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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1481-1491

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1481-1491.)
© 1995 American Heart Association, Inc.

Articles

Effects of Dexamethasone on the Synthesis, Degradation, and Secretion of Apolipoprotein B in Cultured Rat Hepatocytes

Chuen-Neu Wang; Roger S. McLeod; Zemin Yao; David N. Brindley

From the Department of Biochemistry and the Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Alberta, Canada.

Correspondence to Dr D.N. Brindley, Lipid and Lipoprotein Research Group, 328 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada, T6G 2S2.


*    Abstract
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Abstract Oversecretion of apoB and decreased removal of apoB-containing lipoproteins by the liver results in hyperapobetalipoproteinemia, which is a risk factor for atherosclerosis. We investigated how dexamethasone, a synthetic glucocorticoid, affects the synthesis, degradation, and secretion of apoB-100 and apoB-48. Primary rat hepatocytes were incubated with dexamethasone for 16 hours. Incorporation of [35S]methionine into apoB-48 and apoB-100 was increased by 36% and 50%, respectively, with 10 nmol/L dexamethasone, despite a 28% decrease of incorporation into total cell proteins. However, Northern blot analysis revealed that dexamethasone (1 to 1000 nmol/L) did not significantly alter the steady-state concentrations of apoB mRNA, suggesting that the net increase in apoB synthesis may involve increased translational efficiency. The intracellular retention and the rate and efficiency of apoB secretion were determined by pulse-chase experiments in which the hepatocytes were labeled with [35S]methionine for 10 minutes or 1 hour, and the disappearance of labeled apoB from the cells and its accumulation in the medium were monitored. Degradation of labeled apoB-100 after a 3-hour chase in both protocols was decreased from about 50% to 30%, whereas degradation of apoB-48 was decreased from 30% to 10% to 20% by treatment with 10 or 100 nmol/L dexamethasone. Additionally, the half-life of decay (time required for 50% of labeled cell apoB-100 to disappear from the peak of radioactivity following a 10-minute pulse) was increased by treatment with 10 nmol/L dexamethasone from 77 to 112 minutes, and the value for apoB-48 increased from 145 to 250 minutes. Treatment with 100 nmol/L dexamethasone also stimulated secretion of 35S-labeled apoB-100 and apoB-48 by twofold and 1.5-fold, respectively. The increased secretion of apoB-100 and apoB-48 after dexamethasone treatment was confirmed by immunoblot analysis for apoB mass, and the effect was relatively specific since albumin secretion was not significantly changed. We conclude that glucocorticoids promote the secretion of hepatic apoB-containing lipoproteins by increasing the net synthesis of apoB-100 and apoB-48 and by decreasing the intracellular degradation of newly synthesized apoB. An increased action of glucocorticoids coupled with a decreased ability of insulin to suppress these effects in insulin resistance can lead to hyperapobetalipoproteinemia and an increased risk of atherosclerosis.


Key Words: apoB • glucocorticoids • metabolic syndrome • hyperapobetalipoproteinemia • VLDL


*    Introduction
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 *Introduction
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The combined condition of insulin resistance, type II diabetes, android (visceral) obesity, hypertension, and dyslipidemia is often referred to as syndrome X or the metabolic syndrome. This condition represents a powerful association of risk factors for premature atherosclerosis, coronary thrombosis, and stroke.1 2 3 4 The dyslipidemia is characterized by hypertriglyceridemia and low concentrations of HDL. Serum cholesterol concentrations need not necessarily be raised since the LDL are often small and dense. However, hyperapobetalipoproteinemia often occurs, indicating an increased number of potentially atherogenic particles.4 This increase can arise from the oversecretion of VLDL and/or a decreased removal of apoB-containing particles from the circulation; this article concentrates on the regulation of apoB production. ApoB plays a central role in the assembly, secretion, and metabolism of chylomicrons and VLDL and the degradation of LDL.5 There are two forms of apoB in mammals: the larger form, apoB-100, consists of 4536 amino acids; the smaller form, apoB-48, is the amino terminal 48% of apoB-100. ApoB-48 is produced by editing of the apoB-100 transcript; both apolipoproteins are products of the same gene. This process occurs in the intestine, where only apoB-48 is synthesized. Although most mammalian livers produce only apoB-100, rat liver synthesizes both apoB-48 and apoB-100.5

The hepatic production of apoB-containing lipoproteins is subject to hormonal modulation. Insulin decreases apoB secretion by increasing intracellular degradation.6 7 Thyroid hormones regulate apoB secretion by modulating apoB mRNA editing and transcription.8 9 10 Growth hormone decreases apoB-100 synthesis but has no effect on apoB editing in control and hypothyroid rats.11 Hypophysectomy of rats decreases the proportion of apoB that is edited in intact liver and hepatocytes.12 Treatment of the rats with growth hormone alone or combined with tetraiodothyronine and cortisol increases the proportion of apoB editing to that observed in control intact rats. However, experiments with isolated hepatocytes show that a combination of tetraiodothyronine and cortisol with growth hormone is required to increase the proportion of apoB-48. Sjöberg et al12 conclude that there is a complex interaction between growth hormone and thyroid hormones that may explain the discrepancies between the observed effects with intact liver versus isolated hepatocytes. They also suggest that growth hormone may affect apoB editing through effects on triacylglycerol synthesis.12 Glucocorticoids stimulate VLDL secretion in vivo,13 14 15 perfused liver,16 17 and isolated hepatocytes.18 19 20 21 22 Most of these studies have relied on measurements of the lipid components of VLDL, particularly triacylglycerol. Incubation of rat hepatocytes with the synthetic glucocorticoid dexamethasone also stimulates the secretion of apoB by up to fourfold compared with only a 1.5-fold increase in the secretion of triacylglycerol.21 Therefore, dexamethasone causes the hepatocytes to secrete a larger number of smaller VLDL particles.21

