INSULIN-MEDIATED INHIBITION OF APOLIPOPROTEIN-B SECRETION REQUIRES ANINTRACELLULAR TRAFFICKING EVENT AND PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION - STUDIES WITH BREFELDIN-A AND WORTMANNIN IN PRIMARY CULTURES OF RAT HEPATOCYTES

Insulin inhibition of the secretion of apolipoprotein B (apo B) was st udied in primary cultures of rat hepatocytes by using brefeldin A (BFA ), an inhibitor of protein transport from the endoplasmic reticulum (E R) to the Golgi apparatus, and by using the phosphatidylinositol 3-kin ase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 mu g/ml) for 1 h inhibited the subsequent secretion of apo B, albu min and transferrin for up to 3 h. BFA treatment resulted in the time- dependent accumulation in cells of [C-14]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plu s secreted), BFA blocked the insulin-dependent effect. These results s uggest that export of apo B from the ER is a prerequisite for the obse rved insulin effect. Treatment of hepatocytes with wortmannin for 20 m in abolished insulin inhibition of apo B secretion, suggesting that th e insulin effect on the apo B pathway involves activation of PI 3-K. E nzyme inhibitor studies indicate that chymostatin and tylcarbamoyl)-bu tylcarbamoyl]-2-oxiranecarboxylate (E-64-c) partially block insulin ef fects on apo B compared with leupeptin, which had no discernible effec t. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norl eucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat h epatocytes involves (1) vesicular movement of apo B from the ER; (2) a ctivation of PI 3-K and (3) a cellular protease that is either a cyste ine- or calcium-activated neutral protease.