Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide.

Abstract

The oxidant H202 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H202 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the I8 subunit of the insulin receptor was increased 2-fold by treatment of intact cells with 3 mM H202, a concentration that maximally stimulates 2-deoxyglucose uptake. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport.Phosphoamino acid analysis of insulin receptors from H202-treated adipocytes showed that 32p incorporation into phosphotyrosine and phosphoserine residues of the .3 subunit was enhanced. Furthermore, partially purified receptors from H202-treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu8oTyr20. In contrast, the direct addition of H202 to partially purified insulin receptors did not stimulate tyrosine kinase activity or insulin receptor autophosphorylation. This was not due to breakdown of H202 or oxidation of ATP or the required divalent cations. To define the factors involved in H202's effect, we have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H202 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H202 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H202 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H202 on the insulin receptor and requires nonplasma membrane cellular constituents.In intact cells (1) and cell-free systems (2) the insulin receptor is rapidly phosphorylated subsequent to insulin binding. In cell-free systems phosphorylation occurs exclusively on tyrosine residues (2), whereas in intact cells serine is also phosphorylated (1). The f subunit of the receptor contains an ATP binding site (3) and evidence indicates that the insulin receptor itself is a tyrosine-specific protein kinase that undergoes autophosphorylation in an insulin-dependent manner (4). Receptor autophosphorylation enhances the tyrosine kinase activity toward exogenous substrates (5). The intrinsic nature of this kinase activity, as well as the insulin concentration dependence (6) and time course of autophosphorylation (7), has led to the proposal that receptor phosphorylation is an early step in coupling hormone binding to insulin action.The oxidant H202 has many insulin-like effects in rat adipocytes, including stimulation of glucose transport (8, 9), stimulation of glucose utilization (10), and inhibition of hormone-stimulated lipolysis (11). However, H202 has no effect on insulin binding (12). This has led to its use as a tool to ev...

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