Octanoate inhibits very low-density lipoprotein secretion in primary cultures of chicken hepatocytes

The effects of octanoate, a medium-chain fatty acid, on very low-density lipoprotein (VLDL) secretion in primary cultures of chicken hepatocytes were compared with those of palmitate. Palmitate added to the incubation media at concentrations up to 0.36 mM increased intracellular triacylglycerol (TG) accumulation and VLDL-TG secretion in a concentration-dependent manner, whereas the addition of octanoate alone (0.21-0.6 mM) did not change these parameters. VLDL-TG secretion from hepatocytes cultured in media to which 0.6 or 1.0 mM octanoate had been added in the presence of 0.21 mM palmitate was significantly lower than that obtained under control incubation conditions (0.21 mM palmitate only). The addition of 1.0 mM octanoate to the incubation media with or without 0.21 mM palmitate decreased VLDL apolipoprotein B (apoB) secretion. These results demonstrate that the addition of octanoate to primary cultures of chicken hepatocytes reduces VLDL secretion in respect of both TG and apoB secretion. It is suggested that medium-chain fatty acids are a factor modulating VLDL secretion, which plays a key role in fat deposition in chickens.     

Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes

The aim of this study was to investigate the direct effects of growth hormone (GH) on production and secretion of apolipoprotein B (apoB)-containing lipoproteins from hepatocytes. Bovine GH (5-500 ng/ml) was given for 1 or 3 days to rat hepatocytes cultured on laminin-rich matrigel in serum-free medium. The effects of GH were compared with those of 3 nM insulin and 500 microM oleic acid. GH increased the editing of apoB mRNA, and the proportion of newly synthesized apoB-48 (of total apoB) in the cells and secreted into the medium changed in parallel. GH increased total secretion of apoB-48 (+30%) and apoB-48 in very low density lipoproteins (VLDL) more than twofold. Total apoB-100 secretion decreased 63%, but apoB-100-VLDL secretion was unaffected by GH. Pulse-chase studies indicated that GH increased intracellular early degradation of apoB-100 but not apoB-48. GH had no effect on apoB mRNA or LDL receptor mRNA levels. The triglyceride synthesis, the mass of triglycerides in the cells, and the VLDL fraction of the medium increased after GH incubation. Three days of insulin incubation had effects similar to those of GH. Combined incubation with oleic acid and GH had additive effects on apoB mRNA editing and apoB-48-VLDL secretion. In summary, GH has direct effects on production and secretion of apoB-containing lipoproteins, which may add to the effects of hyperinsulinemia and increased flux of fatty acids to the liver during GH treatment in vivo.     

Postnatal development of hepatocellular apolipoprotein B assembly and secretion in the rat.

In this study, we explored the paradox that in suckling rats the serum concentration of LDL is high although the liver secretes only minimal quantities of VLDL, the presumed precursor of LDL. Freshly isolated hepatocytes and hepatocytes in primary culture obtained from adult (90 days old) and suckling (17 days old) rats were used to investigate the synthesis and secretion of apolipoprotein B (apoB) and lipids as well as the density profile of secreted apoB-containing lipoproteins. Furthermore, the effects of dexamethasone and oleate on apoB biogenesis were investigated in primary cultures of hepatocytes from adult and suckling rats. Hepatocytes from suckling rats were unable to assemble mature VLDL but secreted apoB as primordial lipoprotein particles in the LDL-HDL density range. Intracellular degradation of apoB was also reduced in hepatocytes from suckling rats compared with that in hepatocytes from adults. The immaturity in VLDL assembly and apoB degradation of hepatocytes from suckling rats could be overcome by treating the cultures with dexamethasone plus oleate or dexamethasone alone. The lower microsomal triacylglycerol transfer protein (MTP) mRNA concentrations in hepatocytes from suckling rats in comparison with hepatocytes from adult rats were not reflected in lower MTP activity levels. Furthermore, dexamethasone plus oleate treatment had no effect on MTP activity although VLDL assembly and secretion were clearly stimulated. We conclude that, during the suckling period of the rat, serum LDL is directly produced by the liver. This is a result of impaired hepatic VLDL assembly, which is a consequence of low triglyceride synthesis and an inefficient mobilization of bulk lipids in the second step of VLDL assembly.     

Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.     

Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.     

