Inducible nitric-oxide synthase (iNOS) has been implicated in many human diseases including insulin resistance. However, how iNOS causes or exacerbates insulin resistance remains largely unknown. Protein S-nitrosylation is now recognized as a prototype of a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe the mechanism of inactivation of Akt/protein kinase B (PKB) in NO donor-treated cells and diabetic (db/db) mice. NO donors induced S-nitrosylation and inactivation of Akt/PKB in vitro and in intact cells. The inhibitory effects of NO donor were independent of phosphatidylinositol 3-kinase and cGMP. In contrast, the concomitant presence of oxidative stress accelerated S-nitrosylation and inactivation of Akt/PKB. In vitro denitrosylation with reducing agent reactivated recombinant and cellular Akt/PKB from NO donortreated cells. Mutated Akt1/PKB␣ (C224S), in which cysteine 224 was substituted by serine, was resistant to NO donor-induced S-nitrosylation and inactivation, indicating that cysteine 224 is a major S-nitrosylation acceptor site. In addition, S-nitrosylation of Akt/PKB was increased in skeletal muscle of diabetic (db/db) mice compared with wild-type mice. These data suggest that Snitrosylation-mediated inactivation may contribute to the pathogenesis of iNOS-and/or oxidative stress-involved insulin resistance. Nitric oxide (NO)1 is an endogenous cell signaling molecule involved in the regulation of many physiological functions and in the mediation of a variety of pathophysiological processes. NO and NO-related compounds function as both protective and cytotoxic, dependent on the cellular context and the nature of the NO group. The multifaceted actions of the NO group can be classified into two categories: 1) authentic NO-mediated, cGMPdependent, and 2) reactive nitrogen species-mediated, cGMPindependent actions. Nitrosative post-translational modifications, including protein S-nitrosylation and tyrosine nitration, are involved in the cGMP-independent actions. The cGMP-dependent actions play critical roles in a variety of physiological processes, including NO-mediated vasodilation. In contrast, cGMP-independent, nitrosative protein modifications are postulated to be involved in the pathological responses (1-4).Nitric-oxide synthases (NOSs) consist of three distinct genes, inducible nitric-oxide synthase (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). NO is generated by iNOS to a much greater extent, to over 1,000-fold, compared with that produced by the constitutive NOSs, eNOS and nNOS (2, 5). iNOS and nitrosative stress have been implicated in many human diseases, including insulin resistance (6, 7), atherosclerosis (8), inflammation, and neurodegenerative disorders (9). This is largely based on the evidence that iNOS deficiency results in significant amelioration of, or resistance to, these diseases. However, little is known about the molecular mechanisms by which iNOS causes and/or exacerbates these diseases. Furthe...

Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat diet-induced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, L-NIL, in obese diabetic (ob/ob) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5-and 2-fold, respectively, and enhanced IRS-1-and IRS-2-mediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice. Diabetes 54:1340 -1348, 2005 C hronic low-grade inflammation has been proposed to be involved in the pathogenesis in obesity-related insulin resistance and type 2 diabetes. The expression of proinflammatory cytokines, including tumor necrosis factor-␣ (1) and interleukin-6 (2), is upregulated in animal models of and patients with type 2 diabetes. However, limited knowledge is thus far available about the molecular mechanisms by which chronic inflammation mediates insulin resistance and type 2 diabetes.The activation of inhibitor 〉 kinase ␤ (IKK␤)-nuclear factor-〉 (NF-〉), a crucial signaling cascade for inflammatory response, has been highlighted as a mediator of insulin resistance. The pharmacological inhibition or gene disruption of IKK␤ reversed obesity-related insulin resistance and fasting hyperglycemia in rodents and humans (3-5). However, little is known about genes that function as downstream effectors of the IKKb-NF-B pathway to mediate insulin resistance.Inducible nitric oxide synthase (iNOS; also termed NOS2), whose expression is regulated by IKK␤-NF-B (6), is assumed to be one of the candidates that mediate inflammation-involved insulin resistance. Accumulating evidence indicates a close link between iNOS and insulin resistance. Although iNOS was originally identified in macrophages, it is now known that it is widely expressed in many tissues, including insulin-sensitive organs such as skeletal muscle, adipose tissue, and liver, in normal rodents and humans. The expression of iNOS is upregulated by most, if not all, inducers of insulin resistance, including proinflammatory cytokines, obesity (7), free fatty acids (8), hyperglycemia (9,10), endotoxins (6,11), and oxidative stress. In fact, elevated expression of i...

