“SIRT3-AMP–Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved

August 9, 2016 e79 CORRPONDENCE CORRPONDENCE In Response: We certainly agree with Drs Carlström and Lundberg that the nitrate–nitrite–nitric oxide (NO) pathway appears to activate AMP kinase (AMPK) signaling networks in the vascular system and skeletal muscle.1–3 It is all the more remarkable considering the fact that exercise, fasting, and metformin activate these pathways, suggesting that nitrite signaling may represent a convergent signaling pathway underlying these physiological stressors and drug, potentially via the intermediacy of NO synthase–dependent NO generation and secondary oxidation to more stable reservoirs of nitrite and nitrate. Interestingly, Drs Carlström and Lundberg recently reported that short-term treatment with nitrate, the dietary precursor of nitrite, improves glucose disposal via AMPK activation in the liver in A2B receptor knockout mice. 3 We did not observe activation of AMPK and its upstream activator SIRT3 in the livers from rats with multiple features of metabolic syndrome (obese ZSF1) after 14 weeks of oral nitrite supplementation.2 This may be attributable to obese ZSF1 rats having fatty livers and developing more profound insulin resistance, hyperglycemia, glucose intolerance, and dyslipidemia than A2B −/− mice. Nonetheless, the activation of AMPK appears to modulate some of the key signaling pathways for nitrate and nitrite. Additionally, we used nitrite alone in our ZSF1 studies and in our in vitro cultured human skeletal muscle studies, with a combination of nitrate and nitrite tested only in our human treatment studies. We also find that nitrite signaling in skeletal muscle appears to be NO independent and related to oxidation of nitrite by reactive oxygen species (hydrogen peroxide or superoxide) to a putative reactive nitrogen species intermediate such as nitrogen dioxide. Three observations support this thesis. First, our experiments in cultured insulinresistant human skeletal muscle are all performed under atmospheric oxygen tensions, which are supraphysiological in oxygen. The nitrite reductase activity of enzymes that can convert nitrite into NO such as myoglobin requires lower oxygen tensions.4 In contrast, the oxidative reactions of nitrite and myoglobin would be preferred under these oxygenated conditions.5 Second, our nitrite signaling was not inhibited by cPTIO, a direct NO scavenger. Third, with regard to our experiments with superoxide dismutase and catalase, it is critical to note that both nitrite alone and superoxide dismutase/catalase alone increase AMPK-SIRT3 activation, whereas together they inhibit AMPK-SIRT3. This demonstrates an interaction, with superoxide dismutase and catalase blocking nitriteinduced SIRT3 activation and nitrite blocking superoxide dismutase/catalase–induced SIRT3 activation. Although this is a complex interaction, it suggests that nitriteand reactive oxygen species–derived reactive nitrogen species may activate SIRT3. Further studies are clearly required to better understand how nitrite signals under normoxia.