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Triacylglycerol accumulation and secretion in hepatocyte cultures. Effects of insulin, albumin and Triton WR 1339.

ABSTRACT: We have investigated the possibility that the apparent inhibition of very-low-density lipoprotein (VLDL)-triacylglycerol secretion by the addition of insulin to rat hepatocyte cultures may result from insulin-mediated enhancement of hepatic lipase secretion and, consequently, of extracellular triacylglycerol hydrolysis. We have, therefore, studied the effects of the inhibitor of lipase activity, Triton WR 1339, on the secretion of triacylglycerol by cultured rat hepatocytes. Incubation of hepatocyte cultures with increasing concentrations of Triton WR 1339 increased the accumulation of acylglycerol in the medium, suggesting that, in normal incubations, a substantial rate of degradation of secreted triacylglycerol does occur and that it results in an under-estimation of the rate of triacylglycerol secretion. However, Triton did not counteract the inhibitory effects of insulin, suggesting that the observed increased activity of hepatic lipase induced by the hormone cannot account for the inhibition of acylglycerol accumulation in the medium that occurred in the presence of insulin. BSA increased the accumulation of triacylglycerol in culture media by about 2-fold and also decreased the activity of hepatic lipase by 80%. A causative relationship between these two effects was supported by the further observation that Triton abolished the effects of BSA on triacylglycerol accumulation in the medium. The implications of these data for the validity of the use of Triton for the study of hepatic rates of triacylglycerol production in vivo and of secretion by hepatocytes in vitro are discussed.

SUBMITTER: Emmison N

PROVIDER: S-EPMC1132839 | biostudies-other | 1992 Jul

REPOSITORIES: biostudies-other

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Project description:Cytosolic triacylglycerol labelled from [3H]oleate accounted for almost 50% (57 +/- 22 nmol/mg of protein) of the total cellular triacylglycerol which was newly synthesized by cultured hepatocytes during a 24 h incubation. Insulin decreased the export of triacylglycerol as very-low-density lipoprotein (VLDL) during this period. This resulted in a sequestration of newly synthesized triacylglycerol in the cytosol, rather than in the particulate fraction of the cell. Longer periods of incubation with [3H]oleate resulted in increased concentrations of newly synthesized triacylglycerol within the cell, most of which (78 +/- 3% after 48 h; 80 +/- 3% after 72 h) was located within the cytosolic fraction. The quantity of newly synthesized triacylglycerol in the cell cytosol was further increased by insulin. During these periods there were decreases in the amounts of triacylglycerol associated with the particulate fraction of the cell, irrespective of the presence or absence of insulin. In no case was a decrease in VLDL triacylglycerol secretion in response to insulin accompanied by an increased triacylglycerol content in the particulate fraction of the cell. In some experiments, the fate of the cytosolic triacylglycerol was studied by pulse labelling with [3H]oleate. In these cases, when insulin was removed from the medium of cells to which they had previously been exposed, more newly synthesized triacylglycerol was secreted compared with cells which had not been exposed to insulin. This extra triacylglycerol was mobilized from the cytosolic rather than from the particulate fraction of the cell. Subsequent addition of insulin to the medium prevented the mobilization of cytosolic triacylglycerol. These results suggest that insulin enhances the storage of hepatocellular triacylglycerol in a cytosolic pool. Deficiency of insulin in the medium stimulates the mobilization of this pool which is channelled into the secretory pathway, entering the extracellular medium as VLDL.

Project description:The hypolipidemic effect of furan carboxamide derivatives was investigated using the Triton WR-1339 rat model. Nineteen compounds were synthesized, including furan-2-carboxamides of benzophenones and acetophenones (a(1-4)), anilines and amine derivatives (a(5-9)), picolinic-2-carboxamide derivatives of benzophenones and acetophenone (a(10-12)) and furan-2-carboxylate esters of benzophenones and acetophenones, substituted phenols and alcohols (b(1-7)). All the necessary steps were taken to synthesize, purify, and characterize these compounds. They were synthesized by reacting acyl chlorides of the heterocycles with their corresponding amines in the presence of pyridine and tert-butyl acetate. While the conventional heating method yielded acceptable yields for some of the reactions under reflux, the microwave synthesis reactor achieved significantly higher yields for others. Rats with hyperlipidemia were induced with Triton WR-1339 and then subjected to in vivo testing via an intraperitoneal injection of 200 mg kg-1 Triton WR-1339. The model was tested using an oral dose of bezafibrate (100 mg kg-1). After 7 hours of treatment with Triton, the new derivatives represented by compounds a(1-2), a(4-5), a7, and a(10-12) showed significant activity against the complete lipid profile, including a decrease in triglyceride, total cholesterol, and low-density lipoprotein cholesterol and an increase in high-density lipoprotein cholesterol plasma levels. At 20 mg kg-1 dose, these compounds were superior to other lipid-lowering agents in reducing triglyceride levels and slightly increased high-density lipoprotein cholesterol levels. These results indicate a mutual mechanism of action of novel compounds with fibrates, where they have a marked effect on triglyceride and high-density lipoprotein cholesterol levels; for example, a5 causes a significant reduction (p 0.0001) of triglyceride levels by 86%, and a remarkable increase (p 0.0001) in high-density lipoprotein cholesterol plasma levels by 65% as compared to hyperlipidemic rats.

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Triacylglycerol accumulation and secretion in hepatocyte cultures. Effects of insulin, albumin and Triton WR 1339.

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