Lethal hyperkalemic response to succinylcholine continues to be reported, but the molecular mechanisms for the hyperkalemia have not been completely elucidated. In the normal innervated mature muscle, the acetylcholine receptors (AChRs) are located only in the junctional area. In certain pathologic states, including upper or lower motor denervation, chemical denervation by muscle relaxants, drugs, or toxins, immobilization, infection, direct muscle trauma, muscle tumor, or muscle inflammation, and/or burn injury, there is up-regulation (increase) of AChRs spreading throughout the muscle membrane, with the additional expression of two new isoforms of AChRs. The depolarization of these AChRs that are spread throughout the muscle membrane by succinylcholine and its metabolites leads to potassium efflux from the muscle, leading to hyperkalemia. The nicotinic (neuronal) alpha7 acetylcholine receptors, recently described to be expressed in muscle also, can be depolarized not only by acetylcholine and succinylcholine but also by choline, persistently, and possibly play a critical role in the hyperkalemic response to succinylcholine in patients with up-regulated AChRs.

BackgroundDuchenne Muscular Dystrophy (DMD) is characterized by increased muscle damage and an abnormal blood flow after muscle contraction: the state of functional ischemia. Until now, however, the cause-effect relationship between the pathogenesis of DMD and functional ischemia was unclear. We examined (i) whether functional ischemia is necessary to cause contraction-induced myofiber damage and (ii) whether functional ischemia alone is sufficient to induce the damage.Methodology/Principal Findings In vivo microscopy was used to document assays developed to measure intramuscular red blood cell flux, to quantify the amount of vasodilatory molecules produced from myofibers, and to determine the extent of myofiber damage. Reversal of functional ischemia via pharmacological manipulation prevented contraction-induced myofiber damage in mdx mice, the murine equivalent of DMD. This result indicates that functional ischemia is required for, and thus an essential cause of, muscle damage in mdx mice. Next, to determine whether functional ischemia alone is enough to explain the disease, the extent of ischemia and the amount of myofiber damage were compared both in control and mdx mice. In control mice, functional ischemia alone was found insufficient to cause a similar degree of myofiber damage observed in mdx mice. Additional mechanisms are likely contributing to cause more severe myofiber damage in mdx mice, suggestive of the existence of a “two-hit” mechanism in the pathogenesis of this disease.Conclusions/SignificanceEvidence was provided supporting the essential role of functional ischemia in contraction-induced myofiber damage in mdx mice. Furthermore, the first quantitative evidence for the “two-hit” mechanism in this disease was documented. Significantly, the vasoactive drug tadalafil, a phosphodiesterase 5 inhibitor, administered to mdx mice ameliorated muscle damage.

Care of burn-injured patients requires knowledge of the pathophysiologic changes affecting virtually all organs from the onset of injury until wounds are healed. Massive airway and/or lung edema can occur rapidly and unpredictably after burn and/or inhalation injury. Hemodynamics in the early phase of severe burn injury are characterized by a reduction in cardiac output, increased systemic and pulmonary vascular resistance. Approximately 2–5 days after major burn injury, a hyperdynamic and hypermetabolic state develops. Electrical burns result in morbidity much higher than expected based on burn size alone. Formulae for fluid resuscitation should serve only as guideline; fluids should be titrated to physiologic end points. Burn injury is associated basal and procedural pain requiring higher than normal opioid and sedative doses. Operating room concerns for the burn-injured patient include airway abnormalities, impaired lung function, vascular access, deceptively large and rapid blood loss, hypothermia and altered pharmacology.

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Obesity is a major cause of type 2 diabetes, clinically evidenced as hyperglycemia. The altered glucose homeostasis is caused by faulty signal transduction via the insulin signaling proteins, which results in decreased glucose uptake by the muscle, altered lipogenesis, and increased glucose output by the liver. The etiology of this derangement in insulin signaling is related to a chronic inflammatory state, leading to the induction of inducible nitric oxide synthase and release of high levels of nitric oxide and reactive nitrogen species, which together cause post-translational modifications in the signaling proteins. There are substantial differences in the molecular mechanisms of insulin resistance in muscle versus liver. Hormones and cytokines from adipocytes can enhance or inhibit both glycemic sensing and insulin signaling. The role of the central nervous system in glucose homeostasis has also been established. Multi-pronged therapies aimed at rectifying obesity-induced anomalies in both central nervous system and peripheral tissues may prove to be beneficial.

Critical illness is associated with muscle wasting and muscle weakness. Using burn injury as a model of local and systemic inflammatory response, we tested the hypothesis that thermal injury causes apoptosis in muscle. After a 40% body surface area burn to rats, abdominal muscles beneath the burn and limb muscles distant from the burn were examined for apoptosis at varying times after burn. Ladder assay, ELISA, and histological methods showed evidence of apoptosis in the abdominal muscles within 4-12 h with peak changes occurring at 3-7 days. Maximal apoptosis was also evident at distant limb muscles at 3-7 days. Investigation of proapoptotic pathways indicated mitochondrial membrane potential to be altered by 1 h after burn. Starting at 15 min after burn, cytochrome c was released from the mitochondria into the cytosol, followed by increased activity of caspase-3, starting at 6 h after burn. These studies suggest that mitochondria and caspase-mediated apoptotic pathways may be an additional mechanism of muscle weight loss in burns and may be potential therapeutic targets for prevention of muscle wasting.