Methods For Enhancing Exercise Performance

Abstract

Disclosed herein are methods for enhancing one or more effects of exercise in a subject by administering a PPAR.delta. agonist (e.g., GW1516) to the subject in combination with an exercise program. Also disclosed are gene expression profiles unique to the combination of agonist-induced PPAR.delta. activation and exercise. Such profiles are useful, at least, in methods for identifying the use of performance-enhancing drugs in exercised subjects (such as, professional or athletes). Direct interactions between PPAR.delta. and exercised-induced kinases (e.g., AMPK or its subunits, AMPK .alpha.1 and/or AMPK .alpha.2) also are disclosed. Such protein-protein interactions provide new targets for identification of useful compounds.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 11/966,851, filed Dec. 27, 2007, now abandoned, which in turn claims the benefit of U.S. Provisional Application No. 60/882,774 filed Dec. 29, 2006, herein incorporated by reference.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

[0003] The Sequence Listing written in file 92150-876353_ST25.TXT, created on Aug. 28, 2013, 71,772 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.

FIELD

[0004] This disclosure concerns the use of peroxisome proliferator-activated receptor (PPAR).delta. agonists for improving exercise performance in a subject, methods for identifying substance-enhanced exercise performance in a subject, and methods for identifying compounds that affect the interaction of PPAR.delta. with exercise-induced kinases.

BACKGROUND

[0005] Skeletal muscle is an adaptive tissue composed of multiple myofibers that differ in their metabolic and contractile properties including oxidative slow-twitch (type I), mixed oxidative/glycolytic fast-twitch (type IIa) and glycolytic fast-twitch (type IIb) myofibers (Fluck et al., Rev. Physiol. Biochem. Pharmacol., 146:159-216, 2003; Pette and Staron, Microsc. Res. Tech., 50:500-509, 2000). Type I muscle fibers preferentially express enzymes that oxidize fatty acids, contain slow isoforms of contractile proteins and are more resistant to fatigue than are glycolytic muscle fibers (Fluck et al., Rev. Physiol. Biochem. Pharmacol., 146:159-216, 2003; Pette and Staron, Microsc. Res. Tech., 50:500-509, 2000). Type II fibers preferentially metabolize glucose and express the fast isoforms of contractile proteins (Fluck et al., Rev. Physiol. Biochem. Pharmacol., 146:159-216, 2003; Pette and Staron, Microsc. Res. Tech., 50:500-509, 2000).

[0006] Endurance exercise training triggers a complex remodeling program in skeletal muscle that progressively enhances performance in athletes such as marathon runners, mountain climbers and cyclists. This involves changes in metabolic programs and structural proteins within the myofibers that alter the energy substrate utilization and contractile properties that act to reduce muscle fatigue (Fluck et al., Rev. Physiol. Biochem. Pharmacol., 146:159-216, 2003; Pette and Staron, Microsc. Res. Tech., 50:500-509, 2000). Training based adaptations in the muscle are linked to increases in the expression of genes involved in the slow-twitch contractile apparatus, mitochondrial respiration and fatty acid oxidation (Holloszy and Coyle, J. Appl. Physiol., 56:831-838, 1984; Booth and Thomason, Physiol. Rev., 71:541-585, 1991; Schmitt et al., Physiol. Genomics, 15:148-157, 2003; Yoshioka et al., FASEB J., 17:1812-1819, 2003; Mahoney et al., FASEB J., 19:1498-1500, 2005; Mahoney and Tarnopolsky, Phys. Med. Rehabil. Clin. N. Am., 16:859-873, 2005; Siu et al., J. Appl. Physiol., 97:277-285, 2004; Garnier et al., FASEB J., 19:43-52, 2005; Short et al., J. Appl. Physiol., 99:95-102, 2005; Timmons et al., FASEB J., 19:750-760, 2005). Such exercise training-related adaptations can improve performance and protect against obesity and related metabolic disorders (Wang et al., PLoS Biol., 2:e294, 2004; Koves et al., J. Biol. Chem., 280:33588-33598, 2005). Moreover, skeletal muscles rich in oxidative slow-twitch fibers are resistant to muscle wasting (Minnaard et al., Muscle Nerve. 31: 339-48, 2005).

[0007] PPARs are members of the nuclear receptor superfamily of ligand-inducible transcription factors. They form heterodimers with retinoid X receptors (RXRs) and bind to consensus DNA sites composed of direct repeats of hexameric DNA sequences separated by 1 bp. In the absence of ligand, PPAR-RXR heterodimers recruit corepressors and associated histone deacetylases and chromatin-modifying enzymes, silencing transcription by so-called active repression (Ordentlich et al., Curr. Top. Microbiol. Immunol., 254:101-116, 2001; Jepsen and Rosenfeld, J. Cell Sci., 115:689-698, 2002; Privalsky, Ann. Rev. Physiol., 66:315-360, 2004). Ligand binding induces a conformational change in PPAR-RXR complexes, releasing repressors in exchange for coactivators. Ligand-activated complexes recruit the basal transcriptional machinery, resulting in enhanced gene expression. PPARs bind to lower-affinity ligands generated from dietary fat or intracellular metabolism. In keeping with their roles as lipid sensors, ligand-activated PPARs turn on feed-forward metabolic cascades to regulate lipid homeostasis via the transcription of genes involved in lipid metabolism, storage, and transport.

[0008] Three PPAR isotypes exist in mammals: .alpha. (also known as NR1C1), .gamma. (also known as NR1C3), and .delta. (also known as .beta. or NR1C2). PPAR.delta. is expressed in most cell types with relative abundance (Smith, Biochem. Soc. Trans., 30(6):1086-1090, 2002), which led to early speculation that it may serve a "general housekeeping role" (Kliewer et al., Proc. Natl. Acad. Sci. U.S.A., 91:7355-7359, 1994). More recently, PPAR.delta. transgenic mouse models and discoveries aided by the development of high-affinity PPAR.delta. agonists have revealed PPAR.delta. as a key transcriptional regulator with effects in diverse tissues including fat, skeletal muscle, and the heart (for review see, e.g., Barish et al., J. Clin. Invest., 116(3):590-597, 2006).

[0009] Targeted expression of a constitutively active PPAR.delta. receptor (VP16-PPAR.delta.) transgene in rodent skeletal muscle promoted remodeling of skeletal muscle to an oxidative phenotype and increased running endurance in unexercised adult mice (Wang et al., PLoS Biol., 2:e294, 2004). The observed PPAR.delta.-mediated reprogramming of muscle fibers involved the increased expression of genes related to fatty acid oxidation, mitochondrial respiration, oxidative metabolism, and slow-twitch contractile apparatus (Wang et al., PLoS Biol., 2:e294, 2004). These VP16-PPAR.delta. transgenic mice, who had a phenotype similar to endurance-trained athletes, but who had had no exercise training, suggest that pharmacological activation of endogenous PPAR.delta. in an adult, sedentary subject might provide an exercise effect without the actual exercise. Given the numerous benefits of exercise on general health, identification of orally active agents that mimic the effects of exercise is a long standing, albeit elusive medical goal.

SUMMARY

[0010] This disclosure illustrates that, despite expectations to the contrary, pharmacological activation of endogenous PPAR.delta. in adult, sedentary subjects did not promote remodeling of skeletal muscle to an oxidative phenotype or increase running endurance in such subjects. Surprisingly, however, pharmacological activation of PPAR.delta. in combination with at least sub-maximal exercise synergistically modified skeletal muscle architecture (e.g., induced fatigue resistant type I fiber specification and mitochondrial biogenesis) and increased exercise performance (e.g., running endurance). In addition, agonist-induced activation of endogenous PPAR.delta. in combination with exercise led to a unique "gene expression signature" in skeletal muscle, which was distinct from the gene expression profile obtained by either exercise or drug intake alone, and revealed direct interactions between PPAR.delta. and exercise-induced kinases (such as AMPK .alpha.1 and/or AMPK .alpha.2).

[0011] These and other discoveries described herein serve as the basis for disclosed methods. For example, it can now be appreciated that PPAR.delta. agonists (e.g., GW1516) used in combination with exercise can enhance exercise-induced effects, such as to improve exercise endurance (e.g., running endurance) even more than may be achieved by exercise alone. In another example, the expression of one or more genes and/or proteins that are uniquely regulated by the combination of exercise and PPAR.delta. agonist administration can be used to identify subjects using drugs to enhance exercise performance. In still other examples, the newly identified protein complexes, including PPAR.delta. and exercise-induced kinases (such as AMPK .alpha.1 and/or AMPK .alpha.2), can be used to identify agents that have potential to affect PPAR.delta.-regulated gene networks and the corresponding downstream biochemical and/or physiological effects.

[0012] The foregoing and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1A is a series of bar graphs showing the effects of orally administered PPAR.delta. agonist (GW1516) on mRNA expression levels of three biomarkers of fatty acid oxidation, uncoupling protein 3 (UCP3), carnitine palmitoyl-transferase I (mCPT I), and pyruvate dehydrogenase kinase, isoenzyme 4 (PDK4), in quadriceps muscle isolated from sedentary vehicle-treated (V), sedentary GW1516-treated (GW), sedentary VP16-PPAR.delta. transgenic (TG), and sedentary wild-type littermates of VP16-PPAR.delta. transgenic mice (WT). Data are presented as mean.+-.SEM of N=4-9 mice each analyzed in triplicate. * Represents a statistically significant difference between V and GW1516 groups (p<0.05, unpaired student's t-test), or TG and WT groups (p<0.05, unpaired student's t-test).

[0014] FIG. 1B-D are a series of bar graphs showing the regulation of oxidative genes UCP3, mCPT I, and PDK4 by GW1516 (GW) in wild-type (WT) and PPAR.delta. null (KO) primary muscle cells. * represents statistical significance between V and indicated groups (p<0.05, One Way ANOVA; post hoc: Dunnett's Multiple Comparison Test)

[0015] FIG. 1E is a series of bar graphs showing running endurance of vehicle-treated sedentary (V; open bars) and GW1516-treated sedentary (GW; black bars) mice before (Week 0) and after (Week 5) treatment. Running endurance is quantified by the amount of time for which (left panel) or the distance (right panel) animals in each group ran on the treadmill. Data is represented as mean.+-.SD values from N=6 mice.

[0016] FIGS. 2A-C show the effects of administration of a PPAR.delta. agonist, GW1516, on the gastrocnemius muscle of sedentary (V or GW) or trained (Tr or Tr+GW) mice. FIG. 2A shows digital images of representative meta-chromatically stained frozen cross-sections of gastrocnemius muscle from vehicle-treated, sedentary (V), GW1516-treated, sedentary (GW), vehicle-treated, exercised (Tr) and GW1516-treated, exercised (Tr+GW) mice. Type I (slow oxidative) fibers are darkly stained. FIG. 2B is a bar graph showing the percentage of type I fibers (as a percentage of the total fibers) in V, GW, Tr, and Tr+GW gastrocnemius (N=3). FIG. 2C is a bar graph showing the fold change in mitochondrial DNA to nuclear DNA ratio in V (left bar), GW (left center bar), Tr (right center bar), and Tr+GW (right bar) groups of mice (N=9). Data in (B) and (C) are presented as mean.+-.SEM. In each bar graph, * represents a statistical difference between V and the group(s) indicated by asterisk (p<0.05, One-Way ANOVA; post hoc: Dunnett's Multiple Comparison Test).

[0017] FIGS. 3A-C are a series of bar graphs showing gene expression in quadriceps muscle isolated from V, GW, Tr and Tr+GW groups. FIG. 3A shows the relative gene expression levels of biomarkers for fatty acid oxidation (UCP3, mCPT I, PDK4; from left to right). FIG. 3B shows the relative gene expression levels of biomarkers for fatty acid storage (SCD1, FAS, SREBP1c). FIG. 3C shows the relative gene expression levels of biomarkers for fatty acid uptake (FAT/CD36, LPL). Data is presented as mean.+-.SEM of N=9 mice, each analyzed in triplicate. * represents statistically significant difference between V and the group(s) indicated by asterisk (p<0.05, One Way ANOVA; post hoc: Dunnett's Multiple Comparison Test).

[0018] FIG. 3D shows images of Western blots illustrating protein expression levels of oxidative biomarkers (myoglobin, UCP3, CYCS, SCD1) and loading control (tubulin) in protein lysates prepared from quadriceps (N=3).

[0019] FIG. 4 shows a graph of muscle triglyceride levels in gastrocnemius muscle of V, GW, Tr and Tr+GW mice. Data is presented as mean.+-.SEM of N=9 mice, each analyzed in triplicate. * represents statistical significance between V and group(s) indicated by asterisk (*p<0.05, One Way ANOVA; post hoc:Dunnett's Multiple Comparison Test).

[0020] FIGS. 5A and B are bar graphs showing the effects of GW1516 treatment on running endurance in exercise-trained mice. Bar graphs of the (A) time and (B) distance that vehicle-(V; open bars) and GW1516-treated (GW; black bars) mice ran on a treadmill before (Week 0) and after (Week 5) exercise training Data is represented as mean.+-.SD of N=6 mice. *** represents statistically significant difference between V and GW groups (p<0.001; One Way ANOVA; post hoc:Tukey's Multiple Comparison Test).

[0021] FIG. 5C is a bar graph showing epididymal white adipose to body weight ratio in V, GW, Tr and Tr+GW mice. Data is presented as mean.+-.SEM of N=9 mice, each analyzed in triplicate. * represents statistical significance between V and group(s) indicated by asterisk (*p<0.05, One Way ANOVA; post hoc:Dunnett's Multiple Comparison Test).

[0022] FIG. 5D shows digital images of H&E-stained cross-sections of epididymal white adipose from V, GW, Tr and Tr+GW mice. Similar results were obtained from N=3 mice. * represents statistical significance between V and group(s) indicated by asterisk (*p<0.05, One Way ANOVA; post hoc:Dunnett's Multiple Comparison Test).

[0023] FIG. 6 shows a Venn diagram comparing GW, Tr and Tr+GW target genes identified in microarray analysis of quadriceps. Data is an average of N=3 samples in each group. The selection criteria used a p<0.05 on Bonferroni's multiple comparison test and a fold change greater than 1.5.

[0024] FIG. 7A is a series of Western blot images showing AMPK activation by exercise. The levels of phospho-AMPK (phospho-AMPK) and total-AMPK in quadriceps muscle of sedentary (Sed/C57B1) and exercise-trained (Tr/C57B1) mice (N=5-7) are shown.

[0025] FIG. 7B is a series of Western blot images showing AMPK activation by VP16-PPARd over-expression. The levels of phospho-AMPK (phospho-AMPK) and total-AMPK in quadriceps muscle of sedentary wild-type or transgenic mice (Sed/WT or Sed/TG) are shown.

[0026] FIGS. 8A-B show the synergistic regulation of muscle gene expression by PPAR.delta. and AMPK. (A) Venn diagram comparing GW, AI, and AI+GW target genes identified in microarray analysis of quadriceps. Data is an average of N=3 samples in each group. The selection criteria used a p<0.05 on Bonferroni's multiple comparison test and fold change greater than 1.5. (B) Comparison of Tr+GW and AI+GW dependent gene signatures identified in quadriceps. Data is an average of N=3 samples in each group. The selection criteria used is similar to one used in FIG. 8A.

[0027] FIGS. 9A-H show the expression of (A) UCP3, (B) mCPT I, (C) PDK4, (D) SCD1, (E) ATP citrate lyase, (F) HSL, (G) mFABP, and (H) LPL transcripts in quadriceps of mice treated with vehicle (V), GW1516 (GW), AICAR (AI) and the combination of the two drugs (GW+AI) for 6 days. Data is presented as mean.+-.SEM of N=6 mice in each group, analyzed in triplicate. * Indicates statistically significant difference between V and indicated groups (p<0.05, One Way ANOVA; post hoc: Dunnett's Multiple Comparison Test).

[0028] FIGS. 10A-L demonstrate the AMPK-PPAR.delta. interaction. (A-D) show the expression of metabolic genes in wild type and PPAR.delta. null (KO) primary muscle cells treated with V, GW, AI and GW+AI (bars from left to right) for 24 hours. In (E-F, J), AD293 cells were transfected with PPAR.delta.+RXR.alpha.+Tk-PPRE along with control vector, AMPK .alpha.1, .alpha.2 and/or PGC1.alpha. as indicated above. (E) Induction of basal PPAR.delta. transcriptional activity by AMPK .alpha.1 or .alpha.2. (F) Dose-dependent induction of PPAR.delta. transcriptional activity is enhanced by AMPK.alpha.1 (closed circle) or AMPK .alpha.2 (closed square) compared to control (open triangle). In (G-I, K), AD293 cells were transfected and processed as indicated. (G-H) Representative blot showing co-immunoprecipitation of transfected (G) or endogenous (H) AMPK with Flag-PPAR.delta.. (I) Metabolic p32 labeling of PPAR.delta. in AD293 cells transfected as described. (J) Synergistic regulation of basal (V) and ligand (GW) dependent PPAR.delta. transcriptional activity by AMPK .alpha.2 subunit and PGC1.alpha.. (K) Co-immunoprecipitation of PPAR.delta. but not AMPK .alpha.2 subunit with Flag-PGC1.alpha.. (L) Model depicting exercise-PPAR.delta. interaction in re-programming muscle genome.

SEQUENCE INFORMATION

[0029] Nucleic acid and amino acid sequences may be referred to herein by GenBank accession number. It is understood that the sequences given such GenBank accession numbers are incorporated by reference as they existed and were known as of Dec. 29, 2006.

DETAILED DESCRIPTION

I. Introduction

[0030] Disclosed herein are methods for enhancing an exercise effect in a subject including the steps of performing by a subject physical activity (such as aerobic exercise (e.g., running)) sufficient to produce an exercise effect; and administering to the subject an effective amount of a PPAR.delta. agonist (e.g., GW1516). The exercise effect that is enhanced can be, for example, improved running endurance (such as, improved running distance or improved running time or a combination thereof, increased fatty acid oxidation in at least one skeletal muscle of the subject, and/or body fat (e.g., white adipose tissue) reduction). In some method embodiments, a subject is a mammal (such as a racing mammal, like a horse, a dog, or a human), and/or an adult, and/or an exercise-trained subject. In other exemplary methods, the PPAR.delta. agonist is administered on the same day(s) on which the physical activity is performed. In some methods, administration of the PPAR.delta. agonist is by oral administration, intravenous injection, intramuscular injection, and/or subcutaneous injection. In other method embodiments, the effective amount of the PPAR.delta. agonist is from about 1 mg per day to about 20 mg per day in a single dose or in divided doses.

[0031] Also disclosed herein are methods for identifying the use of performance-enhancing substances in an exercise-trained subject, which include determining in a biological sample taken from an exercise-trained subject (e.g, a skeletal muscle biopsy) the expression of the molecules listed in Table 2 or listed in Table 4, or a subset thereof, such as expression of at least 1, at least 5, at least 10, at least 20, at least 40 of the molecules listed in Table 2 or in Table 4.

[0032] In some methods for identifying the use of performance-enhancing substances in an exercise-trained subject, (i) expression is upregulated in one or more of (such as at least 5, at least 10, at least 20, at least 35, or all of) adipose differentiation related protein; stearoyl-Coenzyme A desaturase 2; acetyl-Coenzyme A acetyltransferase 2; ATP citrate lyase; adiponectin, C1Q and collagen domain containing; diacylglycerol O-acyltransferase 2; lipase, hormone sensitive; monoglyceride lipase; resistin; CD36 antigen; fatty acid binding protein 4, adipocyte; lipoprotein lipase; microsomal glutathione S-transferase 1; GPI-anchored membrane protein 1; dual specificity phosphatase 7; homeodomain interacting protein kinase 3; insulin-like growth factor binding protein 5; protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), beta isoform; protein tyrosine phosphatase-like (proline instead of catalytic arginine); member b; CCAAT/enhancer binding protein (C/EBP), alpha; nuclear receptor subfamily 1, group D, member 2(Reverb-b); transferring; archain 1; solute carrier family 1 (neutral amino acid transporter), member 5; RIKEN cDNA 1810073N04 gene; haptoglobin; retinol binding protein 4, plasma; phosphoenolpyruvate carboxykinase 1, cytosolic; cell death-inducing DFFA-like effector c; interferon, alpha-inducible protein 27; carbonic anhydrase 3; cysteine dioxygenase 1, cytosolic; DNA segment, Chr 4, Wayne State University 53, expressed; dynein cytoplasmic 1 intermediate chain 2; Kruppel-like factor 3 (basic); thyroid hormone responsive SPOT14 homolog (Rattus); cytochrome P450, family 2, subfamily e, polypeptide 1; complement factor D (adipsin); and/or transketolase; and/or (ii) expression is downregulated in one or more of gamma-glutamyl carboxylase; 3-oxoacid CoA transferase 1; solute carrier family 38, member 4; annexin A7; CD55 antigen; RIKEN cDNA 1190002H23 gene; fusion, derived from t(12; 16) malignant liposarcoma (human); lysosomal membrane glycoprotein 2; and/or neighbor of Punc E11, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 of these molecules.

[0033] Exemplary methods for identifying the use of performance-enhancing substances in an exercise-trained subject involve determining protein expression and/or determining expression of a gene encoding the protein. Such methods are routine in the art. In some examples, the level of protein or nucleic acid expression is quantified.

[0034] Methods of identifying an agent having potential to enhance exercise performance in a subject also are disclosed herein. Such methods can include (i) providing a first component comprising a PPAR.delta. receptor or an AMPK-binding fragment thereof; (ii) providing a second component comprising an AMP-activated protein kinase (AMPK), AMPK.alpha.1, AMPK.alpha.2, or a PPAR.delta.-binding fragment of any thereof; (iii) contacting the first component and the second component with at least one test agent under conditions that would permit the first component and the second component to specifically bind to each other in the absence of the at least one test agent; and (iv) determining whether the at least one test agent affects the specific binding of the first component and the second component to each other. An effect on specific binding of the first component and the second component to each other identifies the at least one test agent as an agent having potential to enhance exercise performance in a subject.

[0035] In some methods of identifying an agent having potential to enhance exercise performance a third component, i.e., a PPAR.delta. agonist (e.g., GW1516), is involved and the first component, second component, and third component are contacted as described above.

II. Abbreviations and Terms

[0036] AMPK AMP-activated protein kinase

[0037] bps Beats per second

[0038] MAPK Mitogen-activated protein kinase

[0039] mCPT I Muscle carnitine palmitoyl transferase I

[0040] QPCR or qPCR Quantitative PCR

[0041] PDK4 Pyruvate dehydrogenase kinase 4

[0042] PES Performance-enhancing substance(s)

[0043] PPAR Peroxisome proliferator-activated receptors

[0044] UCP3 Uncoupling protein 3

[0045] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and/or Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of various embodiments of the disclosure, the following explanations of specific terms are provided:

[0046] Expression: The process by which the coded information of a nucleic acid transcriptional unit (including, for example, genomic DNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a polypeptide. Gene expression can be influenced by external signals; for instance, exposure of a cell, tissue or subject to an agent that enhances gene expression. Expression of a gene also may be regulated anywhere in the pathway from DNA to RNA to polypeptide. Regulation of gene expression occurs, for instance, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression (for example expression of one or more of the genes listed in Tables 2 and 4) can be measured at the RNA level or the protein level and by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0047] The expression of a nucleic acid may be modulated compared to a control state, such as at a control time (for example, prior to administration of a substance or agent that affects regulation of the nucleic acid under observation) or in a control cell or subject, or as compared to another nucleic acid. Such modulation includes but is not necessarily limited to overexpression, underexpression, or suppression of expression. In addition, it is understood that modulation of nucleic acid expression may be associated with, and in fact may result in, a modulation in the expression of an encoded polypeptide or even a polypeptide that is not encoded by that nucleic acid (such as downstream regulated polypeptide(s)).

[0048] The expression of a polypeptide also may be modulated compared to a control state, such as at a control time (for example, prior to administration of a substance or agent that affects expression of a nucleic acid encoding or regulating the polypeptide) or in a control cell or subject, or as compared to another polypeptide. Modulation of polypeptide expression includes, but is not limited to, overexpression or decreased expression of the polypeptide, alteration of the subcellular localization or targeting of the polypeptide, alteration of the temporally regulated expression of the polypeptide (such that the polypeptide is expressed when it normally would not be, or alternatively is not expressed when it normally would be), alteration in the stability of the polypeptide, alteration in the spatial localization of the protein (such that the polypeptide is not expressed where it would normally be expressed or is expressed where it normally would not be expressed).

[0049] Isolated: An "isolated" biological component (such as a polynucleotide, polypeptide, or cell) has been purified away from other biological components in a mixed sample (such as a cell or tissue extract). For example, an "isolated" polypeptide or polynucleotide is a polypeptide or polynucleotide that has been separated from the other components of a cell in which the polypeptide or polynucleotide was present (such as an expression host cell for a recombinant polypeptide or polynucleotide).

[0050] The term "purified" refers to the removal of one or more extraneous components from a sample. For example, where recombinant polypeptides are expressed in host cells, the polypeptides are purified by, for example, the removal of host cell proteins thereby increasing the percent of recombinant polypeptides in the sample. Similarly, where a recombinant polynucleotide is present in host cells, the polynucleotide is purified by, for example, the removal of host cell polynucleotides thereby increasing the percent of recombinant polynucleotide in the sample. Isolated polypeptides or nucleic acid molecules, typically, comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even over 99% (w/w or w/v) of a sample.

[0051] Polypeptides and nucleic acid molecules are isolated by methods commonly known in the art and as described herein. Purity of polypeptides or nucleic acid molecules may be determined by a number of well-known methods, such as polyacrylamide gel electrophoresis for polypeptides, or agarose gel electrophoresis for nucleic acid molecules.

[0052] Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.

[0053] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences 8:155-165, 1992; Pearson et al., Methods in Molecular Biology 24:307-331, 1994; Tatiana et al., (1999), FEMS Microbiol. Lett., 174:247-250, 1999. Altschul et al. present a detailed consideration of sequence alignment methods and homology calculations (J. Mol. Biol. 215:403-410, 1990).

[0054] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST.TM., Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the internet under the help section for BLAST.TM..

[0055] For comparisons of amino acid sequences of greater than about 30 amino acids, the "Blast 2 sequences" function of the BLAST.TM. (Blastp) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default=5]; cost to extend a gap [default=2]; penalty for a mismatch [default=-3]; reward for a match [default=1]; expectation value (E) [default=10.0]; word size [default=3]; number of one-line descriptions (V) [default=100]; number of alignments to show (B) [default=100]). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method.

[0056] For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST.TM. (Blastn) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default=11]; cost to extend a gap [default=1]; expectation value (E) [default=10.0]; word size [default=11]; number of one-line descriptions (V) [default=100]; number of alignments to show (B) [default=100]). Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method.

[0057] Specific binding: Specific binding refers to the particular interaction between one binding partner (such as a binding agent) and another binding partner (such as a target). Such interaction is mediated by one or, typically, more noncovalent bonds between the binding partners (or, often, between a specific region or portion of each binding partner). In contrast to non-specific binding sites, specific binding sites are saturable. Accordingly, one exemplary way to characterize specific binding is by a specific binding curve. A specific binding curve shows, for example, the amount of one binding partner (the first binding partner) bound to a fixed amount of the other binding partner as a function of the first binding partner concentration. As the first binding partner concentration increases under these conditions, the amount of the first binding partner bound will saturate. In another contrast to non-specific binding sites, specific binding partners involved in a direct association with each other (e.g., a protein-protein interaction) can be competitively removed (or displaced) from such association (e.g., protein complex) by excess amounts of either specific binding partner. Such competition assays (or displacement assays) are very well known in the art.

[0058] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprising" means "including." Hence "comprising A or B" means "including A or B", or "including A and B."

[0059] Materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999).

III. Methods of Enhancing an Exercise Effect

[0060] Exercise is known to have many effects on subjects that perform it. Exercise effects at the molecular, biochemical, and/or cellular levels (e.g., modified regulation of genes and/or gene networks and corresponding proteins involved in energy substrate utilization and contractile properties of muscle) form the basis of physiological effects that are observed at the tissue, organ, and/or whole body levels (e.g., increased cardiorespiratory endurance, muscular strength, muscular endurance, and/or flexibility, and/or improvements in body appearance). Disclosed herein are methods for enhancing one or more exercise effects by combining, at least, physical activity with administration of one or more PPAR.delta. agonists. In some examples, physical activity is replaced with administration of an AMPK activator (e.g., AICAR).

[0061] In general terms, exercise is the performance of some physical activity. A single episode (also referred to as a bout) of physical activity is performed for a particular duration and at a particular intensity. If more than one bout of exercise is performed, separate bouts of exercise may have the same or different durations and/or the same or different intensities.

