The Anti-diabetic Drugs Rosiglitazone and Metformin Stimulate AMP-activated Protein Kinase through Distinct Signaling Pathways

AMP-activated protein kinase (AMPK) is activated within the cell in response to multiple stresses that increase the intracellular AMP:ATP ratio. Here we show that incubation of muscle cells with the thiazolidinedione, rosiglitazone, leads to a dramatic increase in this ratio with the concomitant activation of AMPK. This finding raises the possibility that a number of the beneficial effects of the thiazolidinediones could be mediated via activation of AMPK. Furthermore, we show that in addition to the classical activation pathway, AMPK can also be stimulated without changing the levels of adenine nucleotides. In muscle cells, both hyperosmotic stress and the anti-diabetic agent, metformin, activate AMPK in the absence of any increase in the AMP:ATP ratio. However, although activation is no longer dependent on this ratio, it still involves increased phosphorylation of threonine 172 within the catalytic (α) subunit. AMPK stimulation in response to hyperosmotic stress does not appear to involve phosphatidylinositol 3-phosphate kinase, protein kinase C, mitogen-activated protein (MAP) kinase kinase, or p38 MAP kinase α or β. Our results demonstrate that AMPK can be activated by at least two distinct signaling mechanisms and suggest that it may play a wider role in the cellular stress response than was previously understood. AMP-activated protein kinase (AMPK) is activated within the cell in response to multiple stresses that increase the intracellular AMP:ATP ratio. Here we show that incubation of muscle cells with the thiazolidinedione, rosiglitazone, leads to a dramatic increase in this ratio with the concomitant activation of AMPK. This finding raises the possibility that a number of the beneficial effects of the thiazolidinediones could be mediated via activation of AMPK. Furthermore, we show that in addition to the classical activation pathway, AMPK can also be stimulated without changing the levels of adenine nucleotides. In muscle cells, both hyperosmotic stress and the anti-diabetic agent, metformin, activate AMPK in the absence of any increase in the AMP:ATP ratio. However, although activation is no longer dependent on this ratio, it still involves increased phosphorylation of threonine 172 within the catalytic (α) subunit. AMPK stimulation in response to hyperosmotic stress does not appear to involve phosphatidylinositol 3-phosphate kinase, protein kinase C, mitogen-activated protein (MAP) kinase kinase, or p38 MAP kinase α or β. Our results demonstrate that AMPK can be activated by at least two distinct signaling mechanisms and suggest that it may play a wider role in the cellular stress response than was previously understood. AMP-activated protein kinase 5-amino-4-imidazolecarboxamide AICA riboside dinitrophenol mitogen-activated protein the synthetic peptide corresponding to the amino acid sequence HMRSAMSGL- HLVKRR The AMP-activated protein kinase (AMPK)1 plays a key role in the regulation of metabolism within the muscle cell and has been implicated as a potential target in type 2 diabetes mellitus and in obesity (1Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: 1-10PubMed Google Scholar, 2Moller D.E. Nature. 2001; 414: 821-827Crossref PubMed Scopus (893) Google Scholar, 3Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). AMPK is a heterotrimeric complex consisting of a catalytic (α) subunit and two regulatory subunits (β and γ) (4Woods A. Cheung P.C.F. Smith F.C. Davison M.D. Scott J. Beri R.K. Carling D. J. Biol. Chem. 1996; 271: 10282-10290Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Isoforms of all three subunits have been identified, including two isoforms of the catalytic subunit, α1 and α2 (5Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Previous studies have shown that AMPK is activated following depletion of cellular ATP together with a concomitant rise in AMP (6Corton J.M. Gillespie J.G. Hardie D.G. Curr. Biol. 1994; 4: 315-324Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 7Hardie D.G. Salt I.P. Hawley S.A. Davies S.P. Biochem. J. 1999; 338: 717-722Crossref PubMed Scopus (317) Google Scholar). An increase in the AMP:ATP ratio causes increased phosphorylation of AMPK on threonine residue 172 within the α subunit by an as yet poorly characterized upstream kinase (8Hawley S.A. Davison M.D. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). In response to activation, AMPK switches off ATP-utilizing pathways and switches on ATP-producing pathways. These combined actions have led to the proposal that AMPK acts as a cellular fuel gauge (9Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar, 10Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1276) Google Scholar). A number of physiological and pathophysiological stimuli that lead to an increase in the AMP:ATP ratio within the cell have been demonstrated to activate AMPK, including muscle contraction, heat shock, metabolic poisoning, and ischemia (6Corton J.M. Gillespie J.G. Hardie D.G. Curr. Biol. 1994; 4: 315-324Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 11Fryer L. Hajduch E. Rencurel F. Salt I. Hundal H. Hardie D. Carling D. Diabetes. 2000; 49: 1978-1985Crossref PubMed Scopus (159) Google Scholar, 12Hayashi T. Hirshman M. Fujii N. Habinowski S. Witters L. Goodyear L. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (380) Google Scholar, 13Marsin A. Bertrand L. Rider M. Deprez J. Beauloye C. Vincent M. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Although activation of AMPK appears to be a direct consequence of an increase in the AMP:ATP ratio, it is not clear whether there are other signals, which do not involve changes in adenine nucleotide levels, that can lead to activation of AMPK. Recently, Zhou et al. (14Zhou 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 (4432) Google Scholar) demonstrated the activation of AMPK by metformin in both hepatocytes and skeletal muscle. Metformin, one of the most widely used oral drugs for the treatment of type 2 diabetes, decreases hyperglycemia and has beneficial effects on circulating lipids, without affecting insulin secretion (15Wu M.S. Johnston P. Sheu W.H.H. Hollenbeck C.B. Jeng C.Y. Goldfine I.D. Chen Y.D.I. Reaven G.M. Diabetes Care. 1990; 13: 1-8Crossref PubMed Scopus (210) Google Scholar, 16Stumvoll M. Nurjhan N. Perriello G. Dailey G. Gerich J.E. New Engl. J. Med. 1995; 333: 550-554Crossref PubMed Scopus (1015) Google Scholar). The glucose lowering effects of metformin are attributable to both an increase in muscle glucose uptake (17Hundal H.S. Ramal T. Reyes R. Leiter L.A. Klip A. Endocrinology. 1992; 131: 1165-1173Crossref PubMed Scopus (143) Google Scholar) and a decrease in hepatic glucose production (16Stumvoll M. Nurjhan N. Perriello G. Dailey G. Gerich J.E. New Engl. J. Med. 1995; 333: 550-554Crossref PubMed Scopus (1015) Google Scholar, 18Hundal R.S. Krssak M. Dufour S. Laurent D. Lebon V. Chandramouli V. Inzucchi S.E. Schumann W.C. Petersen K.F. Landau B.R. Shulman G.I. Diabetes. 2000; 49: 2063-2069Crossref PubMed Scopus (816) Google Scholar). Activation of AMPK by metformin was found to be required for the decrease in glucose production and the increase in fatty acid oxidation in hepatocytes and for the increase in glucose uptake in skeletal muscle (14Zhou 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 (4432) Google Scholar). In addition, we have recently shown that the stimulation of fatty acid oxidation in skeletal muscle by leptin occurs following a biphasic activation of AMPK (3Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). For both metformin and leptin, it was not clear whether the mechanism leading to activation of AMPK involved a significant decrease in ATP levels (3Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar,14Zhou 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 (4432) Google Scholar). In this study we report that in muscle cells AMPK can be activated by two distinct pathways: one that involves changes in the AMP:ATP ratio and one that is independent of this ratio. Furthermore, we report the novel finding that AMPK is activated acutely by the thiazolidinedione, rosiglitazone via the AMP:ATP-dependent pathway. These results will aid further work on the potential benefit of therapeutic agents aimed at targeting AMPK in diseases such as type 2 diabetes and obesity. H-2Kb cells were derived from skeletal muscle of heterozygous H-2Kb tsA58 transgenic mice (19Jat P.S. Noble M.D. Ataliotis P. Tanaka Y. Yannoutsos N. Larse L. Kioussis D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5096-5100Crossref PubMed Scopus (628) Google Scholar). Myoblasts were maintained under permissive conditions in Dulbecco's modified medium containing heat-inactivated fetal calf serum (20% (v/v)), chick embryo extract (2% (v/v)),l-glutamine (2% (v/v)), and penicillin/streptomycin (1% (w/v)) at 33 °C in the presence of was following a to conditions by of and incubation at °C J.E. M. Ataliotis P. P.S. Noble M.D. Biol. 1994; PubMed Scopus Google Scholar). For all cells were for containing fetal calf and AMPK α1 and α2 was in H-2Kb cell as previously L. Hajduch E. Rencurel F. Salt I. Hundal H. Hardie D. Carling D. Diabetes. 