Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site

Xue Liang Du, Diane Edelstein, Stefanie Dimmeler, Qida Ju, Chengyu Sui, Michael Brownlee

Research output: Contribution to journalArticle

624 Scopus citations

Abstract

Endothelial nitric oxide synthase (eNOS) is activated by phosphorylation of serine 1177 by the protein kinase Akt/PKB. Since hyperglycemia-induced mitochondrial superoxide overproduction increases O-linked N-acetylglucosamine modification and decreases O-linked phosphorylation of the transcription factor Sp1, the effect of hyperglycemia and the hexosamine pathway on eNOS was evaluated. In bovine aortic endothelial cells, hyperglycemia inhibited eNOS activity 67%, and treatment with glucosamine had a similar effect. Hyperglycemia-associated inhibition of eNOS was accompanied by a twofold increase in O-linked N-acetylglucosamine modification of eNOS and a reciprocal decrease in O-linked serine phosphorylation at residue 1177. Both the inhibition of eNOS and the changes in its post-translational modifications were reversed by antisense inhibition of glutamine: Fructose-6-phosphate amidotransferase, the rate-limiting enzyme of the hexosamine pathway, or by blocking mitochondrial superoxide overproduction with uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD). Immunoblot analysis of cells expressing myc-tagged wild-type human eNOS confirmed the reciprocal increase in O-linked N-acetylglucosamine and decrease in O-linked serine 1177 phosphorylation in response to hyperglycemia. In contrast, when myc-tagged human eNOS carried a mutation at the Akt phosphorylation site (Ser1177), O-linked N-acetylglucosamine modification was unchanged by hyperglycemia and phospho-eNOS was undetectable. Similar changes in eNOS activity and covalent modification were found in aortae from diabetic animals. Chronic impairment of eNOS activity by this mechanism may partly explain the accelerated atherosclerosis of diabetes.

Original languageEnglish (US)
Pages (from-to)1341-1348
Number of pages8
JournalJournal of Clinical Investigation
Volume108
Issue number9
DOIs
StatePublished - Jan 1 2001

ASJC Scopus subject areas

  • Medicine(all)

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