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.     

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.     

Eicosapentaenoic acid inhibits synthesis and secretion of triacylglycerols by cultured rat hepatocytes

Primary cultures of rat hepatocytes were used to study the effects of eicosapentaenoic and oleic acid on synthesis and secretion of triacylglycerols associated with very low density lipoproteins. From the experiments the following was observed. Oleic acid markedly stimulates secretion as well as synthesis of triacylglycerols, whereas eicosapentaenoic acid causes very little or no increase in secretion or synthesis as compared to a fatty-acid-free medium. The effects could already be observed after 15 min incubation. The inhibitory effect of eicosapentaenoic acid is reversible within 1-2 h. Eicosapentaenoic acid inhibits much of the stimulatory effect of oleic acid on synthesis and secretion of triacylglycerols. The cellular uptake of eicosapentaenoic acid is somewhat higher than that of oleic acid and the metabolism of these fatty acids to acid-soluble materials is similar. Eicosapentaenoic acid does not affect the secretory pathway of triacylglycerols per se. From these results it may be concluded that the mechanism for the inhibitory effect of eicosapentaenoic acid on triacylglycerol secretion is probably via reduced triacylglycerol synthesis.     

DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis

Dietary triacylglycerols are a major source of energy for animals. The absorption of dietary triacylglycerols involves their hydrolysis to free fatty acids and monoacylglycerols in the intestinal lumen, the uptake of these products into enterocytes, the resynthesis of triacylgylcerols, and the incorporation of newly synthesized triacylglycerols into nascent chylomicrons for secretion. In enterocytes, the final step in triacylglycerol synthesis is believed to be catalyzed primarily through the actions of acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. In this study, we analyzed intestinal triacylglycerol absorption and chylomicron synthesis and secretion in DGAT1-deficient (Dgat1(-/-)) mice. Surprisingly, DGAT1 was not essential for quantitative dietary triacylglycerol absorption, even in mice fed a high fat diet, or for the synthesis of chylomicrons. However, Dgat1(-/-) mice had reduced postabsorptive chylomicronemia (1 h after a high fat challenge) and accumulated neutral-lipid droplets in the cytoplasm of enterocytes when chronically fed a high fat diet. These results suggest a reduced rate of triacylglycerol absorption in Dgat1(-/-) mice. Analysis of intestine from Dgat1(-/-) mice revealed activity for two other enzymes, DGAT2 and diacylglycerol transacylase, that catalyze triacylglycerol synthesis and apparently help to compensate for the absence of DGAT1. Our findings indicate that multiple mechanisms for triacylglycerol synthesis in the intestine facilitate triacylglycerol absorption.     

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes.

Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.     

The effects of dietary n-3 polyunsaturated fatty acids delivered in chylomicron remnants on the transcription of genes regulating synthesis and secretion of very-low-density lipoprotein by the liver: modulation by cellular oxidative state

The influence of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (PUFA) (derived from fish or corn oil, respectively) on the expression of mRNA for four genes involved in the regulation of the synthesis, assembly, and secretion of very-low-density lipoprotein (VLDL) in the liver was investigated in normal rat hepatocytes and after manipulation of the cellular oxidative state by incubation with N-acetyl cysteine (NAC) or CuSO(4). The four genes investigated were those encoding apolipoprotein B (apoB), the microsomal triacylglycerol transfer protein (MTP), and the enzymes acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2), which play a role in the regulation of triacylglycerol and cholesteryl ester synthesis, respectively. mRNA levels for apoB, MTP, and DGAT were unaffected by either fish or corn oil chylomicron remnants, but the amount of ACAT2 mRNA was significantly reduced after incubation of the hepatocytes with fish oil remnants as compared with corn oil remnants or without remnants. These findings indicate that the delivery of dietary n-3 PUFA to hepatocytes in chylomicron remnants downregulates the expression of mRNA for ACAT2, and this may play a role in their inhibition of VLDL secretion. However, when the cells were shifted into a pro-oxidizing or pro-reducing state by pretreatment with CuSO(4) (1 mM) or NAC (5 mM) for 24 hr, levels of mRNA for MTP were increased by about 2- or 4-fold, respectively, by fish oil remnants, whereas corn oil remnants had no significant effect. Fish oil remnants also caused a smaller increase in apoB mRNA in comparison with corn oil remnants in NAC-treated cells (+38%). These changes would be expected to lead to increased VLDL secretion rather than the decrease associated with dietary n-3 PUFA in normal conditions. These findings suggest that relatively minor changes in cellular redox levels can have a major influence on important liver functions such as VLDL synthesis and secretion.     

Construction of a recombinant human FGF1 expression vector for mammary gland-specific expression in human breast cancer cells

Studies have shown that not only does palmitic acid promote triglyceride (TG) accumulation, but it also affects cell viability in in vitro steatosis models. However, to what degree these effects are mediated by steatosis in goose primary hepatocytes is unknown. In this study, the effects of palmitic acid on the lipid metabolism homeostasis pathway and on apoptosis were determined. The authors measured the mRNA levels of genes involved in TG synthesis, lipid deposition, fatty acid oxidation and the assembly and secretion of VLDL-TG in goose primary hepatocytes. The results indicated that palmitic acid can significantly reduce the activity of goose hepatocytes, and that palmitic acid had a significant effect on TG accumulation; however, with increasing palmitic acid concentrations, the extracellular TG and extracellular VLDL concentration gradually decreased. With increasing palmitic acid concentrations, the gene expression levels of DGAT1, DGAT2, PPARα, CPT-1, FoxO1 and MTTP (which regulate hepatic TG synthesis, fatty acid oxidation and the assembly and secretion of VLDL-TGs) first increased and then decreased; the change in PLIN gene expression was palmitic acid dose-dependent, similar to the regulatory mode of intracellular TG accumulation. In conclusion, this study clearly shows that palmitic acid can promote TG accumulation and induce apoptosis in goose primary hepatocytes, and this effect may be related to the lipid metabolism pathway.     

Lipoprotein lipase-mediated sequestration of long-chain polyunsaturated triacylglycerols in serum LDL from normal and hypothyroid rats

Rat serum VLDL, unlike human, contains significant proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids. Hypothyroidism in this species is characterized by low levels of serum VLDL, the accumulation of LDL, elevated levels of lipoprotein lipase and depressed hepatic lipase activity. The hypothyroid rat thus represents an interesting model in which to study hepatic VLDL metabolism and the substrate specificity of lipoprotein lipase. This report shows that serum IDL and LDL in both euthyroid and hypothyroid rats contain progressively enhanced proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids when compared to VLDL. Hypothyroidism resulted in a decrease in the proportion of 22:6 fatty acid within the serum VLDL triacylglycerols when compared to euthyroid VLDL. Lipolysis of VLDL from euthyroid rats in vitro using the perfused rat heart system resulted in increases or sequestration of triacylglycerols containing long-chain polyunsaturated fatty acids within the IDL fraction similar to those seen in vivo. It is concluded that lipoprotein lipase-mediated hydrolysis of VLDL triacylglycerols and the conversion of VLDL to IDL and LDL in the rat results in a progressive sequestration of the longer-chain polyunsaturated triacylglycerol molecular species with the IDL and LDL.     

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 1990 Borden Award Lecture. Dietary regulation of fatty acid and triglyceride metabolism.

The level of circulating triacylglycerols is determined by the balance between their delivery into the plasma and their removal from it. Plasma triacylglycerols are derived either from dietary fat as chylomicrons or from endogenous hepatic synthesis as very low density lipoproteins. Their removal occurs through the action of lipoprotein lipase after which the fatty acids are either stored in adipose tissue or oxidized, primarily in skeletal muscle and heart. The composition of the diet has been shown to influence many of these processes. Hepatic fatty acid synthesis and triacylglycerol secretion are affected by the quantity and composition of dietary fat, carbohydrate, and protein. Polyunsaturated but not saturated fats reduce hepatic fatty acid synthesis by decreasing the amount of the lipogenic enzymes needed for de novo fatty acid synthesis. Dietary fish oils are particularly effective at reducing both fatty acid synthesis and triacylglycerol secretion and as a result are hypotriacylglycerolemic, particularly in hypertriacylglycerolemic individuals. In addition, dietary fish oils can increase the oxidation of fatty acids and lead to increased activity of lipoprotein lipase in skeletal muscle and heart. It appears that the hypotriacylglycerolemic effect of dietary fish oils is mediated by effects on both synthesis and removal of circulating triacylglycerols.     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.     

Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes

Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze the final committed step of triacylglycerol (TG) synthesis in hepatocytes. After its synthesis in the endoplasmic reticulum (ER) TG is either stored in cytosolic lipid droplets (LDs) or is assembled into very low-density lipoproteins in the ER lumen. TG stored in cytosolic LDs is hydrolyzed by adipose triglyceride lipase (ATGL) and the released fatty acids are converted to energy by oxidation in mitochondria. We hypothesized that targeting/association of ATGL to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Individual inhibition of DGAT1 or DGAT2 in Huh7 hepatocytes incubated with oleic acid did not yield differences in TG accretion while combined inhibition of both DGATs completely prevented TG synthesis suggesting that either DGAT can efficiently esterify exogenously supplied fatty acid. DGAT2-made TG was stored in larger LDs, whereas TG formed by DGAT1 accumulated in smaller LDs. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, the ATGL activator ABHD5/CGI-58, or LD coat proteins PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL was found to preferentially target to LDs generated by DGAT1 and fatty acids released from TG in these LDs were also preferentially used for fatty acid oxidation. Combined inhibition of DGAT2 and ATGL resulted in larger LDs, suggesting that the smaller size of DGAT1-generated LDs is the result of increased lipolysis of TG in these LDs.     

