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.
Proper function of the endothelium, which lines all blood contacting surfaces, is essential in cardiovascular health. Endothelial nitric oxide (NO) production, which is regulated by endothelial nitric oxide synthase (eNOS) phosphorylation, is considered the gold standard measure of endothelial function. eNOS is O‐GlcNAcylated through the hexosamine biosynthetic pathway (HBP), which may inhibit eNOS phosphorylation. Statins, a cholesterol lowering drug class, increase NO production. This study aimed to determine if statins impact NO production through eNOS O‐GlcNAcylation. We hypothesized statins decrease eNOS O‐GlcNAcylation through a concomitant decrease in glucose metabolism and thereby increase NO production. To investigate this hypothesis, human umbilical vein endothelial cells (HUVEC) were treated with a physiologically relevant fluvastatin dose. Total, phosphorylated, and O‐GlcNAcylated eNOS were measured by Western blot. Metabolites were measured by YSI biochemical analyzer or mass spectrometry using U‐13C6 glucose. Fluvastatin increased eNOS protein in addition to decreasing eNOS O‐GlcNAcylation 24 hours post treatment. To determine whether fluvastatin directly decreased O‐GlcNAcylation via the catalytic enzyme O‐GlcNAc transferase (OGT), endothelial cells were treated with statins along with glucosamine, an O‐GlcNAc substrate which feeds directly into the HBP. Glucosamine reversed fluvastatin effects on eNOS O‐GlcNAcylation, suggesting that fluvastatin does not directly affect catalytic addition of O‐GlcNAc to protein targets. Further, OGlcNAcase (OGA) activity, which would catalyze O‐GlcNAc removal, did not increase with statin treatment. Instead, endothelial cell glucose consumption and lactate production decreased 24 and 48 hours after statin treatment. Since the ratio of glucose consumption to lactate production was similar across all samples, statin treatment did not change the preferential use of glucose for anaerobic glycolysis. Metabolic mass spectrometry showed that statin treated HUVEC had markedly reduced glycolytic metabolites after fructose‐1,6‐bisphosphate, suggesting that fluvastatin likely decreases O‐GlcNAcylation by acting at or below phosphofructokinase to decrease glucose metabolism. These studies show that statins may have additional beneficial cardiovascular effects through lowered endothelial glucose metabolism.
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