Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

Regulation of apoprotein synthesis and secretion in the human hepatoma Hep G2. The effect of exogenous lipoprotein

The regulation of lipoprotein assembly and secretion at a molecular level is incompletely understood. To begin to identify the determinants of apoprotein synthesis and distribution among lipoprotein classes, we have examined the effects of chylomicron remnants which deliver triglyceride and cholesterol, and beta very low density lipoprotein (beta VLDL), which deliver primarily cholesterol, on apolipoprotein synthesis and secretion by the human hepatoma Hep G2. Hep G2 cells were incubated with remnants or beta VLDL for 24 h, the medium was changed and the cells then incubated with [35S]methionine. The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and counted. Remnants caused a 14-fold, and beta VLDL a 7-fold, increase in VLDL apoprotein (apo) secretion; the apoB/apoE ratio in this class was unchanged. Preincubation with either of the lipoproteins also stimulated low density lipoprotein apoB secretion. Preincubation with beta VLDL, but not with remnants, significantly increased apoE and apoA-I secreted in high density lipoprotein (HDL). In addition, the apoE/apoA-I ratio precipitated from the HDL of beta VLDL-treated cells by anti-apoE was 2.2-fold higher than that precipitated by anti-apoA-I. There was no difference in the ratios precipitated from control HDL. This was due to the secretion of a lipoprotein, subsequently isolated by immunoaffinity chromatography, that contained predominantly apoE. When Hep G2 cells were preincubated with oleic acid alone, total apoprotein secretion was not altered. However, cholesterol-rich liposomes stimulated secretion of newly synthesized apoE, but not apoB, while apoA-I secretion was variably affected. Cholesterol-poor liposomes had no effect. Thus, lipid supply is a determinant of apoprotein synthesis and secretion, and cholesterol may be of particular importance in initiating apoprotein synthesis.     

Stimulation by oleic acid of incorporation of L-[4,5-3H]leucine into very low density lipoprotein apoprotein by the isolated perfused rat liver

Livers from normal fed or fasted (24h) rats were perfused in vitro to determine whether fatty acid affects the biosynthesis of very low density lipoprotein (VLDL) apoprotein. Oleate stimulated VLDL triacylglycerol output and increased incorporation of L-[4,5-3H]leucine into VLDL apoprotein in both the fed and fasted groups. The increased incorporation of [3H]leucine was mainly into VLDL-apoprotein E. The total mass of VLDL apoprotein secreted was also stimulated by oleate proportionately. These data suggest that fatty acids may stimulate hepatic synthesis and/or secretion of the VLDL apoproteins and that apo E, may be required for the formation and secretion of triacyl-glycerol in the VLDL.     

Effects of oleic acid on the biosynthesis of lipoprotein apoproteins and distribution into the very-low-density lipoprotein by the isolated perfused rat liver.

The effects of oleic acid on the biosynthesis and secretion of VLDL (very-low-density-lipoprotein) apoproteins and lipids were investigated in isolated perfused rat liver. Protein synthesis was measured by the incorporation of L-[4,5-3H]leucine into the VLDL apoproteins (d less than 1.006) and into apolipoproteins of the whole perfusate (d less than 1.21). Oleate did not affect incorporation of [3H]leucine into total-perfusate or hepatic protein. The infusion of oleate, however, increased the mass and radioactivity of the VLDL apoprotein in proportion to the concentration of oleate infused. Uptake of oleate was similar with livers from fed or fasted animals. Fasting itself (24 h) decreased the net secretion and incorporation of [3H]leucine into total VLDL apoprotein and decreased the output of VLDL protein by the liver. A linear relationship existed between the output of VLDL triacylglycerol (mumol/h per g of liver) and secretion and/or synthesis of VLDL protein. Net output of VLDL cholesterol and phospholipid also increased linearly with VLDL-triacylglycerol output. Oleate stimulated incorporation of [3H]leucine into VLDL apo (apolipoprotein) E and apo C by livers from fed animals, and into VLDL apo Bh, B1, E and C by livers from fasted rats. The incorporation of [3H]leucine into individual apolipoproteins of the total perfusate lipoprotein (d less than 1.210 ultracentrifugal fraction) was not changed significantly by oleate during perfusion of livers from fed rats, suggesting that the synthesis de novo of each apolipoprotein was not stimulated by oleate. This is in contrast with that observed with livers from fasted rats, in which the synthesis of the total-perfusate lipoprotein (d less than 1.210 fraction) apo B, E and C was apparently stimulated by oleate. The observations with livers from fed rats suggest redistribution of radioactive apolipoproteins to the VLDL during or after the process of secretion, rather than an increase of apoprotein synthesis de novo. It appears, however, that the biosynthesis of apo B1, Bh, E and C was stimulated by oleic acid in livers from fasted rats. Since the incorporations of [3H]leucine into the VLDL and total-perfusate apolipoproteins were increased in fasted-rat liver when the fatty acid was infused, part of the apparent stimulated synthesis of the VLDL apoprotein may be in response to the increased formation and secretion of VLDL lipid.     