In addition to stimulating secretion of hepatic apoB-containing lipoproteins, glucocorticoids also decrease the expression of hepatic LDL receptors.22 These effects of glucocorticoids on the secretion of VLDL and the uptake and degradation of LDL are antagonized by high concentrations of insulin.18 21 22 Therefore, an increase in cortisol production or sensitivity to cortisol together with insulin insensitivity can increase the secretion of apoB and decrease the removal of IDL and LDL. These combined effects can potentially cause hyperapobetalipoproteinemia, which is a risk factor for atherosclerosis in the metabolic syndrome and in conditions of metabolic stress.23 24

The purpose of the present investigation was to determine the mechanisms by which glucocorticoids modify the secretion of apoB in cultured rat hepatocytes. The rate of secretion of hepatic apoB-containing lipoproteins may be determined at multiple levels,25 26 27 including apoB gene transcription,9 28 29 apoB mRNA translation,29 30 apoB polypeptide translocation across the membrane of the endoplasmic reticulum,31 and apoB-lipid assembly.32 33 34 35 In addition, intracellular degradation of newly synthesized apoB plays a major role in controlling its secretion.6 36 37 38 39 In this study, we demonstrate for the first time that incubation of rat hepatocytes with the glucocorticoid dexamethasone stimulates the net synthesis of apoB-100 and apoB-48 and also decreases their intracellular degradation. These changes enable more apoB-100 and apoB-48 to be secreted from the hepatocytes.


*    Methods
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[35S]Methionine (Tran35S-label) was purchased from ICN. Sheep polyclonal antibodies for human apoB and apoA-I were from Boehringer Mannheim. Protein A conjugated to Sepharose CL 4B, fatty acid–poor bovine serum albumin, and Triton X-100 were from Sigma. All reagents for electrophoresis were from Bio-Rad Laboratories. Leibovitz L-15 medium and newborn calf serum were from GIBCO Laboratories. The enhanced chemiluminescence detection reagents and conjugated anti-rabbit IgG-horseradish peroxidase for immunoblotting were obtained from Amersham. Rabbit polyclonal antibodies directed against rat apoB were provided by Dr R. Davis (San Diego State University, Calif). A rat apoB cDNA fragment (nucleotides 6280 through 6940) was a gift from Dr J. Scott (Royal Postgraduate Medical School, London, England). The cDNA probe for human glyceraldehyde-3-phosphate dehydrogenase was purchased from Clontech Laboratories. Anti-rat albumin antibody was a gift from Dr D.E. Vance (University of Alberta). All other reagents were purchased from Sigma or Fisher Scientific.

Preparation and Treatment of Hepatocytes
Hepatocytes were prepared from male Sprague-Dawley rats (200 to 300 g) by collagenase perfusion.21 40 Approximately 8x105 cells were plated onto 35-mm collagen-coated culture dishes in modified Leibovitz L-15 medium containing 10% (vol/vol) newborn calf serum and 4 mg choline chloride/L. Cells were incubated for 1 hour at 37°C, after which the unattached and nonviable cells were removed. Adherent hepatocytes were incubated 4 more hours in the same medium and then placed in Leibovitz L-15 medium containing 0.2% (wt/vol) fatty acid–poor bovine serum albumin, 4 mg choline chloride/L, and the appropriate concentration of dexamethasone. Unless otherwise indicated, cells were exposed to dexamethasone for 16 hours.

Extraction and Analysis of mRNA
Total cell RNA was isolated from hepatocytes by using the acid guanidinium thiocyanate–phenol-chloroform extraction method,41 fractionated on 1% agarose gels containing 0.67% (vol/vol) formaldehyde, and transferred to nylon membranes.42 The hybridization with cDNA probes and the washing conditions have been described.9 The concentration of apoB mRNA was determined relative to that of glyceraldehyde-3-phosphate dehydrogenase by scanning densitometry.

Labeling of Cells
After the dexamethasone treatment the cells were washed twice with methionine-free Leibovitz medium (labeling medium) and incubated for 40 minutes in the same medium to deplete the intracellular methionine pool. Preincubation and pulse-chase dexamethasone concentrations were the same. To examine protein biosynthesis, the hepatocytes were labeled for 5 to 20 minutes in 0.5 mL labeling medium containing 200 µCi [35S]methionine. At the end of the labeling period, cells were recovered for further analysis as described below. In studies of secretion and degradation hepatocytes were pulse labeled for 10 minutes, after which the medium was removed, and the cells were washed twice with Leibovitz medium containing 10 mmol/L L-methionine and 3 mmol/L L-cysteine (chase medium). Chase medium (1 mL) was then added, and the cells were incubated at 37°C for the times indicated in the legends to Figs 5 through 7DownDownDown. In other pulse-chase experiments (Fig 5Down and TableDown) cells were labeled for 60 minutes with 200 µCi [35S]methionine, washed twice with chase medium, and incubated for 40 minutes before adding fresh chase medium and beginning the chase. At each time point during the chase, cells and media were recovered for analysis as described below.



