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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1229-1235.)
© 1996 American Heart Association, Inc.

Articles

ApoB-100 Secretion by HepG2 Cells Is Regulated by the Rate of Triglyceride Biosynthesis but Not by Intracellular Lipid Pools

Fabienne Benoist; Thierry Grand-Perret

the Laboratoire Glaxo Wellcome, Centre de Recherche, Les Ulis, France.

Correspondence to Dr T. Grand-Perret, Laboratoire Glaxo Wellcome, Centre de Recherche, 25 avenue du Quebec, ZA de Courtaboeuf, 91951 Les Ulis cedex, France. E-mail TGP28876@GGR.CO.UK.

	Abstract

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Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pretreatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

Key Words: lipoprotein assembly • cholesteryl ester • hepatocyte • 5-tetradecyloxy-2-furancarboxylic acid

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Overproduction of apoB-100–containing lipoproteins appears to be a common cause of hyperlipidemia in humans.^{1 2} Therefore, it is of major interest to understand the mechanisms that regulate the synthesis and secretion of apoB-100 from hepatocytes in order to modulate its production. So far, these mechanisms remain poorly understood.

ApoB-100 is synthesized only in the liver. Because it is extremely hydrophobic, apoB-100 cannot be secreted as a free protein. Association of apoB-100 with lipids to form a lipoprotein particle is required for secretion. Most studies that have used primary cultures of rat hepatocytes,³ whole liver,^{4 5 6 7} and HepG2 cells^{8 9 10 11 12 13} suggest that apoB secretion is regulated posttranslationally, since apoB mRNA levels are not significantly modified after treatments that increase or decrease apoB-100 secretion. Among the posttranslational modifications affecting apoB-100, the assembly with lipids seems to play a key regulatory role for the intracellular processing and secretion of apoB-100.¹⁴ Many studies have focused on the role of TGs, cholesterol, CEs, or phosphatidylcholine on the regulation of apoB-100 secretion in various cellular models.^{3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23}

Oleic acid increases TG synthesis and apoB-100 secretion by HepG2 cells.^{15 16 17 18 19 20} The effect of oleic acid on apoB-100 is primarily due to the suppression of intracellular degradation¹⁷ and is not the result of an increase in mRNA levels.^{8 9 10 11 12} Boren et al²¹ have suggested that the cotranslational addition of lipids is critical for the transfer of apoB to the endoplasmic reticulum lumen and subsequent secretion and that apoB is targeted for degradation if it is not associated with core lipids (ie, TGs and CEs). Wu et al²² report that the acyl coenzyme A synthase inhibitor triacsin D inhibits the effect of oleic acid. Arbeeny et al²³ and Kempen et al²⁴ have shown that TOFA, an inhibitor of acetyl coenzyme A carboxylase, inhibits the synthesis of fatty acids and TGs and consequently apoB-100 and TG secretion. Taken together, these results suggest that apoB-100 secretion is modulated by TG synthesis.

Nevertheless, other studies suggest that cholesterol or CE availability controls apoB-100 production.^{25 26 27 28 29} But these results are controversial: Furukawa and Hirano³⁰ and Wu et al²² report that the modulation of CE biosynthesis does not alter apoB secretion or degradation. Moreover, a hydroxymethylglutaryl coenzyme A reductase inhibitor such as pravastatin decreases the synthesis of cholesterol but does not affect apoB-100 secretion in HepG2 cells.²⁸ Phosphatidylcholine synthesis may also regulate apoB secretion by rat hepatocytes.^{31 32 33 34}

The controversy concerning the role of different lipids in the secretion of apoB-100 could be due to a confusion between the size of lipid pools and the availability of the lipids at the site of lipoprotein assembly. The intracellular compartmentalization of lipid droplets may explain their lack of availability for assembly with nascent apoB-100 in the endoplasmic reticulum.

