Our previous data revealed that endothelial dysfunction observed in response to hyperglycemia was partially mediated by increased O-GlcNAcylation. Yet, the activation of endothelial nitric oxide synthase (eNOS), a key enzyme regulating endothelial function, is mainly dependent on its phosphorylation at specific serine residues, which are also key targets of O-GlcNAcylation. Although studies explored the interplay between eNOS phosphorylation and O-GlcNAcylation, none investigated the impact of altering this balance on endothelial function.

To evaluate the impact of modulating the balance between eNOS phosphorylation and O-GlcNAcylation on endothelial function.

To modulate O-GlcNAcylation, isolated rat aorta and cultured endothelial cells (HUVECs) were incubated with high glucose (33mM) or specific inhibitors of O-GlcNAcylation enzymes: Thiamet-G (O-GlcNAcase inhibitor, 0.1μM) or OSMI-2 (O-GlcNAc transferase inhibitor, 50μM). Tissues were also stimulated with insulin (0.1μM) to promote phosphorylation and explore the interplay with O-GlcNAcylation. Western Blot analysis was performed to assess eNOS O-GlcNAcylation and phosphorylation. Endothelial function was evaluated by vascular reactivity in aortic rings and NO production was quantified in tissues and endothelial cells.

Our preliminary data showed that hyperglycemia (HG) increases protein O-GlcNAcylation, which is associated with impaired vasorelaxation in response to acetylcholine. Interestingly, treatment with Thiamet-G, which increases O-GlcNAcylation, also impaired endothelial function. Consistent with these findings, the use of OSMI-2 reversed the alteration induced by HG, emphasizing the critical role of O-GlcNAcylation in vascular dysfunction. This effect seems mediated by the interplay between O-GlcNAcylation and eNOS phosphorylation. In HUVECs, Thiamet-G-induced O-GlcNAcylation reduced eNOS phosphorylation, while OSMI-2 decreased O-GlcNAcylation and increased phosphorylation of this enzyme.

These data suggest that an imbalance between O-GlcNAcylation and phosphorylation of eNOS may contribute to vascular endothelial dysfunction. Further experiments are in progress to better understand the impact of eNOS O-GlcNAcylation in modulating vascular function under stress conditions which is highly dependent on phosphorylation mechanisms.