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  		Figure 5. Line graphs showing pulse-chase analysis of the turnover of apoB-100 and apoB-48. Hepatocytes were incubated in the absence ({circ}) or presence ({bullet}) of 10 nmol/L dexamethasone for 16 hours and then pulse labeled with [35S]methionine (200 µCi/35-mm dish) for 10 minutes. Medium was removed, and hepatocytes were reincubated in medium containing 10 mmol/L L-methionine and 3 mmol/L L-cysteine (chase medium) for the times indicated. Total apoB radioactivities (E and F) represent the summation of that in cells (A and B) and media (C and D). Results are mean values from three independent experiments. P<.01 by ANOVA for all results except medium apoB-48.



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  		Figure 6. Fluorographs of production and secretion of apoB-100 and apoB-48 by rat hepatocytes. Rat hepatocytes were incubated for 16 hours in the presence or absence of 10 nmol/L dexamethasone as indicated. A, Lanes I and III show intracellular apoB-100 and apoB-48 in the hepatocytes after a 10-minute incubation with [35S]methionine as described in the legend to Fig 5Up. Lanes II and IV show results after labeling the cells for 10 minutes and then performing a 10-minute chase (see "Methods"). B, ApoB-100 and apoB-48 secreted into medium from cells that were pulse labeled for 10 minutes with [35S]methionine and chased for the times indicated.



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  		Figure 7. Plots showing disappearance kinetics of apoB labeled with [35S]methionine from hepatocytes in pulse-chase experiments. Curves represent the disappearance of apoB from control ({circ}) and dexamethasone-treated ({bullet}) cells by using the individual results from Fig 5Up. The relative recoveries of labeled apoB-100 and apoB-48 are expressed as percentages of the maximum incorporation of [35S]methionine, which was attained after 10 or 20 minutes in different experiments; this time point is taken as time zero. A polynomial function was used to define the curves; the r2 values for the fit of the experimental points to the function were .96 for apoB-100 and .87 for apoB-48 in the control cells and .95 for apoB-100 and .91 for apoB-48 in cells treated with 10 nmol/L dexamethasone.


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  		Table 1. Effect of Dexamethasone Concentration on the Distribution of 35S-Labeled ApoB-100 and ApoB-48

Imunoprecipitation and Immunoblotting
Hepatocyte monolayers from continuous labeling or pulse-chase experiments were washed twice with ice-cold phosphate-buffered saline. Cells were solubilized immediately by adding 0.2 mL hot (80°C to 90°C) lysis buffer (0.05 mol/L Tris-HCl, pH 8.0, 0.15 mol/L NaCl, 0.015% [wt/vol] phenylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 1% [wt/vol] sodium deoxycholate, and 1% [vol/vol] Triton X-100) containing 1% (wt/vol) sodium dodecyl sulfate. Medium was collected and centrifuged at 10 000g for 10 minutes to remove cell debris. Samples of the medium (0.9 mL) were combined with 0.1 mL 10x lysis buffer containing 1% (wt/vol) sodium dodecyl sulfate. Cell lysates were incubated at 70°C for 15 minutes to ensure complete cell lysis and diluted with lysis buffer to achieve a sodium dodecyl sulfate concentration of 0.1% (wt/vol). Immunoprecipitation of apoB and apoA-I (using sheep polyclonal antibodies) and immunoblot analysis of apoB (using rabbit polyclonal antibodies) and rat serum albumin were performed.43 Immunoprecipitated samples were separated on 3% to 15% polyacrylamide gradient gels and visualized by fluorography. Apolipoprotein radioactivity was measured by liquid scintillation counting after solubilizing the gel with hydrogen peroxide and perchloric acid.35 Changes in apoB and rat albumin mass in cells and medium were determined by immunoblot analysis by using chemiluminescence detection and scanning densitometry. Multiple exposures of the blots were performed and quantified within the linear range of the film. The densitometric changes reflected the mass of apoB-100, apoB-48, and albumin as verified by multiple dilutions of the cell lysate and medium samples. Differences within a 10-fold range could be detected at a single exposure.

Other Methods
Protein was determined by using the bicinchoninic acid (Pierce Chemical Co) method with bovine serum albumin as the standard. The incorporation of [35S]methionine into total cell proteins and the activity of lactate dehydrogenase were determined.21 Results were analyzed by ANOVA with post hoc multiple comparison with a Bonferroni test.


*    Results
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 *Results
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Dexamethasone Stimulates Net Incorporation of [35S]Methionine Into ApoB
The effect on apoB synthesis of pretreating rat hepatocytes with dexamethasone was determined by measuring the rate of [35S]methionine incorporation. After a 16-hour incubation with 10 nmol/L dexamethasone, the initial rate of the net incorporation of [35S]methionine into apoB-100 (Fig 1ADown) and apoB-48 (Fig 1BDown) was significantly greater than in untreated cells. Dexamethasone increased the labeling of apoB-100 and apoB-48 by about twofold and 1.5-fold, respectively, after 20 minutes. Secretion of the labeled apoB into the medium was not detected during this time, and therefore the rate of [35S]methionine incorporation reflects the net synthetic rate. In contrast to apoB, the rate of [35S]methionine incorporation into apoA-I was not affected significantly by treatment with 10 nmol/L dexamethasone (Fig 1CDown).



