Tumor necrosis factor-[alpha ] inhibits insulin-induced increase in endothelial nitric oxide synthase and reduces insulin receptor content and phosphorylation in human aortic endothelial cells

Abstract

Insulin exerts a vasodilatory effect through the release of nitric oxide (NO) from the endothelium. We have recently demonstrated that insulin also inhibits the expression of intracellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), 2 major proinflammatory mediators, by human aortic endothelial cells (HAEC) and the proinflammatory mediator, nuclear factor (NF-[kappa ]B), in the nucleus in parallel with an increase in endothelial nitric oxide synthase (e-NOS) expression. The inhibition of ICAM-1 by insulin is NO dependent. Because tumor necrosis factor-[alpha ] (TNF-a ) is proinflammatory and may thus inhibit the action of insulin at the endothelial cell level, we have now investigated whether TNF-a affects (1) insulin receptor content; (2) insulin receptor (IR) autophosphorylation induced by insulin, and (3) e-NOS expression by the endothelial cells. TNF-[alpha ] (1 to 5 ng/mL) caused e-NOS inhibition in a dose-dependent fashion as measured by Western blotting. This inhibition was reduced with insulin addition. TNF-[alpha ] also inhibited tyrosine autophosphorylation of the IR in HAEC induced by insulin and reduced IR [beta ]-subunit protein expression in HAEC. These effects of insulin and TNF-[alpha ] were independent of cell proliferation, as cell counts did not change with insulin or TNF-[alpha ]. Our data demonstrate that TNF-[alpha ] may exert its effect by inhibiting IR autophosphorylation in HAEC and also by reducing IR protein (IRP) expression. Although the inhibition of IR autophosphorylation by TNF-[alpha ] is known to occur at the adipocyte level, the data on the inhibitory effect of TNF-[alpha ] on insulin-induced e-NOS expression and IRP contents are novel.