© 1995 American Heart Association, Inc.
Articles |
Effects of Dexamethasone on the Synthesis, Degradation, and Secretion of Apolipoprotein B in Cultured Rat Hepatocytes
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.
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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
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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.
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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 7
. In other pulse-chase experiments (Fig 5 and Table) 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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) or presence (
) 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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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.
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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 1A
) and
apoB-48 (Fig 1B) 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 1C).
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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 2A) or apoB-48 (Fig 2B) 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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The concentration-dependent effects of dexamethasone were
similar for apoB-100 (Fig 3A) and apoB-48 (Fig 3B).
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 3A and 3B), 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 1C
and 3C). 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 3D). 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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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 4).
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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 3,
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 5A and 5B).
Fig 6A 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 5A) and apoB-48 (Fig 5B)
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 5A and 5B). The equivalent
values for apoB-48 were 75±12% and 86±8%, respectively.
Cell apoB radioactivities (Fig 5A and 5B) were also used to determine
the intracellular retention times for apoB-100 and apoB-48 by using
polynomial analysis.6 This analysis (Fig 7A) 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 7B)
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 5C). Secretion of apoB-48
appeared to be increased by dexamethasone by approximately
1.5-fold at 3 hours (P=NS; Fig 5D). Fig 6B 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 5E and 5F). 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 5), 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 8). This time course also
revealed that there was no lag phase for the secretion of apoB (compare
Fig 8C and 8D with Fig 5C and 5D). 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 8A and 8B, respectively);
relative apoB-100 and apoB-48 secretions were increased by about
twofold and 1.5-fold, respectively, after a 180-minute chase (Fig 8C and 8D); and total apoB recovery was increased by
dexamethasone treatment (Fig 8E and 8F).
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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 8) and a 60-minute chase, and
radioactivities associated with apoB at the end of the 40-minute wash
were taken as the initial radioactivity (Table).
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 (Table). 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 (Table). This concentration-dependent effect of
dexamethasone agrees well with that shown in Fig 3. 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 9A); results from four independent experiments are
summarized in Fig 9B. 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 5 and the
Table, 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 Table, 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 9), and 1.6, 1.9-,
1.3-, and 1.04-fold from the long-term pulse-chase experiment
(Table).
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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 9A). 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
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Discussion |
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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 1). 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 7) 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 3) 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 8, Table). 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 8, Table). 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.
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Acknowledgments |
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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.
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References |
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