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  		Figure 1. Line graphs showing effect of dexamethasone on the synthesis of apoB-100, apoB-48, and apoA-I. Rat hepatocytes were pretreated in the absence ({circ}) or presence ({bullet}) of 10 nmol/L dexamethasone for 16 hours. Cells were incubated for 40 minutes in methionine-free medium and then labeled with [35S]methionine (200 µCi/35-mm dish) for the times indicated. Intracellular apoB-100, apoB-48, and apoA-I were immunoprecipitated with corresponding antibodies. Radioactivities associated with the apoBs and apoA-I were quantified after resolution of the immunoprecipitates on polyacrylamide gels. Results are mean±SD (when large enough to be shown) from three independent experiments. ***P<=.001 control vs dexamethasone-treated hepatocytes.

The time of preincubation required to demonstrate the effect of dexamethasone on apoB biosynthesis was examined by using a 10-minute labeling protocol. Dexamethasone had no significant effect on apoB-100 (Fig 2ADown) or apoB-48 (Fig 2BDown) biosynthesis at incubation times of 8 hours or less. However, an approximately twofold stimulation was observed after a 16-hour incubation for both apolipoproteins. The stimulatory effect continued for at least 36 hours of incubation, although there may have been some loss of synthetic capacity at 24 and 36 hours. All subsequent studies were therefore performed after the hepatocytes had been exposed to dexamethasone for only 16 hours.



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  		Figure 2. Bar graphs showing time course of dexamethasone on the incorporation of [35S]methionine into intracellular apoB-100 and apoB-48. Rat hepatocytes were incubated in control medium (open bars) or medium containing 10 nmol/L dexamethasone (solid bars) for 2 to 36 hours. At the times indicated the cells were labeled with [35S]methionine (200 µCi/35-mm dish) for 10 minutes, and radioactivities associated with the intracellular apoB-100 and apoB-48 were quantified as described in the legend to Fig 1Up. Results are from two independent experiments and are shown as mean±range when these were large enough to be indicated.

The concentration-dependent effects of dexamethasone were similar for apoB-100 (Fig 3ADown) and apoB-48 (Fig 3BDown). Dexamethasone at 10 nmol/L stimulated the net synthesis of apoB-100 by 50%. Significant stimulations of apoB-48 synthesis of 43% and 36% were obtained at 1 or 10 nmol/L dexamethasone, respectively. However, the effects of 100 nmol/L dexamethasone on the synthesis of apoB-100 or apoB-48 were more variable, reflecting the biphasic nature of the response. There were no significant effects of 100 or 1000 nmol/L dexamethasone on the synthesis of apoB-100 and apoB-48 (Fig 3ADown and 3BDown), although 100 nmol/L dexamethasone consistently stimulated secretion and decreased intracellular degradation of apoB (see below). The effect of dexamethasone was specific for the apoB proteins since analysis of apoA-I biosynthesis showed a distinctly different dexamethasone concentration dependence. Dexamethasone (1 nmol/L) stimulated apoA-I biosynthesis by 43%, but the 10 to 1000 nmol/L concentrations had no significant effect (Figs 1CUp and 3CDown). This stimulation of apoA-I synthesis is probably related to an increase in mRNA for this protein that occurs after hepatocytes are treated with dexamethasone.44 However, 10 to 1000 nmol/L dexamethasone decreased [35S]methionine incorporation into total protein by 28% to 35% (Fig 3DDown). There was no detectable difference in cell protein mass in the hepatocytes during the experiment, and therefore this value was used to normalize the results and compensate for small differences in the number of hepatocytes per dish. There was also no significant release of lactate dehydrogenase (which would have indicated cell lysis) at any concentration of dexamethasone (results not shown).



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  		Figure 3. Bar graphs showing effect of dexamethasone concentration on the incorporation of [35S]methionine into intracellular apoB-100, apoB-48, apoA-I, and total protein after a 10-minute labeling. Experiments were performed essentially as described in the legend to Fig 2Up except that cells were pretreated with 1 to 1000 nmol/L of dexamethasone for 16 hours before labeling. Values are expressed as a percentage of the values for untreated cells. Results are mean±SD of 7 to 14 independent experiments. *P<=.05, **P<=.01, ***P<=.001 control vs dexamethasone-treated cells.

Northern blot analysis was used to examine the steady-state level of apoB mRNA in rat hepatocytes after a 16-hour incubation with dexamethasone. No significant differences in the level of apoB mRNA (normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA) were detected (Fig 4Down).



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  		Figure 4. Analysis of the mRNA for apoB and glyceraldehyde-3-phosphate dehydrogenase (G3P dehydrogenase). Hepatocytes were incubated for 16 hours in the absence or presence of dexamethasone. Total RNA was extracted, fractionated by agarose gel electrophoresis, and subjected to Northern blot analysis by using cDNA probes for apoB and glyceraldehyde-3-phosphate dehydrogenase. A, Representative autoradiograph of the Northern blots showing signals corresponding to apoB and glyceraldehyde-3-phosphate dehydrogenase. The mobilities of 18S and 28S RNA species are shown. Dexamethasone concentrations were 0, 1, 10, 100, and 1000 nmol/L in lanes 1, 2, 3, 4, and 5, respectively. B, Bar graph showing relative apoB mRNA concentrations (mean±SD from four independent experiments), which were calculated as arbitrary absorbance units normalized to the glyceraldehyde-3-phosphate dehydrogenase signal. Relative apoB mRNA abundance was then expressed as a percentage of the signal ratio obtained in control hepatocytes (100% in each experiment).