[0062] In some method embodiments, a single bout of exercise may last for up to 30 minutes, up to 45 minutes, up to 60 minutes, up to 90 minutes, up to 2 hours, up to 2.5 hours, up to 3 hours, or even longer. Typically, in the absence of a prior exercise history, repeated episodes of physical activity are needed to achieve an exercise-induced effect (such as, increased aerobic capacity or increase running endurance). Thus, in some disclosed methods, bouts of physical activity may be repeated within a single day; for instance, up to 2 bouts of exercise per day, up to 3 bouts of exercise per day, up to 4 bouts of exercise per day, up to 5 bouts of exercise per day, or even more bouts per day. Some professional athletes or racing mammals may exercise in repeated bouts for a total of 8 hours or more a day. In other method embodiments, bouts (or repeated bouts) of exercise are performed on a daily basis, 6 times per week, 5 times per week, 4 times per week or 3 times per week. In at least some of the disclosed methods, exercise may continue for at least 2 weeks, for at least 4 weeks, for at least 6 weeks, for at least 3 months, for at least 6 months, for at least 1 year, for at least 3 years, or indefinitely (for the lifetime of the subject).

[0063] Exercise generally is performed at an intensity that is more than the usual (e.g., average, median, normal standard, or normoactive) activity for a subject, and/or at or less than the maximum activity achievable by a subject performing a particular exercise. Any known indicator of physical performance can be used to determine whether a subject is performing more than a usual amount of activity, including, for instance, measuring heart rate, repetition rate (e.g., revolutions per second, minutes per mile, lifts per minute, and many others), and/or force output. In some methods, a bout of exercise is performed at sub-maximal intensity; for instance, at about 10% maximal intensity, 25% maximal intensity, 50% maximal intensity, or 75% maximal intensity. In other methods, a bout of exercise is performed at 40%-50% maximal heart rate, 50%-60% maximal heart rate, 60%-70% maximal heart rate, or 75%-80% maximal heart rate, where maximum heart rate for a human subject is calculated as: 220 bps--(age of the subject).

[0064] Exercise is generally grouped into three types: (i) flexibility exercise (such as, stretching), which is believed to, at least, improve the range of motion of muscles and joints; (ii) aerobic exercise; and (iii) anaerobic exercise (such as, weight training, functional training or sprinting) which is believed to, at least, increase muscle strength and mass.

[0065] Aerobic exercise refers to a physical activity in which oxidative or aerobic metabolism (as compared to glycolytic or anaerobic metabolism) substantially predominates in exercised skeletal muscles. In particular method embodiments, a subject performs one or more aerobic exercises. Exemplary aerobic exercises include, without limitation, aerobics, calisthenics, cycling, dancing, exercise machines (rowing machine, cycling machine (e.g., inclined or upright), climbing machine, elliptical trainers, and/or skiing machines), basketball, football, baseball, soccer, footbag, housework, jogging, martial arts, massage, pilates, rowing, running, skipping, swimming, walking, yoga, boxing, gymnastics, badminton, cricket, track and field, golf, ice hockey, lacrosse, rugby, tennis, or combinations thereof.

[0066] The disclosed methods contemplate enhancing any known or observable effect of exercise (such as an aerobic exercise, like walking or running). In particular methods, running endurance (e.g., running distance and/or running time) is enhanced.

[0067] Enhancing an exercise effect (such as running endurance) means that such effect is improved in a subject more than would have occurred by exercise alone. In some method embodiments, an enhanced exercise effect is determined by discontinuing administration of a PPAR.delta. agonist in the subject and observing (e.g., qualitatively or quantitatively) a reduction in the exercise effect of interest (e.g., aerobic endurance, such as running endurance). In some instances, an exercise effect of interest, the PPAR.delta.-enhanced portion of which is lost upon discontinuance of PPAR.delta. administration, will be reduced by at least about 5%, by at least about 10%, by at least about 20%, by at least about 30%, or by at least about 50% as compared to the magnitude of the effect with exercise alone.

[0068] A. PPAR.delta. Agonists

[0069] The disclosed methods envision the use of any PPAR.delta. agonist. Preferably such agonist would be non-toxic in the subject to which it is administered. Exemplary PPAR.delta. agonists include GW1516, L-165041 (as described by, e.g., Leibowitz et al., FEBS Lett., 473(3):333-336, 2000), any one or more compounds described in PCT Publication Nos. WO/2006/018174, WO/2005/113506, WO/2005/105754, WO/2006/041197, WO/2006/032023, WO/01/00603, WO/02/092590, WO/97/28115, WO/97/28149, WO/97/27857, WO/97/28137, WO/97/27847, and/or WO/98/27974, and/or a published U.S. national phase application or issued U.S. patent corresponding to any of the foregoing (each of which is expressly incorporated herein by reference). Moreover, other PPAR.delta. agonists can be identified using the methods described, for example, in PCT Publication No. WO/1998/049555 or any corresponding published U.S. national phase application or issued U.S. patent (each of which is expressly incorporated herein by reference).

[0070] In a specific example, the PPAR.delta. agonist is GW1516 (also referred to in the art as GW501516). GW1516 is (2-methyl-4(((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)methy- l)sulfanyl)phenoxy)acetic acid as has been shown to be is bioactive in humans (Sprecher et al., Arterioscler. Thromb. Vasc. Biol. 27(2): 359-65, 2007). In specific examples, GW1516 is administered orally, for example 1 mg-20 mg/day, such as 2.5 mg or 10 mg per day.

[0071] B. Subjects

[0072] The disclosed methods can be performed in any subject capable of performing physical activity (e.g., aerobic exercise). In some method embodiments, a subject is a living multi-cellular vertebrate organism (e.g., human and/or non-human animals). In other exemplary methods, a subject is a mammal (including humans and/or non-human mammals such as veterinary or laboratory mammals) or, in more particular examples, a racing mammal (such as a horse, a dog, or a human). In still other methods, a subject is an adult, an exercise-trained subject, or a healthy subject. Some representative adult, human subjects are 16 years old or old, 18 years old or older, or 21 years old or older. Some representative exercised-trained subjects have performed physical activity (such described in detail above) for at least 4 weeks, for at least 6 weeks, for at least 3 months, or for at least 6 months. In some examples the subject is healthy, for example, is a subject in which no known disease or disorder has been diagnosed or would be apparent after reasonable inquiry to an ordinarily skilled physician in the field to which the disease or disorder pertains.

[0073] C. Methods of Administration, Formulations and Dosage

[0074] The disclosed methods envision the use of any method of administration, dosage, and/or formulation of PPAR.delta. agonist that has the desired outcome of enhancing an exercise effect in a subject receiving the formulation, including, without limitation, methods of administration, dosages, and formulations well known to those of ordinary skill in the pharmaceutical arts.

[0075] Modes of administering a PPAR.delta. agonist (or a formulation including a PPAR.delta. agonist) in a disclosed method include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural, intradural, intracranial, intraventricular, and oral routes. In a specific example the PPAR.delta. agonist is administered orally. Other convenient routes for administration of a PPAR.delta. agonist (or a formulation including a PPAR.delta. agonist) include for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal. Administration can be systemic or local. Pulmonary administration also can be employed (for example, by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.

[0076] In specific method embodiments, it may be desirable to administer a PPAR.delta. agonist locally. This may be achieved by, for example, local or regional infusion or perfusion, topical application (for example, wound dressing), injection, catheter, suppository, or implant (for example, implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like.

[0077] In other method embodiments, a pump (such as a transplanted minipump) may be used to deliver a PPAR.delta. agonist (or a formulation including a PPAR.delta. agonist) (see, e.g., Langer Science 249, 1527, 1990; Sefton Crit. Rev. Biomed. Eng. 14, 201, 1987; Buchwald et al., Surgery 88, 507, 1980; Saudek et al., N. Engl. J. Med. 321, 574, 1989). In another embodiment, a PPAR.delta. agonist (or a formulation including a PPAR.delta. agonist) is delivered in a vesicle, in particular liposomes (see, e.g., Langer, Science 249, 1527, 1990; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365, 1989).

[0078] In yet another method embodiment, a PPAR.delta. agonist can be delivered in a controlled-release formulation. Controlled-release systems, such as those discussed in the review by Langer (Science 249, 1527 1990), are known. Similarly, polymeric materials useful in controlled-released formulations are known (see, e.g., Ranger et al., Macromol. Sci. Rev. Macromol. Chem. 23, 61, 1983; Levy et al., Science 228, 190, 1985; During et al., Ann. Neurol. 25, 351, 1989; Howard et al., J. Neurosurg. 71, 105, 1989). For example, a PPAR.delta. agonists may be coupled to a class of biodegradable polymers useful in achieving controlled release of a compound, including polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

[0079] The disclosed methods contemplate the use of any dosage form of PPAR.delta. agonist (or formulation containing the same) that delivers the PPAR.delta. agonist and achieves a desired result. Dosage forms are commonly known and are taught in a variety of textbooks, including for example, Allen et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Eighth Edition, Philadelphia, Pa.:Lippincott Williams & Wilkins, 2005, 738 pages. Dosage forms for use in a disclosed method include, without limitation, solid dosage forms and solid modified-release drug delivery systems (e.g., powders and granules, capsules, and/or tablets); semi-solid dosage forms and transdermal systems (e.g., ointments, creams, and/or gels); transdermal drug delivery systems; pharmaceutical inserts (e.g., suppositories and/or inserts); liquid dosage forms (e.g., solutions and disperse systems); and/or sterile dosage forms and delivery systems (e.g., parenterals, and/or biologics). Particular exemplary dosage forms include aerosol (including metered dose, powder, solution, and/or without propellants); beads; capsule (including conventional, controlled delivery, controlled release, enteric coated, and/or sustained release); caplet; concentrate; cream; crystals; disc (including sustained release); drops; elixir; emulsion; foam; gel (including jelly and/or controlled release); globules; granules; gum; implant; inhalation; injection; insert (including extended release); liposomal; liquid (including controlled release); lotion; lozenge; metered dose (e.g., pump); mist; mouthwash; nebulization solution; ocular system; oil; ointment; ovules; powder (including packet, effervescent, powder for suspension, powder for suspension sustained release, and/or powder for solution); pellet; paste; solution (including long acting and/or reconstituted); strip; suppository (including sustained release); suspension (including lente, ultre lente, reconstituted); syrup (including sustained release); tablet (including chewable, sublingual, sustained release, controlled release, delayed action, delayed release, enteric coated, effervescent, film coated, rapid dissolving, slow release); transdermal system; tincture; and/or wafer.

[0080] Typically, a dosage form is a formulation of an effective amount (such as a therapeutically effective amount) of at least one active pharmaceutical ingredient (such as a PPAR.delta. agonist) with pharmaceutically acceptable excipients and/or other components (such as one or more other active ingredients). The preferred aim of a drug formulation is to provide proper administration of an active ingredient (such as a PPAR.delta. agonist) to a subject. A formulation should suit the mode of administration. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and, more particularly, in humans.

[0081] Excipients for use in exemplary formulations include, for instance, one or more of the following: binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, colorings, preservatives, diluents, adjuvants, and/or vehicles. In some instances, excipients collectively may constitute about 5%-95% of the total weight (and/or volume) of a particular dosage form.

[0082] Pharmaceutical excipients can be, for instance, sterile liquids, such as water and/or oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when a formulation is administered intravenously. Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Oral formulations can include, without limitation, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. A more complete explanation of parenteral pharmaceutical excipients can be found in Remington, The Science and Practice of Pharmacy, 19th Edition, Philadelphia, Pa.:Lippincott Williams & Wilkins, 1995, Chapter 95. Excipients may also include, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like. Other examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A formulation, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0083] A dosage regimen utilizing a PPAR.delta. agonist is selected in accordance with a variety of factors including type, species, age, weight, sex and physical condition of the subject; the route of administration; and/or the particular PPAR.delta. agonist formulation employed. An ordinarily skilled physician or veterinarian can readily determine an effective amount of a PPAR.delta. agonist (or formulation thereof) useful for enhancing an exercise effect in a subject.

[0084] In some method embodiments involving oral administration, oral dosages of a PPAR.delta. agonist will generally range between about 0.001 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day, and such as about 0.01-10 mg/kg/day (unless specified otherwise, amounts of active ingredients are on the basis of a neutral molecule, which may be a free acid or free base). For example, an 80 kg subject would receive between about 0.08 mg/day and 8 g/day, such as between about 0.8 mg/day and 800 mg/day. A suitably prepared medicament for once a day administration would thus contain between 0.08 mg and 8 g, such as between 0.8 mg and 800 mg. In some instance, formulation including a PPAR.delta. agonist may be administered in divided doses of two, three, or four times daily. For administration twice a day, a suitably prepared medicament as described above would contain between 0.04 mg and 4 g, such as between 0.4 mg and 400 mg. Dosages outside of the aforementioned ranges may be necessary in some cases. Examples of daily dosages that may be given in the range of 0.08 mg to 8 g per day include 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, 1 g, 2 g, 4 g and 8 g. These amounts can be divided into smaller doses if administered more than once per day (e.g., one-half the amount in each administration if the drug is taken twice daily).

[0085] For some method embodiments involving administration by injection (e.g., intravenously or subcutaneous injection), a subject would receive an injected amount that would deliver the active ingredient in approximately the quantities described above. The quantities may be adjusted to account for differences in delivery efficiency that result from injected drug forms bypassing the digestive system. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day. Typically, a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, such as for example 0.1 mg/ml, 0.3 mg/ml, or 0.6 mg/ml, and administered in amounts per day equivalent to the amounts per day stated above. For example, an 80 kg subject, receiving 8 ml twice a day of an intravenous formulation having a concentration of active ingredient of 0.5 mg/ml, receives 8 mg of active ingredient per day.

[0086] In other method embodiments, a PPAR.delta. agonist (or a formulation thereof) can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (for example, in which the loading dose is about two to five times a maintenance dose). In some embodiments, the dose is varied during the course of PPAR.delta. agonist usage based on the condition of the subject receiving the composition, the apparent response to the composition, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term administration of a PPAR.delta. agonist (or formulation thereof) is contemplated, for instance in order to effect sustained enhancement of an exercise effect (such as aerobic endurance, e.g., running endurance).

IV. Methods for Determining Drug-Induced Enhancement of Exercise Performance

[0087] The use of performance-enhancing substances (PES), particularly by children and professional athletes, has been in the news because of potential adverse health consequences and the arguable effects that such practices have on moral development of the individual and on fair athletic competition for all (Committee on Sports Medicine and Fitness, Reginald L. Washington, Md., Chairperson, Pediatrics, 115(4):1103-1106, 2005). One of the discoveries provided herein is that certain genes (and/or the proteins encoded thereby) are uniquely regulated by a combination of exercise and a pharmaceutical agent (a PPAR.delta. agonist) that results in enhanced physical performance (see Table 2). In some cases, the particular genes (and/or proteins encoded thereby) were up- or down-regulated by the combined treatment but were not affected by either intervention alone. In other cases, the particular genes (and/or proteins encoded thereby) were not affected by the combined treatment but were up- or down-regulated by one or both intervention when practiced alone. The unique regulation of these genes (and/or the encoded proteins) makes them useful markers (either alone or in any combination) for identifying exercising subjects who are taking (or receiving) PES.

[0088] A PES is any substance taken in nonpharmacologic doses specifically for the purpose of improving sports performance (e.g., by increasing strength, power, speed, or endurance (ergogenic) or by altering body weight or body composition). Exemplary PES include the following: (i) pharmacologic agents (prescription or nonprescription) taken in doses that exceed the recommended therapeutic dose or taken when the therapeutic indication(s) are not present (e.g., using decongestants for stimulant effect, using bronchodilators when exercise-induced bronchospasm is not present, increasing baseline methylphenidate hydrochloride dose for athletic competition); (ii) agents used for weight control, including stimulants, diet pills, diuretics, and laxatives, when the user is in a sport that has weight classifications or that rewards leanness; (iii) agents used for weight gain, including over-the-counter products advertised as promoting increased muscle mass; (iv) physiologic agents or other strategies used to enhance oxygen-carrying capacity, including erythropoietin and red blood cell transfusions (blood doping); (v) any substance that is used for reasons other than to treat a documented disease state or deficiency; (vi) any substance that is known to mask adverse effects or detectability of another performance-enhancing substance, and/or (vii) nutritional supplements taken at supraphysiologic doses or at levels greater than required to replace deficits created by a disease state, training, and/or participation in sports. In one example the PES is GW1516.

[0089] The biomarkers of substance-induced performance enhancement identified herein and useful in a disclosed method include one or more (or any combination of) the genes (and/or proteins encoded thereby) listed in Table 2, and in some examples listed in Table 4. In particular method embodiments, at least 2, at least 3, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, or at least 40 of the genes (and/or proteins encoded thereby) listed in Table 2 (or Table 4) are detected in a disclosed method. In one example at least one gene (and/or protein encoded thereby) from each class listed in Table 2 (e.g., cytokines, fat metabolism) is analyzed.

[0090] In more specific method embodiments, upregulated expression is detected for one or more of the following genes (or proteins encoded thereby): adipose differentiation related protein; stearoyl-Coenzyme A desaturase 2; acetyl-Coenzyme A acetyltransferase 2; ATP citrate lyase; adiponectin, C1Q and collagen domain containing; diacylglycerol O-acyltransferase 2; lipase, hormone sensitive; monoglyceride lipase; resistin; CD36 antigen; fatty acid binding protein 4, adipocyte; lipoprotein lipase; microsomal glutathione S-transferase 1; GPI-anchored membrane protein 1; dual specificity phosphatase 7; homeodomain interacting protein kinase 3; insulin-like growth factor binding protein 5; protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), beta isoform; protein tyrosine phosphatase-like (proline instead of catalytic arginine); member b; CCAAT/enhancer binding protein (C/EBP), alpha; nuclear receptor subfamily 1, group D, member 2(Reverb-b); transferring; archain 1; solute carrier family 1 (neutral amino acid transporter), member 5; RIKEN cDNA 1810073N04 gene; haptoglobin; retinol binding protein 4, plasma; phosphoenolpyruvate carboxykinase 1, cytosolic; cell death-inducing DFFA-like effector c; interferon, alpha-inducible protein 27; carbonic anhydrase 3; cysteine dioxygenase 1, cytosolic; DNA segment, Chr 4, Wayne State University 53, expressed; dynein cytoplasmic 1 intermediate chain 2; Kruppel-like factor 3 (basic); thyroid hormone responsive SPOT14 homolog (Rattus); cytochrome P450, family 2, subfamily e, polypeptide 1; complement factor D (adipsin); and/or transketolase. In particular method embodiments, upregulation of at least 2, at least 3, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, or at least 38 of the foregoing genes (and/or proteins encoded thereby) are detected in a disclosed method.

[0091] In other method embodiments, downregulated expression is detected in one or more of the following genes (and/or proteins encoded thereby): gamma-glutamyl carboxylase; 3-oxoacid CoA transferase 1; solute carrier family 38, member 4; annexin A7; CD55 antigen; RIKEN cDNA 1190002H23 gene; fusion, derived from t(12; 16) malignant liposarcoma (human); lysosomal membrane glycoprotein 2; and/or neighbor of Punc E11. In particular method embodiments, downregulation of at least 2, at least 3, at least 5, or at least 7 of the foregoing genes (and/or proteins encoded thereby) are detected in a disclosed method.

[0092] In still other method embodiments, a combination of upregulated genes (and/or proteins encoded thereby) and downregulated genes (and/or proteins encoded thereby) as described above is detected in a sample from a subject (such as, an exercised or exercise-trained subject).

[0093] Yet other method embodiments involve the detection in a sample of a combination of the above-described upregulated genes (and/or proteins encoded thereby) and/or the above-described downregulated genes (and/or proteins encoded thereby), and/or the above-described exercise-regulated genes that are not affected by exercise combined with PPAR.delta. administration.

[0094] Disclosed methods may be used for detecting PES use in any subject capable of taking or receiving one or more such PES. In some method embodiments, a subject is a living multi-cellular vertebrate organism (e.g., human and/or non-human animals). In other exemplary methods, a subject is a mammal (including humans and/or non-human mammals) or, in more particular examples, a racing mammal (such as a horse, a dog, or a human). In still other methods, a subject is an exercise-trained subject. Some representative exercised-trained subjects have performed physical activity (such described in detail above) for at least 4 weeks, for at least 6 weeks, for at least 3 months, or for at least 6 months. Other exercise-trained subjects may be student athletes and/or professional athletes (including, in some examples, non-human professional athletes, such as race horses and/or racing dogs).

[0095] Any sample from a subject (e.g., a biological sample) in which can be detected one or more genes and/or proteins uniquely regulated by exercise in combination with PPAR.delta. agonist intake (as described in detail throughout this specification) is contemplated for use in a disclosed method. Exemplary samples for use in a disclosed method include blood, saliva, urine, muscle biopsy (e.g., skeletal muscle biopsy), cheek swab, fecal sample, sweat, and/or sperm.

[0096] Methods of detecting the expression of genes and/or proteins in a sample (e.g, biological sample) are very well known (see, e.g., U.S. Pat. Nos. 6,911,307; 6,893,824; 5,972,692; 5,972,602; 5,776,672; 7,031,847; 6,816,790; 6,811,977; 6,806,049; 6,203,988; and/or 6,090,556).

[0097] In particular method embodiments, expression of one or more genes identified herein can be detected by any method of nucleic acid amplification (such as, polymerase chain reaction (PCR) or any adaptation thereof, ligase chain reaction, transcription-based amplification systems, cycling probe reaction, Q.beta. replicase amplification, strand displacement amplification, and/or rolling circle amplification), solid-surface hybridization assays (such as Northern blot, dot blot, gene chips, and/or reversible target capture), solution hybridization assays (such as MAP technology (which uses a liquid suspension array of 100 sets of 5.5 micron probe-conjugated beads, each internally dyed with different ratios of two spectrally distinct fluorophores to assign it a unique spectral address)), and/or in situ hybridization. Various of the foregoing nucleic acid detection methods are described in detail in the review by Wolcott (Clin. Microbiol. Rev., 5(4):370-386, 1992). Other detailed and long-established protocols for practicing some such nucleic acid detection methods are found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); and/or Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th edition, Wiley & Sons, 1999.

[0098] In other method embodiments, expression of one or more proteins encoded by corresponding genes identified herein can be detected by Western blot, immunohistochemistry, immunoprecipitation, antibody microarrays, ELISA, and/or by functional assay (e.g., kinase assay, ATPase assay, substrate (or ligand) binding assay, protein-protein binding assay, or other assay suitable for measuring a particular protein function).

[0099] If the pattern of expression identified in the test subject is similar to that shown in Table 2 (e.g., the genes shown as upregulated and downregulated in Table 2 are observed in the subject to be upregulated and downregulated, respectively), this indicates that the subject is taking a PES, such as a PPAR.delta. agonist (e.g., GW1516). In contrast, If the pattern of expression identified in the test subject is different to that shown in Table 2 (e.g., the genes shown as upregulated and downregulated in Table 2 are observed in the subject to be not differentially expressed or show a different pattern of regulation), this indicates that the subject is not taking a PES, such as a PPAR.delta. agonist (e.g., GW1516).

V. Methods for Identifying Agents of Potential Interest

[0100] This disclosure identifies a previously unknown protein-protein interaction between PPAR.delta. and particular exercise-induced kinases (e.g., AMPK, such as the AMPK.alpha.1 and/or AMPK.alpha.2 subunit(s) of AMPK). The interaction between PPAR.delta. and AMPK may have important functional outcomes, such as enhancing exercise performance (e.g., aerobic exercise performance, such as running endurance) in a subject.

[0101] The foregoing discoveries enable methods for identify agents, e.g., having potential to enhance exercise performance (e.g., aerobic exercise performance, such as running endurance) in a subject. In some such methods, agents that affect (e.g., enhance, weaken, or substantially disrupt) the protein-protein interaction are identified. In other such methods, agents that affect (e.g., increase, decrease, or substantially eliminate) AMPK-dependent phosphorylation of a PPAR.delta. complex are identified.

[0102] A. Exemplary Agents

[0103] An "agent" is any substance or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for modulating a protein activity (e.g., AMPK-dependent phosphorylation of a PPAR.delta. complex), or useful for modifying or affecting a protein-protein interaction (e.g., PPAR.delta.-AMPK interaction). Any agent that has potential (whether or not ultimately realized) to modulate any aspect of the PPAR.delta.-AMPK interaction disclosed herein is contemplated for use in the screening methods of this disclosure.

[0104] Exemplary agents include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries (see, e.g., Lam et al., Nature, 354:82-84, 1991; Houghten et al., Nature, 354:84-86, 1991), and combinatorial chemistry-derived molecular library made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang et al., Cell, 72:767-778, 1993), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab').sub.2 and Fab expression library fragments, and epitope-binding fragments thereof), small organic or inorganic molecules (such as, so-called natural products or members of chemical combinatorial libraries), molecular complexes (such as protein complexes), or nucleic acids.

[0105] Libraries (such as combinatorial chemical libraries) useful in the disclosed methods include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res., 37:487-493, 1991; Houghton et al., Nature, 354:84-88, 1991; PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Natl. Acad. Sci. USA, 90:6909-6913, 1993), vinylogous polypeptides (Hagihara et al., J. Am. Chem. Soc., 114:6568, 1992), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Am. Chem. Soc., 114:9217-9218, 1992), analogous organic syntheses of small compound libraries (Chen et al., J. Am. Chem. Soc., 116:2661, 1994), oligocarbamates (Cho et al., Science, 261:1303, 1003), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem., 59:658, 1994), nucleic acid libraries (see Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nat. Biotechnol., 14:309-314, 1996; PCT App. No. PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522, 1996; U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, January 18, page 33, 1993; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones and methathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514) and the like.

[0106] Libraries useful for the disclosed screening methods can be produce in a variety of manners including, but not limited to, spatially arrayed multipin peptide synthesis (Geysen, et al., Proc. Natl. Acad. Sci., 81(13):3998-4002, 1984), "tea bag" peptide synthesis (Houghten, Proc. Natl. Acad. Sci., 82(15):5131-5135, 1985), phage display (Scott and Smith, Science, 249:386-390, 1990), spot or disc synthesis (Dittrich et al., Bioorg. Med. Chem. Lett., 8(17):2351-2356, 1998), or split and mix solid phase synthesis on beads (Furka et al., Int. J. Pept. Protein Res., 37(6):487-493, 1991; Lam et al., Chem. Rev., 97(2):411-448, 1997). Libraries may include a varying number of compositions (members), such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members, such as up to about 500,000 members, or even more than 500,000 members.

[0107] In one convenient embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (e.g., affectors of AMPK-PPAR.delta. protein-protein interactions). Such combinatorial libraries are then screened in one or more assays as described herein to identify those library members (particularly chemical species or subclasses) that display a desired characteristic activity (such as increasing or decreasing an AMPK-PPAR.delta. protein-protein interaction). The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. In some instances, pools of candidate agents may be identify and further screened to determine which individual or subpools of agents in the collective have a desired activity.

[0108] B. Exemplary Assays

[0109] As disclosed herein, PPAR.delta. forms a protein-protein interaction with AMPK or one or more of its subunits (such as AMPK.alpha.1 and/or AMPK.alpha.2). Agents that affect (e.g., increase or decrease) an AMPK-PPAR.delta. interaction or AMP-dependent phosphorylation of a PPAR.delta. complex may have the effect of enhancing exercise performance (e.g., aerobic exercise performance, such as running endurance) in a subject and, therefore, are desirable to identify.

[0110] In screening methods described here, tissue samples, isolated cells, isolated polypeptides, and/or test agents can be presented in a manner suitable for high-throughput screening; for example, one or a plurality of isolated tissue samples, isolated cells, or isolated polypeptides can be inserted into wells of a microtitre plate, and one or a plurality of test agents can be added to the wells of the microtitre plate. Alternatively, one or a plurality of test agents can be presented in a high-throughput format, such as in wells of microtitre plate (either in solution or adhered to the surface of the plate), and contacted with one or a plurality of isolated tissue samples, isolated cells, and/or isolated polypeptides under conditions that, at least, sustain the tissue sample or isolated cells or a desired polypeptide function and/or structure. Test agents can be added to tissue samples, isolated cells, or isolated polypeptides at any concentration that is not lethal to tissues or cells, or does not have an adverse effect on polypeptide structure and/or function. It is expected that different test agents will have different effective concentrations. Thus, in some methods, it is advantageous to test a range of test agent concentrations.

[0111] Disclosed methods envision, as appropriate, the use of PPAR.delta. or AMPK (such as AMPK.alpha.1 or AMPK.alpha.2) or functional fragments of any thereof as contained, independently, in a subject, one or a plurality of cells or cellular extracts, one or a plurality of tissue or tissue extracts, or as an isolated polypeptide. PPAR.delta. ligand optionally is included (or is omitted) in disclosed methods.

