title: Insulin inhibits the maturation phase of VLDL assembly via a phosphoinositide 3-kinase-mediated event.
abstract: LY 294002 (80 micromol/L), an inhibitor of phosphoinositide 3-kinase, was used to investigate the involvement of this enzyme in the insulin-mediated regulation of very low density lipoprotein (VLDL) apolipoprotein B (apoB) output from cultured rat hepatocytes. Newly synthesized apoB was pulse-labeled with [(35)S]methionine and was then allowed to assemble, via an intermediate precursor stage, into mature VLDL during subsequent chase periods. Brefeldin A (BFA, 0.2 microgram/mL) was used to discriminate between the role of insulin in the regulation of the early, compared with the later, events of VLDL assembly, including apoB degradation. Insulin (78 nmol/L), when present during the pulse-labeling and subsequent chase periods, inhibited the secretion of apoB-100 and apoB-48 as VLDL by 53% and 56%, respectively. Degradation of both was concomitantly increased. Secretion of high density lipoprotein apoB, derived from VLDL precursors, was relatively unaffected under these conditions, as was the net synthesis of apoB-100 and apoB-48. The presence of BFA during the pulse-labeling period and subsequent chase period prevented the maturation of VLDL in the insulin-treated and the non-insulin-treated cells. BFA was then removed, allowing the maturation of VLDL to proceed. Removal of insulin at this stage reversed the overall inhibitory effect of insulin. Furthermore, when insulin remained present during this period, the simultaneous presence of LY 294002 also reversed the inhibitory effect of insulin on VLDL apoB output and abolished the increase in apoB degradation. The results suggest that insulin signaling via phosphoinositide 3-kinase inhibited the maturation phase of VLDL assembly by preventing bulk lipid transfer to a VLDL precursor, thus enhancing the degradation of apoB. There was no inhibition of the conversion of newly synthesized apoB into the VLDL precursor form.
authors.affiliation: Metabolic Research Laboratory, Oxford Centre for Diabetes, Nuffield Department of Clinical Medicine, University of Oxford, Radcliffe Infirmary, Oxford, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Brown
authors.forename: G F
authors.initials: GF
authors.lastname: Gibbons
mesh_terms: D000818:Animals; D001055:Apolipoproteins B / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004336:Drug Antagonism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D008075:Lipoproteins, HDL / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000096:biosynthesis*; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051381:Rats; D014280:Triglycerides / Q000096:biosynthesis
chemical_list: D001055:Apolipoproteins B; D020126:Brefeldin A; D002867:Chromones; D004791:Enzyme Inhibitors; D007328:Insulin; D008075:Lipoproteins, HDL; D008079:Lipoproteins, VLDL; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D014280:Triglycerides; C030106:very low density lipoprotein triglyceride; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one