The aim of this study was to examine the respective roles of intracellular lipid pools and lipid biosynthesis in the regulation of apoB-100 secretion. In a first set of experiments, the contribution of the intracellular lipid mass was evaluated by modulating the lipid content of HepG2 cells by a 3-day treatment with lipid modulators. The rate of apoB-100 secretion was then measured in the absence of these lipid modulators. The secretion of apoB-100 remained unchanged in all conditions even after drastic modulation of the TG or CE pools. In a second set of experiments, HepG2 cells were incubated for 4 hours with L-[³⁵S]methionine and ¹⁴C-labeled lipid precursors in the presence of lipid modulators. Lipid biosynthesis and proteins that were secreted during the labeling period were quantified. We observed that the secretion of apoB-100 was correlated with TG biosynthesis but not with the biosynthesis of other lipids. Our data suggest that in HepG2 cells apoB-100 secretion is regulated by the TG biosynthesis rate rather than by intracellular lipid pools.

	Methods

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Materials
L-[³⁵S]methionine (1000 Ci/mmol) and [1,3-¹⁴C]glycerol (40 mCi/mmol) were purchased from Dupont New England Nuclear and [¹⁴C]acetate (62 mCi/mmol) from Amersham International. Basal medium (Eagle's), RPMI 1640 medium, L-glutamine, penicillin, streptomycin, and FBS were obtained from GIBCO. TOFA and Sandoz 58-035 were synthesized by Glaxo Laboratories. Simvastatin was extracted from Zocor tablets. Oleic acid, BSA (essential fatty acid free), and the high-performance liquid chromatography grade organic solvents used for HPTLC were purchased from Sigma.

Cell Cultures
The HepG2 cell line was obtained from American Type Culture Collection. The cells were seeded into 24-well plates (200 000 cells/170 mm²) containing basal medium (Eagle's) supplemented with penicillin and streptomycin (100 U/mL each) and 10% heat-inactivated FBS and were incubated in a humidified incubator (5% CO₂) at 37°C. Experiments were initiated when the cells had reached 70% to 90% confluence.

Modulation of the Intracellular Pools of Neutral Lipids
HepG2 cells in 24-well plates were treated for 3 days in RPMI 1640 medium supplemented with penicillin and streptomycin (100 U/mL each) and 1% heat-inactivated FBS with either lipid precursors or enzyme inhibitors or a combination of both. Oleic acid complexed to BSA (molar ratio, 9:1)¹⁵ was used at 100 µmol/L, TOFA and 58-035 at 1 µmol/L, and simvastatin at 2 µmol/L; medium and effectors were replaced every day. After the 3-day treatment the cells were washed and incubated for 16 hours without any modulator. Finally, the cells were pulsed for 4 hours with 15 µCi L-[³⁵S]methionine, 2 µCi [1,3-¹⁴C]glycerol, and 4 µCi [¹⁴C]acetate together in 500 µL methionine-free RPMI 1640 without any effector or inhibitor. After the pulse period the medium was removed, and cells were washed. Intracellular lipids and secreted proteins were analyzed separately.

Modulation of TG, Cholesterol, and CE Biosynthesis Rates
HepG2 cells in 24-well plates were labeled for 4 hours with 120 µCi L-[³⁵S]methionine, 5 µCi [1,3-¹⁴C]glycerol, and 20 µCi [¹⁴C]acetate in methionine-free RPMI 1640 medium supplemented with penicillin and streptomycin (100 U/mL each) and 1% heat-inactivated FBS. Oleic acid complexed to BSA (molar ratio, 9:1¹⁵ ; 750 µmol/L), TOFA (5 µmol/L), 58-032 (2 µmol/L), and simvastatin (2 µmol/L) were used alone or in combination. Intracellular lipids and secreted proteins were analyzed separately.

