Secretion of lipids, apolipoproteins, and lipoproteins by human hepatoma cell line, HepG2: effects of oleic acid and insulin

The aim of this study was to determine the effect of oleic acid and insulin on the secretion of lipoproteins by HepG2 cells grown in minimum essential medium. Triglycerides were the major neutral lipid (57% of total) and apoB was the predominant apolipoprotein (56% of total) secreted by these cells. The addition of oleate resulted in a two-fold increase in the concentration of neutral lipids but only a slight to moderate increase in the apolipoprotein (A-I, A-II, B, and E) levels. The secretion of very low density lipoproteins (VLDL) was stimulated by 425%, low density lipoproteins (LDL) by 77%, and high density lipoproteins (HDL) by 68%. Whereas neutral lipid composition of LDL was unchanged, the VLDL particles contained a significantly higher percentage of triglyceride and lower percentages of cholesterol and cholesteryl esters compared with VLDL secreted in the absence of oleate. Oleate had no significant effect on the composition of apolipoproteins in VLDL, LDL and HDL. In basal medium, insulin caused a significant decrease in the secretion of neutral lipids and apolipoproteins, particularly triglycerides and apoB. In addition to a 60-68% reduction in the total concentration of VLDL and LDL, insulin altered their composition by producing particles that had a significantly lower content of triglycerides, contained less apoB, and were deficient in apoE. There were no major changes in the concentration or composition of HDL particles. Insulin had a similar but less pronounced effect on the concentration and composition of lipoproteins secreted in the presence of oleate. The increased accumulation of triglycerides in the HepG2 cells concomitant with their reduced levels in the medium suggests that insulin may affect the secretion rather than synthesis of triglyceride-rich lipoproteins.     

Utilization of ascites plasma very low density lipoprotein triglycerides by Ehrlich cells

Much of the lipid present in the ascites plasma in which Ehrlich cells grow is contained in very low density lipoproteins (VLDL). Chemical measurements indicated that triglycerides were taken up by the cells during in vitro incubation with ascites VLDL. When tracer amounts of radioactive triolein were incorporated into the ascites VLDL, the percentage uptakes of glyceryl tri[1-(14)C]oleate and triglycerides measured chemically were similar. The cells also took up [2-(3)H]glyceryl trioleate that was added to VLDL, but the percentage of available (3)H recovered in the cell lipids was 30-40% less than that of (1 4)C from glyceryl tri[1-(1 4)C]oleate. This difference was accounted for by water-soluble (3)H that accumulated in the incubation medium, suggesting that extensive hydrolysis accompanied the uptake of VLDL triglycerides. Radioactive fatty acids derived from the VLDL triglycerides were incorporated into cell phospholipids, glycerides, and free fatty acids, and they also were oxidized to CO(2). Triglyceride utilization increased as the VLDL concentration was raised. These results suggest that one function of the ascites plasma VLDL may be to supply fatty acid to the Ehrlich cells and that the availability of fatty acid to this tumor is determined in part by the ascites plasma VLDL concentration. Although Ehrlich cells incorporate almost no free glycerol into triglycerides, considerable amounts of [2-(3)H]glyceryl trioleate radioactivity were recovered in cell triglycerides. This indicates that at least some VLDL triglycerides were taken up intact. The net uptake of VLDL protein and cholesterol was very small relative to the triglyceride uptake, suggesting that intact triglycerides are transferred from the ascites VLDL to the Ehrlich cells and that hydrolysis occurs after the triglyceride is associated with the cells.     

ABHD5/CGI-58 facilitates the assembly and secretion of apolipoprotein B lipoproteins by McA RH7777 rat hepatoma cells

Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [¹⁴C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.     

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Lipoprotein secretion in lean and obese Zucker female rats in vivo and in a single-pass-perfused liver preparation

