Role of AMP Kinase and PPARδ in the Regulation of Lipid and Glucose Metabolism in Human Skeletal Muscle

Abstract

The peroxisome proliferator-activated receptor (PPAR)δ has been implicated in the regulation of lipid metabolism in skeletal muscle. Furthermore, activation of PPARδ has been proposed to improve insulin sensitivity and reduce glucose levels in animal models of type 2 diabetes. We recently demonstrated that the PPARδ agonist GW501516 activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake in skeletal muscle. However, the underlying mechanism remains to be clearly identified. In this study, we first confirmed that incubation of primary cultured human muscle cells with GW501516 induced AMPK phosphorylation and increased fatty acid transport and oxidation and glucose uptake. Using small interfering RNA, we have demonstrated that PPARδ expression is required for the effect of GW501516 on the intracellular accumulation of fatty acids. Furthermore, we have shown that the subsequent increase in fatty acid oxidation induced by GW501516 is dependent on both PPARδ and AMPK. Concomitant with these metabolic changes, we provide evidence that GW501516 increases the expression of key genes involved in lipid metabolism (FABP3, CPT1, and PDK4) by a PPARδ-dependent mechanism. Finally, we have also demonstrated that the GW501516-mediated increase in glucose uptake requires AMPK but not PPARδ. In conclusion, the PPARδ agonist GW501516 promotes changes in lipid/glucose metabolism and gene expression in human skeletal muscle cells by PPARδ- and AMPK-dependent and -independent mechanisms. The peroxisome proliferator-activated receptor (PPAR)δ has been implicated in the regulation of lipid metabolism in skeletal muscle. Furthermore, activation of PPARδ has been proposed to improve insulin sensitivity and reduce glucose levels in animal models of type 2 diabetes. We recently demonstrated that the PPARδ agonist GW501516 activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake in skeletal muscle. However, the underlying mechanism remains to be clearly identified. In this study, we first confirmed that incubation of primary cultured human muscle cells with GW501516 induced AMPK phosphorylation and increased fatty acid transport and oxidation and glucose uptake. Using small interfering RNA, we have demonstrated that PPARδ expression is required for the effect of GW501516 on the intracellular accumulation of fatty acids. Furthermore, we have shown that the subsequent increase in fatty acid oxidation induced by GW501516 is dependent on both PPARδ and AMPK. Concomitant with these metabolic changes, we provide evidence that GW501516 increases the expression of key genes involved in lipid metabolism (FABP3, CPT1, and PDK4) by a PPARδ-dependent mechanism. Finally, we have also demonstrated that the GW501516-mediated increase in glucose uptake requires AMPK but not PPARδ. In conclusion, the PPARδ agonist GW501516 promotes changes in lipid/glucose metabolism and gene expression in human skeletal muscle cells by PPARδ- and AMPK-dependent and -independent mechanisms. The peroxisome proliferator-activated receptors (PPARs) 4The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; CPT1, carnitine palmitoyltransferase 1; PDK4, pyruvate dehydrogenase kinase 4; FABP3, fatty acid-binding protein 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; PGC1, PPARγ co-activator 1; FBS, fetal bovine serum; siRNA, small interfering RNA. have been the focus of attention within the field of metabolic research because of their lipid-sensor properties and functional role in transcriptional regulation. PPARs regulate lipid utilization and storage and influence metabolic substrate utilization in relation to energy supply during fasting (1Holst D. Luquet S. Nogueira V. Kristiansen K. Leverve X. Grimaldi P.A. Biochim. Biophys. Acta. 2003; 1633: 43-50Crossref PubMed Scopus (163) Google Scholar) or energy demand during physical exercise (2Watt M.J. Southgate R.J. Holmes A.G. Febbraio M.A. J. Mol. Endocrinol. 2004; 33: 533-544Crossref PubMed Scopus (112) Google Scholar). PPAR isoforms display tissue-specific expression and gene regulatory profiles. PPARγ is a key regulator of adipose development and adipose insulin sensitivity (3He W. Barak Y. Hevener A. Olson P. Liao D. Le J. Nelson M. Ong E. Olefsky J.M. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15712-15717Crossref PubMed Scopus (796) Google Scholar), whereas PPARα regulates genes involved in hepatic lipid oxidation (4Berger J. Moller D.E. Annu. Rev. Med. 2002; 53: 409-435Crossref PubMed Scopus (2096) Google Scholar). PPARδ is the predominant isoform in skeletal muscle, and studies in transgenic mice show that targeted expression of activated PPARδ increases the predominance of oxidative type 1 muscle fibers, enhances whole-body insulin sensitivity, and increases exercise endurance capacity (5Wang Y.X. Zhang C.L. Yu R.T. Cho H.K. Nelson M.C. Bayuga-Ocampo C.R. Ham J. Kang H. Evans R.M. PLoS Biol. 2004; 2: e294Crossref PubMed Scopus (893) Google Scholar). Because pharmacological activation of PPARδ with the specific agonist GW501516 improves insulin sensitivity in aged rhesus monkeys (6Oliver Jr., W.R. Shenk J.L. Snaith M.R. Russell C.S. Plunket K.D. Bodkin N.L. Lewis M.C. Winegar D.A. Sznaidman M.L. Lambert M.H. Xu H.E. Sternbach D.D. Kliewer S.A. Hansen B.C. Willson T.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5306-5311Crossref PubMed Scopus (966) Google Scholar) and rodents (7Tanaka T. Yamamoto J. Iwasaki S. Asaba H. Hamura H. Ikeda Y. Watanabe M. Magoori K. Ioka R.X. Tachibana K. Watanabe Y. Uchiyama Y. Sumi K. Iguchi H. Ito S. Doi T. Hamakubo T. Naito M. Auwerx J. Yanagisawa M. Kodama T. Sakai J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15924-15929Crossref PubMed Scopus (734) Google Scholar, 8Leibowitz M.D. Fievet C. Hennuyer N. Peinado-Onsurbe J. Duez H. Berger J. Cullinan C.A. Sparrow C.P. Baffic J. Berger G.D. FEBS Lett. 2000; 473: 333Crossref PubMed Scopus (296) Google Scholar), PPARδ agonism may offer an efficacious strategy for the management of metabolic disorders. The PPARδ-mediated enhancement in whole-body insulin sensitivity primarily reflects improvements in the lipid profile. PPARδ activation can improve glucose homeostasis by increasing lipid oxidation to lower plasma-free fatty acid levels, which thereby relieves negative feedback on the canonical insulin signaling cascade to enhanced glucose uptake and reduce plasma glucose levels (9Lee C.H. Olson P. Hevener A. Mehl I. Chong L.W. Olefsky J.M. Gonzalez F.J. Ham J. Kang H. Peters J.M. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3444-3449Crossref PubMed Scopus (413) Google Scholar). Skeletal muscle is a key target tissue in orchestrating this scenario, because it is a key organ for lipid oxidation and glucose uptake. Indeed, improvements in skeletal muscle insulin sensitivity are beneficial to control glucose homeostasis (10Björnholm M. Zierath J.R. Biochem. Soc. Trans. 2005; 33: 354-357Crossref PubMed Scopus (158) Google Scholar). Genetic data in humans provide evidence for a role for PPARδ in the regulation of skeletal muscle glucose metabolism. Single nucleotide polymorphisms of the human PPARD gene are associated with enhanced whole-body insulin sensitivity. The use of positron emission tomography to determine tissue-specific glucose disposal rates in vivo that suggest single nucleotide polymorphisms in the PPARD gene mainly affect glucose uptake in skeletal muscle but not adipose tissue (11Vanttinen M. Nuutila P. Kuulasmaa T. Pihlajamaki J. Hallsten K. Virtanen K.A. Lautamaki R. Peltoniemi P. Takala T. Viljanen A.P. Knuuti J. Laakso M. Diabetes. 2005; 54: 3587-3591Crossref PubMed Scopus (50) Google Scholar). Furthermore, single nucleotide polymorphisms in the PPARD gene modify the conversion from impaired glucose tolerance to type 2 diabetes, particularly in combination with single nucleotide polymorphisms identified in PPAR gamma co-activaor (PGC)1α and PPARγ2 (12Andrulionyte L. Peltola P. Chiasson J.L. Laakso M. Diabetes. 2006; 55: 2148-2152Crossref PubMed Scopus (54) Google Scholar). Interestingly, improvements in insulin sensitivity are correlated with increased PPARδ expression in type 2 diabetic patients performing moderate walking exercise (13Fritz T. Krämer D.K. Karlsson H.K. Galuska D. Engfeldt P. Zierath J.R. Krook A. Diabetes Metab. Res. Rev. 2006; 22: 492-498Crossref PubMed Scopus (91) Google Scholar). In addition, PPARδ expression is associated with an increased proportion of insulin-sensitive oxidative skeletal muscle fiber types in human (14Krämer D.K. Ahlsén M. Norrbom J. Jansson E. Hjeltnes N. Gustafsson T. Krook A. Acta Physiologica (Oxf.). 