THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1990 by The American Soaety for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 15, Issue of May 25, pp. 8854-8862, 1990 Printed in U.S. A.

Insulin Modulation of Hepatic Synthesis and Secretion of Apolipoprotein B by Rat Hepatocytes”

(Received for publication, October 24, 1989)

Janet D. Sparks+ and Charles E. Sparks From the Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Insulin inhibition of apolipoprotein B (apoB) secre- tion by primary cultures of rat hepatocytes was inves- tigated in pulse-chase experiments using [35S]methio- nine as label. Radioactivity incorporation into apoBa and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of de- tergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-poly- acrylamide gel electrophoresis.

Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentra- tion of insulin. Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media la- beled apoB were analyzed; > 90% of labeled apoB was present in cells at 10 and 20 min after pulse, and labeled apoB did not appear in the medium until 40 min of chase. Insulin treatment inhibited the incorpo- ration of label into total apoB by 48%, into apoBH by 62%, and into apoBL by 40% relative to other cellular proteins. Insulin treatment favored the more rapid disappearance of labeled cellular apoBa with an intra- cellular retention half-time of 50 min (initial half-life of decay, tw = 25 min) compared with 85 min in control (L = 60 min). Intracellular retention half-times of labeled apoBL were similar in control and insulin- treated hepatocytes and ranged from 80 to 100 min. After 180 min of chase, 44% of labeled apoBL in control and 32% in insulin-treated hepatocytes remained cell associated. Recovery studies indicated that insulin stimulated the degradation of 45 and 27% of newly synthesized apoBH and apoBL, respectively.

When hepatocyte monolayers were continuously la- beled with [35S]methionine and then incubated in chase medium with and without insulin, labeled apoBa was secreted rapidly, reaching a plateau by 1 h of chase, whereas labeled apoBL was secreted linearly over 3-5 h of chase. Insulin inhibited the secretion of immu- noassayable apoB but not labeled apoB.

Results demonstrate that 1) insulin inhibits synthesis of apoB from [35S]methionine, 2) insulin stimulates degradation of freshly translated apoB favoring apoBH over apoBL, and 3) an intracellular pool of apoB, pri- marily apoBL, exists that is largely unaffected by in- sulin. Overall, insulin action in primary hepatocyte

* This work was supported by Research Grant HL 29837 from the National Heart, Lung and Blood Institute, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t To whom correspondence should be addressed: Dept. of Pathol- og$ and Laboratory- Medicine, University of Rochester School of Medicine and Dentistry. Box 608, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-8236.

cultures reduces the secretion of freshly synthesized apoB and favors secretion of preformed apoB enriched in apoBL.

Very low density lipoprotein is synthesized and secreted by liver after a series of complex intracellular events involving the synthesis of apolipoprotein B (apoB)’ and lipid and their assembly into lipoproteins. ApoB is one of the largest mam- malian proteins synthesized in liver as a single polypeptide chain having a molecular weight of 512,000 and is an obligate component for very low density lipoprotein assembly and secretion (1, 2). In rats (3-5) and humans (6), apoB exists in two forms which are metabolically distinct which we have designated as apoBH for the higher molecular weight form and apoBL for the lower molecular weight form (3). In rats apoBH- and apoBL-containing lipoproteins are secreted by liver (3-5), whereas in human liver the higher molecular weight form (apoB-100) predominates (7, 8). In both human and rat, apoBL-containing lipoproteins are secreted by intes- tine (6, 9). Human apoB-100 and apoB-48, corresponding to rat apoBH and apoBL, respectively, are products of a single- copy gene (10, 11). The mechanism of production of both apoB-100 and apoB-48 from a single gene is a result of an RNA-editing process that changes a glutamine codon (CAA) of the mRNA for apoB-100 into a translational stop codon (UAA), thereby generating a shortened protein product, apoB- 48 (12-14). A similar mechanism for the formation of apoBH and apoBL from a single-copy gene in rat liver has been demonstrated (15, 16) making the apoB forms in rat equiva- lent to those in humans. As a consequence of this RNA editing mechanism, the amino acid sequence of apoBL (apoB-48) is identical to that of the corresponding amino-terminal portion of apoBH (apoB-100).

Numerous studies support the concept that hepatic apoB synthesis and secretion are metabolically regulated. ApoB production rates are reduced in fasting (17, 18) and strepto- zotocin-induced diabetes (19, 20) and are increased in carbo- hydrate feeding (21). Moreover, there are marked changes in apoBH and apoBL expression in rats through development (22, 23). Even more recent studies have documented that thyroid hormone in viva influences the RNA-editing process of apoB mRNA in rat liver (24,25). The specific mechanisms involved in the hormonal regulation of hepatic apoB transla- tion, assembly with lipid and lipoprotein secretion, however, remain largely unknown.

Recent studies using primary cultures of rat hepatocytes

’ The abbreviations used are: ape, apolipoprotein; apoBH, apo B of higher molecular weight; apoB L,-apo-B of iower molecular weight; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electropho- resis; BSA, bovine serum albumin; PBS, phosphate-buffered saline.

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indicate there is an insulin receptor-mediated pathway that regulates apoB secretion by rat liver (26-28). This pathway has also been identified in liver perfusion studies (20) and in HepG2 cells (29, 30). Insulin in culture medium stimulates lipid synthesis from acetate (27), causes the accumulation of triglyceride within hepatocytes (26, 27, 29, 31), and reduces triglyceride (26-29, 31) and apoB secretion (26-30). If the mechanism of insulin action were due to inhibition of the apoB secretory pathway the apoB and triglyceride content of hepatocytes would increase concomitantly with the reduction of lipoprotein secretion. As apoB does not accumulate along with the triglyceride within hepatocytes with insulin treat- ment (27, 32) it is unlikely that the mechanism of insulin action is only to inhibit the lipoprotein secretory pathway. The current study demonstrates that insulin in the medium of primary cultures of rat hepatocytes reduces the incorpora- tion of [““Slmethionine label into apoBH and apoBL relative to other cellular proteins and favors the degradation of newly synthesized presecretory apoB. Decreased apoB synthesis and increased apoB degradation stimulated by insulin are respon- sible for reducing the amount of apoB secreted by hepatocytes incubated in the presence of insulin. Evidence is presented for a slower secretory pool of cellular apoB, mostly apoBL, which continues to be secreted in the presence of insulin in the medium.

EXPERIMENTAL PROCEDURES

Materials--Studies were performed in male Sprague-Dawley rats fed ad lib&urn and weighing ZOO-280 g. L-[““S]Methionine (>800 Ci/ mmol) was obtained from Amersham Corp. RPM1 1640 medium, Waymouth’s MB 752/l medium, Amberlite MB-3, Triton X-100, and benzamidine were from Sigma. Immunoprecipitin was purchased from Bethesda Research Laboratories. a-Toluenesulfonyl fluoride and XAR-5 film were from Eastman Kodak Co. Sepharose CL-4B was from Pharmacia LKB Biotechnology Inc. All other reagents were obtained from sources described previously (19).

