzerland Following glucose and insulin infusion there is an increase in oxygen consumption and energy expenditure. To examine the contribution of the/3- and a-adrenergic nervous system to this thermogenic response, 12 healthy volunteers participated in three study protocols: (1) euglycaemic insulin (approxmately 100mU/l) clamp; (2) IV propranolol+insulin clamp; (3) phentolamine+insulin clamp. During the control insulin clamp, total glucose uptake, glucose oxidation and non-oxidative glucose uptake averaged 7.9 _+ 0.4, 2.6 -+ 0.2, and 5.2 -+ 0.5 mg. kg -~. min -1. Following propranolol, total glucose uptake was reduced (6.9 _+ 0.4 mg. kg -1- min-% p < 0.01), primarily due to a decrease in glucose oxidation (1.9 -+ 0.2, p < 0.02); non-oxidative glucose uptake remained unchanged. Phentolamine had no effect on any parameter of insulin-mediated glucose metabolism. The increment in energy expenditure (0.03 < 0.01 versus 0.10 < 0.01 kcal/min) and glucose/insulin-induced thermogenesis (1.5 _+ 0.5 versus 4.9 _+ 1.5%) were reduced by 70% during the propranolol/insulin clamp versus the control insulin clamp (p< 0.001). The increment in energy expenditure (0.12 -+ 0.03 kcal/min) and thermogenesis (5.7 -+ 1.3%) was unaffected by phentolamine. These results indicate that in man fl-adrenergic stimulation plays an important role in the glucose/insulin-mediated increase in energy expenditure and thermogenesis; the a-adrenergic nervous system does not appear to participate in this response.

352. The Quality of Different Insulin Syringes H. Skodda, R Warzecha, I. Mtihlhauser, F. W. Kemmer and V. J6rgens. Department of Medicine University of Dtisseldorf, FRG. Three types of syringes are commonly used for insulin injection: glass syringes a and plastic syringes with detachable b or non-detachable needles c. We evaluated the accuracy of the ejected insulin volume and the dead space of the syringe using 10 different insulin syringes. After a standardized instruction, 50 volunteers drew up 32 U Velasulin ( ]004mg=l ml=40IE) in a randomized order. All syringes were weighed before and after drawing up and after ejection of the insulin.

Ejected Error Dead space (units) (%) (mg)

Diarex ~ 31.58 _+ 1.0 2.63 90 + 24 Inject Solob (2 ml) 33.72--+2.2 6.72 66+17 Omnifix b (1 ml) 32,14+1.0 2.41 67+ 8 Pharma-Plast b (1 ml) 30.24 _+ 1.2 5.69 75 + 12 Primo (1 ml) b 30.50-+0.7 4.69 56+18 Primo (2ml) b 31.58-+1.4 3.62 69+18 Terumo (1 ml) b 32.25 -+ 0.6 1.62 95 + 10 Plastipak (2 ml) b 32.72-+ 0.6 2.38 65 -+ 8 Monoject (1 ml) c 31.25 -+ 0.2 2.31 3 _+ ] Plastipak (1 ml) ~ 32.02+0.5 0.97 5-+ 2

Results expressed as mean _+ SD

One syringe with non-detachable needle showed tolerable precision and small dead space as needed for adaptation and mixing of small insulin doses. Monoject ejected less insulin than Plastipak (p 0.001).

353. Effects of Monensin on Ion Handling and Insulin Secretion in Iso- lated Rat Islets of Langerhans J. E. Smith and S. L. Howell. Department of Physiology, Queen Eliza- beth College, University of London, London UK Monensin (10 -8 mol/1), a sodium selective monovalent ionophore, inhibited glucose-stimulated insulin secretion from isolated rat islets in static incubations by 70% (n=48, p<0.001). In perifusion experiments monensin caused a 50% inhibition of glucose-stimulated secretion when the islets were exposed to 20mmol/1 glucose and l0 8 tool/1 monensin simultaneously (n = 4, p < 0.01). When the islets were pre-exposed to monensin for 20rain before challenging with 20 mmol/1 glucose, this inhibition was more pronounced. The effiux

86 of Rb from preloaded islets was significantly increased by 10 .8 tool/1 monensin (n = 4, p < 0.001), but net 86Rb uptake was inhib-

Abstracts 195

ited by 30% alter I h (n=45 ,p< 0.001) and by over 50% after 2h incubation (n = 45, p < 0.001). Monensin (10 -8 mol/1) had no effect upon glucose-stimulated 32p effiux from preloaded islets (n= 6). The ionophore also stimulated 45Ca efflux from preloaded islets perifused with calcium-free medium containing lmmol/1 EGTA (n=6, p<0.01). Monensin (10 -7 tool/l) inhibited glucose-stimulated insulin secretion completely but had no effect upon the glucose-induced inhibition of 45Ca efflux from preloaded islets (n= 4). However, the same concentration of monensin significantly inhibited glucose-stimulated 45Ca uptake over 5 rain incubation (n = 30, p < 0.01). Exposure to monensin clearly produces extensive changes in ion handling and secretory responses of isolated islets.

354. Effect of Guinea Pig Anti-insulin Serum on Insulin Metabolism in Normal Rats: Autoradiographic and Scintigraphic Studies F. Sodoyez-Goffaux, J. C. Sodoyez and C.J. De Vos. Departments of Paediatrics and of Internal Medicine, University of Liege, Liege, Bel- gium HPLC purified 123I-or 12SI-Tyr A14-Insulin (123I- or 12sI-Ins) was injected IV in rats pretreated with saline or guinea-pig anti-insulin serum (GPAIS). Kinetic analysis of scintigraphic data showed that in saline- injected rats, 123I-Ins was rapidly taken up (30% of total injected at 3 min) and degraded by the liver, by an insulin-receptor mediated mechanism. After GPAIS, ~2~I-Ins was massively concentrated by the liver (90% of total injected at 5 min) and liver radioactivity remained constant during the following 25 min observation period. After 125I- Ins injection, liver autoradiographic studies showed that silver grains were almost exclusively associated with hepatocytes (saline pretreated rats) or with reticulo-endothelial cells (GPAIS pretreated rats). In conclusion, in the presence of specific antibodies, plasma insulin forms immune complexes which are rapidly cleared by the reticulo- endothelial system. If similar mechanisms are operative in insulin-immunized diabetic patients, trapping of administered insulin by the reticulo-endothelial system and ensuing 'insulin resistance' would be better appreciated by scintigraphic measurement of 'antibody flux' rather than by static determination of plasma antibody concentration.

355. Enhanced Electrophoretic Mobility of Low-Density Lipoproteins from Diabetic Patients: Correlation with Metabolic Control M.Songini, G. M. Baule 1, E. Podda, F. Pintus and S. Muntoni. Centre for Metabolic Diseases and Atherosclerosis, Ospedale S.Michele, Cagliari, and 1Second Division of Medicine, Ospedale Civile, Sassari, Italy Post-translational non-enzymatic glycosylation of proteins can occur in diabetes as a consequence of hyperglycaemia. Recent evidence indicates that, besides haemoglobin, apolipoprotein B of LDL may un- dergo glycosylation, leading to changes in metabolic properties and to enhanced electrophoretic mobility of LDL. The present study was carried out to evaluate the influence of metabolic control, assessed by determining stable glycosylated haemoglobin (HbA~o), on electrophoretic mobility of LDL in 45 diabetic patients (21 insulin-dependent and 24 non-insulin-dependent) of both sexes. Electrophoresis of LDL, separated by ultracentrifugation, was conducted on agarose-gel in barbital buffer. The distance of migration of diabetic LDL was compared to control LDL, and the difference measured by a Zeiss Projector for radial immunodiffusion. Patients were divided into two groups: (a) HbAIr > 7% (10 insulin-dependent and 10 non-insulin-dependent); (b) HbAlc~< 7% (l 1 insulin-dependent and 14 non-insulin- dependent). HbA~c was positively correlated with LDL mobility (r= 0.20;p < 0.001) in group a, but not in group b. It is concluded that, in diabetes, poor metabolic control is reflected by enhanced electrophoretic mobility of LDL: such behaviour is very likely to stem from non-enzymatic glycosylation of tysine groups on apolipoprotein 13.

356. Continuous Subcutaneous Insulin Infusion Treatment is Associated with Normalization of Serum Magnesium in Type I (Insulin-Dependent) Diabetic Patients G. E. Sonnenberg, G. Reintges, E. Chantelau, F. Best and M. Berger. Department of Medicine E, University Hospital, Diisseldorf, FRG It has been suggested that hypomagnesaemia may be an additional risk factor in the development of ischaemic heart disease and diabetic retinopathy. This study evaluates the influence of metabolic control achieved by continuous subcutaneous insulin infusion (CSII) on concentration of serum magnesium. Nine Type I diabetic patients treated by CSII were observed during 7 months. Serum magnesium was determined using atomic absorption spectrophotography and haemoglobin A~o using the colorimetric procedure. The parameters were measured at monthly intervals. Mean haemoglobin AI~ de-

creased significantly from 9.9 + 0.8% (mean_+ SEM; normal range: 4.1-7.8% of total haemoglobin) before CSII to 7.0___ 0.4% (p < 0.02) after 4 months and to 7.9 _+ 0.3% after 7 months. Initially all patients had a significant hypomagnesaemia (0.74_+ 0.02 retool/l) compared with age and sex-matched healthy volunteers (0.82_+0.02mmol/1, p< 0.001). Mean serum magnesium increased significantly to 0.81 _+ 0.02 mmol/1 (p<0.001) after I month of CSII and remained within the normal range. There was a highly significant inverse correlation between mean haemoglobin Aic and serum magnesium levels (r= - 0.949, p < 0.001). Achieving near-normoglycaemia by means of CSII is associated with a normalization of serum magnesium in Type I diabetic patients. The normalization of serum magnesium may contribute to preventing the development of macro- and microangiopathy.

357. Therapy of Type 2 (Non-insulin Dependent) Diabete with Enzyme Inducers: A New Approach E. A. Sotaniemi, A.J. Arranto and S. Sutinen. Clinical Research Unit, Departments of Internal Medicine and Pathology, University of Oulu, Oulu, Finland Therapy-resistant patients with Type 2 diabetes have glucose intolerance and hyperglycaemia without insulin deficiency. Liver function, as measured by conventional tests, is normal or slightly altered, whereas hepatic microsomal enzyme activity is reduced. Since post- receptional (intracellular) glucose metabolism is influenced by these enzymes, we added phenobarbital or medroxyprogesterone acetate, known inducers, to the sulphonylurea regimen of 15 Type 2 diabetic patients. The fasting blood glucose was reduced by 30% from the pre- therapy level while on inducers, but not with placebo. In some patients, the blood glucose decrease was marked (from 18 to 5-7 mmol/ 1). Serum immunoreactive insulin decreased in all the subjects, although in one case there was an increase. The change in glucose metabolism was associated with an increase in the microsornal enzyme activity (antipyrine test). Body weight decreased in most cases, although it remained unaltered in two and increased in one subject. Withdrawal of the inducers resulted in blood glucose elevation and reduced antipyrine metabolism. Decrease of hepatic fat and normalization of plasma cell membrane of hepatocytes were visible in diagnostic liver biopsies. Activation of post-receptional events in hepatocytes seems hence to be a new approach in the treatment of therapy resistant Type 2 diabetic patients.

358. Appearance and Disappearance of Complement-Fixing and Other Islet Cell Antibodies Amongst Type I (Insulin-Dependent) Diabetic patient and Their First Degree Relatives K. M. Spencer, J. McNally ~, A.Tarn and D. Doniach ~. Department of Diabetes, St. Bartholomew's Hospital and ~Department of Immunolo- gy, Middlesex Hospital, London, UK The Barts-Windsor Family Study has been in existence for 5 years. 361 parents, 322 non-diabetic siblings and 197 diabetic children have taken part. Complement-fixing islet cell antibody (CF-ICA) has been found in six parents and 14 siblings and has persisted in three parents and six siblings. In all but two subjects, CF-ICA was present at the time of entry to the study. These two, who had been negative for several years, are of great potential importance. Non-complement-fixing islet cell antibody (ICA) has been found in 22 parents and 43 siblings and has persisted in seven parents and 15 siblings. CF-ICA has been found only in subjects who also have ICA. ICA has been found in 14% of HLA-identical siblings, 14% of haplo-identical siblings and 10% of HLA non-identical siblings. Whereas CF-ICA has been found in 9% of HLA-identical siblings, 4% of haplo-identical siblings but only 1% of HLA non-identical siblings. Seven previously non-diabetic subjects have become diabetic and all had persistent ICA before diagnosis. ICA has reappeared in 33/119 diabetics in whom it had disappeared. When ICA appears in a non-diabetic it often reappears simultaneously in the proband.

359. Decrease of Fasting and Late Night Cortisol After Improved Meta- bolic Control in 12 Type 1 (Insulin-Dependent) Diabetic Patients Treat- ed with Continuous Subcutaneous Insulin Infusion A. J. SpijkeI, J. H. H. Thijssen and D.W. Erkelens. Department of In- ternal Medicine, University Hospital, Utrecht, The Netherlands Improved control of glycaemia induces normalization of intermediary metabolism. We wondered whether high-normal serum cortisol in Type 1 diabetes is influenced by continuous subcutaneous insulin infusion (CSII). In 12patients, mean age 33.8 years (range 21-48), whose insulin dependence existed for 18.1 years (range: 2-30), cortisol was measured by radiohnmunoassay at 08.00 (fasting), 16.00 and

196 Abstracts

23.00 h during conventional treatment (CT) with multipIe daily injections and after attaining near normalization of blood glucose established by glycosylated haemoglobin. Fasting cortisol (mean _+ SD) decreased during CSII from 0.59 _+ 0.14 to 0.41 + 0.11 Ixmol/l (normal: 0.20-0.65 gmol/1; p < 0.001). Late night (23.00 h) cortisol decreased also during CSII from 0.17_+0.10 to 0.09+0.08 p.mol/1 (p< 0.025). No change was measured in the 16.00h cortisol level during CT: 0.26+ 13 jxmol/l or during CSII: 0.24_+0.08 gmol/1 (normal: 0.10- 0.351xmol/1; p=0.3). Glycosylated haemoglobin decreased from 13.5 + 2.7 to 9.7 + 1.3% (p < 0.001). There was no difference in total insulin daily dose, intercurrent disease or complication rate before and during CSII. We conclude that fasting and late night cortisol levels decrease after switching from conventional treatment to CSII in Type I diabetes. It has to be substantiated whether this is an epiphe- nomenon or a factor in normalization of intermediary metabolism.

360. Four Year's Prospective Study on the Progression of Background Retinopathy in Relation to Metabolic Control and Platelet Aggregation in Type I (Insulin-Dependent) Diabetes. E. Standl, T. Lander, H.U. Janka, and T. Dexel. Diabetes Research Unit, Munich, FRG The factors involved in the development of diabetic retinopathy are still under debate. The present study was designed. To evaluate prospectively for 4 years the progression of existing background diabetic retinopathy in relation to metabolic control and platelet aggregation in Type-1 diabetes. One hour post-prandial blood glucose and HbAa were assessed every month in 20patients (mean age 34• mean duration of diabetes 16.7 • 1.3 years) taking insulin 2-3 times/ day, performing home glucose monitoring, and without renal disease. Platelet aggregation (ADP-50%-threshold-concentration; umol/ml) was measured in vitro every half-year, fundus photography and fluorescence angiography were performed every year. After 4 years, patients (four drop-outs) were divided into group 1 (five patients with none, three with minimal deterioration of retinopathy) and group 2 (four patients with significant retinal hemorrhages and/or soft exudates, four with proliferative changes necessilating laser photocoag- ulation). Compared with group 2, group 1 showed lower mean blood glucose (9.85 • 0.58 versus 11.76 • 0.31 mmol/1, p < 0.02), and lower mean HbA1 (10.4_+0.2 versus 12.1 • p<0.005), but no significant differences in platelet aggregation, mean age and duration of diabetes. When deterioration of diabetic retinopathy was graded, significant correlations with average HbA1 (r=0.84; p<0.001), blood glucose (r= 0.57; p < 0.05), and platelet aggregation (r= 0.60, p < 0.05) were disclosed. The results suggest a strong linkage of metabolic control, and to some degree also of platelet aggregation, with progression of diabetic retinopathy.