[0112] 1. Agents that Affect a Protein-Protein Interaction

[0113] A "direct association" between two or more polypeptides (such as, PPAR.delta. and AMPK (such as AMPK.alpha.1 or AMPK.alpha.2) is characterized by physical contact between at least a portion of the interacting polypeptides that is of sufficient affinity and specificity that, for example, immunoprecipitation of one of the polypeptides also will specifically precipitate the other polypeptide; provided that the immunoprecipitating antibody does not also affect the site(s) involved in the interaction. A direct association between polypeptides also may be referred to as a "protein-protein interaction." The binding of one polypeptide to another in a protein-protein interaction (e.g., PPAR.delta. to AMPK (or AMPK.alpha.1 and/or AMPK.alpha.2) and vice versa) is considered "specific binding".

[0114] Agents that affect an AMPK-PPAR.delta. interaction can be identified by a variety of assays, including solid-phase or solution-based assays. In an exemplary solid-phase assay, PPAR.delta. or an AMPK-binding fragment thereof and AMPK or a subunit thereof (such as AMPK.alpha.1 and/or AMPK.alpha.2) or a PPAR.delta.-binding fragment thereof are mixed under conditions in which PPAR.delta. and AMPK (or its subunit(s) or functional fragments) normally interact (e.g., co-immunoprecipitate). One of the binding partners is labeled with a marker such as biotin, fluoroscein, EGFP, or enzymes to allow easy detection of the labeled component. The unlabeled binding partner is adsorbed to a support, such as a microtiter well or beads. Then, the labeled binding partner is added to the environment where the unlabeled binding partner is immobilized under conditions suitable for interaction between the two binding partners. One or more test compounds, such as compounds in one or more of the above-described libraries, are separately added to individual microenvironments containing the interacting binding partners. Agents capable of affecting the interaction between the binding partners are identified, for instance, as those that increase or decrease (e.g., increase) retention or binding of the signal (i.e., labeled binding partner) in the reaction microenvironment, for example, in a microtiter well or on a bead for example. As discussed previously, combinations of agents can be evaluated in an initial screen to identify pools of agents to be tested individually, and this process is easily automated with currently available technology.

[0115] In other method embodiments, solution phase selection can be used to screen large complex libraries for agents that specifically affect protein-protein interactions (see, e.g., Boger et al., Bioorg. Med. Chem. Lett., 8(17):2339-2344, 1998); Berg et al., Proc. Natl. Acad. Sci., 99(6):3830-3835, 2002). In one such example, each of two proteins that are capable of physical interaction (for example, PPAR.delta. (or AMPK-binding fragments thereof) and AMPK or AMPK.alpha.1 or AMPK.alpha.2 (or PPAR.delta.-binding fragments of any thereof) are labeled with fluorescent dye molecule tags with different emission spectra and overlapping adsorption spectra. When these protein components are separate, the emission spectrum for each component is distinct and can be measured. When the protein components interact, fluorescence resonance energy transfer (FRET) occurs resulting in the transfer of energy from a donor dye molecule to an acceptor dye molecule without emission of a photon. The acceptor dye molecule alone emits photons (light) of a characteristic wavelength. Therefore, FRET allows one to determine the kinetics of two interacting molecules based on the emission spectra of the sample. Using this system, two labeled protein components are added under conditions where their interaction resulting in FRET emission spectra. Then, one or more test compounds, such as compounds in one or more of the above-described libraries, are added to the environment of the two labeled protein component mixture and emission spectra are measured. An increase in the FRET emission, with a concurrent decrease in the emission spectra of the separated components indicates that an agent (or pool of candidate agents) has affected (e.g., enhanced) the interaction between the protein components.

[0116] Interactions between PPAR.delta. (or AMPK-binding fragments thereof) and AMPK or AMPK.alpha.1 or AMPK.alpha.2 (or PPAR.delta.-binding fragments of any thereof) also can be determined (e.g., quantified) by co-immunoprecipitation of the relevant component polypeptides (e.g., from cellular extracts), by GST-pull down assay (e.g., using purified GST-tagged bacterial proteins), and/or by yeast two-hybrid assay, each of which methods is standard in the art. Conducting any one or more such assays in the presence and, optionally, absence of a test compound can be used to identify agents that improve or enhance (or, in other embodiments, decrease or inhibit) the interaction between PPAR.delta. (or AMPK-binding fragments thereof) and AMPK or AMPK.alpha.1 or AMPK.alpha.2 (or PPAR.delta.-binding fragments of any thereof) in the presence of a test compound as compared to in the absence of the test compound or as compared to some other standard or control.

[0117] In certain method embodiments, one or more AMPK (such as AMPK.alpha.1 and/or AMPK.alpha.2)-binding fragments of PPAR.delta. and/or one or more PPAR.delta.-binding fragments of AMPK (such as AMPK.alpha.1 and/or AMPK.alpha.2) are used. Polypeptide fragments having the desired binding activities can be identified by making a series of defined PPAR.delta. fragments and/or AMPK (such as AMPK.alpha.1 or AMPK.alpha.2) fragments using methods standard in the art. For example, cDNA encoding the protein(s) of interest (e.g., PPAR.delta. or AMPK) can be serially truncated from the 3' or 5' end (provided that a start codon is engineered into 5' truncations) using conveniently located restriction enzyme sites (or other methods) and leaving intact (or otherwise correcting) the proper reading frame. Conveniently, a nucleic acid sequence encoding an epitope tag (such as a FLAG tag) is placed in frame with (and substantially adjacent to) the truncated protein-encoding sequence to produce a nucleic acid sequence encoding an epitope-tagged protein fragment. The epitope-tagged protein fragment can be expressed in any convenient expression system (such as a bacterial expression system), isolated or not, and mixed with a sample containing a protein or other protein fragment to which the epitope-tagged protein fragment may bind. An antibody specific for the tag (or other region of the protein fragment) can be used to immunoprecipitate the fragment of interest together with any protein(s) or protein fragment(s) that bind to it. Protein(s) or protein fragment(s) that bind to the epitope-tagged protein fragment of interest can be particular identified, e.g., by Western blot.

[0118] In particular methods, the formation of a PPAR.delta.-AMPK (such as AMPK.alpha.1 and/or AMPK.alpha.2) complex (including complexes including one or both of PPAR.delta.-binding AMPK fragments and/or AMPK-binding PPAR.delta. fragments) or the affinity of PPAR.delta. (or AMPK-binding fragments thereof) and AMPK (or PPAR.delta.-binding fragments thereof) for each other is increased when the amount of such complex or the binding affinity is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 100% or at least 250% higher than a control measurement (e.g., in the same test system prior to addition of a test agent, or in a comparable test system in the absence of a test agent).

[0119] In other particular methods, the formation of a PPAR.delta.-AMPK (such as AMPK.alpha.1 and/or AMPK.alpha.2) complex (including complexes including one or both of PPAR.delta.-binding AMPK fragments and/or AMPK-binding PPAR.delta. fragments) or the affinity of PPAR.delta. (or AMPK-binding fragments thereof) and AMPK (or PPAR.delta.-binding fragments thereof) for each other is decreased when the amount of such complex or the binding affinity is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 100% or at least 250% lower than a control measurement (e.g., in the same test system prior to addition of a test agent, or in a comparable test system in the absence of a test agent).

[0120] 2. Agents that Affect AMPK-Dependent Phosphorylation

[0121] Disclosed are methods of screening test agents for those that affect (e.g., increase or decrease) AMPK (e.g., AMPK.alpha.1 and/or AMPK.alpha.2)-dependent phosphorylation of the PPAR.delta. complex. Agents that affect AMPK-dependent phosphorylation of the PPAR.delta. complex can be identified by a variety of assays, such adaptations of solid-phase- or solution-based assays described above, where the end point to be detected is phosphorylation of one or more components of the PPAR.delta. complex.

[0122] Methods for detecting protein phosphorylation are conventional (see, e.g., Gloffke, The Scientist, 16(19):52, 2002; Screaton et al., Cell, 119:61-74, 2004) and detection kits are available from a variety of commercial sources (see, e.g., Upstate (Charlottesville, Va., USA), Bio-Rad (Hercules, Calif., USA), Marligen Biosciences, Inc. (Ijamsville, Md., USA), Calbiochem (San Diego, Calif., USA). Briefly, phosphorylated protein (e.g., phosphorylation of one or more components of the PPAR.delta. complex) can be detected using stains specific for phosphorylated proteins in gels. Alternatively, antibodies specific phosphorylated proteins can be made or commercially obtained. Antibodies specific for phosphorylated proteins can be, among other things, tethered to the beads (including beads having a particular color signature) or used in ELISA or Western blot assays.

[0123] In one example, a PPAR.delta. complex (or a fragment thereof containing an AMPK phosphorylation site) and AMPK or one or more of it subunits (such as AMPK.alpha.1 and/or AMPK.alpha.2) or functional fragments thereof that are capable of phosphorylation are mixed under conditions whereby a PPAR.delta. complex is phosphorylated by AMPK. A PPAR.delta. complex is adsorbed to a support, such as a microtiter well or beads. Then, AMPK (or its one or more subunits (such as AMPK.alpha.1 and/or AMPK.alpha.2) or phosphorylation-capable fragments thereof) is added to the environment where the complex is immobilized. A phosphate donor typically is also included in the environment. The phosphate to be donated, optionally, can be labeled. One or more test compounds, such as compounds in one or more of the above-described libraries, are separately added to the individual microenvironments. Agents capable of affecting AMPK-dependent phosphorylation are identified, for instance, as those that enhance (or inhibit) phosphorylation of immobilized PPAR.delta. complex. In embodiments involving a labeled phosphate donor, phosphorylation of immobilized PPAR.delta. complex can be determined by retention or binding of a labeled phosphate in the reaction microenvironment, for example, in a microtiter well or on a bead for example. In other embodiments, such reactions can take place in solution (i.e., with no immobilized components), PPAR.delta. complex can be isolated from the solution (e.g., by immunoprecipitation with PPAR.delta.-specific or phosphate-specific antibodies), and its level of phosphorylation in the presence (and, optionally, absence) of one of more test agents determined as previously discussed.

[0124] In particular methods, the phosphorylation of a PPAR.delta. complex is increased when such posttranslational modification is detectably measured or when such posttranslational modification is at least 20%, at least 30%, at least 50%, at least 100% or at least 250% higher than control measurements (e.g., in the same test system prior to addition of a test agent, or in a comparable test system in the absence of a test agent, or in a comparable test system in the absence of AMPK).

[0125] In particular methods, the phosphorylation of PPAR.delta. complex is decreased when such posttranslational modification is detectably reduced or when such posttranslational modification is at least 20%, at least 30%, at least 50%, at least 100% or at least 250% lower than control measurements (e.g., in the same test system prior to addition of a test agent, or in a comparable test system in the absence of a test agent, or in a comparable test system in the absence of AMPK).

[0126] C. Screening Assay Target(s)

[0127] 1. PPAR.delta.

[0128] A PPAR.delta. polypeptide useful in a disclosed screening method is any known PPAR.delta. receptor. Also useful in the disclosed screening methods are homologs, functional fragments, or functional variants of a PPAR.delta. that retains at least AMPK-binding activity as described herein for a prototypical PPAR.delta. polypeptide (see Example 6).

[0129] The amino acid sequences of prototypical PPAR.delta. polypeptides (and PPAR.delta.-encoding nucleic acid sequences) are well known. Exemplary PPAR.delta. amino acid sequences and PPAR.delta.-encoding nucleic acid sequences are described, for instance, in U.S. Pat. No. 5,861,274, and U.S. Pat. Appl. Pub. No. 20060154335 (each of which is expressly incorporated herein by reference), and in GenBank Accession Nos. NP.sub.--035275 (SEQ ID NO:1) (GI:33859590) (Mus musculus amino acid sequence); NM.sub.--011145.3 (SEQ ID NO:2) (GI:89001112) (Mus musculus nucleic acid sequence); NP.sub.--006229 (SEQ ID NO:3) (GI:5453940) (Homo sapiens amino acid sequence); NM.sub.--006238.3 (SEQ ID NO:4) (GI:89886454) (Homo sapiens nucleic acid sequence); NP.sub.--037273 (SEQ ID NO:5) (GI:6981384) (Rattus norvegicus amino acid sequence); NM.sub.--013141.1 (SEQ ID NO:6) (GI:6981383) (Rattus norvegicus nucleic acid sequence); NP.sub.--990059 (SEQ ID NO:7) (GI:45382025) (Gallus gallus amino acid sequence) or NM.sub.--204728.1 (SEQ ID NO:8) (GI:45382024) (Gallus gallus nucleic acid sequence). In some method embodiments, a PPAR.delta. homolog or functional variant shares at least 60% amino acid sequence identity with a prototypical PPAR.delta. polypeptide; for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with an amino acid sequence as set forth in U.S. Pat. No. 5,861,274, U.S. Pat. Appl. Pub. No. 20060154335, or GenBank Accession No. NP.sub.--035275 (SEQ ID NO:1) (GI:33859590) (Mus musculus amino acid sequence); NP.sub.--006229 (SEQ ID NO:3) (GI:5453940) (Homo sapiens amino acid sequence); NP.sub.--037273 (SEQ ID NO:5) (GI:6981384) (Rattus norvegicus amino acid sequence); or NP.sub.--990059 (SEQ ID NO:7) (GI:45382025) (Gallus gallus amino acid sequence). In other method embodiments, a PPAR.delta. homolog or functional variant has one or more conservative amino acid substitutions as compared to with a prototypical PPAR.delta. polypeptide; for example, no more than 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservative amino acid changes compared to an amino acid sequence as set forth in U.S. Pat. No. 5,861,274, U.S. Pat. Appl. Pub. No. 20060154335, or GenBank Accession No. NP.sub.--035275 (SEQ ID NO:1) (GI:33859590) (Mus musculus amino acid sequence); NP.sub.--006229 (SEQ ID NO:3) (GI:5453940) (Homo sapiens amino acid sequence); NP.sub.--037273 (SEQ ID NO:5) (GI:6981384) (Rattus norvegicus amino acid sequence); or NP.sub.--990059 (SEQ ID NO:7) (GI:45382025) (Gallus gallus amino acid sequence). The following table shows exemplary conservative amino acid substitutions:

TABLE-US-00001 Conservative Original Residue Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0130] Some method embodiments involve a PPAR.delta. functional fragment (such as an AMPK-binding fragment), which can be any portion of a full-length known PPAR.delta. polypeptide, including, e.g., about 20, about 30, about 40, about 50, about 75, about 100, about 150 or about 200 contiguous amino acid residues of same; provided that the fragment retains a PPAR.delta. function of interest (e.g., AMPK binding). PPAR.delta. encompasses known functional motifs (such as ligand-binding domain, a DNA-binding domain, and a transactivation domain).

[0131] 2. AMPK

[0132] Mammalian AMP-activated kinase (AMPK) is a heterotrimeric protein composed of 1 alpha subunit, 1 beta subunit, and 1 gamma subunit. There are, at least, two known isoforms of the alpha subunit (.alpha.1 and .alpha.2). AMPK.alpha.1 and AMPK.alpha.2 have 90% amino acid sequence identity within their catalytic cores but only 61% in their C-terminal tails (see Online Mendelian Inheritance in Man (OMIM) Database Accession No. 602739; publicly available at the following website: ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602739).

[0133] An AMPK (such as AMPK.alpha.1 and/or AMPK.alpha.2) polypeptide useful in a disclosed screening method is any known AMPK protein or subunit thereof (such as AMPK.alpha.1 and/or AMPK.alpha.2). Also useful in the disclosed screening methods are homologs, functional fragments, or functional variants of an AMPK protein or subunit thereof (such as AMPK.alpha.1 and/or AMPK.alpha.2) that retains at least PPAR.delta.-binding activity as described herein (see Example 6).

[0134] The amino acid sequences of prototypical AMPK subunits (such as AMPK.alpha.1 and/or AMPK.alpha.2) (and nucleic acids sequences encoding prototypical AMPK subunits (such as AMPK.alpha.1 and/or AMPK.alpha.2)) are well known. Exemplary AMPK.alpha.1 amino acid sequences and the corresponding nucleic acid sequences are described, for instance, in GenBank Accession Nos. NM.sub.--206907.3 (SEQ ID NO:9) (GI:94557298) (Homo sapiens transcript variant 2 REFSEQ including amino acid and nucleic acid sequences); NM.sub.--006251.5 (SEQ ID NO:10) (GI:94557300) (Homo sapiens transcript variant 1 REFSEQ including amino acid and nucleic acid sequences); NM.sub.--001013367.3 (SEQ ID NO:11) (GI:94681060) (Mus musculus REFSEQ including amino acid and nucleic acid sequences); NM.sub.--001039603.1 (SEQ ID NO:12) (GI:88853844) (Gallus gallus REFSEQ including amino acid and nucleic acid sequences); and NM.sub.--019142.1 (SEQ ID NO:13) (GI:11862979) (Rattus norvegicus REFSEQ including amino acid and nucleic acid sequences). Exemplary AMPK.alpha.2 amino acid sequences and the corresponding nucleic acid sequences are described, for instance, in GenBank Accession Nos. NM.sub.--006252.2 (SEQ ID NO:14) (GI:46877067) (Homo sapiens REFSEQ including amino acid and nucleic acid sequences); NM.sub.--178143.1 (SEQ ID NO:15) (GI:54792085) (Mus musculus REFSEQ including amino acid and nucleic acid sequences); NM.sub.--001039605.1 (SEQ ID NO:16) (GI:88853850) (Gallus gallus REFSEQ including amino acid and nucleic acid sequences); and NM.sub.--214266.1 (SEQ ID NO:17) (GI:47523597) (Sus scrofa REFSEQ including amino acid and nucleic acid sequences).

[0135] In some method embodiments, a homolog or functional variant of an AMPK subunit shares at least 60% amino acid sequence identity with a prototypical AMPK.alpha.1 and/or AMPK.alpha.2 polypeptide; for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with an amino acid sequence as set forth in the GenBank Accession Nos. NM.sub.--206907.3 (SEQ ID NO:9); NM.sub.--006251.5 (SEQ ID NO:10); NM.sub.--001013367.3 (SEQ ID NO:11); NM.sub.--001039603.1 (SEQ ID NO:12); NM.sub.--019142.1 (SEQ ID NO:13); NM.sub.--006252.2 (SEQ ID NO:14); NM.sub.--178143.1 (SEQ ID NO:15); NM.sub.--001039605.1 (SEQ ID NO:16); or NM.sub.--214266.1 (SEQ ID NO:17). In other method embodiments, a homolog or functional variant of an AMPK subunit has one or more conservative amino acid substitutions as compared to a prototypical AMPK.alpha.1 and/or AMPK.alpha.2 polypeptide; for example, no more than 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservative amino acid changes compared to an amino acid sequence as set forth in as set forth in GenBank Accession Nos. NM.sub.--206907.3 (SEQ ID NO:9); NM.sub.--006251.5 (SEQ ID NO:10); NM.sub.--001013367.3 (SEQ ID NO:11); NM.sub.--001039603.1 (SEQ ID NO:12); NM.sub.--019142.1 (SEQ ID NO:13); NM.sub.--006252.2 (SEQ ID NO:14); NM.sub.--178143.1 (SEQ ID NO:15); NM.sub.--001039605.1 (SEQ ID NO:16); or NM.sub.--214266.1 (SEQ ID NO:17). Exemplary conservative amino acid substitutions have been previously described herein.

[0136] Some method embodiments involve a functional fragment of AMPK or a subunit thereof (such as AMPK.alpha.1 and/or AMPK.alpha.2), including a PPAR.delta.-binding fragment or a fragment with PPAR.delta. phosphorylation activity. Functional fragments of AMPK or a subunit thereof (such as AMPK.alpha.1 and/or AMPK.alpha.2) can be any portion of a full-length or intact AMPK polypeptide complex or subunit thereof (such as AMPK.alpha.1 and/or AMPK.alpha.2), including, e.g., about 20, about 30, about 40, about 50, about 75, about 100, about 150 or about 200 contiguous amino acid residues of same; provided that the fragment retains at least one AMPK (or AMPK.alpha.1 and/or AMPK.alpha.2) function of interest (e.g., PPAR.delta. binding and/or PPAR.delta. phosphorylation activity). Protein-protein interactions between PPAR.delta. and AMPK are believed to involve, at least, an AMPK.alpha. subunit (such as AMPK.alpha.1 and/or AMPK.alpha.2). Moreover, because PPAR.delta. specifically binds both AMPK.alpha.1 and AMPK.alpha.2 (see Example 6), such interaction likely is mediated by the portions of these AMPK.alpha. isoforms that share the most sequence homology (as discussed above). Accordingly, in some method embodiments, an AMPK PPAR.delta.-binding fragment includes a functional fragment encompassing (or consisting of) the catalytic core domain of an alpha subunit of AMPK (such as AMPK.alpha.1 and/or AMPK.alpha.2).

EXAMPLES

[0137] The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.

Example 1

Administration of PPAR.delta. Agonist Surprisingly does not Enhance Physical Performance in Non-Exercised Subjects

[0138] Wang et al. previously demonstrated that skeletal muscle-specific expression of a constitutively active form of PPAR.delta. receptor resulted in transgenic mice with skeletal muscles that had an increased number of slow, oxidative (type I) muscle fibers and markedly increased running endurance (Wang et al., PLoS Biol., 2:e294, 2004). This Example demonstrates that administration of a PPAR.delta. agonist (GW1516) to non-transgenic mice also results in the expression in skeletal muscle of some biomarkers of oxidative metabolism. However, in unexpected contrast to the results obtained by genetic activation of the PPAR.delta. pathway, PPAR.delta. activation by pharmacological treatment did not modify fiber-type composition of skeletal muscle, nor improve running endurance in non-transgenic, sedentary (also referred to as "non-exercised" or "untrained") mice.

[0139] Male C57B/6J mice (8 wks old) were obtained from Jackson Laboratory and housed in the Salk Institute animal care facility. The animals were acclimated to their surroundings for one week prior to experimentation, and had access at all times to standard mouse chow and water ad libitum.

[0140] Mice were acclimated to moderate treadmill running (10 m/min for 15 min) every other day for 1 week. After acclimation, basal running endurance was determined by placing each mouse on a treadmill, gradually increasing the speed from 0 to 15 m/min, and maintaining 15 m/min until the mouse was exhausted. The time and distance run until exhaustion were recorded as the basal endurance values (Week 0).

[0141] Mice then were treated once per day for 4 weeks with vehicle or the PPAR.delta. agonist, GW1516 (5 mg/kg). Treatments were administered orally. During the treatment period, mice were housed in standard laboratory cages and received only the amount of physical activity that could be had by normal movements about such cage.

[0142] Animals were euthanized by carbon dioxide asphyxiation 72 hours after the final treatment. Gastrocnemius and quadriceps muscles were isolated, frozen and stored at -80.degree. C. for future analysis. Total RNA was prepared from quadriceps muscle using TRIZOL.TM. reagent (Invitrogen, Calsbad, Calif., USA) in conformance with manufacturer's instructions. Real time quantitative PCR (QPCR) was used to determine expression levels of uncoupling protein 3 (UCP3), muscle carnitine palmitoyl transferase I (mCPT I) and pyruvate dehydrogenase kinase 4 (PDK4) using primers known to those of ordinary skill in the art.

[0143] As shown in FIG. 1A, four weeks of GW1516 treatment induced the expression of UCP3, mCPT I, and PDK4, in quadriceps muscle of treated mice (compare V to GW). These changes in gene expression were detected as early as 4 days after treatment and with drug concentrations ranging from 2-5 mg/kg/day. Moreover, in the gene expression studies, maximal effects of PPAR.delta. activation were detected in pre-dominantly fast-twitch (quadricep and gastrocnemius) but not slow-twitch (soleus) muscles.

[0144] Using primary muscle cells cultured from wild type and PPAR.delta. null mice (Chawla et al., Proc. Natl. Acad. Sci. USA. 100(3): 1268-73, 2003; Man et al., J. Invest. Dermatol. 2007; Rando and Blau, J. Cell. Biol. 125(6): 1275-87, 1994), it was confirmed that the induction of oxidative genes by GW1516 is mediated via activation of PPAR.delta. in skeletal muscles (FIGS. 1B-D). Moreover, this is similar to the expression changes found in the same gene set in muscles from mice expressing the constitutively active VP16-PPAR.delta. transgene (Wang et al., PLoS Biol., 2:e294, 2004) (FIG. 1A, see TG). Collectively, these results indicate that pharmacological activation of PPAR.delta. can initiate an oxidative response in adult skeletal muscle.

[0145] Expression of biomarkers characteristic of an oxidative phenotype in skeletal muscle, typically, has been correlated with increased oxidative performance (e.g., increased running endurance) of such skeletal muscle. This correlation was observed, for instance, in the VP16-PPAR.delta. transgenic mouse (Wang et al., PLoS Biol., 2:e294, 2004). For this and other reasons, it was expected that GW1516 treatment similarly would increase running performance. Accordingly, to determine the functional effects of ligand, age and weight matched cohorts of treated and control mice were subjected to an endurance treadmill performance test before (week 0) and after (week 5) treatment.

[0146] Following four weeks of treatment and housing in standard laboratory cages without additional exercise, the running endurance of GW1516-treated and control mice again was determined as described above. Remarkably, and despite expectations for improvement, GW1516-treated mice did not significantly differ from controls in either the time spent or distance run on the treadmill prior to exhaustion (FIG. 1E). Furthermore, long-term drug treatment of up to 5 months also did not change running endurance.

[0147] These results indicate that although in non-trained adult muscle pharmacological activation of PPAR.delta. induces some transcriptional changes, it fails to alter either fiber type composition or endurance. In summary, pharmacologic activation of the PPAR.delta. genetic program in adult C57B1/6J mice is insufficient to promote a measurable enhancement of treadmill endurance.

Example 2

Administration of PPAR.delta. Agonist Remodels Skeletal Muscle in Exercised-Trained Subjects

[0148] Fiber type proportions in skeletal muscle are believed to be determined by heredity and environmental factors, such as physical activity level (Simoneau and Bouchard, FASEB J., 9(11):1091-1095, 1995; Larsson and Ansved, Muscle Nerve, 8(8):714-722, 1985). Endurance exercise training is known to remodel the skeletal muscle by increasing type I slow-twitch fibers, oxidative enzymes, and mitochondrial density, which progressively alter performance (Holloszy et al., J. Appl. Physiol. 56:831-8, 1984; Booth et al., Physiol Rev. 71:541-85, 1991; Schmitt et al., Physiol. Genomics. 15:148-57, 2003; Yoshioka et al., FASEB J. 17:1812-9, 2003; Mahoney et al., Phys. Med. Rehabil. Clin. N. Am. 16:859-73, 2005; Mahoney et al., FASEB J. 19:1498-500, 2005; Siu et al., J. Appl. Physiol. 97:277-85, 2004; Garnier et al., FASEB J. 19:43-52, 2005; Short et al., J Appl Physiol. 99:95-102, 2005; Timmons et al., FASEB J. 19: 750-60, 2005). This example demonstrates that PPAR.delta. agonist treatment influences skeletal muscle on a molecular level.

[0149] To determine whether co-administration of GW1516 in the context of endurance exercise can enhance changes in fiber type composition and mitochondrial biogenesis, the effect of GW1516 treatment on muscle fiber-type composition was determined by meta-chromatic staining of cryo-sections of gastrocnemius as described by Wang et al. (PLoS Biol., 2:e294, 2004). Meta-chromatic staining was used, following a routine myofibrillar ATPase reaction, to demonstrate quantitative differences in phosphate deposition among different skeletal muscle fiber types and, thereby, differentiate skeletal muscle fiber types (Doriguzzi et al., Histochem., 79(3):289-294, 1983; Ogilvie and Feeback, Stain Technol., 65(5):231-241, 1990). In this assay, muscle fibers with high ATPase activity (e.g., type I (slow oxidative) muscle fibers) are darkly stained.

[0150] As shown in FIG. 2A, there was no significant difference in the proportion of type I (slow, oxidative) muscle fibers in the gastrocnemius muscles of vehicle- and GW1516-treated sedentary mice. In contrast, hindlimb muscles of VP16-PPAR.delta. transgenic mice exhibited an increased number of type I muscle fibers when assayed by monochromatic staining. In trained mice, GW1516 increased the proportion of type I fibers (by .about.38%) compared to the vehicle-treated sedentary mice (FIGS. 2A and 2B). Therefore, administration of a PPAR.delta. agonist (e.g., GW1516) alone to sedentary subjects does not significantly affect the number of type I (slow-twitch, oxidative) muscle fibers in hindlimb muscles, but can increase the number of type I muscle fibers in hindlimb muscles of trained subjects.

