Insulin inhibits the maturation phase of VLDL assembly via a phosphoinositide 3-kinase-mediated event

LY 294002 (80 mu mol/L), an inhibitor of phosphoinositide 3-kinase, was use d to investigate the involvement of this enzyme in the insulin-mediated reg ulation of very low density lipoprotein (VLDL) apolipoprotein B (apoB) outp ut from cultured rat hepatocytes. Newly synthesized apoB was pulse-labeled with [S-35] methionine and was then allowed to assemble, via an intermediat e precursor stage, into mature VLDL during subsequent chase periods. Brefel din A (BFA, 0.2 mug/mL) was used to discriminate between the role of insuli n in the regulation of the early, compared with the later, events of VLDL a ssembly, including apoB degradation. Insulin (78 nmol/L), when present duri ng 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 c onditions, as was the net synthesis of apoB-100 and apoB-48. The presence o f BFA during the pulse-labeling period and subsequent chase period prevente d 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. R emoval of insulin at this stage reversed the overall inhibitory effect of i nsulin. Furthermore, when insulin remained present during this period, the simultaneous presence of LY 294002 also reversed the inhibitory effect of i nsulin on VLDL apoB output and abolished the increase in apoB degradation. The results suggest that insulin signaling via phosphoinositide 3-kinase in hibited the maturation phase of VLDL assembly by preventing bulk lipid tran sfer to a VLDL precursor, thus enhancing the degradation of apoB. There was no inhibition of the conversion of newly synthesized apoB into the VLDL pr ecursor form.