EP2234622A2 - Méthodes améliorant les performances et le tonus musculaires - Google Patents

Méthodes améliorant les performances et le tonus musculaires

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Publication number
EP2234622A2
EP2234622A2 EP08866040A EP08866040A EP2234622A2 EP 2234622 A2 EP2234622 A2 EP 2234622A2 EP 08866040 A EP08866040 A EP 08866040A EP 08866040 A EP08866040 A EP 08866040A EP 2234622 A2 EP2234622 A2 EP 2234622A2
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EP
European Patent Office
Prior art keywords
subject
ampk
pparδ
exercise
agonist
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08866040A
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German (de)
English (en)
Other versions
EP2234622A4 (fr
Inventor
Ronald M. Evans
Vihang A. Narkar
Reuben J. Shaw
Michael Downes
Ruth T. Yu
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Salk Institute for Biological Studies
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Salk Institute for Biological Studies
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Publication date
Priority claimed from US11/966,851 external-priority patent/US20080187928A1/en
Application filed by Salk Institute for Biological Studies filed Critical Salk Institute for Biological Studies
Publication of EP2234622A2 publication Critical patent/EP2234622A2/fr
Publication of EP2234622A4 publication Critical patent/EP2234622A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/06—Anabolic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This disclosure concerns the use of agonists of AMP- activated protein kinase (AMPK) for improving exercise and modifying energy metabolism in a subject.
  • AMPK AMP- activated protein kinase
  • the disclosure also provides methods of treating muscle wasting diseases and disorders and promoting muscle tone in sedentary subjects.
  • the disclosure also relates to a combination of AMPK and peroxisome proliferator- activated receptor (PPAR) ⁇ agonists for improving exercise performance in a subject, and methods for identifying compounds that modulate gene expression profiles associated with muscle tone, endurance or performance.
  • AMPK AMP- activated protein kinase
  • PPAR peroxisome proliferator- activated receptor
  • 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 Ha) and glycolytic fast- twitch (type Hb) 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.
  • 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.
  • the disclosure also provide a method of treating a muscle wasting disease, muscle atrophy or aging comprising administering to a subject a composition comprising an AMPK agonist, wherein the muscle tone, mass or endurance is promoted.
  • the method comprises treating a subject that may have an immobilized limb or which may be immobilized due to other medical treatments to promote or maintain muscle tone in the subject .
  • the disclosure demonstrates that unexpected finding that orally active AMPK agonists are sufficient as a single agent to improve exercise endurance by nearly 45% in non-exercised subjects.
  • the disclosure provides a method for enhancing an exercise effect in a subject, comprising administering to a subject an AMP kinase (AMPK) agonist wherein an exercise effect is enhanced.
  • AMPK AMP kinase
  • the AMPK agonist can be any AMPK agonist, derivatives, salts or esters thereof.
  • the AMPK agonist is AICAR.
  • the method can further comprise administering to the subject an effective amount of a PPAR ⁇ agonist (e.g., GW1516), thereby further enhancing the exercise effect in the subject.
  • the subject can be a racing animal including a human, eguine, or canine .
  • the disclosure further comprises 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 presence of an AMPK agonist and/or expression of one or more molecules listed in Tables 2, 4 or 6.
  • the disclosure also provides a composition comprising an AMPK agonist and a PPAR ⁇ agonist in a pharmaceutically acceptable carrier.
  • the composition may be an energy supplement, beverage, food product or pharmaceutical.
  • the AMPK agonist or PPAR agonist can be a salt, ester, prodrug, precursor or derivative thereof.
  • This disclosure illustrates that, despite expectations to the contrary, pharmacological activation of AMPK or endogenous PPAR ⁇ in adults, promote remodeling of skeletal muscle to an oxidative phenotype or increase running endurance in such subjects.
  • PPAR ⁇ agonists 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.
  • the expression of one or more genes and/or proteins that are uniquely regulated by the combination of exercise and PPAR ⁇ agonist administration can be used to identify subjects using drugs to enhance exercise performance.
  • the newly identified protein complexes including PPAR ⁇ and exercise-induced kinases (such as AMPK ⁇ l and/or AMPK cc2) , can be used to identify agents that have potential to affect PPAR ⁇ -regulated gene networks and the corresponding downstream biochemical and/or physiological effects .
  • FIG. IA is a series of bar graphs showing the effects of orally administered PPAR ⁇ agonist (GW1516) on mRNA expression levels of three biomarkers of fatty acid oxidation, uncoupling protein 3 (UCP3), carnitine palmitoyltransferase 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-PPAR5 transgenic (TG), and sedentary wild-type littermates of VP16-PPAR5 transgenic mice (WT) .
  • V sedentary vehicle-treated
  • GW1516-treated sedentary VP16-PPAR5 transgenic
  • WT sedentary wild-type littermates of VP16-PPAR5 transgenic mice
  • FIG. IB-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 ⁇ null (KO) primary muscle cells. * represents statistical signrficance between V and indicated groups (p ⁇ 0.05, One Way ANOVA; post hoc: Dunnett's Multiple Comparison Test) .
