A deazaadenosine-insensitive methylation of phosphatidylethanolamine is involved in lipoprotein secretion

We have examined the effect of inhibitors of methylation of phosphatidylethanolamine on lipoprotein secretion from cultured rat hepatocytes. The incorporation of [1-3H]ethanolamine into phosphatidylcholine of hepatocytes and secreted lipoproteins was inhibited by greater than 90% by the methylation inhibitors 3-deazaadenosine and Neplanocin. In addition, these compounds strongly inhibited the incorporation of [3-3H]serine into the choline moiety of phosphatidylcholine of the hepatocytes, but had no effect on incorporation of [3-3H]serine into secreted phosphatidylcholine. The results suggest that a pool of phosphatidylcholine targeted for lipoprotein secretion originates from phosphatidylethanolamine made from serine and this methylation reaction has the unique property of being insensitive to 3-deazaadenosine.     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into 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.     

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.     

Inhibition of phosphatidylcholine synthesis via the phosphatidylethanolamine methylation pathway impairs incorporation of bulk lipids into VLDL in cultured rat hepatocytes

Inhibition of phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway was shown to decrease the secretion of VLDL from primary rat hepatocytes (Nishimaki-Mogami et al. 1996. BIOCHIM: Biophys. Acta. 1304: 21-31). To understand further the role of PE methylation, we determined the effect of bezafibrate, an inhibitor of PE methylation, on VLDL assembly within the microsomal lumen. Bezafibrate was shown to decrease VLDL (triacylglycerol) secretion only when cellular PE methylation was active in the presence of methionine. Pulse-chase experiments showed that bezafibrate treatment did not impair the movement of [(35)S]apolipoprotein (apo)B-48 from microsomal membranes into the lumen. However, bezafibrate treatment resulted in reduced VLDL-[(35)S]apoB-48 and increased [(35)S]apoB-48-containing particles in the HDL density range (HDL-[(35)S]apoB-48) within the lumen. Inhibition of PE methylation by bezafibrate or 3-deazaadenosine after the completion of HDL-[(35)S]apoB-48 assembly effectively decreased VLDL-[(35)S]apoB-48 secretion with a concomitant increase in HDL-[(35)S]apoB-48 secretion. These findings suggest that inhibition of PC synthesis via the PE methylation pathway impairs the stage of bulk triacylglycerol incorporation during the assembly of VLDL.     

Role of phosphatidylcholine biosynthesis in the regulation of lipoprotein homeostasis

PURPOSE OF REVIEW: This review summarizes the role of phosphatidylcholine metabolism in plasma lipoprotein homeostasis. RECENT FINDINGS: While it was previously known that phosphatidylcholine biosynthesis was required for normal hepatic VLDL secretion, recent studies have shown that both phosphatidylcholine biosynthetic pathways (the cytidine 5'-diphosphocholine and the phosphatidylethanolamine methylation pathways) are required. In addition, a requirement of acyl-coenzyme A synthetase 3, but not acyl-coenzyme A synthetase 1 or 4, for phosphatidylcholine synthesis and VLDL secretion is now documented. ABCA1 has been implicated in the transfer of phosphatidylcholine to apolipoproteinA-1 both during and after secretion of apolipoproteinA-1. Other studies have introduced the concept of reverse phosphatidylcholine transport in which both HDL and LDL supply phosphatidylcholine to the liver. An unexpected finding is that half of the phosphatidylcholine delivered to liver from lipoproteins is converted into triacylglycerol. SUMMARY: The liver is both a donor of phosphatidylcholine during the assembly and secretion of lipoproteins as well as a recipient of phosphatidylcholine from plasma lipoproteins.     

Isolation and analysis of lipoproteins secreted by rat liver hepatocytes

A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.     

