© 1999 American Heart Association, Inc.
Original Contribution |
Glucose Phosphorylation Is Essential for the Turnover of Neutral Lipid and the Second Stage Assembly of Triacylglycerol-Rich ApoB-Containing Lipoproteins in Primary Hepatocyte Cultures
From the Oxford Lipid Metabolism Group, Metabolic Research Laboratory, Nuffield Department of Clinical Medicine, University of Oxford, Radcliffe Infirmary, Oxford, UK.
Correspondence to Dr G.F. Gibbons, Metabolic Research Laboratory, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK. E-mail geoff.gibbons{at}mrl.ox.ac.uk
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Abstract |
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Abstract—Primary hepatocytes cultured in a medium supplemented with amino acids and lipogenic substrates responded to increased extracellular glucose by increasing the secretion of VLDL apoB. This effect was accompanied by an increased secretion of VLDL triacylglycerol (TAG) derived from endogenous stores. Glucose also stimulated intracellular TAG mobilization via the TAG lipolysis/esterification cycle. All these effects were abolished in the presence of mannoheptulose (MH), an inhibitor of glucose phosphorylation. Glucose also gave rise to a modest (50% to 60%) increase in the incorporation of 35S methionine into newly synthesized apoB (P<0.05) and to a doubling of newly-synthesized apoB secretion as VLDL (P<0.05). The magnitude of these effects was similar for apoB-48 and for apoB-100. MH inhibited apoB-48 and apoB-100 synthesis and VLDL secretion at all glucose concentrations. The effects of glucose and MH on the secretion of newly-synthesized apoB-48 or apoB-100 as small dense particles were less pronounced. Glucose had no effects on the posttranslational degradation of newly-synthesized apoB-100 or apoB-48. However, this process was significantly enhanced by MH. The results suggest that glucose stimulates TAG synthesis, turnover, and output as VLDL. These effects are associated with an increased VLDL output of apoB mediated mainly by an increase in the net synthesis of both apoB-48 and apoB-100. All these changes are prevented by interference with glucose phosphorylation. Output of small, dense, apoB-containing particles is relatively unaffected by the glucose and MH-induced changes in TAG synthesis and lipolysis, an observation which suggests that only the bulk lipid addition step of VLDL assembly is affected by changes in glucose metabolism.
Key Words: primary hepatocytes • apoB • glucose • lipid recruitment • phosphorylation
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Introduction |
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Elevated plasma glucose is a characteristic feature of the hypertriglyceridemia that frequently accompanies conditions such as non–insulin-dependent diabetes mellitus (NIDDM) and obesity.1 2 3 4 The role of glucose in this relationship has been studied for several years and there now seems little doubt that 1 of the direct effects of glucose at the hepatic level is to increase the secretion of VLDL TAG.5 6 7 8 Controversy exists, however, as to whether this effect of glucose is accompanied by a similar increase in the secretion of apoB, and studies with primary cultures of rat hepatocytes7 and the human hepatoma cell line HepG29 showed little effect of glucose in this regard. Recent studies have produced evidence for a glucose-mediated stimulation of apoB output in HepG2,10 11 and it has been suggested that the precise effect of glucose is dependent on the culture conditions in vitro.11
We have previously shown that high rates of VLDL output from primary hepatocytes can be maintained only when the culture medium is supplemented with certain amino acids and fatty acid precursors, which provide an environment conducive to vigorous de novo lipogenesis.12 13 14 It has also been observed in primary hepatocyte cultures that apoB output requires efficient de novo fatty acid synthesis.15 Glucose stimulates fatty acid synthesis in hepatocytes partly by a mechanism that involves increased expression of the lipogenic genes acetylcoenzyme A carboxylase and fatty acid synthase.16 17 This effect was abolished in the presence of inhibitors of glucokinase.16 In view of these relationships, we have studied the effects of glucose and of the glucokinase and fatty acid synthesis inhibitor mannoheptulose (MH) on intracellular apoB metabolism and secretion. Of particular interest was the regulation of the second stage of apoB-containing lipoprotein assembly that involves bulk lipid addition.18 19 20 21 22 It is not yet clear whether this step requires microsomal triglyceride transfer protein (MTP).23 24 Nevertheless, it seems likely that this stage of assembly is dependent on the efficient mobilization and transfer of endogenous neutral lipid25 to appropriate intracellular site(s) in the secretory apparatus.22 26 27 28 29 30 This latter step may require efficient cytosolic TAG lipolysis and reesterification.26 28 31 32 33 34 35 36 37 In view of the potential importance of bulk lipid transfer as a regulatory target for VLDL assembly and secretion, we have examined the relationship between glucose mediated changes in apoB metabolism and the lipolytic mobilization of endogenous, cellular TAG and how this relationship is affected when glucose phosphorylation is prevented.
