title: High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells.
abstract: Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca2+ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKB-mediated eNOS phosphorylation at Ser1177. Therefore, we propose that phosphorylation of eNOS at Ser1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose.
authors.affiliation: Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom. isalt001@udcf.gla.ac.uk
authors.forename: Ian P
authors.initials: IP
authors.lastname: Salt
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Morrow
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Brandie
authors.forename: John M C
authors.initials: JM
authors.lastname: Connell
authors.forename: John R
authors.initials: JR
authors.lastname: Petrie
mesh_terms: D001011:Aorta; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000008:administration & dosage*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D050721:Proto-Oncogene Proteins c-cbl; D015398:Signal Transduction / Q000187:drug effects; D044767:Ubiquitin-Protein Ligases
chemical_list: D003598:Cytoskeletal Proteins; D005947:Glucose; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C526292:IRS1 protein, human; C526297:IRS2 protein, human; C496314:NOS3 protein, human; C067419:CBL protein, human