In vitro characterization of the mechanism of insulin degradation and the effect of chloroquine.
The cultured rat hepatoma cell line, MH1C1, inactivates 125I-labeled insulin by a temperature dependent mechanism. The estimated Km 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 degrees C with 125I-labeled insulin are immunoreactive with anti-insulin antibody, while the iodocompounds released from the cells incubated at 37 degrees 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 degrees 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.
D000818:Animals; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology*; D002850:Chromatography, Gel; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007330:Insulin Antibodies / Q000032:analysis; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000032:analysis
D007328:Insulin; D007330:Insulin Antibodies; D002738:Chloroquine; D011972:Receptor, Insulin