High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser¹¹⁷⁷ phosphorylation in human aortic endothelial cells

Ian P. Salt, Valerie A. Morrow, Fiona M. Brandie, John M. C. Connell, John Petrie

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    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 PKBmediated 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.
    Original languageEnglish
    Pages (from-to)18791-18797
    Number of pages7
    JournalJournal of Biological Chemistry
    Volume278
    Issue number21
    DOIs
    Publication statusPublished - May 2003

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    Phosphorylation
    Nitric Oxide Synthase Type III
    Endothelial cells
    Nitric Oxide
    Endothelial Cells
    Insulin
    Glucose
    Proto-Oncogene Proteins c-akt
    Medical problems
    Type 1 Diabetes Mellitus
    Phosphatidylinositol 3-Kinases
    Adipocytes
    Hyperglycemia
    Type 2 Diabetes Mellitus
    Cultured Cells
    Down-Regulation
    Availability

    Cite this

    Salt, Ian P. ; Morrow, Valerie A. ; Brandie, Fiona M. ; Connell, John M. C. ; Petrie, John. / High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser¹¹⁷⁷ phosphorylation in human aortic endothelial cells. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 21. pp. 18791-18797.
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    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 PKBmediated 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.",
    author = "Salt, {Ian P.} and Morrow, {Valerie A.} and Brandie, {Fiona M.} and Connell, {John M. C.} and John Petrie",
    note = "dc.publisher: American Society for Biochemistry and Molecular Biology This research was originally published in Journal of Biological Chemistry. Salt, Ian P.: Morrow, Valerie A.: Brandie, Fiona M.: Connell, John M. C.: Petrie, John R. High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells. The Journal of Biological Chemistry. 2003. Vol 278:pp.18791-18797. {\circledC} the American Society for Biochemistry and Molecular Biology dc.description.sponsorship: British Heart Foundation Menarini Pharmaceuticals Joint Equipment Initiative",
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    High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser¹¹⁷⁷ phosphorylation in human aortic endothelial cells. / Salt, Ian P.; Morrow, Valerie A.; Brandie, Fiona M.; Connell, John M. C.; Petrie, John.

    In: Journal of Biological Chemistry, Vol. 278, No. 21, 05.2003, p. 18791-18797.

    Research output: Contribution to journalArticle

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    N1 - dc.publisher: American Society for Biochemistry and Molecular Biology This research was originally published in Journal of Biological Chemistry. Salt, Ian P.: Morrow, Valerie A.: Brandie, Fiona M.: Connell, John M. C.: Petrie, John R. High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells. The Journal of Biological Chemistry. 2003. Vol 278:pp.18791-18797. © the American Society for Biochemistry and Molecular Biology dc.description.sponsorship: British Heart Foundation Menarini Pharmaceuticals Joint Equipment Initiative

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