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Methods |
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Materials
Waymouth's medium (glucose free and [glucose plus methionine free]) was obtained from Gibco Ltd. Glucose and methionine were added as required. All radiochemicals were from Amersham International. Bovine serum albumin (fatty acid free), sodium oleate, glycerol, dexamethasone, glutamine, alanine, serine, lactate, pyruvate, MH, anti-sheep IgG-alkaline phosphatase conjugate, and Protein A-sepharose CL4B were obtained from Sigma. The triacylglycerol (TAG) assay kit (GPO-PAP kit) and anti-(human apoB) antiserum were obtained from Boehringer-Mannheim. Anti-rat apoB antiserum was raised in rabbits, as described previously.38 Acrylamide-bisacrylamide (40% wt/vol) was obtained from Bio-Rad Laboratories Ltd. Male Wistar rats were purchased from Harlan UK Ltd.
Maintenance of Animals and Preparation of Hepatocyte
Cultures
Male rats of the Wistar strain were housed in a
thermostatically controlled room (20±2°C) with artificial lighting
(10 hours light/14 hours dark). Animals had unrestricted access to
water and to a commercial pelleted diet (4.3% fat, 51.2% carbohydrate
(mainly starch), 22.3% protein, 4.5% fiber, and 7.7% ash).
Hepatocytes were prepared under sterile conditions from
rats weighing between 220 and 280 g at 10 AM (2
hours into the light phase of the cycle), as described
previously.13 The isolated hepatocytes were
suspended at a concentration of 0.65x106
cells/mL in Waymouth's medium MB752/1 containing glucose (25
mmol/L), methionine (0.33 mmol/L), fetal bovine serum (10% v/v),
penicillin (90 000 U/L), streptomycin (90 000 µg/L), glutamine
(3.6 mmol/L), alanine (0.36 mmol/L), and serine (0.45
mmol/L). The cell suspension (3.0 mL) was plated out onto
collagen-coated dishes,39 and the cells were allowed to
form a monolayer (3 to 4 hours). In experiments requiring prelabeled
cellular TAG, 3H glycerol (0.25 mmol/L;
2.93x104 dpm/nmol) and 14C
oleate (0.75 mmol/L; 0.98x103 dpm/nmol)
bound to bovine serum albumin were present during this
period (see below). Following cell attachment, the medium was removed,
the monolayer was washed with phosphate-buffered saline, and the
cells cultured in serum-free, glucose-free Waymouth's medium
supplemented with the above antibiotics and amino acids and, in
addition, dexamethasone (1.0 µmol/L), pyruvate
(1 mmol/L), and lactate (10 mmol/L). This medium is
subsequently referred to as supplemented medium.13 14 At
this stage, glucose was added to give initial concentrations of 0, 5,
10, 15, 20, or 25 mmol/L. Hepatocytes were cultured
for 24 hours, the medium was removed, and the cells were harvested. In
pulse-chase experiments (see below) cells were cultured for 16 hours
only under the above conditions. The medium was removed, the cells
washed twice with PBS, and supplemented Waymouth's medium (lacking
glucose and methionine) was added. Varying amounts of glucose were
added to give concentrations identical to those present during the
previous 16 hours. Cellular apoB was then labeled and chased as
described below.
Pulse-Chase Experiments With Labeled ApoB
35S-methionine (100 µCi/dish) was added
to the hepatocytes in methionine-free medium, and the cells
were labeled for 1 hour. The medium was removed and the monolayer
washed twice with PBS. At this stage, some dishes were removed to
measure label incorporation into cellular apoB (net synthesis). Three
mL of supplemented medium containing unlabeled methionine (10
mmol/L) and glucose was added to the remaining cells to give
concentrations identical to those present during the pulse period.
The cells were cultured for an additional 8 hours under these
conditions, after which the medium was removed and the cells
harvested.
Separation of VLDL From Dense ApoB-Containing Particles
At the end of each culture period, the cell medium was
centrifuged in a Beckman 50.4 fixed angle rotor for 16
hours at 40 000 rpm. The floating VLDL was separated from the denser
particles (d >1.006) in the infranate by tube-slicing.