Preparation of Hepatocytes for Primary Culture-Hepatocytes were prepared for primary culture in serum-free medium (27) and seeded onto loo-mm diameter sterile Petri dishes coated with rat tail colla- gen. The dishes were incubated for 2-4 h at 37 “C in an atmosphere of 95% air, 5% CO,. after which the medium and nonadherent cells were discarded and adherent cells washed three times and reincubated in Waymouth’s MB 752/l medium or RPM1 1640 medium containing 0.2% (w/v) bovine serum albumin (BSA) (5 ml/lOO-mm dish). Cel- lular protein was determined (33) after washing monolayers three times in phosphate-buffered saline. Cellular protein ranged from 2.5 to 5.0 mg of protein/dish.

Preparation of Rabbit Anti-rat Apolipoprotein B Polyclonal Anti- sera-Rat triglyceride-rich lipoproteins (3-8 mg of protein) were isolated from sera of sucrose-fed rats by ultracentrifugation at d < 1.019 g/ml and were delipidated (34). Apoproteins were dissolved in SDS buffer and apoBH and apoBL were separated by SDS column chromatography on 180-cm columns containing Sepharose CL-4B using a column buffer composed of 0.1 M sodiumph&phate, pH 7.4, 1% (w/v) SDS (35). with added 10 mM dithiothreitol. Eluted fractions containing apoB were evaluated by Coomassie Blue-stained gels after SDS-PAGE of the column fractions. Those fractions containing apoBH and apoBL were pooled and the majority of SDS was removed by dialysis against two changes of 4 liters of 0.1% Triton X-100 containing 25 g of Amberlite MB-3 for 3-5 h at room temperature using a molecular weight 50,000 cut-off membrane. Dialysis was continued overnight at 4 “C against 4 liters of 0.01% (w/v) ammonium formate, 0.01% Triton X-100 without resin. The apoB solution was removed, lyophilized, and dissolved in 1.0 ml of saline, and then 1.0 ml of complete Freund’s adjuvant was added and an emulsion pre- pared. New Zealand White rabbits were immunized by multiple intramuscular injections of the emulsion. Rabbits were boosted sev- eral times at 6-8-wk intervals with a similarly prepared apoB emul- sion using incomplete Freund’s adjuvant. Sera were obtained 2 weeks after each boost. The specificity of the antisera was determined by Western blot analysis on nitrocellulose paper as described previously (36), which demonstrated appropriate immunoreactivity to both apoBH and apoBL.

Pulse-Chase Studies of ApoB Production Using r”S]Methionine- Two types of pulse-chase experiments were used to study the kinetics of apoB synthesis and secretion in primary hepatocyte cultures (Figs. 1A and 6A). In both types of experiments apoB accumulation in the medium during the chase period was monitored by radioimmunoassay (Figs. 1B and 6B). Pulse-chase 1 studies were similar to those de- scribed using HepG2 cells (37, 38) to quantitate hepatic synthesis and secretion of-newly synthesized apoB. Hepatocyte monolayers were incubated in Wavmouth’s MB 752/l medium (5.0 ml/loo-mm dish) containing 0.1 iM insulin (control) or 10 nM ins&n. After overnight incubation (12-14 h), monolayers were washed three times and then incubated for 30-40 min in 3.0 ml of methionine-free RPM1 1640 medium containing 0.2% (w/v) BSA. To each dish, still contain- ing 3.0 ml of medium, was added 0.1 ml of methionine-free RPM1 1640 medium containing 0.2% (w/v) BSA and 1 mCi/ml [““Slmethi- onine (>800 Ci/mmol). Incubation was continued for exactly 10 min, after which the medium from each dish was quickly withdrawn and monolayers were washed three times and then reincubated for 10,20, 40, 90, and 180 min at 37 “C in 5.0 ml of RPM1 1640 medium containing 0.2% (w/v) BSA and 10 mM L-methionine (chase medium). Immunoprecipitation of hepatocellular and medium ““S-labeled apoB was carried out as described below. The time of chase required for 50% of the maximum newly synthesized labeled apoB to disappear from hepatocytes was determined from disappearance curves in order to calculate intracellular retention half-times as described by Yeo et al. (39). The half-life of decay of cellular apoB in the cell was determined from curves plotted where the peak of cellular apoB radioactivity (100%) was set to 0 min and other times during the chase period were adjusted accordingly. The initial half-life was calculated as described by Borchardt and Davis (42). In control studies after 180 min of incubation in chase medium hepatocyte monolayers were washed twice with 5.0 ml of chase medium contain- ing heparin (300 units/ml) or human low density lipoprotein (100 pg/ ml) followed by a third wash with chase medium before detergent solubilization and immunoprecipitation of cellular ““S-labeled apoB.

In pulse-chase 2 studies, after seeding the cells for 2-4 h hepatocyte monolayers were washed three times in methionine-free RPM1 1640 medium containing 0.2% (w/v) BSA and were reincubated for 12-14 h at 37 “C in methionine-free RPM1 1640 medium (5 ml/dish) con- taining added L-methionine (1 KM), [““Slmethionine (100 &i/dish), and 0.1 nM insulin. After incubation, labeled medium was withdrawn, and hepatocyte monolayers were washed three times and reincubated for 0, 0.5, 1, 2, and 3 h (and in some cases 5 h) in 5.0 ml of RPM1 1640 medium containing 0.2% (w/v) BSA and 10 mM L-methionine (chase medium) with and without’10 nM insulin added.

Zmmunoprecipitation-Dishes were terminated at the times indi- cated in the figures by washing hepatocyte monolayers three times with room temperature chase medium. Hepatocytes were immediately solubilized by addition of hot (80-90 “C) solubifization buffer (1.0 ml/ mg cell protein) which consisted of 0.05 M Tris buffer. 0.15 M NaCl. pfi 7.4, containing 1 mM a-toluenesulfonyl fluoride, 2’mM benzami: dine (freshly added), 5.0 mM EDTA, 1% (v/v) Triton X-100, and 0.5% (w/v) SDS. After incubation for 1 h at 65-80 “C, the solubilized cells formed a clear solution which was transferred to tubes and reheated for 5 min at 95 “C. For immunoprecipitation of cellular apoB, 0.5 ml of the solubilized cell extract was mixed with 0.5 ml of 0101 M Tris-HCl, 0.15 M NaCl, pH 7.4, and 0.5 ml of rabbit antiserum to rat apoB diluted in 1% (w/v) BSA/phosphate-buffered saline. For precipitation of apoB from medium samples, 0.5 ml of each medium was mixed with 0.5 ml of solubilization buffer and then with 0.5 ml of diluted rabbit antiserum. The dilution of antiserum used was previously shown to optimally precipitate labeled apoB from solubi- lized cell extracts and media. Immunoprecipitation was carried out overnight at 4 “C and immune complexes were collected by addition of 40 pl of Immuno-precipitin and incubation for 30 min at room temperature. Before use, bacterial cells were purified by washing in a solution of 4% (w/v) SDS, 5% (v/v) 2-mercaptoethanol followed by three washes in 0.01 M Tris-HCl, 0.15 M NaCl, pH 7.4, containing 1% (w/v) BSA. Immunoprecipitates were collected by centrifugation at 2800 rpm and were washed three to five times in 0.01 M Tris-HCl, 0.15 M NaCl, pH 7.4, containing0.15% (w/v) SDS, 0.1% (v/v) Triton X-100, and 2 AM EDTA. Labeled apoB was eluted in 150 pi of 0.0625 M Tris-HCl buffer. PH 6.8. containing 2% (w/v) SDS. 5% (v/v) 2- mercaptoethanol, &?% (v/vi glycerol, and ld ki dithiothreitoi with heating twice to 95 “C for 5 min. Bacterial cells were removed from the eluate by final centrifugation at 2800 rpm for 20 min. Eluted “S- labeled apoB was radioassayed and an aliquot was applied to a 3.5- 24% AcrylaideTM/acrylamide gradient gel cast on GelBondTM PAGE

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8856 Insulin Effects on Rat Hepatocyte ApoB

film. Labeled apoBn and apoBL were separated by electrophoresis for 3-4 h at 150 V (40), and afterwards proteins were fixed in sequential solutions of acetic acid/2-propanol. Fixative was removed from gels by agitation of the gel in 1% (v/v) glycerol for 2-4 h at room temperature and gels were then enhanced by agitation in AutofluorTM for 4 h. Enhanced gels were dried to a thin film in an convection oven at 170 “F for 30 min and placed in a wafer rigid cassette containing preflashed Kodak XAR-5 film. The films were exposed at -80 “C for appropriate exposure times and were developed using an automatic processor. For assessment of radioactivity distribution of fluoro- graphs, each lane was scanned three times and the percent distribu- tion was determined using an automatic integrating densitometer (EDC 1376, Helena Laboratories, Beaumont, TX). The average per-

cent apoBn and apoBL in each lane as determined by scanning was multiplied by actual radioassayed disintegrations/min/mg cell protein in order to calculate apoBn and apoBL radioactivity in the original sample.

Analytical Procedures-The apoB content of media and 0.5% (v/ v) Triton X-100solubilized hepatocytes (26) was assayed using a single monoclonal antibody solid phase radioimmunoassay (27, 36). The antibody used was a ““I-labeled mouse monoclonal antibody equally reactive against rat apoBu and apoBL. The apoB content of cell homogenates from individual hepatocyte culture dishes was de- termined immediately after preparation of the homogenate and each homogenate was assayed in triplicate. For determination of protein synthesis in pulse-chase studies, 0.1 ml of the detergent-solubilized cell extract or medium sample was mixed with 0.4 ml of 1% (w/v) BSA/phosphate-buffered saline and 0.5 ml of 0.1 M DL-methionine. Afterwards, 1.0 ml of 20% (w/v) trichloroacetic acid was added with mixing and the mixture was heated to 95 ‘C for 15 min. Precipitated protein was separated by centrifugation at 2800 rpm for 20 min and aliquots of the supernatant were radioassayed to correct for non- trichloroacetic acid-precipitable radioactivity.

RESULTS

Insulin Effect on Incorporation of pS]Methionine into ApoB in Pulse-Chase 1 Studies- The capacity of hepatocytes to incorporate labeled methionine into apoB and to secrete newly synthesized apoB was examined using a pulse-chase protocol (Fig. 1A) similar to that described for HepG2 cells (37, 38). Hepatocyte monolayers were cultured for 12-14 h in either control medium (0.1 nM insulin) or in medium contain- ing an inhibitory concentration of insulin (10 nM insulin) prior to pulse labeling. Fig. 1B indicates that reduced secretion of apoB by insulin was maintained for 180 min of the chase period. ?S-Labeled apoBH and ?S-labeled apoBL appeared in the medium at 40 min of chase and continued to be secreted throughout the MO-min chase period (Fig. 2).

Greater than 90% of peak label was present in the cell at 20 min (Table I) and apoB had not been secreted into the medium by 20 min into the chase period. These reasons allowed a direct comparison of the amount of label incorpo- rated into cell apoB during the IO-20 min in control and insulin-treated hepatocytes giving an estimation of freshly synthesized apoB in the two conditions (Table II). To compare experiments, the amount of [?S]methionine incorporated into total cellular apoB (apoBa plus apoBL) in control hepatocytes was adjusted to 10,000 dpm/mg cell protein and the same factor was used to adjust the apoB radioactivity in insulin- treated hepatocytes. As shown in Table II, hepatocytes incu- bated for 12-14 h in medium containing 10 nM insulin incor- porated 43% less label into total apoB, 58% less label into apoBh, and 34% less label into apoBL than hepatocytes in- cubated in control medium.

The amount of [35S]methionine incorporated into cell pro- tein during the lo-20-min time interval was measured by trichloroacetic acid precipitation and was somewhat larger in insulin-treated than control hepatocytes (mean percent in- crease over control f S.D., n = 3): 25 f 12%. The increased incorporation into protein corresponded to increased uptake of [““Slmethionine which was reflected by an increase in

A. Pulse-chase protocol 1

to‘m 40 180

12 to 14 h incubation lll i 1 - Cells incubated in ccmtrol medium

t

Chase period of 180 min or in medium containing

IO nM insulin

10 min pulse

B. Apo B mass secreted into medium (Pulse-chase 1)

TIME, min

FIG. 1. Pulse-chase 1 protocol and apoB secretion into chase medium in pulse-chase 1 studies. A, schematic presentation of the pulse-chase 1 protocol. Rat hepatocytes (loo-mm dishes) were incubated with Waymouth’s MB 752/l medium containing 0.2% (w/ v) BSA and either 0.1 nM insulin (control) or 10 nM insulin (insulin- treated) for 12-14 h. After incubation, medium was withdrawn and hepatocytes were labeled by incubation with medium containing 100 &i of [35S]methionine/dish for 10 min. The labeled medium was removed and hepatocytes were reincubated in medium containing 0.2% (w/v) BSA and 10 mM L-methionine (chase medium) for 10,20, 40, 90, and 180 min. B, apoB secretion during the chase period of pulse-chase 1 studies was assayed by monoclonal immunoassay. Con- trol results are adjusted to 150 ng of apoB/mg of cell protein accu- mulated in medium in 180 min. Results of four rat liver experiments are presented as the mean + S.D. at each time point. 0 and 0, apoB secreted by hepatocytes incubated in control medium and in medium containing 10 nM insulin, respectively. *, apoB secreted by insulin- treated hepatocytes is significantly different from that secreted by control hepatocytes (p < 0.05).

trichloroacetic acid-soluble radioactivity during the 10-20- min interval (mean percent increase over control f S.D., n = 3 livers): 38 + 16%. Because of the apparent difference in precursor uptake, label incorporated into cellular apoB was normalized by calculating results relative to label incorporated into total cellular protein as described by Williams and Daw- son (41). After normalization, hepatocytes incubated for 12- 14 h in medium containing 10 nM insulin synthesized 48% less total apoB, 62% less apoBn, and 40% less apoBL than hepatocytes incubated in control medium. In these same studies 35S-labeled apoBH synthesis as a percentage of total 35S-labeled cellular apoB synthesis in control and insulin- treated hepatocytes was (mean f S.D., n = 3): 38 + 4.3% and 28 f 7.5%, respectively, indicating that apoBL synthesis was favored over apoBn synthesis in control and insulin-treated hepatocytes.

Insulin Effect on Time Course of ~5S]Methionine-labeled ApoB Disappearance from Hepatocytes-Intracellular reten- tion half-times for apoBH and apoBL were calculated as de- scribed by Yeo et al. (39) from disappearance curves of labeled cellular apoB. The half-life of decay of apoB in the cell was

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Insulin Effects on Rat Hepatocyte ApoB

Cells

40 90 180

Media

40 so 180

40 90 180

FIG. 2. Fluorograph of enhanced SDS-PAGE gels of immu- noprecipitated [?3]methionine-labeled apoBH and apoBL of detergent-solubilized cells and media. Rat hepatocyte mono- layers incubated in control and insulin medium were pulse-labeled and chased as described in the legend to Fig. 1. A (control hepatocytes) and B (insulin-treated hepatocytes) are fluorographs of cell- and medium-immunoprecipitated ““S-labeled apoB from pulse-chase stud- ies at 40, 90, and 180 min of chase as indicated at the top of each lane. S, the beginning of the separating gel; and arrows, molecular weight 205,000, 96,000, and 68,000 marker proteins.

TABLE I

I:‘“S]Methionine incorporation into cellular apoB during initial IO-20-min chase period

Hepatocytes (loo-mm dishes) were incubated in medium contain- ing 0.1 nM insulin (control) and 10 nM insulin for 12-14 h. Monolayers were then incubated with [““Slmethionine (100 &i/dish) for 10 min and reincubated in chase medium containing 10 mM L-methionine. labeled apoB of detergent-solubilized cells was isolated by immuno- precipitation and radioassayed. The proportion of ‘“‘S-labeled apoBu and ““S-labeled apoBL in immunoprecipitates was determined by scanning densitometry of fluorographs of enhanced SDS gels. ass- labeled apoB in hepatocytes at 10 and 20 min into the chase period was calculated as a percentage of the maximum label incorporated into cellular apoB/mg of cell protein. Results are expressed as the average percent in the cell at 10 and 20 min t S.D. (number of rat livers).

Apolipoprotein Ba Apolipoprotein B,. Time

Control Insulin Control Insulin % o/o

10 min (3) 96.8 + 5.2 81.0 + 12.2 99.2 f 1.3 98.2 + 3.2 20 min (4) 94.9 + 8.2 98.2 + 3.6 90.6 + 16.1 91.3 f 11.4

determined from curves plotted where the peak of cellular radioactivity (100%) was set to 0 min and other times during the chase period were adjusted accordingly. The initial half- life was calculated as described by Borchardt and Davis (42).

TABLE II

Effect of insulin on incorporation of f “S/methionine into apoB Hepatocytes (loo-mm dishes) are incubated in medium containing

0.1 nM insulin (control) and 10 nM insulin for 12-14 h. Hepatocytes are labeled by incubation with [,‘S]methionine (100 pCi/dish) for 10 min and reincubated in chase medium containing 10 mM L-methio- nine. Labeled apoB of detergent-solubilized cells is isolated by im- munoprecipitation and radioassayed. The proportion of ,‘“S-labeled apoBa and “‘S-labeled apoBt in immunoprecipitates is determined by scanning densitometry of fluorographs of enhanced SDS gels. The percent of label in each apoB band is multiplied times the total cellular apoB immunoprecipitated radioactivity per mg of cell protein which is measured by radioassay. Total “S-labeled apoB in the control hepatocytes (apoBn plus apoB,, radioactivity) is adjusted to 10,000 dpm/mg of cell protein and the same factor is used to adjust the apoB radioactivity in corresponding insulin-treated hepatocytes. Results are from three rat liver experiments and are expressed as the average + SD.

APOB 1‘0 I‘AL APO& APO&.

adjusted dpmfmg cell protein” Control 10,000 3,767 f 428 6,233 f 428 Insulin 5,669 k 270” 1,587 + 466h 4,082 +- 319’ % decrease 43 f 2.7 58 f 9.2 34 + 5.4

relatroe dpm/mg cell protein’

Insulin 5,168 + 1,34gh 1,469 f 632* 3,700 + 839’ % decrease 48 + 13.5 62 k 12.6 40 + 17.3

’ Adjusted apoB label (dpm/mg cell protein) is the amount of cell synthesized apoB at 10 plus 20 min of the chase period when greater than 90% of the apoB label is intracellular.

’ Significant difference between control versus insulin-treated he- patocytes at a probability level of at least p < 0.02.

’ Relative apoB label (dpm/mg cell protein) is the adjusted apoB synthesized by insulin-treated hepatocytes calculated relative to the percent change in cellular protein synthesis at 10 plus 20 min of the chase period as determined by trichloroacetic acid precipitation. The increase in label incorporation into cellular protein in insulin-treated cells was (mean + S.D.): 25 f 12% for the three experiments. Ap0B~o7.~i., apoBu, and apoBt as a percent of total protein synthesis in control hepatocytes is 1.07, 0.40, and 0.67% and in insulin-treated hepatocytes is 0.48, 0.16, and 0.32%, respectively.

These two calculations differ, in part, because the intracellular retention half-time calculation includes the delay in reaching the peak of maximum radioactivity within the cell. In Figs. 3 and 4 the decay of cellular apoBn and apoBL in the cell in control and insulin-treated hepatocytes are compared. The average intracellular retention half-time of ,‘“S-labeled apoBu in control hepatocytes was significantly longer (85 min) than in insulin-treated hepatocytes (50 min). The half-life of ‘YS- labeled apoBn in the cell also differed significantly between control and insulin-treated hepatocytes and was 60 and 25 min, respectively. Under both conditions, ““S-labeled apoBn disappeared almost completely by 180 min of chase (6 f 4.2 uersus 3 + 4.2% residual cell-associated radioactivity, respec- tively; n = 4 rat livers). These results indicate that insulin in the medium stimulated the disappearance of newly synthe- sized apoBH from the cell.

In contrast to almost complete disappearance of ““S-labeled apoBn from hepatocytes, at 180 min of chase, 44 f 8.0 and 32 f 15.2% of freshly synthesized ““S-labeled apoBi. in control and insulin-treated hepatocytes, respectively, remained cell- associated (n = 4 rat livers). The intracellular retention half- time of “S-labeled apoBi. was significantly longer than that of ““S-labeled apoBn and averaged 140 min in control and 115 min in insulin-treated hepatocytes. The half-lives of the ini- tial decay of apoBL in control and insulin-treated hepatocytes were similar (Fig. 4) and ranged from 80 to 100 min in individual experiments. The proportion of “‘S-labeled apoBL radioactivity in control and insulin-treated hepatocytes at 180

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8858 Insulin Effects on Rat Hepatocyte ApoB

0 10 20 30 40 50 50 70 80 90 100

TIME, min

FIG. 3. Disappearance kinetics of [““Slmethionine-labeled cellular apoBn in pulse-chase 1 studies. The curues represent the time course of disappearance of labeled apoB from control hepato- cytes (0) and from insulin-treated hepatocytes (0). Hepatocyte apoB radioactivity is the amount of immunoprecipitated “S-labeled apoB found in the hepatocytes during the chase period as a percentage of the maximum [ “Slmethionine incorporated into hepatocellular apoB (100%) which was adjusted to zero time. Data points are calculated from seven separate liver preparations. A polynomial function de- scribed the curves and R’ was 0.96 for control and 0.96 for insulin curves.

