Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press The nuclear receptor PPARb/d programs muscle glucose metabolism in cooperation with AMPK and MEF2 Zhenji Gan,1,3 Eileen M. Burkart-Hartman,2,3,4 Dong-Ho Han,2 Brian Finck,2 Teresa C. Leone,1,2 Emily Y. Smith,1 Julio E. Ayala,1 John Holloszy,2 and Daniel P. Kelly1,2,5 1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Orlando, Florida 32827, USA: 2Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA To identify new gene regulatory pathways controlling skeletal muscle energy metabolism, comparative studies were conducted on muscle-specific transgenic mouse lines expressing the nuclear receptors peroxisome proliferator-activated receptor a (PPARa; muscle creatine kinase [MCK]-PPARa)orPPARb/d (MCK-PPARb/d). MCK-PPARb/d mice are known to have enhanced exercise performance, whereas MCK-PPARa mice perform at low levels. Transcriptional profiling revealed that the lactate dehydrogenase b (Ldhb)/Ldha gene expression ratio is increased in MCK-PPARb/d muscle, an isoenzyme shift that diverts pyruvate into the mitochondrion for the final steps of glucose oxidation. PPARb/d gain- and loss-of-function studies in skeletal myotubes demonstrated that PPARb/d, but not PPARa, interacts with the exercise-inducible kinase AMP-activated protein kinase (AMPK) to synergistically activate Ldhb gene transcription by cooperating with myocyte enhancer factor 2A (MEF2A) in aPPARb/d ligand-independent manner. MCK-PPARb/d muscle was shown to have high glycogen stores, increased levels of GLUT4, and augmented capacity for mitochondrial pyruvate oxidation, suggesting a broad reprogram- ming of glucose utilization pathways. Lastly, exercise studies demonstrated that MCK-PPARb/d mice persistently oxidized glucose compared with nontransgenic controls, while exhibiting supranormal performance. These results identify a transcriptional regulatory mechanism that increases capacity for muscle glucose utilization in a pattern that resembles the effects of exercise training. [Keywords: muscle; exercise; nuclear receptors; glucose metabolism; gene regulation] Supplemental material is available for this article. Received September 2, 2011; revised version accepted November 2, 2011. Muscle performance and resistance to fatigue are deter- from the exercise-stimulated kinase AMP-activated protein mined, in part, by the capacity to burn the chief fuels— kinase (AMPK) and related cellular signaling pathways fatty acids (FAs) and glucose—in order to generate the ATP (Witczak et al. 2008). Delineation of the molecular reg- needed for consistent mechanical function (Coggan 1991; ulatory pathways involved in the beneficial effects of ex- Holloszy et al. 1998; Burke and Hawley 1999; Hawley ercise on muscle glucose metabolism could yield novel 2002; Hargreaves 2004). The capacity for muscle glucose therapeutic targets aimed at the prevention or treatment utilization is an important determinant of muscle fitness. of obesity-related insulin resistance and its consequences. Endurance training enhances insulin-dependent and -in- The capacity of muscle to burn FAs is determined, in part, dependent muscle glucose uptake and utilization (Ivy and at the level of gene expression. A transcriptional regulatory Holloszy 1981; Richter et al. 1982; Hayashi et al. 1997; circuit involved in the control of skeletal muscle FA uti- Holloszy 2005), resulting in improvements in whole-body lization has been delineated. The peroxisome proliferator- insulin sensitivity (DeFronzo et al. 1985; Treadway et al. activated receptors (PPARs) a and b (also known as d), 1989; Goodyear et al. 1995; Wojtaszewski et al. 1997, 2000a). members of the nuclear receptor superfamily, have been Evidence is emerging that the effects of exercise training shown to regulate genes involved in muscle FA uptake and on muscle glucose uptake involve events downstream catabolism (Desvergne and Wahli 1999). The activity of the PPARs is regulated at multiple levels, including availability of activating ligands (endogenous FA/lipid moieties) and 3These authors contributed equally to this work. 4Present address: Abbott Laboratories, Abbot Park, Illinois 60064, USA coactivators such as PPARg coactivator-1a (PGC-1a) 5Corresponding author. (Desvergne and Wahli 1999; Vega et al. 2000; Wende et al. E-mail [email protected]. Article published online ahead of print. Article and publication date are 2007; Madrazo and Kelly 2008; Schupp and Lazar 2010). online at http://www.genesdev.org/cgi/doi/10.1101/gad.178434.111. Most of the work relevant to the control of cellular fuel GENES & DEVELOPMENT 25:2619–2630 Ó 2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11; www.genesdev.org 2619 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Gan et al. metabolism by PPARs has focused on PPARa.PPARa levels of expression ranging from high physiological activates transcription of genes involved in numerous (low expression [LE]) to supraphysiological (medium [ME] steps of cellular FA uptake and oxidation in muscle, liver, and high [HE] expression) (Supplemental Fig. 1A,B), cor- and heart (Gulick et al. 1994; Leone et al. 1999; Finck responding to those of the MCK-PPARa lines (Finck et al. et al. 2002, 2005; Madrazo and Kelly 2008; Montagner 2005). The data shown here represent assessment of the et al. 2011). Thus, activation of PPARa serves to repro- HE lines unless indicated otherwise. Gene expression pro- gram the skeletal myocyte for high-capacity FA burning. filing studies conducted with RNA isolated from muscle of PPARb shares many gene targets with PPARa, includ- the MCK-PPARb and MCK-PPARa mice, compared with ing those involved in cellular FA utilization (Desvergne corresponding nontransgenic littermate (NTG) controls, and Wahli 1999; Gilde et al. 2003; Huss and Kelly 2004; confirmed increased expression of many known PPAR Montagner et al. 2011). Surprisingly, however, transgenic target genes involved in cellular FAO in both lines (Supple- mice with skeletal muscle-specific forced expression of mental Fig. 1C,D). Exercise studies using a motorized either PPARa or PPARb exhibit remarkably different phe- treadmill confirmed that MCK-PPARb mice ran longer notypes (Luquet et al. 2003; Wang et al. 2004; Finck et al. distances than corresponding NTG controls using an en- 2005), suggesting that the two structurally related nuclear durance regimen (Supplemental Fig. 1E), consistent with the receptors regulate a subset of unique downstream genes and original observations by Wang et al. (2004) for the PPARb- biological functions. Specifically, muscle-specific PPARa VP16 transgenic line. In contrast, MCK-PPARa mice ran (muscle creatine kinase [MCK]-PPARa) transgenic mice shorter distances than NTG controls (Supplemental Fig. 1E). exhibit myocyte triacylglyceride (TAG) accumulation, high The gene expression profiling data sets were analyzed to muscle FA oxidation (FAO) rates, glucose intolerance, and identify metabolic genes that were differentially regulated mild insulin resistance (Finck et al. 2005). In striking in the two MCK lines. Comparative analysis identified contrast, muscle-specific PPARb mice (MCK-PPARb) de- several genes involved in glucose utilization pathways velop many features of an exercise-trained phenotype that were differentially regulated (Supplemental Fig. 2). (‘‘marathon mice’’), including increased endurance, in- Of particular interest was the expression pattern of the creased mitochondrial capacity, an oxidative fiber type Ldhb (or Ldh2)andLdha (or Ldh1) genes. Ldhb gene ex- shift, and enhanced insulin sensitivity (Luquet et al. 2003; pression was increased, whereas Ldha gene expression was Wang et al. 2004). modestly decreased, in MCK-PPARb muscle, a pattern that The contrasting phenotypes of the MCK-PPAR lines, was not observed for MCK-PPARa (Supplemental Fig. 2). while clearly representing extremes due to genetic manip- These results were of interest because the LDHB isoenzyme ulation of transcription factor expression, afford a unique favors the reaction that converts lactate to pyruvate, which opportunity to identify new downstream gene regulatory in turn provides substrate for the mitochondrial TCA cycle mechanisms involved in the chronic control of muscle (glucose oxidation), whereas LDHA favors the reverse energy metabolism. Moreover, delineation of the mech- reaction to produce lactate from pyruvate generated by anisms whereby PPARa and PPARb regulate distinct glycolysis (Fig. 1A). These findings were validated by quan- gene targets is an important question for the nuclear titative RT–PCR (qRT–PCR) across several muscle types receptor biology field. Therefore, we embarked on a study (Fig. 1B). The expected LDH isoenzyme activity shifts were to compare and contrast the gene expression profiles and confirmed by activity gel studies (Fig. 1C; Supplemental Fig. metabolic phenotypes of closely matched MCK-PPAR 3; data not shown). lines. Our results indicate that, in addition to controlling muscle FA metabolism, PPARb activates a program in- PPARb cooperates with AMPK and MEF2 to activate volved in muscle glucose utilization in a pattern that is Ldhb gene transcription strikingly similar to the effects of exercise training. Specif- ically, PPARb activates transcription of the gene encoding Evidence has emerged that AMPK serves as a transducer lactate dehydrogenase B (LDHB),