Characterization of Insulin-induced Receptor Loss and Evidence for Internalization of the Insulin Receptor

Abstract

We studied the relationship between insulin-induced receptor loss and insulin internalization in isolated rat adipocytes. When cells were exposed to 100 ng/ml insulin at 37°C for times ranging from 10 min to 6 h, a monoexponential loss of insulin receptors was observed for up to 4 h, with a t½ of 75 min and a 75% maximal loss. The insulin-induced loss of receptors was dose-dependent with a % maximal effective insulin concentration of 7.6 and 1.3 ng/ml in 2- and 6-h insulin- pretreated cells, respectively. The ability of insulin to mediate receptor loss was inhibited by low temperature (16°C) and by the metabolic energy depleters dinitrophenol and NaF, suggesting that receptor loss is mediated by an 125I-insulin compared with control cells, and the relationship between insulin-induced receptor loss and insulin internalization was assessed by comparing these two processes over time and as a function of temperature. Using this approach, a significant linear relationship was observed between these partners at various temperatures (r = 0.99) and times (r = 0.94). These results suggest that the mechanism whereby insulin induces receptor loss in adipocytes is by internalization of insulin-receptor complexes. Insulin receptors remaining on 2-h insulin, pretreated cells had identical dissociation rates and exhibited similar negative cooperative interactions when compared with control cells. Furthermore, anti-insulinreceptor antibodies inhibited binding in both groups of cells to the same extent. In contrast to these similarities, insulin-induced receptor loss was accompanied by a progressive impairment in the ability of cells to accumulate intracellular insulin in the presence ofchloroquine, which we interpret as an insulin-induced slowing of insulin internalization. In conclusion, (1) insulin causes a time-, temperature-, dose-, and energy-dependent loss of cell-surface insulin receptors; (2) the rate of receptor loss and the extent of insulin internalization are closely correlated, suggesting that insulin-induced receptor loss in adipocytes is mediated by endocytosis of insulin-receptor complexes; and (3) insulin-induced receptor loss is accompanied by a reduced ability of residual receptors to internalize insulin.