Fluvastatin Decreases Endothelial Nitric Oxide Synthase O‐Glcnacylation by Decreasing Glucose Uptake

Abstract

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.