Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179).
Vasodilator actions of insulin are mediated by activation of endothelial nitric-oxide synthase (eNOS) and subsequent production of NO. Phosphatidylinositol 3-kinase and Akt play important roles in insulin-signaling pathways leading to production of NO in vascular endothelium. Here we dissected mechanisms whereby insulin activates eNOS by using the fluorescent dye DAF-2 to directly measure NO production in single cells. Insulin caused a rapid increase in intracellular NO in NIH-3T3(IR) cells transiently transfected with eNOS. The stimulation of NO production by lysophosphatidic acid (LPA) was abrogated by pretreatment of cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Remarkably, in the same cells, insulin-stimulated production of NO was unaffected. However, cells expressing the eNOS-S1179A mutant (disrupted Akt phosphorylation site) did not produce detectable NO in response to insulin, whereas the response to LPA was similar to that observed in cells expressing wild-type eNOS. Moreover, production of NO in response to insulin was blocked by coexpression of an inhibitory mutant of Akt, whereas the response to LPA was unaffected. Phosphorylation of eNOS at Ser(1179) was observed only in response to treatment with insulin, but not with LPA. Interestingly, platelet-derived growth factor treatment of cells activated Akt but not eNOS. Results from human vascular endothelial cells were qualitatively similar to those obtained in transfected NIH-3T3(IR) cells, although the magnitude of the responses was smaller. We conclude that insulin regulates eNOS activity using a Ca(2+)-independent mechanism requiring phosphorylation of eNOS by Akt. Importantly, phosphorylation-dependent mechanisms that enhance eNOS activity can operate independently from Ca(2+)-dependent mechanisms.
Montagnani
M
M
Cardiology Branch, NHLBI, and Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Chen
H
H
Barr
V A
VA
Quon
M J
MJ
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
D002118:Calcium; D004533:Egtazic Acid; D007328:Insulin; D008246:Lysophospholipids; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine; C496314:NOS3 protein, human; C496315:Nos3 protein, mouse; C494918:AKT1 protein, human; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid