Sequential insulin degradation in cultured fetal hepatocytes in
relation with chloroquine-dependent events .
Zachayus, Jean-Luc, Shahid Khan, and Christiane Plas.
Laboratoire de Biologie-Odontologie, Universit[acute]e Paris 7,
Institut Biom[acute]edical des Cordeliers, 15 rue de l'Ecole de
M[acute]edecine, 75006 Paris, France
APStracts 3:0089E, 1996.
- Insulin cellular degradation was studied in cultured 18-day old
fetal rat hepatocytes in the presence and absence of insulin
degradation inhibitors which 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 HPLC. Within cells, four components
containing intact [125I](A14)insulin A-chain and part of B-chain (A1
to A4, according to increasing retention times) were found together
with two [125I](B26)insulin B-chain C-terminal fragments (B1 and B2).
Medium degradation intermediates comprised B1 and B2 but not A1 to
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%, while that of A3, A4 and B2 was
not significantly affected. N-ethylmaleimide (NEM) 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 C-terminus, the one sensitive to chloroquine and IDE
inhibitors occurring after endosomal segregation of insulin and its
receptor.
Received 3 January 1996; accepted in final form 5 April 1996.
APS Manuscript Number E2-6.
Article publication pending Am. J. Physiol. (Endocrinol. Metab.).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 1 May 96