Background—Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated.

Methods and Results—We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an ≈3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase–deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Ras had a much smaller effect.

Conclusions—Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.