0 10 20 30 ‘lo 50 60 70 80 90 100

TIME, min

FIG. 4. Disappearance kinetics of [‘“Slmethionine-labeled cellular apoB,, in pulse-chase 1 studies. The curues represent the time course of disappearance of labeled apoB from control hepato- cytes (0) and from insulin-treated hepatocytes (0). Hepatocyte apoB radioactivity is the amount of immunoprecipitated ““S-labeled apoB found in the hepatocytes during the chase period as a percentage of the maximum [““Slmethionine incorporated into hepatocellular apoB (100%) which was adjusted to zero time. Data points are calculated from six separate liver preparations. A polynomial function described the curves and RY was 0.91 for control and 0.91 for insulin curves.

min was not markedly altered by washing hepatocyte mono- layers with medium containing heparin (300 units/ml) or human low density lipoprotein (100 pg/ml) prior to the solu- bilization and immunoprecipitation procedures indicating that the “S-labeled apoBL is within the cells.

Distribution of f’“S]Methionine-labeled ApoBH and ApoB,. at 180 Min in Pulse-Chase 1 Studies-The distribution of ‘“‘S- labeled apoBH and apo BI, between hepatocytes and medium at 180 min is seen in Fig. 5. In control hepatocytes an average of 88% of ‘“S-labeled apoBH and 84% of “S-labeled apoBI, were recovered in media plus cells. In insulin-treated hepa- tocytes an average of 43% of ““S-labeled apoBH and 57% of

CELL MEDIUM

FIG. 5. Insulin effect on the distribution of [%]methionine- labeled apoB between cells and media at 180 min of chase. Rat hepatocyte monolayers incubated in control and insulin medium were pulse-labeled and chased as described in the !.egend to Fig. 1. Cell apoB radioactivity is the amount of immunoprecipitated ““S- labeled apoB found in hepatocytes at 180 min of chase as a percentage of the maximum [““Slmethionine incorporated into apoB. Medium apoB radioactivity is the amount of immunoprecipitated ““S-labeled apoB found in the media after 180 min of chase as a percentage of the maximum. Results are expressed as the mean percent -t S.D. of four separate rat liver experiments. *, significant difference between control and insulin-treated hepatocytes using paired t statistics at a probability level of at least p < 0.05.

“‘S-labeled apoB,, were recovered in media plus cells. Calcu- lation of insulin-dependent degradation (recovery in control minus recovery in insulin-treated hepatocytes) was 45% for apoBH and 27% for apoBL.

Insulin Effect on the Secretion of Immunoassayed ApoB in Pulse-Chase 2 Studies-In order to differentiate insulin ef- fects on newly synthesized apoB uersus the cellular apoB pool we performed pulse-chase 2 studies (Fig. 6A). Hepatocyte monolayers were labeled in medium containing 0.1 nM insulin and [““Slmethionine (100 &i/dish) for 12-14 h to approach steady-state conditions. After the labeling period, medium was withdrawn, and the cells were rinsed and reincubated for 0.5, 1,2, and 3 h (and in some case 5 h) in chase medium with and without insulin (10 nM). The apoB secretory rate assayed by monoclonal immunoassay between 2 and 3 h of incubation was reduced by the presence of insulin in the medium (mean + S.D., n = 4 livers): 46 f 7 and 27 f 7 ng/mg/h, respectively (Fig. 6B).

Ratio of Cellular [‘“SlMethionine-labeled A~oBL to r5S] Methionine-labeled ApoBH in Pulse-Chase 2 Studies-After overnight labeling in control medium and before the chase period began the cellular ratio of labeled apoB,> to apoBH was 5.3 -+ 2.2 (mean f S.D., n = 6 livers). A similar ratio was obtained with [‘“C]L-leucine label used in pulse-chase 2 stud- ies where the ratio of cellular labeled apoBL to labeled apoBH averaged 8.6. These results indicate that the cellular pool of apoB is predominantly apoBL.

Secretion of ~l”S]Methionine-labeled A~oBH and A~oBL in Pulse-Chase 2 Studies-After overnight labeling, the secretion of ““S-labeled apoB into chase medium containing no insulin or 10 nM insulin was examined at various time points during the chase period. ““S-Labeled apoBH was secreted rapidly and reached a plateau at about 1 h (Fig. 7). The presence of insulin in the medium delayed secretion of ““S-labeled apoBL at % and 1 h (Fig. 8), but afterwards the accumulation rate of “S- labeled apoBI. in medium paralleled that of control hepato- cytes. These results suggest that after an initial readjustment (O-l h), there is little additional effect of insulin on the secretion of ““S-labeled apoBI. over the 3-h chase period. The secretion of cellular .‘“S-labeled apoB[. continued to be linear up to 5 h of chase which was the latest time point assayed (results not shown).

Insulin Effect on Cellular ApoB Content-The apoB content

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Insulin Effects on Rat Hepatocyte ApoB 8859

A. Pulse-chase protocol 2

lZto14hincubation i ‘7 ;

3h

zc c

Cells incubated in control me&urn Chase period of 3 to 5 h in medium with labeled amino acid

t with or without IO nM insulin

Chase begins

B. Apo B mass secreted into medium (Pulse-chase 2)

TIME, h

FIG. 6. Pulse-chase 2 protocol and apoB secretion into chase medium in pulse-chase 2 studies. A, schematic presentation of the pulse-chase 2 protocol. Hepatocytes (loo-mm dishes) are incu- bated in methionine-free RPM1 1640 medium (5 ml/dish) containing added L-methionine (1 PM), [‘“Slmethionine (100 &i/dish), and 0.1 nM insulin RPM1 1640 medium for 12-14 h. After incubation, medium is withdrawn and replaced with chase medium containing 10 mM L- methionine and either no insulin or 10 nM insulin for 0.5, 1, 2, and 3 h and, in some experiments, 5 h. B, apoB secretion during the chase period of pulse-chase 2 studies assayed by monoclonal immunoassay. Control results are adjusted to 150 ng of apoB/mg of cell protein accumulated in medium in 180 min. Results of four separate rat liver experiments are presented as the mean k S.D. at each time point. 0 and 0, apoB secreted by hepatocytes into insulin-free medium and medium containing 10 nM insulin, respectively. *, the apoB secreted by insulin-treated hepatocytes is significantly different from that secreted by control hepatocytes (p < 0.05).

of hepatocytes incubated in medium containing 0.1 nM insulin (control) and in 10 nM insulin for 12-14 h was measured by radioimmunoassay of freshly prepared cellular homogenates of primary hepatocytes. The averages of at least 5 dishes each of control and insulin-treated hepatocytes were compared from eight separate rat liver preparations. The apoB content of hepatocytes incubated with 10 nM insulin was modestly reduced compared with that of hepatocytes incubated in con- trol medium (mean f SD.): 226 + 36 uersus 282 f 25 ng/mg of cell protein, respectively, p < 0.003. The percent decrease of cellular apoB by insulin in the eight rat liver preparations averaged 19.8 + 9.3% (mean f S.D.).

