Regulation of very-low-density-lipoprotein lipid secretion in hepatocyte cultures derived from diabetic animals.
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ABSTRACT:
SUBMITTER: Duerden JM
PROVIDER: S-EPMC1133262 | biostudies-other | 1989 Aug
REPOSITORIES: biostudies-other
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Project description:Hepatocytes derived from diabetic rats were cultured in serum-free Waymouth's medium containing various supplements, after an initial 4 h period during which the cells were allowed to attach to the culture dish in the presence of foetal-bovine serum (10%). After removal of serum, these cells secreted much less very-low-density lipoprotein (VLDL) apoprotein B (apoB) and triacylglycerol than those derived from normal rats when cultured for 24 h in the basal medium. Inclusion of oleate (0.75 mM) in the medium initially increased the output of apoB and triacylglycerol, but the rates remained lower than those observed in normal hepatocytes and declined to zero after 72 h. This time-dependent decline in VLDL output was prevented by addition of dexamethasone to the oleate-containing medium. Levels of apoB and triacylglycerol output characteristic of normal hepatocytes could only be completely restored, however, by further addition of a mixture of lipogenic substrates (lactate plus pyruvate) to the medium. Restoration of normal levels of VLDL secretion in diabetic hepatocytes in vitro by this means was accompanied by a normal inhibitory response of apoB and triacylglycerol output to short-term (24 h) treatment with insulin or glucagon. Exposure of the cells to insulin for 72 h enhanced the secretion of VLDL, whereas treatment with glucagon for the same period potentiated the original inhibitory effect. The defective secretion of VLDL apoB observed when diabetic hepatocytes were cultured in the basal medium for 24 h could also be rectified by inclusion of a mixture of oleate (0.75 mM), lactate (10 mM), pyruvate (1 mM), dexamethasone (1 microM) and insulin (78 nM) in the medium during the 4 h attachment period in the presence of serum. Under these conditions, the increase in the secretory response of triacylglycerol was not so pronounced.
| S-EPMC1134580 | biostudies-other
Project description:Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.
| S-EPMC2852959 | biostudies-literature
Project description:Lipid droplets (LDs) serve as crucial hubs for lipid trafficking and metabolic regulation through their numerous interactions with various organelles. While the interplay between LDs and the Golgi apparatus has been recognized, their roles and underlying mechanisms remain poorly understood. Here, we reveal the role of Ras-related protein Rab-2A (Rab2A) in mediating LD-Golgi interactions, thereby contributing to very-low-density lipoprotein (VLDL) lipidation and secretion in hepatocytes. Mechanistically, our findings identify a selective interaction between Golgi-localized Rab2A and 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) protein residing on LDs. This complex facilitates dynamic organelle communication between the Golgi apparatus and LDs, thus contributing to lipid transfer from LDs to the Golgi apparatus for VLDL2 lipidation and secretion. Attenuation of Rab2A activity via AMP-activated protein kinase (AMPK) suppresses the Rab2A-HSD17B13 complex formation, impairing LD-Golgi interactions and subsequent VLDL secretion. Furthermore, genetic inhibition of Rab2A and HSD17B13 in the liver reduces the serum triglyceride and cholesterol levels. Collectively, this study provides a new perspective on the interactions between the Golgi apparatus and LDs.
| S-SCDT-10_1038-S44318-024-00288-X | biostudies-other
Project description:In contrast to water-soluble fuels such as glucose or ketone bodies, the use of lipids as an energy source for tissues has required the development of complex structures for their transport through the aqueous plasma. In the case of endogenously synthesized triacylglycerol this is achieved by the assembly and secretion of hepatic VLDL which provides the necessary stability in an aqueous medium. An essential component of this assembly process is apo B. Dietary changes which require an increase in hepatic VLDL secretion appear to be accompanied by increases in the availability of functional apo B. Interesting questions relate to: (a) the intracellular site(s) of triacylglycerol association with apo B, and (b) the mechanism(s) by which the availability of functional apo B at this site responds to metabolic and hormonal signals which reflect dietary status and, thus, the need to secrete triacylglycerol. As regards the latter, although in some cases changes in apo B synthesis occur in response to VLDL secretion hepatic apo B mRNA levels appear to be quite stable in vitro. Intracellular switching of apo B between the secretory and degradative pathways may be important in controlling VLDL assembly and post-translational modifications of the apoprotein may also play a role by influencing its ability to bind to triacylglycerol. Transport is not the only problem associated with the utilization of a concentrated energy source such as triacylglycerol and the complex