Dexamethasone Increases ApoB Secretion and Decreases Intracellular Degradation
Pulse-chase studies were performed to examine the postsynthetic fate of nascent apoB. Following a 10-minute pulse of [35S]methionine, radiolabeled apoB was recovered from the cells and medium at timed intervals. As expected from Figs 1 through 3UpUpUp, there was a greater incorporation of [35S]methionine into apoB-100 and apoB-48 at the beginning of the chase period in cells pretreated with dexamethasone. There was a 10- to 20-minute delay in reaching the peak of maximum incorporation of [35S]methionine into apoB in both control and dexamethasone-treated cells (Fig 5AUp and 5BUp). Fig 6AUp illustrates further the phenomenon of continued incorporation of radiolabeled precursor into both apoB-100 and apoB-48. The reason for the delay in attaining maximum incorporation into apoB is not clear; it may reflect the elongation of partially completed apoB polypeptides during the chase period or a pool of radiolabeled methionine that does not exchange readily with the exogenous methionine.

The radioactivity of cell apoB-100 (Fig 5AUp) and apoB-48 (Fig 5BUp) disappeared rapidly from untreated cells during the first 60 minutes of the chase period. In cells treated with 10 nmol/L dexamethasone, however, the intracellular retention times of both apoB-100 and apoB-48 were increased compared with those of untreated cells. For example, compared with the maximum incorporation the proportion of apoB-100 remaining in the cells at the 60-minute chase point was 65±10% for control cells and 83±1% for dexamethasone-treated cells (Fig 5AUp and 5BUp). The equivalent values for apoB-48 were 75±12% and 86±8%, respectively.

Cell apoB radioactivities (Fig 5AUp and 5BUp) were also used to determine the intracellular retention times for apoB-100 and apoB-48 by using polynomial analysis.6 This analysis (Fig 7AUp) established that the intracellular half-life of decay for apoB-100 (time required for 50% of labeled cell apoB to disappear from the peak of radioactivity that occurred at 10 or 20 minutes) was 112 minutes in treated cells compared with 77 minutes in untreated cells. Likewise, the half-life of decay of apoB-48 (Fig 7BUp) was prolonged from 145 to 250 minutes by dexamethasone treatment. This dexamethasone-induced increase in the amount of apoB in the hepatocytes could make more apoB available for secretion.

Following an initial lag period of 30 to 40 minutes, apoB-100 secretion was increased by about twofold by dexamethasone treatment at chase times longer than 1 hour (Fig 5CUp). Secretion of apoB-48 appeared to be increased by dexamethasone by approximately 1.5-fold at 3 hours (P=NS; Fig 5DUp). Fig 6BUp shows the representative fluorographs illustrating the stimulation of apoB-100 and apoB-48 secretion during the chase period. The increased retention of cell apoB and higher secretion of apoB mean that the total recovery of apoB was increased by dexamethasone treatment (Fig 5EUp and 5FUp). At the end of the 180-minute chase, 48±10% of apoB-100 radiolabel was recovered from the untreated dishes compared with 66±12% recovered from the treated dishes (mean±SD, n=3). Similarly, the recovery of radiolabel in apoB-48 in dexamethasone-treated dishes was 89±2% after 180 minutes compared with 73±8% in untreated cultures.

Since dexamethasone increased the net synthesis and decreased the degradation of apoB (Fig 5Up), the time required to reach maximum apoB labeling might differ in the presence and absence of the hormone. Consideration was given to the possibility that this difference may have resulted in an overestimation of the effects of dexamethasone in the kinetic analysis of the intracellular degradation of apoB. We therefore repeated the analysis by using a longer pulse period (60 minutes) before the chase. In these experiments, there was a 30- to 40-minute delay in reaching the maximum incorporation of [35S]methionine into apoB-100 and apoB-48 in treated and untreated cells (results not shown). Thus, we started the chase at 40 minutes after removal of the labeling medium and expressed the radioactivity associated with apoB at this time as 100% (Fig 8Down). This time course also revealed that there was no lag phase for the secretion of apoB (compare Fig 8CDown and 8DDown with Fig 5CUp and 5DUp). Results obtained from these longer labeling experiments support those from the 10-minute pulse-labeling experiments. The radiolabel in cell apoB-100 and apoB-48 disappeared more slowly from hepatocytes pretreated with 100 nmol/L dexamethasone than from control hepatocytes during the first 60 minutes of the chase (Fig 8ADown and 8BDown, respectively); relative apoB-100 and apoB-48 secretions were increased by about twofold and 1.5-fold, respectively, after a 180-minute chase (Fig 8CDown and 8DDown); and total apoB recovery was increased by dexamethasone treatment (Fig 8EDown and 8FDown).



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  		Figure 8. Line graphs showing effect of dexamethasone on the longer term retention and secretion of apoB-100 and apoB-48. Hepatocytes were incubated in the absence ({circ}) or presence ({bullet}) of 100 nmol/L dexamethasone for 16 hours. The cells were pulse labeled with [35S]methionine for 1 hour, and the chase began after washing the cells for 40 minutes with chase medium as described in "Methods." Results are given as a percentage of radioactivity in cell apoB at the beginning of the chase. The relative radioactivities of total apoB (E and F) were calculated from the summation of apoB in cells (A and B) and media (C and D). Results are mean±SD from four independent experiments when large enough to be shown. *P<=.05, **P<=.001, ***P<=.0001 control vs dexamethasone-treated cells.