Lipoprotein lipase of cultured mesenchymal rat heart cells. IV. Modulation of enzyme activity by VLDL added to the culture medium.

Lipoprotein lipase activity was studied in rat heart cell cultures grown in the presence of 20% fetal calf and horse serum and a medium concentration of triacylglycerol of 0.03 mg/ml. After 6--8 days, when the enzyme activity had reached high levels, the cells were incubated for 24 h in a medium containing 20% serum derived from fasted or fed rats. No change in enzyme activity occurred in the presence of fasted rat serum, but a 50% fall was observed with fed rat serium. When the complete culture medium was supplemented with rat plasma VLDL (0.075--0.75 mg triacylglycerol) a pronounced decrease in lipoprotein lipase activity occurred after 3--5 h of incubation. Similar extent of enzyme fall was observed also in the presence of triacylglycerol-rich lipoproteins isolated from rat plasma after feeding of safflower oil or lard, even though the fatty acid composition of the triacylgylcerol varied markedly. As the addition of VLDL to the culture medium resulted in a lesser fall of heparin releasable than residual activity it seems that there was no direct inhibition of surface bound enzyme activity and that the transport of the enzyme to the cell surface was not affected. These data indicate that addition of VLDL to the culture medium resulted in a fall in enzyme synthesis, while total protein synthesis as determined by incorporation of [3H]leucine, remained unchanged. This inhibition could be reproduced by increasing free fatty acid concentration of the medium, however addition of excess albumin to VLDL-containing medium did not prevent the fall in enzyme activity. The present results obtained with cultured rat hearts cells suggest that in vivo plasma levels of triacylglycerol-rich lipoproteins could modulate the lipoproteins could modulate the lipoprotein lipase activity of the heart.     

Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.     

The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

The optimum cofactor requirements for triacylglycerol biosynthesis in rat adipose-tissue homogenates containing mitochondrial, microsomal and cytosolic fractions were investigated. In general the optimum concentrations of cofactors for triacylglycerol biosynthesis were found to differ from those for total fatty acid esterification. The results provided further evidence for the key role of phosphatidate phosphohydrolase in the regulation of triacylglycerol biosynthesis. Albumin was included in the incubation medium to permit the use of concentrations of added fatty acids that would swamp the effects of endogenous fatty acids. The addition of albumin had little effect on the incorporation of palmitic acid and stearic acid into lipids including triacylglycerols. By contrast, a critical concentration of albumin (about 60 muM) was required before incorporation of oleic acid or linoleic acid into triacylglycerols occurred. The system was used to study the incorporation of different 1-14C-labelled fatty acids from a mixture of unesterified fatty acids [palmitic acid 30%; stearic acid 10%; oleic acid 40%; linoleic acid 20% (molar percentages)] separately into the positions 1,2 and 3 of triacyl-sn-glycerols. In general the stereo-specific distribution of the labelled fatty acids incorporated into triacylglycerols paralleled the normal distribution of fatty acids within rat adipose-tissue triacylglycerols, suggesting that the specificities of the relevant acyltrasferases have the major role in determining the positional distribution of fatty acids within triacylglycerols.     

Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase.

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.     

Fatty acid specificity for the synthesis of triacylglycerol and phosphatidylcholine and for the secretion of very-low-density lipoproteins and lysophosphatidylcholine by cultures of rat hepatocytes.

1. The synthesis and secretion of glycerolipids by monolayer cultures of rat hepatocytes was measured by using radioactive choline, glycerol and fatty acids and by measuring the concentration of triacylglycerols in the cells. 2. The incorporation of glycerol into triacylglycerol and the accumulation of this lipid in hepatocytes showed little specificity for fatty acids, except for eicosapentaenoate, which stimulated least. Oleate was more effective at stimulating triacylglycerol secretion than were palmitate, stearate, arachidonate and eicosapentaenoate. 3. Linoleate, linolenate, arachidonate and eicosapentaenoate stimulated the incorporation of glycerol and choline into phosphatidylcholine that was secreted into the medium. By contrast, palmitate and stearate produced relatively high incorporations into the phosphatidylcholine that remained in the cells. 4. The incorporation of glycerol and choline into lysophosphatidylcholine in the medium was stimulated 2-3-fold by all of the unsaturated fatty acids tested, whereas palmitate and stearate failed to stimulate if the acids were added separately. When 1 mM-stearate was added with 1 mM-linoleate, the incorporation of linoleate into lysophosphatidylcholine was about 4 times higher than that of stearate. 5. It is proposed that the secretion of lysophosphatidylcholine by the liver could provide a transport system for choline and essential unsaturated fatty acids to other organs.     

Prostaglandins suppress VLDL secretion in primary rat hepatocyte cultures: relationships to hepatic calcium metabolism.

In primary cultures of rat hepatocytes, prostaglandin E2 and prostaglandin D2 (PGE2 and PGD2) inhibited the secretion of very low density lipoprotein (VLDL)-associated apoB, triacylglycerol, and cholesterol. These effects were concentration-dependent and remained apparent for at least 3 days of culture without an effect on the apoB/triacylglycerol ratio of the secreted VLDL. Prostaglandins had no effect on the overall synthesis of triacylglycerol but triacylglycerol accumulated within the cells, without intracellular accumulation of apoB. PGE2, when added to the medium together with glucagon, increased the inhibition of VLDL secretion, compared to that observed with glucagon alone. However, PGE2 did not increase the stimulatory effect of glucagon on ketogenesis. Unlike glucagon, the prostaglandins did not inhibit fatty acid synthesis nor did they stimulate ketogenesis or production of cAMP. Thus, of all the parameters of hepatic lipid metabolism studied, PGE2 and PGD2 selectively affected VLDL. Selective inhibition of VLDL secretion was also observed with the calcium antagonist verapamil. The divalent cation ionophore A23187 also inhibited VLDL release but, in contrast, also inhibited fatty acid and cholesterol synthesis. The results suggest that VLDL secretion is modulated at some optimal cell calcium concentration that may be mediated selectively by agents such as prostaglandins.     

Simultaneous determination of molecular species of monoacylglycerols,diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography

The aim of the present study was to investigate the applicability of a previously developed method for the analysis of triacylglycerol molecular species to the simultaneous determination of triacylglycerols, diacylglycerols and monoacylglycerols of human very-low-density lipoproteins (VLDL). Ten elderly women were recruited for the study. Blood was obtained in fasting conditions and VLDL were isolated by ultracentrifugation. Neutral lipids were separated by solid-phase extraction and were subsequently injected on a reversed-phase HPLC system, with an elution system composed of acetone in acetonitrile. The method allowed the separation of four monoacylglycerols, 18 diacylglycerols and 24 triacylglycerols, including the resolution of positional isomers of diacylglycerols. Monoacylglycerols were composed of oleic, linoleic, palmitic and stearic acids. The major diacylglycerols were 1,2-dilinoleoyl-glycerol and 1,3-dilinoleoyl-glycerol (14.24+/-1.02 and 17.93+/-1.42%, respectively). The main triacylglycerols quantified were dioleoyl-stearoyl-glycerol (OOS), oleoyl-dipalmitoyl-glycerol (OPP), trilinoleoyl-glycerol (LLL) and linoleoyl-distearoyl-glycerol (LSS), accounting for 11.25+/-2.15, 10.14+/-2.05, 9.35+/-2.30 and 8.56+/-1.56%, respectively. An inverse relationship between polarity and fatty acid disappearance from triacylglycerols (r(2)=0.82, P<0.05) and from diacylglycerols (r(2)=0.93, P<0.01) was discovered. In conclusion, the method allowed, for the first time, the easy, rapid and simultaneous determination in a single chromatogram of triacylglycerol, diacylglycerol and monoacylglycerol molecular species of human VLDL by reversed-phase HPLC.     

Overexpression of human diacylglycerol acyltransferase 1, acyl-coa:cholesterol acyltransferase 1, or acyl-CoA:cholesterol acyltransferase 2 stimulates secretion of apolipoprotein B-containing lipoproteins in McA-RH7777 cells

The relative importance of each core lipid in the assembly and secretion of very low density lipoproteins (VLDL) has been of interest over the past decade. The isolation of genes encoding diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) provided the opportunity to investigate the effects of isolated increases in triglycerides (TG) or cholesteryl esters (CE) on apolipoprotein B (apoB) lipoprotein biogenesis. Overexpression of human DGAT1 in rat hepatoma McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of TG. These effects were associated with decreased intracellular degradation and increased secretion of newly synthesized apoB as VLDL. Similarly, overexpression of human ACAT1 or ACAT2 in McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of CE. This led to decreased intracellular degradation and increased secretion of VLDL apoB. Overexpression of ACAT2 had a significantly greater impact upon assembly and secretion of VLDL from liver cells than did overexpression of ACAT1. The addition of oleic acid (OA) to media resulted in a further increase in VLDL secretion from cells expressing DGAT1, ACAT1, or ACAT2. VLDL secreted from DGAT1-expressing cells incubated in OA had a higher TG:CE ratio than VLDL secreted from ACAT1- and ACAT2-expressing cells treated with OA. These studies indicate that increasing DGAT1, ACAT1, or ACAT2 expression in McA-RH7777 cells stimulates the assembly and secretion of VLDL from liver cells and that the core composition of the secreted VLDL reflects the enzymatic activity that is elevated.