Insulin-stimulated production of nitric oxide is inhibited by wortmannin - Direct measurement in vascular endothelial cells

Hypertension is associated with insulin-resistant states such as diabetes and obesity, Nitric oxide (NO) contributes to regulation of blood pressure, To gain insight into potential mechanisms linking hypertension with insulin resistance we directly measured and characterized NO production from human umbilical vein endothelial cells (HUVEC) in response to insulin using an amperometric NO-selective electrode. Insulin stimulation of HUVEC resulted in rapid, dose-dependent production of NO with a maximal response of similar to 100 nM NO (200,000 cells in 2 ml media; ED(50) similar to 500 nM insulin), Although HUVEC have many more IGF-1 receptors than insulin receptors (similar to 400,000, and similar to 40,000 per cell respectively), a maximally stimulating dose of IGF-1 generated a smaller response than insulin (40 nM NO; ED(50) similar to 100 nM IGF-1), Stimulation of HUVEC with PDGF did not result in measurable NO production. The effects of insulin and IGF-1 were completely blocked by inhibitors of either tyrosine kinase (genestein) or nitric oxide synthase (L-NAME). Wortmannin (an inhibitor of phosphatidylinositol 3-kinase [PI S-kinase]) inhibited insulin-stimulated production of NO by similar to 50%, Since PI 3-kinase activity is required for insulin-stimulated glucose transport, our data suggest that NO is a novel effector of insulin signaling pathways that are also involved with glucose metabolism.