problems of waste product disposal and recycling have to be dealt with. In the case of triacylglycerol, potentially toxic waste products include atherogenic remnants and LDL. The overall problem, then, in the long-term, involves the development of a 'safe' means of utilizing triacylglycerol and this requirement accounts for much of the complexity of plasma lipoprotein metabolism. In this area, the rat could teach the human a few tricks. One of these appears to be the utilization of hepatic apo B48 rather than apo B100 for VLDL assembly in response to increases in the extrahepatic utilization of hepatically synthesized triacylglycerol. Under these conditions, the remnants of hepatic triacylglycerol utilization by peripheral tissues are cleared from the plasma much more readily via a process which seems to involve the cycling of more triacylglycerol back to the liver than that which occurs in humans. The means by which this is achieved, though, are obscure and may involve a chylomicron remnant receptor, the nature of which, itself, remains controversial.(ABSTRACT TRUNCATED AT 400 WORDS)
| S-EPMC1131384 | biostudies-other
Project description:High rates of hepatic cellular triacylglycerol synthesis and very-low-density-lipoprotein (VLDL) triacylglycerol output were maintained in vitro for at least 3 days when hepatocytes were cultured in a medium lacking insulin but supplemented with 1 microM-dexamethasone, 10 mM-lactate, 1 mM-pyruvate and 0.75 mM-oleate (supplemented medium). Under these conditions VLDL output remained constant, whereas cell triacyglycerol content increased 10-fold over 3 days, suggesting that the secretory process was saturated. Insulin, present during the first 24 h period, enhanced the storage of cellular triacylglycerol by inhibiting the secretion of VLDL. This stored triacyglycerol was subsequently released into the medium as VLDL if insulin was removed. With the supplemented medium the increased rate of VLDL secretion after insulin removal exceeded that observed under 'saturating' conditions, suggesting that pre-treatment with insulin enhanced the capacity for VLDL secretion. In contrast with the short-term (24 h) effects of insulin, longer-term exposure (greater than 48 h) to insulin enhanced the secretion of VLDL compared with insulin-untreated cultures. Under these conditions, insulin increased the net rates of triacylglycerol synthesis. The results suggest that insulin affects the secretion of VLDL triacylglycerol by two distinct and opposing mechanisms: first, by direct inhibition of secretion; second by increasing triacylglycerol synthesis, which stimulates secretion. The net effect at any time depends upon the relative importance of each of these processes.
| S-EPMC1133520 | biostudies-other
Project description:RationaleSeveral reports suggest that antisense oligonucleotides against miR-33 might reduce cardiovascular risk in patients by accelerating the reverse cholesterol transport pathway. However, conflicting reports exist about the impact of anti-miR-33 therapy on the levels of very low-density lipoprotein-triglycerides (VLDL-TAG).ObjectiveWe test the hypothesis that miR-33 controls hepatic VLDL-TAG secretion.Methods and resultsUsing therapeutic silencing of miR-33 and adenoviral overexpression of miR-33, we show that miR-33 limits hepatic secretion of VLDL-TAG by targeting N-ethylmaleimide-sensitive factor (NSF), both in vivo and in primary hepatocytes. We identify conserved sequences in the 3'UTR of NSF as miR-33 responsive elements and show that Nsf is specifically recruited to the RNA-induced silencing complex following induction of miR-33. In pulse-chase experiments, either miR-33 overexpression or knock-down of Nsf lead to decreased secretion of apolipoproteins and TAG in primary hepatocytes, compared with control cells. Importantly, Nsf rescues miR-33-dependent reduced secretion. Finally, we show that overexpression of Nsf in vivo increases global hepatic secretion and raises plasma VLDL-TAG.ConclusionsTogether, our data reveal key roles for the miR-33-NSF axis during hepatic secretion and suggest that caution should be taken with anti-miR-33-based therapies because they might raise proatherogenic VLDL-TAG levels.
| S-EPMC4108429 | biostudies-literature
Project description:The very-low-density-lipoprotein secretion rate of isolated hepatocytes obtained from rats fed a high-fat diet was half that of cells from control animals. In fat-fed rats, the initial cellular uptake of [l-14C]oleate in vitro was decreased by 25%, its esterification to triacylglycerols and phospholipids by 50% and its incorporation into very-low-density-lipoprotein triacylglycerols by 70%. Exogenous oleate was not the main precursor of very-low-density lipoproteins in these animals. Lipogenesis, a minor source of very-low-density lipoproteins with the control diet in our experimental conditions, was inhibited by 84% after fat-feeding. A short-term inhibition of lipogenesis in vitro did not result in a decrease in very-low-density-lipoprotein secretion rate. The results suggest that fat-feeding decreased availability of exogenous as well as endogenous fatty acids for synthesis of very-low-density lipoproteins.