DISCUSSION

The current study demonstrates that hepatobytes incubated for 12-14 h in medium containing 10 nM insulin incorporate significantly less [““Slmethionine into apoBToT*L, apoBH, and apoBL than hepatocytes incubated in control medium (Table II). We have expressed results relative to cellular protein synthesis to control for precursor availability (41) with the assumption that there is no channeling of amino acid precur- sor in insulin-treated cells and that both apoBH and apoBL are derived from the same labeled amino acid pool. The

1000

. Apolipoprotein BH

1 2 3

Time, h

FIG. 7. Time course of secretion of [‘%]methionine labeled apoBH from steady-state labeled hepatocytes in pulse-chase 2 studies. Rat hepatocyte monolayers incubated in control medium were continuously labeled overnight and then chased in medium containing either no insulin or 10 nM insulin as described in the legend to Fig. 6. 0 and 0, “S-labeled apoBH secreted by hepatocytes into insulin-free medium and medium containing 10 nM insulin, respectively. Results are the average “S-labeled apoBa radioactivity (counts/min/mg of cell protein) secreted into the medium at each time point + S.D. in three rat liver experiments.

loo00 Apolipoprotein BL T

Time, h FIG. 8. Time course of secretion of [36S]methionine-labeled

apoBL from steady-state labeled hepatocytes in pulse-chase 2 studies. Rat hepatocyte monolayers incubated in control medium were continuously labeled overnight and then chased in medium containing either no insulin or 10 nM insulin as described in the legend to Fig. 6. 0 and 0, %-labeled apoBL secreted by hepatocytes into insulin-free medium and into medium containing 10 nM insulin, respectively. Results are the average %-labeled apoBL radioactivity (counts/min/mg of cell protein) secreted into the medium at each time point +. S.D. in three rat liver experiments.

estimate of apoB synthesis is based on the observation that there is a delay in the attainment of the peak of maximum label incorporation into cellular apoB (Table I). The delay is variable and ranges from 10 min in rat hepatocytes (42) to lo-25 min in HepG2 cells (37, 38). The reason for the delay may be due to elongation of preformed labeled apoB on ribosomes (42, 43). We chose to average the lo- and 20-min values as there were some differences in attainment of peak radioactivity; however, the values at 10 and 20 min were similar (Table I), and in both cases more than 90% of apoB radioactivity was cellular. If the calculation were made based on lo-min values, the reduction in apoB synthesis would have been (mean f S.D., n = 3): 55 f 10% for apoBH, and 42 + 11% for apoBL, compared to 62 and 40% as reported in Table II. Whether apoB mRNA levels change in rat hepatocytes under insulin stimulation remains to be determined. Assum- ing the mechanism of insulin inhibition of apoB secretion in rat hepatocytes is similar to that recently described in HepG2

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8860 Insulin Effects on Rat Hepatocyte ApoB

cells where total apoB mRNA is unchanged (30, 44) and its half-life is 16 h (30) our results would suggest that the effect of insulin on apoB synthesis may be related to apoB mRNA activity or to translational efficiency of apoB message.

In pulse-chase 1 studies (Fig. 1) newly synthesized ?S- labeled apoB appears in the medium at 40 min following pulse with label and both ““S-labeled apoB and apoB mass secretion as measured by monoclonal immunoassay continue at a roughly linear rate throughout the chase period in both control and insulin-treated hepatocytes. Our results are similar to previously reported results indicating that 35S-labeled apoB appears in medium after 45 min of chase in rat hepatocytes (42); after 35 min in HepG2 cells (37), and between 30 and 45 min in estrogen-induced chick hepatocytes (45).

Pulse-chase data have been calculated in two ways. The first is according to the method of Yeo et al. (39) using the intracellular retention times or the time required for 50% of each labeled apoB component to disappear from the cell. In order to compare the rate of movement of apoB through the cell in control and insulin-treated hepatocytes a second cal- culation was made where pulse-chase results are adjusted by setting the peak (100%) of cellular apoB radioactivity to zero time of chase. The difference in the two calculations, in part, is the delay in attaining the peak of apoB radioactivity in the cell. The average intracellular retention half-time of 35S- labeled apoBn in control and insulin-treated hepatocytes was 85 and 50 min, respectively. The half-life of decay of apoBn in the cell in control and insulin-treated hepatocytes was 60 and 25 min, respectively. Using either calculation, results demonstrate that insulin in the medium favors the more rapid disappearance of newly synthesized apoBn from hepatocytes and little ‘S-labeled apoBu was retained in hepatocytes after 180 min of chase in either control or insulin-treated hepato- cytes.

The time course of 3”S-labeled apoBL disappearance from control and insulin-treated hepatocytes differed significantly from that of 35S-labeled apoBu. The intracellular retention half-time of 35S-labeled apoBL was 140 min in control hepa- tocytes and 115 min in insulin-treated hepatocytes. After 180 min of incubation in chase medium a significant portion, 44% in control and 32% in the insulin-treated hepatocytes, had not been secreted and remained within hepatocytes. These results suggest that a substantial portion of freshly synthe- sized apoBL enters a presecretory pool. Movement of freshly synthesized apoBL through the cell is not dramatically af- fected by insulin.

Most newly synthesized apoBn (88%) and apoBL (84%) were recovered at 180 min following pulse-labeling in medium and cells in control hepatocytes (Fig. 5). In hepatocytes in- cubated in medium containing 10 nM insulin only 43% of 35S- labeled apoBn and 57% of ““S-labeled apoBL were recovered. Low apoB recoveries in pulse-labeling studies were also ob- tained by Borchardt and Davis (42) using hepatocytes which were cultured in medium containing 1 pg/ml insulin (167 nM). They found only 36% of apoBn and 60% of apoBt, were recovered in cells plus medium. In the current study an average of 45% of 35S-labeled apoBn and 27% of 35S-labeled apoBL degradation was insulin-dependent. In fluorographs of SDS-PAGE of ““S-labeled apoB of detergent-solubilized cel- lular and medium immunoprecipitates few labeled bands are present in gel regions corresponding to proteins smaller than intact apoB (Fig. 2). Smaller pieces of apoB are seen in cellular immunoprecipitates in the studies of Reuben et al. (15) and Davis et al. (46). Quantitatively, these pieces (~5% of the total apoB) are minor which suggests that degradation is rapid and relatively efficient. Although these fragments con-

stitute a small proportion of total cell immunoprecipitates of apoB they may be important metabolically. Recent studies suggest that apoB degradation may occur early in the endo- plasmic reticulum (46) and degradation of freshly translated apoB and its stimulation by insulin may regulate the propor- tion of apoB which enters the secretory pathway.

Although apoB degradation is a potential hypothesis to explain the lack of apoB recovery, an alternative hypothesis is that ‘S-labeled apoBu may have served as a precursor to 35S-labeled apoBL. This is unlikely as disappearance curves of apoBn and apoBL from the cell are not consistent with a precursor-product relationship. In addition, a significant por- tion of 35S-labeled-apoBr, is also degraded indicating that the process is not selective. Furthermore, the mechanism for generation of apoBu and apoBL in rat liver is not believed to be a result of proteolytic cleavage of apoBn but rather the translation from two distinct mRNAs (15, 16).