[0151] In addition to its effects on the fiber type, exercise training increased skeletal muscle mitochondrial biogenesis, which can be measured as a function of mitochondrial DNA expression levels using quantitative real time PCR (QPCR). Mitochondrial DNA expression levels were determined in muscles of V, GW, Tr, and GW+Tr subjects using quantitative real time PCR. As shown in FIG. 2C, similar to type I fiber changes, mitochondrial DNA expression was not changed by drug alone but was increased by approximately 50% with the combination of exercise and GW1516 treatment (FIG. 2C). Such an increase is known to contribute to enhanced endurance capacity (e.g., Holloszy, Med. Sci. Sports 7:155-64, 1975).

[0152] Slow-twitch and fast-twitch muscle fiber types also can be distinguished by myosin isoform expression (Gauthier and Lowey, J. Cell Biol. 81:10-25, 1979; Fitzsimons and Hoh, Biochem. J. 193:229-33, 1981). Myosin isoform expression in skeletal muscle adapts to various conditions, such as changes in muscle mechanics, muscle innervation, or exercise paradigm (for review, see, e.g., Baldwin and Haddad, J. Appl. Physiol., 90(1):345-57, 2001; Baldwin and Haddad, Am. J. Phys. Med. Rehabil., 81(11 Suppl):540-51, 2002; Parry, Exerc. Sport Sci. Rev., 29(4):175-179, 2001). The effect of GW1516 administration on myosin heavy chain (MHC) expression (MHC I, MHC IIa, MHC IIb) was determined by methods known to those of ordinary skill in the art.

[0153] GW1516 treatment in sedentary mice increased the expression of MHC I (a marker of slow-twitch, oxidative muscle fibers) and decreased the expression of MHC IIb (a marker of fast-twitch, glycolytic muscle fibers) as compared to vehicle-treated, control mice. In comparison, GW1516 treatment did not alter the expression of MHC IIa (a marker of fast-twitch oxidative/glycolytic muscle fibers) in sedentary mice. Therefore, at least at the transcriptional level, the PPAR.delta. agonist was capable of inducing some proteins characteristic of a slow-twitch muscle fiber phenotype.

[0154] In summary, expression of constitutively active PPAR.delta. in the skeletal muscles of VP16-PPAR.delta. transgenic mice resulted in a "long-distance running phenotype" with "profound and coordinated increases in oxidative enzymes, mitochondrial biogenesis and production of specialized type I fiber contractile proteins-the three hallmarks of muscle fiber type switching" (Wang et al., PLoS Biol., 2:e294, 2004). In contrast, pharmacological activation of PPAR.delta. in normal subjects only partially recapitulated VP16-PPAR.delta. transgenesis by regulating some metabolic genes. Markedly, administration of a PPAR.delta. agonist to sedentary subjects did not lead to a change in fiber type specification (as measured by monochromatic staining) or enhance exercise endurance. Transgenic over-expression of activated PPAR.delta. at birth pre-programs the nascent myofibers to trans-differentiate into slow-twitch fibers, thus imparting a high basal endurance capacity to adult transgenic mice. In contrast, since fiber type specification is completed prior to exposure of adults to PPAR.delta. agonist, the potential plasticity of muscle to drug treatment alone is virtually non-existent.

[0155] This example illustrates that the genetic or pharmacologic activation of the PPAR.delta. regulatory program in skeletal muscles of adult, sedentary subjects does not have the same outcome. The ability to genetically manipulate skeletal muscle specification by activation of the PPAR.delta. receptor in a transgenic mouse from early development in the absence of exercise is not necessarily predictive of the result of pharmacologically activating the PPAR.delta. program in the sedentary, normal adult. The cellular "template" for PPAR.delta. effects on skeletal muscle is very different in a normal subject as compared to a genetically engineered transgenic subject. For example, in a normal adult, muscle fiber specification of individual muscle groups is already determined and the connections between muscle fibers and spinal motor neurons are established prior to pharmacological activation of the PPAR.delta.-regulated program. In the transgenic mouse, the constitutively active PPAR.delta. transgene is active all the while muscle fiber specification is being determined and connections between muscle fibers and motor neurons are being made. In addition, the effects of activation of endogenous PPAR.delta. receptor by a single daily dose of a PPAR.delta. agonist, which is expect to have a transient peak exposure followed by clearance, likely are much different from the effects of the constitutive activation of a VP16-PPAR.delta. transgene.

Example 3

The Combination of PPAR.delta. Agonist Treatment and Exercise Training Significantly Affected Fatty Acid Metabolism and Markers of Fatty Acid Oxidation

[0156] In addition to affecting the contractile apparatus of skeletal muscle, exercise training also increases skeletal muscle mitochondrial density (e.g., Freyssenet et al., Arch. Physiol. Biochem., 104(2):129-141, 1996). This Example illustrates that PPAR.delta. agonist treatment (e.g., GW1516) in exercise-trained subjects affected fatty acid metabolism in exercised muscle.

[0157] The effects of GW1516 treatment and exercise, alone or in combination, on components of the oxidative metabolism of fatty acids were determined by measuring gene expression levels of selective biomarkers for fatty acid .beta.-oxidation (FAO). Male C57B/6J mice (8-10 wks old) were randomly divided into four groups (nine per group): (i) vehicle-treated and sedentary (V), (ii) GW1516-treated and sedentary (GW), (iii) vehicle-treated and exercise trained (Tr) and (iv) GW1516-treated and exercise trained (GW+Tr). Mice in all groups were acclimated to moderate treadmill running and basal running endurance was determined as described in Example 1. Thereafter, mice in the exercise-trained groups received four weeks (5 days/week) of exercise training on a treadmill inclined at 5 degrees. Intensity and time of training were gradually increased. At the end of four weeks, all exercise-trained mice were running for 50 min/day at 18 m/min. Vehicle or GW1516 was administered to the respective exercise-treated or sedentary groups as described in Example 1. Unless otherwise noted, V, GW, Tr and GW+Tr subjects described in this and the examples below were similarly treated. At the end of the drug treatment and/or training protocol (Week 5) 6 mice per group were subjected to the running test. These interventions do not affect body weight and food intake in mice. RNA was prepared real time quantitative PCR performed as described in Example 1.

[0158] Confirming the results obtained in Example 1, UCP3, mCPT I, and PDK4 were upregulated by GW1516 but showed no further induction with exercise (see FIGS. 1A and 3A). Unexpectedly, a second set of genes were identified that showed no response to exercise or GW1516 alone but were robustly induced by the combination. This intriguing response profile includes a series of genes involved in the regulation of fatty acid storage [such as steroyl-CoA-desaturase (SCD1), fatty acyl coenzyme A synthase (FAS) and serum response element binding protein 1c (SREBP 1c)] and fatty acid uptake [such as the fatty acid transporter (FAT/CD36) and lipoprotein lipase (LPL)] adding a new set of target genes to exercise and drug treated mice (FIGS. 3B, 3C and 6A-C).

[0159] In addition to gene expression, protein expression was determined for selective oxidative biomarkers including myoglobin, UCP3, cytochrome c (CYCS) and SCD1, using Western blotting. Protein homogenates were prepared from quadriceps muscle, separated by SDS polyacrylamide gel electrophoresis, transferred to blotting membrane and probed with antibodies specific for myoglobin (Dako), UCP3 (Affinity Bioreagents), cytochrome c (Santacruz) SCD1 (Santacruz), and, as a loading control, tubulin (Sigma). A robust up regulation of myoglobin, UCP3, cytochrome c, and SCD1 protein expression was observed with combined exercise and GW1516 treatment in comparison to treatment with the PPAR.delta. agonist or exercise alone (FIG. 3D).

[0160] Altered triglycerides can be used to access changes in muscle oxidative capacity. Muscle triglyceride (mTG) content was measured as previously described (Wang et al., PLoS Biol., 2:e294, 2004) using a kit from Thermo Electron Corporation. As shown in FIG. 4, mTG content was comparable between vehicle and GW1516-treated sedentary mice and was substantially increased in muscle of mice receiving only exercise training. In contrast, dramatic increase in triglycerides in exercised muscle was completely reversed in GW1516-treated exercise trained mice, indicating increased fat utilization (FIG. 4).

[0161] Gene and/or protein expression that is induced by a combination of exercise and drug treatment (e.g., PPAR.delta. agonist administration) but not by either input alone is believed to be a new discovery. This type of response can be used to further characterize the intersection of pharmacologic and physiologic genetic networks. For example, one or more genes and/or proteins uniquely regulated by one or more drugs (e.g., PPAR.delta. agonists) and exercise can be used as markers, for instance, of illicitly boosting performance in professional and/or amateur athletes.

Example 4

Administration of PPAR.delta. Agonist Enhances the Physical Performance of Exercise-Trained Subjects

[0162] As described in Example 1, although GW1516 treatment induces wide-spread genomic changes associated with oxidative metabolism, nonetheless alone it failed to increase running endurance. This finding was unexpected because it was known that constitutive activation of the PPAR.delta. gene network (in the VP16-PPAR.delta. transgenic mouse) lead to a distance-running phenotype (familiarly, a "marathon mouse"). On the other hand, as surprisingly shown in Example 3, PPAR.delta. agonist (e.g., GW1516) treatment in conjunction with exercise produced an enriched remodeling program that included a series of transcriptional and posttranslational adaptations in the skeletal muscle. This indicates that exercise training serves as a trigger to unmask a set of PPAR.delta. target genes. This Example provides methods used to demonstrate that administration of a PPAR.delta. agonist (e.g., GW1516) surprisingly improves physical performance in exercised (trained) subjects.

[0163] Male C57B/6J mice (8-10 wks old) were randomly divided into four groups (nine per group): (i) vehicle-treated and sedentary (V), (ii) GW1516-treated and sedentary (GW), (iii) vehicle-treated and exercise trained (Tr) and (iv) GW1516-treated and exercise trained (GW+Tr), acclimated to moderate treadmill running as described in Example 1, and exercise-trained as described in Example 3. At the end of the drug treatment and/or training protocol (Week 5) 6 mice per group were subjected to the running test.

[0164] At the end of the drug treatment and/or training protocol (Week 5), running endurance of six mice per group was determined in the same manner as was basal running endurance. No follow-up endurance tests were performed on three mice in each group to confirm that changes observed in the skeletal muscle were not due to the acute run, but were related to the exercise training.

[0165] As shown in FIGS. 5A and 5B, the same dose and duration of GW1516 treatment that failed to alter running endurance in sedentary mice, when paired with 4 weeks of exercise training, increases running time by 68% and running distance by 70% over vehicle-treated trained mice (FIGS. 5A and 5B, compare Week 5). Comparison of running time and distance before (week 0) and after (week 5) exercise and drug treatment revealed a 100% increment in endurance capacity for individual mice, underscoring the robustness of the combination paradigm (FIGS. 5A and 5B). In contrast, the same exercise protocol without concurrent GW1516 treatment did not significantly increase running endurance in C57B1/6J mice.

[0166] Hematoxylin and eosin (H&E) staining of white adipose tissue paraffin sections was performed as previously described (Wang et al., PLoS Biol., 2:e294, 2004; Wang et al., Cell, 113:159-70, 2003). As shown in FIG. 5C, GW1516 treatment in combination with exercise produced a significant (32%) reduction in the epididymal fat to body weight ratio, which was further evident in the decreased cross-sectional area of the adipocytes in the same group (FIG. 5D). Therefore, the combined effects of GW1516 and exercise are not restricted to muscle.

[0167] Using the methods described in Example 2, it was also demonstrated that the combination of GW1516 treatment and exercise training significantly increased the number of type I muscle fibers in exercised muscle. However, combining GW1516 treatment with exercise did not induce additional changes in MHC I and MHC IIb expression. Therefore, although orally administered PPAR.delta. agonist (GW1516) alone is capable of inducing the expression of at least some of the contractile proteins in the PPAR.delta.-regulated gene network (see Example 5) the transcriptional effect observed was not sufficient to induce a post-transcriptional change in fiber-type composition as was observed by meta-chromatic staining in GW1516-treated, exercised mice.

[0168] This Example illustrates that PPAR.delta. agonist (e.g., GW1516) treatment unexpectedly augments the performance of aerobic exercise (e.g., running distance and endurance) in an exercised subject. Endurance exercise is known to channel extra-muscular fat to muscle triglyceride stores by inducing adipose tissue lipolysis to meet increased oxidative demands (Despres et al., Metabolism, 33:235-9, 1984; Mauriege et al., Am. J. Physiol., 273:E497-506, 1997; Mader et al., Int. J. Sports Med., 22:344-9, 2001; Schmitt et al., Physiol. Genomics, 15:148-57, 2003; Schrauwen-Hinderling et al., J. Clin. Endocrinol. Metab., 88:1610-6, 2003). In addition, the induction of FAO components and selective up-regulation of fatty acid storage and up-take components in GW1516-treated, exercised mice described in Example 3 indicate enhanced mobilization of fat as fuel in skeletal muscle. Therefore, combined exercise and GW1516 treatment dramatically increases muscle oxidative capacity in subjects, for example by increasing local fatty acid synthesis and/or mobilizing fatty acid stores from adipose tissue.

[0169] This is the first demonstration of how an orally active PPAR.delta. agonist and exercise can co-operatively re-program the muscle genome and raise endurance limits.

Example 5

The Combination of PPAR.delta. Agonist Treatment and Exercise Training Produced a Unique Gene Expression Signature in Exercised Muscle

[0170] A comprehensive study of the skeletal muscle transcriptional program in V, GW, Tr and Tr+GW mice was conducted using microarray analysis. Affymetrix.TM. high-density oligonucleotide array mouse genome 430A 2.0 chips were used. Preparation of in vitro transcription products, oligonucleotide array hybridization, and scanning were performed in conformance with Affymetrix.TM.-provided protocols. To minimize discrepancies due to variables, the raw expression data were scaled by using Affymetrix.TM. MICROARRAY SUITE.TM. 5.0 software, and pairwise comparisons were performed. The trimmed mean signal of all probe sets was adjusted to a user-specified target signal value (200) for each array for global scaling. No specific exclusion criteria were applied. Additional analyses were performed using the freeware program BULLFROG 7 (available on the internet Barlow-LockhartBrainMapNIMHGrant.org) and the Java-based statistical tool VAMPIRE (Hsiao et al., Bioinformatics, 20:3108-3127, 2004).

[0171] Genome-wide analysis of the quadriceps muscle revealed that GW1516 treatment, exercise, and the combination regulated 96, 113 and 130 genes, respectively (FIG. 6). Approximately 50% of the target genes regulated by GW1516 or exercise alone were the same, demonstrating that PPAR.delta. activation of the gene network partially mimics exercise effects on the same network.

[0172] The 130 genes regulated by the combination of GW1516 treatment and exercise training and a classification of each such gene are shown in Table 1. The 130 regulated genes included 30 fat metabolism genes, 5 oxygen carriers, 5 mitochondrial genes, 3 carbohydrate metabolism genes, 15 signal transduction genes, 16 transcription genes, 10 transport genes, 3 steroid biogenesis genes, 5 heat shock genes, 2 angiogenesis genes, 5 proliferation and apoptosis genes, 2 cytokines, and 29 others. The majority of the genes in the exercise-trained/GW1516-treated (GW+Tr) gene signature shown in Table 1 were induced (109/130). The 109 upregulated genes are shown in non-bold font in Table 1 (final column>1). Down-regulated genes are shown in bold italics in Table 1 (final column<1).

TABLE-US-00002 TABLE 1 Genes regulated by GW1516 treatment and exercise training FEATURE LOCUS DESCRIPTION GW + Tr ANGIOGENESIS 1417130_s_at Angptl4 angiopoietin-like 4 5.495 CARBOHYDRATE METABOLISM 1449088_at Fbp2 fructose bisphosphatase 2 2.808 1423439_at Pck1 phosphoenolpyruvate carboxykinase 1, cytosolic 3.518 1434499_a_at Ldhb lactate dehydrogenase B 2.541 PROLIFERATION & APOPTOSIS 1425621_at Trim35 tripartite motif-containing 35 1.856 1448272_at Btg2 B-cell translocation gene 2, anti-proliferative 1.601 1452260_at Cidec cell death-inducing DFFA-like effector c 4.771 1417956_at Cidea cell death-inducing DNA fragmentation factor, alpha 49.625 subunit-like effector A CYTOKINES 1426278_at Ifi27 interferon, alpha-inducible protein 27 1.714 1421239_at Il6st interleukin 6 signal transducer 1.972 FAT METABOLISM 1448318_at Adfp adipose differentiation related protein 2.009 1424729_at BC054059 cDNA sequence BC054059 5.08 1424937_at 2310076L09Rik RIKEN cDNA 2310076L09 gene 1.868 1450010_at Hsd17b12 hydroxysteroid (17-beta) dehydrogenase 12 2.376 1415965_at Scd1 stearoyl-Coenzyme A desaturase 1 6.494 1415822_at Scd2 stearoyl-Coenzyme A desaturase 2 1.849 1423828_at Fasn fatty acid synthase 6.323 1455061_a_at Acaa2 acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3- 1.926 oxoacyl-Coenzyme A thiolase) 1448987_at Acadl acetyl-Coenzyme A dehydrogenase, long-chain 2.549 1422651_at Adipoq adiponectin, C1Q and collagen domain containing 3.082 1422820_at Lipe lipase, hormone sensitive 3.032 1449964_a_at Mlycd malonyl-CoA decarboxylase 1.781 1426785_s_at Mgll monoglyceride lipase 1.907 1420658_at Ucp3 uncoupling protein 3 (mitochondrial, proton carrier) 2.943 1425326_at Acly ATP citrate lyase 2.606 1460409_at Cpt1a carnitine palmitoyltransferase 1a, liver 2.753 1422677_at Dgat2 diacylglycerol O-acyltransferase 2 2.784 1425834_a_at Gpam glycerol-3-phosphate acyltransferase, mitochondrial 2.207 1417273_at Pdk4 pyruvate dehydrogenase kinase, isoenzyme 4 2.27 1449182_at Retn resistin 4.114 1435630_s_at Acat2 acetyl-Coenzyme A acetyltransferase 2 1.625 1425829_a_at Abcb1a ATP-binding cassette, sub-family B (MDR/TAP), member 10.322 1A 1423166_at Cd36 CD36 antigen 1.584 1422811_at Slc27a1 solute carrier family 27 (fatty acid transporter), member 1 3.58 1416023_at Fabp3 fatty acid binding protein 3, muscle and heart 1.833 1424155_at Fabp4 fatty acid binding protein 4, adipocyte 2.189 1431056_a_at Lpl lipoprotein lipase 1.659 1422432_at Dbi diazepam binding inhibitor 1.936 1422811_at Slc27a1 solute carrier family 27 (fatty acid transporter), 1 3.58 HEAT SHOCK RESPONSE 1448881_at Hp haptoglobin 1.679 1427126_at Hspa1b heat shock protein 1B 8.845 1438902_a_at Hsp90aa1 heat shock protein 90 kDa alpha (cytosolic), class A 1.513 member 1 1431274_a_at Hspa9a heat shock protein 9A 1.61 1416755_at Dnajb1 DnaJ (Hsp40) homolog, subfamily B, member 1 3.59 MISCELLANEOUS 1460256_at Car3 carbonic anhydrase 3 2.339 1415841_at Dync1i2 dynein cytoplasmic 1 intermediate chain 2 1.705 1432344_a_at Aplp2 amyloid beta (A4) precursor-like protein 2 1.937 1416429_a_at Cat catalase 1.82 1418306_at Crybb1 crystallin, beta B1 2.457 1448842_at Cdo1 cysteine dioxygenase 1, cytosolic 3.266 1453527_a_at Neurl neuralized-like homolog (Drosophila) 1.941 1451603_at Rtbdn retbindin 2.32 1453724_a_at Serpinf1 serine (or cysteine) peptidase inhibitor, clade F, member 1 7.765 1427285_s_at Surf4 surfeit gene 4 2.091 1424737_at Thrsp thyroid hormone responsive SPOT14 homolog (Rattus) 2.685 1431609_a_at Acp5 acid phosphatase 5, tartrate resistant 3.91 1448538_a_at D4Wsu53e DNA segment, Chr 4, Wayne State University 53, 1.586 expressed 1425552_at Hip1r huntingtin interacting protein 1 related 1.75 1429360_at Klf3 Kruppel-like factor 3 (basic) 1.901 1449413_at Mpv17l Mpv17 transgene, kidney disease mutant-like 1.988 1451667_at C530043G21Rik RIKEN cDNA C530043G21 gene 1.5 1425865_a_at Lig3 ligase III, DNA, ATP-dependent 2.693 1415994_at Cyp2e1 cytochrome P450, family 2, subfamily e, polypeptide 1 2.941 1417867_at Cfd complement factor D (adipsin) 2.828 1451015_at Tkt transketolase 2.256 1432344_a_at Aplp2 amyloid beta (A4) precursor-like protein 2 1.937 1419487_at Mybph Myosin binding protein H 1.578 MITOCHONDRIAL PROTEINS 1415897_a_at Mgst1 microsomal glutathione S-transferase 1 1.916 1423109_s_at Slc25a20 solute carrier family 25 (mitochondrial 1.865 carnitine/acylcarnitine translocase), member 20 OXYGEN CARRIERS 1448348_at Gpiap1 GPI-anchored membrane protein 1 1.83 1451203_at Mb myoglobin 1.578 1428361_x_at Hba-a1 hemoglobin alpha, adult chain 1 1.632 1417184_s_at Hbb-b2|Hbb-y hemoglobin, beta adult minor chain|hemoglobin Y, beta- 1.626 like embryonic chain SIGNAL TRANSDUCTION 1455918_at Adrb3 adrenergic receptor, beta 3 3.83 1452097_a_at Dusp7 dual specificity phosphatase 7 1.661 1419191_at Hipk3 homeodomain interacting protein kinase 3 1.694 1448152_at Igf2 insulin-like growth factor 2 1.635 1422313_a_at Igfbp5 insulin-like growth factor binding protein 5 1.772 1428265_at Ppp2r1b protein phosphatase 2 (formerly 2A), regulatory subunit A 2.509 (PR 65), beta isoform 1449342_at Ptplb protein tyrosine phosphatase-like (proline instead of 2.38 catalytic arginine), member b 1422119_at Rab5b RAB5B, member RAS oncogene family 1.603 1425444_a_at Tgfbr2 transforming growth factor, beta receptor II 2.13 1431164_at Rragd Ras-related GTP binding D 2.101 1420816_at Ywhag 3-monooxygenase/tryptophan 5-monooxygenase activation 1.87 protein, gamma polypeptide STEROID BIOGENESIS 1418601_at Aldh1a7 aldehyde dehydrogenase family 1, subfamily A7 3.862 1426225_at Rbp4 retinol binding protein 4, plasma 2.065 TRANSCRIPTION 1417794_at Zfp261 zinc finger protein 261 1.847 1424731_at Nle1 notchless homolog 1 (Drosophila) 1.831 1454791_a_at Rbbp4 retinoblastoma binding protein 4 2.865 1460281_at Asb15 ankyrin repeat and SOCS box-containing protein 15 1.78 1449363_at Atf3 activating transcription factor 3 1.802 1418982_at Cebpa CCAAT/enhancer binding protein (C/EBP), alpha 2.168 1417065_at Egr1 early growth response 1 2.577 1415899_at Junb Jun-B oncogene 1.792 1421554_at Lmx1a LIM homeobox transcription factor 1 alpha 4.106 1416959_at Nr1d2 nuclear receptor subfamily 1, group D, member 2(Reverb-b) 1.794 1450749_a_at Nr4a2 nuclear receptor subfamily 4, group A, member 2 (NURR1) 1.776 1460215_at Rpo1-4 RNA polymerase 1-4 2.498 1420892_at Wnt7b wingless-related MMTV integration site 7B 4.449 1423100_at Fos FBJ osteosarcoma oncogene 3.9 TRANSPORT PROTEINS 1425546_a_at Trf transferrin 1.907 1423743_at Arcn1 archain 1 1.617 1451771_at Tpcn1 two pore channel 1 2.842 1416629_at Slc1a5 solute carrier family 1 (neutral amino acid transporter), 1.939 member 5 1420295_x_at Clcn5 chloride channel 5 2.333 1417839_at Cldn5 claudin 5 1.545 1434617_x_at 1810073N04Rik RIKEN cDNA 1810073N04 gene 2.326 Data is average of N = 3 samples in each group (p < 0.05).

[0173] Surprisingly, the combination of GW1516 treatment and exercise established a unique gene expression pattern that was neither an amalgamation nor a complete overlap of the two interventions (FIG. 6). This unique signature included 48 new target genes (Table 2) not regulated by GW1516 and exercise alone and excluded 74 genes regulated by GW1516 or exercise alone (a selected few of which are shown in Table 3). This signature for the combination of GW1516 treatment and exercise (Table 2) was highly enriched in genes encoding regulatory enzymes for energy homeostasis, angiogenesis, oxygen transport, signal transduction, transcription and substrate transport, which are processes that are involved in endurance adaptation. Particularly, a predominance of genes involved in oxidative metabolism, is selectively up-regulated by combined exercise and drug treatment (see unbolded genes in Tables 1 and 2). In addition, several stress-related genes activated by either intervention, including heat shock proteins, metallothioneins and other stress biomarkers (Table 3) are not changed by the combination possibly reflecting a potential lessening of exercise-based damage.

TABLE-US-00003 TABLE 2 Gene targets unique to combined GW1516 treatment and exercise training. DESCRIPTION LOCUS GW + Tr ANGIOGENESIS CARBOHYDRATE METABOLISM phosphoenolpyruvate carboxykinase 1, cytosolic Pck1 3.518 CYTOKINES interferon, alpha-inducible protein 27 Ifi27 1.714 FAT METABOLISM adipose differentiation related protein Adrp 2.009 stearoyl-Coenzyme A desaturase 2 Scd2 1.849 acetyl-Coenzyme A acetyltransferase 2 Acat2 1.625 ATP citrate lyase Acly 2.606 adiponectin, C1Q and collagen domain containing Adipoq 3.082 diacylglycerol O-acyltransferase 2 Dgat2 2.784 lipase, hormone sensitive Lipe 3.032 monoglyceride lipase Mgll 1.907 resistin Retn 4.114 CD36 antigen Cd36 1.584 fatty acid binding protein 4, adipocyte Fabp4 2.189 lipoprotein lipase Lpl 1.659 HEAT SHOCK RESPONSE haptoglobin Hp 1.679 MITOCHONDRIAL PROTEINS microsomal glutathione S-transferase 1 Mgst1 1.916 OTHERS carbonic anhydrase 3 Car3 2.339 cysteine dioxygenase 1, cytosolic Cdo1 3.266 DNA segment, Chr 4, Wayne State University 53, expressed D4Wsu53e 1.586 dynein cytoplasmic 1 intermediate chain 2 Dync1i2 1.705 Kruppel-like factor 3 (basic) Klf3 1.901 thyroid hormone responsive SPOT14 homolog (Rattus) Thrsp 2.685 cytochrome P450, family 2, subfamily e, polypeptide 1 Cyp2e1 2.941 complement factor D (adipsin) Cfd 2.828 transketolase Tkt 2.256 OXYGEN CARRIERS GPI-anchored membrane protein 1 Gpiap1 1.83 PROLIFERATION & APOPTOSIS cell death-inducing DFFA-like effector c Cidec 4.771 SIGNAL TRANSDUCTION dual specificity phosphatase 7 Dusp7 1.661 homeodomain interacting protein kinase 3 Hipk3 1.694 insulin-like growth factor binding protein 5 Igfbp5 1.772 protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), beta Ppp2r1b 2.509 isoform protein tyrosine phosphatase-like (proline instead of catalytic arginine), Ptplb 2.38 member b STEROID BIOGENESIS retinol binding protein 4, plasma Rbp4 2.065 TRANSCRIPTION CCAAT/enhancer binding protein (C/EBP), alpha Cebpa 2.168 nuclear receptor subfamily 1, group D, member 2(Reverb-b) Nr1d2 1.794 TRANSPORT transferrin Trf 1.907 archain 1 Arcn1 1.617 solute carrier family 1 (neutral amino acid transporter), member 5 Slc1a5 1.939 RIKEN cDNA 1810073N04 gene 1810073N04Rik 2.326 Down-regulated genes are in bold italics. (N = 3, each pooled from 3 mice, p < 0.05).