| S-EPMC1163258 | biostudies-other
Project description:Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation. The transmembrane 6 superfamily member 2 (TM6SF2) E167K genetic variant associates with NAFLD and with reduced plasma triglyceride levels in humans. However, the molecular mechanisms underlying these associations remain unclear. We hypothesized that TM6SF2 E167K affects hepatic very low-density lipoprotein (VLDL) secretion and studied the kinetics of apolipoprotein B100 (apoB100) and triglyceride metabolism in VLDL in homozygous subjects. In 10 homozygote TM6SF2 E167K carriers and 10 matched controls, we employed stable-isotope tracer and compartmental modeling techniques to determine apoB100 and triglyceride kinetics in the 2 major VLDL subfractions: large triglyceride-rich VLDL1 and smaller, less triglyceride-rich VLDL2. VLDL1-apoB100 production was markedly reduced in homozygote TM6SF2 E167K carriers compared with controls. Likewise, VLDL1-triglyceride production was 35% lower in the TM6SF2 E167K carriers. In contrast, the direct production rates for VLDL2-apoB100 and triglyceride were not different between carriers and controls. In conclusion, the TM6SF2 E167K genetic variant was linked to a specific reduction in hepatic secretion of large triglyceride-rich VLDL1. The impaired secretion of VLDL1 explains the reduced plasma triglyceride concentration and provides a basis for understanding the lower risk of cardiovascular disease associated with the TM6SF2 E167K genetic variant.
| S-EPMC7819740 | biostudies-literature
Project description:The effects of different calcium-antagonists on secretion of very-low-density lipoprotein (VLDL) from cultured rat hepatocytes were examined. Verapamil (an inhibitor of voltage-dependent calcium channels) and EGTA (a calcium chelator) decreased VLDL-triacylglycerol secretion in a concentration-dependent manner, with maximum inhibition (about 90%) at 0.2 mM-verapamil and 5 mM-EGTA. Inorganic calcium-antagonists such as lanthanum, nickel, cobalt and manganese decreased secretion of VLDL-triacylglycerol by 55-95%, whereas the calcium-agonist barium did not affect secretion. Inhibition of VLDL-triacylglycerol secretion appeared within 30 min, without inhibition of triacylglycerol synthesis. Pulse-chase experiments revealed that verapamil and cobalt inhibited the secretory pathway itself. Cobalt showed a concentration-dependent inhibition of VLDL-triacylglycerol secretion, with maximal effect at 8 mM. Although inhibition by cobalt was not completely reversible, Trypan Blue exclusion and lactate dehydrogenase leakage indicated that the hepatocytes were not injured by cobalt or any of the other calcium-antagonists tested. Inhibition of protein synthesis by cycloheximide did not affect triacylglycerol secretion (up to 2 h), and the observed effects were therefore probably not due to impaired production of apolipoproteins. Taken together, these results suggest that calcium is important for secretion of VLDL particles.
| S-EPMC1148427 | biostudies-other
Project description:To study potential effects of hepatic cholesterol concentration on secretion of very-low-density lipoprotein (VLDL) by the liver, male rats were fed on unsupplemented chow, chow with lovastatin (0.1%), or chow with lovastatin (0.1%) and cholesterol (0.1%) for 1 week. Livers were isolated from these animals and perfused in vitro, with a medium containing [2-14C]acetate, bovine serum albumin and glucose in Krebs-Henseleit buffer, and with an oleate-albumin complex. With lovastatin feeding, the hepatic concentrations of cholesteryl esters and triacylglycerols before perfusion were decreased, although free cholesterol was unchanged. However, hepatic secretion of all the VLDL lipids was decreased dramatically by treatment with lovastatin. Although total secretion of VLDL triacylglycerol, phospholipid, cholesterol and cholesteryl esters was decreased, the decrease in triacylglycerol was greater than that in free cholesterol or cholesteryl esters, resulting in secretion of a VLDL particle enriched in sterols relative to triacylglycerol. In separate studies, the uptake of VLDL by livers from control animals or animals treated with lovastatin was measured. Uptake of VLDL was estimated by disappearance of VLDL labelled with [1-14C]oleate in the triacylglycerol moiety, and was observed to be similar in both groups. During perfusion, triacylglycerol accumulated to a greater extent in livers from lovastatin-fed rats than in control animals. The depressed output of VLDL triacylglycerols and the increase in triacylglycerol in the livers from lovastatin-treated animals was indicative of a limitation in the rate of VLDL secretion. Addition of cholesterol (either free cholesterol or human low-density lipoprotein) to the medium perfusing livers from lovastatin-fed rats, or addition of cholesterol to the diet of lovastatin-fed rats, increased the hepatic concentration of cholesteryl esters and the output of VLDL lipids. The concentration of cholesteryl esters in the liver was correlated with the secretion of VLDL by the liver. These data suggest that cholesterol is an obligate component of the VLDL required for its secretion. It is additionally suggested that cholesteryl esters are in rapid equilibrium with a small pool of free cholesterol which comprises a putative metabolic pool available and necessary for the formation and secretion of the VLDL. Furthermore, the specific radioactivity (d.p.m./mumol) of the secreted VLDL free cholesterol was much greater than that of hepatic free cholesterol, suggesting that the putative hepatic metabolic pool is only a minor fraction of total hepatic free cholesterol.
| S-EPMC1138436 | biostudies-other