Immunoprecipitation, Sodium Dodecyl
Sulfate-Polyacrylamide Gel Electrophoresis, and
Radioassay of Labeled ApoB-48 and ApoB-100
Labeled apoB was immunoprecipitated from the cells, and from the
VLDL and d>1.006 fractions of the medium, using a polyclonal anti-rat
apoB antibody raised in rabbits as described previously.38
The resulting Protein-A Sepharose bead suspension was heated with
sample buffer containing 20 µg of rat plasma VLDL apoB. After
centrifugation, the supernatant containing the
immunoprecipitated labeled apoB-48 and apoB-100 was subject to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis in a
3% to 20% gradient polyacrylamide gel.38 Bands
containing apoB-48 and apoB-100 were visualized with Coomassie
Brilliant Blue R (Bio-Rad) and the dried gel was exposed to
x-ray film for 48 hours. Bands containing labeled B-48 and B-100 were
excised from the gel and solubilized with 30%
H2O2 and NCS-IL
tissue solubilizer. After neutralizing the solubilizer with glacial
acetic acid, scintillation fluid (Optiphase) was added and
radioactivity determined in a Beckman LS-6500 scintillation counter
(Wallac Scintillation).
Other Analytical Methods
Cellular protein was determined
colorimetrically by the method of Lowry et
al.40 Total ketone body production was determined
enzymatically on the infranate (d>1.006) using the method of
Williamson et al.41 Cellular and VLDL TAG mass were
determined enzymatically after Folch extraction42 using
the GPO-PAP kit from Boehringer Mannheim.
Newly-synthesized cellular and VLDL TAG and phospholipid were
determined by scintillation counting after Folch
extraction42 and thin-layer chromatography
of labeled lipids on silica plates (Unichem, Silica Gel G, 20x20 cm,
250 µm) with n-hexane-diethyl ether-glacial acetic acid
70:30:1.6 (vol/vol/vol).36 Bands were visualized using
Rhodamine 6G. VLDL apoB (total mass) was determined using an
enzyme-linked immunoabsorbent assay with anti-sheep IgG antibody
(Sigma) and anti-human apoB antiserum
(Boehringer-Mannheim).14
Calculation of Intracellular TAG Turnover
(Lipolysis/Reesterification)
Further culture of cells in which the intracellular TAG had been
doubly labeled with 3H glycerol and
14C oleate gives rise to a relative decline in
the specific radioactivity of the 3H label
compared with that of the 14C
label.37 The 3H glycerol and
14C fatty acid moieties thus appear to become
distinct entities and undergo differential metabolism in
cells subsequently cultured in the absence of exogenous oleate and
glycerol. This situation could arise if the original, prelabeled TAG
pool was subjected to lipolysis followed by reesterification of the
resultant 14C labeled fatty acid with a diluted
3H glycerol pool of lower specific radioactivity.
The extent of the relative decline in the 3H
specific radioactivity (ie, the decrease in the
3H:14C specific activity
ratio) of the VLDL and cellular TAG is a measure of the amount of
unlabeled glycerol-containing moiety that has entered the TAG glycerol
pool without a corresponding entry of unlabeled fatty acid. The excess
dilution of the TAG-glycerol pool, therefore, reflects the degree of
TAG lipolysis and reesterification of the 14C
labeled oleate with 3H glycerol of a lower
specific radioactivity. Thus, if the initial
3H:14C specific activity
ratio of the cellular TAG is "
", this may change to a value
"
" after cell culture. If =, then no excess unlabeled
glycerol has entered the TAG pool and lipolysis is zero. If, however,
< excess unlabeled glycerol has entered the pool, the amount of
which is represented by
(/-1) pools. This is
equal to the fractional turnover of cellular TAG. Because the mass of
TAG at the end of the culture period is known (ie, the TAG pool size),
the absolute TAG turnover may be calculated as the product of the
fractional turnover times pool size.
In practice, cells were prelabeled with 3H
glycerol and 14C oleate during the plating period
(see above). After cell attachment, the labeled medium was removed and
the monolayer washed twice with PBS. At this stage, some dishes were
removed for measurement of the "initial"
3H:14C specific activity ratio of the cellular
TAG. Three ml of medium lacking glycerol and oleate was added to the
remaining dishes, and the cells were cultured in the presence or
absence of various concentrations of glucose for an additional 24
hours. During this period, some of the original, dual labeled cellular
TAG will be secreted as VLDL. The "final" TAG pool will thus
consist of the sum of the remaining cell TAG and that secreted as VLDL.