Treatment with high-dose simvastatin reduces secretion of apolipoprotein B-lipoproteins in patients with diabetic dyslipidemia

HMG-CoA reductase inhibitors (statins) are effective lipid-altering drugs for the treatment of dyslipidemia in patients with type 2 diabetes mellitus. We conducted a randomized, double-blind, placebo-controlled, crossover design trial to determine the effects of simvastatin, 80 mg/day, on plasma lipid and lipoprotein levels and on the metabolism of apolipoprotein B (apoB) in VLDL, intermediate density lipoprotein (IDL), and LDL and of triglycerides (TGs) in VLDL. Simvastatin therapy decreased TG, cholesterol, and apoB significantly in VLDL, IDL, and LDL. These effects were associated with reduced production of LDL-apoB, mainly as a result of reduced secretion of apoB-lipoproteins directly into the LDL density range. Statin therapy also reduced hepatic production of VLDL-TG. There were no effects of simvastatin on the fractional catabolic rates of VLDL-apoB or -TG or LDL-apoB. The basis for decreased VLDL-TG secretion during simvastatin treatment is not clear, but recent studies suggest that statins may activate peroxisomal proliferator-activated receptor alpha (PPARalpha). Activation of PPARalpha could lead to increased hepatic oxidation of fatty acids and less synthesis of TG for VLDL assembly.     

Myristic acid increases dense lipoprotein secretion by inhibiting apoB degradation and triglyceride recruitment

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B-100 (apoB-100) levels. To identify mechanisms at the level of production, rat hepatoma cells, McA-RH7777, were incubated with [(35)S]methionine and either fatty acid-BSA complexes or BSA alone. There were increases in labeled apoB-100 secretion with saturated fatty acids palmitic and myristic (MA) (153 +/- 20% and 165 +/- 11%, respectively, relative to BSA). Incubation with polyunsaturated docosahexaenoic acid (DHA) decreased secretion to 26 +/- 2.0%, while monounsaturated oleic acid (OA) did not change it. In pulse-chase studies, MA treatment resulted in reduced apoB-100 degradation, in agreement with its promotion of secretion. In triglyceride (TG) studies, synthesis was stimulated equally by OA, MA, and DHA, but TG secretion was relatively decreased with MA and DHA. With OA, the majority of newly secreted apoB100-lipoproteins was d < or = 1.006, but with MA, they were much denser (1.063 < d). Furthermore, the relative recruitment of newly synthesized TG to lipoproteins was impaired with MA. We conclude that mechanisms for effects of specific dietary fatty acids on plasma lipoprotein levels may include changes in hepatic production. In turn, hepatic production may be regulated by specific fatty acids at the steps of apoB-100 degradation and the recruitment of nascent TG to lipoprotein particles.     

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.     

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.     

Synthesis and secretion of very low density lipoproteins by isolated rat hepatocytes in suspension: Role of diacylglycerol acyltransferase

Isolated rat hepatocytes in suspension secrete very low density lipoproteins (VLDL) at a rate comparable with that of the perfused liver. The apoproteins of these lipoproteins are mainly of the B and E type. The amount of apoprotein C in VLDL secreted by hepatocytes is much less than that present in VLDL obtained from rat serum. Incubation of hepatocytes in the presence of fatty acids stimulates the intracellular synthesis of triacylglycerols and their secretion in VLDL. This stimulation is a linear function of the palmitic acid concentration up to 1.6 mm, the highest concentration tested. Colchicine (50 μm) reduced VLDL secretion by 90%. The stimulation of triacylglycerol synthesis and VLDL secretion upon incubation of hepatocytes with fatty acids is paralleled by an enhanced activity of microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), the only enzyme exclusively involved in the synthesis of triacylglycerols. A mixture of oleic (0.2 mm) and palmitic (0.2 mm) acid added to the cell medium stimulates the activity of DGAT by 354%. This increase in enzyme activity persisted during cell homogenization and subsequent preparation of microsomes to assay the enzyme. It is concluded that freshly isolated hepatocytes in suspension represent a good system to study triacylglycerol synthesis and VLDL secretion, and that the stimulatory effects of fatty acids on these processes are, at least partially, mediated by enhanced activities of DGAT.     