Studies were performed to determine if the cellular retention and secretion of apoB were also dose dependent. The 60-minute labeling was followed by a 40-minute wash (Fig 8Up) and a 60-minute chase, and radioactivities associated with apoB at the end of the 40-minute wash were taken as the initial radioactivity (TableUp). Dexamethasone at 10 to 1000 nmol/L decreased the proportion of labeled apoB-100 that was degraded from about 47% to about 17% to 19%, and 10 or 100 nmol/L dexamethasone increased the proportion of apoB-100 that was secreted from 11% to 26%. A similar effect of dexamethasone on apoB-48 degradation was observed at 10 or 100 nmol/L (TableUp). The increased apoB secretion was related to decreased degradation and is probably a consequence of increased apoB availability.

In addition, 1 or 10 nmol/L dexamethasone increased the total amount of [35S]methionine in apoB-100 and apoB-48 in the cells (TableUp). This concentration-dependent effect of dexamethasone agrees well with that shown in Fig 3Up. The increased net synthesis of apoB could contribute to a stimulation in the absolute rather than relative secretion of 35S-labeled apoB. For example, incorporation of [35S]methionine into apoB-100 was increased at 10 nmol/L dexamethasone by 1.38-fold (8800 dpm in control cells versus 12 000 dpm in treated cells), and the relative secretion of apoB-100 was increased by 2.55-fold (11% and 28% in control and treated cells, respectively). Thus, there were on average 1.7-, 3.5-, 2.8-, and 1.4-fold increases in the absolute secretion of apoB-100 at 1, 10, 100, and 1000 nmol/L dexamethasone. The equivalent increases for apoB-48 were 1.6-, 1.9-, 1.3-, and 1.04-fold.

Changes in ApoB Radiolabel Reflect Changes in the Mass of ApoB in Cells and Medium
Changes in the steady-state mass of apoB in cells and secretion into the medium were determined by immunoblot analysis (Fig 9ADown); results from four independent experiments are summarized in Fig 9BDown. Dexamethasone (10 nmol/L) increased the mass of intracellular apoB-100 and apoB-48 by averages of 2.2- and 1.2-fold, respectively. However, these results did not reach significance in the four experiments because of the relatively large interexperimental variation. The relative lack of significant change for apoB-48 mass in the cells may result from an equilibrium between the increased synthesis and decreased degradation versus the increased secretion. As expected from the results reported in Fig 5Up and the TableUp, the secretions of apoB-100 and apoB-48 were increased significantly by 10 nmol/L dexamethasone. The average apparent increases in secretion for apoB-100 were 2.2-, 3.9-, 2.6-, and 1.5-fold at 1, 10, 100, and 1000 nmol/L dexamethasone, respectively. These changes in secretion for apoB-100 were similar to the increases of absolute secretion as calculated from the TableUp, which were 1.7-, 3.5-, 2.8-, and 1.4-fold, respectively, for apoB-100. The equivalent values for apoB-48 secretion were 1.2-, 1.5-, 1.3-, and 1.1-fold from Western blot analysis (Fig 9Down), and 1.6, 1.9-, 1.3-, and 1.04-fold from the long-term pulse-chase experiment (TableUp).



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  		Figure 9. Effect of dexamethasone concentration on the secreted and intracellular mass of apoB-100 and apoB-48. Rat hepatocytes were preincubated with 0 to 1000 nmol/L dexamethasone for 16 hours; the medium was replaced with equivalent medium for 4 hours, after which the cells and medium were collected. A, Immunoblots of cell and medium apoB (top) and albumin (bottom). Lanes 1 through 5 show results from cells treated with 0, 1, 10, 100, and 1000 nmol/L dexamethasone, respectively. B, Bar graphs showing relative mass of apoB-100 and apoB-48 in the cells and medium. Results are mean±SD of four independent experiments. *P<=.05, ***P<=.001 control vs dexamethasone-treated cells.

In contrast to the apoB mass levels, neither the mass of intracellular albumin nor the accumulation of albumin in the medium was changed significantly by 1 to 100 nmol/L dexamethasone (Fig 9AUp). These results agree with our previous work, in which dexamethasone alone did not alter the secretion of albumin labeled with [3H]leucine, whereas the secretion of apoB (as measured by immunotitration) was increased.21


*    Discussion
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 *Discussion
down arrowReferences
 
We analyzed the effects of dexamethasone on the steady-state level of apoB mRNA, the net rate of apoB synthesis, intracellular degradation of newly synthesized apoB, and the rate and efficiency of apoB secretion. Although dexamethasone increases the secretion of apoB from cultured primary rat hepatocytes,21 the underlying mechanisms have not been elucidated. Dexamethasone increased the net synthesis of apoB-100 and apoB-48 by up to twofold. This effect is specific since dexamethasone decreased the overall synthesis of proteins, a result that is consistent with the well-known effects of glucocorticoids in inhibiting protein synthesis and directing amino acids into gluconeogenesis.21 45 We do not know for certain why 100 and 1000 nmol/L dexamethasone failed to stimulate apoB synthesis, but it may result from alterations in the intracellular specific radioactivity of [35S]methionine resulting from changes in amino acid uptake or increased degradation of cell proteins. The increased rate of apoB synthesis at 1 or 10 nmol/L dexamethasone did not seem to result from an altered transcription rate, since the steady-state concentrations of the apoB mRNA did not change significantly. Pulse-chase experiments demonstrated that pretreatment of hepatocytes with dexamethasone decreased the intracellular degradation of both apoB-100 and apoB-48 and increased their secretion into the medium. These combined results demonstrate that glucocorticoids are important stimulators of the hepatic secretion of apoB-containing lipoproteins.

