[Insulin degrading system on isolated rat hepatocytes; enzymatic and immunologic characteristics of insulin-degrading activities].
Insulin degradation by isolated rat hepatocytes was investigated. Using the preincubation method, the extracellular insulin-degrading activity was removed from the medium over 120 min at 15 degrees C and 60 min at 37 degrees C. The degradation of insulin was assayed by the ability of binding to specific receptors on isolated rat hepatocytes (rebound method) and the precipitability with trichloroacetic acid (TCA method). The degrading activities measured by the rebound method showed twice those by the TCA method, however, a positive correlation with high coefficient (r = 0.87, P less than 0.001) was demonstrated between both methods. The insulin-degrading activity by hepatocytes depended on time, temperature and cell concentrations, and the optimum pH was 7.0. As a result of kinetic analysis of insulin binding and degradation, the degradation velocity of 125I-insulin was inhibited by 50% at native insulin concentration of 7 X 10(-8) M, whereas the half-maximum inhibition of 125I-insulin binding was demonstrated at that of 4 X 10(-9) M. The Km for insulin degradation was 170 nM. In order to characterize the enzymatic properties of insulin-degrading activity by isolated hepatocytes, the effects of various compounds and anti-insulin-degrading enzyme (IDE) rabbit serum on insulin degradation were examined. N-ethylmaleimide and anti-IDE serum significantly inhibited the insulin-degrading activity, while dithiothreitol stimulated it, whereas chloroquine and NH4Cl had little effect on insulin-degrading activity. Finally, the immunoenzymatic labelling of hepatocytes by anti-IDE serum showed the presence of cell surface IDE on isolated hepatocytes. These results suggest that most of insulin-degrading activity by isolated rat hepatocytes is identical to pig muscle IDE. Therefore, it would seem that IDE plays an important role in insulin metabolism by isolated rat hepatocytes rather than lysosomes.
D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
D007328:Insulin; D005033:Ethylmaleimide; D004229:Dithiothreitol