Inhibition of Inducible Nitric-oxide Synthase by Activators of AMP-activated Protein Kinase

AMP-activated protein kinase (AMPK), an energy-sensing 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), an energy-sensing 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) 1The abbreviations used are: AMPK, AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide-riboside; TNF-α, tumor necrosis factor-α; NO, nitric oxide; iNOS, inducible nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; LPS, lipopolysaccharide; ACC, acetyl-CoA carboxylase; BW, body weight; IRS-1, insulin receptor substrate-1; PI, phosphatidylinositol; IFN, interferon; RT, reverse transcription; siRNA, small interfering RNA; PPAR, peroxisome proliferator-activated receptor; PG, prostaglandin; eNOS, endothelial nitric oxide synthase; 15d PGJ2, 15-deoxy-Δ12,14-prostaglandin J2. 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 (1Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (720) Google Scholar). 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 α-subunit and two regulatory subunits, β and γ. 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 (2Hong S.P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (481) Google Scholar). 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 (3Winder W.W. J. Appl. Physiol. 2001; 91: 1017-1028Crossref PubMed Scopus (326) Google Scholar, 4Sakamoto K. Goodyear L.J. J. Appl. Physiol. 2002; 93: 369-383Crossref PubMed Scopus (193) Google Scholar). 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 (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1036) Google Scholar). 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 (6Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1691) Google Scholar, 7Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang C.C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (852) Google Scholar, 8Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3483) Google Scholar). Chronic treatment of animal models of type 2 diabetes with the AMPK activator AICAR improves glucose homeostasis and insulin sensitivity (9Winder W.W. Diabetes Technol. Ther. 2000; 2: 441-448Crossref PubMed Scopus (53) Google Scholar, 10Halseth A.E. Ensor N.J. White T.A. Ross S.A. Gulve E.A. Biochem. Biophys. Res. Commun. 2002; 294: 798-805Crossref PubMed Scopus (58) Google Scholar, 11Fiedler M. Zierath J.R. Selen G. Wallberg-Henriksson H. Liang Y. Sakariassen K.S. Diabetologia. 2001; 44: 2180-2186Crossref PubMed Scopus (53) Google Scholar). 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 activate AMPK, and this is believed to contribute to their insulin-sensitizing actions in diabetic subjects (12Musi N. Hirshman M.F. Nygren J. Svanfeldt M. Bavenholm P. Rooyackers O. Zhou G. Williamson J.M. Ljunqvist O. Efendic S. Moller D.E. Thorell A. Goodyear L.J. Diabetes. 2002; 51: 2074-2081Crossref PubMed Scopus (672) Google Scholar, 13Hawley S.A. Gadalla A.E. Olsen G.S. Hardie D.G. Diabetes. 2002; 51: 2420-2425Crossref PubMed Scopus (582) Google Scholar, 14Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 15Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Invest. 2001; 108: 1167-1174Crossref PubMed Scopus (4472) Google Scholar). However, the potential effects of AMPK activation on proinflammatory mediators of insulin resistance have not been investigated yet. Indeed, an inflammatory component also is present in obesity-linked diabetes (16Pickup J.C. Crook M.A. Diabetologia. 1998; 41: 1241-1248Crossref PubMed Scopus (833) Google Scholar, 17Marette A. Curr. Opin. Clin. Nutr. Metab. Care. 2002; 5: 377-383Crossref PubMed Scopus (123) Google Scholar) as reflected by increased systemic and tissue concentrations of the proinflammatory cytokines TNF-α and interleukin-6 in obese human subjects (18Hotamisligil G.S. Arner P. Caro J.F. Atkinson R.L. Spiegelman B.M. J. Clin. Investig. 1995; 95: 2409-2415Crossref PubMed Scopus (2991) Google Scholar, 19Yudkin J.S. Stehouwer C.D. Emeis J.J. Coppack S.W. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 972-978Crossref PubMed Scopus (2164) Google Scholar) and several animal models of obesity (20Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-91Crossref PubMed Scopus (6193) Google Scholar, 21Hotamisligil G.S. Spiegelman B.M. Diabetes. 1994; 43: 1271-1278Crossref PubMed Google Scholar, 22Perreault M. Marette A. Nat. Med. 2001; 7: 1138-1143Crossref PubMed Scopus (431) Google Scholar). Furthermore, we recently have reported that inducible nitric-oxide synthase (iNOS), a cytokine-inducible proinflammatory mediator in several pathological conditions, is overexpressed in muscle and fat of genetic and dietary models of obesity and type 2 diabetes (22Perreault M. Marette A. Nat. Med. 2001; 7: 1138-1143Crossref PubMed Scopus (431) Google Scholar). Targeted disruption of the iNOS gene was found to protect high fat-fed obese mice from developing insulin resistance and to significantly improve glucose tolerance (22Perreault M. Marette A. Nat. Med. 2001; 7: 1138-1143Crossref PubMed Scopus (431) Google Scholar). A role for iNOS in mediating insulin resistance also is supported by the finding that pharmacological inhibition of iNOS reverses impaired insulin action in cultured myocytes exposed to cytokines and the endotoxin LPS (23Bedard S. Marcotte B. Marette A. Biochem. J. 1997; 325: 487-493Crossref PubMed Scopus (143) Google Scholar). Because an inflammatory component that includes iNOS is involved in the pathogenesis of muscle insulin resistance in obesity-linked type 2 diabetes, we asked whether AMPK activation might improve insulin action in muscle cells through inhibition of iNOS, and if so, whether AMPK is a key iNOS inhibitory pathway in other cell types where increased iNOS expression is pathogenic. Materials—AICAR was purchased from Toronto Research Chemicals (Toronto, Canada). Troglitazone and 15-deoxy PGJ2 were from Cayman Chemical Co. (Ann Arbor, MI). Interferon-γ and TNF-α were from Research Diagnostics (Flanders, NJ) and RD Systems (Minneapolis, MN), respectively. 1400W was from Biomol Research Laboratories (Plymouth, PA), and Oligofectamine was from Invitrogen. ADP-Sepharose 4B beads were from Amersham Biosciences, and the Renaissance enhanced chemiluminescence kit was from PerkinElmer Life Sciences (Boston, MA). All other chemicals were from Sigma. Monoclonal antibodies against iNOS and eNOS and a polyclonal antibody against nNOS were obtained from Transduction Laboratories (Mississauga, Canada). Polyclonal antibodies against AMPK (which recognizes both α1- and α2-AMPK), phospho-AMPK, and phospho-ACC (acetyl-CoA carboxylase) were purchased from New England Biolabs (Beverly, MA). IRS-1 antibody (C-20) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Culture—L6 myoblasts and 3T3-L1 fibroblasts were grown in α-minimum Eagle's medium (10% fetal bovine serum) or α-Dulbecco's modified Eagle's medium (20% calf serum) containing 1% antibiotic/antimycotic solution and differentiated into myotubes and adipocytes as described (23Bedard S. Marcotte B. Marette A. Biochem. J. 1997; 325: 487-493Crossref PubMed Scopus (143) Google Scholar, S. Marcotte B. Marette A. J. Physiol. 1999; Google Scholar). were a of M. Research Canada). and of the from was as described G. S. S. D. M. Eur. J. 2001; PubMed Scopus Google Scholar). of iNOS by cytokines LPS in 3T3-L1 adipocytes, and and the effects of were as described in the The of in the medium was used as an of was as described (23Bedard S. Marcotte B. Marette A. Biochem. J. 1997; 325: 487-493Crossref PubMed Scopus (143) Google Scholar). RNA interfering were by Research and to the The of the 2 were as siRNA, myotubes in were to and LPS treatment with α1- and by the Oligofectamine were with was by the and of purchased from were used in were with a of AICAR metformin or to LPS BW, or were and and adipose tissues were and of and and and levels in skeletal and adipose tissue were by A. J.F. Res. 1998; PubMed Scopus Google Scholar). was in containing and for to were in a The tissue was in of containing a were for and protein of the was by the protein and tissue were in an was to and the system as described A. J.F. Res. 1998; PubMed Scopus Google Scholar). The was of and of of and and adipose tissue of were used to nitric-oxide synthase as described (22Perreault M. Marette A. Nat. Med. 2001; 7: 1138-1143Crossref PubMed Scopus (431) Google Scholar). were to and was as described S. S. Marcotte B. Marette A. Diabetes. 