Pancreatic trophism in experimental liver cirrhosis

International Journal ofPancreatology, vol. 14, no. 2, 157-166, October 1993 �9 Copyright 1993 by Humana Press Inc. All fights of any nature whatsoever reserved. 0169-4197/93/14:157-166/53.00

Pancreatic Trophism in Experimental Liver Cirrhosist

Istvdn Nagy, *,I Ferenc Hajnal, 1 Gdbor Mohflcsi/ J6zsef N~meth, I

Zoltdn Ldszik, Z and Akos Pap 3

11st Department of Medicine, and 2Department of Pathology, Albert Szent-Gy6rgyi Medical University, Szeged, Hungary, 32nd Department of Medicine, St. Imre Hospital, Budapest, Hungary

Summary

Pancreatic trophism and pancreatic enzyme composition, and plasma levels of cholecystokinin, insulin, glucagon, and glucose in liver cirrhosis induced by chronic thioacetamide administration (0.02% in the drinking water for 12 mo) were studied in rats. Advanced liver cirrhosis was evident in all thioacetamide- treated rats. The weight of the pancreas and its contents of DNA, protein, trypsinogen, chymotrypsinogen, proelastase, secretory trypsin inhibitor, and amylase were significantly increased as compared to the controls. The pancreatic secretory enzyme content changes showed a nonparallelism, characteristic of a cholecystokinin effect. Light and electron microscopy revealed a normal pancreatic architecture. Bioassayed plasma cholecystokinin levels in both fed and 24-h-fasted cirrhotic rats were significantly higher than in the corresponding controls. The plasma glucose, insulin, and glucagon levels demonstrated hypoglycemic tendencies with a glucagon predominance. These findings indicate that advanced liver cirrhosis in the rat is accompanied by pancreatic hypertrophy and hyperplasia, which might be attributed, at least in part, to elevated circulating cholecystokinin levels.

Key Words: Thioacetamide; liver cirrhosis; pancreas; rats; cholecystokinin; insulin; glucagon.

Introduction Pancreatic hypersecretion has frequently been

found in patients with cirrhosis of the liver (2-5). However, the cause of this phenomenon is not clearly understood. Previous studies in our laboratory have shown signs of pancreatic hypertrophy (6) and hypersecretion (7) in rats rendered cirrhotic

Received December 21, 1992; Revised March 18, 1993; Accepted April 5, 1993.

*Author to whom all correspondence and reprint requests should be addressed: First Department of Medicine, Albert Szent-Gy6rgyi Medical University, Szeged, P.O. Box 469, H- 6701, Hungary.

t Some parts of this work were presented at the 21 st Meeting of the European Pancreatic Club, Glasgow, UK, September 20- 23, 1989 (ref. 1).

by thioacetamide (TAA) treatment. The liver is known to be a major site of the elimination of gastrointestinal hormones, including cholecystokinin (CCK) peptides (7-12), which are possibly involved in the regulation of pancreatic secretion and growth (13-15). Elevated plasma CCK levels and a prolonged half-life of CCK-octapeptide in patients with liver cirrhosis were recently reported (16-18), but the pancreatic function was not tested in these studies.

In order to widen our understanding of the influence of advanced liver cirrhosis on pancreatic lrophisrn, the present study was performed to examine the pancreatic size, its contents of DNA, protein, and diges- tive enzymes, and the circulating levels of CCK, insulin, and glucagon in TAA-induced cirrhotic rats.

157

158 Nagy et al.

Material and Methods

Animals

Female R-Amsterdam rats with an initial body weight of 140-150 g were used. (Prolonged treatment with TAA has been found to be better tolerated by female than male rats [19].) They were kept at 24~ with a 12-h light cycle from 6:00 to 18:00, and both experimental and control animals were fed the same commercial laboratory chow diet containing 69% carbohydrate, 20% protein, and 2.7% fat (LATI, G6d/5116, Hungary) ad libitum throughout. To produce liver cirrhosis, 25 rats were given drinking water containing 0.02% TAA (Fluka AG, Buchs, Switzerland) ad libitum for 12 mo. The animals were studied 1 wk after withdrawal of TAA administration. Controls (n = 20) received tap water. At the end of the experiment, half of the animals from both the TAA-treated and the control groups were fasted for 24 h before being killed; the other half of the animals were studied without food restriction. Between 8:00 and 10:00 AM, the rats were anesthetized with 50 mg/kg pentobarbital sodium ip. Blood samples were taken from the aorta with a heparinized syringe and stabilized with EDTA-Na 2 (1.5 mg/mL of blood) and approtinin (Gordox R, Richter, Budapest, Hungary; 1000 KIU/mL) for plasma CCK and glucagon determinations. Other measurements were carried out on the heparinized plasma. Portions of the plasma were used immediately for glucose assay (20). For hormone assays, plasma samples were stored at -20~ until use.

