AMP-activated protein kinase (AMPK), an energysensing enzyme that is activated in response to cellular stress, is a critical signaling molecule for the regulation of multiple metabolic processes. AMPK has recently emerged as an attractive novel target for the treatment of obesity and type 2 diabetes because its activation increases fatty acid oxidation and improves glucose homeostasis. Here we show that pharmacological activation of AMPK by insulin-sensitizing drugs markedly inhibits inducible nitric-oxide synthase (iNOS), a proinflammatory mediator in endotoxic shock and in chronic inflammatory states including obesity-linked diabetes. AMPK-mediated iNOS inhibition was observed in several cell types (myocytes, adipocytes, macrophages) and primarily resulted from post-transcriptional regulation of the iNOS protein. AMPK activation in vivo also blunted iNOS induction in muscle and adipose tissues of endotoxin-challenged rats. Reduction of AMPK expression by small interfering RNA reversed the inhibitory effects of AMPK activators on iNOS expression and nitric oxide production in myocytes. These results indicate that AMPK is a novel anti-inflammatory signaling pathway and thus represents a promising therapeutic target for immune-inflammatory disorders. AMP-activated protein kinase (AMPK)1 is emerging as an important energy-sensing/signaling system in mammalian tissues. It is a member of a metabolite-sensing protein kinase family that acts as a fuel gauge by monitoring cellular energy levels (1). When AMPK "senses" decreased energy storage, it acts to switch off ATP-consuming pathways and switch on alternative pathways for ATP regeneration. AMPK is a heterotrimer consisting of a catalytic ␣-subunit and two regulatory subunits,  and ␥. In response to cellular energy depletion, as reflected by an increase in the AMP/ATP ratio, AMPK is phosphorylated and activated by a still uncharacterized upstream AMPK kinase (2). It also can be activated allosterically by increases in the AMP/ATP and creatine/creatine-P ratios. The metabolic function of AMPK perhaps has been documented best in exercising skeletal muscle, where its activation seems to contribute to increased glucose transport and fatty acid oxidation (3, 4). AMPK can be activated chemically with 5-aminoimidazole-4-carboxamide riboside (AICAR), which is taken up by cells and phosphorylated by adenosine kinase to form 5-aminoimidazole-4-carboxamide ribonucleoside, a nucleotide that mimics the effect of AMP (5). More recent studies also have identified AMPK as the mediator of the metabolic effects of the adipose-derived peptidic hormones leptin and adiponectin in skeletal muscle and liver (6 -8).Chronic treatment of animal models of type 2 diabetes with the AMPK activator AICAR improves glucose homeostasis and insulin sensitivity (9 -11). These beneficial effects are thought to be explained mainly by the well known actions of AMPK on glucose metabolism and lipid oxidation in muscle and liver. Accordingly, the anti-diabetic drugs metformin and rosiglitazone also activat...
OBJECTIVE-Synthetic ligands for peroxisome proliferatoractivated receptor-␥ (PPAR-␥) improve insulin sensitivity in obesity, but it is still unclear whether inflammatory signals modulate their metabolic actions. In this study, we tested whether targeted disruption of inducible nitric oxide (NO) synthase (iNOS), a key inflammatory mediator in obesity, modulates the metabolic effects of rosiglitazone in obese mice. RESEARCH DESIGN AND METHODS-iNOSϪ/Ϫ and iNOS ϩ/ϩ were subjected to a high-fat diet or standard diet for 18 weeks and were then treated with rosiglitazone for 2 weeks. Whole-body insulin sensitivity and glucose tolerance were determined and metabolic tissues harvested to assess activation of insulin and AMP-activated protein kinase (AMPK) signaling pathways and the levels of inflammatory mediators.RESULTS-Rosiglitazone was found to similarly improve whole-body insulin sensitivity and insulin signaling to Akt/PKB in skeletal muscle of obese iNOS Ϫ/Ϫ and obese iNOS ϩ/ϩ mice. However, rosiglitazone further improved glucose tolerance and liver insulin signaling only in obese mice lacking iNOS. This genotype-specific effect of rosiglitazone on glucose tolerance was linked to a markedly increased ability of the drug to raise plasma adiponectin levels. Accordingly, rosiglitazone increased AMPK activation in muscle and liver only in obese iNOS Ϫ/Ϫ mice. PPAR-␥ transcriptional activity was increased in adipose tissue of iNOS Ϫ/Ϫ mice. Conversely, treatment of 3T3-L1 adipocytes with a NO donor blunted PPAR-␥ activity. CONCLUSIONS-Our results identify the iNOS/NO pathway as a critical modulator of PPAR-␥ activation and circulating adiponectin levels and show that invalidation of this key inflammatory mediator improves the efficacy of PPAR-␥ agonism in an animal model of obesity and insulin resistance.
BackgroundIt is believed that the endotoxin lipopolysaccharide (LPS) is implicated in the metabolic perturbations associated with both sepsis and obesity (metabolic endotoxemia). Here we examined the role of inducible nitric oxide synthase (iNOS) in skeletal muscle insulin resistance using LPS challenge in rats and mice as in vivo models of endotoxemia.Methodology/Principal FindingsPharmacological (aminoguanidine) and genetic strategies (iNOS−/− mice) were used to counter iNOS induction in vivo. In vitro studies using peroxynitrite (ONOO−) or inhibitors of the iNOS pathway, 1400 W and EGCG were conducted in L6 myocytes to determine the mechanism by which iNOS mediates LPS-dependent insulin resistance. In vivo, both pharmacological and genetic invalidation of iNOS prevented LPS-induced muscle insulin resistance. Inhibition of iNOS also prevented insulin resistance in myocytes exposed to cytokine/LPS while exposure of myocytes to ONOO− fully reproduced the inhibitory effect of cytokine/LPS on both insulin-stimulated glucose uptake and PI3K activity. Importantly, LPS treatment in vivo and iNOS induction and ONOO− treatment in vitro promoted tyrosine nitration of IRS-1 and reduced insulin-dependent tyrosine phosphorylation.Conclusions/SignificanceOur work demonstrates that iNOS-mediated tyrosine nitration of IRS-1 is a key mechanism of skeletal muscle insulin resistance in endotoxemia, and presents nitrosative modification of insulin signaling proteins as a novel therapeutic target for combating muscle insulin resistance in inflammatory settings.
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