Effects of Lysosomal Inhibitors on ¹²⁵I-Insulin and ¹²⁵I-Asialofetuin Degradation by the Isolated, Perfused Rat Liver and Isolated Rat Hepatocytes

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.