1997; PubMed Scopus Google Scholar). were by the enhanced chemiluminescence RNA cellular RNA was and iNOS was by reverse as described (23Bedard S. Marcotte B. Marette A. Biochem. J. 1997; 325: 487-493Crossref PubMed Scopus (143) Google Scholar, S. Marcotte B. Marette A. J. Physiol. 1999; Google Scholar). were with 2 of to protein was as described (22Perreault M. Marette A. Nat. Med. 2001; 7: 1138-1143Crossref PubMed Scopus (431) Google Scholar). are as The effects of the were by an of by were to be AMPK iNOS and in the effect of the AMPK activator AICAR on iNOS induction in myocytes exposed to cytokines and LPS, which is a of chronic with insulin resistance (23Bedard S. Marcotte B. Marette A. Biochem. J. 1997; 325: 487-493Crossref PubMed Scopus (143) Google Scholar, S. S. Marcotte B. Marette A. Diabetes. 1997; PubMed Scopus Google Scholar). in treatment of muscle cells with AMPK as reflected by increased of AMPK on known to activate the enzyme S.A. M. Woods A. S.P. Carling D. Hardie D.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google as well as by its to of ACC, a target of AMPK and a of its activation W.W. Hardie D.G. J. Physiol. 1999; 277: Google Scholar). The anti-diabetic metformin and the and also activated AMPK in cells Chronic of myocytes to cytokines and and LPS markedly production as by the of the in the medium effect was explained by a induction of iNOS and protein levels and eNOS nNOS is in or and production was by with the iNOS 1400W F. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) not of AICAR a of a inhibition with metformin or also decreased the effect of on All AMPK activators production through the of cellular iNOS protein levels AMPK activators to the cellular expression of the protein in used as an not Reduction of iNOS protein expression was not to of iNOS because iNOS levels were not by AICAR or the anti-diabetic drugs metformin and However, high concentrations of iNOS levels in In we also whether AICAR production through in or by inhibition of iNOS by muscle cells was not found to be by AICAR for and Furthermore, AICAR not iNOS and to iNOS catalytic in as by the of from A and in It has been reported that AICAR increases adenosine by tissue H.E. T.A. R.L. PubMed Scopus Google and this contribute to iNOS However, adenosine to iNOS in and the inhibitory effect of AICAR on production was not decreased by adenosine with and in role for adenosine in iNOS whether iNOS inhibition reverses the insulin effects of we the effects of AMPK activators on insulin action in myocytes. to activation of an mediator of the metabolic actions of glucose because the is increased by AMPK through in A and cytokines and LPS treatment insulin resistance as by a in the of insulin to as with However, of AICAR, or to cells insulin action on AMPK to increase in the of insulin The of insulin action by was to that obtained with the iNOS 1400W AMPK iNOS in and we whether AMPK activation inhibits iNOS in other cell have reported that adipose tissue is a of iNOS expression in and that chronic treatment of adipocytes with cytokines and this inflammatory response in S. Marcotte B. Marette A. J. Physiol. 