It is this combined value that is used to calculate the final
3H:14C specific
radioactivity ratio of TAG. For instance, in 1 of the experiments used
to compile Figure 6
, the initial
3H:14C specific activity
ratio of the cellular TAG was 1.70. After culture of these cells in the
presence of 25 mmol/L glucose and absence of MH for 24 hours, the
final specific activity ratio of the sum of the cellular and VLDL TAG
was 0.78. Thus the fractional turnover was
(1.70/0.78-1)=1.18 pools.
The total pool size of TAG was 347 µg/mg protein. Thus the absolute
turnover of TAG at 25 mmol/L glucose was 1.18x347=409 µg
· mg-1 · 24
hours-1.
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and
Statistical Analysis
Values are presented as the means±SEM of several
independent experiments (n). Significant differences were calculated
using a paired or unpaired Student's t test. In some cases,
statistical tests were carried out by ANOVA (2 factor with replication
and 1-way t-tests: 2 samples assuming equal variances).
These tests were done using the data analysis package in
Microsoft Excel for Windows 95, Version 7.
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Results |
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Glucose Enhances, and Mannoheptulose Suppresses, ApoB Net Synthesis and Secretion as VLDL
After removal of the serum-containing medium, hepatocytes were cultured overnight (16 hours) in supplemented Waymouth's medium either in the absence or in the presence of glucose at concentrations of 5 and 25 mmol/L. In each case MH was absent or present at a concentration of 15 mmol/L. Cells were then pulse-labeled for 1 hour as described in the Methods section. Some dishes from each group were removed for measurement of 35S label incorporation into apoB; the remainder were chased for 8 hours either in the presence or absence of glucose and MH, as above. Amounts of labeled apoB remaining within the cell or secreted as VLDL (d<1.006) and as denser particles (d>1.006) were measured at the end of this period. Figure 1
shows that compared with the response
observed in the absence of glucose, glucose (25 mmol/L) resulted
in a 55.3±21.6% increase (P<0.02) in labeled apoB-100 at
the end of the pulse. At 5 mmol/L glucose, the smaller
increase in apoB-100 label (18.8±14.7%) was not significant. MH
suppressed the appearance of labeled cellular apoB-100 at both
concentrations of glucose, but the effect was most pronounced at 5
mmol/L glucose. Similar effects of glucose and MH were also observed on
the appearance of labeled apoB-48. Thus, at 25 mmol/L, glucose
stimulated labeled apoB-48 formation by 40.8±12.2%
(P<0.01), whereas at 5 mmol/L, glucose had no
significant effect (20.4±16.5%). Again, MH inhibited labeled apoB-48
formation at both concentrations of glucose (Figure 1).
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The glucose-mediated stimulation of labeled apoB production
occurred in association with an increase in the output of labeled apoB
into the VLDL fraction of the medium during the chase period (Figure 2a). This increase amounted to 112±26%
(P<0.01) between 0 and 25 mmol/L glucose for apoB-48
and 93±27% (P<0.01) for apoB-100. Since 5 mmol/L
glucose had little effect, most of these changes occurred between 5 and
25 mmol/L glucose. MH suppressed the VLDL secretion of
newly-synthesized apoB-100 and apoB-48, an effect which became
attenuated at 25 mmol/L glucose (Figure 2a). Thus, whereas
in the absence of glucose MH inhibited VLDL apoB-48 secretion by
81±1.8% (P<0.001), at 25 mmol/L the corresponding
value was 56.3±9.6% (P<0.01). The corresponding values
for VLDL apoB-100 were 85.2±2.4% (P<0.001) and
68.8±6.2% (P<0.01).
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Secretion of newly-synthesized apoB as small particles of density
>1.006 g/mL was less sensitive to stimulation by glucose (Figure 2b
). This was the case both for labeled apoB-100 and apoB-48.
Neither did inhibition of glucose phosphorylation by MH
inhibit the secretion of these small particles to the same extent as
that observed for VLDL apoB. This differential effect of glucose led to
an increase in the proportions of secreted apoB that appeared as VLDL
at 25 mmol/L glucose compared with those at 0 and 5 mmol/L
glucose (Figure 3) (P<0.05).
In the d>1.006 infranate, the apoB-48:apoB-100 ratio was higher than
that observed in the VLDL. Thus, at each concentration of glucose, a
larger proportion of the total secreted apoB-100 appeared in the VLDL
compared with that of total secreted apoB-48. This effect was largely
abolished in the presence of MH (Figure 3).
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Because glucose and MH had relatively little effect on the secretion of
the smaller particles, the overall response of total (VLDL+d>1.006)
apoB secretion to MH and glucose was relatively small compared with
their effects on the secretion of VLDL apoB alone (Table 1). For instance, instead of the 2-fold
increase in VLDL apoB-48 and B-100 secretion mediated by 25 mmol/L
glucose, the corresponding values for total apoB-48 and B-100 were
1.38-fold and 1.23-fold, respectively. In the presence of MH, there was
virtually no response of total apoB secreted to the presence of glucose
in the medium (Table 1).