Analysis of Intracellular Lipids by Quantitative HPTLC
After the incubation HepG2 cells were washed and dried. Lipids were extracted over 24 hours at 4°C by adding 2-propanol (250 µL/well). Samples (40 µL) were sprayed on HPTLC on silica gel 60 F₂₅₄ (Merck) by using a Camag ATS 3 sample application. Phospholipids and neutral lipids were resolved by using the multiple development principle with a Camag AMD tank.³⁴ The 25-step universal automated multiple development gradient program³⁵ was used without preconditioning. The first 15 steps consist of a 100% methanol to 100% dichloromethane gradient, and the last 10 steps a gradient of n-hexane, n-heptane, diethylether, and acetic acid (63:18:18:1) and 100% n-hexane. Each lipid was identified by using purified standards from Sigma revealed by phosphomolybdic acid staining. The radioactivity associated with the lipids was quantified by using a Phosphor-Imager (Molecular Dynamics), and the mass of intracellular lipids was quantified by using a laser densitometer (personal densitometer SI, Molecular Dynamics) with oleyl ester lipids or cholesterol as standards.

Analysis of ApoB-100, ApoA-I, Fibronectin, and Albumin Secretion
After the labeling period, the medium was centrifuged for 1 minute at 8000g to remove detached cells and debris. The proteins were analyzed by using SDS-PAGE under reducing conditions according to Laemmli³⁶ using resolving gels containing a 5% to 12% gradient and a stacking gel of 4% acrylamide. Dried gels were exposed to a PhosphorImager screen, and the radioactivities of apoB-100, fibronectin, apoA-I, and albumin were determined. For the experiments on the modulation of the intracellular pools of neutral lipids, the secretion rates of apoB-100 and apoA-I were expressed as the ratio of radioactivity of apoB-100 or apoA-I versus albumin. This allowed us to correct the small variation in the number of cells on day 4 due to slight cell growth differences.

2D Gel Electrophoresis of Secreted Proteins
2D gel electrophoresis was performed¹⁵ with the following modifications. Briefly, samples were applied to native agarose acrylamide gel (Lipofilm; Sebia). After 1.5 hours at 150 V, a 1.5x1.5x70-mm piece of gel was cut, equilibrated 10 minutes in SDS-Laemmli buffer, applied to 5% to 12% acrylamide gradients, and separated by SDS-PAGE. Radioactivity was revealed by using a PhosphorImager screen.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Modulation of Intracellular TG and CE Pools Does Not Significantly Affect the Rate of ApoB-100 Secretion
HepG2 intracellular lipid pools were upregulated or downregulated by a 3-day treatment with lipid precursors or inhibitors of enzymes involved in the lipid synthesis pathways. The cells were then washed and incubated for 16 hours without any modulator both to remove the modulators and to restore biosynthesis rates that were equivalent to the control. Finally, cells were pulsed for 4 hours with radioactive precursors without any effector. After the pulse period, radiolabeled secreted proteins were analyzed by using SDS-PAGE, and intracellular lipids were extracted from the cells. To obtain a better resolution of phospholipids and neutral lipids, cellular lipid mass was analyzed by using HPTLC with the multiple development principle. The four major lipids were quantified by scanning the HPTLC using oleyl ester lipids and cholesterol as standards after a phosphomolybdic acid staining.

Several modulators of lipid synthesis were used for the pretreatment: simvastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, affects cholesterol synthesis; TOFA, a potent acetyl coenzyme A carboxylase inhibitor, inhibits fatty acid synthesis²³ ; 58-035 inhibits ACAT and decreases cholesterol esterification²² ; and oleic acid, which is a precursor of several lipids. To check the efficiency of washing and the 16-hour incubation without any effector, we verified that incorporation of [1,3-¹⁴C]glycerol and [¹⁴C]acetate into each lipid was nearly equivalent in all conditions, which would indicate that biosynthesis rates were restored. TG biosynthesis rates were 97% and 101% of control after TOFA and oleic acid pretreatment, respectively. At the end of the experiment, strong changes in the TG and CE contents of HepG2 cells were observed, whereas phospholipids and free cholesterol were not affected (Fig 1 and Table 1). Simvastatin decreased CE mass and increased TG mass. Similarly, the ACAT inhibitor 58-035 strongly decreased CEs. By contrast, oleic acid increased both CEs and TGs. A combination of oleic acid and 58-035 increased TGs and decreased CEs. TOFA decreased CE and TG mass, particularly the latter. These different treatments modified the intracellular lipid mass of HepG2 cells in various ways, but none of them affected the secretion of apoB-100 or apoA-I (Table 1). We conclude that the secretion rates of apoB-100 and apoA-I are not regulated by the cellular pool of CEs or TGs.