TABLE-US-00004 TABLE 3 Gene targets regulated by GW1516 treatment or exercise training alone. FEATURE LOCUS DESCRIPTION GW Tr GW + Tr Hspb1 heat shock protein 1 1.815 1.965 -- 1451284_at Hspb7 heat shock protein family, 7 (cardiovascular) 3.414 1.753 -- 1422943_a_at Dnaja1 DnaJ (Hsp40) homolog, subfamily A, 1 -- 1.545 -- 1421290_at Hsp110 heat shock protein 110 -- 1.587 -- 1416288_at Serpinh1 serine (or cysteine) peptidase inhibitor, H, 1 -- 2.198 -- 1423566_a_at Dnaja4 DnaJ (Hsp40) homolog, subfamily A, 4 1.756 1.545 -- 1417872_at Mt1 metallothionein 1 2.364 -- -- 1424596_s_at Mt2 metallothionein 2 2.151 -- -- 1416157_at Cryab crystallin, alpha B 1.561 1.52 -- 1423139_at Crygf crystallin, gamma F 1.801 3.56 -- 1448830_at Smad3 MAD homolog 3 (Drosophila) 1.841 1.886 -- 1450637_a_at Ankrd1 ankyrin repeat domain 1 (cardiac muscle) 4.235 -- -- 1416029_at Tnfrsf12a TNF receptor superfamily, 12a 1.759 1.782 -- 1426464_at Jun Jun oncogene -- 1.521 -- Data is average of N = 3 samples in each group (p < 0.05)

[0174] Thirty-two percent of the GW+Tr-regulated genes encode enzymes of metabolic pathways such as fatty acid biosynthesis/storage (e.g., FAS, SCD 1 & 2), uptake [e.g., FAT/CD36, fatty acid binding proteins (FABP) and LPL] and oxidation [e.g., adiponectin, hormone sensitive lipase (HSL), PDK4, UCP3]; and carbohydrate metabolism [e.g., fructose bisphosphate 2 (FBP2), phosphoenolpyruvate carboxykinase 1 (PEPCK1), lactate dehydrogenase B], which along with oxygen transporters and mitochondrial proteins form the largest class of genes directly linked to muscle performance (Ikeda et al., Biochem. Biophys. Res. Commun. 296:395-400, 2002; Achten and Jeukendrup, Nutrition. 20:716-27, 2004; Hittel et al., J. Appl. Physiol. 98: 168-79, 2005; Civitarese et al., Cell Metab. 4:75-87, 2006; Nadeau et al., FASEB J. 17:1812-9, 2006; Kiens, Physiol. Rev. 86:205-43, 2006; Yamauchi et al., Nat. Med. 8:1288-95, 2006). Unexpectedly, established PPAR.alpha. target genes fatty acyl-CoA oxidase and medium chain acyl-CoA dehydrogenase (MCAD) were not represented in the signature. All but four of these metabolic genes were induced, which indicated a general increase in oxidative capacity of skeletal muscle in exercise-trained subjects that received GW1516 treatment.

[0175] Other genes regulated in quadriceps muscle by the combination of exercise and GW1516 treatment encoded proteins involved in pathways such as angiogenesis (e.g., angiopoietin-like 4 protein/also a known regulator of lipid metabolism), (e.g., adrenergic receptor .beta.3, insulin-like growth factor, insulin-like growth factor binding protein 5), transcription (e.g., C/EBP .alpha., Reverb .beta., NURR1) and substrate transport (e.g., transferrin, chloride channel 5) (Nagase et al., J. Clin. Invest. 97:2898-904, 1996; Singleton and Feldman, Neurobiol. Dis. 8:541-54, 2001; Adams, J. Appl. Physiol. 93:1159-67, 2002; Centrella et al., Gene. 342: 13-24, 2004; Lundby et al., Eur. J. Appl. Physiol. 96: 363-9, 2005; Mahoney et al., FASEB J. 19:1498-500, 2005; Mahoney et al., Phys. Med. Rehabil. Clin. N. Am. 16: 859-73, 2005; Ramakrishnan et al., J. Biol. Chem. 280:8651-9, 2005). Without wishing to be bound to a particular theory, such other genes are likely involved, at least in part, in muscle remodeling and increased endurance observed in GW1516-treated, exercise-trained subjects.

[0176] Interestingly, comparative expression analysis of the 48 gene subset of the endurance signature (Table 2), but not of either intervention alone, revealed a striking similarity to `untrained` VP16-PPAR.delta. transgenic mice. This observation confirms the primary dependence of the 48 genes on PPAR.delta. and indicates that exercise-generated signals may function to synergize PPAR.delta. transcriptional activity to levels comparable to transgenic over-expression. Therefore, exercise cues along with PPAR.delta. agonist may function to hyper-activate receptor transcriptional activity to re-program of adult muscle.

[0177] Genes and/or proteins uniquely affected (e.g., up-regulated or down-regulated or not substantially regulated) by exercise in the presence of one or more pharmaceutical agents (e.g., PPAR.delta. agonists) can be used as markers, for instance, of "drug doping" in exercise-trained subjects (e.g., athletes). It is expected that the unique set of 48 genes regulated by GW+Tr, but not GW1516 treatment or exercise training alone, can be used to identify exercised subjects who have received a variety performance-enhancing drugs.

Example 6

PPAR.delta. Directly Interacts with Exercise-Activated Kinases, p44/42 MAPK and AMPK

[0178] Exercise training is known to activate kinases, such as p44/42 MAPK and AMPK, which regulate gene expression in skeletal muscle (Chen et al., Diabetes, 52:2205-12, 2003; Goodyear et al., Am. J. Physiol., 271:E403-8, 1996). AMPK affects skeletal muscle gene expression and oxidative metabolism (Chen et al., Diabetes. 52: 2205-12, 2003, Reznick et al., J. Physiol. 574: 33-9, 2006). The interaction between exercise-regulated kinases and PPAR.delta. signaling is described in this Example.

[0179] The levels of phospho-p44/42 MAPK and phospho-AMPK .alpha. subunit and total AMPK were determined in protein homogenates of quadriceps muscle by Western blot. Antibodies specific for phospho-p44/42 MAPK, phospho- and total-AMPK .alpha.1 antibodies were obtained from Cell Signaling. The phospho-specific AMPK .alpha.1 antibody recognizes the key activating threonine in the activation loop.

[0180] Active forms of both kinases (phospho-p44/42 MAPK and phospho-AMPK .alpha. subunit) were expressed at higher levels in the quadriceps muscles of exercised mice relative to the sedentary controls (FIG. 7A). Previous reports claim that PPAR.delta. is not required for activation of AMPK by GW1516 in cultured cells (Kramer et al, Diabetes. 54(4):1157-63, 2005 and Kramer et al., J. Biol. Chem. 282(27):19313-2, 2007). In contrast, it was observed that GW1516 failed to activate p44/42 or AMPK in either sedentary or trained muscles, which indicated that PPAR.delta.-regulated effects are downstream to the exercise-induced signals that activate these kinases. Furthermore, AMPK appears to be constitutively active in muscles of VP16-PPAR.delta. transgenic mice in absence of exercise or drug (FIG. 7B). These results indicate that synergy is AMPK and PPAR.delta. co-dependent.

[0181] If synergy is AMPK and PPAR.delta. co-dependent, selective co-activation of AMPK and PPAR.delta. would induce gene expression changes that mimic those triggered by combined exercise and PPAR.delta. as well as VP16-PPAR.delta. over-expression. To demonstrate this, transcriptional changes induced in skeletal muscle by combined exercise and GW1516 treatment (as described in Example 5) were compared to that of combined AMPK activator (the cell permeable AMP analog AICAR; 250 mg/kg/day, i.p.) and GW1516 (5 mg/kg/day, oral gavage) treatment for 6 days. Genome analysis was performed using the methods described in Example 5.

[0182] Simultaneous GW1516 and AICAR treatment for 6 days created a unique gene expression signature in the quadriceps of untrained C57B1/6J mice (FIG. 8A, which includes target genes associated with translation, protein processing, amino acid metabolism, fat metabolism, oxygen carriers, carbohydrate metabolism, signal transduction, transcription, transport, steroid biogenesis, heat shock response, angiogenesis, proliferation and apoptosis, cytokines, contractile proteins, stress, and others) that shares 40% of the genes with that of combined GW1516 treatment and exercise (FIG. 8B). Classification of the 52 genes common to the two signatures (combined PPAR.delta. activation and exercise or PPAR.delta. and AMPK co-activation) (listed in Table 4) revealed that the majority of the targets were linked to oxidative metabolism.

TABLE-US-00005 TABLE 4 Targets common to exercise-PPAR.delta. and AMPK-PPAR.delta. gene signatures. DESCRIPTION LOCUS Tr + GW AI + GW ANGIOGENESIS angiopoietin-like 4 Angptl4 5.495 2.917 APOPTOSIS cell death-inducing DFFA-like effector c Cidec 4.771 1.838 cell death-inducing DNA fragmentation factor, alpha Cidea 49.625 1.842 subunit-like effector A CARBOHYDRATE METABOLISM lactate dehydrogenase B Ldhb 2.541 1.917 fructose bisphosphatase 2 Fbp2 2.808 2.478 FAT METABOLISM stearoyl-Coenzyme A desaturase 1 Scd1 6.494 1.78 fatty acid binding protein 3, muscle and heart Fabp3 1.833 1.5 pyruvate dehydrogenase kinase, isoenzyme 4 Pdk4 2.27 2.486 uncoupling protein 3 (mitochondrial, proton carrier) Ucp3 2.943 2.792 adiponectin, C1Q and collagen domain containin Adipoq 3.082 1.56 diacylglycerol O-acyltransferase 2 Dgat2 2.784 2.14 solute carrier family 27 (fatty acid transporter), member 1 Slc27a1 3.58 2.195 lipase, hormone sensitive Lipe 3.032 1.746 solute carrier family 25 (mitochondrial Slc25a20 1.704 1.697 carnitine/acylcarnitine translocase), member 20 CD36 antigen Cd36 1.584 1.513 phosphoenolpyruvate carboxykinase 1, cytosolic Pck1 3.518 1.781 fatty acid synthase Fasn 6.323 2.24 fatty acid binding protein 4, adipocyte Fabp4 2.189 1.81 monoglyceride lipase Mgll 1.907 1.51 acetyl-Coenzyme A acetyltransferase 2 Acat2 1.625 1.563 acetyl-Coenzyme A dehydrogenase, long-chain Acadl 2.549 1.992 resistin Retn 4.114 1.756 malonyl-CoA decarboxylase Mlycd 1.781 1.962 transketolase Tkt 2.256 1.983 ATP citrate lyase Acly 2.458 1.91 HEAT SHOCK heat shock protein 90 kDa alpha (cytosolic), class A member 1 Hsp90aa1 1.455 0.616 DnaJ (Hsp40) homolog, subfamily B, member 1 Dnajb1 3.59 0.604 CYTOKINES interferon, alpha-inducible protein 27 Ifi27 1.714 1.537 OTHER sarcolipin Sln 0.363 4.576 thyroid hormone responsive SPOT14 homolog (Rattus) Thrsp 2.685 1.766 RIKEN cDNA 2310076L09 gene 2310076L09Rik 1.868 2.117 myosin, heavy polypeptide 2, skeletal muscle, adult Myh2 2.194 1.797 surfeit gene 4 Surf4 2.091 0.654 acid phosphatase 5, tartrate resistant Acp5 3.91 1.477 serine (or cysteine) proteinase inhibitor, clade A, member 1a Serpina1a 0.396 3.891 cysteine dioxygenase 1, cytosolic Cdo1 3.266 1.678 erythroid differentiation regulator 1 0.619 1.805 RIKEN cDNA 1810073N04 gene 1810073N04Rik 2.326 1.628 superoxide dismutase 3, extracellular Sod3 1.606 1.617 complement factor D (adipsin) Cfd 2.828 1.5 cytochrome P450, family 2, subfamily e, polypeptide 1 Cyp2e1 2.941 1.743 catalase Cat 1.728 1.902 early growth response 1 Egr1 2.577 0.65 OXYGEN CARRIER hemoglobin, beta adult minor chain|hemoglobin Y, beta- Hbb-b2|Hbb-y 1.626 1.503 like embryonic chain STEROID BIOGENESIS retinol binding protein 4, plasma Rbp4 2.065 2.225 SIGNAL TRANSDUCTION adreneyrgic receptor, beta 3 Adrb3 3.83 1.56 protein tyrosine phosphatase-like (proline instead of catalytic Ptplb 2.38 1.569 arginine), member b dual specificity phosphatase 7 Dusp7 1.661 1.672 TRANSCRIPTION nuclear receptor subfamily 4, group A, member 2 Nr4a2 1.776 0.437 TRANSPORT solute carrier family 1 (neutral amino acid transporter), Slc1a5 1.939 1.511 member 5 two pore channel 1 Tpcn1 2.842 1.487 seminal vesicle secretion 5 Svs5 0.095 2.243 Data is average of N = 3 samples in each group (p < 0.05).

[0183] Quantitative expression analysis of selective oxidative genes (eight of those listed in Table 4) was determined in quadriceps of mice treated with vehicle (V), GW1516 (GW, 5 mg/kg/day), AICAR (AI, 250 mg/kg/day) and the combination of the two drugs (GW+AI) for 6 days using the methods described in Example 1. As shown in FIGS. 9A-H, several of these biomarkers including PDK4, SCD1, ATP citrate lyase, HSL, mFABP and LPL were induced in a synergistic fashion by GW1516 and AICAR in the quadriceps (FIGS. 9C-9H). Intriguingly, synergism was undetectable in UCP3 and mCPT I (FIGS. 9A and B). These genes were induced in quadriceps of untrained VP16-PPAR.delta. mice, where AMPK is constitutively active (Table 5).

TABLE-US-00006 TABLE 5 Selective oxidative genes induced in muscle by combined PPAR.delta. and AMPK activation as well as VP16-PPAR.delta. over-expression Description Locus GW + AI VP-PPAR.delta. ATP citrate lyase Acly 1.648 3.095 carnitine palmitoyltransferase 1b, Cpt1b 1.371 1.678 muscle fatty acid binding protein 3, muscle Fabp3 1.447 5.904 and heart fatty acid synthase Fasn 2.24 2.749 lipoprotein lipase Lpl 1.113 1.72 lipase, hormone sensitive Lipe 1.746 2.203 pyruvate dehydrogenase kinase, Pdk4 2.486 5.06 isoenzyme 4 stearoyl-Coenzyme A desaturase 1 Scd1 1.78 7.353 uncoupling protein 3 Ucp3 2.792 4.107

[0184] Collectively, these results demonstrate that while interaction between AMPK and PPAR.delta. may substantially contribute to re-programming of the skeletal muscle transcriptome during exercise, additional changes may involve cross-talk between other components of the exercise signaling network and PPAR.delta..

[0185] In summary, PPAR.delta. and exercise synergistically regulate running endurance. Although not bound by theory, kinase activation may influence PPAR.delta. signaling during exercise in establishing an "endurance gene expression signature" that effectively enhances performance.

Example 7

AMPK Increases Transcriptional Activation by PPAR.delta.

[0186] The genetic synergism described in Example 6 indicates that AMPK directly regulates the transcriptional activity of PPAR.delta. in skeletal muscles. To demonstrate this, an analysis of the effects of GW1516 and AICAR on gene expression in primary muscle cells isolated from wild type and PPAR.delta. null mice was performed.

[0187] Primary muscle cells were isolated from wild type and PPAR.delta. null mice as previously described (Rando and Blau, J. Cell. Biol. 125(6):1275-87, 1994). Skeletal muscle C2C12 cells were cultured in DMEM containing 20% serum and penicillin/streptomycin cocktail. For differentiation, cells at 80% confluence were switched to a differentiation medium (DMEM+2% serum) for 4 days to obtain differentiated myotubules. Cells were treated with vehicle, GW1516, AICAR, or GW1516+AICAR (GW: 0.1 .mu.M; AICAR: 500 .mu.M) for 24 hours. RNA expression of UCP3, PDK4, LPL, and HSL was determined using real time quantitative PCR as described in Example 1.

[0188] As shown in FIGS. 10A-D, synergism is dependent on PPAR.delta. and lost in the null cells. Similar synergistic regulation of gene expression by GW1516 and AICAR was also observed in differentiated C2C12 cells. These results show that AMPK activation may enhance ligand-dependent transcriptional effects of PPAR.delta. in muscles.

[0189] To more directly address this, reporter gene expression assays were utilized. AD 293 cells were cultured in DMEM containing 10% serum and an antibiotic cocktail. Cells were transfected with one or more of CMX-Flag, CMX-Flag PPAR.delta., CMX-Tk-PPRE, or CMX-.beta.GAL, or an hAMPK (.alpha.1 and .alpha.2 subunits, Origene) expression vector using Lipofectamine.TM. 2000 in accordance with the manufacturer's instructions. Anti-Flag antibody-conjugated beads were incubated overnight at 4.degree. C. with lysates from transfected cells. Flag-tagged protein or protein complexes were immunoprecipitated by separating the beads from non-bound materials. The beads were washed in ice-cold lysis buffer followed by extraction in Laemmli buffer. For co-immunoprecipitation experiments SDS was excluded from the lysis buffer. Western blotting was performed with antibodies specific for the Flag tag or AMPK .alpha. subunit(s).

[0190] Co-transfection of either catalytic AMPK .alpha.1 or .alpha.2 subunits, but not control vector, with PPAR.delta. increased the basal (FIG. 10E) and GW1516-dependent transcriptional activity (FIG. 10F) of PPAR.delta. in inducing a PPRE-driven reporter gene in AD293 cells. AMPK over-expression or GW1516 treatment did not change reporter activity in transfections excluding the PPAR.delta. expression vector negating the possibility of an effect via RXR. Additional results indicate that AMPK may modulate PPAR.delta. transcriptional activity by directly interacting with the receptor. In AD293 cells co-transfected with Flag-PPAR.delta. and with either catalytic AMPK .alpha.1 or .alpha.2 subunits, both of the subunits co-immunoprecipitated as a complex with Flag-PPAR.delta. (FIG. 10G). Furthermore, Flag-PPAR.delta. also co-immunoprecipitated endogenous AMPK.alpha. subunits from AD293 cells confirming a direct physical interaction between the nuclear receptor and the kinase (FIG. 10H). Despite physical interaction, AMPK failed to increase PPAR.delta. phosphorylation.

[0191] While potential AMPK phosphorylation sites were found in PPAR.delta., none of these sites were phosphorylated by AMPK in in vitro kinase assays. This was further confirmed by measuring the p32 labeling of PPAR.delta. in AD 293 cells in the presence or absence of AMPK. AD 293 cells were transfected with PPAR.delta. and hAMPk (.alpha.1 or .alpha.2 subunit) expression vectors as described above. Forty-eight hours after transfection, the cells were washed three times with phosphate-free DMEM and incubated with .sup.32P-orthophosphate in phosphate-free DMEM for 20 hours (100 .mu.Ci/5 ml). Cells were washed three times with ice-cold phosphate-free DMEM and lysed in ice-cold lysis buffer.

[0192] As shown in FIG. 10I, overall PPAR.delta. phosphorylation is not increased by AMPK in vivo. However, co-transfection of AMPK.alpha.2 and co-activator PGC1.alpha. (a known phosphorylation target of AMPK) co-operatively interact to further induce both the basal and ligand-dependent transcriptional activity of PPAR.delta. (FIG. 10J). Strikingly, no significant physical interaction between Flag-PGC1.alpha. and AMPK (FIG. 10K) was detected, both of which independently interacted with PPAR.delta.. Collectively, these observations indicate that AMPK may be present in a transcriptional complex with PPAR.delta. where it can potentiate receptor activity via direct protein-protein interaction and/or by phosphorylating co-activators such as PGC1.alpha..

[0193] These results indicate that AMPK directly interacts with PPAR.delta. and dramatically increases basal and ligand-dependent transcription via the receptor. Despite physical interaction, AMPK does not phosphorylate PPAR.delta.. AMPK and its substrate PGC1.alpha. synergistically increased PPAR.delta. transcription, indicating indirect regulation of receptor by AMPK via co-regulator modification.

[0194] The conclusion that exercise-activated AMPK interacts with PPAR.delta. in regulating gene expression in vivo is strengthened by the observation that treatment of animals with AICAR (AMPK activator) and GW1516 creates a gene signature in skeletal muscle that replicates up to 40% of the genetic effects of combined exercise and GW1516 treatment (see Table 4). Moreover, several candidate genes from this signature are synergistically induced by GW1516 and AICAR in wild type but not in PPAR.delta. null primary muscle cells, demonstrating that the interactive effects of the two drugs are mediated through PPAR.delta.. While 45% of the commonly regulated genes are linked to oxidative metabolism, additional common targets relevant to muscle performance include angiogenic, signal transduction and glucose sparing genes (Table 4). It is possible that the portion of the PPAR.delta.-exercise signature that is independent of PPAR.delta.-AMPK interaction (FIG. 8B) may depend on cross-talk between the receptor and other exercise signal transducers such as MAPK, calcineurin/NFAT and SIRT 1. These possibilities are summarized in FIG. 10L, where AMPK and additional components of the signaling network are proposed to interact with liganded PPAR.delta. to generate a muscle endurance gene signature and enhanced endurance adaptation.

[0195] In summary, it is shown herein that synthetic PPAR.delta. activation alone induces a set of genomic changes that fail to alter the preset muscle architecture and endurance levels in adult mice. However, the combination of PPAR.delta. activation with exercise brings about novel transcriptional changes, potentially via interaction with kinases such as AMPK (as depicted in FIG. 10L), re-setting the muscle transcriptome to a phenotype that dramatically enhances muscle performance.

Example 8

Enhancing Exercise Effect in a Subject

[0196] This example describes methods that can be used to increase or enhance an exercise in a healthy mammalian subject. Although specific conditions are described, one skilled in the art will appreciate that minor changes can be made to such conditions.

[0197] Healthy adult human subjects perform aerobic exercise (e.g., running) for at least 30 minutes (e.g., 30-90 minutes) for at least 3-4 days per week (e.g., 3-7 days per week) for at least 2 weeks (e.g., at least 4-12 weeks). The exercise is performed at 40%-50% maximal heart rate, 50%-60% maximal heart rate, 60%-70% maximal heart rate, or 75%-80% maximal heart rate, where maximum heart rate for a human subject is calculated as: 220 bps--(age of the subject).

[0198] During or after performing aerobic exercise as described above, the subjects are orally administered GW1516 [(2-methyl-4(((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)meth- yl)sulfanyl)phenoxy)acetic acid] at a dose of 1 to 20 mg per day, such as 2.5 or 10 mg per day. Subjects can continue to perform aerobic exercise while receiving GW1516. The subject can receive GW1516 for a period of at least 2 weeks, such as at least 4 weeks.

[0199] The exercise effect achieved in the treated subjects (e.g., running endurance) can be compared to such an effect in untreated subjects. Exercise effect can be measured using methods known in the art, such as measuring aerobic or running endurance (for example measuring distance run until exhaustion or amount of time to run a particular distance). In some instances, the exercise effect of interest is increased in treated subjects by at least 5%, such as at least 10% as compared to untreated subjects.

Example 9

Identifying Performance Enhancing Substances in an Exercise-Trained Subject

[0200] This example describes methods that can be used to identify performance-enhancing substances in an exercised-trained subject.

[0201] A biological sample obtained from a healthy adult human is analyzed to determine if the subject is taking a PES (e.g., GW1516) by analyzing expression of one or more of the molecules (nucleic acids or proteins) listed in Table 2 or Table 4. Suitable biological samples include samples containing genomic DNA or RNA (including mRNA) or proteins obtained from cells of a subject, such as those present in peripheral blood, urine, saliva, tissue biopsy, or buccal swab. For example, a biological sample of the subject can be assayed for a change in expression (such as an increase or decrease) of any combination of at least four molecules (nucleic acids or proteins) listed in Table 2 or 4, such as any combination of at least 10, at least 20, at least 30, or at least 40 of those listed in Table 2 or 4, for example all of those listed in Table 2 or 4.

Analyzing Nucleic Acid Molecules

[0202] Methods of isolating nucleic acid molecules from a biological sample are routine, for example using PCR to amplify the molecules from the sample, or by using a commercially available kit to isolate mRNA or cDNA. However, nucleic acids need not be isolated prior to analysis. Nucleic acids can be contacted with an oligonucleotide probe that will hybridize under stringent conditions with one or more nucleic acid molecule listed in Table 2 or 4. The nucleic acids which hybridize with the probe are then detected and quantified. The sequence of the oligonucleotide probe can bind specifically to a nucleic acid molecule represented by the sequences listed in Table 2 or 4.

[0203] Increased or decreased expression of the molecules listed in Table 2 or 4 can be detected by measuring the cellular levels of mRNA. mRNA can be measured using techniques well known in the art, including for instance Northern analysis, RT-PCR and mRNA in situ hybridization. Details of mRNA analysis procedures can be found, for instance, in provided examples and in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0204] Oligonucleotides specific to sequences listed in Table 2 or 4 can be chemically synthesized using commercially available machines. These oligonucleotides can then be labeled, for example with radioactive isotopes (such as .sup.32P) or with non-radioactive labels such as biotin (Ward and Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-57, 1981) or a fluorophore, and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis. These specific sequences are visualized, for example by methods such as autoradiography or fluorometric (Landegren et al., Science 242:229-37, 1989) or colorimetric reactions (Gebeyehu et al., Nucleic Acids Res. 15:4513-34, 1987).

Analyzing Proteins

[0205] Proteins in the biological sample can also be analyzed. In some examples, proteins are isolated using routine methods prior to analysis.

[0206] In one example, surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry is used to detect changes in differential protein expression, for example by using the ProteinChip.TM. (Ciphergen Biosystems, Palo Alto, Calif.). Such methods are well known in the art (for example see U.S. Pat. No. 5,719,060; U.S. Pat. No. 6,897,072; and U.S. Pat. No. 6,881,586). SELDI is a solid phase method for desorption in which the analyte is presented to the energy stream on a surface that enhances analyte capture or desorption. Therefore, in a particular example, the chromatographic surface includes antibodies that recognize proteins listed in Table 2 or 4. Antigens present in the sample can recognize the antibodies on the chromatographic surface. The unbound proteins and mass spectrometric interfering compounds are washed away and the proteins that are retained on the chromatographic surface are analyzed and detected by SELDI-TOF. The MS profile from the sample can be then compared using differential protein expression mapping, whereby relative expression levels of proteins at specific molecular weights are compared by a variety of statistical techniques and bioinformatic software systems.

[0207] In another examples, the availability of antibodies specific to the molecules listed in Table 2 or 4 facilitates the detection and quantification of proteins by one of a number of immunoassay methods that are well known in the art, such as those presented in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). Methods of constructing such antibodies are known in the art. Any standard immunoassay format (such as ELISA, Western blot, or RIA assay) can be used to measure protein levels. Immunohistochemical techniques can also be utilized for protein detection and quantification. For example, a tissue sample can be obtained from a subject, and a section stained for the presence of the desired protein using the appropriate specific binding agents and any standard detection system (such as one that includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in Bancroft and Stevens (Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

[0208] For the purposes of detecting or even quantifying protein or nucleic acid expression, expression in the test sample can be compared to levels found in cells from a subject who has not taken a PES. Alternatively, the pattern of expression identified in the test subject can be compared to that shown in Table 2 or 4.

[0209] For example, if the test sample shows a pattern of expression similar to that in Table 2 or 4 (e.g., the genes shown as upregulated and downregulated in Table 2 or 4 are observed in the subject to be upregulated and downregulated, respectively), this indicates that the subject is taking a PES, such as a PPAR.delta. agonist (e.g., GW1516). In contrast, If the pattern of expression identified in the test subject is different to that shown in Table 2 or 4 (e.g., the genes shown as upregulated and downregulated in Table 2 or 4 are observed in the subject to be not differentially expressed or show a different pattern of regulation), this indicates that the subject is not taking a PES, such as a PPAR.delta. agonist (e.g., GW1516).

[0210] A significant increase in the non-bolded proteins listed in Table 2 in the cells of a test subject compared to the amount of the same protein found in normal human cells is usually at least 2-fold, at least 3-fold, at least 4-fold or greater difference. Substantial overexpression of the non-bolded proteins listed in Table 2 in the subject's sample can be indicative of the subject taking a PES. Similarly, a significant decrease in the bolded proteins listed in Table 2 in the cells of a test subject compared to the amount of the same protein found in normal human cells is usually at least 2-fold, at least 3-fold, at least 4-fold or greater difference. Substantial underexpression of the bolded proteins listed in Table 2 in the subject's sample can be indicative of the subject taking a PES.