361. Treatment of Lipoatrophy in Diabetes Mellitus J. Stfinescu and I. Mincu. Centre of Nutrition and Metabolic Diseases, Bucharest, Rumania Lipoatrophy may occur in Type 1 (insulin-dependent) diabetic patients, predominantly in women particularly during the reproductive period (20-45 years), and have aesthetic consequences. It may be due to be the protein impurities in some of the commercial insulins that produce a local immunological reaction resulting in atrophy of the SC tissue at the site of insulin injection. An novel method of treatment was used, consisting of purified (monocomponent) insulin and local peri-lesional alpha-chymotrypsin and xylocaine infiltrations. We studied 34 insulin-dependent diabetic patients (32 women, two men), mean age 34 years, mean duration of disease 4 years, who had received previous treatment with lente insulin (Novo Industries) in 97% of cases, and with long-acting insulin (Hoechst) in 3% of cases. Addi- tion of local treatment with alpha-chymotrypsin to the use of monocomponent insulin increased the improvement rate from 11.8% to 94.1%, with only two failures (5.9%).

362. The Effect of ICI 128436, a Novel Aldose Reductase Inhibitor, on the Development of Diabetic Complications in the Rat D. Stribling, D.J. Mirrlees and D.C.N. Earl. ICI Pharmaceuticals, Macclesfield, UK It has been suggested that inappropriate production of sorbitol by aldose reductase in insulin-independent tissues contributes to the development of diabetic complications. ICI 128436 (2-(2-fiuoro-4-bromo- benzyl)-l,2-dihydro-l-oxophthalazin-4-ylactic acid) is a chemically novel, potent inhibitor of aldose reductase from a wide range of species. Kinetic studies indicate that it is a mixed non-competitive reversible inhibitor with respect to glucose with an IC50 in vitro of 2 x 10 -8 mol/ l for enzyme from either rat or human lens. Once daily

oral administration of ICI 128436 (20 mg/kg) to streptozotocin-diabetic rats does not affect plasma glucose levels, but reduces elevated levels of nerve sorbitol and fructose to the normal range. Similar effects are seen in other tissues, such as lens and retina, which also suffer pathological changes in chronic diabetes. ICI 128436 has a plasma half-life of some 10 h in the diabetic rat, yet polyol levels do not begin to rise until > 48 h after the last dose of compound. Chronic administration of ICI 128436 to diabetic rats prevents the development of sugar cataracts in the lens and the deterioration of sciatic nerve conduction velocity. It is anticipated that, if the Sorbitol hypothesis is correct, ICI 128436 will ameliorate at least some diabetic complications.

363. Insulin Absorption Kinetics: Comparison of Disappearance of Iodinated Insulin and Appearance of 'Free' Insulin in Blood H. Strom, S. Lorentzen-Lund, K. Dahl-Jorgensen, P.Torjesen and K. Hansen. Medical Department B and Hormone Laboratory, Aker Hospital, Oslo, Norway The relationship between absorption of insulin from SC tissue and appearance of insulin in blood of insulin-dependent diabetic patients is complex. Furthermore, uncertainty exists about the current available 'free' insulin measurement and the importance of SC degradation. We therefore compared the two methods. Absorption was measured in eight patients by disappearance of 125I-semi-synthetic human insulin, (Nordisk; 8 IU injected on the abdomen) and appearance of 'free' insulin in plasma after pre-incubation for 2 h at room temperature. A correlation of 0.99 was found between the two methods. The half-life of the depot was 109 rain and Tmax appeared after 1 h. The absorption for the first 2 h was best described by zero order kinetics (r=0,999) and then a first order process (r=0,999). 'Free' insulin measurements following 2 h incubation at room temperature was closely associated with the disappearance of iodinated insulin from SC tissue. These findings indicate that SC degradation is without clinical importance, and that the best fitting kinetic model of the absorption is a zero order process for the first 2 h and then a first order process. These results indicate that the initial phase of insulin absorption is different from that described earlier.

364. Pattern of Glucagon Release in Response to Various Doses of Somatostatin-28 in Ducks M-T. Strosser, D. Di Scala-Guenot and P. Mialhe. Institute of PhYsiol- ogy, Universitb L. Pasteur, Strasbourg, France We have reported previously in ducks, that glucagon release was stimulated during somatostatin-14 infusions; this increase was secondary to the drop in plasma non-esterified-fatty acids (NEFA). Thirty min infusions of somatostatin-28 elicited different glucagon responses related to the amount infused: (1) with 50 ng. kg-1-min-1, an increase in glucagon plasma level, similar to the profile obtained with somatostatin-14; (2) with 200 ng- kg ~- min -1, a steady glucagon level during infusion followed by an important rebound release (from 1.37 ng/ml at the end of the infusion to 3.65 and 3.44 ng/ml respectively at 5 and 15 rain thereafter); (3) with 400 ng. kg 1. rain-l, an inhibition of glucagon (from 1.27 ng/ml to 0.75 after a 20 rain infusion) and a rebound secretion up to 4 ng/ml lasting 15 rain after the infusion was stopped. Substantial hypoglycaemia due to glucagon inhibition was observed. Therefore, it appears that somatostatin-28 is more potent in inhibiting A cell activity than somatostatin-14. The dose-dependent inhibition of insulin and NEFA levels was nearly similar compared with somatostatin-14. In ducks somatostatin-28 acts on glucagon regulation by at least two opposite mechanisms: (1) it increases glucagon release indirectly by decreasing plasma NEFA levels; (2) it inhibits glucagon release by a direct effect on pancreatic A cells.

365. A Disturbed Immediate Heart Rate Acceleration to Tilt Correlates with Sympathetic Adreno-Medullary Denervation in Type1 (Insulin- Dependent) Diabetes G. Sundkvist, B.Lilja, P. Manhem, and L.-O.Alm~r. Departments of Medicine, Endocrinology, and Clinical Physiology, University of Lund and Malta6 General Hospital, Maim6, Sweden The immediate heart rate response to tilt is mainly regulated by the vagal nerve, although sympathetic nerve activity might contribute to the immediate acceleration. The present study was designed to examine whether a disturbed immediate acceleration to tilt correlates with sympathetic denervation. Accordingly, a 2-s and 90 ~ tilt was performed in 13 male patients (mean age 45 years, range 31-61 years) with long-standing Type 1 diabetes and the heart rate, blood pressure, and plasma adrenaline and plasma noradrenaline responses were assessed. The immediate acceleration was disturbed in five patients as shown by a previously described abnormal acceleration index. Plas-

Abstracts

ma adrenaline fell or was unchanged during the first minute after tilt in four of these patients. In contrast, plasma adrenaline increased in all patients with a normal immediate acceleration. This difference in behaviour was significant (p < 0.014). The diastolic blood pressure fell 8 min after tilt in six patients. The diastolic pressure fall was minor (5 mmHg) in five of these patients. Patients with diastolic pressure falls showed a significantly lower increase in plasma noradrenaline (between 1 and 8 min after tilt) than those without (38 +9 versus 116_+16%, p<0.003). A disturbed immediate acceleration to tilt correlates with sympathetic adreno-medullary denervation and even a minor diastolic blood pressure fall indicates peripheral sympathetic denervation.

366. Re-instatement of Glucose Responsiveness of Rat Insulinoma Cells Maintained in Tissue Culture: Demonstration of Acute Glucose-In- duced Insulin Release Without Stimulation of 4SCa Uptake S. K. Swanston-Flatt and P.R. Flatt. Department of Biochemistry, University of Surrey, Guildford, Surrey, UK Acute and long-term effects of glucose on pancreatic B cells from an X-ray-induced transplantable rat insulinoma were examined in vitro. Fresh tumour cells failed to respond acutely to glucose or glucose plus

45 theophylline with increased Ca uptake and insulin release. During culture in RPMI-1640, blood cells and necrotic tumour cells rapidly disappeared. Cultured secretory cells did not proliferate but exhibited viability >95% and consumed substantial amounts of glucose (-1.6 lamol/106 cells per 7 days). Glucose (1.4-22.2 mmol/l) did not affect insulin content (73-911xg/106 cells) or insulin release (0.49-0.60 lag/106 cells per 7 days) in 7-day cultures, but both variables decreased by 5 ! % and 80% compared with 2-day values. Insulin output represented 1% of the fall in insulin content, suggesting substantial cellular insulin degradation. Acute exposure of 2-day cultured cells to glucose (16.7 mmol/1) did not affect insulin release (64-88 rig/ 106 cells per h) or 45Ca uptake (1.4-2.0 nmol/106 cells per h). However, combination with theophylline (10 mmol/1) evoked a 22-30% stimulatory insulin response. Cells cultured for 7 days also failed to respond acutely to glucose with increased ~SCa uptake, but secretion was stimulated by 22% 34% after maintenance at glucose concentrations of 5.6-22.2, but not 1.4 mmol/l. Addition of theophylline potentiated the insulin response irrespective of 45Ca uptake. This study demonstrates re-instatement of acute glucose responsiveness of tumour B cells, which exhibit increased efficiency of cellular Ca 2+ utilization.

367. Effect of the Genetic Background on the Capacity for Islet Cell Replication in Mice I. Swenne and A.Andersson. Department of Medical Cell Biology, University of Uppsala, Uppsala, Sweden Proliferation of pancreatic B cells may compensate for B cell destruction and an increased peripheral insulin resistance. Since the capacity for regeneration may be genetically determined, we have estimated glucose stimulated islet cell replication in inbred C57BL/6J and C57BL/KsJ mice. Islets isolated from 1, 3 and 9 month old mice were cultured in low (2.7 mmol/1) or high (16.7 mmol/1) glucose concentrations and islet cell replication was measured by means of 3H-thymidine incorporation as detected by autoradiography. To measure islet cell replication in vivo islets were implanted intrasplenically into normal or alloxan diabetic syngeneic mice, replicatory activity being estimated autoradiographically after 1 week. Glucose was found to stimulate islet cell replication in vitro in all age groups in both strains by a factor of two, but the proliferative rate decreased with increasing age. The rate of islet cell replication in C57BL/6J mice was, however, twice as high as in C57BL/KsJ mice, irrespective of the age. More- over, the alloxan-diabetic C57BL/6J mice displayed a more than twofold higher rate of replication in the implanted islets than the corresponding C57BL/KsJ mice. The results indicate that the genetic background modulates the proliferative response to glucose of the islets and may play a role in the development of diabetic syndromes.

368. Metabolic Effects of Diet Guar Gum Supplementation on Glucose Production, Metabolic Clearance Rate of Glucose, Insulin Binding, and Lipid Profile in Type 2 (Non-Insulin-Dependent) Diabetic Patients V.Tagliaferro, C. Bozzo, A. Bruno, A. Lombardi, M. Cassader and L. Cravero. Institute of Internal Medicine, University of Turin, Italy Insulin resistance is a pathogenic mechanism of hyperglycaemia in Type 2 diabetes. Fibre in the diet is reported to ameliorate hyperglycaemia in diabetes. To study this problem, we have treated eight newly diagnosed, normal weight, Type 2 diabetic patients, mean age 45 _+ 5 years, for 6 weeks on their usual diet plus guar-gum 8 g/day. The fol-

197

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lowing parameters were studied before and after guar treatment: glucose production (mg.kg -1 .min -1) by 3H-glucose infusion, insulin secretion during hyperglycaemic clamp (100mg/dl on fasting gl~- caemia), metabolic clearance rate of glucose ( M C R = m l . k g - . m i n - l = M-coefficient of DeFronzo/glycaemia), insulin binding to isolated monocytes (% specific binding) and ultracentrifugation of plasma lipid. Guar treatment produced the following variations: a significant decrease of fasting glycaemia (from 132 _+ 8 to 116 _+ 7 mg/dl) and insulinaemia (from 21 _+ 4 to 16 _+ 3 mU/l), a non-significant decrease of glucose production (from 3.17 + 0.7 to 2.44_+ 1.2), an increase in glucose MCR (from 1.55 _+ 03 to 2.0 _+ 0.31), an increase in % insulin binding (from 2.51 _+ 0.45 to 2.73 _+ 0.3), a decrease of total triglycerides and cholesterol, and a decrease of LDL-cholesterol (from 139 _+ 21 to 121 _+ 12 mg/dl), an increase in HDL-phospholipids (from 102 _+ 10 to 114 _+ 8 mg/dl). These results confirm that guar-gum, even at low but well tolerated doses, is able to produce an amelioration in glucose and lipid metabolism in mild Type 2 diabetes.

369. A Possible Hierarchy of Vulnerability of Nerve Fibres in Type t (Insulin-Dependent) Diabetic Patients. L.Tallarigo, B. Rossi, L.Benzi, F.Sartucci, R. Miccoli, P.Marchetti, S. Lenzi, M. Tognarelli and R. Navalesi. Cattedra di Malattie del Ri- cambio, Clinica Medica II e Istituto di Clinica Neurologica, Pisa, Italy To evaluate the clinical utility of some measurements of nerve conduction velocity in the diagnosis of early abnormalities in peripheral nerve function in diabetes, we studied motor conduction velocities (MCV) in ulnar and posterior tibial nerve, F-index from the abductor hallucis (in the Dll popliteal-region tract), sensory conduction velocities (SCV) in sural nerve and H-index from soleus in 20 fairly well- controlled (HbA1, 10.3 + 2.2%) Type 1 diabetic patients without clinical signs of neuropathy (mean age 22.8 -+ 5.8 years; mean duration of diabetes 6_+ 4 years) and 20 normal subjects. Ulnar nerve MCV was reduced significantly in diabetic versus control subjects (55.2 + 5.1 versus 60.9_+_ 6.7 ms, p< 0.02). Tibial nerve MCV was strongly reduced in diabetic versus control subjects (43.1 _+ 3.7 versus 49.2_+ 3.8 ms, p < 0.001). In contrast, F-index and sural SCV were similar in diabetic and control subjects. Nevertheless H-index was significantly reduced in diabetic versus control subjects (53.5_+ 5.2 versus 58.3 _+ 3.7 ms, p < 0.02). In summary we found early impairment of MCV in the distal tracts of nerves both in upper and lower limbs while the proximal tracts were spared. In fact we observed normal values of the F-index. In contrast early changes in SCV affected the proximal nerve tracts, as showed by H-index and sural nerve results. Our data suggest a hierarchy of vulnerability of motor and sensory fibres in diabetes.

370. Metabolic Fate of Palmitate in IsOlated Islets of Fed and Starved Rats J.Tamarit-Rodriguez, E.Vara and J.Tamarit. Department of Bio- chemistry, Faculty of Medicine, Madrid, Spain We have shown previously that 2-bromostearate, an inhibitor of fatty- acid oxidation, restores a normal insulin response to glucose in fasted islets. Therefore, we investigated starvation effects on islet palmitate metabolism. Palmitate oxidation was measured as 14CO2 produced from 0.5 mmol/1 (U-14C)-palmitate (pmol/ixh). Incorporation of (U- 14C)-palmitate (0.5 mmol/1) into islet lipids (TG + PL) was studied by measuring the radioactivity in triacylglycerol and phospholipid fractions after Folch lipid extraction and chromatographic separation (pg.h 1.txDNA-1). In fed islets a 3-20mmol/1 change in glucose concentration reduced palmitate oxidation by 50% and doubled its incorporation into TG and PL. Starvation (48 h) induced a higher rate (x 2) of palmitate oxidation at 3 mmol/1 glucose, not inhibited by 20 mmol/l glucose. The latter also failed to increase palmitate incorporation into TG + PL, the amount of labelled TG being significantly lower than in fed islets. 2-bromostearate (0.25 mmol/1) strongly decreased islet palmitate oxidation irrespective of either nutritional state or glucose concentration. It also restored 20 mmol/1 glucose ability to stimulate palmitate incorporation into TG + PL in fasted islets but had no significant effect on fed islets. Starvation makes islet palmitate metabolism insensitive to glucose. 2-bromostearate antagonism to the effect of starvation on islet secretion and lipid metabolism suggests a possible link between these two phenomena.

371. Assessment of Methods of Achieving and Measuring Long-Term Metabolic Control in Animal Models of Insulin-Dependent Diabetes Mellitus F. J. Tames, A.J. Bone, and J. D. Baird. Metabolic Unit, University De- partment of Medicine, Western General Hospital, Edinburgh, Sot- land

198 Abstracts

Use of animal models for investigating the development of diabetic microangiopathy in insulin-dependent diabetes depends on adequate methods for measuring, achieving and maintaining various degrees for metabolic control for long periods. Using both streptozotocin-induced and spontaneously diabetic BB Wistar rats, we have investigated the use of fast haemoglobin (fast Hb) and compared the degree of metabolic control achieved by Azlet miniosmotic pumps implanted for periods of up to 4 weeks. Electrophoresis on agarose and citrate agar gels showed no abnormal fractions interfering with rapid determination (Coming) of stable fast Hb in saline-washed erythrocytes (10 jxl blood, interassay coefficient of variation: < 3.5%). Mean_+ SD fast Hb was 2.73 -+ 0.11% for non-diabetic Wistar rats and 6.3 -+ 0.24% for untreated diabetic animals. Pumps gave significantly better metabolic control than conventional treatment as measured by mean-+ SD fast Hb (3.1 _+ 0.02 versus 4.1 + 0.4% p < 0.002), random plasma glucose (6.4 + 0.62 versus 7.6 + 0.08 mmol/1, p < 0.02), 24 h urinary glucose excretion (2.8_+ 1.3 versus 4.8 + 1.6 g, p < 0.01) MAGE value (1.7-+0.8 versus 3.8 + 0.5) and serum cholesterol (2.3 + 0.26 versus 2.65 -+ 0.28 mmol/1, p < 0.01). There was no difference between treatment groups in respect of body weight, food and water intake, plasma lactate, serum triglyceride, and MODD values. Use of fast Hb measurements and osmotic pumps make valid long-term studies of the development of microangiopathy feasible.

372. Adipocyte Insulin Binding and Insulin Sensitivity in Brittle Diabe- tes R. Taylor, D.J. Husband, S. M. Marshall and K.G.M.M. Alberti. De- partment of Clinical Biochemistry and Metabolic Medicine, Royal Victoria infirmary, Newcastle upon Tyne, UK Insulin binding and in vitro insulin sensitivity was assessed using adipocytes from seven female patients with severe brittle diabetes (age 24-+2 years, 120+5% ideal body weight, mean daily insulin dose 137 _+ 14 units). All patients were in stable metabolic control on a standard diabetic diet when studied. For comparison, a group of female diabetics patients (mean age 23 +2 years, mean daily insulin dose 51 + 3 units, 124% ideal body weight and a group of non-diabetic subjects were studied). Maximal insulin binding was markedly reduced in the brittle diabetic patients (1.74%, 2.23% and 2.52%/10cm 2 cell membrane for brittle, non-brittle and normal subjects, respectively, p< 0.01 brittle versus normal subjects and p< 0.05 brittle versus non- brittle subjects). Scatchard analysis showed predominantly decreased receptor number and also reduced affinity in the brittle group. Degra- dation of tracer insulin was similar in the three groups. Basal lipogenesis was slightly higher in the brittle patients adipocytes (81 _+ 13 versus 56 + 11 and 64+ 9 pmol/10 cm 2 cell membrane) as was maximal lipogenesis (193 +_ 28, 110_+ 20 and 132 + 19 pmol/10 cm 2, respectively; p < 0.05 brittle versus non-brittle and normal subjects). However, half maximal stimulation of lipogenesis was observed at similar insulin concentrations (1.4, 2.0 and 1.7 x 10 -11 tool/l). Hence in this condition, low insulin receptor number and affinity is associated with no impairment of in vitro insulin sensitivity as assessed by measuring lipogenesis.

373. Effects of Fasting and Changes in Non-Esterified Fatty-Acid Con- centrations on Leucine-Carbon Flax and Oxidation in Vivo P. Tessari, S. Nissen, E. Tsalikian and M. W. Haymond. Patologia Me- dica e Malattie del Ricambio, University of Padua, Italy, and Endo- crine Research Unit, Mayo Clinic, Rochester MN, USA. As fat may have a protein-sparing effect during fasting, it is of interest to know whether changes in non-esterified fatty acid (NEFA) concentrations affect leucine-carbon flux and oxidation in vivo. Dogs fasted for 14 or 46 h were infused with L-(4,5)-3H-Leucine and 1-14C-a - ketoisocaproate. Flux and oxidation rates were calculated at steady- state using a dual-isotope model. After baseline-sampling, animals received one of the following infusions for 150-180 min: (1) intralipid+heparin; (2) glycerol (as control for intralipid+heparin); (3) nicotinic acid, in order to generate different NEFA concentrations. In 14 h-fasted dogs (NEFA 0.5 mmol/l) basal leucine-carbon flux and oxidation were higher (+ 30%, p < 0.01, and + 40%, p < 0.05, respectively) in comparison with 46 h-fasted dogs (NEFA = 1.4 mmol/1). In 14h-fasted animals, intralipid+heparin infusion increased NEFA

!r~ 3 O70470~.2mto ~ ; ! ~ ; ~ 2 ; i 7 )mC ~nne- d climb ~ 1 & ~ r bf whn? !l~y~0r ~6

controls showed no changes. In 46 h-fasted dogs, nicotinic acid infusion decreased NEFA ( A = -- 1.1 mmol/1) and increased leucine-carbon flux from 3.6 _+ 0.4 to 4.4 _. 0.5 #mol. kg -1. min -1 (p < 0.01) and leucine-carbon oxidation from 0.4+0.1 to 1.1 +0.4#mol-kg -1.

rain-1 (p < 0.02). In summary: (1) in short-term fasting leucine-carbon flux and oxidation are lower than in the post-absorptive period; (2) acute changes in NEFA concentrations induce opposite changes in both leucine-carbon flux and oxidation and may modulate leucine metabolism in vivo.

374. Effect of Induced Glycosuria on Uric Acid Clearance N.C.Thalassinos, A.Vassiliou, N.Tountas, G.Chalevelakis and S. Raptis. Second Department of Internal Medicine-Propaedeutic, Athens, Greece Gout is encountered less frequently in diabetic than non-diabetic subjects probably due to increased uric acid clearance (CUA), as we have shown in a recent report where the degree of glycosuria in diabetic subjects was parallel to CUA. Attempting to clarify further the aetiology of increased CUA, glycosuria was induced by a 12-h glucose infusion in 10 non-diabetic normal volunteers, while a similar degree of polyuria was achieved by a normal saline infusion on a separate day. Uric acid, glucose and creatinine were determined in 4-h blood samples and urine collections and the CUA, creatinine clearance and their ratio was subsequently calculated. The results obtained showed that during both infusions there was a highly significant increase (p < 0.001) in CUA and a significant decrease (p< 0.01) in serum uric acid while there was no statistical difference in these changes between the two infusions. It is concluded that increased CUA and reduced serum uric acid are a feature of glycosuria probably due to the resulting polyuria as similar changes were observed in the polyuria following normal saline infusion.

375. Calcium-Dependent Stimulation of Protein Phosphorylation by Calmodulin and Phosphatidylserine in Mouse Pancreatic Islets P. Thams, K. Capito and C.J. Hedeskov. Department of Biochemistry A, University of Copenhagen, Denmark In a search for the mediator of Ca2+-induced exocytosis and insulin release, the effects of calmodulin and phosphatidylserine on protein phosphorylation were studied. Homogenates and subcellular fractions of mouse pancreatic islets were incubated with 32P-ATP in the presence of Mg 2+, and with various combinations of Ca 2+, calmodulin, and phosphatidylserine. Phosphorylated proteins were separated by SDS-polyacrylamide gel elctrophoresis and radioactive bands were localized b~( autoradiography. Islet protein phosphorylation was enhanced by Ca-+-calmodulin; the major endogenous substrates in a homogenate were two proteins of mol.wt, around 53 and 100 kdalton. The 100 kdalton phosphoprotein was localized to a 27 000 g supemat- ant fraction, whereas the 53 kdalton phosphoprotein was present in a 27 000 g particulate fraction of mouse islets. In the presence of Ca 2+, phosphatidylserine stimulated phosphorylation of 12 proteins in the mol.wt, range from 30-200 kdalton in a 27 000 g supernatant fraction. No effect of Ca ~+ plus phosphatidylserine was observed in a 27 000 g particulate fraction of mouse islets. The results demonstrate that Ca 2 +-dependent phosphorylation of endogenous proteins in pancreatic islets in regulated independently by phospholipid and calmodulin. The extensive stimulatory effect of phosphatidylserine suggests that the Ca2+-phopholipid-sensitive protein kinase may prove to be a prominent phosphorylation system in pancreatic islets.

376. Saturability of Insulin Degradation in Normal and Type I (Insulin- Dependent) Diabetic Men B.Thorsteinsson, S.Fugleberg, K.Ellemann, B. Feldt-Rasmussen, O. Ortved Andersen and C. Binder. Steno Memorial Hospital, Gen- torte, and Frederiksborg County Hospital, Hillerod, Denmark Both non-saturable and saturable insulin degradation mechanisms have been proposed in animals and man. A kinetic model allowing the possible existence of both mechanisms of insulin degradation was constructed on the basis of these presumptions: Iex=--Iend + A + B = - I~nd + kl' C + (k2" c)/(k3 + C), where Iex = the exogenous insulin infusion rate, I~nd=the endogenous insulin release rate, A= the non-saturable component, B=the saturable component, C= the steady state plasma insulin concentration, k~ =the clearance of insulin in A, k2=the maximal insulin degradation rate in B, and k3 = the plasma insulin concentration at which the insulin degradation rate in B equals kz/2. The model was tested in 14 normal and six Type I diabetic men (without insulin antibodies) by means of constant infusion of porcine insulin to steady-state (plasma insulin range: 0.01-17.6 nmol/1) using euglycaemic glucose clamp. In conclusion (1) existence of non-saturable insulin degradation could not be demonstrated in either group (weighted non-linear regression, method of least squares). The median value of k2, k3, and k2/k3 were 0.115nmol.kg-l .min -1, 4.31nmol/1, and 26.5ml-kg-l-min -1 in

Abstracts 199

normal men compared with 0.179 nmol. kg -1. min -~, 12.4 nmol/1, and 18.7 ml. kg- I. min -1 in diabetic patients (p = 0.0082, p = 0.0010, and p = 0.0022, respectively)�9 (2) At physiological levels insulin is degraded at a lower rate in diabetic compared with normal men.

377. Visualization of Insulin Binding and Internalization Ch.Thun, R. D. Ful3ggnger, X. Bihr and E.F. Pfeiffer. Department of Internal Medicine I, University of Ulm, FRG Colloidal gold is easily detected by transmission electron microscopy. Insulin can be linked with colloidal gold, and the resulting complexes behave like the native insulin. Colloidal gold was prepared according to Frens and coupled with insulin to generate insulin-gold-complexes (IGC). Our studies showed that: (1) IGC competitively displaced 125I- insulin bound to IM-9 lymphocytes; (2) IGC stimulated r4C-glucose incorporation into fat cells. Compared with unlabelled insulin this activity was > 20%. Living blood cells were incubated with IGC and processed for transmission electron microscopy. Human monocytes and neutrophils in particular bind and internalize IGC specifically. Binding and internalization is time-, temperature- and pH-dependent. Internalization proceeded in consecutive steps: formation of coated pits, coated vesicles and secondary lysosomes. IGC was not detected in the nucleus but war often close to the Golgi region. Distribution histograms indicates two phases of the internalization process at 37~ Non-nucleated human red blood cells only bind IGC. In contrast, nucleated red blood cells from chicken, trout and frog, as well as human reticulocytes, bind and internalize IGC. In condusion, IGC may be of value in studying intracellular processing of insulin, and internalization of insulin appears to be limited to nucleated cells.

378. Impairment of Insulin Sensitivity by Alcohol in Normal Subjects A. Tiengo, A. Avogaro, P. Fontana, F. Mollo, A. Lapolla, C. Marescotti and E. Iori. Malattie deI Ricambio, Patologia Medica deil'Universitfi di Padova, Italy In order to explain the impairment of glucose tolerance induced by alcohol, we evaluated the effects of different doses of alcohol on peripheral insulin sensitivity, expressed as both glucose disposal and as modifications of insulin-dependent intermediary metabolites during euglycaemic clamp. At time 0, saline or alcohol at 9 g/h or 17 g/h were infused randomly to five and four normal males respectively. At time 180 min an exogenous insulin infusion of 33 mU-m 2-1. min -1 was started. Blood glucose was clamped at 4.8 mmol/l and the same insulin plateau of 70 + 2 mU/1 was achieved both with and without alcohol insulin sensitivity was calculated as M (mmol. kg-4. min-~ value, where M is the glucose metabolized. With alcohol (17 g/h) M was 0.39 + 0.04; without 0.46.0.08 (p < 0.02). With alcohol (9 g/h) M was 0.41 + 0.03; without 0.49 • 0.04 (p < 0.02). During alcohol infusion a marked increase in lactate/pyruvate (p<0.02) and 3-hydroxy butyrate/acetoacetate (p < 0.02) ratio was observed without varying during clamp. After exogenous insulin administration a lack of increment in blood lactate and pyruvate and a normal decrement of non-esterified fatty acids, glycerol and ketone-bodies at both rates of alcohol infusion were observed. In conclusion, alcohol administration at different doses impairs insulin-mediated glucose disposal probably through an impairment of glycolitic pathway. Alcohol does not interfere with the antilipolitic and antiketogenetic effects of insulin�9

379. Amelioration of Insulin Sensitivity Induced by Physical Exercise in Type 2 (Non-Insulin-Dependent) Diabetes Mellitus M. Trovati, F. Cavalot, S. Vitali, L. Ricchiardi, M. Cottafavi, G. Pagano and G. Lenti. Institute of Internal Medicine, University of Turin, Italy Exercise increases insulin sensitivity and decreases insulin secretion in animals and in healthy man. Since insulin resistance and insulin hypersecretion are important features of Type 2 diabetes, we planned the present study in order to investigate the effects of a mild physical activity in Type 2 diabetic patients. Five Type 2 diabetic subjects (aged 40-62 years, not pharmacologically treated) were submitted to an euglycaemic (about 5.55mmol/1) hyperinsulinaemic (about 100mU/1) clamp in order to measure the steady-state glucose infusion rate (SSGIR) before and after 2 months of programmed physical activity (2 hours a week of run or cycle at the pulse rate of about 120beats/ min). No significant change was observed in body weight (75 _+ 6 and 74__6kg) or in fasting plasma glucose (8.5+0.5 and 8.33+ 1.05 mmol/1) Basal insulin levels decreased from 17• to 11 • 2mU/1 (p<0.05), and SSGIR increased from 29• to 42• 5.9 umol. kg -1 �9 min -~. The increase of SSGIR is significant (p< 0.05) when we consider only the patients with a reduced SSGIR before the study, since the only patient with an elevated SSGIR (53 umol. k~ -1 �9 mm ) did not show a further increase. In conclusmn, mild physical

activity, also advisable for middle-aged patients, can reduce the insulin resistance and the insulin hypersecretion in Type 2 diabetic patients independently of body weight changes. The reduction of hyperinsulinaemia could be useful also for the prevention of atherosclerosis.