Head group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes

We have demonstrated that hepatic very low density lipoprotein (VLDL) secretion requires active phosphatidylcholine (PC) synthesis via either the CDP-choline pathway or phosphatidylethanolamine (PE) methylation pathway (Yao, Z., and Vance, D.E. (1988) J. Biol. Chem. 263, 2998-3004). In the present work, the head group specificity of phospholipid synthesis required for lipoprotein secretion was investigated in cultured hepatocytes isolated from choline-deficient rats. When N-monomethylethanolamine (0.1 mM) or N,N-dimethylethanolamine (0.1 mM) was added to the culture medium, the cells synthesized correspondingly phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). However, the synthesis of PDME could correct the impaired VLDL secretion only to a limited extent, whereas the synthesis of PMME inhibited VLDL secretion. Although dimethylethanolamine did not promote VLDL secretion as well as choline, dimethylethanolamine altered the increased triacylglycerol synthesis in the choline-deficient cells as effectively as choline. Supplementation of the culture medium with ethanolamine (0.1 mM) had little effect on cellular PE or PC levels, nor was normal VLDL secretion resumed. However, the amounts of cellular PC and PE were both decreased when the medium was supplemented with N-monomethylethanolamine or N,N-dimethylethanolamine. These results suggest that the choline head group moiety of PC is specifically required for normal VLDL secretion and cannot be replaced with ethanolamine, monomethylethanolamine, or dimethylethanolamine. In addition, the impaired VLDL secretion from the choline-deficient hepatocytes could also be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilization of a methyl group from betaine for PC formation via methylation of PE.     

Synthesis and secretion of VLDL by rat hepatocytes--modulation by cholesterol and phospholipids.

The synthesis and secretion of apoB, the major protein component of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), were studied using rat hepatocytes maintained in primary culture. Supplementation of hepatocytes with rat serum VLDL and LDL increased the production of apoB while delipidated lipoproteins had no significant effect, suggesting a role for lipids in the production of apoB. Addition of cholesterol to the culture medium also increased the production of apoB in a concentration-dependent manner. Pulse labelling followed by chase in presence of cholesterol indicated enhancement in apoB secretion. Mevinolin which inhibits cholesterol synthesis significantly reduced the secretion of apoB. The presence of phosphatidylcholine and phosphatidylethanolamine in the culture medium also increased the secretion of apoB into the medium. These data suggest that availability of lipids, particularly cholesterol, is an important determinant of apoB synthesis and secretion as VLDL.     

Fatty acids inhibit N-methylation of phosphatidylethanolamine in rat hepatocytes and liver microsomes

Supplementation of rat hepatocytes with various fatty acids in the culture medium reduced the conversion of [3H]phosphatidylethanolamine into phosphatidylcholine. Unsaturated fatty acids were the most effective inhibitors of phospholipid methylation. The inhibition of phosphatidylethanolamine methylation by oleate (2 mM) was reversed within 1 h after replacement with fatty acid-deficient medium. Fatty acids and their CoA derivatives (0.15-0.5 mM) produced 50% inhibition of phosphatidylethanolamine methyltransferase in rat liver microsomes. The first methylation reaction was the site of fatty acid inhibition, as methylation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was not reduced in the presence of oleate. The inhibition by oleate was reversed by inclusion of bovine serum albumin or by addition of phospholipid liposomes. Thus, while fatty acids stimulate phosphatidylcholine biosynthesis in hepatocytes via the CDP-choline pathway, the methylation pathway is inhibited.     

Lipoprotein synthesis and secretion by cultured rat hepatocytes. Parallel inhibition of secretion of VLDL, HDL and albumin by monensin

The biosynthesis and secretion of very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by cultured normal rat hepatocytes was investigated with particular emphasis on its modification by monensin. This acidic ionophore coordinately inhibited the rates of secretion of the several VLDL apolipoproteins and the VLDL lipids, suggesting an effect late in the process of biosynthesis and secretion, probably at the stage of exiting from the Golgi apparatus. The secretion of immunoreactive albumin into the medium was comparably inhibited, implying that the pathway and mechanisms involved in albumin secretion may be closely similar to those for VLDL synthesis and secretion. Secretion of phospholipids and of apolipoproteins E and A-I in the HDL fraction increased progressively with time over 18 h in control incubations but was strongly inhibited by monensin. During extended incubation with monensin at high concentrations (10 microM), there was a net release to the medium of a number of hepatocyte proteins, including some that comigrated with apolipoprotein A-I and apolipoprotein C, making it appear that monensin increased the secretion of these apolipoproteins. However, using labeled amino acids, it was shown by autoradiography and by immunoprecipitation that secretion of newly-synthesized, radioactive apolipoprotein A-I and apolipoprotein C was actually inhibited by monensin. These results are compatible with the conclusion that HDL synthesis and secretion may occur by mechanisms closely related to those for synthesis and secretion of albumin and VLDL.     