2006; 188: 207-216Crossref PubMed Scopus (82) Google Scholar) and rodent (5Wang Y.X. Zhang C.L. Yu R.T. Cho H.K. Nelson M.C. Bayuga-Ocampo C.R. Ham J. Kang H. Evans R.M. PLoS Biol. 2004; 2: e294Crossref PubMed Scopus (893) Google Scholar) skeletal muscle. The effects of PPARs on metabolic responses may involve the AMP-activated protein kinase (AMPK). Indirect evidence to support this hypothesis arises from the observation that the metabolic profile achieved in response to AMPK activation overlaps with the metabolic phenotype observed with PPARδ activation. AMPK is a heterotrimeric protein kinase that participates in cellular energy homeostasis (15Long Y.C. Zierath J.R. J. Clin. Investig. 2006; 116: 1776-1783Crossref PubMed Scopus (768) Google Scholar). Once activated under conditions of low energy status, AMPK enhances cellular nutrient uptake, activates ATP-producing catabolic pathways, and down-regulates energy-consuming processes (15Long Y.C. Zierath J.R. J. Clin. Investig. 2006; 116: 1776-1783Crossref PubMed Scopus (768) Google Scholar). AMPK-mediated effects on lipid and glucose metabolism partly involve activation of PPARs through diverse signaling pathways (16Lee W.J. Kim M. Park H.-S. Kim H.S. Jeon M.J. Oh K.S. Koh E.H. Won J.C. Kim M.-S. Oh G.T. Biochem. Biophys. Res. Commun. 2006; 340: 291-295Crossref PubMed Scopus (314) Google Scholar, 17Ravnskjaer K. Boergesen M. Dalgaard L.T. Mandrup S. J. Mol. Endocrinol. 2006; 36: 289-299Crossref PubMed Scopus (72) Google Scholar) and/or direct phosphorylation (18Leff T. Biochem. Soc. Trans. 2003; 31: 224-227Crossref PubMed Google Scholar). The PPARδ agonist GW501516 increases basal and insulin-stimulated glucose uptake in cultured primary human myotubes, C2C12 cells, and 3T3-L1 adipocytes, with effects correlated with AMPK phosphorylation in human skeletal myotubes (19Krämer D.K. Al-Khalili L. Perrini S. Skogsberg J. Wretenberg P. Kannisto K. Wallberg-Henriksson H. Ehrenborg E. Zierath J.R. Krook A. Diabetes. 2005; 54: 1157-1163Crossref PubMed Scopus (112) Google Scholar). The contribution of AMPK to the described effects of PPAR agonists on the regulation of lipid and glucose metabolism is unknown. The aim of this study was to determine whether PPARδ has a direct role in the regulation of glucose and lipid metabolism in skeletal muscle. In addition, we endeavored to determine whether AMPK contributes to the enhanced metabolic phenotype observed in skeletal muscle conferred by PPARδ agonists. Materials—Dulbecco's modified Eagle's medium (DMEM), Ham's F-10 medium, fetal bovine serum (FBS), penicillin, streptomycin, and fungizone from and from and from Skeletal muscle from of the metabolic cells and primary muscle L. Krämer D. Wretenberg P. Krook A. Acta 2004; PubMed Scopus Google Scholar). The the the in with and in human myotubes, with a of in with for 2 to and in with for 2 the cells for the of myotubes and uptake and glucose in primary human muscle cells was described L. Kannisto K. Zhang J. G.D. Ehrenborg E. Zierath J.R. Krook A. Mol. Sci. 2003; PubMed Scopus Google Scholar). of fatty acid uptake and oxidation described K. A. Al-Khalili L. Zhang Krook A. Zierath J.R. Metab. 2006; PubMed Scopus Google Scholar). and cultured in and the was myotubes for in the and and with GW501516 for or for with GW501516 in the medium for the of the cells with the in an the of the cells with and for was was and was the was for of specific and data and by was for expression GW501516 and to that in control cells with and from of AMPK, acetyl-CoA PPARγ co-activator and by the AMPK and PPARδ was from The phosphorylation of AMPK and was with and for in 1 1 1 1 and 1 and to for of in and by to with with and with the primary with and with an and from by and by in described and with The was described L. Yu M. C. Zierath J.R. Krook A. J. 2004; PubMed Scopus (54) Google Scholar). in to on 2 of in with and 2 of was to the cells used for The for the control and PPAR isoforms of from The for AMPK and from and described L. K. S. Krook A. Mol. Endocrinol. 