The plasma lipoprotein composition as well as lipoprotein synthesis and secretion were studied in vivo and in a single-pass-perfused liver preparation in lean and obese Zucker rats. Compared with their lean littermates the levels in the plasma of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) + low density lipoprotein (LDL) and high density lipoprotein (HDL) were increased 4-, 2- and 2.5 fold, respectively, in obese rats. In these rats both VLDL and IDL + LDL were enriched in triglycerides, while the HDL were enriched in cholesterol. Although the VLDL and IDL + LDL protein concentrations were the same in lean and obese rats, the HDL protein concentration was 3-fold greater in the obese rats. Both the lean and obese rats incorporated similar amounts of [14C]leucine into total liver protein. However, obese rats incorporated 2.5-fold and 6-fold more [14C]leucine into VLDL and HDL in vivo, 2.7-fold and 1.7 fold more [35S]methionine in VLDL and HDL present in the perfusate, than did lean rats. The perfusate [35S]S-labelled apoproteins (apo-B100, B48; apo-E, apo-AI, apo-AIV and apo-C) were separated by gel electrophoresis and identified by autoradiography. Incorporation of [3H]glycerol into liver, VLDL, IDL + LDL and HDL triglycerides was 2-, 48-, 13- and 1.5-fold higher in obese than in lean rats, respectively. The [3H]-labelled triglycerides in VLDL and IDL + LDL present in the perfusate was 5.4-fold and 4.4-fold more in obese rat. There was no difference in the incorporation of [3H]glycerol into triglycerides of perfusate HDL between the two genotypes of rats. Thus, the hypertriglyceridaemia observed in obese Zucker rats results from very high synthetic rates of both the lipid and protein moieties of plasma lipoproteins. Before this study, no report of the simultaneous triglycerides and protein synthesis in vivo and in a single-pass-perfused liver preparations had been reported.     

Overexpression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolism in rat primary hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.     

The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes

Hepatocytes obtained from rats fed a choline-deficient diet for 3 days were cultured in a medium +/- choline (100 microM) or methionine (200 microM). We investigated how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis, and lipoprotein secretion. The mass of triacylglycerol and phosphatidylcholine secreted was increased about 3-fold and 2-fold, respectively, by the addition of either choline or methionine to the cultured cells. Similarly, a 3-fold stimulation in the secretion of [3H]triacylglycerol and [3H]phosphatidylcholine derived from [3H]oleate was observed after the addition of choline or methionine. Fractionation of secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of triacylglycerol and phosphatidylcholine from choline-deficient cells was mainly due to impaired secretion of very low density lipoproteins (VLDL) (but not high density lipoproteins (HDL)). Fluorography of L-[4,5-3H]leucine-labeled lipoproteins showed a remarkable inhibition of VLDL secretion by choline deficiency. The addition of choline or methionine stimulated the synthesis of phosphatidylcholine and increased the cellular phosphatidylcholine levels to that in normal cells. While there was little effect of choline on the synthesis and amount of cellular phosphatidylethanolamine, the addition of methionine diminished cellular phosphatidylethanolamine levels. Choline deficiency did not change the rate of incorporation of L-[4,5-3H]leucine into cellular VLDL apolipoproteins, nor the rate of disappearance of radioactivity from L-[4,5-3H]leucine-labeled cellular apoB, apoE, and apoC. These results suggest that hepatic secretion of VLDL, but not HDL, requires active phosphatidylcholine biosynthesis. Secondly, the inhibitory effect of choline deficiency on VLDL secretion can be compensated by the methylation of phosphatidylethanolamine.     

Lipid and lipoprotein synthesis in isolated and cultured hepatocytes from lean and obese Zucker rats

Hepatocytes were isolated by EDTA perfusion of livers from lean (Fa/-) and obese (fa/fa) Zucker rats. Triacylglycerol (TG) and sn-glycerol 3-phosphate were increased in fa/fa hepatocytes, but free fatty acids, cholesterol and phospholipid concentrations were similar in both groups. In spite of an identical fatty acid uptake rate, glycerolipid synthesis was higher in obese compared to lean rat hepatocytes, and this difference remained for at least 2-3 days of culture. Triacylglycerol mass secretion was 2-fold higher in obese than in lean rat hepatocytes. This was confirmed by the higher incorporation of labeled glycerol and oleic acid into the medium TG fraction floating at density 1.006 g/ml. Density gradient ultracentrifugation of [14C]oleate-labeled lipoproteins showed that fa/fa hepatocytes secreted more TG-rich lipoproteins, and that 87% of the label was in the VLDL fraction compared with 67% in the medium of Fa/- hepatocytes. Decreased utilisation of leucine for protein synthesis in obese rat compared to lean rat hepatocytes was associated with enhanced leucine oxidation to CO2. [35S]Methionine incorporation showed an identical cell protein synthesis rate. Autoradiography after PAGE separation of secreted apolipoproteins (apoBh, Bl, apoA-VI, apoE, apoA-I, apoC) showed an identical pattern in both cell types.     