  • FIG. IE 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.
  • FIGS. 2A-C show the effects of administration of a PPAR ⁇ 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
  • Data in (B) and (C) are presented as mean ⁇ SEM.
  • * 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) .
  • FIGS. 3A-D are a series of bar graphs showing gene expression in guadriceps 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) .
  • B shows the relative gene expression levels of biomarkers for fatty acid storage (SCDl, FAS, SREBPIc) .
  • 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.
  • FIGS. 8A-C show the synergistic regulation of muscle gene expression by PPAR ⁇ and AMPK.
  • C Classification of 52 targets that were common to Tr+GW and AI+GW gene signatures. [0026] FIGS.
  • 10A-L demonstrate the AMPK-PPAR ⁇ interaction.
  • A- D show the expression of metabolic genes in wild type and PPAR ⁇ null (KO) primary muscle cells treated with V, GW, AI and GW+AI (bars from left to right) for 24 hours.
  • E-F, J AD293 cells were transfected with PPAR ⁇ +RXR ⁇ +Tk-PPRE along with control vector, AMPK ⁇ l, cc2 and/or PGC l ⁇ as indicated above.
  • E Induction of basal PPAR ⁇ transcriptional activity by AMPK ⁇ l or cc2.
  • FIG. 11A-I shows that AICAR increases running endurance.
  • A-F C57B1/6J mice were treated with vehicle (open bars or thin lines) or AICAR (500 mg/kg/day, 4 weeks) (closed bars or thick lines) .
  • A Representative immunoblots showing levels of UCP3, phospho-acetyl CoA carboxylase (ACC) , phospho-AMPK, and total-AMPK in quadriceps.
  • B Average body weight.
  • GenBank accession number The sequences given such GenBank accession numbers are incorporated by reference as they existed and were known as of December 29, 2007.
  • resveratrol The aerobic effects of resveratrol are thought to depend on activation of SIRTl-PGCIa coactivator complex in skeletal muscle.
  • downstream transcriptional factor (s) targeted by SIRTl/PGCla in mediating these effects are not known.
  • both SIRTl/PGCla and resveratrol activate multiple targets, and thus whether there is a specific signaling pathway that can be selectively activated by a synthetic drug to improve endurance is not known.
  • Exercise training activates a number of transcriptional regulators and serine-threonine kinases in skeletal muscles that contribute to metabolic reprogramming (Bassel-Duby and Olson, 2006) .
  • Overexpression of a constitutively active PPAR ⁇ (VP16-PPAR5) in skeletal muscles of transgenic mice preprograms an increase in oxidative muscle fibers, enhancing running endurance by nearly 100% in untrained adult mice (Wang et al., 2004).
  • AMPK AMP-activated protein kinase
  • AMP kinase agonists such as AICAR have been studied for insulin regulation, diabetes and obesity. However, AMP kinases have not previously been demonstrated to promote muscle tone or to improve endurance or exercise. The disclosure provides that AMPK agonists provide a beneficial effect for muscle wasting diseases and disorders, benefits to sedentary subject or immobilized limbs and in combination with PPARd agonist an unexpected synergistic effect.
  • PPAR ⁇ agonist such as, for example, GW1516 (shown to be bioactive in humans) enables mice to run 60%-75% longer and further than the nontreated controls only; however, such an effect is only seen when administration of a PPAR ⁇ agonist is combined with exercise training.
  • This "super- endurance phenotype" is linked to a transcriptional boost provided by exercise-activated AMPK resulting in a novel endurance gene signature (see, e.g., Figure 10L)
  • AMP-activated protein kinase (AMPK) and AMPK kinase (AMPKK) comprise a protein kinase cascade.
  • the AMPK cascade regulates fuel production and utilization intracellularly. For example, low cellular fuel (e.g., an increase in AMP concentration) increase AMPK activity.
  • AMPK functions either to conserve ATP or to promote alternative methods of ATP generation.
  • AMP-activated protein kinase or AMPK consists of three proteins (subunits) that together make a functional enzyme that plays a role in cellular energy homeostasis. It is expressed in a number of tissues, including the liver, brain, and skeletal muscle. Activation of AMPK has been shown to activate hepatic fatty acid oxidation and ketogenesis, inhibit cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibit adipocyte lipolysis and lipogenesis, stimulate skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulate insulin secretion by pancreatic beta-cells.
  • Triggering the activation of AMPK can be carried out with increasing concentrations of AMP.
  • the ⁇ subunit of AMPK undergoes a conformational change so as to expose the active site (Thr-172) on the ⁇ subunit.
  • the conformational change of the y subunit of AMPK can be accomplished under increased concentrations of AMP.
  • Increased concentrations of AMP will give rise to the conformational change on the ⁇ subunit of AMPK as two AMP bind the two Bateman domains located on that subunit.
  • the AMPK agonist comprises an AICAR compound.