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The effects of glucose and MH on the posttranslational degradation of
newly-synthesized apoB-48 and apoB-100 were assessed by comparing the
amounts of labeled cellular apoB after the 1 hour pulse with the sum of
those remaining in the cell and those secreted into the medium at the
end of the 8-hour chase period (Table 2).
Compared with the proportion observed in its absence, glucose, either
at 5 or 25 mmol/L, had no effect on the proportion of labeled
apoB-100 remaining after 8 hours. Neither was there any effect
on labeled apoB-48. It would appear, therefore, that the same
proportion of labeled apoB synthesized during the 1 hour pulse was
subsequently degraded irrespective of the glucose concentration in the
medium. At each concentration of glucose, however, the
simultaneous presence of MH decreased the total recovery of
labeled apoB-48 and labeled apoB-100 (Table 2)
(P<0.02, by ANOVA). Thus, inhibition of glucose
phosphorylation resulted in an increased
posttranslational degradation of newly-synthesized apoB.
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The effects of glucose and MH on the secretion of newly-synthesized
apoB as VLDL were paralleled by similar effects on the secretion of
VLDL apoB measured as total mass. In these experiments
hepatocytes were cultured with oleate (0.75 mmol/L)
and glycerol (0.25 mmol/L) for 4 hours immediately after plating
into serum-containing medium. After transfer into serum-free medium and
removal of oleate and glycerol, they were then cultured for an
additional 24 hours in the presence of glucose at concentrations of 0,
5, 10, 15, 20, and 25 mmol/L. MH (15 mmol/L) was present
or absent in each case. Under these conditions, glucose had little
effect up to 5 mmol/L, but between 5 and 25 mmol/L glucose,
apoB mass output increased almost 2-fold from 1680±120 ng/mg protein
to 3100±402 µg/mg protein. MH suppressed VLDL apoB output at all
concentrations of glucose and effectively prevented the normal
glucose-mediated increase in VLDL apoB secretion (Figure 4).
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Mobilization and Transport of Intracellular TAG is Enhanced by
Glucose and Suppressed by MH
The above effects of glucose and MH on newly-synthesized apoB and
apoB mass were accompanied by an increased lipolytic mobilization of
endogenous, cellular TAG for VLDL synthesis. The protocol
for these experiments was identical to that described above for
measurement of VLDL apoB output as total mass except that during the
initial 4 hours plating period in the presence of serum, oleate
(0.75 mmol/L) and glycerol (0.25 mmol/L) were labeled with
14C and 3H, respectively.
After this time the medium was removed and replaced with serum-free
medium lacking oleate and glycerol. This procedure resulted in a
prelabeled pool of intracellular TAG, the mobilization and
secretion of which was determined over the next 24 hours in the
presence or absence of glucose (0 to 25 mmol/L) and/or MH (15
mmol/L). Figure 5 shows that glucose gave
rise to a concentration-dependent increase in the VLDL secretion of the
intracellular pool of TAG measured either as total mass or
14C label. MH suppressed VLDL TAG output at all
glucose concentrations (Figure 5).
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Endogenous hepatic TAG undergoes a continuous cycle
of lipolysis and reesterification. The lipase(s) involved have not yet
been characterized, but it appears that most of the secreted VLDL TAG
is derived from fatty acids released by lipolysis of
endogenous cellular TAG stores.31 32 33 34 35 36 37
Furthermore, under some conditions, the rate of intracellular TAG
lipolysis correlates with the rate of VLDL TAG
secretion26 37 raising the possibility that these 2
processes are, in some way, metabolically related. We have
previously described a method for measuring the rate of hepatic
TAG/fatty acid "cycling" based on the relative decline in the
specific radioactivity of the glycerol moiety of TAG prelabeled with
14C oleate and 3H glycerol
or with 3H oleate and 14C
glycerol.32 37 We applied this method in the current work
to determine whether the glucose and MH-related changes in apoB
metabolism and VLDL assembly were associated with changes
in the lipolytic transfer of intracellular TAG into the secretory
pathway and with overall changes in the rate of operation of the
lipolysis/reesterification cycle. Figure 6 shows the results of this experiment
that utilized the same batches of cultured cells as those used to
determine the effects of glucose and MH on intracellular TAG secretion
(Figure 5). In this case, however, after the initial 4-hours
prelabeling of intracellular TAG with 14C oleate
and 3H glycerol, cells from some dishes were
harvested for determination of the initial
3H:14C specific activity
ratio of intracellular TAG. The remaining hepatocytes were
cultured for an additional 24 hours at glucose concentrations ranging
from 0 to 25 mmol/L either in the presence or absence of MH. At
the end of this period the
3H:14C specific activity
ratios of the remaining cellular TAG and of the secreted VLDL TAG were
determined and the relative loss of 3H glycerol
was measured in each case. These values were then used to calculate the
entry of unlabeled glycerol into the prelabeled TAG glycerol pool, and
thus the turnover of TAG during the 24-hour culture period (see
Methods). Figure 6 shows that glucose, particularly at
concentrations >10 mmol/L, led to a maximum 2-fold increase in
TAG turnover (lipolysis/reesterification). This effect of glucose was
almost completely abolished in the presence of MH. However, unlike its
effects on VLDL apoB and TAG secretion (Figures 4 and 5),
MH had no inhibitory effect on cellular TAG turnover in the
absence of glucose.