Lipoprotein secretion by the human hepatoma cell line Hep G2: differential rates of accumulation of apolipoprotein B and lipoprotein lipids in tissue culture media in response to albumin, glucose and oleate

Serum low-density lipoprotein (LDL) concentration is a major determinant of susceptibility to the development of atherosclerosis. A major component of the protein moiety of LDL and its precursor very-low-density lipoprotein is apolipoprotein B (apo B). The human hepatoma cell line, Hep G2, was used as a model for the investigation of mechanisms which control hepatic secretion of the apo B and lipid components of lipoproteins. Using a sensitive immunoradiometric assay for apo B developed in this laboratory, we showed that bovine serum albumin inhibited and glucose, and fatty acids enhanced the rate of accumulation of apo B in the culture medium of Hep G2 cells. However, these substances did not necessarily affect LDL lipids in the same way as apo B. This finding appeared to be due to Hep G2 cells expressing lipase activities which led to triacylglycerol and phospholipid hydrolysis and lipid reuptake. Reuptake of apo B also occurred, but its rate of accumulation in the culture medium suggested it was a closer reflection of its true secretory rate.     

Effect of apolipoproteins E and C-III on the interaction of chylomicrons with parenchymal and non-parenchymal cells from rat liver.

[3H]Triacylglycerol-labelled chylomicrons were isolated from intestinal lymph, obtained from rats made hypolipidaemic by treatment with pharmacological amounts of 17 alpha-ethynyloestradiol. Oestrogen treatment results in a large reduction in the content of apolipoproteins (apo) E and C of lymph chylomicrons. Upon incubation in vitro with freshly isolated parenchymal and non-parenchymal cells the apo E-, apo C-poor chylomicrons became readily cell-associated. With increasing chylomicron concentrations this cell-association was saturable and half-maximal cell-association was achieved at about 0.55 mg of triacylglycerol/ml. The cell-association was time- and temperature-dependent. A more than 90% inhibition of the cell-association of the [3H]triacylglycerol moiety was observed with both parenchymal and non-parenchymal cells when pure apo C-III (12.6 micrograms/mg of triacylglycerol) was incorporated into the chylomicrons. These data indicate that apo E-, apo C-poor chylomicrons are bound to both parenchymal and non-parenchymal liver cells at a high-affinity site of limited capacity and that binding to this site is strongly inhibited by apo C-III. With apo C-III-enriched chylomicrons simultaneous determination of the cell-association of the 125I-apo C-III and the [3H]triacylglycerol moiety indicated that more 125I-apo C-III becomes associated to the cells than expected on the basis of [3H]triacylglycerol radioactivity measurements. It is suggested that upon cell-association of apo C-III its binding to the chylomicron particles is lost. Consequently the occupation of the cellular recognition site by apo C-III prevents further chylomicron binding and thus leads to a decrease of the cell-association level of the [3H]triacylglycerol moiety. Apo E enrichment of the chylomicrons led to an increased cell-association rate with parenchymal cells and to a marked increase of the cell-association level with non-parenchymal cells. The cell-association of the apo E radioactivity followed closely the [3H]triacylglycerol radioactivity, indicating that the particle-apo E complex is bound as a unity. The apo E effects were opposed by apo C-III. With apo E-, apo C-III-enriched chylomicrons more 125I-apo E became associated with the cells than could be expected on the basis of the [3H]triacylglycerol measurements. It is concluded that apo C-III can weaken the interaction of apo E with the chylomicrons leading to the cell-association of free apo E. It appears that subtle changes in the apo E and/or apo C-III content of chylomicrons can influence the interaction with both parenchymal and non-parenchymal liver cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of apolipoprotein secretion in very-low-density and high-density lipoproteins from cultured rat hepatocytes by dexamethasone.

The effects of dexamethasone (a synthetic glucocorticoid) and insulin on the secretion of very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) were investigated. Rat hepatocytes in monolayer culture were preincubated for 15 h in the presence or absence of combinations of 100 nM-dexamethasone and 2 nM-, 10 nM- or 50 nM-insulin. Dexamethasone increased [3H]oleate incorporation into secreted triacylglycerol by 2.7-fold and the mass of triacylglycerol secreted by 1.5-fold. Insulin alone decreased these parameters and antagonized the effect of dexamethasone. Dexamethasone increased the secretion of [3H]leucine in apolipoprotein (apo) E, and in the large (BH) and small (BI) forms of apo B in VLDL by about 7.1-, 3.6- and 4.0-fold respectively. Insulin alone decreased the secretion of these 3H-labelled apolipoproteins in VLDL. However, 2 nM-insulin with dexamethasone increased the secretion of 3H-labelled apo BH and apo BL by a further 0.8- and 3.2-fold respectively; 50 nM-insulin decreased the secretions of apo E, apo BH and apo BL in VLDL. Similar effects for dexamethasone or insulin alone were also obtained for the masses of apo E and apo BL + H secreted in VLDL. Albumin secretion was not significantly altered by either dexamethasone or insulin alone, but in combination they stimulated by 2.1-2.6-fold. Insulin or dexamethasone alone had little effect on the secretion of apolipoproteins in the HDL fraction. However, dexamethasone plus 2 nM-insulin increased the incorporation of [3H]leucine into apo AI, apo AH plus apo C, apo AIV and apo E of HDL by about 1.8-, 1.6-, 1.7- and 2.0-fold respectively. The apo E in the bottom fraction represented about 69% of the total 3H-labelled apo E secreted. The responses in the total secretion of apo E from the hepatocytes resembled those seen in HDL. The interactions of insulin and dexamethasone are discussed in relation to the general regulation of lipoprotein metabolism, the development of hyperlipidaemias and the predisposition to premature atherosclerosis.     

Regulation of apolipoprotein secretion by long-chain polyunsaturated fatty acids in newborn swine enterocytes

Long-chain polyunsaturated fatty acids (LC-PUFA) are important in the development of the immature nervous system, and adding these fatty acids to infant formula has been proposed. To determine the effect of n-3 LC-PUFA on apolipoprotein secretion and lipid synthesis in newborn swine enterocytes, differentiated IPEC-1 cells were incubated for 24 h with docosahexaenoic acid (DHA; 22:6) or eicosapentaenoic acid (EPA; 20:5) complexed with albumin at a fatty acid concentration of 0.8 mM or albumin alone (control) added to the apical medium. Oleic acid (OA; 18:1) was used a control for lipid-labeling studies. Both DHA and EPA reduced apolipoprotein (apo) B secretion by one-half, whereas EPA increased apo A-I secretion. The increased apo A-I secretion occurred primarily in the high-density lipoprotein fraction. These changes in apoprotein secretion were not accompanied by significant changes in synthesis. Modest decreases in apo B mRNA levels were observed for DHA and EPA, whereas there were no changes in apo A-I mRNA abundance. EPA reduced cellular triacylglycerol labeling by one-half, and DHA and EPA decreased cellular phospholipid labeling compared with OA. Labeled triacylglycerol secretion was decreased 75% by EPA, and DHA doubled labeled phospholipid secretion. If present in vivo, these effects should be considered before supplementing infant formula with these fatty acids.     

7-ketocholesterol inhibits VLDL secretion by cultured human and rabbit hepatocytes

7-ketocholesterol, one of the major product of autoxidation of dietary cholesterol, was found to inhibit secretion of very low density lipoprotein [14C]cholesterol, [14C]triacylglycerol and [35S]apoprotein B,E,C by cultured human and rabbit hepatocytes. A parallel inhibition (about 35%) of cholesterol synthesis but not of triacylglycerol formation was observed. Incubation with 10 micrograms/ml of oxysterol also reduced the total apo-B secretion measured by ELISA and increased intracellular apo-B mRNA level. These results seem to indicate that 7-ketocholesterol decreases secretion of very low density lipoprotein (VLDL) particles and exerts inhibitory effects on apo-B production at the co-translational or posttranslational level.     

