Abstract-Endothelial nitric oxide synthase (eNOS) plays a central role in maintaining cardiovascular homeostasis by controlling NO bioavailability. The activity of eNOS in vascular endothelial cells (ECs) largely depends on posttranslational modifications, including phosphorylation. Because the activity of AMP-activated protein kinase (AMPK) in ECs can be increased by multiple cardiovascular events, we studied the phosphorylation of eNOS Ser633 by AMPK and examined its functional relevance in the mouse models. Shear stress, atorvastatin, and adiponectin all increased AMPK Thr172 and eNOS Ser633 phosphorylations, which were abolished if AMPK was pharmacologically inhibited or genetically ablated. The constitutively active form of AMPK or an AMPK agonist caused a sustained Ser633 phosphorylation. Expression of gain-/loss-of-function eNOS mutants revealed that Ser633 phosphorylation is important for NO production. The aorta of AMPK␣2 Ϫ/Ϫ mice showed attenuated atorvastatin-induced eNOS phosphorylation. Nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) confirmed that eNOS Ser633 was able to compete with Ser1177 or acetyl-coenzyme A carboxylase Ser79 for AMPK␣ phosphorylation. Nano-LC/MS/MS confirmed that eNOS purified from AICAR-treated ECs was phosphorylated at both Ser633 and Ser1177. Our results indicate that AMPK phosphorylation of eNOS Ser633 is a functional signaling event for NO bioavailability in ECs.

Shear stress imposed by blood flow is crucial for maintaining vascular homeostasis. We examined the role of shear stress in regulating SIRT1, an NAD + -dependent deacetylase, and its functional relevance in vitro and in vivo. The application of laminar flow increased SIRT1 level and activity, mitochondrial biogenesis, and expression of SIRT1-regulated genes in cultured endothelial cells (ECs). When the effects of different flow patterns were compared in vitro, SIRT1 level was significantly higher in ECs exposed to physiologically relevant pulsatile flow than pathophysiologically relevant oscillatory flow. These results are in concert with the finding that SIRT1 level was higher in the mouse thoracic aorta exposed to atheroprotective flow than in the aortic arch under atheroprone flow. Because laminar shear stress activates AMP-activated protein kinase (AMPK), with subsequent phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser-633 and Ser-1177, we studied the interplay of AMPK and SIRT1 on eNOS. Laminar flow increased SIRT1-eNOS association and eNOS deacetylation. By using the AMPK inhibitor and eNOS Ser-633 and -1177 mutants, we demonstrated that AMPK phosphorylation of eNOS is needed to prime SIRT1-induced deacetylation of eNOS to enhance NO production. To verify this finding in vivo, we compared the acetylation status of eNOS in thoracic aortas from AMPKα2 −/− mice and their AMPKα2 +/+ littermates. Our finding that AMPKα2 −/− mice had a higher eNOS acetylation indicates that AMPK phosphorylation of eNOS is required for the SIRT1 deacetylation of eNOS. These results suggest that atheroprotective flow, via AMPK and SIRT1, increases NO bioavailability in endothelium.NAD + -dependent deacetylase | AMP-activated protein kinase | endothelial nitric oxide synthase | NO bioavailability | endothelial homeostasis S IRT1, also known as Sirtuin 1 (silent mating type information regulation 2 homolog), contributes to the caloric restriction (CR)-induced increase in lifespan in species ranging from yeast to mammals (1-3). Functioning as an NAD + -dependent class III histone deacetylase (4), SIRT1 deacetylates multiple targets in mammalian cells, including tumor suppressor p53, Forkhead box O1 and 3 (FOXO1 and FOXO3), peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α), liver X receptor, and hypoxia-inducible factor 2α (5-14). By regulating these molecules involved in cell survival and in carbohydrate and lipid metabolism, SIRT1 functions as a master regulator of stress response and energy homeostasis.SIRT1 is also an important modulator in cardiovascular functions in health and disease. The beneficial effects of SIRT1 on endothelial cell (EC) biology were demonstrated by several previous studies. Ota et al. (15) showed that overexpression of SIRT1 prevented oxidative stress-induced endothelial senescence, whereas inhibition of SIRT1 led to premature senescence. Treatment of human coronary arterial ECs with resveratrol (RSV), an SIRT1 activator, increased the mitochondrial mass and ke...

Summary c-Myc is known to promotes glutamine usage by up-regulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis, and suggest a novel molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.