The whole pancreas and liver were rapidly removed, cleaned, and weighed. For light microscopy, small weighed pieces of liver and pancreas were fixed in 4% neutral buffered formalin. For electron microscopy, pieces of the pancreas were fixed in 3 % phosphate-buffered glu- taraldehyde and embedded in Durcupan ACM (Fluka AG) after postfixation in 1% OsO4. Sections were double- stained with uranyl acetate and lead citrate, and examined by a Tesla BS-500 electron microscope. For biochemical analysis, the pancreas was immediately frozen in liquid nitrogen, and stored at-20~ until assayed.

Biochemical Analyses The pancreas was homogenized in 9 vol (w/v) of

ice-cold buffer containing 0.02M Tris-HC1, pH 7.8, 0.15M NaC1, and 0.1% Triton X- 100. Portions of the

homogenate were immediately used for the extrac- tion of pancreatic secretory trypsin inhibitor (PSTI), DNA, and insulin (21). Enzyme measurements were carried out on supernatant fractions of homogenates after centrifugation at 20,000g for 20 min. DNA, PSTI, amylase, trypsinogen, chymotrypsinogen, proelastase, and protein were assayed by colorimetric methods (22-28). Lipase activity was determined at 25~ pH 8.0, by a pH-stat method (29), modified for the assay of rat pancreatic lipase. The reaction mixture contained 1 mL of Sigma Lipase Substrate (Sigma Chemical Co., St. Louis, MO), 0.15M NaC1, and 2 mM CaC1 z in a final volume of 10 mL. The details of these pro- cedures as well as the activation of trypsinogen, chymotrypsinogen, and proelastase have been described in a previous paper (30). Enzyme activities are expressed as total pancreatic contents and given in SI units (lakat [microkat] or nkat [nanokat] per pancreas; 1 ~kat = 1000 nkat = 1/60 international unit [IU]).

Plasma levels of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), urea nitrogen, and total bilirubin were measured by standard clinical methods.

Hormone Assays

Plasma CCK was estimated by bioassay as described by Liddle et al. (31). Briefly, CCK was ex- tracted from the plasma by adsorption onto SEP-PAK C-18 cartridges (Waters Associates, Millipore Corp., Milford, MA). After elution and drying under nitrogen stream, the CCK in the extracts was quantitated by its ability to release amylase from dispersed rat pancreatic acini. Standard curves were obtained by using HPLC-purified CCK-8, synthetized by G. K. T6th and B. Penke, Dept. of Medical Chemistry, A. Szent-Gytirgyi Medical University, Szeged, Hungary (32). The assay is sensitive to plasma levels of CCK as low as 0.25 pM (33).

Insulin was determined with a radioimmunoassay kit (Izinta, Budapest, Hungary), using human insulin as the standard. Glucagon was estimated with the radioimmunoassay kit of Biodata, Rome, Italy.

Statistics

All results are expressed as means _+ SD. For statistical evaluation, regression analysis, Mann- Whitney's test or Student's t-test was used, as

International Journal of Pancreatology Volume 14, 1993

Liver Cirrhosis and Pancreas 159

appropriate. Any p values of less than 0.05 were accepted as significant.

Results

Nine out of 25 TAA-treated animals died before the end of the experiment; all 16 survivors were examined. From the control group, 2 animals were lost during the 12-mo period; 16 of the remaining 18 were randomly selected for examination.

The liver function tests showed unaltered plasma ASAT and ALAT activities, whereas AP and total bilirubin were slightly elevated in the drug-treated rats (Table 1).