View larger version (62K): [in this window] [in a new window]   Figure 1. Autoradiograph shows modulation of intracellular lipid mass as determined by quantitative HPTLC. HepG2 cells were treated for 3 days with either a lipid precursor and/or inhibitors of enzymes involved in lipid synthesis pathways, after which the cells were washed and incubated for 16 hours without any modulator. Cells were then pulse-labeled for 4 hours with L-[35S]methionine, [1,3-14C]glycerol, and [14C]acetate without any effector or inhibitor. Intracellular lipids were extracted by 2-propanol. After spraying on HPTLC, lipids were separated by using the multiple development principle with a Camag AMD tank. The mass of intracellular lipids was quantified by using a laser densitometer after phosphomolybdic acid staining with oleyl ester lipids or cholesterol as standards. Lanes a and b, control conditions; c, 1 µmol/L TOFA; d, 100 µmol/L oleic acid; e, 100 µmol/L oleic acid combined with 1 µmol/L 58-035; f, 1 µmol/L 58-035; and g, 2 µmol/L simvastatin. This represents a typical experiment reproduced three times.

View this table: [in this window] [in a new window]   Table 1. Modulation of Neutral Lipid Intracellular Pools Does Not Significantly Affect Rate of ApoB-100 Secretion

Modulation of TG Biosynthesis Regulates ApoB-100 Secretion
In this experiment, lipid modulators were added during the 4-hour radiolabeling period. After this pulse period, radiolabeled secreted proteins were analyzed by using SDS-PAGE, and biosynthesized lipids in the cells were analyzed by using HPTLC. The radioactivities associated with cholesterol, CEs, TGs, and the secreted apoB-100 were quantified by using a PhosphorImager screen. Under control conditions, a small percentage of radiolabeled lipids is secreted into lipoproteins: 0.5%, 0.3%, and 0.02% of TGs, CEs, and cholesterol, respectively, were secreted.

Such a short treatment had almost no effect on the mass of intracellular lipids (data not shown). The radioactivity associated with the lipids was widely modulated by treatments, indicating that the biosynthetic rates of several lipids were modified (Table 2). Simvastatin inhibited both intracellular cholesterol and CE biosynthesis but did not affect apoB-100 secretion. The ACAT inhibitor 58-035 reduced CE synthesis without affecting the secretion of apoB-100. Oleic acid strongly stimulated apoB-100 secretion, and concomitant increases of TG and CE synthesis were observed. Moreover, oleic acid in combination with 58-035 still stimulated both apoB-100 secretion and TG synthesis, whereas CE synthesis was inhibited. This suggests that the stimulation of apoB-100 secretion induced by adding exogenous fatty acids, such as oleic acid, is not due to an increase of CE synthesis.

View this table: [in this window] [in a new window]   Table 2. Modulation of TG Biosynthesis Regulates ApoB-100 Secretion

When an inhibitor of endogenous fatty acid synthesis, such as TOFA, was used, a decrease in the biosynthetic rates of both esterified lipids was observed; this decrease was more pronounced for TGs. Treatment of HepG2 cells with TOFA also reduced apoB-100 secretion to <25% of control. Oleic acid totally reversed all the effects of TOFA, suggesting that the modulation of apoB-100 secretion or lipid synthesis was a consequence of the reduced free fatty acid availability within the cell.

