SEQUENTIAL INSULIN DEGRADATION IN CULTURED FETAL HEPATOCYTES IN RELATION TO CHLOROQUINE-DEPENDENT EVENTS

Sequential insulin degradation in cultured fetal hepatocytes in relati on to chloroquine-dependent events. Am. J. Physiol. 271 (Endocrinol. M etab. 34): E417-E425, 1996.-Insulin cellular degradation was studied i n cultured 18-day-old fetal rat hepatocytes in the presence and absenc e of insulin degradation inhibitors that decrease the glycogenic respo nse to insulin. After cell incubation with 3 nM [I-125]A14 or -B26 ins ulin, hormone degradation products associated with cells or present in the medium were analyzed by high-performance liquid chromatography. W ithin cells, four components containing intact [I-125]A14 insulin A-ch ain and part of the B-chain (A(1)-A(4), according to increasing retent ion times) were found together with two [I-125]B26 insulin B-chain COO H-terminal fragments (B-1 and B-2). Medium degradation intermediates c omprised B-1 and B-2 but not A(1)-A(4). Cellular insulin fragments A(3 ) and B-2 exhibited a maximal transient accumulation after 2 min, wher eas the others increased progressively to plateau after 10 min. Chloro quine inhibited the formation of A(1), A(2), and B-1 by 70-80%, wherea s that of A(3), A(4), and B-2 was not significantly affected. N-ethylm aleimide and bacitracin, two inhibitors of insulin-degrading enzyme (I DE), decreased the formation of chloroquine-dependent cellular peptide s. Thus cell-associated insulin degradation implied primarily two clea vages in B-chain near the COOH-terminus, the one sensitive to chloroqu ine and IDE inhibitors occurring after endosomal segregation of insuli n and its receptor.