[0211] While this disclosure has been described with an emphasis upon particular embodiments, it will be obvious to those of ordinary skill in the art that variations of the particular embodiments may be used and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the following claims:

Sequence CWU 1

1

171440PRTMus musculus 1Met Glu Gln Pro Gln Glu Glu Thr Pro Glu Ala Arg Glu Glu Glu Lys 1 5 10 15 Glu Glu Val Ala Met Gly Asp Gly Ala Pro Glu Leu Asn Gly Gly Pro 20 25 30 Glu His Thr Leu Pro Ser Ser Ser Cys Ala Asp Leu Ser Gln Asn Ser 35 40 45 Ser Pro Ser Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly Ala 50 55 60 Ser Gly Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp Lys Ala 65 70 75 80 Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe 85 90 95 Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Asp Arg 100 105 110 Ile Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg 115 120 125 Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg Phe 130 135 140 Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Leu Thr 145 150 155 160 Ala Ser Glu Gly Cys Gln His Asn Pro Gln Leu Ala Asp Leu Lys Ala 165 170 175 Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met Thr 180 185 190 Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ser Ser His Asn Ala 195 200 205 Pro Phe Val Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys Gly 210 215 220 Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Asn Glu Ile 225 230 235 240 Ser Val His Val Phe Tyr Arg Cys Gln Ser Thr Thr Val Glu Thr Val 245 250 255 Arg Glu Leu Thr Glu Phe Ala Lys Asn Ile Pro Asn Phe Ser Ser Leu 260 265 270 Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu Ala 275 280 285 Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Leu Val 290 295 300 Ala Asn Gly Ser Gly Phe Val Thr His Glu Phe Leu Arg Ser Leu Arg 305 310 315 320 Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val Lys 325 330 335 Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile Ala 340 345 350 Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro Gln 355 360 365 Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu Glu Phe His Leu 370 375 380 Gln Val Asn His Pro Asp Ser Gln Tyr Leu Phe Pro Lys Leu Leu Gln 385 390 395 400 Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met Met 405 410 415 Gln Trp Leu Lys Lys Thr Glu Ser Glu Thr Leu Leu His Pro Leu Leu 420 425 430 Gln Glu Ile Tyr Lys Asp Met Tyr 435 440 23240DNAMus musculus 2gccgcaggcc gcggcggacc tggggattaa tgggaaaagt tttggcagga gctgggggat 60tctgcggagc ctgcgggacg gcggcagcgg cgcgagaggc ggccgggaca gtgctgtgca 120gcggtgtggg tatgcgcatg ggactcactc agaggctcct gctcactgac agatgaagac 180aaacccacgg taaaggcagt ccatctgcgc tcagacccag atggtggcag agctatgacc 240aggcctgcag gcgccacgcc aagtgggggt cagtcatgga acagccacag gaggagaccc 300ctgaggcccg ggaagaggag aaagaggaag tggccatggg tgacggagcc ccggagctca 360atgggggacc agaacacacg cttccttcca gcagctgtgc agacctctcc cagaattcct 420ccccttcctc cctgctggac cagctgcaga tgggctgtga tggggcctca ggcggcagcc 480tcaacatgga atgtcgggtg tgcggggaca aggcctcggg cttccactac ggggtccacg 540cgtgcgaggg gtgcaagggc ttcttccgcc ggacaatccg catgaagctc gagtatgaga 600agtgcgatcg gatctgcaag atccagaaga agaaccgcaa caagtgtcag tactgccgct 660tccagaagtg cctggcactc ggcatgtcgc acaacgctat ccgctttgga cggatgccgg 720aggccgagaa gaggaagctg gtggcggggc tgactgccag cgaggggtgc cagcacaacc 780cccagctggc cgacctgaag gccttctcta agcacatcta caacgcctac ctgaaaaact 840tcaacatgac caaaaagaag gcccggagca tcctcaccgg caagtccagc cacaacgcac 900cctttgtcat ccacgacatc gagacactgt ggcaggcaga gaagggcctg gtgtggaaac 960agctggtgaa cgggctgccg ccctacaacg agatcagtgt gcacgtgttc taccgctgcc 1020agtccaccac agtggagaca gtccgagagc tcaccgagtt cgccaagaac atccccaact 1080tcagcagcct cttcctcaat gaccaggtga ccctcctcaa gtatggcgtg cacgaggcca 1140tctttgccat gctggcctcc atcgtcaaca aagacgggct gctggtggcc aacggcagtg 1200gcttcgtcac ccacgagttc ttgcgaagtc tccgcaagcc cttcagtgac atcattgagc 1260ccaagttcga gtttgctgtc aagttcaatg cgctggagct cgatgacagt gacctggcgc 1320tcttcatcgc ggccatcatt ctgtgtggag accggccagg cctcatgaat gtgccccagg 1380tagaagccat ccaggacacc attctgcggg ctctagaatt ccatctgcag gtcaaccacc 1440ctgacagcca gtacctcttc cccaagctgc tgcagaagat ggcagacctg cggcagctgg 1500tcactgagca tgcccagatg atgcagtggc taaagaagac ggagagtgag accttgctgc 1560accccctgct ccaggaaatc tacaaggaca tgtactaagg ccgcagccca ggcctcccct 1620caggctctgc tgggcccagc cacggactgt tcagaggacc agccacaggc actggcagtc 1680aagcagctag agcctactca caacactcca gacacgtggc ccagactctt cccccaacac 1740ccccaccccc accaaccccc ccattccccc aacccccctc ccccaccccg ctctccccat 1800ggcccgtttc ctgtttctcc tcagcacctc ctgttcttgc tgtctcccta gcgcccttgc 1860tccccccctt tgccttcctt ctctagcatc cccctcctcc cagtcctcac atttgtctga 1920ttcacagcag acagcccgtt ggtacgctca ccagcagcct aaaagcagtg gggcctgtgc 1980tggcccagtc ctgcctctcc tctctatccc cttcaaagac atgagccatc caaagaaaca 2040ctacgctctc tctgggccca gctttccaag aagcctggcc tggaccaact gccatcccag 2100cttgtggtca ccaccacagg gttcctcctc cagagagcaa gtgggcaggg agcctgggcc 2160gggagccata ttcccaggct gtctcagccc taggcacacc actctctgac acttcctttc 2220ttctcgccgg cgtcctaggt cattgtcaca gatgaccctt gtgctgccta ggagatgacc 2280cctccagatg tcccctccag atgcggtcca acggccccac tgaagggaag ggggtagagg 2340caggccggaa ggagcagcgg cacacttagg tcccagggtc agaagctaga cagcgagtgg 2400gcaggccctc catcagcacc cctcctctac cctgtagcag catccagact ggcagatccc 2460agtaccagga actggaccat agctgttctt tcttctcctg ggagatgctg gcacacctgc 2520cccccccccc cccttgcagc tgccccggtg tagccatgac actggctcac ctctcggtca 2580ccacagagtc cctcccattc cctccccaag gccactgggg tacagctatg gccctgttct 2640taggactggt gatctgtgag caggcaggga tatcctacca ggtcacccct gccagctcac 2700aggcagagtt gctagggttc ctctgaccct gtcctctctc ccactcactt gtaccagtag 2760ctctgtggcc ttctcttctt ttgcctggct ggtcacctgc tcccatctgc tgcttcaagt 2820ggcttgaaac ttgctgggtg ctcccatact cagccccagc ccggcagatc ctgcctctag 2880gcccataggt gatcagccca ggctcggctc ctgccaacac agaatgctgc cagattcccc 2940gctagcacac tccctgcccc tcacctctac tgatcaggtc ttggggtgtt ccttgtgggg 3000cccacccagg ctgagaatgg agctacatca cccgccctgc ccccacctgc ccagccccgc 3060ccaggtctgg tgctgaggat gcagctcctc tcagggtctg aagtctccaa atctgaaatg 3120tatatttttg ctaggagccc cagcttcccg tgtttttaat ataaatagtg tatacagact 3180gacggaactt taaataaatg ggaattacgt atttaagaaa aaaaaaaaaa aaaaaaaaaa 32403441PRTHomo sapiens 3Met Glu Gln Pro Gln Glu Glu Ala Pro Glu Val Arg Glu Glu Glu Glu 1 5 10 15 Lys Glu Glu Val Ala Glu Ala Glu Gly Ala Pro Glu Leu Asn Gly Gly 20 25 30 Pro Gln His Ala Leu Pro Ser Ser Ser Tyr Thr Asp Leu Ser Arg Ser 35 40 45 Ser Ser Pro Pro Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly 50 55 60 Ala Ser Cys Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp Lys 65 70 75 80 Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly 85 90 95 Phe Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Glu 100 105 110 Arg Ser Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys 115 120 125 Arg Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg 130 135 140 Phe Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Leu 145 150 155 160 Thr Ala Asn Glu Gly Ser Gln Tyr Asn Pro Gln Val Ala Asp Leu Lys 165 170 175 Ala Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met 180 185 190 Thr Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ala Ser His Thr 195 200 205 Ala Pro Phe Val Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys 210 215 220 Gly Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu 225 230 235 240 Ile Ser Val His Val Phe Tyr Arg Cys Gln Cys Thr Thr Val Glu Thr 245 250 255 Val Arg Glu Leu Thr Glu Phe Ala Lys Ser Ile Pro Ser Phe Ser Ser 260 265 270 Leu Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu 275 280 285 Ala Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Leu 290 295 300 Val Ala Asn Gly Ser Gly Phe Val Thr Arg Glu Phe Leu Arg Ser Leu 305 310 315 320 Arg Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val 325 330 335 Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile 340 345 350 Ala Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro 355 360 365 Arg Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu Glu Phe His 370 375 380 Leu Gln Ala Asn His Pro Asp Ala Gln Tyr Leu Phe Pro Lys Leu Leu 385 390 395 400 Gln Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met 405 410 415 Met Gln Arg Ile Lys Lys Thr Glu Thr Glu Thr Ser Leu His Pro Leu 420 425 430 Leu Gln Glu Ile Tyr Lys Asp Met Tyr 435 440 43734DNAHomo sapiens 4gcggagcgtg tgacgctgcg gccgccgcgg acctggggat taatgggaaa agttttggca 60ggagcgggag aattctgcgg agcctgcggg acggcggcgg tggcgccgta ggcagccggg 120acagtgttgt acagtgtttt gggcatgcac gtgatactca cacagtggct tctgctcacc 180aacagatgaa gacagatgca ccaacgaggc tgatgggaac caccctgtag aggtccatct 240gcgttcagac ccagacgatg ccagagctat gactgggcct gcaggtgtgg cgccgagggg 300agatcagcca tggagcagcc acaggaggaa gcccctgagg tccgggaaga ggaggagaaa 360gaggaagtgg cagaggcaga aggagcccca gagctcaatg ggggaccaca gcatgcactt 420ccttccagca gctacacaga cctctcccgg agctcctcgc caccctcact gctggaccaa 480ctgcagatgg gctgtgacgg ggcctcatgc ggcagcctca acatggagtg ccgggtgtgc 540ggggacaagg catcgggctt ccactacggt gttcatgcat gtgaggggtg caagggcttc 600ttccgtcgta cgatccgcat gaagctggag tacgagaagt gtgagcgcag ctgcaagatt 660cagaagaaga accgcaacaa gtgccagtac tgccgcttcc agaagtgcct ggcactgggc 720atgtcacaca acgctatccg ttttggtcgg atgccggagg ctgagaagag gaagctggtg 780gcagggctga ctgcaaacga ggggagccag tacaacccac aggtggccga cctgaaggcc 840ttctccaagc acatctacaa tgcctacctg aaaaacttca acatgaccaa aaagaaggcc 900cgcagcatcc tcaccggcaa agccagccac acggcgccct ttgtgatcca cgacatcgag 960acattgtggc aggcagagaa ggggctggtg tggaagcagt tggtgaatgg cctgcctccc 1020tacaaggaga tcagcgtgca cgtcttctac cgctgccagt gcaccacagt ggagaccgtg 1080cgggagctca ctgagttcgc caagagcatc cccagcttca gcagcctctt cctcaacgac 1140caggttaccc ttctcaagta tggcgtgcac gaggccatct tcgccatgct ggcctctatc 1200gtcaacaagg acgggctgct ggtagccaac ggcagtggct ttgtcacccg tgagttcctg 1260cgcagcctcc gcaaaccctt cagtgatatc attgagccta agtttgaatt tgctgtcaag 1320ttcaacgccc tggaacttga tgacagtgac ctggccctat tcattgcggc catcattctg 1380tgtggagacc ggccaggcct catgaacgtt ccacgggtgg aggctatcca ggacaccatc 1440ctgcgtgccc tcgaattcca cctgcaggcc aaccaccctg atgcccagta cctcttcccc 1500aagctgctgc agaagatggc tgacctgcgg caactggtca ccgagcacgc ccagatgatg 1560cagcggatca agaagaccga aaccgagacc tcgctgcacc ctctgctcca ggagatctac 1620aaggacatgt actaacggcg gcacccaggc ctccctgcag actccaatgg ggccagcact 1680ggaggggccc acccacatga cttttccatt gaccagccct tgagcacccg gcctggagca 1740gcagagtccc acgatcgccc tcagacacat gacacccacg gcctctggct ccctgtgccc 1800tctctcccgc ttcctccagc cagctctctt cctgtctttg ttgtctccct ctttctcagt 1860tcctctttct tttctaattc ctgttgctct gtttcttcct ttctgtaggt ttctctcttc 1920ccttctccct tgccctccct ttctctctcc accccccacg tctgtcctcc tttcttattc 1980tgtgagatgt tttgtattat ttcaccagca gcatagaaca ggacctctgc ttttgcacac 2040cttttcccca ggagcagaag agagtggggc ctgccctctg ccccatcatt gcacctgcag 2100gcttaggtcc tcacttctgt ctcctgtctt cagagcaaaa gacttgagcc atccaaagaa 2160acactaagct ctctgggcct gggttccagg gaaggctaag catggcctgg actgactgca 2220gccccctata gtcatggggt ccctgctgca aaggacagtg ggcaggaggc cccaggctga 2280gagccagatg cctccccaag actgtcattg cccctccgat gctgaggcca cccactgacc 2340caactgatcc tgctccagca gcacacctca gccccactga cacccagtgt ccttccatct 2400tcacactggt ttgccaggcc aatgttgctg atggccccct gcactggccg ctggacggca 2460ctctcccagc ttggaagtag gcagggttcc ctccaggtgg gcccccacct cactgaagag 2520gagcaagtct caagagaagg aggggggatt ggtggttgga ggaagcagca cacccaattc 2580tgcccctagg actcggggtc tgagtcctgg ggtcaggcca gggagagctc ggggcaggcc 2640ttccgccagc actcccactg cccccctgcc cagtagcagc cgcccacatt gtgtcagcat 2700ccagggccag ggcctggcct cacatccccc tgctcctttc tctagctggc tccacgggag 2760ttcaggcccc actccccctg aagctgcccc tccagcacac acacataagc actgaaatca 2820ctttacctgc aggctccatg cacctccctt ccctccctga ggcaggtgag aacccagaga 2880gaggggcctg caggtgagca ggcagggctg ggccaggtct ccggggaggc aggggtcctg 2940caggtcctgg tgggtcagcc cagcacctgc tcccagtggg agcttcccgg gataaactga 3000gcctgttcat tctgatgtcc atttgtccca atagctctac tgccctcccc ttccccttta 3060ctcagcccag ctggccacct agaagtctcc ctgcacagcc tctagtgtcc ggggaccttg 3120tgggaccagt cccacaccgc tggtccctgc cctcccctgc tcccaggttg aggtgcgctc 3180acctcagagc agggccaaag cacagctggg catgccatgt ctgagcggcg cagagccctc 3240caggcctgca ggggcaaggg gctggctgga gtctcagagc acagaggtag gagaactggg 3300gttcaagccc aggcttcctg ggtcctgcct ggtcctccct cccaaggagc cattctgtgt 3360gtgactctgg gtggaagtgc ccagcccctg cccctacggg cgctgcagcc tcccttccat 3420gccccaggat cactctctgc tggcaggatt cttcccgctc cccacctacc cagctgatgg 3480gggttggggt gcttcctttc aggccaaggc tatgaaggga cagctgctgg gacccacctc 3540cccctccccg gccacatgcc gcgtccctgc cccgacccgg gtctggtgct gaggatacag 3600ctcttctcag tgtctgaaca atctccaaaa ttgaaatgta tatttttgct aggagcccca 3660gcttcctgtg tttttaatat aaatagtgta cacagactga cgaaacttta aataaatggg 3720aattaaatat ttaa 37345440PRTRattus norvegicus 5Met Glu Gln Pro Gln Glu Glu Thr Pro Glu Ala Arg Glu Glu Glu Lys 1 5 10 15 Glu Glu Val Ala Thr Gly Asp Gly Ala Pro Glu Leu Asn Gly Gly Pro 20 25 30 Glu His Thr Leu Pro Ser Ser Ser Cys Thr Asp Leu Ser Gln Asn Ser 35 40 45 Ser Pro Ser Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly Ala 50 55 60 Ser Gly Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp Lys Ala 65 70 75 80 Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe 85 90 95 Phe Arg Arg Thr Ile Arg Met Lys Leu Lys Tyr Glu Lys Cys Asp Arg 100 105 110 Ile Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg 115 120 125 Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg Phe 130 135 140 Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Leu Thr 145 150 155 160 Ala Ser Glu Gly Cys Gln Gln Asn Pro Gln Leu Ala Asp Leu Lys Ala 165 170 175 Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met Thr 180 185 190 Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ser Ser His Asn Ala 195 200 205 Pro Phe Ile Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys Gly 210 215 220 Leu Val Trp Lys Gln Leu Val Asn Gly Pro Pro Pro Tyr Asn Glu Ile 225 230 235 240 Ser Val His Val Phe Tyr Arg Cys Gln Ser Thr Thr Val Glu Thr Val 245 250 255 Arg Glu Leu Thr Glu Phe Ala Lys Asn Ile Pro Asn Phe Ser Ser Leu 260 265 270 Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu Ala 275 280 285 Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Leu Val 290 295 300 Ala Asn Gly Ser Gly Phe Val Thr His Glu Phe Leu Arg Ser Ile Arg 305 310 315 320 Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val Lys 325 330 335 Phe Asn

Ala Leu Glu Leu Val Asp Ser Asp Leu Ala Leu Phe Ile Ala 340 345 350 Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro Gln 355 360 365 Val Glu Ala Ile Gln Asp Thr Ile Leu Gln Ala Leu Glu Phe His Leu 370 375 380 Gln Val Asn His Pro Asp Ser Gln Tyr Leu Phe Pro Lys Leu Leu Gln 385 390 395 400 Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met Met 405 410 415 Gln Trp Leu Lys Lys Thr Glu Ser Glu Thr Leu Leu His Pro Leu Leu 420 425 430 Gln Glu Ile Tyr Lys Asp Met Tyr 435 440 62054DNARattus norvegicus 6ggcacgagct gggggattct gcggagcgtg cgggacggcg gcggcggcgg cggcggcggc 60ggcggcggcg gcgggagagg cggctggaac agcgctgtgc agcagcgggg ttatgcgtgt 120gggactcgcc cagaggctcc tgctcactga cagatgagga caaacccacg gtaaaggcgg 180tccatctgcg ctcagaccca gatggtggca gagctatgac caggcctgca ggcgccacgc 240caagtggggt cagtcatgga acagccacag gaggagaccc ctgaggcccg ggaagaggag 300aaagaggaag tggccacggg tgacggagcc ccagagctca acgggggacc agagcacacc 360cttccttcca gcagctgcac agacctctcc cagaattcct ccccttcctc cctgctggac 420cagctgcaga tgggctgtga tggggcctca ggtggcagcc tcaacatgga gtgccgggtg 480tgcggagaca aggcctcagg cttccactac ggggtccacg cgtgtgaggg gtgcaagggc 540ttcttccgcc ggacaatccg catgaagctc aagtacgaga agtgcgatcg gatctgcaag 600atccagaaga agaaccgcaa caagtgtcag tactgccgct tccagaagtg cctggcgctc 660ggcatgtccc acaacgctat ccgctttgga aggatgccgg aggccgagaa gaggaagctg 720gtggcggggc tgacggccag cgagggatgc cagcaaaacc cccagctggc cgacctgaag 780gccttctcca agcacatcta caatgcctac ctgaaaaact tcaacatgac caaaaagaag 840gcccggagca tcctcactgg caagtccagc cataacgcac ccttcatcat ccacgacatt 900gagacgctgt ggcaggcaga gaagggcctg gtgtggaagc agctggtgaa cgggccgccg 960ccctacaacg agatcagcgt gcatgtgttc taccgctgcc agtccaccac cgtggagaca 1020gtgcgggagc tcaccgagtt cgccaagaac atccccaact tcagcagcct cttcctcaac 1080gaccaggtga ccctcctcaa gtacggcgtg catgaggcca tctttgccat gctggcctcc 1140attgtcaaca aagacggact gctggtggcc aacggcagtg gcttcgtcac ccatgagttc 1200ttgcgcagta tccgcaagcc cttcagtgac atcattgagc ccaagttcga gtttgctgtc 1260aagttcaatg ctctggagct ggtcgacagt gatctggccc ttttcattgc cgccatcatt 1320ctgtgcggag accggccagg cctcatgaac gtgccacagg tggaagccat ccaggacacc 1380atcctgcagg ctctagaatt ccatctgcag gtcaaccacc ccgacagcca gtacctcttc 1440cccaagctgc tgcagaaaat ggccgacctg cggcagctgg tcactgaaca cgcgcagatg 1500atgcagtggc tgaagaagac ggagagtgag accttgctgc accccctgct ccaggagatc 1560tacaaggaca tgtactaagg ctgcacgcag ccagcctccc gcagctccgc tgggcccagc 1620cacggactgt tcagaggacc cgcccacagg cactggccac agcccacgca gctagagcca 1680ctcacaacac tccagacacg gcccagactc tcaccctctc cgcccgccct cggcacccgg 1740ttctccccag cacttcctgt tcatgctgtc tccccagcac ccttgctcct ccacctggcc 1800ttctctagca tcctgcgcct ccccgcctgt ccccacatct gtctgattca cgccagtgag 1860cccattagtc cgctcaccag cagcctagaa gcagtgaggc ctgcactggc ccggccctgc 1920ctgtctctgt cccctcttca aggacatgag ccatccaaag aaacactatg ttctctctga 1980gtccgacttt ccaagaaact tgcctggact gactgccatc ccaggctgtc tgaaccctag 2040gcacccctcg tgcc 20547443PRTGallus gallus 7Met Glu Gln Leu Gln Glu Glu Val Pro Glu Val Arg Glu Glu Glu Glu 1 5 10 15 Glu Glu Glu Glu Ala Val Thr Val Pro Ser Gly Ala Ser Asp Pro Ser 20 25 30 Ala Gly Pro Asp Ser Ser Leu Pro Ser Ser Ser Tyr Thr Asp Leu Ser 35 40 45 Gln Ser Ser Ser Pro Ser Leu Ser Asp Gln Leu Gln Met Gly Cys Glu 50 55 60 Glu Thr Ala Ser Gly Ala Leu Asn Val Glu Cys Arg Val Cys Gly Asp 65 70 75 80 Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys 85 90 95 Gly Phe Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys 100 105 110 Glu Arg Ser Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr 115 120 125 Cys Arg Phe Gln Lys Cys Leu Ser Leu Gly Met Ser His Asn Ala Ile 130 135 140 Arg Phe Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly 145 150 155 160 Leu Thr Ala Ser Glu Ile Ser Cys Gln Asn Pro Gln Val Ala Asp Leu 165 170 175 Lys Ala Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn 180 185 190 Met Thr Lys Lys Lys Ala Arg Gly Ile Leu Thr Gly Lys Ala Ser Ser 195 200 205 Thr Pro Gln Pro Phe Val Ile His Asp Met Asp Thr Leu Trp Gln Ala 210 215 220 Glu Lys Gly Leu Val Trp Lys Gln Leu Val Asn Gly Ile Pro Pro Tyr 225 230 235 240 Lys Glu Ile Gly Val His Val Phe Tyr Arg Cys Gln Cys Thr Thr Val 245 250 255 Glu Thr Val Arg Glu Leu Thr Glu Phe Ala Lys Ser Ile Pro Ser Phe 260 265 270 Ile Gly Leu Tyr Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val 275 280 285 His Glu Ala Ile Phe Ala Met Leu Ala Ser Ile Met Asn Lys Asp Gly 290 295 300 Leu Leu Val Ala Asn Gly Asn Gly Phe Val Thr Arg Glu Phe Leu Arg 305 310 315 320 Thr Leu Arg Lys Pro Phe Asn Glu Ile Met Glu Pro Lys Phe Glu Phe 325 330 335 Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ser Leu 340 345 350 Phe Val Ala Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn 355 360 365 Val Lys Gln Val Glu Glu Ile Gln Asp Asn Ile Leu Arg Ala Leu Glu 370 375 380 Phe His Leu Gln Ser Asn His Pro Asp Ala Gln Tyr Leu Phe Pro Lys 385 390 395 400 Leu Leu Gln Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala 405 410 415 Gln Leu Val Gln Lys Ile Lys Lys Thr Glu Thr Glu Thr Ser Leu His 420 425 430 Pro Leu Leu Gln Glu Ile Tyr Lys Asp Met Tyr 435 440 81756DNAGallus gallus 8cgggagcggg ccatcccggg agatcgcttt ggtccccggc tccccgtgct gacatggtaa 60cggaacggcg gctctcagcg gcgggcagac agggacagct gcggagaggc tagtgcaact 120ggcagggtcc atctcggata gacgcggatg atgccagagc tatgacaggg attgcagtct 180tggcactgag gggtgatcag ttatggaaca actacaggag gaagtacctg aggtcaggga 240agaggaggag gaagaggaag aggcagtgac agtgcccagc ggagcctcgg acccaagtgc 300aggaccagac agctcgctgc cctcgagcag ctacaccgac ctttcgcaga gctcctctcc 360ctccctgtca gaccaactgc agatgggctg cgaggagacg gcctcggggg cactgaatgt 420ggagtgcaga gtctgtggag acaaagcctc gggcttccac tacggtgtgc acgcctgcga 480gggctgcaag ggtttcttcc gccggacgat ccgcatgaag ctggaatatg agaagtgtga 540gcgaagctgc aagattcaga agaagaaccg gaataagtgc cagtactgcc gcttccagaa 600atgcctctcg ttgggcatgt cacataacgc aatccgcttt ggccgcatgc cggaggcaga 660gaagaggaag ctggtagcgg ggctgacagc aagcgagatc agctgccaga acccacaggt 720ggccgacctg aaagctttct ccaagcacat ctacaacgcc tacctgaaaa acttcaacat 780gaccaaaaag aaagcgagag gtatcttgac cgggaaggcc agcagcaccc cacagccttt 840tgtgatccat gacatggaca ccctgtggca agcagaaaag gggctggtct ggaaacagct 900ggtgaatggg atccccccct acaaggagat cggggtgcac gtcttctacc gctgccagtg 960cactacagta gaaactgtgc gggagctcac cgagtttgcc aagagcatcc ccagcttcat 1020aggcctctac ttgaatgacc aagtgactct gctgaagtat ggggtgcacg aggccatctt 1080tgccatgctg gcctctatca tgaacaagga tgggctgctg gtggccaacg ggaatggctt 1140tgtgacccgt gagttcctgc gtacgttgcg caagcccttc aacgagatca tggagcccaa 1200gtttgagttt gctgtgaagt tcaacgcact ggagctggat gacagcgacc tgtctctgtt 1260tgtggctgcc attatcctgt gtggagatcg tcccggcctg atgaacgtga agcaggtgga 1320ggagatccaa gacaacatcc tccgagcact ggagttccac ctgcagtcca accacccgga 1380cgcccagtac ctcttcccca agttgctgca gaagatggct gacctgcggc agctggtgac 1440agagcacgcc cagctggtgc agaagatcaa gaagacagag acagagacct ctctgcaccc 1500gcttctgcag gagatctaca aggacatgta ctaaggacct cttatttatg agaatgaagc 1560catggattcc aagcgagact ctttgtacaa aaacaaagaa aacctttccc catgtcttcc 1620atttcttcta ctggaaactc ttttctacat gactccaatg tacggtacac agggcaaggt 1680gctgtaaggt attgcagcca cctcctggta gcttgggact tccattaaca cactaacgaa 1740tttacaaagg caaatc 175695130DNAHomo sapiens 9agcgccatgc gcagactcag ttcctggaga aagatggcga cagccgagaa gcagaaacac 60gacgggcggg tgaagatcgg ccactacatt ctgggtgaca cgctgggggt cggcaccttc 120ggcaaagtga aggttggcaa acatgaattg actgggcata aagtagctgt gaagatactc 180aatcgacaga agattcggag ccttgatgtg gtaggaaaaa tccgcagaga aattcagaac 240ctcaagcttt tcaggcatcc tcatataatt aaactgtacc aggtcatcag tacaccatct 300gatattttca tggtgatgga atatgtctca ggaggagagc tatttgatta tatctgtaag 360aatggaagga aatctgatgt acctggagta gtaaaaacag gctccacgaa ggagctggat 420gaaaaagaaa gtcggcgtct gttccaacag atcctttctg gtgtggatta ttgtcacagg 480catatggtgg tccatagaga tttgaaacct gaaaatgtcc tgcttgatgc acacatgaat 540gcaaagatag ctgattttgg tctttcaaac atgatgtcag atggtgaatt tttaagaaca 600agttgtggct cacccaacta tgctgcacca gaagtaattt caggaagatt gtatgcaggc 660ccagaggtag atatatggag cagtggggtt attctctatg ctttattatg tggaaccctt 720ccatttgatg atgaccatgt gccaactctt tttaagaaga tatgtgatgg gatcttctat 780acccctcaat atttaaatcc ttctgtgatt agccttttga aacatatgct gcaggtggat 840cccatgaaga gggccacaat caaagatatc agggaacatg aatggtttaa acaggacctt 900ccaaaatatc tctttcctga ggatccatca tatagttcaa ccatgattga tgatgaagcc 960ttaaaagaag tatgtgaaaa gtttgagtgc tcagaagagg aagttctcag ctgtctttac 1020aacagaaatc accaggatcc tttggcagtt gcctaccatc tcataataga taacaggaga 1080ataatgaatg aagccaaaga tttctatttg gcgacaagcc cacctgattc ttttcttgat 1140gatcatcacc tgactcggcc ccatcctgaa agagtaccat tcttggttgc tgaaacacca 1200agggcacgcc atacccttga tgaattaaat ccacagaaat ccaaacacca aggtgtaagg 1260aaagcaaaat ggcatttagg aattagaagt caaagtcgac caaatgatat tatggcagaa 1320gtatgtagag caatcaaaca attggattat gaatggaagg ttgtaaaccc atattatttg 1380cgtgtacgaa ggaagaatcc tgtgacaagc acttactcca aaatgagtct acagttatac 1440caagtggata gtagaactta tctactggat ttccgtagta ttgatgatga aattacagaa 1500gccaaatcag ggactgctac tccacagaga tcgggatcag ttagcaacta tcgatcttgc 1560caaaggagtg attcagatgc tgaggctcaa ggaaaatcct cagaagtttc tcttacctca 1620tctgtgacct cacttgactc ttctcctgtt gacctaactc caagacctgg aagtcacaca 1680atagaatttt ttgagatgtg tgcaaatcta attaaaattc ttgcacaata aacagaaaac 1740tttgcttatt tcttttgcag caataagcat gcataataag tcacagccaa atgcttccat 1800ttgtaatcaa gttatacata attataaccg agggctggcg ttttggaatg caatttgcac 1860agggattgga acatgattta tagttaaaag cctaatatgc agaaatgaat taagatcatt 1920ttgttgttca ttgtgcagta tgtatatagc ataatataca cagtgaatta taggtctcag 1980gcttacttga tttttggcta ttttatattt agtgtacaca gggctttgaa atattaattt 2040acataaaggc cttcatatat tattacgtgt tatatattac gtgttataaa tttattcaat 2100aaatatttgc ctagaattcc caagaccttt ataggtgatt ttgttttctg ggctccttaa 2160cttcataaat agctagtatc ttccagcagt agtaacagtc tggataactt cttccatatc 2220cctccctctt tgtttttttg agacagtgtc actttgtcac ccaggctgga gtgcaatggt 2280gtggtctcgg ctcactgcaa cctccacctc ccgggttcaa gtgattctcc cgcctcagct 2340tcctgagtag ctggaactac aggcgtgtgc caccacaccc ggctaatttt tcgtattttt 2400agtgtagacg gggtttcact atgttgccca ggctggtctc gaactcctga ccgcgtgatc 2460caccacctca gcttcccaaa gtggtgggat tacaggcgtg agccaccgca cccggcctcc 2520atatccccct tttaaaattc tgtagtgtat ggtaagtcat atcagatatc agacctaatt 2580taaatttcat tttagcttta caagtccaaa aacacagaat ttatatattc agatactcta 2640gcactaattt tagtcttaaa atattcccac gatattctgt acacaaaatg ttctttttgt 2700tacaagagct gagttgcata tactgtagat aaatcatatt atttttgcca atttcacaaa 2760ttcctctggc ccatcatgtc agtcattatt gagtatatgc acacattgct acttatttga 2820ttatgtatct tttaaattga ttcagtgcat agaaaactat ctcttacaaa ctttaagtgc 2880tctgatatga cttccccccc aaattttatt atgaacattt ttaaaaacag aaaaattgaa 2940aaactgtttg gtaagcacat gtatatctac catttagatt cagcagttgt taatgttttg 3000tcatttgttt tctctatacc tatatatgta tagatacagc tagttatgca tatatatgca 3060tatatgtgtt tgtttgtgta tgtatatatg cttttttccc cctgaaccat ttggatgtta 3120cagacatact tatcaccgtg aaaatacttc aagtatctcc tacagataat gacattctcc 3180taaaaatccg taataccatt gtaaaagtaa taattcccca atatcatcta atcaagccat 3240atttaaattt ctgaagttaa ctccaaattt ctttatagct gattatttca aactaggatc 3300caattaaagt ttacatatga cacttggtta taactcttta gttggatata acattattat 3360tattttgata aaatatggaa caaatcaatt ctattaataa gtggtcacat ttgttttggg 3420cttaaattac tttttaaaga tactggattt tcctaagatt tctgatttac actgatattt 3480ttttttgtca ttcttaattg catcacacaa tagatgtaaa tgaagatgta gtcacctcag 3540ataaaattgg tatcgtgtat gataatattg tatcatttat atttgcctta tgttaacttt 3600aagaaattga tttttttgta ttaatcattt tcccattgca acagagctat attttttcta 3660ttttaagaat catattttag gattattttt ggcaaataca gtgagcactt atgtaaccag 3720atgataatga actcaaatgt catgatagct tgcataaatg gtgactctag tagatttgac 3780tcaagcactt ctagaatcat gcactgaatt caaaagaaaa atcttgctgc tttttgtcca 3840gggcttgttc tattcaactt ctaatttgaa agctgtacaa agtaatagaa gttccattta 3900aatatgagtt caaaactgta tttacttttt atgtggccct ctctttaggg gattctaatt 3960ttacttaggg tctctaagtg cagcataatg ttcctgatgt taacagaaga ctgtattttt 4020aaagttacaa atttgtatat ggaattaagt aatggcgcta tatacgctgt tgtggggagg 4080ggggaagaaa aggaggaacc aattaaatag gaccttttaa aaattgttaa ttttgtaaac 4140tttgcttctc ttataagtta ttgtgattca ttttagttac tgtgttttat tttgaaaata 4200tttaaatatt gcacttctat aaatagtatg ataaatgcac agacaattgc agtaaattct 4260tttttaagct aggatatttg aaatgacaac ctttggttaa gtgtgtcaag gttgcaacag 4320aattttcaca atttttttgt tgtttgcaaa ttgttactaa tattgaagag gtaagggagg 4380caatgcaaat gatttttaat ctttttttat tatcttttca gcagtttata ttttttgtga 4440ctttatgcaa ccatattttt actttgtctt gacaactgaa agatgtataa ggttttttgc 4500cagaaatgta ctgtatacat agttttaagt ataacagatt ttactgatat gtaaaaattt 4560tgccattaaa ataaatgatt tctcactgag aggaactttt ctaccaggtt ggggcatatg 4620ggagcttaat atatcatatc taatttaaaa taatttcact gaaataaact ccattgcttt 4680tacctaattt ttttcttgag atgcttttgt agtttttcag agttttagat gattttatac 4740aaaatcctct gcctagcact gctctttttg atgttgtagt gacaccattt acattgaatt 4800aatgcttggt agcctggggc tagatgtgga actccatgga tctgtgttct gactggcacc 4860tttggaatga aagaaaagtg tgtgctgtcc aaattttttc cccttaattc tttccctcat 4920cttctcaccc ataatagaaa ttttatttcc attgtgagtt ctgacaagaa tgaaattcca 4980catacaacat aactgtaaat tgttggtagg tagaagttaa tatttgtggt tcatgtatat 5040tttgaccaga gtatatttaa gtatataatt tcagcttcct tgatttagaa atatgatata 5100ataaagaaaa actccattta tcatctgtta 5130105085DNAHomo sapiens 10agcgccatgc gcagactcag ttcctggaga aagatggcga cagccgagaa gcagaaacac 60gacgggcggg tgaagatcgg ccactacatt ctgggtgaca cgctgggggt cggcaccttc 120ggcaaagtga aggttggcaa acatgaattg actgggcata aagtagctgt gaagatactc 180aatcgacaga agattcggag ccttgatgtg gtaggaaaaa tccgcagaga aattcagaac 240ctcaagcttt tcaggcatcc tcatataatt aaactgtacc aggtcatcag tacaccatct 300gatattttca tggtgatgga atatgtctca ggaggagagc tatttgatta tatctgtaag 360aatggaaggc tggatgaaaa agaaagtcgg cgtctgttcc aacagatcct ttctggtgtg 420gattattgtc acaggcatat ggtggtccat agagatttga aacctgaaaa tgtcctgctt 480gatgcacaca tgaatgcaaa gatagctgat tttggtcttt caaacatgat gtcagatggt 540gaatttttaa gaacaagttg tggctcaccc aactatgctg caccagaagt aatttcagga 600agattgtatg caggcccaga ggtagatata tggagcagtg gggttattct ctatgcttta 660ttatgtggaa cccttccatt tgatgatgac catgtgccaa ctctttttaa gaagatatgt 720gatgggatct tctatacccc tcaatattta aatccttctg tgattagcct tttgaaacat 780atgctgcagg tggatcccat gaagagggcc acaatcaaag atatcaggga acatgaatgg 840tttaaacagg accttccaaa atatctcttt cctgaggatc catcatatag ttcaaccatg 900attgatgatg aagccttaaa agaagtatgt gaaaagtttg agtgctcaga agaggaagtt 960ctcagctgtc tttacaacag aaatcaccag gatcctttgg cagttgccta ccatctcata 1020atagataaca ggagaataat gaatgaagcc aaagatttct atttggcgac aagcccacct 1080gattcttttc ttgatgatca tcacctgact cggccccatc ctgaaagagt accattcttg 1140gttgctgaaa caccaagggc acgccatacc cttgatgaat taaatccaca gaaatccaaa 1200caccaaggtg taaggaaagc aaaatggcat ttaggaatta gaagtcaaag tcgaccaaat 1260gatattatgg cagaagtatg tagagcaatc aaacaattgg attatgaatg gaaggttgta 1320aacccatatt atttgcgtgt acgaaggaag aatcctgtga caagcactta ctccaaaatg 1380agtctacagt tataccaagt ggatagtaga acttatctac tggatttccg tagtattgat 1440gatgaaatta cagaagccaa atcagggact gctactccac agagatcggg atcagttagc 1500aactatcgat cttgccaaag gagtgattca gatgctgagg ctcaaggaaa atcctcagaa 1560gtttctctta cctcatctgt gacctcactt gactcttctc ctgttgacct aactccaaga 1620cctggaagtc acacaataga attttttgag atgtgtgcaa atctaattaa aattcttgca 1680caataaacag aaaactttgc ttatttcttt tgcagcaata agcatgcata ataagtcaca 1740gccaaatgct tccatttgta atcaagttat acataattat aaccgagggc tggcgttttg 1800gaatgcaatt tgcacaggga ttggaacatg atttatagtt aaaagcctaa tatgcagaaa 1860tgaattaaga tcattttgtt gttcattgtg cagtatgtat atagcataat atacacagtg 1920aattataggt ctcaggctta cttgattttt ggctatttta tatttagtgt acacagggct 1980ttgaaatatt aatttacata aaggccttca tatattatta cgtgttatat attacgtgtt 2040ataaatttat tcaataaata tttgcctaga attcccaaga cctttatagg tgattttgtt 2100ttctgggctc cttaacttca taaatagcta gtatcttcca gcagtagtaa cagtctggat 2160aacttcttcc atatccctcc ctctttgttt ttttgagaca gtgtcacttt gtcacccagg 2220ctggagtgca atggtgtggt ctcggctcac tgcaacctcc acctcccggg ttcaagtgat 2280tctcccgcct cagcttcctg agtagctgga actacaggcg tgtgccacca cacccggcta

2340atttttcgta tttttagtgt agacggggtt tcactatgtt gcccaggctg gtctcgaact 2400cctgaccgcg tgatccacca cctcagcttc ccaaagtggt gggattacag gcgtgagcca 2460ccgcacccgg cctccatatc ccccttttaa aattctgtag tgtatggtaa gtcatatcag 2520atatcagacc taatttaaat ttcattttag ctttacaagt ccaaaaacac agaatttata 2580tattcagata ctctagcact aattttagtc ttaaaatatt cccacgatat tctgtacaca 2640aaatgttctt tttgttacaa gagctgagtt gcatatactg tagataaatc atattatttt 2700tgccaatttc acaaattcct ctggcccatc atgtcagtca ttattgagta tatgcacaca 2760ttgctactta tttgattatg tatcttttaa attgattcag tgcatagaaa actatctctt 2820acaaacttta agtgctctga tatgacttcc cccccaaatt ttattatgaa catttttaaa 2880aacagaaaaa ttgaaaaact gtttggtaag cacatgtata tctaccattt agattcagca 2940gttgttaatg ttttgtcatt tgttttctct atacctatat atgtatagat acagctagtt 3000atgcatatat atgcatatat gtgtttgttt gtgtatgtat atatgctttt ttccccctga 3060accatttgga tgttacagac atacttatca ccgtgaaaat acttcaagta tctcctacag 3120ataatgacat tctcctaaaa atccgtaata ccattgtaaa agtaataatt ccccaatatc 3180atctaatcaa gccatattta aatttctgaa gttaactcca aatttcttta tagctgatta 3240tttcaaacta ggatccaatt aaagtttaca tatgacactt ggttataact ctttagttgg 3300atataacatt attattattt tgataaaata tggaacaaat caattctatt aataagtggt 3360cacatttgtt ttgggcttaa attacttttt aaagatactg gattttccta agatttctga 3420tttacactga tatttttttt tgtcattctt aattgcatca cacaatagat gtaaatgaag 3480atgtagtcac ctcagataaa attggtatcg tgtatgataa tattgtatca tttatatttg 3540ccttatgtta actttaagaa attgattttt ttgtattaat cattttccca ttgcaacaga 3600gctatatttt ttctatttta agaatcatat tttaggatta tttttggcaa atacagtgag 3660cacttatgta accagatgat aatgaactca aatgtcatga tagcttgcat aaatggtgac 3720tctagtagat ttgactcaag cacttctaga atcatgcact gaattcaaaa gaaaaatctt 3780gctgcttttt gtccagggct tgttctattc aacttctaat ttgaaagctg tacaaagtaa 3840tagaagttcc atttaaatat gagttcaaaa ctgtatttac tttttatgtg gccctctctt 3900taggggattc taattttact tagggtctct aagtgcagca taatgttcct gatgttaaca 3960gaagactgta tttttaaagt tacaaatttg tatatggaat taagtaatgg cgctatatac 4020gctgttgtgg ggagggggga agaaaaggag gaaccaatta aataggacct tttaaaaatt 4080gttaattttg taaactttgc ttctcttata agttattgtg attcatttta gttactgtgt 4140tttattttga aaatatttaa atattgcact tctataaata gtatgataaa tgcacagaca 4200attgcagtaa attctttttt aagctaggat atttgaaatg acaacctttg gttaagtgtg 4260tcaaggttgc aacagaattt tcacaatttt tttgttgttt gcaaattgtt actaatattg 4320aagaggtaag ggaggcaatg caaatgattt ttaatctttt tttattatct tttcagcagt 4380ttatattttt tgtgacttta tgcaaccata tttttacttt gtcttgacaa ctgaaagatg 4440tataaggttt tttgccagaa atgtactgta tacatagttt taagtataac agattttact 4500gatatgtaaa aattttgcca ttaaaataaa tgatttctca ctgagaggaa cttttctacc 4560aggttggggc atatgggagc ttaatatatc atatctaatt taaaataatt tcactgaaat 4620aaactccatt gcttttacct aatttttttc ttgagatgct tttgtagttt ttcagagttt 4680tagatgattt tatacaaaat cctctgccta gcactgctct ttttgatgtt gtagtgacac 4740catttacatt gaattaatgc ttggtagcct ggggctagat gtggaactcc atggatctgt 4800gttctgactg gcacctttgg aatgaaagaa aagtgtgtgc tgtccaaatt ttttcccctt 4860aattctttcc ctcatcttct cacccataat agaaatttta tttccattgt gagttctgac 4920aagaatgaaa ttccacatac aacataactg taaattgttg gtaggtagaa gttaatattt 4980gtggttcatg tatattttga ccagagtata tttaagtata taatttcagc ttccttgatt 5040tagaaatatg atataataaa gaaaaactcc atttatcatc tgtta 5085114655DNAMus musculus 11gcggcgccat gcgcagactc agttcctgga gaaagatggc gacggccgag aagcagaagc 60acgacgggcg ggtgaagatc ggccactaca tcctggggga cacgcttggt gtcggcacct 120tcgggaaagt gaaggtgggc aagcacgagt tgaccggaca taaagtggct gtgaagatac 180tcaaccggca gaagattcgg agccttgacg tggtgggaaa aatccgccgg gagattcaga 240acctgaagct gttcaggcac cctcacatca tcaaactgta ccaggtcatc agtacaccat 300ctgatatttt catggtgatg gaatatgtct ctggaggaga gctatttgat tatatctgta 360aaaatggaag gttggacgaa aaggaaagcc gccgtctgtt ccagcagatc ctttccggtg 420tggattattg tcacaggcat atggtggtcc acagagattt gaaacctgag aacgtcctgc 480ttgatgcaca catgaatgca aagatagccg actttggtct ttcaaacatg atgtcagatg 540gtgaattttt aagaacaagc tgtggctcac ccaattatgc cgcaccagaa gtcatttcag 600gaagattgta cgcaggcccc gaggtggaca tctggagcag cggggtcatt ctctatgctt 660tgctgtgtgg aaccctccct tttgatgatg accatgtgcc aactcttttt aagaagatat 720gtgatgggat cttttatacc cctcagtact taaacccttc agtaatcagc cttttgaaac 780atatgctgca ggtggatccc atgaagaggg ccgcaataaa agatatcagg gaacacgagt 840ggtttaaaca ggaccttccg aagtatctct ttcctgagga cccatcttat agttcaacca 900tgatcgatga cgaagccttg aaagaagtgt gtgagaagtt cgagtgttcg gaggaggagg 960tcctcagctg cctgtacaac agaaaccacc aggacccact agccgtcgcc taccacctca 1020tcatagacaa caggagaata atgaatgaag ccaaagattt ctacctagca accagcccac 1080ctgactcttt cctggacgac caccatttaa ctcggcctca ccctgaaaga gtaccgttct 1140tggttgccga aacaccacgg gcccggcaca ccctggatga attaaaccca cagaaatcca 1200aacaccaagg tgtacggaag gcaaaatggc atttgggaat tcgaagtcaa agccgaccca 1260atgatatcat ggcagaagtt tgtagagcaa tcaagcagtt ggattatgaa tggaaggttg 1320taaaccccta ttatttgcgt gtacgaagga agaatcctgt gacaagcaca ttttccaaaa 1380tgagtctaca gctataccaa gtggatagta ggacttactt gttggatttc cgtagtattg 1440atgatgagat tacagaagcc aaatcaggga ctgctactcc acagagatcg ggatccatca 1500gcaactatcg atcttgccaa aggagtgact ctgatgccga agctcaagga aagccctcag 1560acgtctccct tacctcatct gtcacctccc tcgactcctc ccctgtcgac gtagctccaa 1620gaccaggaag tcatacaata gaattttttg aaatgtgtgc aaatctaatt aaaattcttg 1680cacagtaaac ctaaaatttt gcttatttct ttgatgcaat aagcatgcat aataaataat 1740agccaaatac ttccacttac caagttatat atatgtgtgt atataactga ggattggcct 1800tttggaatgc agtttgcaca gggactggaa aacgattgat cgttaaaagc ctgatgtgca 1860gaattgaatg aggatcactc tgttatcccg tattcagtag gtacacacag cgtaacacac 1920acacaatgga ctgtaggtct caagcttact tgacttttgg ccatttgaat ttgctgtaac 1980aggtctcagt cgaccataaa tttaccataa agccttcaca tattagtaca tgttgatgta 2040ttttatatta gcttcatgaa tttattgagt tgcctggaat cctcatagac cttattagat 2100aatgctgttt tctgggctcc ttaacttcca aaatagctac tagtttccat ttttggttac 2160attgtgaact atttccatat tcctcttaac atgctatttt tgtgtagtaa gtcatttcag 2220agagcagtct ggatttaaca tttttctttg gcttgtgagc ccccaaagcc ccagaattta 2280tatgttcaga ccctccaagc cctgaattta ctttttcagg tttgcccttt tatatatgaa 2340gtgctgcttt ggatccaaga gccgagttgc tcacactgta gtgaatcgtg ttgcttctgt 2400cagtgcacag agacgctatc atgtcagcca gtgttgaatg tctttggtat tttctaagcc 2460aatccagtgc atagaaagga cagtgtttat acgctctgaa tgctaggatt ggggttttag 2520cccctgattt tattctggac aatttttaaa gaaagaaaag ttgcaagaat ttagtgactg 2580catgtgtatt tactacttag ctcctacaac tactgtttgg ccatatttgc tctctagatc 2640cacacatgta taatatacag atatgcacat atattcgagt atatgtttgc ttttattctg 2700aaccactgag atgttaaggt atatatatat atatatatat accagccctg aatacttcag 2760cacttcctaa aaataataat aatgtccttt agaaaccttc tgaaaccatt ataaaatcaa 2820taatttccag atagtgcctg gttttccaga ttagctgtaa ctgcccagaa ttccatttaa 2880gttacagcct gattttattt gcagttcttt aatcaggtta ataacactat tttgaaaaga 2940tgtagaagaa atcctttctt caaactggcc aagtttattt caggttttaa ttcaaaataa 3000tgagtggcta aagaagtgtg atttttcttc aatctctgat ttatatgcct ctctcctttt 3060agtggtagca tagcataatg ggtgtaagtg aagatttaag tcacgctaga caaagccggt 3120attgtgtgag ctaataatta atgtcatatt ggccatattt ggtttccttt aagtttagga 3180aattggtcag aggctctagc ccagtggtga ggctgggtca tctccagcaa cgtgaaacaa 3240aatgcatttt ctttaaacat taaccatccc ccctttgtaa gagagacttt tccccccact 3300gcattcattt aaaaatattt tgccccatcc ccaaacctgt ccagcactta tgtaagaacg 3360catttggagg acatgatagc ttgcataaaa gatgacgcta tagtttatgc actgatttcc 3420ggacaaaaat gatgcttctt tttgttcggg gcttgtttta ttcaactccg aattgaatgc 3480tgtataaagt aatggagact ccatttacct gagttcctga ctgtatttat acctgtgtgt 3540ttgtgccctc tgtgtggggc tccctgatgt cacgtatgct gtctgaatgc agcgctgtat 3600tcccgctgtg tagaacagaa ggctgcattt gtaaagttat agatttgtaa ataaaactga 3660ggaatatctc tataaactgt tggggggacg gggcacaaaa ggaggagcca actaaatagg 3720accttctaaa atttgttaat tttgtaatct ttgcttcttt tataagttat tctgtgattc 3780cttttagtta ccgagtttta ttttgaagac attaaaatat tgcacttgta taaatagtat 3840gataaatgca gactattgca gtcaaatttt ttaaaagcta ggatatctga aatgacaact 3900ttcagttaag tatgtcaagc ccaggacagg attttcacaa atgtattgta gtttgcaaat 3960tcttacactg aaaatgtaaa gggagtcaat gcaaatgatt tttaatcttt tttttattat 4020cttttcagca atttatattt tcttgtgatt ttatgcaacc atatttttac tttgtcttga 4080caactgaaag ctgtgtgatg tttttgacag aaaagtactg tatacatagt tttaaatata 4140acagattttg ctgatatgta aaaattttgc cattaaagtt gatttctcat gaaaggaact 4200tttctaccaa gtcgggagac gcaagagctt aatatatcta atttaaaata atttcactga 4260aataaacacc attgctttta ctttgattct atttttttct taagatgctt ttatagtgtt 4320tgggagtttt agataatttt atatgaattc tctccctacc ttgccccatc ctttggatct 4380ttgataccat tcacaaataa actcatgtgg ctgggtgtgt aaagatatgt accccaaggg 4440gcatcttggg aaagaaaacc ataaattctt ctccttaact cctccctctg cctgttcatt 4500gtgggctctg acatgatgaa tggaatgcca cacacagcct gactgtaagc cgttgttggg 4560aggagtccac cattgtggct tatgtatgtt tgcccagagt aacctgattt agaaagatgg 4620cataataaag aaaaaagttc gtttttcatt tgtta 4655123786DNAGallus gallus 12gccacttctg cgttaagagc aggtaggcag cgaagatatc cccgcacgac agtgccgtga 60aagacgacgc gagcgcacgc caccactcaa cccggaagta gcagcgcaca ccgcctccac 120ccctgtaccg gaaatagttc gctccggcgg aaggcgggaa aggagaactc tggcactcgc 180ttccgggggc tggctgcggg cgggcgctcc tcctcaccgg aggcccggcc caccactatg 240cgcagactca gctgttggtt caagatggcg gcggcagata aacagaagca cgagcacggg 300cgagtgaaga ttgggcatta cattctgggc gacacgcttg gcgtcggcac tttcggcaaa 360gttaaggttg gcaagcatga gttaactgga cataaagttg cagtgaagat cctgaatcga 420caaaagattc gcagccttga cgttgtagga aaaattcgca gggagattca gaacctcaaa 480ctcttcagac atcctcatat aattaagctg taccaggtca tcagtacccc aactgacatt 540ttcatggtga tggaatacgt ttctggagga gagctgtttg attatatctg taaaaatgga 600aggcttgatg agaaggaaag tagacgtctg ttccagcaaa tcctttctgg tgtggattac 660tgtcacaggc acatggtagt tcacagagat ctaaagcctg aaaatgtgtt gcttgatgca 720cacatgaatg ccaagatagc tgactttggt ctatcaaata tgatgtctga tggagaattt 780ttaagaacaa gctgtggttc ccctaactat gctgcaccgg aagtaatttc aggaaggtta 840tatgcaggcc cagaagtaga tatttggagc agtggggtta ttctctatgc actgttatgt 900ggaacacttc catttgatga tgatcacgtg ccaacacttt ttaagaagat atgtgatggt 960atcttttata cccctcagta cctgaatcca tctgtcatta gtcttctgaa acatatgctg 1020caagtggatc caatgaagag agcaacaatc cgagacatta gggaacacga atggtttaag 1080caggaccttc ccaaatactt gtttcctgaa gacccttcgt atagttctac catgattgat 1140gacgaagcct taaaagaagt atgtgagaag tttgaatgta ccgaagagga agtgttaagt 1200tgtctgtata gccgaaatca tcaggatcct ttagcagttg cttatcacct cattatagat 1260aatagaagaa taatgaacga ggccaaagac ttctacctgg ctacaagccc gccagattct 1320tttcttgacg atcaccatct gtctcgccct catcctgaga gagtgccatt cttagtagct 1380gaagcaccac ggcctcgcca tactcttgat gagctgaatc cacagaaatc aaaacaccaa 1440ggtgtaagaa gagcgaagtg gcatttgggg atacggagtc agagtcgacc gaatgatata 1500atggctgaag tttgtcgagc aattaagcag ctggattatg aatggaaggt tgtaaatcca 1560tactacttgc gtgttcgacg gaagaatcca gtgacaagtg catattctaa aatgagttta 1620caactgtatc aagtggacag cagaacatat ttactagact tccgtagcat tgatgatgaa 1680atcattgaaa cgaagtctgg gactgcaact ccacagagat caggttctgt gagcaattac 1740agatcctgtc agaaagattc agatggtgat gcacaaggaa aatctgcaga tacatccctt 1800acctcatcaa tgacctcttc acttgattct tcgacagctg acttaactcc aagaccaggc 1860agtcatacaa tagaattttt tgagatgtgt gcaaatctta ttaaaatact tgcacaataa 1920ctttgaaggt gggcttattt cttttacagc aataagcatg cataaattgt agtcagatgc 1980ttccagttgt aatcaattta aattaacaaa ggcgctgaaa aaactagctg caaaatgcaa 2040tttacctggg gattaaagct attatgagca gttaggtata ttgctttgaa gctggagtgg 2100attctgattt tctgctatcc agtagcttaa tacaggtaca gaaagtcaca tgccccttgg 2160gcagtgcttg taacattttc attgaatgca cgttagtatt tatataacat ttatattaca 2220tggtccttct tatccttctt gttggtgatg catacatctg gagcactgta tgaaaatgtg 2280cacttacagg ttcaatgacc taagtatgac ttgttggtca ctgtctacat ttgtgcctca 2340gttgatcaga acagttaatg tctcctaaca gtaaagtgtt aaaactgctg ttccttcccc 2400ctttattcag cagtatatga cattttaaac aaagtttgat gtgctataat ataacattca 2460ataattgata attttgatta aatgttggca aatccaacca attggaattt tcagtacacg 2520taagttctct gcagcacatt tatgagtaaa attctctgag gtgtagaaca gttgaagcca 2580cagcagtgcc aagagagatg ttgctgagag gatatgccac gcaatggcct gaagcaaatt 2640actgctctga agcactgaga gcaagctcct gatctgggca ggtttgctag ctcataccat 2700agtgtagtag cagcacagct cctgcagact gagaatcctt agaagtatat gcatgtagtt 2760aagagcacag cctaggggtt gttgcatgtt attccacaat ctgaaattga cacacctttt 2820ttaaaaaaag aagttaatac ttagatatgt ctgcttacat cacatgttaa cttactatat 2880gctgtataga attcaattgc agttgaaata tgtctatagg ctaagttttt ccgggctacc 2940cttacacaaa tttttgcatt taaattgtag aaattaatag tgcagttttt caattgagta 3000tttaaagctg aaattgtcag ctaaagtgtt aatagttagg aaatcttgaa gtgtaggtcg 3060cagttatgga taacacagaa accataactg cagaagaaat gtttaagaaa atgaccttaa 3120atgctgaatc agatatattt gctcaaaaat gttaaatatg ttgtaaattg gatgtacata 3180cccacagttc cgttttcatc tagcactaat ggatgtaaat attaatcagt aaattgccat 3240aaaacatttt tatttcttca ttggggattt ttcttttcag aatagacttc atggcttagt 3300ttgaaagatt gttctcccat ttagaaactg gaaatcaagg cattaatata aacagtgaaa 3360cttggacttt gttggaatgg atgggacttc ttgccaatga tgggtctaag attccatcct 3420tgacacactg ctaactacag atgacaagaa cagtctgatg cagttcaatt ggcttcagtc 3480cagaaattcc tttgttacaa atttgaagaa atttacatat gcttaagcct cagaactgct 3540gtagctcttc tcaactgggc aactgtgtgg cctgtcttgt tttttgtcct caagtagtgt 3600ctcgcacggt ccctgtaacg caaggttata ttactgcagc gcagagtggg aggattctca 3660gataaaactt tgcatttaaa ctattgagat tgagatggca gtacgtggga ccagctatca 3720gtcatacttt taagtccgtt cttcagccat gtaaggaaag gttctgatgt tctgaataaa 3780taggtg 3786131647DNARattus norvegicus 13atggccgaga agcagaagca cgacgggcgg gtgaagatcg gccactacat cctgggggac 60acgctgggcg tcggcacctt cgggaaagtg aaggtgggca agcacgagtt gactggacat 120aaagttgctg tgaagatact caaccggcag aagattcgaa gcctggacgt ggtcgggaaa 180atccgcagag agatccagaa cctgaagctt ttcaggcacc ctcatataat caaactgtac 240caggtcatca gtacaccgtc tgatattttc atggtcatgg aatatgtctc aggaggagag 300ctatttgatt atatctgtaa aaatggaagg ttggacgaaa aggagagtcg acgtctgttc 360cagcagatcc tttctggtgt ggactattgt cacaggcata tggtggtcca cagagatttg 420aaacctgaaa acgtcctgct tgatgcacac atgaatgcaa agatagccga cttcggtctt 480tcaaacatga tgtcagatgg tgaattttta agaacgagct gtggctcgcc caattatgct 540gcaccagaag taatttcagg aagattgtac gcaggccctg aagtagacat ctggagcagc 600ggggtcattc tctatgcttt gctgtgtgga actctccctt ttgatgatga ccacgtgcca 660actcttttta agaagatatg tgacgggata ttttataccc ctcagtattt gaatccctct 720gtaataagcc ttttgaagca tatgctgcag gtagatccta tgaagagggc cacaataaaa 780gatatcaggg aacatgaatg gtttaagcag gaccttccaa aatatctctt tcctgaagac 840ccgtcttata gttcaaccat gattgatgat gaagccttaa aagaagtgtg tgagaagttc 900gagtgctcag aggaggaggt cctcagctgc ctgtacaaca gaaaccacca ggacccactg 960gcagttgcct accacctcat aatagacaac aggagaataa tgaacgaagc caaagatttc 1020tacttggcaa caagcccacc cgattctttc ctcgatgatc accatttaac tcggcctcac 1080cctgagagag taccattctt ggttgccgaa acaccaaggg cccgacacac cctagatgaa 1140ttaaacccac agaaatccaa acaccaaggc gtacggaagg caaagtggca tttggggatt 1200cgaagtcaaa gccgacccaa tgacatcatg gcagaagtgt gtagagcaat caagcagttg 1260gactatgaat ggaaggttgt aaacccctat tatttgcgtg tgcgaaggaa gaaccctgtg 1320acaagcacat tttccaaaat gagtctacag ctataccaag tggatagtag gacttactta 1380ttggatttcc gaagtattga tgatgagatt acagaagcca aatcagggac tgctactcca 1440cagagatcgg gatccatcag caactatcga tcttgccaaa ggagcgactc cgacgccgag 1500gctcaaggaa agccctcaga agtctctctt acctcatccg tgacctccct cgactcctct 1560cctgttgacg tagctccaag accaggaagt cacacgatag aattttttga aatgtgtgca 1620aatctaatta aaattcttgc acagtaa 1647142435DNAHomo sapiens 14gcggagcggc aggcggtgga gcgaggccgc gcgcgccgaa gatggctgag aagcagaagc 60acgacgggcg ggtgaagatc ggacactacg tgctgggcga cacgctgggc gtcggcacct 120tcggcaaagt gaagattgga gaacatcaat taacaggcca taaagtggca gttaaaatct 180taaatagaca gaagattcgc agtttagatg ttgttggaaa aataaaacga gaaattcaaa 240atctaaaact ctttcgtcat cctcatatta tcaaactata ccaggtgatc agcactccaa 300cagatttttt tatggtaatg gaatatgtgt ctggaggtga attatttgac tacatctgta 360agcatggacg ggttgaagag atggaagcca ggcggctctt tcagcagatt ctgtctgctg 420tggattactg tcataggcat atggttgttc atcgagacct gaaaccagag aatgtcctgt 480tggatgcaca catgaatgcc aagatagccg atttcggatt atctaatatg atgtcagatg 540gtgaatttct gagaactagt tgcggatctc caaattatgc agcacctgaa gtcatctcag 600gcagattgta tgcaggtcct gaagttgata tctggagctg tggtgttatc ttgtatgctc 660ttctttgtgg caccctccca tttgatgatg agcatgtacc tacgttattt aagaagatcc 720gagggggtgt cttttatatc ccagaatatc tcaatcgttc tgtcgccact ctcctgatgc 780atatgctgca ggttgaccca ctgaaacgag caactatcaa agacataaga gagcatgaat 840ggtttaaaca agatttgccc agttacttat ttcctgaaga cccttcctat gatgctaacg 900tcattgatga tgaggctgtg aaagaagtgt gtgaaaaatt tgaatgtaca gaatcagaag 960taatgaacag tttatatagt ggtgaccctc aagaccagct tgcagtggct tatcatctta 1020tcattgacaa tcggagaata atgaaccaag ccagtgagtt ctacctcgcc tctagtcctc 1080catctggttc ttttatggat gatagtgcca tgcatattcc cccaggcctg aaacctcatc 1140cagaaaggat gccacctctt atagcagaca gccccaaagc aagatgtcca ttggatgcac 1200tgaatacgac taagcccaaa tctttagctg tgaaaaaagc caagtggcat cttggaatcc 1260gaagtcagag caaaccgtat gacattatgg ctgaagttta ccgagctatg aagcagctgg 1320attttgaatg gaaggtagtg aatgcatacc atcttcgtgt aagaagaaaa aatccagtga 1380ctggcaatta cgtgaaaatg agcttacaac tttacctggt tgataacagg agctatcttt 1440tggactttaa aagcattgat gatgaagtag tggagcagag atctggttcc tcaacacctc 1500agcgttcctg ttctgctgct ggcttacaca gaccaagatc aagttttgat tccacaactg 1560cagagagcca ttcactttct ggctctctca ctggctcttt gaccggaagc acattgtctt 1620cagtttcacc tcgcctgggc agtcacacca tggatttttt tgaaatgtgt gccagtctga 1680ttactacttt agcccgttga tctgtctcta gtttctttct gttattgcac tatgaaaatc 1740agttatattc tttaaatttt tatcttactt ttggataata tccactgcaa tactaattga 1800gaaacatgaa ttatttccag gggcacacaa tgctattgaa attactgaaa acaaaatatc 1860tgacatctta tttacttgta gaaatctgta attctattgt gcctatgata aattcacata 1920ggcaatatct ttaataggtt aatatcaatg aagattttta attacaataa tgagttcact 1980acagacgatt aacacaccac actggcgaac