TOFA Specifically Inhibits ApoB-100 Secretion
Inhibition of fatty acid biosynthesis by TOFA strongly decreased apoB-100 secretion in correlation with the inhibition of TG biosynthesis. The profile of proteins secreted by HepG2 cells after a 4-hour radiolabeling period in the presence of increasing doses of TOFA was analyzed by using SDS-PAGE. ApoB-100 was the only secreted protein affected by TOFA (Fig 2A). Quantification of the radiolabeled secreted proteins showed that the IC₅₀ of TOFA on apoB-100 secretion was 2.5 µmol/L, whereas the secretion of fibronectin, albumin, and apoA-I was not inhibited or only marginally affected at doses up to 16 µmol/L (Fig 2B). This inhibition of apoB-100 secretion was correlated with TG biosynthesis, since the IC₅₀ for the latter was 2.0 µmol/L (data not shown). Oleic acid was able to completely reverse the effect even at high doses of TOFA. Moreover, the stimulation of apoB-100 by oleic acid was maintained regardless of the TOFA dose. An 18-fold difference in apoB-100 secretion was observed between conditions with enhanced (by oleic acid) or decreased (by 16 µmol/L TOFA) TG biosynthesis (Fig 2A, lanes e and f).

View larger version (89K): [in this window] [in a new window]   Figure 2. HepG2 cells were labeled for 4 hours with L-[35S]methionine in the presence of varying concentrations of TOFA with or without 0.75 mmol/L oleic acid complexed to BSA. Secreted proteins were analyzed by using SDS-PAGE (5% to 12%) under reducing conditions. Radioactivity was revealed by using a PhosphorImager screen. The migration of standard molecular weights are indicated. A, PhosphorImager screen autoradiography. Lanes a and f, control conditions; b and g, 0.6 µmol/L TOFA; c and h, 1.7 µmol/L TOFA; d and i, 5 µmol/L TOFA; and e and j, 16 µmol/L TOFA. + or - indicates incubation with or without oleic acid, respectively. B, Line graph shows autoradiography scanning. ApoB-100 (), fibronectin (), albumin (), and apoA-I (*) were quantified and expressed as percent of control without TOFA. ApoB-100 secretion in the presence of oleic acid () is expressed as percent of control without oleic acid and without TOFA. This represents a typical experiment reproduced four times.

The specificity of TOFA for apoB-100 appears to be very high, as we could not detect any other modulated protein by 2D gel analysis of the proteins secreted by TOFA-treated HepG2 cells (Fig 3). Fibronectin, which corresponds to the location above 200 kD, was slightly decreased in this experiment (70% of control), but this effect was not reproducible and was not observed during SDS-PAGE, which suggests a loss of fibronectin, probably due to its adhesive property. Most of the apoB-100–containing lipoproteins secreted by HepG2 cells have the size of LDL, whereas a small proportion have VLDL size.¹⁵ These apoB-100–containing lipoproteins, LDL and VLDL, are well resolved by a native agarose-acrylamide gel that separates the lipoproteins with respect to their diameter. This gel was used as the first dimension of the 2D gel. Both VLDL and LDL apoB-100 were decreased by TOFA (Fig 3). Thus, inhibition of TG biosynthesis by TOFA reduced the secretion of both lipid-rich VLDL and relatively lipid-poor LDL. This is in accordance with the finding that TG secretion was also decreased to 40% of control at 5 µmol/L TOFA.

View larger version (60K): [in this window] [in a new window]   Figure 3. PhosphorImager screen autoradiography of 2D gel electrophoresis. HepG2 cells were labeled as in Fig 2, and secreted proteins were first separated on native agarose-acrylamide gel designed for lipoprotein analysis (Lipofilm). A small piece of the gel was cut, equilibrated in SDS-Laemmli buffer, and applied to SDS-PAGE as in Fig 2. Arrows indicate apoB-100 into LDL; dashed arrows, apoB-100 into VLDL; and - and +, control conditions and 5 µmol/L TOFA, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The various cellular models and multiple experimental conditions that have been used to study the role of lipids in the regulation of apoB-100 secretion have generated conflicting results.^{22 23 24 25 26 27 28 29 30 31 32} We reinvestigated this question in HepG2 cells by using several modulators of lipid synthesis. The size of the CE and TG pools was upregulated or downregulated by a 3-day treatment with oleic acid and/or 58-035 (an ACAT inhibitor), simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), or TOFA (an inhibitor of fatty acid synthesis). These modulators of lipid synthesis were eliminated by washing the HepG2 cells and incubating them overnight in fresh medium. Such treatment was sufficient to restore lipid synthesis rates equivalent to the control condition. Either through inhibition by 58-035 or stimulation by oleic acid, the cellular content of CE could be modulated over a ninefold range between the two conditions. However, the apoB-100 secretion rate was remarkably stable. Similarly, in the presence of TOFA or a combination of oleic acid and 58-035, the TG content of HepG2 varied over a 12-fold range, and no effect was observed on apoB-100 secretion. Moreover, the TG/CE ratio could be as high as 90 (after treatment with oleic acid and 58-035) or as low as 3 (in the presence of TOFA) without any effect on apoB-100 secretion.