1999; Google Scholar). in of AICAR or anti-diabetic drugs to cultured 3T3-L1 adipocytes activated AMPK as reflected by enhanced of AMPK production and cellular iNOS protein not iNOS levels and are of iNOS in or inflammatory production of with or against several and tumor it also can proinflammatory effects on cells J. C. 1997; PubMed Scopus Google Scholar). of AMPK by AICAR and metformin iNOS induction of to and The also increased AMPK and a inhibition of production in cells and The inhibitory effects of AMPK activators also were to in iNOS protein induction through a post-transcriptional and However, concentrations of a in iNOS levels was also of AMPK by in of AICAR and iNOS in and of whether AMPK also iNOS induction in we the of AICAR and metformin to iNOS in skeletal muscle and adipose tissue of with the endotoxin LPS, a of shock with insulin resistance S. S. Marcotte B. Marette A. Diabetes. 1997; PubMed Scopus Google Scholar). of with AICAR or metformin increased and significantly production in the as well as in muscle and and adipose tissues AICAR and metformin induction of iNOS protein expression in muscle and fat tissues of In eNOS were by LPS or AMPK activators in muscle and fat was not in the effect of AMPK on iNOS protein. observed for cell the inhibitory effects of AMPK activators on production was explained by post-transcriptional because iNOS expression was not by AICAR or metformin However, is which a small in iNOS in adipose RNA of the of AMPK on iNOS and whether AICAR and anti-diabetic drugs iNOS through AMPK we used RNA to AMPK that of for the and catalytic expression to of that of cells with treatment resulted in a inhibition of AMPK and by AMPK activators In the of AICAR, and to production and iNOS protein expression was significantly as with cells with These results that iNOS expression and production in through activation of of the multiple of in and states has where of production be of therapeutic iNOS induction and production is an important and of C. M. A. 2000; PubMed Scopus Google high of also can to in chronic and other by activation of the system C. J. Clin. Investig. 1997; 100: PubMed Scopus Google Scholar, J.F. B. J. Med. 1997; PubMed Scopus Google Scholar, J.C. J. 1998; PubMed Scopus Google Scholar, S. S. A. S. J. A. G. R. J. 2003; PubMed Scopus Google Scholar). iNOS induction also is in chronic metabolic as J.M. H. Res. 1999; 43: PubMed Scopus Google Scholar, D. A. S. E. S. A. B. 2000; PubMed Scopus Google Scholar) and obesity-linked diabetes (22Perreault M. Marette A. Nat. Med. 2001; 7: 1138-1143Crossref PubMed Scopus (431) Google Scholar, M. M. Y. J. Clin. 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Diabetes. 2002; 51: 2420-2425Crossref PubMed Scopus (582) Google Scholar, 14Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 15Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Invest. 2001; 108: 1167-1174Crossref PubMed Scopus (4472) Google Scholar, N. Goodyear L.J. Curr. 2002; 2: PubMed Google Scholar). These anti-diabetic effects mainly have been to the and metabolic effects of AMPK However, that inhibition of iNOS induction and a novel by which AMPK improves insulin action in Indeed, we show that AMPK activation by several drugs iNOS induction in myocytes exposed to cytokines and LPS, an in of insulin resistance for glucose transport (23Bedard S. Marcotte B. Marette A. Biochem. J. 1997; 325: 487-493Crossref PubMed Scopus (143) Google Scholar). Accordingly, we also found that in activation can be reversed by AMPK Furthermore, the inhibitory effect of AMPK activation on iNOS was in vivo in a of shock with insulin resistance S. S. Marcotte B. Marette A. Diabetes. 1997; PubMed Scopus Google Scholar). studies be to whether AMPK activation also can or iNOS induction in chronic inflammatory with insulin resistance as and obesity-linked diabetes. AMPK activation also iNOS protein expression and production in adipocytes and in adipose tissue of rats. the role of in adipose cells still is not it is thought to be involved in the of lipid metabolism in S. Marcotte B. Marette A. J. Physiol. 1999; Google Scholar) and be a mediator of skeletal muscle insulin resistance in obesity-linked diabetes (22Perreault M. Marette A. Nat. 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