Glucose-induced Phosphorylation of the Insulin Receptor Functional Effects and Characterization of Phosphorylation Sites

Elevated glucose concentrations have been reported to in-hibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts trans-fected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids ( D CT). In both cell lines, 25 mM glucose impaired re-ceptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuri-dine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor pep-tide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is indepen-dent of activation of the MAP kinase cascade. Proportion-ately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase acti-vation and mitogenic signaling.