380. Vascular Reactivity in Newly Diagnosed Diabetic Children T. Tuvemo, M, Kobbah and U. Ewald. Department of Pediatrics, Uni- versity Hospital, Uppsala, Sweden Recently, reduced vascular reactivity measured by a new non-invasive technique could be demonstrated in children with Type I diabetes of 2-8 years' duration. This study describes vascular reactivity in newly diagnosed diabetic children and its correlation to carbohydrate control. Eight diabetic children were studied twice (on days 3-9 and on days 18-31) after the diabetic disorder was diagnosed. Post-ischaemic hyperaemia, a functional vascular reaction, was studied using a conventional transcutaneous oxygen (tcPO2) electrode at 37 ~ Twenty- four-hour glucose excretion, glycosylated haemoglobin (HbA0, plasma glucose and mean plasma glucose during 24h were analysed by standard methods. Post-ischaemic tcPO2 increased (1.7___ 1.0-3.2_+ 1.2 kPa, p < 0.01). HbA1, plasma glucose and urinary glucose excretion decreased. No significant correlation was found between the parameters of carbohydrate metabolism and vascular reactivity, neither on days 3-9 nor on days 18-31. In conclusion, vascular reactivity on days 3-9 after diagnosis of Type 1 diabetes was strongly reduced compared to days 18-31, when values were in the range found in healthy children. During insulin treatment both vascular reactivity and metabolic control normalizes in parallel. The mechanism for the reversible reduced vascular reactivity in recently diagnosed diabetic children is probably complex and is not explained by disturbances in carbohydrate metabolism only.

381. Adrenaline Inhibition of Insulin Release is not Mediated by Lower- ing of Islet Cycle AMP S. Ulrich and C. B. Wollheim. Institute of Clinical Biochemistry, Uni- versity of Geneva, Geneva, Switzerland It has been suggested that a2-adrenergic inhibition of insulin release results from decreased generation of cyclic AMR We investigated whether lowering of cyclic AMP always accompanies adrenaline inhibition of insulin release. When freshly isolated rat islets were incubated for 30 min, 8-bromo-cyclic AMP (5 mmol/1) doubled insulin release at non-stimulatory glucose concentrations. Adrenaline (10 .6 mol/1) abolished the stimulation due to exogenous cyclic AMR Alterations of endogenous cyclic AMP and insulin release were measured simultaneously in the presence of forskolin, a specific stimula- tor of adenylate cyclase. At 3 min, forskolin (30 #mol/1) augmented islet cyclic AMP sixfold at glucose concentrations of both 2 and 20 mmol/1, but enhanced insulin release only at 20 mmol/l glucose. Adrenaline (]0-7mol/1) abolished stimulated insulin release while greatly potentiating forskolin-induced rise in cyclic AMR Both inhibition of insulin release and enhancement of islet cyclic AMP by adrenaline were overcome (by 65%) by the a2-adrenergic blocker 5(o_ himbine. At 15 min, cyclic AMP was further augmented by forskolin but adrenaline no longer potentiated the effect, although still inhibiting insulin release. Thus, in the presence of forskolin, the inhibition of insulin release by adrenaline is not associated with a lowering of cyclic AMP levels. This, and the inhibition of 8-bromo-cyclic AMP- stimulated insulin release, strongly suggest that adrenaline inhibits insulin release at a step distal to cyclic AMP generation.

382. Echocardiographic Evaluation of Left Ventricular Performance and Hypertrophy in Newly Diagnosed Non-Insulin Dependent Diabetic Subjects M. Uusitupa, O. Siitonen, A. Aro and K. Py6r~il~i. Department of Med- icine, University of Kuopio, Kuopio, Finland An echocardiographic study to assess left ventricular performance and hypertrophy (LVH) was carried out in 133 newly diagnosed diabetic patients (aged 45-64 years) and in 144 randomly selected non- diabetic subjects of the same age. A reliable recording was obtained in 59 male and 48 female diabetic patients and in 52 male and 70 female non-diabetic subjects. Male diabetic patients showed significantly lower ejection fraction than male controls (66.7 + 1.3 versus 72.6 + 1.4%; mean + SEM; p= 0.001), but no significant differences were found in variables reflecting LVH. Female diabetic patients showed slightly lower ejection fraction than female controls (71.8 • 1.8 versus 73.5 + 0.9%; p ~ 0.270), but the calculated left ventricular mass/m 2 of body surface area was significantly greater in female diabetic patients than controls (171 +10 versus 142+4g/m2; p=0.027). After adjust-

200 Abstracts

ment for age, body mass index, coronary heart disease and hypertension, male diabetic patients still had significantly lower ejection fraction than male controls indicating depressed myocardial contractility attributable to diabetes itself. LVH found in female diabetic patients could be ascribed to the frequent occurrence of hypertension, but there was some evidence of a synergistic effect of diabetes and hypertension on LVH in women.

383. Improvement in Long-Term Diabetic Control After High Fibre (Bran and Gua0 Diets S. Vaaler, K.F. Hanssen, K. Dahl-Jorgensen, W. Frolich, J. Aaseth, B. Odegaard and 121. Aagen~es. Pediatric Department, Medical Depart- ment B, Clinical Chemical Department, Aker Hospital, Oslo and Na- tional Institute of Technology, Oslo, Norway While high fibre diets improve diabetic control in short-term studies, evidence from long-term studies are lacking. Twenty-eight insulin-dependent diabetic patients underwent three dietary periods, each lasting 3 months. First (normal period) they used a white flour bread, then their bread rations were enriched with wheat bran (bran period - mean daily supplementation: 33 g and then with guar gum (guar period - mean daily supplementation: 29 g) in a randomized crossover pattern. No other efforts were taken to improve their diabetic control. Fasting and post-prandial blood glucose levels were measured at home once weekly (filter paper method), other parameters were measured monthly. No improvement in diabetic control was seen during the normal period. Mean blood glucose decreased from 11.8+ 3.5 mmol/1 (mean + SD) in the normal period to 9.9 +2.6 mmol/1 (p < 0.025) in the bran period and 9.9 _+ 2.8 mmol/ l (p < 0.025) in the guar period. HbA1 decreased from 10.5 +_ 2.1% in the normal period to 9.9+1.2% (NS) in the bran period and 9.6+1.5% (/)<0.05) in the guar period. Total cholesterol increased from 4.9 + 0.8 mmol/1 in the normal period to 5.4 + 1.0 mmol/1 (p < 0.025) in the bran period, but decreased to 4.5_+ 0.8 mmol/ l (p < 0.05) in the guar period. Conclu- sion: in this long-term study bran and guar gave a sustained decrement in mean blood glucose.

384. Risk Factors for Atheroselerosis in Individuals with Impaired Glu- cose Tolerance O. Vaccaro, B. Capaldo, L. Tutino, G. Riccardi and A. Rivellese. De- partment of Internal Medicine, 2nd Medical School, University of Naples, Italy Individuals with impaired glucose tolerance (IGT) are considered to be at risk for cardiovascular disease. This study attempts to evaluate whether this increased risk might be explained by the clustering of other major cardiovascular risk factors after controlling for the excess of obesity in these subjects. Among 1300 participants in a health examination survey (age range: 40-59years), all IGT individuals (n = 65) plus 125 age, sex and weight matched control subjects (EASD criteria) participated in the study. Treated hypertensive and hyperlip- idaemic patients were excluded. Only blood pressure (both systolic and diastolic) was signficantly higher in the IGT group ('136 + '16 versus 127 + 15 mmHg, p < 0.00'1 ; 87 + '10 versus 84 + 8 mmHg, p < 0.05; mean_+SD), while fasting blood lipids were similar (cholesterol 5.54 + 0.88 versus 5.67 + '1.04; triglycerides '1.65 _+ 0.67 versus '1.53 + 0.72 mmol/l) as was serum insulin both fasting (24_+ 16 versus 28 + 4'1 mU/1) and 2 h after 75 g of oral glucose (80_+ 52 versus 67_+ 49 mU/1). In a random subgroup of 20 IGT and 44 control subjects plasma lipoprotein composition was also evaluated. No difference was found in VLDL-triglycerides ('1.06_+ 0.61 versus 0.80_+ 0.39 mmol/1). LDL-cholesterol (3.96 + 0.57 versus 3.9'1 _+ 0.83 mmol/1) or HDL-cholesterol ('1.32 _+ 0.57 versus '1.37 _+ 0.31 mmol/1).In conclusion, when controlling for obesity, IGT is not associated with any lipid abnormality; blood pressure is the only cardiovascular SK factor associated with IGT independent of matched variables.

385. Treatment of Pregnant Type I (Insulin-Dependent) Diabetic Pat- ients with Continuous Subcutaneous Insulin Infusion: Several Obstetri- cal Complications Despite Tight Metabolic Control E.van Ballegooie, G. H.A.Visser and R.N. Laurini. Departments of Clinical Endocrinology Obstetrics and Gynaecology and Pathology, University Hospital, Groningen, The Netherlands Twenty-one patients with Type 1 diabetes (White Class B-D) were treated with continuous SC insulin infusion (CSII) during 24 pregnancies. In nine patients CSII was started before conception. Good metabolic control could be achieved in all patients with a mean blood glucose of 5.3 mmol/l. HbA1 (mean_+ SD) fell from 10.3 +2.2% to 8.5 __ 1.3% after 1 month CSII, and decreased to 7.2 _+ '1.0% at term (normal range: 6-8.5%). Technical and patient-related pump-prob-

lems occurred once a month. None of the patients developed ketoacidosis; seven episodes of severe hypoglycaemia occurred. These results were significantly better than those previously obtained with conventional insulin treatment. Twenty out of 21 infants, born after 28 weeks of gestation, survived with a decreased perinatal morbidity compared with conventional treatment. Only one infant had a birth weight > 97.7 percentile. Despite the generally favourable outcome, several obstetrical complications were encountered, including spontaneous preterm labour (n = 3), severe intrauterine growth retardation (n=2), ante-partum asphyxia (n=3) and unexplained intrauterine death (n = 1, week 37, birth weight 95-97.7 percentile). It is concluded that long-term (near) normoglycaemia can be achieved with CSII. This reduces the duration of hospitalization as well as perinatal morbidity. The obstetric risks, however, are still considerable, even if pump treatment is started before conception:

386. Comparison of Reagent Strips and Reflectance Meters for Blood Glucose Estimation R Van Crombrugge, R. Rottiers and A. Vermeulen. Department of En- docrinology, University Hospital, Gent, Belgium We compared three reagent strips (Dextrostix, Hemo-glukotest 20-800R ( = HG 20-800R) and Visidex) and two reflectance meters (Glucometer/Dextrostix and Reflolux/HG 20-800R) with a laboratory method. 100 unexperienced patients visually measured their capillary blood glucose concentration with all strips and an investigator used the reflectance meters independently with the same strips. The correlation coefficient with a conventional laboratory method (capillary plasma) for the visual reading of Dextrostix was + 0.684, of HG 20-800R+ 0.851 and of Visidex+ 0.794. The correlation coefficient for the Glucometer was + 0.866 and for the Reflolux + 0.955. In a second part of the study, experienced operators tested Visidex, HG 20-800R and Reflolux on 100 venous blood samples. The reference method was a glucose-oxidase method (venous plasma). The correlation coefficient was+0.912 for Visidex,+0.967 for HG 20-800R and + 0.989 for Reflolux. Slope and intercept from the least square regression line were for Visidex were + 1.274 a n d - 46.2 mg/dl, respectively, for HG 20-800R+1.057 a n d - 1 0 mg/dl, and for Reflo- lux+ 1.109 and-21 .9 mg/dl. Visual reading of HG 20-800R and Visidex (both having a dual colour zone) seems superior to glucose estimation by Dextrostix. Reflolux (with (HG 20-800R) was more reliable than Glucometer (with Dextrostix).

387. Regulation of Insulin Receptors in the Heart: Studies on Adult Rat Cardiac Myocytes in Primary Culture E.van Echten, J. Eckel, A. Stocks and H. Reinauer. Diabetes Research Institute, Dtlsseldorf, FRG Regulation of insulin receptors by insulin has been shown in different cell systems. This relationship, however, has not been studied in the heart. Therefore, attempts have been made to establish a system of adult cardiac myocytes in primary culture and to investigate the regulation of insulin receptors in these cells. Cardiocytes were kept in culture in serum free medium for at least 4 days. Under these conditions cells maintained a rod-shaped morphology, unaltered ultrastructure and a constant level of ATP (241 + 9 nmol/106 cells, n = 5). Cultured cells exhibited a nearly constant level of steady-state insulin binding (60 rain at 37 ~ up to 3 days. Curvilinear Scatchard plots of insulin binding were observed during this culture period. Exposure of cardiocytes to high concentrations of insulin (1.7 7 x 10 .6 tool/l) for different periods of time (4-30 h) resulted in an increasing down-regulation of the insulin receptor reaching a maximal decrease in insulin binding by approximately 50%. This effect was reversible after removal of the hormone from the culture medium. In conclusion, our data show that heart myocytes in primary culture represent a new model for the study of cardiac insulin receptors; moreover, it appears that insulin can inversely regulate its own receptor in this tissue.

388. Diabetic Nephropathy and Continuous Subcutaneous Insulin Infu- sion P.Vannini, A.Ciavarella, M.Flammini, L.C.Borgnino ~ and L.Bacci. Department of Metabolic Diseases and 1Department of Nephrology and Dialysis, Bologna, Italy The aim of our research was to evaluate the effect of the strict glycaemic control achieved by means of continuous subcutaneous insulin infusion (CSII - Microjet Miles) on the progression of diabetic nephropathy. Ten Type 1 diabetic patients, mean age 33 + 10 year, mean duration of diabetes 15.5 + 5.0 year, mean dose of insulin 45.5 U/day, were studied. All had clinical proteinuria, 3 decreased glomerular filtration rate, elevated serum creatinine levels and arterial hyperten-

Abstracts 201

sion, 3 had proliferative and 7 background retinopathy. They were subdivided in two groups of 5 patients. Group A received the conventional insulin treatment, while group B was treated with CSII. Meta- bolic assessment was by means of mean blood glucose (MBG), 24 h urinary glucose excretion and HbA1 (Bio-Rad). Kidney function was studied by evaluation of urinary excretion rates of albumin, IgG (single radial immunodiffusion method), fl-2-microglobulin (Phadebas fl- 2-micro test), lysozyme (turbidimetric method) and of IgG/transfer- rine clearance ratio (selectivity index of proteinuria). The study lasted 8 months. Results: Better metabolic control was observed in patients treated by means of CSII than in those receiving conventional treatment. In both groups kidney function deteriorated progressively which resulted in increased urinary excretion rates of all proteins and in decreased selectivity of proteinuria. In group B kidney function deteriorated less than in groupA (albumin 18versus ~tg.min -1-- month- l ; IgG 3.1 versus 16.0 ~tg. rain <. month<; fl-2-microglobulin

1 1 21.2 versus 66 .0ng .min- .month- ; lysozyme 8.4 versus 67.0ng-- min -1. month-l). Strict metabolic control seems to be able to delay, but not to reverse, the progression of diabetic nephropathy.