The daily food intake of the rats was reduced throughout the administration of TAA; in the 12th mo, it was as low as 10.6 + 0.5 g/d, whereas that of the controls was 15.7 + 1.1 g/d (p < 0.05). One week after the cessation of TAA administration, the food intake of treated rats reached that of the controls. At the end of experiment, the body weight of TAA- treated rats was much less (Table 2); their overall weight gain was about half that of the controls.

Morphological Findings

The liver was markedly enlarged (Table 2), and advanced liver cirrhosis of macronodular type was evident in all TAA-treated rats (Figs. 1 and 2). Micro- scopy showed that the liver architecture had been replaced by nodules of varying sizes, with intense fibrous enlargement and inflammatory infiltration of the portal tracts, marked bile duct proliferation, hepatocellular degeneration (more prominently in the periportal zone), areas of focal liver cell necro- sis, and regeneration. A few cholangiomas and hemangiomas were also present. On the basis of these findings, we hereafter refer to the TAA-treated rats as cirrhotic.

Light microscopic examination of the pancreas of the cirrhotic rats revealed a normal pancreatic architecture with intense basophilia in the basal region of acinar cells (Fig. 3). Electron microscopy also demonstrated normal acinar cells with a well- developed rough endoplasmic reticulum (Fig. 4). The light microscopic picture of the kidneys of TAA-treated rats also appeared to be normal, except for scattered bilirubin pigment accumulation in a few tubules.

Table 1 Liver Function Tests and Plasma Urea Nitrogen

in Control and TAA-Treated Rats

Control, TAA-treated, n= 16 n= 16

ASAT, lakat/L 1.51 + 0.14 1.59 + 0.90

ALAT, lakat/L 0.83 + 0.12 0.74 + 0.15

AP, lakat/L 0.65 + 0.16 1.12 + 0.19" Total bilirubin,

gmol/L 4.08 + 1.53 12.9 + 3.51" Urea nitrogen,

mmol/L 6.18 + 0.56 6.43 + 0.92

Values are means + SD. ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase, AP, alkaline phosphatase. *p < 0.05 vs controls.

No significant morphological alterations were seen in the liver, pancreas, and kidneys of the control rats.

Pancreatic Trophism and Enzyme Composition

The wet weight and DNA content of the pancreas were not significantly modified by starvation for 24 h before the rats were killed, and these data from both fed and fasted animals were therefore utilized for statistical evaluation. On the other hand, the pancreatic protein and enzyme contents of cirrhotic rats fasted for 24 h before examination were about 10-15% lower than those of normally fed cirrhotics (data not shown); accordingly, only the data on the fed (drug-treated or control) animals were used to evaluate these latter parameters.

The wet weight of the pancreas and its content of DNA, protein, trypsinogen, chymotrypsinogen, proelastase, PSTI, and amylase in the cirrhotic rats were significantly higher than those of the controls (Tables 2 and 3). Analysis of the 16 pairs of data on the cirrhotic rats showed a significant linear correlation between the weights of the pancreas and liver (Fig. 5); the weight of the pancreas increased in parallel with that of the liver. On the other hand, there was not a significant correlation between the body weight and the liver weight, or between the body weight and the pancreas weight in the cirrhotic


160 Nagy et al.

Table 2 Body Weight, Liver and Pancreas Wet Weights, and Pancreatic DNA Content

in Control and TAA-Treated Rats

Control, TAA-treated, % of n = 16 n = 16 control

Body wt, g initial 148 • 10.2 final 234 • 15.8

Liver wt, g 6.76 • 0.77 Liver wt per

final body wt, mg/g 28.1 • 4.0 Pancreas wt, mg 769 • 69 Pancreas wt per

final body wt, mg/g 3.27 • 0.23 DNA, mg/pancreas 3.43 • 0.31

146 • 9.3 187 • 19.2" 80

19.1 • 4.9* 275

102 • 35* 363 936 • 150' 122

5.07 + 1.07" 155 4.04 + 0.37* 118

Means + SD. *p < 0.05 vs controls.

Fig. 1. Caudal aspect of liver of rat treated with TAA for 12 mo. Note the coarsely nodular surface.

rats. (In the control group, as expected, there were significant linear correlations between the body weight and the pancreatic weight, between the body weight and the liver weight, and between the pancreatic weight and liver weight; data not shown). With the DNA content as a measure of the cell num- ber, and the ratio of the protein content to the DNA content as an index of hypertrophy, the results indicate that TAA-induced liver cirrhosis was associated with pancreatic hyperplasia and hypertrophy.