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Discussion |
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Glucose Increases Hepatic TAG Transport by Increasing VLDL Particle Number Rather Than VLDL Particle Size
The profound hormonal and metabolic disturbances associated with diabetes and obesity have made it impossible to establish the primary causes of the dyslipidemia, which frequently accompany these diseases. In the case of hypertriglyceridemia, a major contributory factor is increased VLDL secretion by the liver. Many of the above disturbances probably contribute to the overall increase in hepatic VLDL output, including increased influx of nonesterified fatty acids,28 43 44 45 46 hepatic insulin resistance,47 48 49 50 51 and hyperglycemia.5 6 7 8 44 The precise effects of glucose on VLDL secretion are, however, controversial. In particular, it remains to be firmly established as to whether glucose, in addition to promoting the output of VLDL TAG, also increases that of VLDL apoB.7 8 9 10 11 Clarification of this point is important in view of the consequences for VLDL particle size, which is a major determinant both of LDL synthesis and of the proportion of smaller, more potently atherogenic LDL particles.52 53 54 55 Thus it is important to determine whether glucose increases VLDL TAG output predominantly by increasing VLDL particle size (ie, no increase in apoB) or by increasing particle number (ie, increased apoB). The current results suggest that at glucose concentrations >5 to 10 mmol/L there is a concentration-dependent increase in the output of both VLDL TAG and of apoB. The magnitude of the increase is similar in both cases so that there is little, if any, change in VLDL particle size and composition as evidenced by the near constancy of the apoB:TAG ratio (Figure 7
). The apparent small increase in the
ratio at 5 mmol/L glucose is anomalous but is not statistically
significant. Thus it would appear that glucose increases TAG transport
out of the liver by increasing the number of VLDL particles rather than
by simply increasing their size. It is noteworthy that the
glucose-mediated increase in total apoB secreted into the medium (ie,
the sum of that associated with VLDL and the d>1.006 fraction; Table 1) was much less pronounced than that observed for VLDL alone.
This was because apoB in the d>1.006 fraction was relatively
unaffected by glucose. In an earlier study with primary rat
hepatocytes, Davis and colleagues7 observed
little or no effect of glucose on the total amounts of apoB-48 or
apoB-100 secreted into the d>1.21 fraction of the medium that
represented the sum of apoB appearing both as VLDL and
smaller particles. Culture conditions also modify the effects of
glucose on apoB metabolism11 ; in particular,
the expression of the heat shock protein hsp 70, a putative chaperone
of apoB,22 is affected by the composition of the
medium.56 These observations suggest that a regulatory
effect of glucose on apoB metabolism may, under some
circumstances, remain undetected in vitro unless permissive factors are
present in the culture medium.