The method used to modulate the intracellular lipid pools in this study was unusual; in several other studies, the lipid content of the cells in culture has been modulated by incubation with lipoproteins such as TG-rich VLDL or CE-rich LDL.^{11 22 37} These lipids, internalized via a receptor-dependent uptake, are released into the cell after hydrolysis in lysosomes. Incorporation of these lipids into intracellular pools can be differently regulated with respect to endogenously synthesized lipids. The fact that lipids from lysosomal origin could induce different effects cannot be ruled out.

In a second set of experiments, apoB-100 secretion was measured at the end of a 4-hour radiolabeling period in the presence of lipid modulators; this protocol allowed us to show that apoB-100 could be modulated over an 18-fold range. ApoB-100 secretion correlated with the TG synthesis rate, whereas the CE synthesis rate did not. Indeed, inhibition of the cholesterol and/or CE biosynthesis by simvastatin or 58-035 had no effect on apoB-100 secretion. However, when oleic acid was added in combination with the ACAT inhibitor, CE biosynthesis decreased and TG biosynthesis increased, together with a stimulation of apoB-100 secretion. Moreover, the TOFA-induced inhibition of fatty acid synthesis and TGs was associated with a decreased apoB-100 secretion rate. All the effects of TOFA were reversed by oleic acid. Thus, the present study clearly indicates that in HepG2 cells the rate of secretion of apoB-100 is regulated by the rate of TG biosynthesis and not by the rate of CE biosynthesis.

Minor modifications of some lipids have also been observed and can be explained by indirect consequences of the primary effect. For example, simvastatin increased the mass of TGs; this could be attributed to an increased availability of fatty acids that were not used for cholesterol esterification or because acetyl coenzyme A is diverted from the cholesterol synthetic pathway to the fatty acid synthetic pathway.

Microscopic observation of HepG2 cells after oil red O staining³⁸ indicates that TG and CE intracellular pools are stored as cytoplasmic lipid droplets. Modulation of the mass of TGs and CEs by a 3-day treatment with TOFA or oleic acid modified the number and size of the lipid droplets. However, these lipids were not directly available for assembly with apoB-100 since the modulation of these pools had no effect on lipoprotein secretion. This strongly suggests that neutral lipid synthesis is compartmentalized: TGs available for secretion are synthesized at the proximity of lipoprotein assembly sites, whereas stored TGs are not directly available for the assembly of apoB-100–containing lipoproteins. This is in accordance with the results of Gibbons et al,³⁹ who report that the mobilization of these droplets for VLDL assembly is impaired in HepG2 cells. Indeed, HepG2 cells secreted <1% per day of lipids biosynthesized from acetate (data not shown).

We observed that the inhibition of TG biosynthesis by TOFA reduced the secretion of both lipid-rich VLDL and relatively lipid-poor LDL. This suggests that lipid secretion was also decreased, which was confirmed by measuring TG secretion. This is in accordance with a rate-limiting role of core lipid assembly for apoB-100–containing lipoprotein production. From the clinical point of view, inhibition of fatty acid and TG synthesis can be a very interesting way to decrease apoB-100 secretion and is very specific since it does not affect the secretion of other proteins. However, TOFA would probably not be active in vivo because the liver constantly receives a flux of fatty acids by the action of lipoprotein lipase or hepatic lipase or by adipocyte lipolysis.