2006; PubMed Scopus Google Scholar). of and by a with a was with with a of 1 was D. H. N. Leverve J. Biochem. PubMed Scopus Google Scholar). of and identified with was of in with animal models that activation of PPARδ to increased lipid metabolism in skeletal muscle. determine the effects of PPARδ activation in human muscle, primary myotubes to GW501516 for 1 or uptake, by intracellular accumulation of was increased in response to GW501516 GW501516 not increase uptake the that transcriptional effects are required to metabolic the for PPARδ for the GW501516 effect on fatty acid uptake, the expression of PPARα or PPARδ was and of the on the expression of the targeted gene was expression was for PPARα and for with control expression of PPARδ was in and confirmed by The in PPARδ the of uptake induced by whereas the in PPARα expression was Furthermore, the of AMPK was effect on GW501516-mediated uptake in PPARδ and with for uptake, incubation of human myotubes for with GW501516 in a increase in whereas effect was observed the to the agonist The of PPARδ or the of the of PPARα not this effect GW501516 of in by a PPARδ-dependent of human myotubes for with GW501516 in a increase in and expression with cells In expression of GAPDH, and by GW501516 not The GW501516-mediated transcriptional effect on CPT1, PDK4, and was PPARδ expression was The in PPARα or not the GW501516-mediated of these that activation of PPARδ in cultured primary human skeletal muscle increases lipid metabolism with an increase in of key of the fatty acid transport and oxidation and GW501516 of of activated PPARδ in skeletal muscle increases the expression of and a in skeletal muscle fiber type to an oxidative type phenotype (5Wang Y.X. Zhang C.L. Yu R.T. Cho H.K. Nelson M.C. Bayuga-Ocampo C.R. Ham J. Kang H. Evans R.M. PLoS Biol. 2004; 2: e294Crossref PubMed Scopus (893) Google Scholar). expression of PGC1, a target that is involved in P. U. Zhang C. V. A. S. 98: PubMed Scopus Google Scholar) and the of muscle J. H. Zhang P. E. Olson R. 2002; PubMed Scopus Google Scholar), was by GW501516 of the was a for protein expression of in of AMPK Furthermore, protein expression of and of the was also by to the PPARδ agonist for 1 or AMPK GW501516 by a and in the (19Krämer D.K. Al-Khalili L. Perrini S. Skogsberg J. Wretenberg P. Kannisto K. Wallberg-Henriksson H. Ehrenborg E. Zierath J.R. Krook A. Diabetes. 2005; 54: 1157-1163Crossref PubMed Scopus (112) Google Scholar), AMPK phosphorylation was increased by the specific PPARδ agonist GW501516 in primary cultured human skeletal muscle. of primary human muscle with GW501516 for 1 or in a and AMPK In cells PPARα and was to PPAR the AMPK phosphorylation was that this effect was of PPARα or PPARδ. The in AMPK expression the GW501516 effect on AMPK the phosphorylation of ACC, a target of AMPK, was increased and 1 or of GW501516 of PPARα or PPARδ expression by not this whereas of expression to a in the effect of GW501516 on the underlying mechanism of the effect of GW501516 on AMPK we nucleotide by in human skeletal myotubes to determine the cellular and a to levels and levels increased in a with control cells to the not was associated with a in for of the of the PPARδ agonist used 1 and In addition, intracellular low and to the of a an increase in levels in the of GW501516 was not to a increase in these provide evidence that the increase in AMPK phosphorylation observed GW501516 was because of a in cellular energy in the cultured human The of AMPK but (19Krämer D.K. Al-Khalili L. Perrini S. Skogsberg J. Wretenberg P. Kannisto K. Wallberg-Henriksson H. Ehrenborg E. Zierath J.R. Krook A. 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Wallberg-Henriksson H. Ehrenborg E. Zierath J.R. Krook A. Diabetes. 2005; 54: 1157-1163Crossref PubMed Scopus (112) Google Scholar). The for these effects to be to the acid proposed by Diabetes Metab. Rev. PubMed Scopus Google Scholar), enhanced utilization of is to to a in the effects of enhanced lipid and glucose utilization in response to activation of PPARδ are evidence in primary from mice an increase in the conversion of glucose that PPARδ activation increases the utilization of glucose in hepatic (9Lee C.H. Olson P. Hevener A. Mehl I. Chong L.W. Olefsky J.M. Gonzalez F.J. Ham J. Kang H. Peters J.M. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3444-3449Crossref PubMed Scopus (413) Google Scholar). Furthermore, studies suggest that PPARδ polymorphisms may an role in glucose metabolism in skeletal muscle and may be in the conversion from impaired glucose tolerance to (12Andrulionyte L. Peltola P. Chiasson J.L. Laakso M. Diabetes. 2006; 55: 2148-2152Crossref PubMed Scopus (54) Google Scholar). We have demonstrated that GW501516 has direct effects on glucose transport in human skeletal muscle cells (19Krämer D.K. Al-Khalili L. Perrini S. Skogsberg J. Wretenberg P. Kannisto K. Wallberg-Henriksson H. Ehrenborg E. Zierath J.R. Krook A. Diabetes. 2005; 54: 1157-1163Crossref PubMed Scopus (112) Google Scholar). we provide evidence that the effect of GW501516 on glucose uptake is of PPARδ activation. Indeed, of PPARδ or PPARα expression not the GW501516-mediated increase in glucose uptake. However, a in the expression of AMPK the GW501516 effect on glucose The effect of the PPARδ agonist on the of glucose transport is AMPK by Because we observed an increase in the in cells with the phosphorylation of AMPK be to a of cellular energy in a to a of K. N. A. R. M. E. H. W. C. Diabetes. 2004; 53: PubMed Scopus Google Scholar, M. E. J. 2006; PubMed Scopus Google Scholar) or Nogueira V. E. N. M. Leverve X. J. Biol. 2000; PubMed Scopus Google Scholar), direct and on Indeed, the in levels be to a specific of or of the and/or to an effect on the nucleotide and/or Furthermore, an effect of GW501516 on the oxidative phosphorylation for the observed effects on thereby the of and to AMPK activation K. J. K. V. C. H. W. C. 2006; PubMed Scopus Google Scholar). effect on metabolism is by the that AMPK phosphorylation was with a in levels an incubation with The PPARδ agonist GW501516 has been to increase glucose uptake in muscle In and muscle with effect on glucose uptake was S. S. J. 2006; PubMed Scopus Google Scholar). However, muscle to 1 GW501516 for on the or of fatty in the incubation glucose transport rates have been to be increased in the of and in the of fatty K. J. K. V. C. H. W. C. 2006; PubMed Scopus Google Scholar). Interestingly, we that of serum from the incubation enhances the response to GW501516 on glucose uptake in cultured human muscle cells not the of fatty in serum may influence the of GW501516 on glucose uptake in human skeletal muscle The mechanism by which fatty affect the GW501516-mediated and/or AMPK phosphorylation remains to be Using in cultured myotubes, effects of GW501516 to Zhang X. S. 2005; PubMed Scopus Google Scholar). has recently been to the effect of PPAR on R. P. 2004; PubMed Scopus Google Scholar). effects have been described for of PPARγ D. A. N. C. A. 2005; PubMed Scopus Google Scholar). in increased in the to activation of AMPK D. A. N. C. A. 2005; PubMed Scopus Google Scholar, K. K.A. D.A. FEBS Lett. 2005; PubMed Scopus Google Scholar). activation of AMPK has been an strategy for of the metabolic associated with type 2 (15Long Y.C. Zierath J.R. J. Clin. Investig. 2006; 116: 1776-1783Crossref PubMed Scopus (768) Google Scholar, M. E. J. 2006; PubMed Scopus Google Scholar). AMPK activation in skeletal muscle may a for the effects of in skeletal muscle M. E. J. 2006; PubMed Scopus Google Scholar). In to GW501516 is a specific of PPARδ are for PPARγ M.L. A. E. D. M.L. C. Xu H.E. Lambert M.H. Willson T.M. Jr., W.R. Sternbach D.D. Med. Lett. 2003; PubMed Scopus Google Scholar), the required for activation of PPARδ may be the required to In with PPARδ mice on a are to the PPARδ agonist GW501516 and to metabolic with type control that effects are required for metabolic improvements Y.X. C.H. S. Yu R.T. Ham J. Kang H. Evans R.M. 2003; PubMed Scopus Google Scholar). The effects of GW501516 on glucose uptake in cultured cells may a accumulation of the in the with responses in The effects of GW501516 on AMPK In we provide evidence that the specific PPARδ GW501516 enhances lipid uptake and utilization in primary human skeletal muscle effects are in transcriptional GW501516 also We have demonstrated that changes in the cellular energy status, by an increase in the an GW501516 we have shown that activation of AMPK a in the of the effects of the PPARδ agonist GW501516 on glucose metabolism in skeletal muscle. We Zierath for of the