2000; 49: 1978-1985Crossref PubMed Scopus (159) Google Scholar). cells were for at °C in containing glucose in the presence or absence of rosiglitazone, 2 metformin, 5-amino-4-imidazolecarboxamide dinitrophenol or as in the this cells were in and by addition of of was by at for the and protein the AMPK were from and of protein by incubation with an to protein for 2 at AMPK were from the of this incubation by with an to protein For AMPK were a to protein AMPK in the was by the peptide S.P. Carling D. Hardie D.G. Eur. J. Biochem. PubMed Scopus Google Scholar). of protein as were by on or and to were in for at were with an or an in this at °C and with The were for at with by were and a was H-2Kb cells were in addition of of acid was by at for 2 acid was from the by three with of a of and were by on a on a The was in and with a from to containing at a of were by at and with the of under the AMP and ATP were by and used to the AMP:ATP have previously shown that AMPK is activated in H-2Kb muscle cells in response to a number of L. Hajduch E. Rencurel F. Salt I. Hundal H. Hardie D. Carling D. Diabetes. 2000; 49: 1978-1985Crossref PubMed Scopus (159) Google and results have other of AMPK in including metformin (14Zhou 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 (4432) Google Scholar) and leptin (3Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). Here we show that incubation of H-2Kb muscle cells with the thiazolidinedione, rosiglitazone, leads to a activation of AMPK. the of of rosiglitazone on the of and AMPK in H-2Kb muscle Activation was at with activation at a and rosiglitazone following a a stimulation by rosiglitazone to with However, the activation of to rosiglitazone, with a stimulation at with for the and the for activation of AMPK was for of AMPK by rosiglitazone A 2 that activation of AMPK is by an increase in the phosphorylation of threonine 172 within the α subunit, an that the of this residue J. M. Biol. 2001; Google Scholar). Previous studies have shown that threonine 172 is the phosphorylation within AMPK (8Hawley S.A. Davison M.D. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). also that phosphorylation of was increased in with AMPK and phosphorylation 2 These demonstrate that activation of AMPK by rosiglitazone leads to effects on of activation of AMPK and phosphorylation of AMPK and in response to H-2Kb cells were with rosiglitazone for and AMPK in and was shown are the from two independent which by than C, protein of H-2Kb cell with rosiglitazone for the were by and with for 172 within the AMPK α subunit or In addition to a number of cellular stresses that ATP production D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1276) Google AMPK has recently been shown to be activated in response to metformin (14Zhou 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 (4432) Google Scholar). The finding that rosiglitazone also AMPK to the of whether stimulation of AMPK in muscle occurs the in response to by the possibility of effects on AMPK activation in response to we have previously both and are activated following incubation of H-2Kb muscle cells with AICA in response to hyperosmotic stress in the presence of and following incubation with the agent, activation of both and by was at a of the not in any further increase in AMPK not of cells in the presence of AICA riboside and not increase AMPK to cells with In treatment of cells with and in a and increase in the of both and the stimulation with stress a on the stimulation of AMPK by AICA riboside AICA riboside is within the cell to the which in cells can to levels and the actions of AMP on AMPK J.E. F. Carling D. Beri R.K. 1994; PubMed Scopus Google Scholar, J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; PubMed Scopus