Effects of probucol on lipid metabolism and secretion in long-term cultures of adult rat hepatocytes

To study the effects of probucol on hepatic lipid metabolism, we used adult rat hepatocytes cultured on a feeder layer of 3T3 cells lethally treated with mitomycin C. These cultures synthesize and secrete for at least 2 weeks various lipids from [14C]acetate and [14C]oleate precursors. Treatment with 20 micrograms/ml of probucol for 7 and 14 days decreased the secretion of various radiolabeled lipid species to the culture medium and produced an intracytoplasmic accumulation of triacylglycerol droplets. The lipids whose secretion was most decreased were free and esterified cholesterol (50-70% reduction). Secretion of triacylglycerols and phospholipids was also reduced but to a lower extent. Intracytoplasmic triacylglycerols accumulated and the activity of glycerol phosphate dehydrogenase, a marker enzyme of glycerolipid synthesis, also increased (35-56%). The total incorporation of both radioactive precursors into free and esterified cholesterol and phospholipids was reduced 20-60%. Our data show that 2-week treatment of 3T3-hepatocyte cultures with pharmacological concentrations of probucol reduces significantly lipid secretion and suggest that at least part of the in vivo hypolipidemic effect of probucol could be attributed to a decrease in the secretion of lipids (i.e., lipoproteins) by hepatocytes.     

Bile acids inhibit secretion of very low density lipoprotein by rat hepatocytes

The influence of taurocholate on very low density lipoprotein (VLDL) triacylglycerol synthesis and secretion was studied by isolated rat liver-parenchymal cells. The incorporation of [3H]glycerol into cell-associated and VLDL triacylglycerols were measured after incubation in medium containing 0.75 mM oleate. Taurocholate caused a maked decrease in VLDL [3H]triacylglycerol secretion from the hepatocytes: 50-150 microM taurocholate inhibited secretion of VLDL [3H]triacylglycerols by 70-90%. Similar results were obtained when the mass of secreted VLDL triacylglycerols was measured. Taurocholate caused a decreased secretion of VLDL [3H]triacylglycerols after 15-30 min incubation. A higher amount of cellular triacylglycerols was found in taurocholate-supplemented cells. Furthermore taurocholate did not change the intracellular lipolysis of triacylglycerols. These results suggest that bile acids interfere more probably with the assembly and/or secretion of VLDL-particles and not with earlier stages of VLDL formation, e.g. triacylglycerol synthesis.     

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.     

Metabolism of very low density lipoproteins--effect of sardine oil.

The effect of feeding fish oil on the metabolism of lipoproteins was studied in rats. Rats were fed diet containing 10% sardine or groundnut oil for 6 weeks. There was a significant decrease in the total cholesterol, phospholipids and triglycerides as well as the amount of the lipids associated with VLDL and LDL in serum in fish oil-fed rats. The synthesis and secretion of lipoproteins particularly apoB containing lipoproteins by primary cultures of hepatocytes from these rats were studied by 14(C)-acetate or 3(H)-leucine labelling. Primary cultures of hepatocytes derived from sardine oil-fed rats showed reduced incorporation of 3(H)-leucine into apoB containing lipoproteins secreted into the medium when compared to those fed groundnut oil, indicating a decreased synthesis and secretion of apoB. This was further confirmed by significantly lower incorporation of 14(C)-radioactivity into total and individual lipids of VLDL secreted into the medium, as well as that associated with different lipids in cell layer. The activity of lipoprotein lipase in adipose tissue and aorta was significantly higher in rats fed sardine oil which may cause an increased clearance of triglyceride-rich lipoproteins from circulation. These results indicate that the fish oil exerts hypolipidemic effect particularly by decreasing the synthesis and secretion of VLDL by liver and possibly by an increased clearance of triglyceride-rich lipoproteins from circulation.     

Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes

Because excess triacylglycerol (TAG) in nonadipose tissues is closely associated with the development of insulin resistance, interest has increased in the metabolism of long-chain acyl-CoAs toward beta-oxidation or the synthesis and storage of TAG. To learn whether a mitochondrial isoform of glycerol-3-phosphate acyltransferase (mtGPAT1) competes with carnitine palmitoyltransferase I (CPT I) for acyl-CoAs and whether it contributes to the formation of TAG, we overexpressed rat mtGPAT1 13-fold in primary hepatocytes obtained from fasted rats. When 100, 250, or 750 microM oleate was present, both TAG mass and the incorporation of [14C]oleate into TAG increased more than twofold in hepatocytes overexpressing mtGPAT1 compared with vector controls. Although the incorporation of [14C]oleate into CO2 and acid-soluble metabolites increased with increasing amounts of oleate in the media, these metabolites were approximately 40% lower in the Ad-mtGPAT1 infected cells, consistent with competition for acyl-CoAs between CPT I and mtGPAT1. A 50-60% decrease was also observed in [14C]oleate incorporation into cholesteryl ester. With increasing amounts of exogenous oleate, [14C]TAG secretion increased appropriately in vector control-infected hepatocytes, suggesting that the machinery for VLDL-TAG biogenesis and secretion was unaffected. Despite the marked increases in TAG synthesis and storage in the Ad-mtGPAT1 cells, however, the Ad-mtGPAT1 cells secreted the same amount of [14C]TAG as the vector control cells. Thus, in isolated hepatocytes, mtGPAT1 may synthesize a cytosolic pool of TAG that cannot be secreted.     

