Sequential insulin degradation in cultured fetal hepatocytes in relation to chloroquine-dependent events.
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.
Zachayus
J L
JL
Laboratoire de Biologie-Odontologie, Université Paris 7, Institut Biomédical des Cordeliers, France.
Khan
S
S
Plas
C
C
D000818:Animals; D002478:Cells, Cultured; D002738:Chloroquine / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007328:Insulin / Q000378:metabolism*; D007339:Insulysin / Q000378:metabolism*; D008099:Liver / Q000196:embryology / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
D007328:Insulin; D002738:Chloroquine; D007339:Insulysin