Insulin activation of NADH ferricyanide reductase in human erythrocytes is mediated by the insulin receptor tyrosine kinase: a comparative study in normal and diabetic states.

Abstract

We have previously reported that NADH ferricyanide reductase in human erythrocytes is stimulated by insulin. Hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors like vanadate, indicating the involvement of glycolysis in the mechanism by which insulin stimulates ferricyanide reduction. Activation of erythrocyte metabolism in response to insulin could be a result of hormone binding to its receptor, inducing phosphorylation of band 3 (at a site for reversible association of glycolytic enzymes) and/or other membrane proteins like the Na(+)/H(+) antiport. Activation of the antiporter protein by insulin can stimulate glycolysis by an increase in intracellular pH, an effect which is prevented by amiloride. Evidence for a role for tyrosine phosphorylation in triggering the reductase activation came from studies with protein kinase inhibitors. Genistein, sphingosine and acridine orange have been shown to prevent insulin-stimulated ferricyanide reduction, implicating tyrosine phosphorylation as an important signal for activation of the enzyme by insulin. To evaluate activation of the enzyme by insulin stimulated phosphorylation, a comparative study was done using erythrocytes from healthy and diabetic humans. We measured ferricyanide reductase activities in basal and insulin stimulated states. Basal activities were lower in diabetics than in normal humans. Nevertheless, hormone stimulated activities were similar, despite earlier reports of decreased receptor phosphorylation of exogenous substrates in type 2 diabetics. These observations, together with previous ones, suggest that insulin-receptor kinase interaction may mediate the action of insulin on human erythrocytes by phosphorylation of cellular proteins like band 3 and/or the Na(+)/H(+) antiport.