Triglyceride but not apoB accumulates within insulin- treated hepatocytes (26-30) coincident to enhanced apoB degradation now reported. Our results suggest that insulin may prevent or uncouple lipid assembly with apoB rendering the unassembled and presumably membrane-bound nascent apoB polypeptide chain more susceptible to degradation in the endoplasmic reticulum. Alternatively, insulin action may directly target apoB for degradation. A protein phosphoryla- tion-dephosphorylation mechanism is an obvious possibility considering the known role of insulin in the dephosphoryla- tion of regulatory enzymes in intermediary metabolism and in the activation of cellular protein kinases (47). In this context, rat apoB has been shown to be phosphorylated on serine (40, 48) and tyrosine residues (40) and vanadate, a phosphotyrosine phosphatase inhibitor, can mimic insulin action by inhibiting apoB secretion by primary cultures of rat hepatocytes (32). Moreover, in primary cultures of hepato- cytes from streptozotocin-induced diabetic rats, apoB secre- tion and cellular content of apoB is markedly reduced (19) and more highly phosphorylated forms of apoB may be pres- ent (40). The extent to which insulin alters the phosphoryla- tion state of apoB and/or its fragments is currently being investigated.

In pulse-chase 2 studies where hepatocyte apoB is prela- beled, the presence of insulin in the chase medium has little effect on the 3 h accumulation of ?S-labeled apoBL and apoBn in the medium. The time course of secretion of labeled rat apoBn was similar to that of prelabeled cellular apoB-100 by HepG2 cells (38). In both studies, the secretion of labeled higher molecular weight apoB plateaued by 1 h. In rat hepa- tocytes, which synthesize both the higher and lower molecular weight forms of apoB, the secretion of 35S-labeled apoBL continues to be secreted even as long as 5 h after the start of the chase period. By the 3rd h the apoB secretory rate (2-3- h interval) as estimated by monoclonal immunoassay is sig- nificantly inhibited by the presence of insulin in the chase medium. Lack of synchrony between “S-labeled apoB secre- tion and immunoassayable apoB reflects the contribution of newly synthesized apoB to the system. This is because during the chase period newly synthesized and secreted apoB is unlabeled due to the large molar excess of methionine in the chase medium whereas both labeled and unlabeled apoB are detectable by immunoassay. We have interpreted the lack of synchronous secretion of label and mass to be an indication of the predominant effect of insulin on newly synthesized apoB (unlabeled apoB) compared with a small effect of insulin on the secretion of prelabeled cellular apoB. Consistent with this interpretation is the implication that the hepatic pool of apoB (mostly apoBL) is available for lipoprotein secretion and

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Insulin Effects on Rat Hepatocyte ApoB 8861

is not affected acutely by the presence of insulin in the medium.

The results of the current study are compatible with the presence of a cellular pool of apoBL. Several lines of evidence support this finding. First, in pulse-chase 1 studies, 44% of newly synthesized ““S-labeled apoBL in control hepatocytes and 32% in insulin-treated hepatocytes is still present within hepatocytes after 180 min of incubation in chase medium. Second, in overnight labeling studies (pulse-chase 2) the ratio of cellular “‘S-labeled apoBL to ““S-labeled apoBn is 5.3 to 1. The calculated molar ratio of apoBL to apoBn (based on reported molecular weights) is on the order of 11 to 1, which supports the idea that apoBL forms a larger pool than apoBn. Furthermore, in pulse-chase 2 studies ““S-labeled apoBL con- tinues to be secreted over 5 h compared with 35S-labeled apoBn which is rapidly secreted. Consistent with the presence of a cellular apoBL pool in liver are studies by Swift et al. (49) who demonstrated the differential labeling of plasma very low density lipoprotein apoBL and apoBu in pulse-labeling studies using rat liver perfusions.

If the reduction in apoB secretion by insulin were due to simple inhibition of the apoB secretory pathway the apoB content of hepatocytes would theoretically increase with con- comitant reduction of apoB secretion. Previous studies from our laboratory suggested that cellular apoB was somewhat reduced (27) or relatively unchanged (32) by insulin in the medium. These previous results were consistent with the studies of Patsch et al. (26) in that the total apoB in the system (medium plus cells) was reduced with insulin treat- ment. Considering that the current study demonstrates that insulin in the medium leads to a 48% decrease in label incorporation into total apoB and a substantial increase in apoB degradation, we were interested in determining whether cellular apoB levels were altered. In more rigorously controlled experiments using eight liver preparations and multiple dishes of hepatocytes we were able to show that there is a 20% reduction in cellular apoB with insulin using a monoclonal antibody which has been shown to react to an epitope on both apoBn and apoBL (36). It is surprising to observe such a small reduction in the cellular apoB pool with insulin considering the magnitude of the alterations in both synthesis and deg- radation; however, we do not know the factors that regulate entry into or exit out of the cellular pool.

If the amino acid sequence of apoBL and the amino-terminal portion of apoBn are identical, what accounts for differences in protein movement through the cell? What signal allows apoBu to be rapidly secreted and/or degraded and apoBL to be delayed in secretion? Differential rates of secretion of various hepatic secretory proteins have been reported (39,43, 50) which have been attributed to the variability in rate of transport from the endoplasmic reticulum to the Golgi (50) and to differences in retention of specific proteins within the Golgi (39). Specific transport receptors and conversely specific retention signals have been postulated mechanisms to explain differences in protein transport through the cell. The current study implicates the carboxyl-terminal domain of apoBn as important in intracellular transport. ApoBi,, which lacks this domain is not as rapidly transported as apoBn and forms the majority of cellular apoB. The nature of the signals for specific transport and degradation is the subject of ongoing studies.

In summary, our results demonstrate that insulin inhibits hepatic synthesis of apoBn and apoBL while stimulating in- tracellular degradation of apoB, a process which favors apoBn. The hepatic pool of apoB, which is predominantly apoBL, is relatively resistant to insulin and continues to be secreted. The overall result is a reduction in apoB-containing lipopro-

tein secretion with secretion of particles enriched in apoBL. We speculate that under insulin stimulation as in the post- prandial state the rat liver secretes lipoproteins more similar in composition to intestinal lipoproteins. The significance of this effect lies in differences in turnover of hepatically syn- thesized apoBi,-containing lipoproteins compared with apoBn since the initial half-life of rat hepatic apoB1-containing lipoproteins is very rapid and plasma residence time is very short (51). Considering that there is also a reduction in number of particles secreted an additional effect of insulin is to minimize competition with clearance pathways common to hepatic and intestinal triglyceride-rich lipoproteins in the post-prandial period.

Acknowledgments-We gratefully acknowledge Joanne F. Cianci, Mary Bolognino, and Cindi L. Swicegood for their excellent technical assistance.

REFERENCES

5.

6.

