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

Insulin inhibition of apolipoprotein B (apoB) secretion by primary cultures of rat hepatocytes was investigated in pulse-chase experiments using [35S]methionine as label. Radioactivity incorporation into apoBH and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of detergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentration of insulin. Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media labeled apoB were analyzed; greater than 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 incorporation 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 apoBH with an intra-cellular retention half-time of 50 min (initial half-life of decay, t1/2 = 25 min) compared with 85 min in control (t1/2 = 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 labeled with [35S]methionine and then incubated in chase medium with and without insulin, labeled apoBH 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 immunoassayable 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, primarily apoBL, exists that is largely unaffected by insulin. Overall, insulin action in primary hepatocyte cultures reduces the secretion of freshly synthesized apoB and favors secretion of preformed apoB enriched in apoBL.


Insulin
inhibition of apolipoprotein B (apoB) secretion by primary cultures of rat hepatocytes was investigated in pulse-chase experiments using [35S]methionine as label. Radioactivity incorporation into apoBa and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of detergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentration of insulin.
Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media labeled 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 incorporation 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 intracellular 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 insulintreated 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  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 mammalian 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)(4)(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 intestine (6,9). Human apoB-100 and apoB-48, corresponding to rat apoBH and apoBL, respectively, are products of a singlecopy 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 equivalent 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 streptozotocin-induced diabetes (19,20) and are increased in carbohydrate 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 indicate there is an insulin receptor-mediated pathway that regulates apoB secretion by rat liver (26)(27)(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)(27)(28)(29)31) and apoB secretion (26)(27)(28)(29)(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 treatment (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 incorporation 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 responsible 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.

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 collagen. 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). Cellular 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. 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 containing 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) containing 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 indicated 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 solubilized 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.
Labeled apoB was eluted in 150 pi of 0.0625 M Tris-HCl buffer. PH 6.8. containing &?% (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 "Slabeled apoB was radioassayed and an aliquot was applied to a 3.5-24% AcrylaideTM/acrylamide gradient gel cast on GelBondTM PAGE 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 fluorographs, each lane was scanned three times and the percent distribution was determined using an automatic integrating densitometer ( 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 containing 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 incorporated 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 insulintreated hepatocytes. As shown in Table II, hepatocytes incubated for 12-14 h in medium containing 10 nM insulin incorporated 43% less label into total apoB, 58% less label into apoBh, and 34% less label into apoBL than hepatocytes incubated in control medium. trichloroacetic acid-soluble radioactivity during the 10-20min 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 Dawson (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 insulintreated 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 retention half-times for apoBH and apoBL were calculated as described by Yeo et al. (39)   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 halflife was calculated as described by Borchardt and Davis (42). 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 'YSlabeled 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, respectively; n = 4 rat livers). These results indicate that insulin in the medium stimulated the disappearance of newly synthesized 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 cellassociated (n = 4 rat livers). The intracellular retention halftime 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 initial decay of apoBL in control and insulin-treated hepatocytes were similar (Fig. 4)  min was not markedly altered by washing hepatocyte monolayers with medium containing heparin (300 units/ml) or human low density lipoprotein (100 pg/ml) prior to the solubilization 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 '"'Slabeled 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 hepatocytes an average of 43% of ""S-labeled apoBH and 57% of were pulse-labeled and chased as described in the !.egend to Fig. 1. Cell apoB radioactivity is the amount of immunoprecipitated ""Slabeled 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. Calculation 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 effects 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 studies 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 "Slabeled apoBI. in medium paralleled that of control hepatocytes. 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 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 control 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 precursor in insulin-treated cells and that both apoBH and apoBL are derived from the same labeled amino acid pool. 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. 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. Assuming the mechanism of insulin inhibition of apoB secretion in rat hepatocytes is similar to that recently described in HepG2 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 ?Slabeled 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 calculation 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 35Slabeled 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 hepatocytes.
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 hepatocytes 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 synthesized apoBL enters a presecretory pool. Movement of freshly synthesized apoBL through the cell is not dramatically affected 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 incubated in medium containing 10 nM insulin only 43% of 35Slabeled apoBn and 57% of ""S-labeled apoBL were recovered. Low apoB recoveries in pulse-labeling studies were also obtained 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 cellular 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 endoplasmic reticulum (46) and degradation of freshly translated apoB and its stimulation by insulin may regulate the proportion 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 portion 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 insulintreated 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 phosphorylation-dephosphorylation mechanism is an obvious possibility considering the known role of insulin in the dephosphorylation 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 hepatocytes from streptozotocin-induced diabetic rats, apoB secretion and cellular content of apoB is markedly reduced (19) and more highly phosphorylated forms of apoB may be present (40). The extent to which insulin alters the phosphorylation state of apoB and/or its fragments is currently being investigated.
In pulse-chase 2 studies where hepatocyte apoB is prelabeled, 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 hepatocytes, 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 (2-3h interval) as estimated by monoclonal immunoassay is significantly inhibited by the presence of insulin in the chase medium. Lack of synchrony between "S-labeled apoB secretion 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 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 continues 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 concomitant 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 treatment. 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 degradation; 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 intracellular 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 postprandial 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 synthesized 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.