The increases in pancreatic secretory enzyme contents showed a nonparallelism, characteristic of a CCK effect: proteases (+98 to +113%) > amylase (+60%) > lipase (unchanged). The marked increase in pancreatic trypsinogen content was paralleled by a similar increase in PSTI content.

There was no significant difference in pancreatic insulin content (measured in the 24-h-fasted animals) between the control and cirrhotic animals (1.34 + 0.44 and 1.36 + 0.35 IU/pancreas, respectively).

Plasma Levels o f Hormones and Glucose

The circulating CCK concentrations in both fed and 24-h-fasted cirrhotic rats were significantly higher than those in the corresponding controls (Table 4). The plasma glucose was significantly lower in the fasted cirrhotic rats than in the fasted controls. The levels of plasma insulin were significantly lower in both fed and fasted cirrhotic rats. Plasma glucagon concentrations displayed only in- significant alterations.

Discussion

The main findings from this study are that advanced liver cirrhosis in the rat is accompanied by hypertrophy and hyperplasia of the exocrine pancreas, elevations in plasma CCK levels, and nonparallel increases in pancreatic secretory enzyme contents, resembling those accompanying CCK action.



Fig. 2. Light microscopic appearance of TAA-induced liver cirrhosis in the rat. Intense fibrous enlargement of the portal tracts; replacement of liver architecture by nodules of varying sizes. Hematoxylin and eosin. Bar = 200 ~n.

Fig. 3. Section of pancreas from a cirrhotic rat stained with hematoxylin and eosin. Note the regular tissue architecture. Bar -- 100 ~rn.

TAA-induced liver cirrhosis has previously been found to be a suitable experimental model of cirrhosis in humans (19, 34-36). The treatment regimen used in this study resulted in advanced nodular cirrho-

sis. Our observations concerning the survival rate, body weight gain, food intake, liver morphology, and liver function tests of the TAA-treated rats agree well with those reported earlier (6,19,34-36). Despite


162 Nagy et al.

Fig. 4. Electron micrograph of the pancreas from a TAA-cirrhotic rat. Acinar cells showing a normal cytoarchitecture with well-developed rough endoplasmic reticulum. Bar = 2 Jam.

Table 3 Pancreatic Protein and Enzyme Contents in Control

and Cirrhotic Rats Fed Ad Libitum

Control, Cirrhotic, % of n = 8 n = 8 Control

Soluble protein mg/pancreas 104 + 13 mg/mgDNA 30.1 + 2.4

Amylase, lakat/pancreas 212 + 40 Lipase, lakat/pancreas 65.9 + 17.8 Trypsinogen, nkat/pancreas 274 + 44 PSTI, nkat/pancreas 3.85 + 0.77 PSTI/trypsinogen, % 1.39 + 0.09 Chymotrypsinogen,

nkat/pancreas 219 + 30 Proelastase, nkat/pancreas 36.5 + 6.4

158 + 15' 152 38.6 + 4.2* 129

340 + 67* 160 78.0 + 22.6 118

614 + 87* 224 8.20 + 0.95* 213 1.34 + 0.12 96

427 + 70* 195 72.1 + 11.9' 198

Means + SD. p < 0.05 vs controls.

the reduced body weight gain, the values of the main trophic parameters of the pancreas (wet weight, DNA, and protein contents) in the cirrhotic rats were found to exceed those in the control group. Nakamura et al. failed to observe an increase in the weight of the pancreas of rats with carbon tetrachloride-induced.

cirrhosis, but they too demonstrated a significant rise in the ratio of pancreas weight to body weight (37). Similarly to their results, in less severe forms of experimental cirrhosis (induced with the same dose of TAA as used in this study, but given for shorter periods of time), we also failed to observe signifi-

International Journal o f P ancreatology Volume 14, 1993


P a n c r e a :

1200-

1000

800-

wt (mg)

6 0 0 - , ,

10 15 20 25 30

Liver w t (g)

Fig. 5. Correlation between liver and pancreas weights in the cirrhotic rats. r -- 0.800; p < 0.001; y = 21.9x + 516.

cant increases in the absolute weight of the pancreas (6). It should be noted that, in this model, the severity of cirrhosis correlates with the increase in liver weight (36). We have now found a positive correlation between the liver and pancreas weights in cirrhotic rats. This means that pancreatic trophism increases with the severity of liver cirrhosis.