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Glucose Enhances Predominantly the Bulk Lipidation Step of
VLDL Assembly
At the intracellular level, some recent important advances
in our understanding of the molecular assembly of VLDL have shed light
on the mechanisms involved in the regulation of TAG output in terms of
the interaction of apoB with neutral lipid at various points in the
secretory apparatus of the cell. The extent to which these
interactions occur determine the proportion of newly-synthesized apoB
molecules that acquire secretory competence and the proportions that
are channeled into a proteolytic degradation pathway (for reviews, see
References 22, 57, 5822 57 58 ). Selection of apoB for secretory potential is
ensured by its cotranslational folding into a conformation that confers
resistance to proteolysis. This may be achieved either by acquisition
of a small quantity of lipid (the first stage of VLDL assembly) during
translocation18 20 21 59 or by interaction with the
membrane bilayer of the endoplasmic reticulum.19
Secretory competent forms of apoB represent the maximum
potential, or capacity, of the cell to secrete VLDL TAG. Whether this
full capacity is actually realized in practice depends on (1) the
extent of subsequent lipidation of each secretory competent molecule of
apoB at the so-called second stage of VLDL assembly, which involves
bulk lipid transfer18 19 20 21 22 and (2) the extent to which
particles unable to complete this step are degraded or
secreted.22 60 61 Although MTP is essential for neutral
lipid transfer to apoB,62 63 it is not yet clear as to
whether MTP is required at only 1 or at both of the above lipid
acquisition steps.23 24 64 Nevertheless, the presence of
at least 2 such lipid interaction steps, each at a critical phase in
VLDL assembly, implies that they may have some regulatory significance
as possible foci for hormones or regulatory metabolites. In particular,
impairment of the efficient transfer of neutral lipids, especially of
TAG, to these crucial intracellular assembly sites may impede the
normal production of mature VLDL.26 27
In addition to that secreted as VLDL, lipid-poor apoB-containing
particles are secreted by perfused rat liver,65 rat
hepatocytes,39 43 66 67 and the rat hepatoma
cell line McA-RH7777 cells.18 19 It has been suggested
that these denser particles are formed during cotranslational assembly,
stabilized by the acquisition of a small quantity of
lipid22 and may represent the products of the
stage 1 lipid transfer process. Some of these particles are secreted
directly, and some become the substrate for the intracellular bulk
lipidation step resulting in mature VLDL.18 19 21 In
McArdle cells, it is predominantly apoB-48 that is secreted as denser
particles. In the current work, with primary hepatocytes, a
significantly larger proportion of apoB-48 was secreted as denser
particles (Figure 3) compared with apoB-100. Nevertheless,
smaller, denser apoB-100-containing particles are secretion-competent
in primary rat hepatocytes (Figure 2b), a property
that they share with the human hepatoma cell line HepG2.20
Glucose increases the proportion of both apoB-100 and apoB-48 that are
secreted as VLDL and these increases are reversed by the glucokinase
inhibitor MH (Figure 3). Thus, in addition to
playing a major regulatory role in the intracellular targeting of fatty
acids into catabolic (oxidative) or anabolic (esterification) pathways
within the cell, glucose also affects intracellular lipid
metabolism in such a way as to increase the availability of
functional apoB for the assembly of VLDL. This is achieved in 2 ways.
First, by facilitating an increase in the net synthesis of apoB.
Second, by ensuring that most of this additional increment in apoB is
made available for the bulk TAG transfer step rather than being
secreted as small, dense particles. Thus glucose increases the
proportion of newly-synthesized apoB, which is able to undergo the
Stage 2 bulk transfer of lipid resulting in an increased mass transfer
of apoB and TAG out of the cell as VLDL (Figures 4 and 5). The absolute secretion of newly-synthesized apoB as small
particles, which may represent the products of the Step 1
lipid transfer process,18 19 20 21 is relatively unaffected by
glucose in the extracellular medium (Figure 2b).
The increased incorporation of labeled
35S-methionine into apoB in the presence of
glucose requires further investigation. We consider this incorporation
to represent net synthesis during the 1 hour pulse. Because, in
HepG2 at least, some cotranslational degradation of apoB
occurs,22 we cannot rule out the possibility that glucose
acts, at least in part, by protecting apoB from degradation during
chain elongation. Nevertheless, it seems likely that glucose has little
or no effect on the posttranslational degradation of either apoB-100 or
apoB-48 because the amount of label remaining in each of the isoforms
as a proportion of that formed at the end of the pulse remained the
same, irrespective of the glucose concentration (Table ).
However, inhibition by MH of the phosphorylation of
glucose, either produced endogenously or added exogenously,
led to a small but significant increase in the posttranslational
degradation of both apoB-100 and apoB-48. It was of some interest that
MH affected various aspects of apoB metabolism even in the
absence of exogenous glucose. A possible explanation is that MH may
suppress net endogenous glucose-6-phosphate
production from glycogen by interfering with the cycling of
glucose-6-phosphate and glucose catalyzed by glucose-6-phosphatase and
glucokinase.68
It might be argued that the observed effects of glucose are a
nonspecific consequence of the increased osmotic load resulting from
the higher glucose concentrations. If this had been the case, it might
have been expected that the observed stimulatory effects of glucose
would be reinforced in the presence of a high concentration of MH.