389. Insulin Receptor: A Tyrosine Protein Kinase for Exogenous Pro- tein Substrates E.Van Obberghen, H. Gazzano and A. Kowalski. INSERM U 145, Faculty of Medicine, Nice, France We have previously shown that the/3 subunit (Mr 95,000) of the insulin receptor is its own protein kinase and that insulin increases phosphorylation of its own receptor. We now report on the ability of insulin receptors to phosphorylate exogenous substrates and on the nature of the aminoacid modified. To investigate the kinase activity of insulin receptors toward exogenous substrates, histone and casein were incu-

3 2 bated with (?'- P)ATP and purified insulin receptors together with or without insulin. Finally, the labelled proteins were analyzed by SDS- PAGE. Both histone and casein were heavily phosphorylated by the insulin receptor-kinase. More important, insulin increased several- fold the substrates' phosphorylation. To identify the aminoacid(s) phosphorylated, the substrates were subjected to acid hydrolysis and phosphoaminoacids were separated by thin layer electrophoresis. In absence of insulin, both substrates were phosphorylated on tyrosine, threonine and serine residues�9 However, insulin-stimulated increase in pho~phorylation was mainly due to an increment in 32p-tyrosine. Similar results were found with the insulin receptor itself. Indeed, insulin-induced increase in autophosphorylation of the/3 subunit was

32 largely accounted for by an increase in P-tyrosine. In conclusion, (1) the insulin receptor-kinase is not only a kinase toward itself, but also toward exogenous substrates; (2) this receptor kinase phosphorylates tyrosine residues in both the receptor and exogenous substrates.

390. Glucose is Involved in the Regulation of Glucose Uptake in 3T3 Adipocytes J.P.M.van Putten, Tj.Wieringa and H.M.J.Krans. Department of Endocrinology and Metabolic Diseases, Diabetes Research Labora- tory, University Hospital, Leiden, The Netherlands In diabetes mellitus, glucose uptake is diminished due to a decrease in number of functional glucose transporters. Knowledge of the regulation of glucose transport is crucial for better understanding of the pathophysiology of diabetes�9 We examined the role of glucose in the regulation of its own uptake and of the effect of insulin. Cultured 3T3- L~ adipocytes were exposed to various concentrations of glucose. Subsequently insulin-sensitive 2-deoxyglucose was measured. Glu- cose concentrations above 5 mmol/l did not affect uptake of 2-desoty- glucose. Glucose deprivation for 48 h, however, resulted in increased Vmax (four times) indicating an increase in number of transporters, parallelled by a loss of insulin effect, not explicable by altered insulin binding. Cycloheximide (1 ~tg/ml) prevented the enhancement of 2- deoxyglucose-uptake but not the stimulatory effect of insulin, indicating that the increase in number of transporters by insulin and by glucose deprivation are probably mediated by different mechanisms. Addition of glucose to deprived cells rapidly 'down-regulates' the number of transporters and restores the stimulatory effect of insulin. Hexoses, competitively transported with 2-deoxyglucose can replace glucose in this respect. Our results indicate that, in adipocytes, glucose can 'down-regulate' its number of own transporters and can affect the stimulatory effect of insulin�9

391. Vascular Complications in Newly-Diagnosed Type 2 (Non-Insulin- Dependent) Diabetic Patients and in Subjects with Impaired Glucose Tolerance A. Verrillo, A. de Teresa, L. Lucibelli, R. Golia, P. Prevete, F. Civetta and G. Mossetti. Clinica Medica, II Policlinico, Naples, Italy

To evaluate the prevalence of retinal, renal and ECG abnormalities in Type 2 diabetes and in subjects with impaired glucose tolerance (IGT) at the time of diagnosis, 54 newly-diagnosed diabetic patients (age 59 +_ 1.6 years mean +_ SEM), 57 subjects with IGT (mean age 57.6 + 1.8 years) and 549 subjects with normal glucose tolerance (mean age 50 _+ 0.7 years) were examined. The three groups were from an Italian rural community (San:a, Southern Italy), in the course of an epidemiological investigation for diabetes and the subjects were divided on the basis of WHO criteria�9 The following parameters were investigated: resting ECG, fundus examination and 24h proteinuria. The frequency of coronary heart disease (ECG-positive for ischaemia by Minnesota code) was 37.1% (20) in diabetic patients, 40.4% (23) in IGT subjects and 18% (102) in controls. Twenty-one diabetic patients (35.5%) and eight subjects with IGT (14%) bad evidence of nephropathy. Retinal changes (microaneurysms and/or exudates) were observed in six diabetic patients (11.1%) and three IGT subjects (5.2%). These results support the conclusion that Type 2 diabetic patients already show vascular complications at the time of diagnosis. Moderate impairment of glucose tolerance seems to be associated with a raised frequency of microvascular abnormalities.

392. Further Evidence that Carbohydrate-Induced Hypertriglyc- eridaemia in the Rat is Caused Primarily by Increased Secretion of Triglycerides L. Verschoor, E.P. Reaven and G.M. Reaven. Erasmus University, Rotterdam, The Netherlands and Stanford University, Palo Alto, Cal- ifornia, USA High carbohydrate diets lead to hypertriglyceridaemia. Fructose feeding leads to more severe hypertriglyceridaemia than glucose feeding by stimulating very low density lipoprotein-triglyceride (VLDL-TG) secretion to a higher extent. Others have claimed an additional decrease in VLDL-TG removal in fructose feeding to explain this difference. However, contradictory results have been reported on the activity of lipoprotein lipase. We focussed our attention on the substrate itself, and determined the rate of TG removal from VLDL particles obtained from rats fed different diets. Rats were fed regular laboratory chow, or a diet containing 66% of total calories as either glucose or fructose. After one week, fasting TG concentrations for rats fed glucose and fructose were 2.60 + 0.37 and 3.95 + 0.59 mmol/1, respectively compared with 1.25 + 0.13 retool/1 in the control rats. An increase in size and slight albeit significant increase in TG: protein ratio and increased total cholesterol and phospholipid content were observed in VLDL, isolated from rats fed high carbohydrate diets�9 Half-life time of TG removal in vivo from VLDL particles obtained from glucose (2.5 _+ 0.2 min) and fructose fed donor rats (3.1 + 0.3 min) did not differ, but were significantly shorter than for VLDL-TG derived from chow fed donors (4.2 + 0.2 min). In conclusion, both glucose and fructose feeding lead to the secretion of larger, TG-rich VLDL particles of which TG is removed faster than from control particles. These results provide further evidence for stimulated VLDL-TG secretion as the major explanation for carbohydrate-induced hypertriglyceridaemia.

393. Decreased (125I) Insulin Binding to Pancreatic Islets of Genetically Obese Ora/fa Zucker) Rats E. J. Verspohl, M.C.M. Melien and H.P.T. Ammon. Department of Pharmacology, Institute of Pharmaceutical Sciences, University of Tfibingen, Tfibingen, FRG The hypothesis was tested whether hyperinsulinaemia is associated with a decreased inhibitory action of insulin in islets offa/fa rats and whether such an effect might be explained by a decrease of insulin binding to islets in vitro. Using collagenase-isolated islets offa/fa and normal Wistar rats (control), we studied the effect of insulin on glucose (16.7 mmol/1)-induced insulin secretion during a 90-min incuba-

�9 " 1 2 5 tion period and performed binding studies (displacement of ( I) in- 125 sulin binding by unlabelled insulin) within 20-rain incubations. ( I)

Insulin 'specifically' bound was 50% lower in islets offa/fa rats (p < 0.01). Scatchard plots of displacement studies revealed a parallel left- ward shift of curvilinear plots. Insulin release was more pronounced in the presence of glucose 16.7 mmol/1 in islets offa/fa rats (745 +39 versus 581 _+ 41 mU/1 per five islets per 90 rain; p < 0.001). The inhibitory effect of increasing concentrations of exogenous insulin on glucose-mediated insulin release was less in islets offa/fa rats than in islets of controls. In conclusion, our data indicate a decrease in high-affinity receptor number in islets offa/fa rats. It is possible that hyperinsulinaemia in fa/fa rats is due to a diminished negative feedback of insulin on its secretion in vitro as a result of a decreased number of high-affinity receptors in pancreatic islets.

202 Abstracts

394. Activation of Kallikrein-Kinin-Prostaglandins System During Muscular Exercise in Normal and Diabetic Rats R. Vettor, C. De Palo, E. De Palo, L. Cal6 ~, A. BorsattP and G. Feder- spil. Institutes of Semeiotica Medica and ~Clinica Medica I, Universi- ty of Padua, Italy In recent years, the kallikrein-kinin-prostaglandins system has been thought to have an insulin-like effect both in vivo and in vitro at muscular and adipose tissue levels. However, few data are available on quantitative variations of this system during muscular exercise. In 54 normal rats, 28 of which were forced to swim for 60 rain, blood glucose and total plasma kallikrein activity (PK) were measured. Muscu- lar phospholipase A2 activity was also determined in tricipital femoral muscle. A similar experimental procedure was used in 20 diabetic rats. A significant decrease in blood glucose levels was found in normal rats after exercise (3.7 _+ 0.18 versus 3.1 ___ 0; 14 mmol/ l ; p < 0.05). PK was found to be significantly higher in resting diabetic rats than in control rats (0.38 _+ 0.03 versus 0.64 + 0.13 U/ml ; p < 0.02). After exercise a significant increase in PK was observed in both groups (0.38 _+ 0.03 versus 0.62 _+ 0.03 U/ml ; p < 0.001 in normal rats; 0.64 _+ 0.13 versus 1.18 _+ 0.37 U/ml ; p < 0.001 in diabetic rats). A similar pattern was observed in muscular phospholipase A2. A significant correlation was found between PK and phospholipase A2 in normal rats. Our data indicate that an activation of the kallikrein-kinin-prostaglandins system exists during muscular exercise. These results agree with the hypothesis of Dietze that this system regulates peripheral glucose uptake. However data obtained in diabetic rats show that plasma glucose levels do not decrease in spite of a large PK increase, showing that a par- allelism between kallikrein activity and tissue glucose uptake does not exist in diabetes.

395. HLA Genotype and Islet Cell Antibodies and Cytotoxic Antibodies in Type (Insulin-Dependent) Diabetic Patients and Their First Degree Relatives P. Vexiau l, I. Deschamp 3, F. Mamoun 2, J. C. Homberg 4, F. Jansen 4 and J. Hors s. ~Service du Pr Canivet, 2INSERM U. 93 H6pital Saint Louis, 3INSERM U. 83 H6pital H6rold, 4Service du Pr Homberg H6pital Saint Antoine, Paris, France. Type I diabetic patients (n = 107) (59 recent onset: < 2 years; 48 long- duration: > 2 years) and 250 healthy first-degree relatives and control subjects were genotyped for HLA-A, B, C and DR antigens. Sera from most of these subjects were tested for islet all antibodies (ICA) and complement fixing antibodies (CF-ICA), by indirect immunofluorescence using human pancreas; for detection of surface antibodies (ICSA) and cytotoxic antibodies (ICA-CT) using suspensions of living mouse cells and a microcytotoxicity technique on Terasaki plates. The prevalence of ICA and CF-ICA was 64% and 41% in recent onset and 25% and 6% in long-duration patients. The prevalence in the healthy relatives was 9% and < 1%. The prevalence of ICSA and ICA-CT in a preliminary study (41 Type 1 patients and 35 relatives) was: diabetic patients, 66% respectively for each type of antibody; healthy relatives, 27% and 17%, with a strong correlation between the two types of antibodies (p < 0.001 for diabetics and p < 0.01 for relatives). No correlation was observed between ICSA, ICA-CT and ICA, CF-ICA. Correlations were studied between the different types of antibodies and HLA groups. A prospective study of these families should permit us to determine the prevalence of these antibodies, in addition to the predictive value of HLA genotype.

396. Tubulin antibodies in recent onset Type I (Insulin Dependent) Dia- betes: A New Serological Marker Separate from Islet Cell Antibodies B.Vialettes ~, B. Rousset 2, Ph. Vague 1 and R.Mornex 2. Department of Diabetology, University of Marseille and 2INSERM U 197, Universi- ty of Lyon, France Islet cell antibodies found in Type 1 diabetes react with either cytoplasmic (ICA) or cell surface (ICSA) antigens. We investigated whether cytoskeletal proteins, tubulin and actin, could represent auto-antigens in Type 1 diabetes. Tubulin and actin were purified from rat brain and labelled by conjugation with the Bolton-Hunter reagent. Antitubulin (Tub-Ab) and antiactin (Act-Ab) antibodies were detected by their ability to bind the labelled protein. Immune-complexes were isolated using a goat anti-human Ig antibody. Sera from 17 early-onset Type I diabetics (21.9 _+ 1.7 years, duration: 3.76 _+ 1.1 months) and 32 long-standing Type I diabetics (39.4_+2.8 years, duration 14.5 _+ 1.8 years) among which 6 presented a concomitant autoimmune disease and were studied for Tub-Ab, Act-Ab, ICA and CSA. Frequency of Tub-Ab was 52.9% in recent-onset and 9.4% in long-standing diabetes. Frequency of Act-Ab was respectively 11.8 and 6.25%. There was no concordance between Tub-Ab and ICA or ICSA in recent-on-

set Type 1 diabetes. In long-duration Type i diabetes, Tub-Ab were found in 3 subjects with persisting ICA or ICSA. Absorption of serum on rat islet did not modify Tub-Ab in contrast to ICSA. Conclusion: Tub-Ab are frequent in recently diagnosed Type I diabetes, rare in long-standing Type i diabetes (only found in some patients with persisting islet-cell antibodies). Lack of association and absorption studies confirm that Tub-Ab and islet-cell antibodies are separate serological markers.

397. Correction of Hyperglyeaemia is Associated with Sustained Reduc- tion of Microalbuminuria in Insulin-Dependent Diabetes The Kroc Collaborative Study Group, Chicago, Illinois; London, UK; London, Ontario, Canada; New Haven; Connecticut and Roch- ester, Minesota, USA In a randomised, prospective, multicentre trial of diabetic control and complications, the effect of correction of hyperglvcaemia on 24-h urinary albumin excretion rate (AER) and serum creatinine was compared in insulin-dependent diabetic patients without persistent Albus- tix-positive proteinuria, one group allocated to continuous subcutaneous insulin infusion (CSII; n=30) and the other to conventional insulin therapy (CIT; n = 29). Groups matched closely for age, weight, duration of diabetes, insulin dose, glycaemia and AER. Assessments were made at 0, 4 and 8 months. Highly significant glycaemic separation was achieved between the two groups. Baseline AER was within normal limits (1-12 Ixg/min) in 20 CSII and 19 CIT diabetic patients, but exceeded them in 10 CSII and 10 CIT patients. These two subgroups retained close matching. AER fell in both CSII and CIT patients with elevated AER at 4 months (p < 0.05, 0 versus 4 months), but returned to baseline values in the CIT group by 8 months, while falling further (p < 0.02) in the CSII group. AER was unchanged in normoalbuminuric patients. Serum creatinine remained normal in all. Correction of hyperglycaemia is associated with a sustained reduction of AER in diabetic patients with elevated AER. Longer-term studies will establish whether this prevents development of Albustix-positive proteinuria.