Identification and partial characterization of discrete apolipoprotein B containing lipoprotein particles produced by human hepatoma cell line HepG2

The purpose of this study was to test the use of human hepatocarcinoma HepG2 cells as a model for studying the formation and secretion of human hepatic lipoproteins. To this end, we determined the rate of accumulation and percent composition of neutral lipids and apolipoproteins in the culture medium of HepG2 cells and isolated and partially characterized the apolipoprotein B (ApoB) containing lipoprotein particles. The rates of accumulation in the medium of HepG2 cells, grown in minimum essential medium during a 24-h incubation, of triglycerides, cholesterol, and cholesterol esters expressed as microgram/(g of cell protein X h) were 373 +/- 55, 167 +/- 14, and 79 +/- 10, respectively; the secretion rates for apolipoproteins B, A-I, E, A-II, and C-III were 372 +/- 36, 149 +/- 14, 104 +/- 13, 48 +/- 4, and 13 +/- 1 microgram/(g of cell protein X h), respectively. The major portion of ApoB was present in very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) (84%), with the remainder occurring in high-density lipoproteins (HDL) (16%). Approximately 10-13% of ApoA-I and ApoA-II were present in VLDL and LDL, while 60% of ApoE occurred in HDL and 40% in VLDL and LDL. To separate ApoB-containing lipoproteins, secreted lipoproteins were fractionated by either sequential immunoprecipitation or immunoaffinity chromatography with antibodies to ApoB and ApoE. Results showed that 60-70% of ApoB occurred in the culture medium as lipoprotein B (LP-B) and 30-40% as lipoprotein B:E (LP-B:E). Both ApoB-containing lipoproteins represent polydisperse systems of spherical particles ranging in size from 100 to 350 A for LP-B and from 200 to 500 A for LP-B:E. LP-B particles were identified in VLDL, LDL, and HDL, while LP-B:E particles were only present in VLDL and LDL. The major neutral lipid of both ApoB-containing lipoproteins was triglyceride (50-70% of the total neutral lipid content); cholesterol and cholesterol esters were present in equal amounts. The LP-B:E particles contained 70-90% ApoB and 10-30% ApoE. The ApoB was identified in both types of particles as B-100. A time study on the accumulation of ApoB-containing lipoproteins showed that LP-B particles were secreted independently of LP-B:E particles.     

Perinatal hepatocyte/hepatoma hybrids: construction of clones that express the developmentally regulated monoacyglycerol acyltransferase activity

Microsomal monoacyglycerol acyltransferase is a developmentally expressed enzyme that catalyzes the synthesis of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and palmitoyl-CoA. The activity is present in liver from fetal and suckling rats but is absent in the adult. In order to obtain a stable permanent cell line that expresses this activity, Fao rat hepatoma cells and hepatocytes from 8-day-old baby rats were hybridized and clones were selected. Two hybrids (HA1 and HA7) expressed monoacylglycerol acyltransferase activity. Like fetal hepatocytes, but unlike hepatocytes from postnatal rats, the HA cells had high rates of [14C]acetate incorporation into glycerolipids, cholesterol, and cholesteryl esters, and they secreted triacylglycerol into the media. Monoacylglycerol acyltransferase specific activity increased 2.5-fold as the cells divided in culture, suggesting growth-dependent regulation. The specific activities of glycerol-P acyltransferase, the committed step of the microsomal pathway of glycerolipid synthesis, and diacylglycerol acyltransferase, the activity unique to triacylglycerol biosynthesis, were comparable to the levels of the corresponding activities in fetal hepatocytes. Addition of insulin or dexamethasone to the media increased the incorporation of [14C]oleate into triacyglycerol about 1.7-fold within 2 h, but had little effect on [14C]oleate incorporation into phospholipid. These hormonally responsive rat-hepatoma/hepatocyte hybrids reflect the fetal stage of hepatocyte development in five major aspects of lipid metabolism: sterol, fatty acid, and triacylglycerol biosynthesis, glycerolipid secretion, and the presence of the developmentally expressed monoacylglycerol pathway.     

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.     

Lipolysis is a prerequisite for lipid accumulation in HepG2 cells induced by large hypertriglyceridemic very low density lipoproteins.