Google Scholar). The effects of hyperosmotic stress and or AICA riboside suggest that mechanisms of AMPK the finding that there is no on AMPK with AICA riboside and treatment is with both a In with a report (14Zhou 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 (4432) Google Scholar) we found that incubation of muscle cells with 2 metformin led to a significant increase in the of both and with the we also found that in muscle cells metformin a on than The results to that pathways for activation of AMPK further this possibility we the levels of adenine in cells with stimuli that activate AMPK. can be from the shown in incubation with causes a increase in the of AMP with A increase in AMP is following incubation of cells with rosiglitazone In to hyperosmotic stress and metformin do not lead to a in nucleotide levels with cells The intracellular AMP:ATP following were by of the AMP and ATP and are in the possibility that the effects of hyperosmotic stress and metformin on AMPK activation were to a increase in the AMP:ATP ratio, we the of required to an increase in AMPK to and the AMP:ATP ratio under the of at which AMPK is stimulated to the as following hyperosmotic stress the AMP:ATP ratio than to at the of required to activate AMPK to the as by metformin, the AMP:ATP ratio was that of the with studies in muscle cells L. Hajduch E. Rencurel F. Salt I. Hundal H. Hardie D. Carling D. Diabetes. 2000; 49: 1978-1985Crossref PubMed Scopus (159) Google incubation with AICA riboside in the of a from the at the as AMP in we were to the AMP:ATP ratio following incubation with AICA However, as can be from the AICA riboside treatment does not a in the levels of ATP and with in H-2Kb cells following AICA from acid of were by The under the AMP and ATP were and used to the AMP:ATP ratio. In the are the S.E. from three to independent was not to the for cells with AICA riboside the the AMP in a from acid of were by The under the AMP and ATP were and used to the AMP:ATP ratio. In the are the S.E. from three to independent was not to the for cells with AICA riboside the the AMP of threonine 172 within the α subunit of AMPK was increased following treatment with and rosiglitazone both of which increase the AMP:ATP ratio. In addition, hyperosmotic stress and metformin, which do not nucleotide levels, also increased threonine 172 that both the and mechanisms of AMPK involve phosphorylation at this stress in addition to a increase in phosphorylation with the direct of the not any for this the effects of hyperosmotic stress and on AMPK appear to be suggest that phosphorylation may be involved in the activation of AMPK. the mechanisms leading to activation of AMPK following an increase in the AMP:ATP ratio are D.G. Salt I.P. Hawley S.A. Davies S.P. Biochem. J. 1999; 338: 717-722Crossref PubMed Scopus (317) Google Scholar, D. Hardie D.G. Eur. J. Biochem. PubMed Scopus Google S.A. Carling D. Hardie D.G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google the mechanisms that to activate AMPK in response to hyperosmotic stress or metformin, that do not the intracellular levels of adenine are the effects of metformin on intracellular signaling pathways have not been hyperosmotic stress has been shown previously to activate a number of of signaling including phosphatidylinositol J. M. A. C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google protein kinase A. H. J. Eur. J. Clin. Invest. 2000; PubMed Scopus Google mitogen-activated protein (MAP) kinase, and MAP kinase kinase in P. Biol. 1997; Full Text PDF PubMed Scopus Google Scholar). the of a number of of pathways on the activation of AMPK by hyperosmotic was used to signaling phosphatidylinositol to the MAP kinase pathway, to the p38 α and MAP kinase isoforms and the to protein kinase S.P. H. M. P. Biochem. 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Our results activation of AMPK by rosiglitazone a number of the mechanisms the beneficial effects of the in the treatment of type 2 AMPK has been shown to be involved in the regulation of in the (14Zhou 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 (4432) Google Scholar, A. D. M. S. P. P. F. Carling D. Biol. 2000; PubMed Scopus Google Scholar). In skeletal activation of AMPK E. J. Physiol. 1999; PubMed Scopus Google Scholar) or of a of AMPK F. S.A. Woods A. Carling D. Biochem. J. 2002; PubMed Scopus Google Scholar) has been shown to increase the of a number of that lead to an increase in insulin These effects with of the actions of the including an increase in glucose levels in muscle cells S. 1994; Full Text PDF PubMed Scopus Google Scholar). Furthermore, rosiglitazone has been to decrease in a number of cell in a M. T. Diabetes. 1999; PubMed Scopus Google Scholar). a key in the of is and by AMPK and was one of the for the kinase D. Hardie D.G. PubMed Scopus Google Scholar). Here we show that rosiglitazone both and AMPK and this leads to a increase in the phosphorylation of there have been no to suggest that the activation of AMPK occurs in the absence of it that the phosphorylation of all will be increased following stimulation of AMPK by In this study we have the effects of rosiglitazone on AMPK, and it to be the longer effects on the kinase However, stimulation of AMPK by AICA riboside has been shown to have effects in muscle E. J. Physiol. 1999; PubMed Scopus Google and it is that results be with Our the possibility that a number of the beneficial effects of the thiazolidinediones may be mediated activation of AMPK. The classical for activation of AMPK involves an increase in the intracellular AMP:ATP ratio (9Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar, 10Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1276) Google Scholar). However, a number of studies have that other pathways also lead to activation of the Activation of in 1998; PubMed Scopus Google Scholar) or in cells T. M. M. A. H. Kemp B.E. Witters L.A. Y. Biochem. 2000; PubMed Scopus Google Scholar) have been demonstrated to increase AMPK Recently, leptin was found to AMPK in a biphasic both and mechanisms (3Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). Activation of AMPK in hepatocytes by metformin was shown to without changing ATP levels, although AMP levels were not in this study (14Zhou 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 (4432) Google Scholar). In study we demonstrate that both hyperosmotic stress and metformin activate AMPK without the AMP:ATP ratio. These results the that AMPK can be activated by that do not changes in the of the Although hyperosmotic stress and metformin both activate AMPK without the AMP:ATP ratio, we are to whether the or it is that stimuli that activate AMPK in with an increase in the AMP:ATP ratio may also activate although the results of the at least for the effects of hyperosmotic stress and In addition to regulation by AMP and AMPK is activated by phosphorylation by as yet upstream kinase AMPK kinase (8Hawley S.A. Davison M.D. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). The phosphorylation within AMPK has been as threonine 172 within the activation of the α subunit (8Hawley S.A. Davison M.D. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). Previous studies have shown that phosphorylation at this is for AMPK B.E. J. Kemp B.E. Witters L.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Woods A. Davison M.D. Carling D. Biochem. J. 2000; PubMed Scopus Google Scholar). with we found that all the stimuli we an increase in phosphorylation at this However, we were not to increased phosphorylation of this in cells to both hyperosmotic stress and cells with that other phosphorylation may be involved in the activation In a study Woods A. Davison M.D. Carling D. Biochem. J. 2000; PubMed Scopus Google Scholar) we that in addition to threonine phosphorylation of other on both the α and subunits are involved in the regulation of AMPK the of the upstream in the AMPK poorly and we whether the protein kinase, or distinct threonine 172 in response to of the upstream is for the mechanisms regulation of AMPK by which in will of the pathways of Our of of signaling that activation of AMPK by hyperosmotic does not activation of phosphatidylinositol p38 α or MAP kinase kinase, or protein kinase C. The results of study have significant for the of the mechanisms leading to activation of AMPK. The finding that AMPK is activated by stimuli that do not increase the AMP:ATP ratio the possibility that other signaling pathways may the AMPK Activation of AMPK by metformin and rosiglitazone, two widely used anti-diabetic via mechanisms may have for the treatment of type 2 diabetes and the that of AMPK could agents for the that in the metabolic for the of