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

Regulation of sex hormone-binding globulin secretion in human hepatoma G2 cells.

Our purpose was to examine the roles of natural (estradiol (E2) and estrone (E1)) and synthetic estrogens (ethinyl estradiol (EE), moxestrol (MOX), and tamoxifene (TAM)) in regulating production of sex hormone-binding globulin (SHBG) by human hepatoma G2 (Hep G2) cells, the rationale being that synthetic estrogens are less rapidly metabolized than natural estrogens and, thus, may alter SHBG levels more readily. In Hep G2 cells, E2, E1, and EE at 10(-7) M did not result in significantly greater SHBG secretion compared to control cells. The synthetic estrogens, MOX and TAM, caused significant, P < 0.001, increases of 30% and 51% in SHBG secretion at 10(-7) M compared to controls. However, when TAM and E2 were added together, each at 10(-7) M, no increase in SHBG secretion was noted. We conclude that natural estrogens at physiologic concentrations do not increase SHBG secretion by Hep G2 cells, but the increase of SHBG secretion caused by MOX and TAM suggests that the lack of effect of E2 and E1 may, in part, be due to their rapid metabolism. In addition, TAM stimulates SHBG secretion by interaction with the genome that is different, in certain respects, from that of E2.     

Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters

Male rats with thoracic duct cannulae were intubated with corn oil or fatty acid methyl esters and the lymph was collected over the next 2-72 h. The apoprotein (apo) composition of the chylomicrons, isolated by conventional ultracentrifugation, was determined by sodium dodecyl sulfate - polyacrylamide - glycerol gel electrophoresis and isoelectric focusing. The lipid content and composition was assessed by gas--liquid chromatography. The particle size was obtained by calculation and confirmed by electron microscopy. The study demonstrates that both the monoacylglycerol (corn oil feeding) and the phosphatidic acid (methyl ester feeding) pathways of triacylglycerol biosynthesis yield chylomicrons with closely similar apoprotein profiles representing apo B-48, apo A-IV, apo E, apo A-I, and the apo C components. A protein band corresponding to apo B-100 was occasionally observed as a minor component of the chylomicrons from both groups of animals. The chylomicrons from corn oil feeding had about two times larger diameters than those from methyl ester feeding. There were no significant differences in the composition of the apoproteins, although the smaller particles had two times higher apoprotein/triacylglycerol ratios. It was calculated that the amount of apo B per lipid particle for the ester fed rats ranged from one to eight molecules and was closely correlated with the particle size. The corn oil fed rats yielded about three molecules apo B per lipid particle regardless of the particle size. It is concluded that the pathway of intestinal triacylglycerol biosynthesis has a significant effect on the apoprotein mass and to a lesser extent on the apoprotein and lipid composition of the chylomicrons. The phosphatidic acid pathway produces smaller particles and transfers to the bloodstream twice as much apoprotein per gram of fat than the monoacylglycerol pathway, which yields the larger particles. Possible variations in the site and rate of biosynthesis of the triacylglycerols could not be entirely excluded as contributing factors.     

The effect of HMG-CoA reductase inhibitor (CS-514) on the synthesis and secretion of apolipoproteins B and A-1 in the human hepatoblastoma Hep G2

Human hepaotoblastoma cells (Hep G2) were cultured with a competitive inhibitor of HMG-CoA reductase, CS-514. The synthesis of cholesterol was markedly inhibited after 1 h preincubation with CS-514. The synthesis and secretion of apolipoprotein (apo) B and A-1, however, were not affected. A long-term incubation (21–24 h) of cells with CS-514 did not change apo B synthesis and secretion, although a slight depression of apo A-1 synthesis was observed. Hep G2 cells were found to secrete LDL- and HDL-like lipoproteins which were poor in cholesterol when cells were incubated with the drug. These results suggest that the modulation of cholesterol synthesis affects neither the synthesis and secretion of apo B and A-1 nor the formation of lipoproteins.     

Secretion of VLDL, but not HDL, by rat hepatocytes is inhibited by the ethanolamine analogue N-monomethylethanolamine.