The chronic administration of exogenous CCK or its analogs is well known to evoke pancreatic hypertrophy and hyperplasia (13-15). Recent studies involving CCK antagonists have demonstrated that endogenous CCK released by food intake plays a significant role in the maintenance of physiological pancreatic trophism in the rat (38-40). The liver is an important site for the elimination of circulating bioactive CCK peptides containing eight or less amino acids (7-12). In liver cirrhosis, the hepatic clearance of several other peptide hormones (insulin, glucagon, and so on) has been shown to be impaired (41-44), and a prolonged half-life of CCK-octapeptide has also been reported (17). Therefore, the increases in plasma CCK levels observed in our cirrhotic rats or in patients with liver cirrhosis (16,18) are most probably a result of an impaired hepatic clearance of small CCK peptides. Indeed, the main circulating form of CCK in human cirrhotics has been found to be CCK-8 (18).

The kidney has been considered to be another important site of CCK metabolism (9,45). As regards

the normal plasma urea nitrogen levels and renal histology, a contribution of renal impairment to the development of elevated plasma CCK concentrations in our cirrhotic rats seems unlikely.

The nonparallel increases in pancreatic secretory enzyme contents in the cirrhotic rats closely resemble those evoked by the prolonged administration of exogenous CCK peptides, by inducing endogenous CCK release with trypsin inhibitor feeding, or with pancreatobiliary diversion in rats (see reviews 13,14). Using a CCK antagonist, it has recently been verified that prolonged moderate (1-1.5-fold) increases in plasma CCK levels, comparable to those found by us in the cirrhotic rats, are responsible for the trophic responses of the pancreas to chronic pancreatobiliary diversion in rats (46). With regard to both the elevated circulating CCK levels and the characteristic changes in the pancreatic enzyme pattern, it may be concluded that the development of pancreatic hypertrophy and hyperplasia in experimental liver cirrhosis might be attributed, at least partially, to an enhanced CCK action affecting the pancreas.

In this study, the plasma levels of secretin, another gastrointestinal hormone considered to play a role in the regulation of pancreatic growth (13,14), were not examined. Although a diminished hepatic secretin elimination may be assumed in cirrhotic rats (47), the enzyme pattern of the pancreas was inconsis- tent with prolonged secretin or secretin + CCK action (13,14,48). In summary, our results suggest a significant contribution of CCK to the development of pancreatic hypertrophy/hyperplasia in cirrhotic rats, but the modulatory role of other factors (secretin? other gastrointestinal hormones? growth factors/cytokines derived from the liver injury? and so on) may also be assumed.

The liver plays a central role in glucose homeosta- sis, too. Human liver cirrhosis is usually associated with a diminished glucose tolerance, accompanied by hyperinsulinemia and hyperglucagonemia, whereas hypoglycemia is more common in severe acute liver injuries (41-43). In TAA-treated rats, we found a hypoglycemic tendency with a glucagon predominance, characterized by decreased plasma insulin and unaltered plasma glucagon levels. In this respect, therefore, this model of liver


164 Nagy et al.

Table 4 Plasma Glucose, Insulin, Glucagon, and CCK Levels in Fed and 24-h-Fasted Control and Cirrhotic Rats

Fed rats Rats fasted for 24 h

Control Cirrhotic Control Cirrhotic

Glucose, mmol/L 6.84 + 0.36 6.72 + 0.41 6.01 + 0.39 4.83 + 0.23* Insulin, l.tU/mL 55.3 + 13.0 32.9 + 6.6* 23.2 + 7.4 11.7 + 2.1' Glucagon, pg/mL 43.2 + 10.2 51.1 + 8.6 41.1 + 7.3 47.7 + 13.5 CCK, pmol/L 1.64 + 0.77 4.78 + 2.09* 0.39 + 0.15 1.06 + 0.50*

Values are means + SD. For all groups, n = 8. *p < 0.05 vs the corresponding control value.

cirrhosis differs from the human disease. In accord with our results, advanced liver injury caused by a similar regimen of TAA administration has been found to be associated with decreased fasting serum glucose concentrations both during treatment and even for a long period after the cessation of TAA administration (36). This hypoglycemia may be explained by the toxic effect of chronic TAA poisoning affecting the periportal zone of the liver lobules, which has a predominant role in gluconeogenesis and thereby in the maintenance of blood glucose levels (49). Since the pancreatic insulin content was found to be normal, the decreased circulating insulin level cannot be explained by an impaired insulin production of the pancreatic islets; rather, it may be a consequence of the hypoglycemic tendency. Further studies are in progress to clarify the features of glucose homeosta- sis in TAA-cirrhotic rats.

Although TAA-induced experimental cirrhosis is not completely analogous to human liver cirrhosis, our observations are consistent with some of those found in cirrhotic patients: increased plasma CCK levels (16-18) and a predominant increase in trypsin output have been demonstrated in human cirrhotics (5), too. It should be noted that the prolonged administration of CCK to humans likewise results in a predominant increase in trypsin output (50,51). These observations suggest a possible role of elevated circulating CCK levels in the development of pancreatic changes associated with human liver cirrhosis.

Acknowledgments

The authors wish to thank G. K. T6th and B. Penke (Dept. of Medical Chemistry, A. Szent-Gy6rgyi

Medical University, Szeged, Hungary) for providing synthetic CCK-8. This study was partly supported by grants from the Hungarian Ministry of Social Wel- fare (ETI" No. T-89/1990 and No. M-14/1990).

References

1 Nagy I, Pap A, Hajnal F. Pancreatic hypertrophy and hyperplasia associated with elevated circulating CCK levels in experimental liver cirrhosis [Abstract]. Digestion 1989; 43: 165.

2 Van Goidsenhoven GE, Henke WJ, Vacca JB, Knight WA. Pancreatic function in cirrhosis of the liver. Am J Dig Dis 1963; 8: 160-173.

3 Dreiling DA, Greenstein AJ, Bordalo C. The hyper- secretory states of the pancreas. Gastroenterology 1973; 75: 1083-1089.

4 Justus PG, Wheeldon SG, Kolts BE. Secretin half-life in cirrhotics with high secretory volumes to a secretin test. Proc Soc Exp Biol Med 1979; 162: 351-353.

5 Renner IG, Rinderknecht H, Wisher JR, Pancreatic secretion after secretin and cholecystokinin stimulation in chronic alcoholics with and without cirrhosis. Dig Dis Sci 1983; 28: 1089-1093.

6 Pap A, Van6 V. Pancreatic amylase and protein concentrations associated with thioacetamide-induced cirrhosis of the liver in rats.ActaMedAcadSciHung 1978; 35: 311-316.

7 Berger Z, Pap A, Ungi I, Varr6 V. Inactivation of cholecystokinin octapeptide by normal and cirrhotic liver in rats. lnt J Pancreatol 1986; 1: 279-289.

8 Debas HT, Grossman MI. Hepatic inactivation of gastrointestinal hormones, in Proceedings of the Fifth World Congress of Gastroenterology, Mexico City, 1974; p. 494.

9 Lonovics J, Hajnal F, Suddith RL, Rayford PL, Thompson JC. Metabolism of different molecular forms of cholecystokinin, in Advances in Physiological Science, Vol. 12, Nutrition, Digestion, G~ti T, Szollfir LG, Ungv:iry G, eds., Akad6mia, Budapest, 1981; 383-390.

10 Doyle JW, Wolfe MM, McGuigan JE. Hepatic clearance of gastrin and cholecystokinin peptides. Gastroenterology 1984; 87: 60--68.



11 Eysselein VE, B&tcher W, Kaufmann GL Jr, Walsh JH. Molecular heterogeneity of canine cholecystokinin in portal and peripheral plasma. Regul Pept 1984; 9: 173-185.

12 Sakamoto T, Fujimura M, Newman J, Zhu XG, Greenley GH, Johnson JC. Comparison of hepatic elimination of different forms of cholecystokinin in dogs. Bioassay and radioimmunoassay comparison of cholecystokinin-8- sulfate and -33-sulfate. J Clin Invest 1985; 75: 280-285.