398. Effect of a New Alpha Amylase Inhibitor Trestatin (Ro 09-0154) on Glucose, Insulin and Non-Esterified Fatty Acid after a Starch Load in Type 2 (Non-Insulin-Dependent) Diabetic Patients G. L. Viviani, G. Carta, G. Cortassa and L. Adezati. Department of Internal Medicine, University of Genoa, Italy Inhibition of carbohydrate absorption appears to be helpful for diabetic control. Trestatin, a new specific alpha amylase inhibitor, has been shown to reduce post-prandial blood glucose and insulin levels in animals and healthy volunteers. In order to evaluate similar activity in diabetic patients the drug was administered to six Type 2 diabetic patients. After a standardized starch load (75 g) increasing doses of Trestatin (10, 30, 50 mg and placebo) were administered daily. Blood samples for glucose, IRI and non-esterified fatty acid (NEFA) were taken at the time 0, 30, 60, 90, 120, 180 min after the load. Increases in blood glucose during the test after the starch load were 90.1 _+ 17.2mg/ml (mean _+ SEM) without drugs, 70.0+12.8 with 10mg; 27.4_+4.2 with 30rag; 9.1 _+2.5 with 50mg and 81.0_+3.7 with placebo. The respective mean increments in insulin were: 18.4_+2.6; 18.9 _+ 3.4; 8.5 _+ 1.3; 5.2 + 0.9; 24.4 + 2.8 mU/1. Maximal NEFA percentage decrements were 73.9 _+ 2.1 ; 67.3 _+ 2.8; 49.7 + 5.9; 36.5 _+ 2.9; 69.4 _+ 3.0% respectively. General tolerance was good. Some flatulence was observed. In conclusion, Trestatin reduces blood glucose, IRI and NEFA responses not only in animals and healthy subjects, but also in Type 2 diabetic patients. Ro 09-0154 may thus become a useful auxil- iary measure for diabetic control.

399. Diabetogenic Action of Cortisol and Adrenaline on Oral Glucose Tolerance Assessed by Hepatic Venous Catheterization in Healthy Man W.Waldhfiusl, P. Bratusch-Marrain, S.Gasi6 and M. Komjati. l .Me- dizinische Universit~tsklinik, Division of Clinical Endocrinology and Diabetes Mellitus, Wien, Austria To compare cortisol and adrenaline action on oral glucose tolerance (75 g), seven healthy men were infused with either cortisol (0.1 mg- kg- J- h - 1 ; _ 720 to 150 min), adrenaline (6~tg/min; - 30 to 150 min) or saline. Splanchnic exchange of substrates, insulin and C-peptide was determined. In the basal state adrenaline increased more than cortisol arterial concentrations and splanchnic output of glucose, fl- hydroxybutyrate and non-esterified-fatty acids (NEFA), whereas the response of insulin and C-peptide was reduced. A more marked hyperglycaemia (area under the curve; mmol- 150 rain -1.1-1 occurred following the oral glucose tolerance test during adrenaline (1902 _+ 79; normal 1117+70; m e a n + S E M ; 2p<0.001) than during cortisol

Abstracts 203

(1437+65; 2p versus normal<0.01) administration, although an identical 30% increase in splanchnic glucose output was observed (normal, 44.2+3.4; adrenaline, 58.9+6.9; cortisol, 57.4+3.9g/ 150 min, 2p < 0.05). In parallel, only cortisol but not adrenaline increased the splanchnic output and arterial concentrations of insulin above control values. Both hormones induced identical responses of pyruvate and acetoacetate, whereas splanchnic lactate uptake was more augmented by adrenaline than by cortisol. The post-prandial splanchnic glucose output/S-C-peptide ratio was normal (0.89 + 0.09) during adrenaline (0.95_+0.13), but reduced during cortisol (0.43 + - 0.05) administration. In conclusion, the more marked post-prandial hyperglycaemia following adrenaline overcomes adrenaline's suppressive effect on insulin secretion, but fails to normalize splanchnic glucose retention. Increased insulin secretion by cortisol also fails to overcome hepatic insulin resistance, but may suffice to equalize peripheral insulin resistance.

400. Successful Early Dietary Therapy Reduces Insulin Requirements for Normoglycaemia in Type 2 (Non-Insulin-Dependent) Diabetes K. Walshe, J. Andrews and D. R. Hadden. Metabolic Unit, Royal Vic- toria Hospital, Belfast, UK The amount of exogenous insulin required to achieve normoglycaemia in diabetic patients who are not necessarily insulin deficient is uncertain. Six previously untreated symptomatic Type 2 diabetic patients (age: 40-64 years, weight: 89.7 + 4.9 kg (mean + SEM), body mass index 30.4 + 1.1 kg/m 2) were maintained on 2 000 kcal for 3 days before a 75 g oral glucose tolerance test. On the following day, 1500 kcal were consumed as three standard meals. Using a Biostator during this time, plasma glucose was maintained between 4.4 and 5.5 mmol/1, and the cumulative requirements of intravenously administered insulin required to maintain this were measured. For the next 3 months intensive outpatient dietetic supervision was maintained. The protocol outlined above was repeated. Mean weight had fallen to 82.1 + 3.9 kg, body mass index to 27.9 + 0.9 kg/m 2. One patient did not keep to the diet program and showed increased insulin requirement despite weight loss. The remaining five patients showed a fall in 2 h plasma glucose during an oral glucose tolerance test from 16.9_+7.5 to 9.3 _+ 4.1 mmol/1. While the mean integrated insulin response during the oral glucose tolerance test was unchanged (initial 479.4_+165.8 to 393 _+ 61.2 mU- min- l . 1 1); on the Biostator protocol the cumulative insulin requirements fell from 35.3 _+ 4.8 to 24.8 _+ 4.9 U. We conclude that successful early dietary management inproves glucose tolerance and reduces insulin requirements in Type 2 diabetes.

401. Production and Evaluation of a Video Cassette Programme for Diabetes Education J. D. Ward, P. Isaac, J. F. Garlant and C. Bradley. Royal Hallamshire Hospital and University Department of Psychology, Sheffield, UK Twenty video cassette programmes have been made (average length 12 min) covering all aspects of diabetic life and management. Patients make substantial contributions to the films. A practising physician, rather than a professional narrator, appears throughout the series. The style and atmosphere were considered to be as important as the factual content. Presentations were designed to arouse interest and motivate patients to concentrate. While the topic of 'hypoglycaemia' lent itself to a stylish production, a lecture style presentation was felt to be more appropriate for the topic of 'insulin treatment regimes'. These two stylistically different films were evaluated using knowledge and attitude questionnaires with groups of insulin requiring diabetic patients who completed the measures before and after viewing the films. Knowledge of diabetes increased significantly following the fihns and discussion (t= 5.53, d.f. 30, p < 0.001). All patients reported that they had gained new information from both films. The 'insulin treatment' film was said to provide significantly more information than the 'hyperglycaemia' film (t= 3.77, d. f. 35, p < 0.001) and was reported to be less clear (t= 3.81, d.f. 35, p < 0.001) and more confusing (t = - 2.45, d. f. 35, p < 0.02). Mean ratings for both films indicated interest and clarity without confusion.

402. Binding of Biosynthetic Human Growth Hormone to Isolated Human Adipocytes C. A. Waugh and D. G. Johnston. University Department of Medicine, Royal Victoria Infirmary, Newcastle-upon-Tyne, UK Human growth hormone (hGH) has previously been demonstrated to bind to certain human tissues such as post-mortem liver slices and cultured IM-9 lymphocytes, but not to normal living human tissue. In this study, specific hGH binding to isolated human adipocytes has been demonstrated and biological activity sought. Fat specimens were ob-

tained during elective abdominal surgery and dispersed by collagenase yielding adipocytes capable of binding insulin and responsive to lipolytic stimuli. Using three different native hGH preparations, a small but variable amount of specific binding was observed. 1251-Met- hGH (Kabi) consistently showed 1-4% specific binding to cell suspensions of 100 gl packed cell equivalent. Non-specific binding was < 10% of total radioactivity bound. Maximum binding was achieved in 4 6 h at 37 ~ and in 12 16 b at 25 ~ Standard curves obtained after 4h incubation at 37~ showed maximal binding inhibition at 25-50 ng/ml unlabelled hGH. Lipolysis, assessed by glycerol release, was unaffected by Met-hGH concentrations of 0.3, 1.0 and 10.0 gg/ ml. Specific hGH binding, presumably to hGH receptors, has thus been demonstrated in normal human tissue permitting the study of growth hormone-receptor interactions and subsequent biological effects for the first time in man.

403. Impaired Somatostatin and Counter-regulatory Hormone Re- sponses in Diabetic Patients With and Without Autonomic Neuropathy S. Webb, M. Fern/tndez Castafier, I. Levy, M. Bergua, R. Casamitjana, J. Gaya, M.J. Martinez and F. Rivera. Diabetic Department and Hor- monal Laboratory, Clinic Hospital, Barcelona, Spain Cortisol, growth hormone, adrenaline, noradrenaline, glucagon and somatostatin responses to insulin induced hypoglycaemia were studied in eight diabetic patients with autonomic neuropathy (AN), six diabetics without AN and six normal volunteers. The diabetics were maintained euglycaemic by artificial pancreas the day before the test. Blood samples were drawn basally and at intervals after 0.15 IU/kg body weight of insulin IV for 120 min. Basal glucagon values were higher in diabetics with AN (209.2 + 26.7 pg/ml, mean + SEM) than in diabetics without AN (122.6 + 26.6, p< 0.05). Somatostatin peak was greater in diabetics without AN (72.6+13.6 versus 36.1+ 3.2 pg/ml, p < 0.05). The response of adrenaline was blunted in the four patients with sympathetic neuropathy. The remaining basal and peak hormone values were similar in the two diabetic groups. Normal subjects showed significantly greater growth hormone (31.7 _+ 7.4 versus 10.3 _+ 0.8 ng/ml) and glucagon (451.5 _+ 26.1 versus 213.2 + 26.0) peak responses to insulin-induced hypoglycaemia than diabetics (p < 0.001 ). Basal (56.4 + 9.3 versus 27.0 + 3.0) and peak (112.9 _+ 12.9 versus 50.2_+ 7.2) somatostatin responses were higher in control subjects (p<0.001). Conclusions: (1)growth hormone and glucagon responses to insulin-induced bypoglycaemia decrease in diabetes, (2) adrenaline is low in sympathetic AN, (3) somatostatin is low in controlled diabetes, and (4) somatostatin secretion is mediated by the autonomic nervous system.

404. Effect of Adrenaline on Ketone Body Kinetics in Normal and Insulin-Deficient Man M. Weiss, U. Keller, S. Brunner and W. Stauffacher. Division of En- docrinology and Metabolism, University Hospital, Basel, Switzerland The effect of adrenaline on ketone body production, peripheral uptake and clearance was measured using primed-continuous infusions of 3J4C-acetoacetate in normal subjects. Infusion of adrenaline (60 ng. kg -1. min-1 during 170 min, n = 6, plasma adrenaline levels ---800pg/ml) resulted in a transient increase in total ketone body production exceeding uptake (11 + 1 versus. 6 + 1 ~tmol-kg -1- min-a In control subjects, n = 9, p < 0.01), causing a 2.8-fold increase in total ketone body concentration: ketone body clearance remained unchanged. During adrenaline, plasma glucose and insulin levels increased gradually. When adrenaline was infused combined with somatostatin (n=6) to prevent adrenaline-related plasma insulin elevation, the increase in ketone body production was augmented and sustained (15 _+ 1 versus 9 -+ 2 txmol �9 kg -1. rain -1, p < 0.01 during somatostatin alone, n=7). The increase in ketone body concentrations was augmented (9.2-fold basal) due to a somatostatin-related decrease of ketone body metabolic clearance by 47%. Plasma non-esterified fatty acids and glycerol increased significantly during adrenaline and paralleled the course of ketone body production. Thus, elevation of adrenaline levels as observed in diabetic ketoacidosis results in normal subjects in transient hyperketonaemia due to increased ketone body production and enhanced lipolysis, but no change of ketone body metabolic clearance. The effect is sustained during insulin deficiency when ketosis is further enhanced by diminished clearance of ketone bodies.

405. Purification and Isolation of Monoiodinated Insulin Tracers Using Reversed-phase High-performance Liquid Chromatography B.S.Welinder, S.Linde, B.Hansen, and O.Sonne 1. Hagedorn Re- search Laboratory, Gentofte, and ~University of Aarhus, Aarhus, Denmark

204 Abstracts

125I-insulins are intensively used in radioimmunological analysis, in binding studies and as biological tracers. Separation and isolation of insulin monoiodinated at Tyr-A14, A19, B16, and B26 are needed due to different binding kinetics. Recent work at our laboratory has shown that this separation can be performed within 2 h using reversed-phase high-performance liquid chromatography (RP-HPLC). Three different C-18 columns eluted with buffers containing different amine salts and acetonitrile all resulted in baseline separation of the 4 isomers. Buffer substances were removed by lyophilization, gel filtration or SEP-PAK purification. All HPLC-purified monoiodoinsulins were essentially homogenous, but the binding affinities to adipocytes were reduced by approximately the same rates percent when compared with similar tracers purified by low-pressure methods (disc-electrophoresis/ion exchange chromatography). Optimization of buffer system and isolation procedure resulted in A14 monoiodoinsulin having 95% binding affinity relative to that of A14 purified by low-pressure methods. This reflects the fact that the biological activity of a polypeptide or protein may be damaged by the conditions used in HPLC: high pressure, hydrophobic milieu, organic solvents and shear forces. The activity of a HPLC-purified polypeptide/protein should therefore always be compared with that of the same compound purified to a comparable degree using low-pressure methods.

406. Insulin Absorption from Dialysate during Peritoneal Dialysis in Diabetic and Non-diabetic Patients T.-E. Wider6e, k C. Smeby 1, S. J6rstad, K.J. Berg and T.M. Svart~s. Department of Nephrology and l Institute of Biophysics, University of Trondheim, Norway Insulin added to the bags of dialysate significantly improves blood glucose regulation in diabetic patients during continuous ambulatory

195 peritoneal dialysis (CAPD). In this study, - I-insulin was mixed with conventional insulin to investigate: (1) adsorption of insulin onto the plastics of the dialysate bags (n=28); (2) intraperitoneal insulin absorption as a function of dwell-time in diabetic patients (n = 3) and non-diabetic patients (n = 3) during CAPD. Insulin absorption was also studied in four different patients with peritonitis. Results: (1) 65 + 4% of supplied insulin was retained on the plastic bags; (2) diabetic patients absorbed significantly more insulin than non-diabetic patients for dwell-time of 30 min, but for dwell-time of 1-8 h there was no significant difference between patient groups, giving insulin adsorption: 33 + 8% for dwell-time of 5 h and 45 + 6% for dwell-time of 8 h. During peritonitis, insulin adsorption for dwell-time of> 2 h was significantly increased (65 + 3% for dwell-time of 5 h). Individual differences in insulin adsorption within the diabetic group corresponded with the insulin dose needed during CAPD treatment. The results indicate improved biological effect of insulin adsorption compared with SC insulin and that all insulin can be supplied via the dialysate for diabetic patients on CAPD, giving improved glucose regulation without SC injections. The insulin dose added to the dialysate bag should be individualized and careful monitoring of blood glucose during peritonitis is important.

