Nitric oxide (NO) is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. In various disease conditions, all three types of NOS (neuronal, inducible, and endothelial) are reported to generate oxidants through unknown mechanisms. We present here the first evidence that peroxynitrite (ONOO -) releases zinc from the zinc-thiolate cluster of endothelial NOS (eNOS) and presumably forms disulfide bonds between the monomers. As a result, disruption of the otherwise SDS-resistant eNOS dimers occurs under reducing conditions. eNOS catalytic activity is exquisitely sensitive to ONOO -, which decreases NO synthesis and increases superoxide anion (O 2 .-) production by the enzyme. The reducing cofactor tetrahydrobiopterin is not oxidized, nor does it prevent oxidation of eNOS by the same low concentrations of OONO -. Furthermore, eNOS derived from endothelial cells exposed to elevated glucose produces more O 2 .-, and, like eNOS purified from diabetic LDL receptor-deficient mice, contains less zinc and fewer SDS-resistant dimers. Hence, eNOS exposure to oxidants including ONOO -causes increased enzymatic uncoupling and generation of O 2 .-in diabetes, contributing further to endothelial cell oxidant stress. Regulation of the zinc-thiolate center of NOS by ONOO -provides a novel mechanism for modulation of the enzyme function in disease.

A MP-activated protein kinase (AMPK) is a phylogenetically conserved heterotrimer protein consisting of three subunits, ␣, ␤, and ␥, each of which has at least two isoforms (1-3). AMPK is activated by physiological stimuli such as exercise and by hormones, including adiponectin and leptin, as well as by pathological stresses such as glucose deprivation, hypoxia, oxidant stress, and osmotic shock (1-3). Increases in the ratio of AMP to ATP activate AMPK by a number of mechanisms, including direct allosteric activation and covalent modification due to activation by an AMPK kinase (1-3), which phosphorylates the ␣-subunit on threonine-172 (Thr172) (4,5). Once activated, AMPK phosphorylates multiple targets both in vivo and in vitro (1-3), including several biosynthetic enzymes such as acetyl-CoA carboxylase, hydroxymethylglutaryl-CoA reductase, glycogen synthase, and both neuronal and endothelial nitric oxide (NO) synthase (eNOS) (2,6).NO, a free radical gaseous molecule synthesized by the action of the enzyme eNOS, is the most important factor in maintaining vascular homeostasis (7). Endothelium-derived NO promotes vasodilation and inhibits platelet aggregation, leukocyte adherence, and vascular smooth muscle proliferation (7). It has been reported that regulation of eNOS activity is regulated by reversible phosphorylation (8,9) and its interaction with other proteins, such as heat shock protein (hsp)-90 (10,11). For example, AMPK has been demonstrated to phosphorylate eNOS at serine-1179 (Ser1179; equal to human Ser1179) with concomitant activation of eNOS in ischemic heart (7) and in intact human aortic endothelial cells activated with oxidants (peroxynitrite [ONOO Ϫ ]) (12), 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (13), and adiponectin (14,15). However, whether AMPK regulates eNOS in vivo remains to be determined.Metformin, a bioguanide derivative (dimethylbiguanide), is one of the most commonly used drugs for the treatment of type 2 diabetes (16,17). Metformin improves insulin sensitivity, decreases insulin levels, and reduces elevated plasma glucose (16,17). In addition to its insulinsensitizing effects, metformin has also been shown to have direct vascular effects (18 -22). Most importantly, met- AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, 5Ј-AMPactivated kinase; AMPK-CA, constitutively active AMPK kinase; BAEC, bovine aortic endothelial cell; cGMP, cyclic GMP; eNOS, endothelial nitric oxide synthase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; hsp, heat shock protein; ICAM, intracellular adhesion molecule; L-NAME, L-nitro-arginine methyl ester; 3-NT, 3-nitrotyrosine; Ser1179, serine-1179; SOD, superoxide dismutase; Thr172, threonine-172; PDK, phosphoinositide-dependent kinase; VCAM, vascular cell adhesion molecule.

OBJECTIVEAutophagy is a critical cellular system for removal of aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in the development of heart failure, the role of autophagy in the development of diabetic cardiomyopathy has not been studied. We investigated whether chronic activation of the AMP-activated protein kinase (AMPK) by metformin restores cardiac function and cardiomyocyte autophagy in OVE26 diabetic mice.RESEARCH DESIGN AND METHODSOVE26 mice and cardiac-specific AMPK dominant negative transgenic (DN)-AMPK diabetic mice were treated with metformin or vehicle for 4 months, and cardiac autophagy, cardiac functions, and cardiomyocyte apoptosis were monitored.RESULTSCompared with control mice, diabetic OVE26 mice exhibited a significant reduction of AMPK activity in parallel with reduced cardiomyocyte autophagy and cardiac dysfunction in vivo and in isolated hearts. Furthermore, diabetic OVE26 mouse hearts exhibited aggregation of chaotically distributed mitochondria between poorly organized myofibrils and increased polyubiquitinated protein and apoptosis. Inhibition of AMPK by overexpression of a cardiac-specific DN-AMPK gene reduced cardiomyocyte autophagy, exacerbated cardiac dysfunctions, and increased mortality in diabetic mice. Finally, chronic metformin therapy significantly enhanced autophagic activity and preserved cardiac functions in diabetic OVE26 mice but not in DN-AMPK diabetic mice.CONCLUSIONSDecreased AMPK activity and subsequent reduction in cardiac autophagy are important events in the development of diabetic cardiomyopathy. Chronic AMPK activation by metformin prevents cardiomyopathy by upregulating autophagy activity in diabetic OVE26 mice. Thus, stimulation of AMPK may represent a novel approach to treat diabetic cardiomyopathy.

Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus.