Desensitization of hepatoma cells to insulin action. Evidence for a post-receptor mechanism

We have previously reported that incubation of rat hepatoma cells with insulin causes a complete and reversible loss of responsiveness to insulin. In order to determine the role of the insulin receptor in desensitization, we have examined the effect of insulin on insulin binding. Exposure of rat hepatoma cells to insulin causes a time-dependent decrease in insulin binding capacity which is detectable at 30 min and maximal at 4-6 h, after which time insulin binding remains 40-50% that of untreated cells. Scatchard analysis indicates that insulin causes a decrease in the number of receptors with little change in the binding affinity. Insulin-induced down regulation of receptors, observable at insulin concentrations as low as 3 ng/ml, is half-maximal at 10-20 ng/ml and is maximal at 100 ng of insulin/ml. When insulin is removed from the culture medium, the cells slowly recover insulin binding capacity; recovery is minimal at 2-4 h but nearly complete after 24 h. Recovery of insulin responsiveness, in contrast, is complete as early as 2 h after insulin is removed. The extent of down regulation of receptors (50-60%) is not sufficient to account for the complete insulin desensitization. In addition, recovery of maximal responsiveness to insulin occurs long before recovery of insulin binding. Therefore, insulin-induced desensitization to insulin is not caused by down regulation of receptors but must involve a post-receptor mechanism.