Identification of Hypoglycemia-dependent Endothelial Nitric Oxide Synthase O-GlcNAcylation Sites and Regulation of O-GlcNAcylation and Nitric Oxide Production by Hypoglycemia

Background: O-GlcNAcylation, an energy-sensitive post-translational modification, plays a major role in endothelial nitric oxide synthase (eNOS) activity regulation. However, the effect of hypoglycemia on eNOS O-GlcNAcylation and whether eNOS exists the novel O-GlcNAcylation sites under hypoglycemia is unknown. Hence, we endeavored to determine the effects of hypoglycemia on eNOS O-GlcNAcylation and the novel O-GlcNAcylation sites of eNOS. Method: Bovine aortic endothelial cells (BAECs) and Sprague-Dawley rats were treated by hypoglycemia, and using immunoblotting to measure their eNOS O-GlcNAcylation. eNOS and transfected eNOS were purified by pull-down assay and immunoprecipitation respectively. Novel O-GlcNAcylation sites of eNOS were predicted by HPLC-MS and MS/MS Ion, and determined by immunoblotting. eNOS activity were detected by Elisa and isotope labelling method. Results: In BAECs and rats` thoracic aorta, hypoglycemia-associated activation of eNOS was accompanied by an increase in O-GlcNAcylation and had no effect on O - linked serine phosphorylation at residue 1179/1177. Changes in this post-translational modification were associated with increased O-GlcNAc transferase (OGT) activity, and were reversed by AMPK knockdown. Immunoblot analysis of cells expressing His-tagged wild-type human eNOS and human eNOS carrying a mutation at the Ser1177 phosphorylation site confirmed the increase in O-GlcNAcylation in response to hypoglycemia. The observed increase in O-GlcNAcylation indicated that eNOS contains novel O-GlcNAcylation sites that are activated by hypoglycemia. Immunoblot analysis of cells expressing His-tagged human eNOS carrying a mutation at Ser738 and Ser867 confirmed the increase in O-GlcNAcylation in response to hypoglycemia. Contrastingly, in His-tagged human eNOS carrying a mutation at Thr866, O-GlcNAcylation was unaffected by hypoglycemia. Differences among culture conditions were identified using two-way analysis of variance (ANOVA), one-way ANOVA, or unpaired Student’s t -test. Conclusions: Hypoglycemia increases eNOS O-GlcNAcylation and activity, potentially via AMPK-OGT pathway, thereby showing the Thr866 as a novel O-GlcNAcylation site involved in hypoglycemia-mediated eNOS activation.