13 Solomon TE. Regulation of exocrine pancreatic cell proliferation and enzyme synthesis, in Physiology of the Gastrointestinal Tract, Johnson LR, ed., Raven, New York, 1981; 873-892.

14 F61sch UR. Regulation of pancreatic growth. Clin Gastroenterol 1984; 13: 679-699.

15 Solomon TE. Control of exocrine pancreatic secretion, in Physiology of the Gastrointestinal Tract, Johnson LR, ed., Raven, New York, 1987; 1173-1209.

16 Himeno S, Tarui S, Kanayama S, Kuroshima T, Shinomura Y, Hayashi C, Tateishi K, Imagawa K, Hashimura E, Hamaoka T. Plasma cholecystokinin responses after ingestion of liquid meal and intraduodenal infusion of fat, amino acids, or hydrochloric acid in man: Analysis with region specific radioimmunoassay. Am J Gastroenterol 1985; 80: 703-707.

17 Kanayama S, Himeno S, Kurokawa M, Shinomura Y, Kuroshima T, Okuno M, Tsuji T, Higashimoto Y, Ikei N, Hashimura E, Tateishi K, Hamaoka T, Tarui S. Marked prolongation in disappearance half-time of plasma cholecystokinin-octapeptide in patients with hepatic cirrhosis. Am J Gastroenterol 1985; 80: 557-560.

18 Kanayama S, Himeno S, Higashimoto Y, Yamasaki Y, Kitani T, Tarui S. Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis. Life Sci 1987; 41: 1915-1920.

19 Gupta DN. Nodular cirrhosis and metastasing tumours produced in the liver of rats by prolonged feeding with thioacetamide. J Path Bact 1956; 72: 415-425.

20 HyvErinen A, Nikkil~i EA. Specific determination of blood glucose with o-toluidine. Clin ChimActa 1962; 7: 140-144.

21 F61sch UR, Grieb N, Caspary WF, Creutzfeldt W. Influence of short- and long-term feeding of an or-amylase inhibitor (BAY e 4609) on the exocrine pancreas of the rat. Digestion 1981; 21: 74-82.

22 Giles KW, Myers A. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (London) 1965; 206: 93.

23 Fritz H, Trautschold I, Werle E. Protease inhibitors, in Methods of Enzymatic Analysis, vol. 2, 2nd ed., Bergmeyer HU, ed., Verlag Chemie, Weinheim, Academic, New York, 1974; 1064-1080.

24 Ceska M, Brown B, Birath K. A new rapid method for the clinical determination of or-amylase activities in human serum and urine. Optimal conditions. Clin Chim Acta 1969; 26:437 444.

25 Erlanger BF, Kokowsky W, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961; 95: 271-278.

26 Bundy HF. Chymotrypsin-catalyzed hydrolysis of N- acetyl- and N-benzoyl-L-tyrosine p-nitroanilides. Arch Biochem Biophys 1963; 102: 416--422.

27 Bieth J, Spiess B, Wermuth CG. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem Med 1974; 11: 350-357.

28 Goa J. Micro biuret method for protein determination; determination of total protein in cerebrospinal fluid. Scand J Clin Lab Med 1953; 5: 218-222.

29 Honegger J, Hadom B. The determination oflipase activity in human duodenal juice. Biol Gastroentero11973; 6: 217-223.

30 Nagy I, Pap ,~, Varr6 V. Time-course of changes in pancreatic size and enzyme composition in rats during starvation, lnt J Pancreatol 1989; 5: 35--45.

31 Liddle RA, Goldfine ID, Williams JA. Bioassay of plasma cholecystokinin in rats: effects of food, trypsin inhibitor, and alcohol. Gastroenterology 1984; 87: 542-549.

32 T6th GK, Penke B, Zarrandi M, Kov~s K. Comparison and optimization of synthetic methods for preparing cholecystokinin peptides, lntJPeptide Protein Res 1985; 26: 630-638.

33 Dilorenzo C, Williams CM, Hajnal F, Valenzuela JE. Pectin delays gastric emptying and increases satiety in obese subjects. Gastroenterology 1988; 95:1211-1215.

