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

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.

Figure 1

Effect of hyperglycemia and increased glucosamine on eNOS activity in BAECs. Cells were incubated with either 5 mM glucose, 30 mM glucose, 5 mM glucose plus two concentrations of glucosamine (a), or 30 mM glucose plus antisense, inverse, or scrambled oligonucleotides for GFAT (b). *P < 0.01 compared with cells incubated in 5 mM glucose alone. n = 5 for each group.

Figure 2

Effect of GFAT antisense oligonucleotides on GFAT expression. Cells were incubated with either 5 mM glucose, 30 mM glucose, 30 mM glucose plus antisense, inverse, or scrambled oligonucleotides for GFAT, or 30 mM glucose after infection with adenoviral vectors expressing either UCP-1 or MnSOD; then Western blots for GFAT were performed as described in Methods. (a) Representative Western blot. (b) GFAT expression (relative densitometric means of 4 blots). *P < 0.01 compared with cells incubated in 5 mM glucose.

Figure 3

Digital micrographs of BAECs labeled with JC-1. (a) Cells incubated in 5 mM glucose. (b) Cells incubated in 30 mM glucose. (c) Cells incubated in 30 mM glucose after infection with UCP-1–expressing adenovirus. (d) Cells incubated in 30 mM glucose after infection with LacZ-expressing adenovirus.

Figure 4

Effect of genes that alter mitochondrial superoxide production on eNOS activity in BAECs. Cells were incubated in 5 mM or 30 mM glucose alone, or in 30 mM glucose plus either UCP-1– or MnSOD-expressing adenoviral vectors, or control vector. Cells expressing UCP-1 and MnSOD were also incubated in 30 mM glucose supplemented with 5 mM glucosamine. eNOS activity was determined after immunoprecipitation as described in Methods. *P < 0.01 compared with cells incubated in 5 mM glucose. n = 5 for each group.

Figure 5

Mitochondrial localization of recombinant human MnSOD. Confocal microscopic images of mitochondrial staining viewed through a Texas Red channel (a), and MnSOD signal viewed through the FITC channel (b). Colocalization of the Texas Red and FITC signals is indicated by the yellow color in the combined images (c).

Figure 6

Effect of hyperglycemia, GFAT antisense oligonucleotides, and genes that alter mitochondrial superoxide production on eNOS O-linked GlcNAc and phospho-eNOS(Ser1177). Cells were incubated in 5 mM glucose alone, 5 mM glucose plus wortmannin, 30 mM glucose alone, and 30 mM glucose plus either GFAT antisense, GFAT inverse, or GFAT scrambled oligonucleotides; or 30 mM glucose plus UCP-1– or MnSOD-expressing adenoviral vectors, as indicated. (a) Representative IP-Western blot. (b) eNOS O-linked GlcNAc blotted with RL2 antibody (relative densitometric means of three IP-Western blots). (c) Phospho-eNOS(Ser1177) (relative densitometric means of three IP-Western blots). *P < 0.01 compared with cells incubated in 5 mM glucose.

Figure 7

Effect of hyperglycemia on O-linked GlcNAc and phospho-eNOS(Ser1177) in myc-tagged wild-type human eNOS and myc-tagged human eNOS mutated at the Akt site. Cells were transfected as described in Methods and incubated in either 5 mM glucose or 30 mM glucose. Western blots were performed for O-linked GlcNAc and phospho-eNOS(Ser1177) and normalized by blotting for myc. (a) Representative IP-Western blot. (b) eNOS O-linked GlcNAc/myc expressed as percent of wild-type eNOS in 5 mM glucose (relative densitometric means of three IP-Western blots). (c) Phospho-eNOS(Ser1177)/myc expressed as percent of wild-type eNOS in 5 mM glucose (relative densitometric means of three IP-Western blots). *P < 0.01 compared with wild-type transfected cells incubated in 5 mM glucose.

Figure 8

Effect of diabetes on aortic eNOS O-linked GlcNAc and phosphoserine. Aortae were dissected from age-matched nondiabetic and 16-week streptozotocin diabetic rats. (a) Representative IP-Western blot. (b) eNOS O-linked GlcNAc (relative densitometric means of three IP-Western blots). (c) eNOS O-linked phosphoserine (relative densitometric means of three IP-Western blots). *P < 0.01 compared with cells incubated in 5 mM glucose. n = 5 for each group.

Figure 9

Effect of diabetes on aortic eNOS activity. Aortae were dissected from age-matched nondiabetic and 16-week streptozotocin diabetic rats, and eNOS activity was determined using an immunoprecipitation assay. *P < 0.01 compared with cells incubated in 5 mM glucose. n = 5 for each group.