407. Insulin-like Growth Factors Stimulate Glycogen Synthesis in Chick Embryo Hepatocytes U.Widmer, Ch. Schmid, A. Bulgheroni, J.Zapf and E.R. Froesch. Metabolic Unit, University Hospital, Zurich, Switzerland Liver cells have specific insulin-like growth factor (IGF) receptors, but no biological actions of IGF on liver cells have been shown. In this study, we investigated the effects of IGF and insulin on glycogen synthesis in chick embryo hepatocytes and compared them with receptor binding. Cells were cultivated for 24 h in defined Williams' E medium containing 11 mmol/1 glucose without serum and hormones and subsequently exposed to insulin or IGFs for 3 h in the presence of 1 uCi (U-14-C)-glucose. Insulin, IGF I and II stimulate the incorporation of labelled glucose into glycogen in a dose-dependent manner. Maximal stimulation is identical: 210_+10% of the control value (mean_+SD, n=12). Half maximal stimulation is achieved at 0.1-0.25 nmol/I with all three hormones (insulin: 30 mU/l). Binding experiments with iodinated hormonesshow that (1) unlabelled IGF I and II behave identically, (2) IGFs compete with (125)I-insulin for binding but their potency is 10% of that of insulin, (3) insulin dis- places ~2sq-IGFs with 1% of the potency of unlabelled IGFs. This study shows for the first time that IGFs stimulate glycogen synthesis in liver ceils. On a molar basis, IGFs and insulin are equipotent and seem to act via their own separate receptors.

408. Impaired Vagal Heart Rate Control in Diabetic Patients: Evalua- tion of Tests and Relationship to Other Long-Term-Complications W. Wieling, C. Borst and A. J. Dunning. Department of Internal Medi- cine, Academic Medical Centre, University of Amsterdam, Amster- dam, The Netherlands We examined the heart rate changes induced by forced breathing and by standing in 84 diabetic patients with a variety of long-term complications in order to assess the presence of autonomic cardiac vagal neuropathy. A previously established age-adjusted lower limit of normal (P0.a0) and an uncertainty range (values between P0.~0 and P0.025) were used for classification of the patients. Twenty-one patients scored below and seven patients scored in or below the uncertainty range for both tests. The correlation between both tests was fair (r=0.74). Symptomatic autonomic neuropathy was found only in 11/21 patients with scores below the uncertainty range on both tests. Definitely abnormal test scores were strongly associated with the presence of retinopathy and nephropathy. Complete cardiac vagal denervation (n = 14) was associated with very severe complications. Vi- bration thresholds on the great toes were abnormal in all patients with definitely abnormal test scores. The correlation between vagal neuropathy and vibration thresholds on the great toes was fair (r= 0.70). Single abnormal tests scores were found to be of little or no importance. It is concluded that cardiac vagal neuropathy is closely associated with the other long-term diabetic complications. Definite cardiac vagal neuropathy can only reliably be evaluated by a combination of tests.

409. Chlorpropamide Alcohol Flush: A Comparison of the Single Chal- lenge and 14-day Tests in 40 Non-Insulin-Dependent Diabetic Patients P. G. Wiles and D.A. Pyke. Diabetic Clinic, King's College Hospital, London, UK To compare the chlorpropamide alcohol flush (CPAF) response after 1 and 14 days' chlorpropamide treatment, we tested 40 non-insulin- dependent diabetic patients (30 male, age 59.5 + 1.3 years, duration of diabetes 9.6 + 1.2 years) with 8 g ethanol, (1) after 250 mg chlorpropamide or (2) after 14 days' chlorpropamide 250 mg daily. Patients assessed independently by subject and observer were classed as 'flushers' or 'non-flushers' if they agreed, and 'intermediate' if they differed. At 12 h there were 12 'flushers' (30%), 3 'intermediates' (7.5%), and 25 'non-flushers' (62.5%); temperature rises (with basal temperatures) were: 'flushers': 1.7 + 0.2 ~ (33.1 ___ 0.3 ~ 'intermediates': 1.0_+ 0.3 ~ (33.4 + 0.6 ~ and 'non-flushers: 0.7 + 0.1 ~ (32.7 _+ 0.4 ~ After 14 days there were 29 'flushers' (72%), four 'intermediate' (10%) and seven 'non-flushers' (18%) (all 12h 'intermediates' becoming 14day 'flushers'); temperature rises (and basal temperatures) were: 'flushers': 2.2 + 0.2 ~ (32.5 + 0.3 ~ 'intermediates': 1.3 -+ 0.1 ~ (32.5 _+ 0.9 ~ and 'non-flushers': 0.7 _+ 0.1 ~ (33.8 _+ 0.2 ~ Plasma chlorpropamide levels were similar in all groups at 12 h and after 2 weeks' treatment. We conclude: (1) CPAF is commoner after 14 than 1 day's chlorpropamide, (2) 'flushers' and 'non-flushers' cannot be distinguished by differences in chlorpropamide levels, (3) basal malar temperatures are similar at day 1 but may be higher in 'non- flushers' after 14 days chlorpropamide, and (4) observer and subject assessment may be the best method of determining CPAF.

410. Functional Studies in Non-immunosuppressed Conscious Mice Carrying Pancreatic Islet Allografts J.D.Wilson, S.J.Prowse and S.P. Haynes. Endocrine Unit, Woden Valley Hospital and Transplantation Biology Unit, Australian Na- tional University, Canberra, Australia CBA/H (H-2 k) male mice were made diabetic by giving streptozotocin 300 mg/kg IV. They were transplanted beneath the renal capsule with seven clusters of BALB/c (H-2 d) adult islets cultured for 7 days in 95% 02 and 5% COz. Basal blood glucose and weight gain returned to normal within 10 days. The increase in blood glucose following intragastric glucose was more marked in the transplanted animals than in the control animals of both the donor and recipient strains. The difference was abolished by pretreatment with intra-peritoneal phentolamine and propranolol. This treatment also increased the serum insulin response to intragastric glucose in the transplanted animals but not to the level of the control animals. The glucose responses to arginine and theophylline were similar in all groups of animals but the insulin responses were considerably lower in the transplanted animals. A prompt and sustained rise in blood glucose, which could be abolished by c~-adrenergic blockade, occurred after SC adrenaline in all groups of animals. Beta-blockade did not affect the response in CBA mice but led to a reduced response in BALB/c mice. This study demon-

Abstracts 205

strates that, in the mouse, adult pancreatic islet allografts can respond to stimuli which require functional adaptation by the islets.

411. Dissociation Between Renal Function and Size in Response to Near-Normoglycaemia in Non-Newly Diagnosed Insulin-Dependent Diabetic Patients with Hyperfiltration and Nephromegaly M. J. Wiseman, G. C. Viberti, A. Saunders and H. Keen. Unit for Meta- bolic Medicine, Guy's Hospital Medical School, London, UK The effect of 6 months near-normoglycaemia on glomerular filtration rate (GFR) and kidney volume was studied in five insulin-dependent diabetics of 2-12 years duration allocated to continuous subcutaneous insulin infusion (CSII) and in six insulin-dependent diabetic patients matched for age, sex and duration of diabetes allocated to conventional insulin treatment (CIT). All patients had elevated GFR ( > 135 ml /min per 1.73 m 2) and kidney volume ( > 300 ml/1.73 m 2) at baseline. 51Cr-EDTA GFR, ultrasound kidney volume, glycosylated haemoglobin and plasma glucose were measured at 0, 1, 3 and 6 months, and were closely matched at baseline in the two groups. Throughout the study, the CSII group had significantly better glycaemic control. GFR fell continuously with time in the CSII group from baseline to 6 months (151 _+ 13.1 versus 126_+9.9 ml /min per 1.73 m2; p < 0.001). The CIT group showed no significant change in GFR from baseline to 6 months (152 _+ 15.6 versus 146 + 21.0 ml /min per 1.73 m3; NS). In contrast, kidney volume did not change from baseline to 6 months in either treatment group (CSII: 352+ 58.5 versus 345 +_ 47.5; CIT: 334_+ 16.8 versus 317 + 47.4 ml per 1.73 m2). Six months near-normoglycaemia normalises the elevated GFR of established diabetes but does not reverse nephromegaly. Nephromegaly is unlikely to play a primary causative role in the genesis of hyperfiltration.

412. Stimulation of Insulin Release Is Accompanied by an Increase in Cytosolic Free Ca 2+ C. B. Wollheim, R.Y.Tsien and T. Pozzan. Institute of Clinical Bio- chemistry, University of Geneva, Geneva, Switzerland The hypothesis that secretagogue-induced insulin release is mediated by a rise of cytosolic free Ca e+ (Ca2+i) was tested. Ca2+ i was measured in an insulin-producing cell line, RINm5F, using the fluorescent Ca 2+ indicator quin 2. After exposure of cell suspensions to the hydrophobic quin 2 acetoxymethylester, the hydrolysed, hydrophilic

" 2 + quin 2 is trapped in the cell cytosol. Resting Ca i was estimated as about 100 nmol/1. The Ca 2+ ionophore ionomycin caused a large increase in Ca2+i. Since many secretagogues depolarize islet cells, membrane potential was monitored with the fluorescent probe bis-oxonol. K + (24 mmol/ l) or L-glutamine (3 mmol/l) depolarized the cells to a similar extent and raised Ca2+ i several-fold. Verapamil (20 p,mol/1), a blocker of voltage-dependent Ca 2+ channels, abolished the Ca2+i response to K + or glutamine but not that induced by ionomycin. All three agents stimulated insulin release from quin 2 loaded cells. In conclusion, these results provide direct experimental evidence that stimulation of insulin release is accompanied by an increase in cytosolic free Ca 2+. A rise in Ca2+ i due to membrane depolarization is overcome by Ca 2+ channel blockade, which attests to the importance of Ca 2+ channels in the control of cytosol Ca 2+.

413. Is the Granular Ca 2+ Content Regulated by the Intracellular So- dium Concentration? G.Wolters, M.Vonk, W.Konijnendijk, and A.Pasma. Department Experimental Endocrinology, University of Groningen, The Nether- lands The metallochromic indicator glyoxal-bis-(2-hydroxyanil) (GBHA) stains an ionized calcium fraction in rat pancreatic islets. This Ca- fraction is localized in the B cell granules. Effects of low Na + (26 retool/l) or K+-removal on GBHA-Ca was investigated. In the presence of calcium, stimulation with 15 retool/1 glucose rapidly decreased GBHA-Ca followed by a strong rise between 30 and 60 rain. At 2.5 retool/1 glucose, GBHA-Ca slightly decreased and then remained constant. Low Na + rapidly increased GBHA-Ca to high plateau values independent of the glucose concentration, whereas high intracellular Na + (induced by removal of extracellular K +) decreased GBHA-Ca similarly at high and low glucose levels. Withdrawal of Ca 2+ at 2.5 mmol/ l glucose caused a fall of GBHA-Ca, which was inhibited by 15 mmol/1 glucose. Simultaneous removal of Na + prevented the Ca 2+ withdrawal-induced GBHA-Ca decrease. These effects of Na + manipulation did not depend on the glucose concentration. Conclusion: decreasing or increasing intracellular Na + abolishes the effects of glucose stimulation on GBHA-Ca. The GBHA-Ca fraction in the secretory granules seems to be strongly regulated by the intracellular sodium concentration.

414. Longitudinal Changes in Glycosylated Haemoglobin in Normal Pregnancy: Studies with Two Independent Methodologies R. Worth, J.M. Potter, J. Drury, R. B. Fraser and D.R. Cullen. Royal Hallamshire and Northern General Hospitals, Sheffield, UK In diabetic pregnancy individual blood glucose concentrations may fail to reflect overall control and thus glycosylated haemoglobin (HbA1), is often measured to give a supplementary index of glycaemia. Evaluation of results is compromised by the lack of longitudinal studies of changes in HbA1, in normal pregnancy. Twenty-one normal pregnant women provided samples for HbA1, (colorimetric and column analysis) at booking ( < 12 weeks), 16, 24 and 32 weeks, delivery and post-partum. At 16weeks, 32weeks and post-partum 75g oral glucose tolerance tests were performed. Results (mean + SD) were compared with the non-pregnant (post-partum) values. Non-pregnant HbA1 levels were 36.9 + 1.9 mmol hydroxymethylfurfural/mol haemoglobin (colorimetric) and 6.7 _+ 0.5% (column). Values fell at booking (34.9 + 2.0, p < 0.02 and 6.5 + 0.4, NS), reached a nadir at 16 weeks (34.4 + 1.9, p < 0.002 and 6.0 + 0.5, p < 0.001) but then rose to a peak at delivery (37.9_+2.3, NS and 7.1_+0.7, NS). Non-pregnant fasting blood glucose concentrations were 3.6_+0.4 but fell to 3.2+ 0.4 mmol/1 at 16 weeks (p < 0.002). Non-pregnant 2 h blood glucose levels were 3.5 _+ 0.6, rising to 4.4 -+ 0.8 mmol/1 at 32 weeks (p < 0.002). Non-pregnant mean corpuscular volume was 84.3 -+ 4.6 88.6 _+ 4.3 fl at 16 weeks (p < 0.02) and 85.8 -+ 5.5 fi at 32 weeks (NS). These physiological changes in HbA1, confirmed by two independent analytical techniques, must be considered when interpreting HbA1, levels in diabetic pregnancy, particularly in the first trimester.

415. Interrelationships of Lipid Intermediary Metabolites and Changes in Some Trace Elements During Intravenous Glucose Tolerance Test in Diabetes C. S. Yajnik, N. Ward, S. Humphreys, B. Pim and T.D.R. Hockaday. Radcliffe Infirmary, Oxford, and Imperial College Reactor Centre, Silwood Park, Ascot, UK Neutron activation analysis of specially collected plasma samples revealed that 55 non-insulin-dependent diabetic patients (mean age 60.2 years) had higher vanadium concentration (223.5 + 10.2 nmol/1, mean-+ SEM) than 22 non-diabetic subjects (188+4nmol/1) while chromium, zinc and copper levels did not differ. 15 of these patients underwent an IV glucose tolerance test (glucose 20 g/m2). Plasma Cr and Zn rose while V and Cu fell during the test. Initial V (255 -+ 20 nmol/1) related weakly and inversely to 3-hydroxybutyrate + acetoacetate (X BA, 0.11 _+ 0.03 mmol/1, r~ = - 0,46, p < 0.1) while the fall in V at 90 rain (to 196 _ 20 nmol/1) related to the fall in non-esterified fatty acids (NEFA) as well as to ZBA at 90 rain (rs=0.62, 0.55; p = 0.04 and 0.08, respectively). V and Cr are known biological antagonists, as are Zn and Cu. The rise in Cr at 60 rain (from 127_+ 8 to 144 _+ 8 nmol/1) correlated with the fall in ZBA at 60 rain (to 0.08 -+ 0.03, rs=0.84, p<0.001). The Zn rise at 60min (from 17.2-+0.9 to 18.3 _+ 0.8 gmol/l) related to the fall in NEFA at 90 min (& = 0,77, p < 0.01) and to the fall in Cu at 60min (from 16.4+0.8 to 16.1_+ 0.6 ~tmol/1, rs = 0.66, p < 0.03) while the fall in Cu at 60 min related to the fall in NEFA at 90 min (r~ = 0.58, p < 0.04).