34 Brodehl J. Thioacetamid in der experimentellen Leberforschung. Klin Wschr 1961; 39: 956-962.

35 Zimmermann T, Franke H, Dargel R. Studies on lipid and lipoprotein metabolism in rat liver cirrhosis induced by different regimens of thioacetamide administration. Exp Pathol 1986; 30: 109-117.

36 Zimmermann T, Mtiller A, Machnik G, Franke H, Schubert H, Dargel R. Biochemical and morphological studies on production and regression of experimental liver cirrhosis induced by thioacetamide in Uje:WIST rats. Z Versuchstierkd 1987; 30: 165-180.

37 Nakamura T, Otsuki M, Tani S, Okabayashi Y, Fujii M, Oka T, Fujisawa T, Baba S. Pancreatic endocrine function in cirrhotic rats. Metabolism 1988; 37: 892-899.

38a Takfics T, Nagy I, Pap ,~, Varr6 V. Effect of a new CCK receptor antagonist on pancreatic secretion and growth in rat [Abstract], Digestion 1987; 38: 60-61.

38b Tak~ics T, Nagy I, Pap/~, Varr6 V. The effect of long- term administration of lorglumide (CR 1409) on rat pancreatic growth and enzyme composition. Pancreas 1990; 5: 606-610.

39 Wisner JR, McLaughlin RE, Rich KA, Ozawa S, Renner IG. Effects of L-364,718, a new cholecystokinin receptor antagonist, on camostate-induced growth of the rat pancreas. Gastroenterology 1988; 94:109-113.

40 Nylander A-G, Chen D, Ihse I, Rehfeld JF, HLkansson R. Pancreatic atrophy in rats produced by the cholecystokinin- A receptor antagonist devazepide. Scand J Gastroenterol 1992; 27: 743-747.

41 Tygstrup N, Iversen J. Carbohydrate metabolism in relation to liver physiology and disease, in The Liver- Annual I, Arias IM, Frenkel M, Wilson JHP, eds., Excerpta Medica, Amsterdam, 1981; 1-30.

42 Petrides SA, Strohmeyer G. Insulinresistenz bei Leberer- krankungen. Z Gastroenterologie 1986; 24: 403--415.


166 Nagy et al.

43 MiJller MJ, Willmann O, Rieger A, Fenk A, Selberg O, Lautz HU, Biirger M, Balks HJ, von zur Mtihlen A, Schmidt FW. Mechanism of insulin resistance associated with liver cirrhosis. Gastroenterology 1992; 102: 2033-2041.

44 Shankar TP, Drake S, Solomon SS. Insulin resistance and delayed clearance of peptide hormones in cirrhotic rat liver. Am JPhysiol 1987; 252: E772-E777.

45 Go VLW. The physiology of cholecystokinin, in Gut Hormones, Bloom SR, ed. Churchill Livingstone, Edinburgh, 1978; 203-207.

46 Watanapa P, Efa EF, Beardshall K, Calam J, Sarraf CE, Alison MR, Williamson RCN. Inhibitory effect of a cholecystokinin antagonist on the proliferative response of the pancreas to pancreatobiliary diversion. Gut 1991; 32: 1049-1054.

47 Chey WY, Hendricks J, Lorber SH. Inactivation of secretin by the liver. Clin Res 1971; 19: 389.

48 Nagy I, Tak~ics T, Moh~icsi G, Pap/~, Varr6 V. Effects of long-term administration of cholecystokinin-octapeptide (CCK-8) with or without secretin on the rat pancreas [Abstract], in Abstracts of the WORLD Congresses of Gastroenterology, Sydney 1990. The Medicine Group (UK), Abingdon, 1990; PP 376.

49 Schmidt E, Schmidt FW. Funktionelle Morphologie der Leber. Z Gastroenterologie 1987; Verh Bd 22: 16-38.

50 Pap ,~, Berger Z, Varr6 V. Trophic effect of cholecystokinin-octapeptide in man--A new way in the treatment of chronic pancreatitis? Digestion 1981; 21: 163-168.

51 Pap/~, Varr6 V. Effect of ceruletide treatment on exocrine pancreatic function in chronic pancreatitis [Abstract]. Digestion 1984; 30:118.


Documents

Pancreatic trophism in experimental liver cirrhosis