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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:321-329

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:321-329.)
© 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

Anna-Marie Brown; David Wiggins; Geoffrey F. Gibbons

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


*    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


*    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.


*    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 "{chi}", this may change to a value "{gamma}" after cell culture. If {chi}={gamma}, then no excess unlabeled glycerol has entered the TAG pool and lipolysis is zero. If, however, {gamma}<{chi} excess unlabeled glycerol has entered the pool, the amount of which is represented by ({chi}/{gamma}-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 6Down, 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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  		Figure 6. Effects of glucose ({square}-{square}) and MH ({circ}–{circ}) on intracellular TAG lipolysis and reesterification. Hepatocytes were cultured as described in Figure 4Up. After 4 hours in the presence of 14C oleate (0.75 mmol/L) and 3H glycerol (0.25 mmol/L) cells from some dishes were harvested and the 3H and 14C specific activity of the cellular TAG determined. The remaining dishes were cultured for an additional 24 hours in the absence of oleate and glycerol. At the end of this period, the 14C and 3H specific activity of TAG was calculated as described in the Methods section. Values represent the mean±SEM of 5 independent hepatocyte preparations. Values marked ** are significantly different (P<0.01) from the corresponding value obtained in the absence of glucose. Values marked {dagger} and {dagger}{dagger} are significantly different (P<0.05 and P<0.01, respectively) from the corresponding values in the absence of MH.

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.


*    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 1Down 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 1Down).



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  		Figure 1. Response of apoB-48 and apoB-100 net synthesis to glucose and MH. After removal of serum-containing medium hepatocytes were cultured for 16 hours in the absence or presence (5 and 25 mmol/L) of glucose. In each case MH (15 mmol/L) was either present or absent. The medium was removed and the cells pulsed for 1 hour with 35S-methionine as described in the Methods section. Cells were harvested and incorporation of label into apoB-100 and apoB-48 was determined. The above values represent the mean±SEM of 3 independent hepatocyte preparations. Values marked * are significantly different from those obtained in the absence of glucose (P<0.05). Values marked {dagger} and {dagger}{dagger} are significantly different from the corresponding values obtained in the absence of MH (P<0.05 and P<0.01, respectively). Values represent the mean±SEM of 3 independent hepatocyte preparations.

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 2aDown). 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 2aDown). 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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  		Figure 2. The effect of glucose and MH on the secretion of newly-synthesized apoB as (a) VLDL and (b) d>1.006 particles. Hepatocytes were cultured for 16 hours in the presence or absence of glucose and MH and pulsed for 1 hour with 35S-methionine. At the end of the pulse period, the medium was removed, the cells were washed, and labeled apoB was chased for 8 hours in the presence of medium containing 10 mmol/L unlabeled methionine. The VLDL fraction was separated from the d>1.006 particles, and the amounts of label associated with apoB-48 and apoB-100, in each fraction, were determined. Values represent the mean±SEM of 3 independent hepatocyte preparations. a, Values marked * and ** are significantly different from those obtained in the absence of glucose (P<0.05 and P<0.01, respectively). Values marked {dagger}{dagger} and {dagger}{dagger}{dagger} are significantly different (P<0.01 and P<0.001, respectively) from the corresponding values obtained in the absence of MH. b, Values marked {dagger} are significantly different from those obtained in the absence of MH (P<0.05).

Secretion of newly-synthesized apoB as small particles of density >1.006 g/mL was less sensitive to stimulation by glucose (Figure 2bUp). 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 3Down) (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 3Down).



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  		Figure 3. Relative distribution of apoB-100 and apoB-48 between the VLDL and the d>1.006 infranate. Using the data obtained in Figure 2Up, the total amount of apoB-48 and apoB-100 secreted into the medium were determined and the proportions associated with VLDL were calculated. Values marked * and ** for apoB-48 are significantly different (P<0.05 and P<0.01, respectively) from the corresponding values for apoB-100. Values marked {dagger}, {dagger}{dagger}, and {dagger}{dagger}{dagger} are significantly different (P<0.05, P<0.01, and P<0.001, respectively) from those obtained at 0 mmol/L glucose. Values marked {blacktriangleup}, {blacktriangleup}{blacktriangleup}, and {blacktriangleup}{blacktriangleup}{blacktriangleup} are significantly different (P<0.05, P<0.01, and P<0.001, respectively) from the corresponding values in the absence of MH.

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 1Down). 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 1Down).


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  		Table 1. Effects of Glucose and MH on Overall Secretion of ApoB-48 and ApoB-100

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 2Down). 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 2Down) (P<0.02, by ANOVA). Thus, inhibition of glucose phosphorylation resulted in an increased posttranslational degradation of newly-synthesized apoB.


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  		Table 2. Effects of Glucose and MH on Posttranslational Degradation of Newly-Synthesized ApoB

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 4Down).



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  		Figure 4. Response of VLDL apoB (total mass) secretion to the presence of glucose ({square}-{square}) and/or MH ({circ}–{circ}). Hepatocytes were cultured for 4 hours in the presence of serum and of glycerol and oleate. They were then cultured for 24 hours after removal of both serum and labeled substrates from the medium. During this period glucose was present at concentrations ranging from 0 to 25 mmol/L. At each glucose concentration MH (15 mmol/L) was either present or absent. After 24 hours the medium was removed, the VLDL fraction isolated, and the total apoB content determined by ELISA as described in the Methods section. Values marked * are significantly different (P<0.05) from the corresponding value in the absence of glucose. Values marked {dagger} are significantly different (P<0.05) from the corresponding values in the absence of MH. (n=5 for glucose, n=4 for glucose+MH).

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 5Down 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 5Down).



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  		Figure 5. Response of VLDL TAG secretion to the presence of glucose ({square}-{square}) and/or MH ({circ}–{circ}). Hepatocytes were cultured as described in Figure 4Up. After 24 hours the VLDL was isolated and its TAG content (mass and 14C label) determined. a, Shows the output of VLDL TAG; b, The output of prelabeled TAG synthesized during the initial 4 hours in the presence of 14C oleate. Values marked *, **, and *** are significantly different (P<0.05, P<0.01, and P<0.001, respectively) from the corresponding values in the absence of glucose. Values obtained in the presence of MH were significantly decreased at all glucose concentrations.

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 6Up 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 5Up). 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 6Up 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 4Up and 5Up), MH had no inhibitory effect on cellular TAG turnover in the absence of glucose.


*    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 7Down). 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 1Up) 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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  		Figure 7. The VLDL TAG:apoB ratio remains constant in the presence of glucose. Hepatocytes were cultured as described in Table 2Up. The data are calculated from the values determined for VLDL TAG output (Figure 5Up) and VLDL apoB output (Figure 4Up).

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 3Up) compared with apoB-100. Nevertheless, smaller, denser apoB-100-containing particles are secretion-competent in primary rat hepatocytes (Figure 2bUp), 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 3Up). 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 4Up and 5Up). 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 2bUp).

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 (TableUp ). 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 1Up), VLDL apoB output (Figures 2bUp and 4Up), and the output of VLDL TAG (Figure 5Up). 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 6Up) 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 {approx}150 nmol/mg protein (Figure 6Up). 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 1Up and 3Up 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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