Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

Abstract

Rationale: In diabetic animals as well as high glucose cell culture conditions, eNOS (endothelial NO synthase) is heavily O-linked-N-acetylglucosaminylated (O-GlcNAcylated), which inhibits its phosphorylation and NO production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells were exposed to either laminar flow (20 dynes/cm 2 shear stress) or oscillating disturbed flow (4±6 dynes/cm 2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT [O-GlcNAc transferase], OGA [O-GlcNAcase], or GFAT [glucosamine-fructose-6-phosphate aminotransferase]). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway activity, thereby reducing UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting hexosamine biosynthetic pathway metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent endothelial cell dysfunction in cardiovascular disease.