In vitro characterization of the mechanism of insulin degradation and the effect of chloroquine

Abstract

The cultured rat hepatoma cell line, MH 1C 1, inactivates 125I-labeled insulin by a temperature dependent mechanism. The estimated K m for insulin degradation is 1.4 · 10 −7 M and the V is 2.5 · 10 −10 mol/10 6 cells/h. The iodocompounds released from cells preincubated at 0°C with 125I-labeled insulin are immunoreactive with anti-insulin antibody, while the iodocompounds released from the cells incubated at 37°C only reacts to a very small degree with anti-insulin antibody. The degradation products were analyzed by Sephadex gel chromatography. Sephadex G-75 gel filtration of iodocompounds derived from cells incubated at 37°C with 125I-labeled insulin for 5, 20 min and 1 h showed a progressive decrease in intact insulin, and an increase in the peak representing insulin breakdown products. Treatment with chloroquine, a lysosomal enzyme inhibitor, resulted in a large increase of cell-associated insulin compared to control cells. However, chromatographic studies of iodocompounds extracted from cells incubated with or without chloroquine show a similar pattern but differ in the size of the peak which represents the degradation products of 125I-labeled insulin. Furthermore, the iodocompounds released from the chloroquine treated cells were not immunoreactive with anti-insulin antibody. These results suggest that chloroquine inhibits the release of insulin degradation products from the cells.