The role of phospholipids in the assembly and secretion of very low density lipoproteins (VLDL) has been investigated by incubation of monolayer cultures of rat hepatocytes with monomethylethanolamine, an analogue of ethanolamine and choline. The cellular concentration of phosphatidylmonomethylethanolamine was increased 17-fold in response to treatment of hepatocytes with monomethylethanolamine. The secretion of phosphatidylcholine, triacylglycerol, and the apolipoproteins BH, BL, and E into VLDL was inhibited by approximately 50% in hepatocytes incubated with monomethylethanolamine, compared to untreated cells. Cell viability was unaffected by treatment with the ethanolamine analogue, as was cellular protein synthesis. The mechanism by which monomethylethanolamine reduced VLDL secretion was examined. Since monomethylethanolamine is a structural analogue of ethanolamine and choline, an obvious hypothesis for explanation of the effect on VLDL secretion was that phosphatidylcholine biosynthesis, which is required for VLDL secretion (Z. Yao and D. E. Vance. 1988. J. Biol. Chem. 263: 2998-3004) was inhibited. However, the biosynthesis of phosphatidylcholine from [3H]choline or from [3H]glycerol was not significantly reduced in the analogue-treated, compared with the untreated, hepatocytes. Nor was the incorporation of [3H]glycerol into cellular triacylglycerol altered in the monomethylethanolamine-treated cells. Furthermore, addition of monomethylethanolamine to hepatocytes did not reduce the rate of biosynthesis of phosphatidylethanolamine either from CDP-ethanolamine or from phosphatidylserine, nor was phosphatidylserine biosynthesis from [3-3H]serine affected. The 50% inhibition of VLDL secretion elicited by monomethylethanolamine was apparently specific for VLDL because there was no difference in secretion of HDL (lipid or apoprotein moieties) or albumin by cells incubated with or without the ethanolamine analogue. The experiments showed that inhibition of VLDL secretion by monomethylethanolamine was not the result of decreased biosynthesis of phospholipids, triacylglycerols, or cholesteryl esters. More subtle effects of the ethanolamine/choline analogue, for example interference by the increased amount of phosphatidylmonomethylethanolamine, in the process of assembly of lipids with apoB remain a possibility.     

NB-598: a potent competitive inhibitor of squalene epoxidase

NB-598, (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bith iophen-5-yl)methoxy]benzene-methanamine, was found to inhibit human microsomal squalene epoxidase (from Hep G2 cells) in a competitive manner. NB-598 inhibited cholesterol synthesis from [14C]acetate dose dependently in Hep G2 cells and increased the intracellular radioactivity of squalene. A single oral administration of NB-598 inhibited cholesterol synthesis from [14C]acetate in rats. Moreover, multiple oral administration of NB-598 to dogs decreased serum total and low density lipoprotein cholesterol levels and increased serum squalene levels. After termination of treatment, the reduced serum cholesterol and increased squalene levels returned to their control values.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Effect of ethanol on lipid metabolism in cultured hepatocytes.

Isolated rat hepatocytes were cultured in a modified HI-WO/BA medium for 16 h. In the following 24 h oleate or oleate plus ethanol was added to the medium. After this period the medium was changed again and the cultures were further incubated with [1-14C]oleate alone or with [1-14C]oleate plus ethanol for 6 h. This allowed a comparison of effects of short-term (6 h) and long-term (24 + 6 h) exposure to ethanol on fatty acid metabolism. The increased intracellular accumulation of triacylglycerol in the presence of ethanol was quantitatively accounted for by increased fatty acid uptake, by decreased fatty acid oxidation in the tricarboxylic acid cycle and by decreased VLDL (very-low-density lipoprotein)-triacylglycerol secretion. Ketone-body production was not affected. After short-term exposure the rate of accumulation of triacylglycerol was increased by 50%. This increase was accounted for by increased fatty acid uptake (44%), decreased tricarboxylic acid-cycle activity (49%) and decreased VLDL-triacylglycerol secretion (7%). After long-term exposure, the rate of accumulation of triacylglycerol was increased by 74%. This increase was accounted for by increased fatty acid uptake (34%), decreased tricarboxylic acid-cycle activity (34%) and decreased VLDL-triacylglycerol secretion (32%). The larger increase in accumulation of triacylglycerol after long-term exposure to ethanol was entirely accounted for by increased inhibition of secretion of VLDL-triacylglycerol. The biochemical mechanisms underlying the observations are discussed.