catctcaatg taagggtggt ttggcaacac 2040ctccttgctt tgctgtttgg tgtaggtaaa tctagtttac ttcctaaatt tcagtaggct 2100ttatgctgtg tttatgcccc caatttattt taacaaaaga agattaaaaa gtaaaagaac 2160cacgagtaag atattattta aatgttgaaa tcttaaaaac ctgcctccaa gatttcagaa 2220gccaagtttt tctaacagta tttgtacaaa tactgcctag tgtattcaac agaaggactg 2280tggtcatgta acaggtaacc acaattttca ggtttcttaa aaacagctgt aactaactca 2340ggatttttat cttgagattt ccctgaataa tatatttatc ttaagagcct tcaagtttca 2400aattaatatt ggaacatctg gaattgcaac aactt 2435152832DNAMus musculus 15ggtagcggcg gcggcagcgg tagcggcggc ggcgctcaga gcccgcggca gggggaggcc 60gtgcgccgaa catggctgag aagcagaagc acgacgggcg ggtgaagatc ggacactacg 120tcctggggga caccctgggc gtcggcacct tcggcaaagt gaagattgga gaacaccaat 180tgacaggcca taaagtggca gttaagatct taaatagaca gaagattcgc agtttagatg 240ttgttggaaa aataaaacga gaaattcaaa atcttaaact ctttcgtcat cctcatatta 300tcaaactcta ccaggtgatc agcactccga cagacttttt tatggtaatg gaatatgtgt 360ctggaggtga attgttcgac tacatctgca aacatgggcg ggttgaagag gtggaagcgc 420gccggctctt ccagcagatc ctgtctgccg tggattactg tcacaggcat atggttgtcc 480atagggacct gaagccagag aatgtgctgc tggatgccca gatgaacgct aagatagctg 540actttggact atctaatatg atgtcagatg gtgaatttct acgaactagc tgtggatcgc 600caaattatgc agcacctgag gtcatctcag gaaggctgta tgcaggtccc gaggtcgata 660tctggagctg tggtgtcatc ctgtatgccc ttctctgtgg caccctccct ttcgatgatg 720agcacgtgcc tacgctcttc aagaagatcc gagggggtgt gttttacatc ccagactatc 780tcaaccgttc tgtcgccact ctgctgatgc acatgctcca ggtggacccc ctgaagcgag 840cgactatcaa agacatacga gaacatgaat ggtttaaaca ggatttgccc agctacctat 900ttcctgaaga cccctcctac gatgcgaatg tcattgtcga tgaggctgtg aaggaagtct 960gtgagaaatt cgagtgtaca gagtcagaag tgatgaatag tctgtatagt ggtgaccctc 1020aagaccagct tgcagtggct tatcatctta tcattgacaa tcggagaata atgaaccaag 1080ccagtgagtt ctacctcgcc tctagtcctc catcaggttc ttttatggat gacagcgcca 1140tgcatattcc tccaggcttg aaaccacatc cagaaaggat gccgcctctc atcgcagaca 1200gccccaaagc acgctgtcca ctggatgcac tcaacacaac gaagcccaag tctctagctg 1260tgaaaaaagc caagtggcat cttggaatcc gaagccagag caaagcgtgt gacattatgg 1320ctgaagtgta ccgagctatg aagcagctgg gttttgaatg gaaggtagtg aatgcatacc 1380atcttcgagt aagaagaaaa aacccagtga ctggcaacta tgtgaaaatg agcttacagc 1440tttacctggt agacagtcgg agctatcttc tggacttcaa aagcatcgat gatgaggtgg 1500tggagcagag gtctggttct tcaacacccc agcgctcctg ttctgctgcg ggcctccaca 1560gagcacggtc aagttttgat tccagcacag ctgagaacca ctccctttct ggctctctca 1620ctggctcttt gactggcagc actttgtcct cggcatcccc gcgcctgggc agtcacacca 1680tggatttttt tgaaatgtgc gccagtctta tcactgcttt agctcgttga ttatccaccc 1740ctggtctctg tctttctgtc acccccctgt gaaatcacat acactcttta aattattatc 1800tcactcggta ttacgtgctc tgcaatagaa gttggtgtga acattcccag gtgacatgca 1860gtgttattga aatcaccgaa aacacagaaa tccagcattc tgtttactcg tagaactctg 1920taactctcct gtgcctgtga caagtttgca tgggtagtac tgggtgagtg tggtggcgct 1980tgctaactta cacctgtgaa ttcgctgtag atggcaagca cacctcactg atgaacgaac 2040ccgctgaggt aggggtggtt caatgggcct ccttccttca tgtttcctgt acgtaaatcc 2100ggtttacctc ctaaacttca gcagctgtta ggctgcctgg gcactcttgc acaagaagat 2160taagatatag aataactgtt agggaaatat tatttcaatg tagaaatctg aaaatcctgt 2220ccccctaaat attagaaacc aaagtctttt aaattatttc tgcaaatact gcctagtatt 2280agccattaag actgtgtttc tgataacagg agcctaatct cccagcttcc tgacagttgt 2340gcaaggcgcc tagttaaatc acaccaccac caagcaaaac aaaaccatgc aaaattagta 2400cacagttcaa tgagacaagc aaaataccaa caacagtctc ttaaaagaaa attctactct 2460atgatcttgt ttgtgtttat cagactgggg tagctgggag gagggccggc agtcatggcc 2520ctctggcatg tttgctaatt cccttaacat ttgttatcga tgccacctcc tccctccgtc 2580ttcctcagga gcctctgtct tgctcaatgc ttttcccttt cattaatcct ttagctaaaa 2640accttgcttt ggccatcatg tctttgtata cttccaaagc ttgatctctg tgaccttcac 2700tgttgaacct gattggacag ggaagcctta aatatttaaa agtatattct cctggaatac 2760gtatatgtgt tgtttacata tatatacata tgtattgaca catcagtgct tttaatcaaa 2820aaaaaaccaa ac 2832163437DNAGallus gallus 16tgccggccct gcgcattgtg gtcctccgcc ggggggcgaa gatggccgag aagcagcaga 60agcacgacgg gagggtgaag atcggccact acgtgctggg ggatacgctg ggggtcggca 120ccttcggcaa agtcaaggtt ggcgaacatc agctgacggg gcacaaagta gcagtaaaaa 180tcctaaacag acagaaaata cgcagcctgg atgtggttgg gaagatcaaa agagaaattc 240aaaacctgaa actcttccgg caccctcata ttatcaaact gtaccaggtc atcagcacgc 300caacagactt cttcatggtc atggaatacg tatctggcgg tgaattattt gattacatct 360gtaagcatgg acgtgttgaa gaggcagagg ctcgacgcct tttccagcag attctctccg 420cggtggatta ctgtcaccga cacatggttg tccacaggga cctgaaacca gagaacgtgc 480tgctggatgc acacatgaat gcgaagatag ctgattttgg attgtccaac atgatgtctg 540atggtgaatt tctacgcacc agctgcggtt ccccaaatta tgcagcccct gaagtcatct 600ctggaaggct gtatgctggc ccagaggtgg acatctggag ctgcggtgtc atcctctatg 660ccctcctctg tggcactttg ccctttgatg atgagcatgt gcccaccctc ttcaagaaga 720tccggggagg cgtgttttac atccctgagt acctcaaccg ctccgttgcc actctgctca 780tgcacatgct gcaggttgac cctctcaagc gtgcaaccat caaggacatc agggaacatg 840agtggtttaa ggaggagctg cccagctacc tcttcccaga ggacccttcc tacgatgcca 900ccgtcatcga tgatgacgcg gttcgggagg tctgtgagaa gtttgagtgc accgagtcag 960aagtgatgaa cagtctgtac agcggcgacc cccaggacca actagcggtc gcctaccacc 1020ttgtcattga caaccggagg atcatgaacc aagccagcga gttctaccta gcctccagcc 1080ccccaactgg ctccttcatg gacgacacca tgcatatccc tcccggtgtg aagccgcacc 1140ctgagcggat gccacccttg atagcagaca gtcccaaggc acgctgccct ttggatgcac 1200tcaacaccac caagcccaaa cctctgactg tcaaaaaggc caagtggcac ctggggatcc 1260gcagccagag caagccctat gacattatgg ccgaggtgta ccgagctatg aaacagttgg 1320attttgaatg gaaggtggtg aactcctacc acctcagagt gcgtcggaag aacccggtga 1380cgggcaatta cgtgaaaatg agtctgcaac tgtaccaggt ggacaaccgc agctacctcc 1440tggacttcaa aagcattgat gacgaggtga tggagcagag gtccggctca tccaccccgc 1500agcgctcgtg ctcagccgct ggcttgcacc ggccaagact aagcattgat gctgcagcag 1560ccgctgagtg ccagtcactg atggggtccc tgagtggctc cttcgtgggc agcatctcct 1620cagtgccccc acgcctgggc agccacacca tggacttctt cgagatgtgt gccagcctga 1680tcatggctct ggctcgctga gccgggcacc agactccaag cacatggcgt tgcactgtga 1740ggaccagctc caacatggcc attctgttgt tcttctgctt ccttccttct cctcaccact 1800ccacagcctg cggcacagaa cctgttcctc tgttacgaga tcccaagtgc actcaaagga 1860cacagcccct caaacaaggc attgaggaaa tcaggaatga cttcgtttca ctcgttagac 1920cagagtgaaa tatatatgag ctttttttta tcctgaaccc aatgggatac gtgggtcagg 1980gctgtaaagt agcaaacata tcactgactt ttggtagaga actccctcct ggctgatcct 2040gaattcacag aaccatgggc aagtatttct ttctgcaaca cacattggtt caggttgtac 2100ctcccacaca gcctgaagca gggggaggac agggctgaac ctctggcaat gctttcaccc 2160taaaacaagc agctgcttca tgtaccaccc caccctctgt tctttttccc ctactaaaga 2220agggtgaggg gtggcatgtg caaagctccc attgcagaca tggggcagga tgctgctgag 2280tcccccagat ccggctctct gcgtcacagc ttcggtgtgc ttacctctca cctttcccat 2340aggaagattt tgcccattgt caccacccca aaatctctgc caaagctgcc tgccttcacc 2400tgccaaggat ccaagaatga agagctctgc tgtcctgata cagaatgggt tgggacctcg 2460ctgcgacatg gagaaggcag ggatatcaca agcatttcac tgtttctttg ctactggctt 2520tctgaaggac agggttttat attttaagac gaactatgag gatttttcag gttaaaattc 2580ttgttattcg agggtaaact ctagtgcatg tgtgacagtt tctttgttta aaagaattat 2640tacttctcat tacataggct taatctaatg gacagagttg gcatctgctt ggctttgcta 2700cggtatttaa ttccatttac ttgattgcaa aggaacttaa tagtctgatc agtcaaggaa 2760caaatgcttt atgaaaacgt tgccaaaaaa atcctgggag ggttattttc ttcctttttt 2820ggttttgctt taagaaacaa aacaaaggag agtgttagca atgttgctgg ttccaggtat 2880gcagtcattc aactttaaaa acaaccctga acccattctt ttgatagttc ttatgtccct 2940ggaagttttt tgctaaggat tgtttttttt ttttgtacag ttgcacttta atttatactt 3000tctgaaagtt tctataagca gcaaggagga gaatgagaca ttaaaccttt ttgtactaat 3060tgttttagat actgaagaca aatacgggat atttttgcca tctactggca gataagtgga 3120cgtttttcag tgtgaagtcc ccctttttta agaacacaaa attgccttac ttttgttaca 3180ggtgacactt cgattctcac taatgtgtat ttccagctcc agttatatca gaagccgcac 3240aaagtagaaa cctgccaaaa aagcatcagt cttgctgttg agttcttgta tgcagaatcc 3300aacacagcag tgttcggatt gcaaacagca gaacaatctc gtttttaaag gaaacactgg 3360aaatctttgt attaatccta ggttttgtaa tgggttttta ttatattatt gttatgaaat 3420aaataccaat ggctgga 3437172145DNASus scrofa 17gactgaggag tccgccgcgg cggcggcgga gcgaggctgc gcgcgccgaa catggctgag 60aagcagaagc acgacgggcg ggtgaagatt ggacactacg tgctggggga cacgctgggt 120gtcggcacct tcggcaaagt gaagattgga gagcatcaac tgacaggcca taaagtggca 180gttaaaattt taaataggca gaagattcgc agtttagatg ttgttggaaa aataaaacga 240gaaattcaaa atctaaaact cttccgtcat cctcatatta tcaaactata ccaggtgatc 300agcactccga cagatttttt tatggtaatg gaatatgtgt ctggtggtga attatttgat 360tacatctgta aacatggacg ggttgaagag atggaagcca ggcggctctt tcagcagatt 420ctctctgccg tggattactg tcacaggcat atggttgttc atcgagacct gaaaccagag 480aatgtgctat tggatgcaca gatgaatgcc aagatagcag attttggatt atctaatatg 540atgtcagatg gtgagtttct gcgaactagt tgtgggtctc caaattacgc agcacctgaa 600gtcatctcag gcagattgta tgcaggtcct gaggtcgata tctggagctg cggtgttatt 660ttgtatgctc ttctttgtgg cacactccca tttgatgatg agcatgtgcc tacattgttt 720aagaagatcc gagggggtgt tttttatatt ccagaatatc tcaatcgttc tgttgccact 780ctcctgatgc atatgctgca ggttgacccc ctgaaacgag caactatcaa agacataaga 840gagcatgaat ggtttaaaca agatttgccc agttacttat ttcctgaaga cccctcctat 900gatgctaacg tcattgatga tgaggctgtg aaagaagtgt gtgaaaaatt tgagtgtaca 960gaatcagaag taatgaacag tttgtatagt ggtgaccctc aagaccagct tgcagtggct 1020tatcatcttg tcattgacaa tcggagaata atgaaccaag ccagtgagtt ctacctcgcc 1080tctagtcccc caactggttc ttttatggat gatagtgcca tgcatattcc cccaggcctg 1140aaacctcatc cagaaaggat gccacctctt atagcagaca gccctaaagc aagatgtcca 1200ttggatgcac tgaatacaac caagcccaaa tctttagctg tgaaaaaagc caagtggcat 1260cttgggattc gaagtcaaag caaaccatat gacattatgg ctgaagttta ccgagctatg 1320aagcagctgg attttgaatg gaaggtagtg aatgcatacc atctacgtgt aagaagaaaa 1380aatccagtga ctggcaatta tgtgaaaatg agcttacagt tgtacctggt tgacaataga 1440agctatcttt tggactttaa aagcattgat gatgaggtgc tggagcagag gtctggttcc 1500tcaacacctc agcggtcctg ctctgctgct ggcttgcaca gaccgcggtc aagcttggat 1560tctgtcactg cggagagcca ctcactctct ggctctctct ctggctcctt gaccggaagc 1620atgctgcctt cagttccacc tcgcctgggc agtcacacca tggatttttt cgaaatgtgt 1680gccagtctga tcactacttt agcccgttga taatctttcc ctagtttcta tcttctgtta 1740ttgcactgtg aaaatgagat atattcttta aattattgtt ttacctattg aaatcacata 1800ctctgcagta gtggttgaga gacgtaacta tctccagagg acactcaata cctttgaaat 1860gactgaaaac aaatctgacg ttttatttac tgtagacatc tgtaattctg ttttgcctac 1920gataaaaatc acataggcgg tgtctttact aggtgaacat cgacgaagat tgttgttcat 1980ctaaaaccgt ggattcactg cagatgatta agacatcgtt actggtgaac catcaacgaa 2040gagtcgcttg gcatcacctt cttgctttac tgtgtagtgt aggtgaatcc agtttgcttc 2100ctaaatttcg gcaggcttta cgctgtgttt atgcccaaga agttt 2145

Claims

1. A method for enhancing an exercise effect in a subject, comprising performing by a subject physical activity sufficient to produce an exercise effect; and administering to the subject an effective amount of a PPAR.delta. agonist, thereby enhancing the exercise effect in the subject.

2. The method of claim 1, wherein the subject is a mammal.

3. The method of claim 2, wherein the subject is a racing mammal.

4. The method of claim 3, wherein the racing mammal is a horse, a dog, or a human.

5. The method of claim 1, wherein the subject is an adult.

6. The method of claim 1, wherein the subject is an exercise-trained subject.

7. The method of claim 1, wherein the PPAR.delta. agonist is GW1516.

8. The method of claim 1, wherein the PPAR.delta. agonist is administered on the same day(s) on which the physical activity is performed.

9. The method of claim 1, wherein the physical activity is an aerobic exercise.

10. The method of claim 9, wherein the aerobic exercise is running.

11. The method of claim 9, wherein the exercise effect is improved running endurance.

12. The method of claim 11, wherein improved running endurance is improved running distance or improved running time or a combination thereof.

13. The method of claim 1, wherein the effective amount is from about 5 mg/kg per day to about 10 mg/kg per day in a single dose or in divided doses.

14. The method of claim 1, wherein administration comprises oral administration, intravenous injection, intramuscular injection, or subcutaneous injection.

15. The method of claim 1, wherein the exercise effect is increased fatty acid oxidation in at least one skeletal muscle of the subject.

16. The method of claim 1, wherein the exercise effect is body fat reduction.

17. The method of claim 16, wherein the body fat is white adipose tissue.

18. A method for identifying the use of performance-enhancing substances in an exercise-trained subject comprising determining in a biological sample taken from an exercise-trained subject the expression of one or more molecules listed in Tables 2 or 4.

19. The method of claim 18, wherein: (i) expression is upregulated in one or more of adipose differentiation related protein; stearoyl-Coenzyme A desaturase 2; acetyl-Coenzyme A acetyltransferase 2; ATP citrate lyase; adiponectin, C1Q and collagen domain containing; diacylglycerol O-acyltransferase 2; lipase, hormone sensitive; monoglyceride lipase; resistin; CD36 antigen; fatty acid binding protein 4, adipocyte; lipoprotein lipase; microsomal glutathione S-transferase 1; GPI-anchored membrane protein 1; dual specificity phosphatase 7; homeodomain interacting protein kinase 3; insulin-like growth factor binding protein 5; protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), beta isoform; protein tyrosine phosphatase-like (proline instead of catalytic arginine); member b; CCAAT/enhancer binding protein (C/EBP), alpha; nuclear receptor subfamily 1, group D, member 2(Reverb-b); transferring; archain 1; solute carrier family 1 (neutral amino acid transporter), member 5; RIKEN cDNA 1810073N04 gene; haptoglobin; retinol binding protein 4, plasma; phosphoenolpyruvate carboxykinase 1, cytosolic; cell death-inducing DFFA-like effector c; interferon, alpha-inducible protein 27; carbonic anhydrase 3; cysteine dioxygenase 1, cytosolic; DNA segment, Chr 4, Wayne State University 53, expressed; dynein cytoplasmic 1 intermediate chain 2; Kruppel-like factor 3 (basic); thyroid hormone responsive SPOT14 homolog (Rattus); cytochrome P450, family 2, subfamily e, polypeptide 1; complement factor D (adipsin); and/or transketolase; or (ii) expression is downregulated in one or more of gamma-glutamyl carboxylase; 3-oxoacid CoA transferase 1; solute carrier family 38, member 4; annexin A7; CD55 antigen, RIKEN cDNA 1190002H23 gene; fusion, derived from t(12; 16) malignant liposarcoma (human); lysosomal membrane glycoprotein 2; and/or neighbor of Punc E11; or (iii) a combination of (i) and (ii).

20. The method of claim 18, wherein determining expression comprises determining protein expression, determining expression of a gene encoding the protein, or a combination thereof.

21. The method of claim 20, comprising determining expression of a gene encoding the protein.

22. The method of claim 18, wherein the biological sample is a skeletal muscle biopsy.

23. A method of identifying an agent having potential to enhance exercise performance in a subject, comprising: providing a first component comprising a PPAR.delta. receptor or an AMPK-binding fragment thereof; providing a second component comprising an AMP-activated protein kinase (AMPK), AMPK.alpha.1, AMPK.alpha.2, or a PPAR.delta.-binding fragment of any thereof; contacting the first component and the second component with at least one test agent under conditions that would permit the first component and the second component to specifically bind to each other in the absence of the at least one test agent; and determining whether the at least one test agent affects specific binding of the first component and the second component to each other, wherein an effect on specific binding identifies the at least one test agent as an agent having potential to enhance exercise performance in a subject.

24. The method of claim 23, further comprising providing a third component comprising a PPAR.delta. agonist; and contacting the first component, second component, and third component.

25. The method of claim 24, wherein the PPAR.delta. agonist is GW1516.