A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels

Nonalcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and elevated free fatty acids are present in the majority of patients with metabolic syndrome and type 2 diabetes mellitus and are strongly associated with hepatic insulin resistance. In the current study, we tested the hypothesis that an increased rate of fatty acid oxidation in liver would prevent the potentially harmful effects of fatty acid elevation, including hepatic triglyceride (TG) accumulation and elevated TG secretion. Primary rat hepatocytes were transduced with adenovirus encoding carnitine palmitoyltransferase 1a (Adv-CPT-1a) or control adenoviruses encoding either beta-galactosidase (Adv-beta-gal) or carnitine palmitoyltransferase 2 (Adv-CPT-2). Overexpression of CPT-1a increased the rate of beta-oxidation and ketogenesis by approximately 70%, whereas esterification of exogenous fatty acids and de novo lipogenesis were unchanged. Importantly, CPT-1a overexpression was accompanied by a 35% reduction in TG accumulation and a 60% decrease in TG secretion by hepatocytes. There were no changes in secretion of apolipoprotein B (apoB), suggesting the synthesis of smaller, less atherogenic VLDL particles. To evaluate the effect of increasing hepatic CPT-1a activity in vivo, we injected lean or obese male rats with Adv-CPT-1a, Adv-beta-gal, or Adv-CPT-2. Hepatic CPT-1a activity was increased by approximately 46%, and the rate of fatty acid oxidation was increased by approximately 44% in lean and approximately 36% in obese CPT-1a-overexpressing animals compared with Adv-CPT-2- or Adv-beta-gal-treated rats. Similar to observations in vitro, liver TG content was reduced by approximately 37% (lean) and approximately 69% (obese) by this in vivo intervention. We conclude that a moderate stimulation of fatty acid oxidation achieved by an increase in CPT-1a activity is sufficient to substantially reduce hepatic TG accumulation both in vitro and in vivo. Therefore, interventions that increase CPT-1a activity could have potential benefits in the treatment of NAFLD.     

Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with ApoB,as well as for ApoB lipidation

The assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranes in vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by approximately 45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes.     

A targeted apoB38.9 mutation in mice is associated with reduced hepatic cholesterol synthesis and enhanced lipid peroxidation

Familial hypobetalipoproteinemia (FHBL) due to truncation-specifying mutations of apolipoprotein B (apoB), which impair hepatic lipid export in very low-density lipoprotein (VLDL) particles, is associated with fatty liver. In an FHBL-like mouse with the apoB38.9 mutation, fatty liver develops despite reduced hepatic fatty acid synthesis. However, hepatic cholesterol contents in apoB38.9 mice are normal. We found that cholesterogenic enzymes (3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol-C5-desaturase, and 7-dehydrocholesterol reductase) were consistently downregulated in two separate expression-profiling experiments using a total of 19 mice (n = 7 each for apob(+/+) and apob(+/38.9), and n = 5 for apob(38.9/38.9)) and Affymetrix Mu74Av2 GeneChip microarrays. Results were confirmed by real-time PCR. Cholesterol synthesis rates in cultured hepatocytes were reduced by 35% and 25% in apob(38.9/38.9) and apob(+/38.9), respectively, vs. apob(+/+). Hepatic triglycerides and lipid peroxides, the latter measured by thiobarbituric acid-reactive substances (TBARS) assay, were significantly elevated in apob(+/38.9) (117%) and apob(38.9/38.9) (132%) vs. apob(+/+) (100%), as were mRNA expression of the microsomal lipid peroxidizing enzymes Cyp4A10 and Cyp4A14. Hepatic lipid peroxide levels were positively correlated with triglyceride contents (r = 0.601, P = 0.0065). Thus the fatty liver due to a VLDL secretion defect is associated with insufficient adaptation to triglyceride accumulation and with increased lipid peroxidation. In contrast, apoB38.9 mice effectively maintain cholesterol homeostasis in the liver, at least in part, by reducing hepatic cholesterol synthesis.     