However, this was not the case, as evidenced by the
inhibitory effects of this monosaccharide on apoB
net synthesis (Figure 1), VLDL apoB output (Figures 2b and 4), and the output of VLDL TAG (Figure 5).
Furthermore, 25 mmol/L glucose had little or no effect on the
secretion of newly-synthesized albumin (results not shown).
Glucose Enhances, and MH Suppresses, Intracellular TAG/Fatty
Acid Cycling
It has previously been shown that the major pool of intracellular
TAG, stored in the cytosol of the hepatocyte, is not in
simple equilibrium with VLDL TAG.31 69 70 We have
previously suggested based on the results of experiments with primary
hepatocytes32 33 and HepG226 27
that the bulk addition of TAG represented by the second
stage of VLDL assembly is, in some way, associated with the lipolytic
mobilization of cytosolic TAG. TAG resynthesized from the fatty acids
produced via this lipolytic step are able to access the site
responsible for Stage 2 TAG transfer to maturing VLDL, whereas intact
cytosolic TAG that has avoided lipolysis is not. Other work, both in
vivo35 and in vitro,36 37 supports the idea
of a lipolytic transfer of cytosolic TAG into secreted VLDL. The
current results suggest that glucose enhances the lipolytic turnover of
cellular TAG (Figure 6) as determined by the relative decrease
in the specific radioactivity of the 3H glyceride
glycerol moiety compared with that of the
14C-labeled TAG fatty acid. However, there are
other possible explanations for the glucose mediated increase in the
loss of 3H glyceride glycerol. For instance, it
might be argued that endogenous TAG lipolysis is
constitutive and that glucose merely affects the proportion of fatty
acids which are reesterified rather than merely lost from the cell via
the oxidative pathway. It is generally agreed that glucose suppresses
endogenous ketogenesis in the livers of starved rats as
first shown by Krebs and Hems71 almost 30 years ago.
However, endogenous ketogenesis in hepatocytes
from fed rats is low25 and in the current work the
difference between ketone body production at 0 and 25
mmol/L glucose was sufficient to account for a maximum diversion of
only 23 nmol endogenous TAG/mg cell protein away from the
ketogenic pathway at the higher glucose concentration (results not
shown). This value is far too low to account for the large increase in
lipolytic TAG turnover of 
150 nmol/mg protein (Figure 6).
Again, glucagon stimulates endogenous ketogenesis but has
no effect on the lipolytic turnover of cellular TAG as measured by the
relative decrease in specific radioactivity of the glyceride
glycerol.32 Finally, inhibition of endogenous
ketogenesis by lysosomotropic agents had no effect on the apparent rate
of intracellular TAG/fatty acid recycling.32
Glucose is an essential energy source for hepatic
metabolism, and in the current work, it was necessary to
maintain a basal level of glucose (5 mmol/L) for this purpose. On
the other hand, in vivo, certain physiological and
pathophysiological conditions result in elevated
plasma glucose concentrations and 1 of the objectives of the current
work was to assess whether these increased concentrations could
directly influence apoB metabolism. We attempted to
simulate this situation by raising the concentrations of glucose in
vitro to a maximum of 25 mmol/L. These higher concentrations are
similar to those found in the portal blood after a high glucose meal
(Bülow et al, unpublished data, 1998). In general, 5
mmol/L glucose, which corresponds to normoglycemia in vivo, had little
effect on the parameters studied compared with those
observed in the absence of glucose (see, eg, Figures 1 and 3 to 6). By contrast, 25 mmol/L
glucose showed consistently significant effects.
We conclude, therefore, that increased glycolysis is associated with (1) an enhanced transport of TAG out of the cell as VLDL, (2) an increased output of VLDL apoB, (3) an increase in the net synthesis of apoB-48 and apoB-100, and (4) an increased recruitment of newly-synthesized apoB for the second-stage assembly of TAG-rich lipoproteins resulting in the production of larger numbers of mature, VLDL particles. These glucose mediated effects are also associated with an increased mobilization of endogenous TAG fatty acids resulting from an enhancement of intracellular TAG/fatty acid cycling. Furthermore, suppression of glucose phosphorylation by glucokinase inhibitors such as MH reverses all the above glucose-dependent effects on lipid mobilization and VLDL assembly.
|
Acknowledgments |
|---|
We would like to thank R. Hems for preparing hepatocytes and M. Barber for typing the manuscript. This work was supported by a Program Grant (G9224993) from the Medical Research Council to G.F.G. We thank the Council for its continued support.
Received April 6, 1998; accepted July 3, 1998.
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