Insulin binding and degradation in short time incubation at 37 C.

Studies on insulin-receptor binding in a short time incubations at 37 C have shown that neither internalization nor receptor-mediated insulin degradation are demonstrable during the first minutes. In the present study insulin receptor binding at 37 C in short time incubation periods was studied in mouse-hepatocytes, simultaneously determinating the proportion of degradation due to the cell activity. Degradation in the incubation buffer after cell separation was abolished during the experiment (900 sec) by a careful wash of the cells. 7.5 cells/ml were incubated with a tracer concentration (14.17 pM) of 125I-insulin and a pharmacological concentration (16.6 microM) of native insulin plus tracer. In the case of tracer insulin, 50% binding was reached in 55 sec and steady state in 160 sec. Once reached, steady state persisted along the experimental time. Binding follows a second order kinetics with k+1: 5 649 X 10(6) M-1 sec-1. In the presence of pharmacological insulin there is competitive inhibition of the tracer which reduces to zero the percent of binding. Binding increases along the time taking positive values, and the slope of binding versus time intersects the abscissa at 102 sec (r: 0.864). As long as binding of the tracer takes place, no degradation occurs until 635 sec, when a degradation slope abruptly appears (r: 0.722). Dissociation studies were followed previous incubation at 37 C during 200 sec with tracer and pharmacological doses. Specific dissociation follows a monoexponential kinetics with k-1: 3 067 X 10(-3) sec-1 and t 1/2: 226 sec. Eighty percent of bound insulin is dissociated with no changes in the slope (r: 0.820), thus suggesting that insulin-receptor binding in the present experimental conditions is basically a reversible process. No degradation was observed during dissociation, which demonstrates that insulin-receptor binding does not degrade insulin if internalization is not performed. At steady state, competitive inhibition curves showed two components: high and low affinity. Doses of 1.66 microM produce a 98% inhibition in the binding of 125I-insulin. The high affinity slope shows two components in the physiological range of insulin concentrations. The first one of very high affinity has a dissociation constant Ko: 7 075 X 10(-10), and a binding capacity of 1.5 X 10(-10). This study demonstrates that, with physiological concentrations of insulin, internalization is the only mechanism of insulin degradation in mouse-hepatocytes.