Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells

Background-Previously, we demonstrated that insulin stimulates production o f nitric oxide (NO) in endothelial cells. However, specific insulin-signali ng pathways mediating production of NO have not been elucidated. Methods and Results-We developed methods for transfection of human umbilica l vein endothelial cells (HUVECs) and direct measurement of NO to begin def ining insulin-signaling pathways related to NO production. HUVECs were cotr ansfected with enhanced Green Fluorescent Protein (eGFP) and another gene o f interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximate to 3-fold increase in production of NO in response to insul in. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant i nsulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kina se (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulat ed production of NO. Overexpression of an inhibitory mutant of Rns had a mu ch smaller effect. Conclusions-Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly t o this process, whereas the contribution of Ras is small.