Lipoprotein kinetic studies have demonstrated that a large proportion of Sf 60-400 very low density lipoprotein (VLDL) is cleared directly from the circulation in Type IV hypertriglyceridemic subjects, at an unknown tissue site. The present studies were designed to investigate the role of hepatocytes in this process and to define the conditions, whereby Type IV Sf 60-400 VLDL would induce lipid accumulation in HepG2 cells. Type IV VLDL (Sf 60-400) failed to augment the total cholesterol, esterified cholesterol, or triglyceride content of HepG2 cells following 24-h incubations. Coincubation of bovine milk lipoprotein lipase (LPL) and Type IV VLDL with HepG2 cells induced a 3-fold increment in cellular esterified cholesterol mass (p less than 0.005) and a 7-fold increase in cellular triglyceride mass (p less than 0.005), compared to VLDL alone. The increased cellular lipid mass was associated with increased oleate incorporation into cellular cholesterol esters and triglycerides. Exogenous LPL hydrolyzed 76% of the VLDL triglyceride over 24 h. LPL action on Type IV VLDL was sufficient to promote cellular uptake of these lipoproteins, while elevated media-free fatty acid levels were not. Although HepG2 cells secrete apolipoprotein (apo) E, we assessed the role of VLDL-associated apoE in the lipid accumulation induced by VLDL plus LPL. ApoE-rich and apoE-poor Type IV VLDL subfractions induced similar increments in cellular esterified cholesterol in the presence of LPL, despite a 4-fold difference in apoE content. Sf 60-400 VLDL, from subjects homozygous for the defective apoE2, plus LPL, behaved identically to Type IV VLDL plus LPL. Type IV VLDL plus LPL, preincubated with anti-apoE (1D7) and apoB (5E11) monoclonal antibodies, known to block the binding of apoE and -B, respectively, to the LDL receptor failed to block lipid accumulation. In contrast, apoE-poor Type IV VLDL, apoE2 VLDL, and VLDL plus 1D7 were taken up poorly by J774 cells, cells that secrete LPL, but not apoE. These studies suggest that lipolytic remodeling of large Type IV VLDL by LPL is a prerequisite for their uptake by HepG2 cells and that HepG2 cell-secreted apoE rather than VLDL-associated apoE is the ligand involved in uptake.     

Effect of mevinolin and glycyrrhizinic acid on cholesterol and bile acid metabolism in cultured rabbit hepatocytes]

A comparison of effects of two hypocholesterolemic drugs--mevinolin and glycyrrhizinic acid, on cholesterol and bile acid metabolism in cultured rabbit hepatocytes has been carried out. The following parameters have been determined: i) cholesterol synthesis from [2-14C]acetate; ii) bile acid production from newly synthesized and [4-14C]-labeled HDL2 cholesterol, and, iii) total cholesterol efflux into the incubation medium Mevinolin (0.5 microgram/ml) inhibited [2-14C] acetate incorporation into cholesterol by more than 90%. Conversely, glycyrrhizinic acid did not influence cholesterol synthesis even when used at high (100 micrograms/ml) concentrations but stimulated the conversion of endogenous (by 37%) and exogenous (by 18%) cholesterol into bile acids and increased, in addition, the proportion of bile acids in the total sterol pool released from hepatocytes into the incubation medium. At the same time, mevinolin used at 0.5 microgram/ml decreased the bile acid production by endogenous (by 27%) and exogenous (by 40%) cholesterol. The data obtained suggest that glycyrrhizinic acid exerts hypocholesterolemic action by stimulation of cholesterol conversion into bile acids without any effect on cholesterol synthesis. As for mevinolin, it has a cholesterol-suppressing effect via a mechanism of cholesterol synthesis inhibition only.     

Low density lipoproteins isolated from the blood of patients with ischemic heart disease induce accumulation of lipids in human aortic intima cells

Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were isolated from the blood of healthy subjects and CHD patients. LDL from the blood of healthy individuals did not raise the intracellular lipid values within 24 h of cultivation. During intracellular lipid values within 24 h of cultivation. During the same incubation period. LDL obtained from the blood of CHD patients caused a 2- to 5-fold rise in cholesterol esters as well as a 1.5- to 3-fold rise in free cholesterol and triglycerides, while the intracellular phospholipid levels remained unchanged. In one of the three cases, the ability to raise the intracellular level of cholesterol esters was demonstrated by VLDL (500 micrograms/ml) derived from CHD patients blood. HDL did not affect the lipid levels in smooth muscle cells cultured from unaffected intima. The obtained data suggests that circulating LDL and, possibly, VLDL in the blood of CHD patients are capable of inducing the accumulation of fat in vascular wall cells.