Effects of chylomicron remnants and beta-VLDL on the class and composition of newly secreted lipoproteins by HepG2 cells

The regulation of lipoprotein secretion in the cell line HepG2 was studied. HepG2 cells were preincubated with chylomicron remnants (triglyceride- and cholesterol-rich) or with beta very low density lipoproteins (beta-VLDL) (cholesterol-rich). The medium was removed and the cells were incubated for and additional 24 hr in a lipoprotein-free medium that contained either [2-3H]glycerol or DL-[2-3H]mevalonate. Cells and media were harvested, and lipoproteins were separated and fractionated. The mass and radioactivity of the lipids in cells and in the lipoproteins were measured. The activities of cellular acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase were also determined. Preincubation with chylomicron remnants induced an increase in cellular triglyceride and stimulated both HMG-CoA reductase and ACAT. Preincubation with beta-VLDL induced an increase in cellular free and esterified cholesterol, inhibited HMG-CoA reductase and stimulated ACAT. Although the absolute amount of VLDL is small, chylomicron remnants induced large relative increases in the amount of triglyceride and phospholipid secreted in VLDL and decreases in the amount of triglyceride secreted in low density (LDL) and high density (HDL) lipoproteins as well as a decrease in the amount of phospholipid secreted in HDL. In contrast, preincubation with beta-VLDL did not affect triglyceride secretion, but markedly stimulated the amount of phospholipid secreted in HDL. Comparison of the mass of glycerolipid actually secreted with that calculated from the cellular specific activity suggested that glycerolipids are secreted from single, rapidly equilibrating pools. Cholesterol and cholesteryl ester secretion were affected differently. Preincubation with chylomicron remnants increased the amount of free cholesterol secreted in both VLDL and LDL, but did not alter cholesteryl ester secretion. Preincubation with beta-VLDL increased free cholesterol secretion in all lipoprotein fractions and increased cholesteryl ester secretion in VLDL and LDL, but not HDL. Comparison of isotope and mass data suggested that the cholesteryl ester secreted came primarily from a preformed, rather than an newly synthesized, pool. In summary, these data provide insight to the mechanism whereby a liver cell regulates the deposition of exogenous lipid.     

A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion

Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a choline-supplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDP-choline pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K for the cytidylyltransferase. Moreover, the amount and m activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.     

Biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine route is an important pathway in isolated rat hepatocytes

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.     

Inhibition by chloroquine of the secretion of very low density lipoproteins by cultured rat hepatocytes

Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.     

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.     

Incorporation of [2-3H] ethanolamine into rat muscle phosphoglycerides

The contribution of phosphatidylethanolamine methylation to phosphatidylcholine biosynthesis in rat muscle was investigated by studying the incorporation of [2-3H] ethanolamine. The specific radioactivities of individual molecular species of muscle phosphoglycerides were measured by a combination of argentation thin-layer chromatography and countercurrent distribution. The specific radioactivity of phosphatidylethanolamine was approximately one thousand times that of phosphatidylcholine. Amongst individual phosphatidylethanolamines, hexaenoic species possessed the highest specific radioactivities and tetraenoic the lowest. Because of the very low incorporation into phosphatidylcholine, the specific radioactivities of combined rather than of individual fractions were measured. The results indicate that the contribution of phosphatidylethanolamine methylation to the overall biosynthesis of phosphatidylcholine in muscle is of minor importance.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Hepatocyte damage induced by carbon tetrachloride: inhibited lipoprotein secretion and changed lipoprotein composition

Changes of lipoprotein secretion and composition in response to CCl4 treatment were studied in monolayer cultures of rat primary hepatocytes. (1) CCl4 decreased secretion of very low density lipoproteins (VLDL) by about 85%, while high density lipoprotein (HDL) secretion was less affected (about 40%). The effect was concentration-dependent. (2) CCl4 significantly inhibited secretion of VLDL- and HDL-associated triglycerides and cholesterol esters. VLDL- and HDL-associated cholesterol was not affected, while secretion of phospholipids was increased. (3) Hepatocytes secreted the apolipoproteins B48, B100, E, C, and A-I. CCl4 reduced secretion of apoproteins associated with VLDL by almost 20%, and by about 75% when associated with HDL. The de novo synthesis of apolipoproteins was attenuated by CCl4. (4) CCl4 caused variations in the apolipoprotein composition in VLDL and HDL. CCl4 intoxication of the liver affected the morphology and/or function of the lipoproteins, which drastically impaired their ability to act as transport vehicles for lipids from the liver to the circulation.