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The AMP-activated protein kinase (AMPK) plays critical roles in growth regulation and metabolism reprogramming. AMPK activation protects cells against apoptosis from injury in different cell and animal models. However, its function in necroptosis remains largely unclear.In the current study, we demonstrated that AMPK was activated upon necroptosis induction and protected mouse embryonic fibroblasts (MEFs) and cardiomyocytes from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and reactive oxygen species (ROS) induced necroptosis. Activation of AMPK with chemicals A-769662, 2-deoxyglucose (2-DG), and metformin or constitutively active (CA) AMPK markedly decreased necroptosis and cytotoxicity induced by MNNG. In contrast, AMPK inhibitor compound C, dominant negative (DN) AMPK, as well as AMPK shRNAs increased necroptosis and cytotoxicity induced by MNNG. We further showed that AMPK physically associated with a protein complex containing PGAM5 and Keap1 whereby facilitating Keap1-mediated PGAM5 ubiquitination upon necroptosis induction. The AMPK agonist metformin ameliorated myocardial ischemia and reperfusion (IR) injury and reduced necroptosis through down-regulating the expression of PGAM5 in the Langendorff-perfused rat hearts.Activation of AMPK protects against necroptosis via promoting Keap1-mediated PGAM5 degradation. Metformin may act as a valuable agent for the protection of myocardial ischemia and reperfusion injury by activating AMPK and reducing necroptosis.

Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine–citrulline conversion assay and HPLC analysis, respectively. Over a period of 4 h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization., Graphical abstract Highlights ► Ascorbate can enhance the activity of endothelial NO synthase (eNOS) within minutes. ► This effect occurs independent of stabilization of tetrahydrobiopterin. ► Ascorbate modulates eNOS phosphorylation in a PP2A- and AMPK-dependent manner.