To further evaluate the role of the lysosomal system in insulin degradation, we have compared the effects of inhibitors of lysosomal function on the degradation of 125I-insulin with 125I-asialofetuin, a lysosomally targeted molecule, by the intact, perfused rat liver and the isolated rat hepatocyte. The inhibitors employed were chloroquine (125 μJLM), NH4CI (10 mM), and leupeptin (50 μg/ml). In the intact, perfused liver the observed inhibition of 125I-asialofetuin degradation at 30 min was as follows: chloroquine, 38%; NH4CI, 32%; and leupeptin, 86%. Chloroquine also inhibited 125Nnsulin degradation in the intact, perfused liver (29%), but NH4CI and leupeptin had no effect. Using the isolated hepatocyte, the observed values for inhibition of 125I-asialofetuin at 60 min were: chloroquine, 85%; NH4CI, 76%; and leupeptin, 81%. Chloroquine produced a 28% inhibition of 125I-insulin degradation, while NH4CI and leupeptin had no effect. Chloroquine and NH4CI decreased cell-associated radioactivity when isolated hepatocytes were incubated with 125I-asialofetuin (leupeptin had no effect), whereas chloroquine caused a 107% increase in cellassociated radioactivity when 125I-insulin was added to the incubation media (NH4CI and leupeptin had no effect), These results indicate that the effects of chloroquine on insulin degradation are an extralysosomal action and that lysosomes appear not to be involved in the physiologic degradation of the insulin molecule.

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