Mechanisms of hepatic very low density lipoprotein overproduction in insulin resistance. Evidence for enhanced lipoprotein assembly, reduced intracellular ApoB degradation, and increased microsomal triglyceride transfer protein in a fructose-fed hamster model

A novel animal model of insulin resistance, the fructose-fed Syrian golden hamster, was employed to investigate the mechanisms mediating the overproduction of very low density lipoprotein (VLDL) in the insulin resistant state. Fructose feeding for a 2-week period induced significant hypertriglyceridemia and hyperinsulinemia, and the development of whole body insulin resistance was documented using the euglycemic-hyperinsulinemic clamp technique. In vivo Triton WR-1339 studies showed evidence of VLDL-apoB overproduction in the fructose-fed hamster. Fructose feeding induced a significant increase in cellular synthesis and secretion of total triglyceride (TG) as well as VLDL-TG by primary hamster hepatocytes. Increased TG secretion was accompanied by a 4.6-fold increase in VLDL-apoB secretion. Enhanced stability of nascent apoB in fructose-fed hepatocytes was evident in intact cells as well as in a permeabilized cell system. Analysis of newly formed lipoprotein particles in hepatic microsomes revealed significant differences in the pattern and density of lipoproteins, with hepatocytes derived from fructose-fed hamsters having higher levels of luminal lipoproteins at a density of VLDL versus controls. Immunoblot analysis of the intracellular mass of microsomal triglyceride transfer protein, a key enzyme involved in VLDL assembly, showed a striking 2.1-fold elevation in hepatocytes derived from fructose-fed versus control hamsters. Direct incubation of hamster hepatocytes with various concentrations of fructose failed to show any direct stimulation of its intracellular stability or extracellular secretion, further supporting the notion that the apoB overproduction in the fructose-fed hamster may be related to the fructose-induced insulin resistance in this animal model. In summary, hepatic VLDL-apoB overproduction in fructose-fed hamsters appears to result from increased intracellular stability of nascent apoB and an enhanced expression of MTP, which act to facilitate the assembly and secretion of apoB-containing lipoprotein particles.     

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.     

Inhibition of fatty acid synthesis decreases very low density lipoprotein secretion in the hamster.

The hamster was developed as a model to study very low density lipoprotein (VLDL) metabolism, since, as is the case in humans, the hamster liver was found to synthesize apoB-100 and not apoB-48. The effect of inhibiting fatty acid synthesis on the hepatic secretion of VLDL triglyceride (TG) and apolipoprotein (apo) B-100 in this model was then investigated. In an in vivo study, hamsters were fed a chow diet containing 0.15% TOFA (5-tetradecyloxy-2-furancarboxylic acid), an inhibitor of acetyl-CoA carboxylase. After 6 days of treatment, plasma triglyceride and cholesterol levels were decreased by 30.2% and 11.6%, respectively. When the secretion of VLDL-TG by the liver was measured in vivo after injection of Triton WR 1339, TOFA treatment was found to decrease VLDL-TG secretion by 40%. In subsequent in vitro studies utilizing cultured primary hamster hepatocytes, incubation with 20 microM TOFA for 4 h resulted in 98% and 76% inhibition in fatty acid and triglyceride synthesis, respectively; VLDL-TG secretion was decreased by 90%. When hepatocytes were pulsed with [3H]leucine, incubation with TOFA resulted in a 50% decrease in the incorporation of radiolabel into secreted VLDL apoB-100. The results of this study indicate that inhibition of intracellular triglyceride synthesis decreases the secretion of VLDL-TG and apoB-100, and does not result in the secretion of a dense, triglyceride-depleted lipoprotein.     

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.     

An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine (PC). We investigated whether there was diminished secretion of lipoproteins from hepatocytes derived from mice that lacked PEMT (Pemt(-/-)) compared with Pemt(+/+) mice. Hepatocytes were incubated with 0.75 mm oleate, the media were harvested, and triacylglycerol (TG), PC, apolipoprotein (apo) B100, and apoB48 were isolated and quantified. Compared with hepatocytes from Pemt(+/+) mice, hepatocytes from Pemt(-/-) mice secreted 50% less TG, whereas secretion of PC was unaffected. Fractionation of the secreted lipoproteins on density gradients demonstrated that the decrease in TG was in the very low density lipoprotein (VLDL)/low density lipoprotein fractions. The secretion of apoB100 was decreased by approximately 70% in VLDLs/low density lipoproteins, whereas there was no significant decrease in apoB48 secretion in any fraction. Transfection of McArdle hepatoma cells (that lack PEMT) with PEMT cDNA enhanced secretion of TG in the VLDLs. Because the levels of PC in the hepatocytes and hepatoma cells were unaffected by the lack of PEMT expression, there appears to be an unexpected requirement for PEMT in the secretion of apoB100-containing VLDLs.