We used the synthetic glucocorticoid dexamethasone in our studies because this compound is much less susceptible to degradation compared with the natural hormones corticosterone and cortisol.21 The high stability of dexamethasone in hepatocyte cultures enabled us to measure long-term effects (16 to 36 hours) on hepatic apoB metabolism. Our technique for the culture of rat hepatocytes was chosen because the cells remain metabolically competent, retain lactate dehydrogenase (a marker for cell lysis), and exhibit stable rates of total protein turnover.46 47 Moreover, the system has been characterized extensively in terms of the production of lipoproteins and responsiveness to hormones.16 19 48 49 50 Incubation of hepatocytes with 1 to 100 nmol/L dexamethasone increases the secretion of VLDL into the medium,18 21 22 decreases the expression of LDL receptor,17 50 and enhances the activities of phosphatidate phosphohydrolase (a key enzyme in glycerolipid synthesis) and tyrosine aminotransferase (a regulatory enzyme in amino acid catabolism and gluconeogenesis).50 51 These results are compatible with the physiological effects of glucocorticoids in experimental animals and human beings and demonstrate that this culture system with rat hepatocytes is a suitable model for investigating the hormonal regulation of lipid and lipoprotein metabolism. The effects on apoB metabolism that are reported here have also been observed at 1 to 100 nmol/L dexamethasone, a concentration at which other physiologically relevant responses are observed. The concentration of the natural hormone, corticosterone, in rats is normally in the 50- to 2000-nmol/L range.51 52 We therefore consider that the effects of dexamethasone reported in this work are physiological rather than pharmacological in nature.

The lack of a significant change in the concentration of apoB mRNA in the hepatocytes after dexamethasone treatment is not surprising as relatively constant steady-state levels of the apoB message occur in various metabolic conditions under which apoB secretion can be altered by severalfold.53 However, small (less than twofold) changes in apoB mRNA concentrations have been found in HepG2 cells treated with 25-hydroxycholesterol29 or high concentrations of amino acids,54 but the physiological relevance of these changes in apoB mRNA levels remains to be established. We cannot exclude entirely the possibility of small increases (about 17%) in the concentration of apoB mRNA of hepatocytes treated with dexamethasone. Nevertheless, Inui et al55 report that treatment of rats with dexamethasone does not significantly affect apoB mRNA concentrations or the extent of apoB mRNA editing.

Other investigators have also reported changes in the rate of apoB synthesis in the absence of significant alterations in steady-state concentrations of apoB mRNA.6 17 30 Apparently the level of apoB mRNA does not always reflect the rate of apoB translation. Although little is known about the factors that may change the rate of translation of apoB mRNA, our results suggest that increased translation efficiency may contribute to the increase in apoB synthesis. One possible explanation for the dexamethasone-induced increase in apoB synthesis is that the interaction of apoB mRNA and the polysome complex is altered. Hepatic polysomes containing apoB mRNA have unusual sedimentation properties,56 and conceivably a critical high-order structure of the apoB mRNA is required for efficient translation. Dexamethasone may alter this structure, thus increasing translational efficiency without changing apoB mRNA concentrations. Furthermore, dexamethasone may decrease apoB degradation very early (<10 minutes), thus producing an increase in the net synthetic rate. Such an early decrease in degradation is compatible with the apparent increased labeling of apoB with longer incubation times. The mean stimulations in apoB-100 labeling were 1.3-, 1.6-, 1.6-, and 2.0-fold at incubation times of 5, 10, 15, and 20 minutes, respectively (Fig 1Up). The equivalent increases for apoB-48 were 1.2-, 1.4-, 1.3-, and 1.5-fold.

Changes in the rate of apoB degradation appear to be important in controlling its secretion.26 This degradation occurs in cultured rat hepatocytes,36 57 58 although under some experimental conditions it becomes marked only when the cells are stimulated with insulin.6 By using two independent pulse-chase protocols we have shown that pretreatment of hepatocytes with 10 or 100 nmol/L dexamethasone significantly decreases the degradation of apoB. This effect of dexamethasone in decreasing apoB degradation is relatively specific since glucocorticoids are well known to stimulate overall protein catabolism.45 The decreased degradation of apoB could also contribute to the increase in the intracellular half-lives of decay for apoB-100 and apoB-48 (Fig 7Up) and/or the retention of apoB in the secretory pathway. This latter effect is compatible with the observed increases in apoB secretion. It also follows that dexamethasone will produce a more pronounced effect on apoB secretion in those culture systems in which there is a marked basal degradation of apoB. Our combined results (Figs 1 through 3UpUpUp) also demonstrate that although 1 or 10 nmol/L dexamethasone can stimulate net apoB synthesis, concentrations of 10 nmol/L or greater are required to produce significant decreases in degradation (Figs 5 through 8UpUpUpUp, TableUp). Despite intensive study, the mechanism by which newly synthesized apoB proteins are degraded remains unclear. However, accumulating evidence suggests that apoB is sensitive to cysteine protease(s): administration of N-acetyl-leucyl-leucyl-norleucinal (an inhibitor of cysteine proteases) effectively inhibits degradation of apoB in HepG2 cells,59 rat hepatocytes treated with insulin,27 and Chinese hamster ovary cells expressing recombinant human apoB.60 The effect of dexamethasone in decreasing apoB degradation in primary rat hepatocytes results from a decreased activity of a cysteine protease or proteases (C.-N.W., D.N.B., unpublished data, 1995). Dexamethasone may either decrease the expression of this protease by modifying its turnover or induce the synthesis of factors that inhibit protease activity. These suggestions are compatible with the mechanisms of action for glucocorticoids, which regulate the expression of a subset of steroid-responsive genes by interacting with specific intracellular receptors.61 62