7.

a.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Gotto, A. M., Levy, R. I., John, K., and Fredrickson, D. S. (1971) N. Engl. J. Med. 284, 813-818

Malloy, M. J., and Kane, J. P. (1982) Med. Clin. N. Amer. 66, 469-484

Sparks, C. E., and Marsh, J. B. (1981) J. Lipid Res. 22, 519-527 Krishnaiah, K. V., Walker, L. F., Borensztajn, J., Schonfeld, G.,

and Getz. G. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3806- 3810

Elovson, J., Huang, Y. O., Baker, N., and Kannan, R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78. 157-161

Kane, J. P., Hardman, D. A., and Paulus, H. E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2465-2469

Rash, J. M.. Rothblat, G. H., and Sparks, C. E. (1981) Biochim. Biophys. Acta 666, 294-298

Edee. S. B.. Hoea. J. M.. Schneider. P. D.. and Brewer. H. B.. Jr. (yi85) Metabocsm 34; 726-730 ’ ’

I I

Sparks, C. E., Hnatiuk, O., and Marsh, J. B. (1981) Can. J. Biochem. 59,693-699

Blackhart, B. D., Ludwig, E. M., Pierotti, V. R., Caiati, L., Onasch, M. A., Wallis, S. C., Powell, L., Pease, R., Knott, T. J.. Chu. M.-L.. Mahlev. R. W.. Scott. J.. McCarthv. B. J.. and Levy-Wilson, &. (1986) J. Bioi. Chem. 261, 15364Li5367

Higuchi, K., Monge, J. C., Lee, N., Law, S. W., and Brewer, H. B., Jr. (1987) Biochem. Biophvs. Res. Commun. 144, 1332- 1339

. -

Chen, S.-H., Habib, G., Yang, C.-Y., Gu,Z.-W., Lee,B. R., Weng, S.-A., Silberman, S. R., Cai, S.-J., Deslmere, J. P., Rosseneu. M., Gotto, A. M:, Jr., Li, W.-H., and &an, L. (1987) Science 238,363-366

Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 56,831-840

Hosaattankar. A. V.. Hiauchi. K.. Law. S. W.. Meelin. N.. and Brewer, H. ‘B., Jr: (1687) kochem. ‘Biophyk R& kom’mun. 148,279-285

Reuben, M. A., Svenson, K. L., Doolittle, M. H., Johnson, D. F., Lusis, A. J., and Elovson, J. (1988) J. Lipid Res. 29, 1337-1347

Tennyson, G. E., Sabatos, C. A., Higuchi, K., Meglin, N., and Brewer, H. B., Jr. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 500-504

Marsh, J. B., and Sparks, C. E. (1982) Proc. Sot. Exp. Biol. Med. 170, 178-181

Davis, R. A., Boogaerts, J. R., Borchardt, R. A., Malone-McNeal, M., and Archambault-Schexnavder. J. (1985) J. Biol. Chem. 260,14137-14144

”

Sparks, J. D., Sparks, C. E., Bolognino, M., Roncone, A. M., Jackson, T. K., and Amatruda, J. M. (1988) J. Clin. Inuest. 82, 37-43

Sparks, J. D., Sparks, C. E., and Miller, L. L. (1989) Biochem. J. 261,83-88

Boogaerts, J. R., Malone-McNeal, M., Archambault-Schexnay- der, J., and Davis, R. A. (1984) Am. J. Physiol. 246, E77-E83

Imaizumi, K., Lu, Y.-F., Sugano, M. (1985) Biochim. Biophys. Acta 837,345-348

Coleman, R. A., Haynes, E. B., Sand, T. M., and Davis, R. A. (1988) J. Lipid Res. 29, 33-42

Davidson, N. O., Powell, L. M., Wallis, S. C., and Scott, J. (1988)

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

8862 Insulin Effects on Rat Hepatocyte ApoB

25.

26.

27.

28.

29. 30.

31.

32.

33.

34.

35. 36.

37.

J. Biol. Chem. 263, 13482-13485 Davidson, N. O., Carlos, R. C., Drewek, M. J., and Parmer, T. G.

(1988) J. Lipid. Res. 29. 1511-1522 Patsch, W., Franz, S., and Schonfeld, G. (1983) J. Clin. Invest,

Patsch, W., Gotto, A. M., Jr., and Patsch, J. R. (1986) J. Biol.

Sparks, C. E., Sparks, J. D., Bolognino, M., Salhanick, A.,

Chem. 261, 9603-9606

71,1161-1174

Strumph, P. S., and Amatruda, J. M. (1986) Metabolism 35, 1128-1136

Dashti, N., and Wolfbauer, G. (1987) J. Lipid Res. 28, 423-436 Pullinger, C. R., North, J. D., Teng, B.-B., Rifici, V. A., Ronhild

de Brito, A. E., and Scott, J. (1989) J. Lipid Res. 30, 1065- 1077

Durrington, P. N., Newton, R. S., Weinstein, D. B., and Stein- berg, D. (1982) J. Clin. Inuest. 70, 63-73

Jackson, T. K., Salhanick, A. I., Sparks, J. D., Sparks, C. E., Bolognino, M., and Amatruda, J. M. (1988) Diabetes 37, 1234- 1240

Markwell, M. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. (1978) Anal. Biochem. 87, 206-210

Lux, S. E., John, K. M., and Brewer, H. B., Jr. (1972) J. Biol. Chem. 247, 7510-7518

Sparks, C. E., and Marsh, J. B. (1981) J. Lipid Res. 22, 514-518 Sparks, J. D., Bolognino, M., Trax, P. A., and Sparks, C. E.

(1986) Atherosclerosis 61, 205-211 Wettesten, M., Bostrom, K., Bondjers, G., Jarfeldt, M., Norfeldt,

P.-I., Carrella, M., Wiklund, O., Borbn, J., and Olofsson, S.-O. (1985) Eur. J. Biochem. 149, 461-466

38. Bostrom, K., Wettesten, M., Boren, J., Bondjers, G., Wiklund,

Biol. Chem. 260,7896-7902

O., and Olofsson, S.-O. (1986) J. Biol. Chem. 261,13800-13806 39. Yeo. K.-T., Parent, J. B., Yeo, T.-K., and Olden, K. (1985) J.

40. Sparks. J. D.. Soarks. C. E.. Roncone. A. M.. and Amatruda. J. -M. (1988) b. kol. dhem. i63,5001-5004

41. Williams, D. L., and Dawson, P. A. (1986) Methods Enzymol. 129,254-271

42. Borchardt, R. A., and Davis, R. A. (1987) J. Biol. Chem. 262, 16394-16402

43. Bamberger, M. J., and Lane, M. D. (1988) J. Biol. Chem. 263, 11868-11878

44. Dashti, N., Alaupovic, P., and Williams, D. L. (1988) Circulation 78, II-96

45. Suita-Mangano. P., Howard, S. C., Lennarz. W. J.. and Lane, M. D. (1982f5. I&01.’ Chem. 257,4292-4300

46. Davis. R. A.. Prewett. A. B.. Chan. D. C. F.. Thomnson. J. J.. Borchardt,‘R. A., and Gallaher, i. R. (1989) J. Lidid R&. 30; 1185-1196

47. Czech, M. P., Klarlund, J. K., Yagaloff, K. A., Bradford, A. P., and Lewis, R. E. (1988) J. Biol. Chem. 263,11017-11020

48. Davis, R. A., Clinton, G. M., Borchardt, R. A., Malone-McNeal, M., Tan, T., and Lattier, G. R. (1984) J. Biol. Chem. 259, 3383-3386

49. Swift, L. L., Padley, R. J., and Getz, G. S. (1987) J. Lipid Res. 28,207-215

50. Lodish, H. F., Kong, N., Snider, M., and Strous, G. J. A. M. (1983) Nature 304, SO-83

51. Sparks, C. E., Rader, D. J., and Marsh, J. B. (1983) J. Lipid Res. 24,156-166

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J D Sparks and C E Sparkshepatocytes.

Insulin modulation of hepatic synthesis and secretion of apolipoprotein B by rat

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