Insulin-Receptor Autophosphorylation and Endogenous Substrate Phosphorylation in Human Adipocytes From Control, Obese, and NIDDM Subjects

Abstract

We identified a possible endogenous substrate (pp185) of the insulin-receptor kinase in human adipocytes by treating intact cells with insulin and immunoblotting the cellular extracts with polyclonal antiphosphotyrosine antibody. This 185,000-Mr protein was phosphorylated on tyrosine residues in response to insulin in both rat and human adipocytes. The time course of pp185 phosphorylation at 37°C was rapid and corresponded closely to insulin-receptor autophosphorylation but preceded insulin-stimulated glucose transport. Unlike many growth factor receptors, including the insulin receptor, pp185 was not adsorbed to wheat-germ agglutinin. We found that pp185 phosphorylation occurred at 12°C and that the phosphoprotein was associated with both cytoplasmic and membrane fractions at this temperature. Furthermore, pp185 phosphorylation was induced to the same extent as insulin by vanadate and hydrogen peroxide, compounds previously shown to mimic the biologic effects of insulin. In addition, dose-response analysis of insulin-stimulated glucose transport, receptor autophosphorylation, and pp185 phosphorylation resulted in ED50 values of 0.3, 12, and 12 ng/ml, respectively. These results demonstrate the magnitude of “spare” autophosphorylation and pp185 phosphorylation with respect to glucose transport stimulation in human adipocytes. To determine whether the insulin resistance characteristic of non-insulin-dependent diabetes mellitus (NIDDM) and obesity is associated with a defect in receptor autophosphorylation and/or endogenous substrate phosphorylation, we estimated the extent of β-subunit and pp185 phosphorylation in adipocytes from NIDDM, obese, and healthy subjects. Although the efficiency of coupling between receptor activation and pp185 phosphorylation was normal in obesity and NIDDM, the capacity for insulin-receptor autophosphorylation was ∼50% lower in NIDDM subjects compared with nondiabetic obese or lean subjects. Thus, the absolute level of pp185 phosphorylation in response to insulin stimulation would be lower in adipocytes from NIDDM subjects. We conclude that pp185 is directly phosphorylated by the insulin-receptor kinase in vivo and may serve as a mediating step to the biologic effects of insulin in human adipocytes. Furthermore, the extent of pp185 phosphorylation is compromised in adipocytes from NIDDM subjects and may contribute xto the insulin resistance associated with this disorder.