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, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (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 (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 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, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) 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 HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

“…In AML12 cells, azaserine decreased UDP-GlcNAc by 50% ( Figure 3 C), which is similar to that observed in three endothelial cell lines. 33 , 34 , 35 …”

Direct cardiac effects of SGLT2 inhibitors

“…SGLT2i effects on eNOS activity are also not always observed, which also could be related to differences in mechanical and metabolic conditions applied to the ECs in the separate studies. For example, hyperglycaemic or oscillating flow conditions induce O-GlcNAcylation of the eNOS enzyme, thereby preventing that increases in enzyme activity can occur [ 84 ]. Finally, differences in measurement sensitivity between research laboratories may also have its impact.…”

Steady Laminar Flow Decreases Endothelial Glycolytic Flux While Enhancing Proteoglycan Synthesis and Antioxidant Pathways

“…Finally, overall glycolytic flux could be altered by changes in flux down glycolysis side branch pathways, including glycogen synthesis and the PPP. We previously showed that flow decreases endothelial glucose flux down the hexosamine biosynthetic pathway, which impacts nitric oxide production [ 38 ]. In this study, we observed increased glucose flux down the uronic acid pathway, which could be used to create more proteoglycans and glycosaminoglycans.…”

“…A custom-built cone-and-plate device was used to apply shear stress to HUVECs [ 38 ]. The device generates flow by rotating the cone around an axis perpendicular to the plate [ 47 ].…”