Not only is the intracellular retention of apoB enhanced by dexamethasone, but the secretion efficiency for apoB (the proportion that is secreted compared with the total cell apoB) is also increased (Fig 8Up, TableUp). The latter effect is unlikely to be an artifact resulting from decreasing reuptake of newly secreted apoB because cultured hepatocytes do not take up apoB to a significant extent.63 We verified this conclusion in our experimental system by demonstrating that incubating hepatocytes with conditioned medium containing 35S-labeled apoB did not result in a significant loss of apoB from the medium over 2 hours. It is not known whether the stimulation of apoB secretion by dexamethasone can be attributed entirely to the decreased degradation of apoB. Earlier experimental data from hepatoma cell lines suggest that supplementation with oleate stimulates apoB secretion by preventing apoB degradation.26 Sakata et al64 have demonstrated that protection of newly synthesized apoB against intracellular degradation by N-acetyl-leucyl-leucyl-norleucinal is necessary but not sufficient to stimulate apoB secretion without the supply of oleate. These combined results illustrate the importance of lipid availability for the secretion of apoB-containing lipoprotein. However, dexamethasone does not significantly change the level of intracellular triacylglycerol.16 Dexamethasone increases the secretion of apoB more than that of triacylglycerol, which is reflected in increased numbers of smaller VLDL particles.21 Therefore, the increase in apoB secretion in dexamethasone-treated cells may not result simply from an increase in cell triacylglycerol availability. However, changes in triacylglycerol turnover could be a factor since dexamethasone increases the activity of phosphatidate phosphohydrolase, which is a key regulatory enzyme in hepatic triacylglycerol synthesis.18 48 49 Moreover, in addition to the neutral lipid availability that affects the efficiency of apoB secretion, other factors, including the lipid composition of the endoplasmic reticulum membrane,35 the microsomal triacylglycerol transfer protein that is presumably involved in lipid recruitment during lipoprotein assembly,65 66 the specific amino acid sequences within the apoB molecule that mediate apoB translocation across the endoplasmic reticulum membrane,67 and (less specifically) the length of the apoB protein68 all have profound effects on the secretion of apoB-containing lipoproteins. Modification of the first two parameters by dexamethasone could change the secretion efficiency of apoB, and more experiments are required to elucidate exactly how glucocorticoids stimulate hepatic apoB synthesis and secretion.

The effects of dexamethasone on net apoB synthesis, degradation, and secretion that we have shown are all opposite to those reported for insulin.6 7 These observations may explain why high concentrations of insulin counteract the effects of dexamethasone in apoB secretion.21 In stress and diabetes there is a complicated interrelation between the effects of glucocorticoids and insulin with other hormones. Glucocorticoids promote insulin insensitivity in the liver and adipose tissue23 24 and sensitize adipose tissue to the lipolytic actions of catecholamines and growth hormone.69 Consequently, the liver receives an increased supply of fatty acids from adipose tissue that aggravates hepatic insulin insensitivity.3 23 24 Fatty acids are also a major driving force for the secretion of triacylglycerol in apoB-containing lipoproteins.22 Therefore, the relative inability of insulin to decrease the supply of fatty acids from adipose tissue and to reverse the effects of glucocorticoids on hepatic VLDL secretion could contribute to the hypertriglyceridemia of stress and non–insulin-dependent diabetes.3 23 24 This latter observation is compatible with the relative lack of suppression of apoB secretion from the liver in obese subjects with insulin resistance.70 Increased secretion of VLDL particles coupled with decreased uptake of IDL and LDL by the hepatic LDL receptor could contribute to the hyperapobetalipoproteinemia that can be observed in insulin resistance.3 4 23 24 These changes are potentially atherogenic, and the involvement of glucocorticoids is supported by the strong correlation between increased serum cortisol in human beings and the extent of coronary artery disease.23 24 71 72 Furthermore, prolonged glucocorticoid therapy can accelerate the development of atherosclerosis.73 The present work provides the first mechanistic explanation for how glucocorticoids can contribute to hyperapobetalipoproteinemia through increasing the net synthesis and decreasing the degradation of apoB, thus stimulating the hepatic secretion of apoB-containing lipoproteins.


*    Acknowledgments
 
This work was supported by grants from the Alberta Heart and Stroke Foundation, the Canadian Diabetes Foundation, and the Medical Research Council of Canada. The Alberta Heritage Foundation for Medical Research provided a postdoctoral fellowship to Dr McLeod, a scholarship to Dr Yao, and a medical scientist award to Dr Brindley. We thank Drs R. Davis for anti-rat apoB antibody, D.E. Vance for anti-rat albumin antibody, J. Scott for the apoB cDNA probe, and M.D. McArthur for advice on statistical analysis.

Received November 14, 1994; accepted May 30, 1995.


*    References
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