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.

Loss of the modulatory role of the endothelium may be a critical initial factor in the development of diabetic vascular diseases. Exposure of human aortic endothelial cells (HAECs) to high glucose (30 or 44 mmol/l) for 7-10 days significantly increased the release of superoxide anion in response to the calcium ionophore A23187. Nitrate, a breakdown product of peroxynitrite (ONOO ؊ ), was substantially increased in parallel with a decline in cyclic guanosine monophosphate (GMP). Using immunochemical techniques and high-performance liquid chromatography, an increase in tyrosine nitration of prostacyclin (PGI 2 ) synthase (PGIS) associated with a decrease in its activity was found in cells exposed to high glucose. Both the increase in tyrosine nitration and the decrease in PGIS activity were lessened by decreasing either nitric oxide or superoxide anion, suggesting that ONOO ؊ was responsible. Furthermore, SQ29548, a thromboxane/prostaglandin (PG) H 2 (TP) receptor antagonist, significantly reduced the increased endothelial cell apoptosis and the expression of soluble intercellular adhesion molecule-1 that occurred in cells exposed to high glucose, without affecting the decrease in PGIS activity. Thus, exposure of HAECs to high glucose increases formation of ONOO ؊ , which causes tyrosine nitration and inhibition of PGIS. The shunting of arachidonic acid to the PGI 2 precursor PGH 2 or other eicosanoids likely results in TP receptor stimulation. These observations can explain several abnormalities in diabetes, including 1) increased free radicals, 2) decreased bioactivity of NO, 3) PGI 2 deficiency, and 4) increased vasoconstriction, endothelial apoptosis, and inflammation via TP receptor stimulation. Diabetes 51: 198 -203, 2002

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.

. release.Mammalian AMP-activated protein kinase (AMPK) 1 belongs to a family of protein kinases that has been highly conserved in animals, plants, and yeast and that plays a key role in the regulation of energy homeostasis (1, 2). AMPK is a heterotrimeric enzyme, consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥) (3, 4). AMPK is activated by cellular stresses such as shock, hypoxia, and ischemia, that deplete ATP and that in turn elevate the ratio of AMP to ATP (2, 5). In addition to allosteric activation by AMP, AMPK is phosphorylated and activated by an upstream kinase, termed AMPK kinase (AMPKK) (6). Activation of AMPK results from phosphorylation of Thr 172 in the activation loop of the catalytic ␣-subunit, although other phosphorylation sites have been reported (7). Whether or not AMPK is regulated by mechanisms other than the AMP/ATP ratio remains elusive.Once activated, AMPK phosphorylates multiple downstream substrates aimed at conserving existing ATP levels. AMPK reduces further ATP expenditure by inhibiting key enzymes in biosynthetic pathways such as acetyl-CoA carboxylase (ACC), which is important in fatty acid synthesis, and 3-hydroxy-3-methyl-CoA reductase in cholesterol synthesis (2, 8 -10). Despite the recent observation that AMPK phosphorylates eNOS on Ser 1179 (based on the bovine eNOS sequence and equivalent to human eNOS-Ser 1177 ) and activates rat cardiac eNOS in vitro (11,12), the mechanism and functional implications of AMPK-mediated eNOS phosphorylation remains unknown.Peroxynitrite (ONOO Ϫ ), a highly reactive oxidant formed by the diffusion-controlled reaction of superoxide anion (O 2 . ) and nitric oxide (NO), is formed during sepsis, inflammation, diabetes, ischemia-reperfusion, and atherosclerosis and contributes to these pathophysiological processes (13)(14)(15)(16)(17). In a recent study we showed that ONOO Ϫ oxidizes the zinc-thiolate cluster of eNOS, inhibiting its NO synthetic activity but increasing the NADPH oxidase activity and O 2 . production by the enzyme (17).In the present study, we further examined how ONOO Ϫ regulates eNOS activity. We show that in BAEC cells, ONOO

Background: Mutant-selective IDH1 inhibitors are potential cancer therapeutics, but the mechanistic basis for their selectivity is not yet well understood. Results: Inhibitor binding modes and kinetic mechanisms were characterized. Conclusion:The inhibitors selectively inhibit mutant IDH1 by interacting with a magnesium-binding residue. Significance: Targeting metal-binding residues with drug-like small molecules is a feasible strategy for IDH1 inhibition.

The TP antagonist inhibits inflammation and accelerated atherogenesis caused by diabetes, most likely by counteracting effects on endothelial function and adhesion molecule expression of eicosanoids stimulated by the diabetic milieu.

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific singlestranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo.Implications: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.

Objectives-To understand the mechanism by which oxidants are linked to insulin resistance, bovine aortic endothelial cells were exposed to oxidized low-density lipoproteins (oxLDL) or peroxynitrite. Methods and Results-OxLDL transiently increased phosphorylation of Erk and Akt within 5 minutes, but 60 minutes later, resulted in decreased insulin-induced Akt phosphorylation. OxLDL promoted a 2-to 5-fold increase in oxidant generation as measured by dihydrorhodamine or dihydroethidium oxidation that was ascribed to peroxynitrite. Exogenous peroxynitrite (25 to 100 mol/L) or oxidized glutathione mimicked the effects of oxLDL. OxLDL increased the S-glutathiolation of p21ras, and adenoviral transfection with either a mutant p21ras (C118S) lacking the predominant site of S-glutathiolation or a dominant-negative mutant restored insulin-induced Akt phosphorylation. The requirement for oxidant-mediated S-glutathiolation and activation of p21ras in mediating insulin resistance was further implicated by showing that insulin signaling was restored by Mek inhibitors or by overexpression of glutaredoxin-1. Furthermore, oxLDL increased Erk-dependent phosphorylation of insulin receptor substrate-1 serine-616 that was prevented by inhibiting oxidant generation, Erk activation, or by the p21ras C118S mutant. Conclusions-This study provides direct evidence for a novel molecular mechanism by which oxidants can induce insulin resistance via S-glutathiolation of p21ras and Erk-dependent inhibition of insulin signaling. Key Words: p21ras Ⅲ peroxynitrite Ⅲ S-glutathiolation Ⅲ glutathione Ⅲ insulin resistance Ⅲ oxidized LDL T he metabolic syndrome is recognized as one of the major causes of human morbidity and mortality. 1 Skeletal muscle cells from 30% of type 2 diabetic patients demonstrate decreased glucose uptake associated with a decrease in insulin receptor substrate (IRS)-1 tyrosine phosphorylation as well as decreased phosphatidylinositol (PI)-3 kinase/Akt activity, 2,3 indicating that abnormalities in the insulin signaling pathway itself are involved. Abnormal function of insulin receptors, IRS-1, PI-3 kinase, and downstream signaling elements including Akt have been demonstrated in cellular models of insulin resistance 3,4 produced by exposing adipocytes, 5 fibroblasts, 6 hepatocytes, 7 or endothelial cells 8 to elevated glucose, 9 fatty acids, 10 inflammatory cytokines, 11 lysophosphatidylcholine, 12 or oxidized low-density lipoprotein (oxLDL). 6 Activation of mitogen-activated protein kinase cascades, including Erk and Jun kinase, has been implicated in mediating degradation of the insulin receptor as well as IRS-1 because of phosphorylation of multiple serine residues. 8 Exactly how these signaling mechanisms are activated in insulin resistant states is controversial.Hyperlipidemia, 13 hyperglycemia, 14,15 oxLDL, 6 lysophosphatidyl choline, 16 and other factors that are elevated in metabolic syndrome increase the generation of oxidants in vascular cells from multiple sources including mitochondria, 17 NADPH oxidase, 18 -20...