416. Insulin Secretion by Exoeytosis from Permeabilized Islets of Lan- gerhans M. A. Yaseen, J. E. Smith, N. Doolabh and S. L. Howell. Queen Eliza- beth College, University of London, London, UK A high voltage discharge technique was used to investigate a possible chemiosmotic mechanism of granule extrusion in insulin secretion. Isolated islets of Langerhans were exposed to electric discharge and their insulin secretion was examined in perifusion as described previously. In pulsation media containing 140 retool/1 NaC1 or KC1 insulin release was significantly inhibited, whereas it was significantly stimulated in 140retool/1 Na isethionate, compared with control (70/ 70retool/1 KC1/Na isethionate, pH 7.0, p < 0.05, n=9). Similarly NaSCN (140 retool/l) was stimulatory whereas KSCN was inhibitory (p < 0.001, n = 9). The magnitude of stimulation in the presence of Na- isethionate or NaSCN was similar and was evoked by I p, mol/1 but not by 10 nmol/1 Ca 2+ - it was drastically and reversibly reduced at room temperature or in the presence of 180 retool/1 added sucrose (p < 0.001, n = 6). This Ca2+-evoked insulin secretion was significantly inhibited at pH 6 and significantly stimulated at pH 8 (each p < 0.05, n = 4). Secretion was massively stimulated in the presence of 1 gmol / l Ca 2+, 20 ~mol/1 cytochalasin B and NaSCN but not by KSCN in the same medium (n = 4). Thus insulin secretion from permeabilized islets was inhibited by chloride ( > 70 retool/l), but stimulated by Na + or isethionate; the process was pH and osmotic pressure dependent.

206 Abstracts

This may suggest the involvement of a cation-osmotic mechanism in exocytosis.

417. High Aerobic Power and Female Sex are Associated with Enhanced Insulin Sensitivity H. Yki-J~rvinen and V. Koivisto. Third Department of Medicine, Uni- versity of Helsinki, Helsinki, Finland Since muscle tissue is responsible for > 90% of glucose utilization during hyperinsulinaemia, we examined the influence of body composition on glucose uptake during euglycaemic insulin clamp at I mU/1. Four groups were studied: runners (n=8), weight lifters (n=8), untrained men (n=7), and women (n=8). Runners had 30%-35% higher maximal aerobic power (71 ml-kg -a. min -1) than the other groups (p < 0.001). Weight lifters had 25%-45% higher relative muscle mass (54% of body weight) than the other groups (p< 0.001). Women had a higher proportion of fat (17%, p < 0.02) and lower proportion of muscle (30%, p < 0.001) than the others. Glucose metabolism during insulin clamp, expressed as mg.kg body weight - l - rain -1, was higher (p < 0.03) in runners (10.0 + 0.9) and weight lifters (10.2+1.0) than in untrained men (7.1 +0.7) or women (7.4_+0.4). Glucose metabolism was related directly to relative muscle mass (r= 0.58, p < 0.001) and inversely to proportion of fat (r= - 0.61, p < 0.001). When expressed per kg muscle tissue, glucose metabolism was similar in runners (25 _+ 2 rag. kg muscle -1. rain -1) and women (25 _+ 1), and 25%-30% higher (p < 0.05) than in weight lifters (19 _+ 2) or untrained men (18_+ 1). In conclusion (1) glucose uptake is related directly to the proportion of muscle and inversely to that of fat, (2) glucose uptake by muscle tissue is elevated in women and runners, whereas greater muscle mass in weight lifters explains their high glucose metabolism, and (3) when insulin sensitivity is determined, body composition should be taken into account and glucose uptake measured per kg glucose consuming tissue rather than per kg total weight.

418. Alcohol Consumption and Diabetic Retinopathy R. J. Young, D. K. McCullnch, R. J. Prescott and B. F. Clarke. Universi- ty Department of Medicine, Royal Infirmary of Edinburgh and De- partment of Medical Statistics, University of Edinburgh, Scotland, UK 296 male diabetic patients (aged 20-59 years with no initially obvious ophthalmoscopic retinopathy) have been followed prospectively for 5 years. Urinalysis for protein, peripheral vascular and neuropathic symptoms, glycaemic control, smoking habit and alcohol consumption were also recorded. Linear logistic, two tailed, statistical analysis was used. 66 developed retinopathy during follow-up; 47 background, 10 maculopathy and nine proliferative. As previously recognised, duration of diabetes (p < 0.001) and poor glycaemic control (p= 0.02) were predictive of the development of retinopathy. Impo- tence (p= 0.001) at the outset was also associated with more frequent development of retinopathy. However, unexpectedly, heavy alcohol consumption was another factor predictive of subsequent retinopathy (p= 0.02). Thus, 21/70 (30%) of those who admitted to drinking > 20 measures of alcohol per week developed retinopathy, compared with 45/226 (20%) among the remainder. The development of maculopathy/proliferative retinopathy showed a particular association occurring in 9/70 (13%) of heavy drinkers, compared with 10/226 (4.5%) of the rest (p= 0.01). Alcohol consumption may therefore be an important independent factor associated with the development of visually threatening diabetic retinopathy.

419. Total and Free Pancreatic Polypeptide and Antibodies in Type 1 (Insulin-Dependent) Diabetic Patients R. Zandomeneghi, A. Luciani and A. Tarozzi. Cattedra Patologia Me- dica 3, University of Modena, Italy Total and free pancreatic polypeptide (PP) and a25I-PP serum binding capacity were determined in 25 Type 1 diabetic patients (aged 45 _+ 10 years) and in 10 control subjects (aged 38 _+ 8 years) to investigate PP levels and insulin capacity to induce PP antibodies. Heparinized venous blood samples (10 ml) were taken on fasting and plasma was divided into three fractions: (a) corresponding to free PP was obtained by precipitating plasma (1 ml) with ethanol (2 ml); (b) corresponding to total PP was obtained by treatment of fraction (a) with 0.1 ml HC1 1 N for 30 min; (c) corresponding to PP antibodies was obtained by treatment of fraction (b) with 1% dextran-charcoal (2 ml). PP was measured by radioimmunoassay on fractions (a) and (b), where 125I-PP serum binding capacity was determined on fraction (c). The control subjects showed free and total PP: 78-+25 and 65-+ 14 pg/ml, respectively (p> 0.05) and no PP binding capacity. Diabetic patients showed higher free PP levels than controls (294 + 85 pg/ml,

p < 0.01). Total PP levels varied in two groups of patients: 19 patients showed no statistical difference between free and total PP, whereas six patients showed higher values for total than free PP (326 + 81 versus 189 _+ 51 pg/ml; p < 0.01). PP binding capacity was present only in this latter group and was not related to duration of treatment or nature of the insulin. In conclusion, diabetic patients thus have higher circulating PP levels than control subjects; insulin therapy induced PP antibodies in 24% of cases.

420. Acute In Vivo Effects of Insulin-like Growth Factors I and II in Normal and Hypophysectomized Rats J. Zapf, Ch. Hauri and E. R. Froesch. Department of Medicine, Uni- versity Hospital, Zurich, Switzerland Intravenous injection of pure insulin-like growth factors (IGF) I or II (20 pg ~ 6 mU) together with insulin antiserum and 1 p.Ci of (U-14C)- glucose caused a rapid (t5 rain) fall of blood glucose in normal (40 and 26%) and hypophysectomized rats (50 and 45%) similar to that by 6 mU of insulin (50%). Concomitantly, the decrease of the (14-C)-serum radioactivity was enhanced. 'Basal' (14-C)-incorporation into di- aphragm glycogen was higher ( x 2), into adipose tissue lipids lower (x 6) in hypophysectomized than in normal rats at identical blood glucose levels. IGF I and II stimulated glycogen synthesis 12- and sev- enfold in hypophysectomized rats, but only five- and 1.2-fold in normal rats (tenfold stimulation each by insulin). In contrast to insulin (fivefold stimulation), they barely stimulated (14-C)-incorporation in- to lipids in normal rats. In hypophysectomized rats the stimulatory effect of insulin on lipid synthesis was largely reduced ( x 2) and similar to that of IGF I, whereas IGF II was ineffective. Results are compatible with and explained by corresponding findings in vitro. In contrast to long-term subcutaneous IGF infusion (action on growth), IGFs as a bolus exert acute effects on glucose homeostasis. Compared with insulin, they have little effect on lipid synthesis from glucose, but prefer- entially favour glucose utilization by muscle, above all in the hypophysectomized state.

421. Evidence for an Inverse Relationship Between HDL-Cholesterol and Insulin I. Zavaroni, E. Dall'Aglio, F. Bruschi, O. Alpi, E. Bonora, A. Pezzaros- sa and U. Butturini. Clinica Medica Generale, Parma University Med- ical School, Parma, Italy HDL-cholesterol appears inversely related to obesity and triglycerides. Since hyperinsulinaemia is the primary metabolic defect of obesity and endogenous hypertriglycefidaemia, we examined the relationship between HDL-cholesterol and triglycerides, insulin, body weight in a working population of 389 men and 218 women. An oral glucose tolerance test (75 g) was performed. Glucose, insulin, triglycerides and HDL-cholesterol were determined. Height and weight were measured. Alcohol intake, smoking, physical activity were estimated by a standard questionnaire. Results: in men, HDL-cholesterol (48 _+ 12 mg/dl) correlated with triglycerides (r= -0.41, p< 0.001), fasting insulin (r= - 0.25, p < 0.001), 1-h insulin (r= - 0A 6, p < 0.001), 2-h insulin (r=-0.10, p<0.05), insulin area ( r=-0 .17 , p<0.001), and body weight (r= -0.20, p< 0.001). In women, HDL-cholesterol (58 _+ 12 mg/dl) correlated with triglycerides (r= - 0.16, p < 0.01), 1-h insulin (r= - 0.14, p < 0.05), 2-h insulin (r= - 0.23, p < 0.001), insulin area ( r=-0 .18 , p<0.01), and body weight ( r=-0 .25 , p<0.001). Once body weight and triglycerides had been controlled for, significant partial correlation coefficients were found between HDL-cholesterol and fasting insulin in men (r= -0.13, p<0.01), and between HDL-cholesterol and 2-h insulin in women (r= - 0.12, p < 0.05). Step- wise regression showed that the best predictors for HDL-cholesterol are triglycerides, fasting insulin, alcohol intake, smoking, body weight in men and body weight, smoking, 2-h insulin, age in women. These results provide evidence for an inverse and independent relationship between HDL-cholesterol and insulin.

422. Assessment of Human Adipocyte Glucose Transport Using the Physiological Substrate D-Glucose S.Zeuzem and R.Taylor. Department of Clinical Biochemistry and Metabolic Medicine, Royal Victoria Infirmary, Newcastle~upon- Tyne, UK and Centre for Internal Medicine, Department of Endo- crinology, Frankfurt-am-Main, FRG Glucose transport into adipocytes is usually assessed using the non- metabolised sugar 3-0-methylglucose which has a different affinity for the transport system than D-glucose. To examine the relationship of D-glucose transport to intracellular glucose metabolism, we have examined the validity of direct D-glucose transport estimation. The method was based upon that of Pedersen and Gliemann, except that

Abstracts 207

U-Ca4-D-glucose (20 gmol/1) was used as tracer. The time course of U-C14-D-glucose uptake into human adipocytes at 37 ~ was linear over the initial 30 s (0.055 pmol/s basally and 0.093 pmol/s with maximal insulin stimulation). Subsequently, a 20 s glucose incubation period was used. Half-maximal insulin stimulation in normal subjects occurred at 3.28 + 0.36 mU/1 and the mean percentage insulin stimulation was 134%. The calculated membrane permeability was 2.64 x 10 .7 basally and 6.40 x 10 7 cm/s for maximal insulin stimulation. Glucose uptake at 20 ~ was decreased by 48% basally and by 53% at maximal insulin stimulation. The coefficient of variation of the assay was 9% for basal uptake and 7% for maximal insulin stimulation. Cell associated U-C~4-D-glucose did not change within 5 rain after stopping glucose uptake with phloretin, suggesting that metabolism of the D-glucose tracer does not affect this measurement of glucose transport. This method not only avoids the use of a non- physiological substrate, but is also simple and reliable.

423. A New Animal Model for Type I Diabetes: Induction of Autoim- mune Response Directed to ]/-Cells by Low Doses of Streptozotocin Combined with Polyclonal B-Lymphocyte Activation M. Ziegler, B. Ziegler, S. Noack, and B. Hehmke. Central Institute of Diabetes, Karlsburg, GDR Diabetes induction in response to multiple low doses of streptozotocin (STZ) in mice is characterized by ]3-cell necrosis, insulitis and a delayed onset of hyperglycaemia. The finding that continued administration of anti-lymphocyte serum in combination with nicotinamide blocked STZ-induced hyperglycaemia suggests an autoimmune component in pathogenesis. However, prior to appearance of insulitis, a major loss in islet mass is produced by direct cytotoxicity of STZ. Therefore, it remains a major experimental problem to distinguish the direct B-cell cytotoxic action of STZ from any secondary cell damage. In our investigation, the effect of polyclonal B-lymphocyte activation (PBA) on the diabetogenic action of low doses of STZ was studied. The results demonstrate that non-diabetogenic doses of STZ cause se-

vere diabetes in Wistar rats by additional PBA (plasma glucose < 25 nmol/l). Pancreatic insulin content was reduced < 3% by combined treatment and plasma of these rats showed cytotoxicity to syngeneic islet cells measured by 51Cr-release. PBA or low doses of STZ alone were without any effect on plasma glucose. It is suggested that STZ-induced structural alterations of B-cells evoke an immune response which is amplified by PBA resulting in B-cell destruction and insulin-dependent diabetes.

424. Cytotoxic/Suppressor T Cells and Soluble Immune Complexes in Diabetic patients with Microangiopathy O.Zuccarini, P.Pozzilli, U. Di Mario, M. Iavicoli, D.Andreani and M. Sensi. Prima Cattedra Endocrinologia, Universit/t degli Studi di Roma 'La Sapienza', Rome, Italy and Department of Diabetes and Immunogenetics, St. Bartholomew's Hospital, London, UK T cell subpopulations, as defined by monoclonal antibodies, and soluble immune complexes, as detected by two methods differing in principle, were evaluated in 19 long-standing Type I and 17 Type 2 diabetic patients with various degrees of microangiopathy. In Type 1 diabetic patients, total T and helper T cells were significantly decreased (p<0.01), whereas cytotoxic/suppressor T cells were increased (p < 0.05). In Type 2 diabetic patients, no significant variation in T cell subpopulation numbers was observed. Immune complex positivity was significantly higher in Type 1 diabetic patients than in Type 2 or normal control subjects. An inverse correlation between cytotoxic/suppressor T cells and soluble immune complexes (p < 0.005) was found when microangiopathy was taken into consideration: normal levels of cytotoxic/suppressor T cells and raised levels of immune complexes being found in patients with retinopathy (p < 0.01). The relationship found between cytotoxic/suppressor T cells, soluble immune complexes and the presence of retinopathy suggests that these cells may have a connection with the increase of immune complexes in circulation and in their deposition into the vessel walls.