Preliminary experiments have confirmed the report by Dashti¹¹ that shows that 25-hydroxycholesterol stimulated both apoB-100 secretion and CE synthesis. Nevertheless, when the ACAT inhibitor 58-035 was added in combination with 25-hydroxycholesterol, apoB-100 secretion was still increased, whereas CE was inhibited (data not shown). In accordance with the results published by Furukawa and Hirano,³⁰ our studies suggest that the stimulation of apoB-100 secretion by 25-hydroxycholesterol was not due to a CE increase but resulted from another mechanism that remains unclear.

Fig 4 summarizes the results we obtained for apoB-100 regulation by lipids in HepG2 cells. We have shown that the TG biosynthesis rate controls apoB-100 secretion, and we have suggested that neutral lipid synthesis was compartmentalized in storage pools and secretory pools. Secretory pools of lipids were transported from their site of synthesis to apoB-100 nascent polypeptides, probably by MTP. This model is in agreement with the fact that MTP is absolutely required for apoB secretion.⁴⁰ Indeed, a defect in the MTP is the cause of abetalipoproteinemia (abetalipoproteinemic patients have only trace amounts of plasma apoB-containing lipoproteins).⁴¹ In vitro, purified MTP preferentially transfers TGs and CEs between membranes.⁴² In HepG2 cells, apoB-100 is secreted as a lipoprotein containing mainly TGs and is very poor in CEs, even if the lipid/protein ratio is lower than it is in plasma VLDL. This confirms the major role of TG availability at the site of synthesis of apoB-100, probably with the contribution of MTP as a shuttle. However, as discussed by Gibbons,⁴³ HepG2 cells have impaired mobilization of lipids stored in cytosolic droplets. For this reason, only biosynthesized TGs are available for lipoprotein assembly. It also explains why HepG2 cells secrete mainly lipid-poor lipoproteins with the size of plasma LDL. By contrast, primary hepatocytes can mobilize the cytosolic pool of lipids and produce mainly lipid-rich lipoprotein with the density of plasma VLDL. In primary cells, the storage pool of lipids can be hydrolyzed to form fatty acids, glycerol, and cholesterol, which can be reesterified at the site of lipoprotein assembly. This explains why we observe that the secretion of apoB-100 by primary human hepatocytes is not affected by short treatment with TOFA or oleic acid (data not shown). In conclusion, this HepG2 cellular model allows us to confirm that TG availability at the site of lipoprotein assembly is rate limiting in the production of apoB-100–containing lipoproteins. Our current studies on the effect of a specific MTP inhibitor on the secretion of apoB-100–containing lipoproteins strongly support this hypothesis.⁴⁴

View larger version (35K): [in this window] [in a new window]   Figure 4. Diagram of the apoB-100–containing lipoprotein secretory pathway. This model shows the point of action of the different lipid modulators used. Zigzag arrows indicate lipid synthesis inhibitors; thick arrows, the pathway important for regulation and assembly of lipoproteins containing apoB-100; thin arrows, the lipid synthesis pathway; dotted arrows, lipid remobilization pathway; dashed arrows, apoB-100 degradation pathway; DAGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; and AcetylCoA, acetyl coenzyme A.

	Selected Abbreviations and Acronyms

 

ACAT = acyl coenzyme A cholesterol acyltransferase BSA = bovine serum albumin CE = cholesteryl ester FBS = fetal bovine serum HPTLC = high-performance thin-layer chromatography MTP = microsomal TG transfer protein SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis TOFA = 5-tetradecyloxy-2-furancarboxylic acid TG = triglyceride 2D = two-dimensional

	Acknowledgments

 
We would like to thank the Glaxo chemists who synthesized TOFA and 58-035.

Received October 18, 1995; revision received April 1, 1996;

	References

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