Sequential insulin degradation in cultured fetal hepatocytes in relation to chloroquine-dependent events
Zachayus, J.L.; Khan, S.; Plas, C.
American Journal of Physiology 271(3 Pt 1): E417-E425
1996
ISSN/ISBN: 0002-9513 PMID: 8843733 Document Number: 466606
Insulin cellular degradation was studied in cultured 18-day-old fetal rat hepatocytes in the presence and absence of insulin degradation inhibitors that decrease the glycogenic response to insulin. After cell incubation with 3 nM (125I)A14 or -B26 insulin, hormone degradation products associated with cells or present in the medium were analyzed by high-performance liquid chromatography. Within cells, four components containing intact (125I)A14 insulin A-chain and part of the B-chain (A-1-A-4, according to increasing retention times) were found together with two (125I)B26 insulin B-chain COOH-terminal fragments (B-1 and B-2). Medium degradation intermediates comprised 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, whereas the others increased progressively to plateau after 10 min. Chloroquine inhibited the formation of A-1, A-2, and B-1 by 70-80%, whereas that of A-3, A-4, and B-2 was not significantly affected. N-ethylmaleimide and bacitracin, two inhibitors of insulin-degrading enzyme (IDE), decreased the formation of chloroquine-dependent cellular peptides. Thus cell-associated insulin degradation implied primarily two cleavages in B-chain near the COOH-terminus, the one sensitive to chloroquine and IDE inhibitors occurring after endosomal segregation of insulin and its receptor.