AJP - Endocrinology and Metabolism, Vol 271, Issue 3 E417-E425, Copyright © 1996 by American Physiological Society
Sequential insulin degradation in cultured fetal hepatocytes in relation to chloroquine-dependent events
J. L. Zachayus, S. Khan and C. Plas
Laboratoire de Biologie-Odontologie, Universite Paris 7, Institut Biomedical des Cordeliers, France.
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 (A1-A4,
according to increasing retention times) were found together with two
[125I]B26 insulin B-chain COOH-terminal fragments (B1 and B2). Medium
degradation intermediates comprised B1 and B2 but not A1-A4. Cellular
insulin fragments A3 and B2 exhibited a maximal transient accumulation
after 2 min, whereas the others increased progressively to plateau after 10
min. Chloroquine inhibited the formation of A1, A2, and B1 by 70-80%,
whereas that of A3, A4, and B2 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.