  • Other compounds useful in the method of the disclosure include analogs of AICAR (such as those disclosed in U. S. Patent No. 5,7 7 7,100, hereby incorporated by reference herein) and prodrugs or precursors of AICAR (such as those disclosed in U. S. Patent No.
  • AMPK AMPK-activating compounds
  • AMPK-activating compounds include, in addition to the aforementioned leptin, adiponectin, and metformin, AICAR (5-aminoimidazole-4- carboxamide) .
  • Other AMPK agonists include, but are not limited to, DRL-16536 (Dr.
  • AICAR for example, is taken into the cell and converted to ZMP, an AMP analog that has been shown to activate AMPK.
  • ZMP acts as an intracellular AMP mimic, and, when accumulated to high enough levels, is able to stimulate AMPK activity (Corton, J. M. et.al. Eur. J. Biochem. 229: 558 (1995)).
  • ZMP also acts as an AMP mimic in the regulation of other enzymes, and is therefore not a specific AMPK activator (Musi, N. and Goodyear, L. J. Current Drug Targets--Immune, Endocrine and Metabolic Disorders 2:119 (2002) ) .
  • the disclosure provides methods for stimulating an "exercise conditioned state" in a subject.
  • the method includes administering to a subject an AMPK agonist in an amount sufficient to simulate an energy deficient state in a subject.
  • energy deficient state refers to a state in which the ⁇ subunit of AMPK undergoes a conformation change, there is increased catabolism of fat stores in a subject or there is conservation of ATP energy stores or a metabolic state found in an exercising individual.
  • the exercise conditioned state can be accomplished in the absence of exercise using the AMPK agonist of the disclosure (an "exercise- free conditioning”) .
  • Stimulating and exercise conditioned states not only has benefits to athletic training, but also provides benefits to subject who, do to injury, disease or disorder, are unable to exercise a limb/muscle or where the subject is sedentary or immobilized. By stimulating an exercise conditioned state the subject can maintain muscle tone and/or mass in the limb or muscle and promote health or recovery.
  • 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) .
  • 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.
  • 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.
  • repeated episodes of physical activity are needed to achieve an exercise-induced effect (such as, increased aerobic capacity or increase running endurance) for a PPAR ⁇ agonist to be effective.
  • an AMPK agonist eliminates that need for exercise to see an effective exercise-induced or promoting effect.
  • no exercise is needed when an AMPK agonist is taken alone or prior to an PPAR ⁇ agonist.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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 (in) anaerobic exercise (such as, weight training, functional training or sprinting) which is believed to, at least, increase muscle strength and mass.
  • flexibility exercise such as, stretching
  • aerobic exercise such as, aerobic exercise
  • anaerobic exercise such as, weight training, functional training or sprinting
  • 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.
  • 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.
  • the disclosed methods contemplate enhancing any known or observable effect of exercise (such as an aerobic exercise, like walking or running) .
  • running endurance e.g., running distance and/or running time
  • the methods and compositions are useful for treating a subject having muscle immobility, muscle wasting disease or disorder or a sedentary subject.
  • muscle tone or mass is improved or maintained in a subject having muscle immobility, muscle wasting disease or disorder or a sedentary activity compared to a subject that does not receive a composition of the disclosure (e.g., an AMPK agonist, or a combination of an AMPK agonist or PPAR agonist) .
  • a composition of the disclosure e.g., an AMPK agonist, or a combination of an AMPK agonist or PPAR agonist
  • the compositions and methods of the disclosure can reduce muscle loss or the rate of muscle loss in a subject having a muscle wasting disease by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a subject not receiving a composition of the disclosure.
  • Muscle weakness, tone, and atrophy result from a number of diseases and disorders including denervation or prolonged muscle disuse. When deprived of regular exercise, muscle fibers lose both bulk and length, producing a visible loss of muscle size and contour and apparent emaciation or deformity in the affected area. Even slight atrophy usually causes some loss of motion or power. Atrophy usually results from neuromuscular disease or injury.
  • Muscle wasting disease includes muscle weakness and atrophy that typically begin in a limb (e.g., hand, arm, or leg). Eventually, weakness and atrophy spread to the trunk, neck, tongue, larynx, pharynx, and legs; progressive respiratory muscle weakness leads to respiratory insufficiency. Other findings include muscle flaccidity, fasciculations, hyperactive deep tendon reflexes, slight leg muscle spasticity, dysphagia, impaired speech, excessive drooling, and depression.
  • Fibrous scar tissue formation, pain, and loss of serum proteins from severe burns can limit muscle movement, resulting in atrophy.
  • Muscle atrophy is a late sign of irreversible ischemia, along with contractures, paralysis, and loss of pulses.
  • Herniated disk can cause muscle weakness, disuse, and ultimately, atrophy.
  • Hypercortisolism may cause limb weakness and eventually atrophy.
  • Hypothyroidism can cause weakness and atrophy of proximal limb muscles. Injuries that result in immobilization of a limb can lead to muscle atropy and muscle wasting including, but not limited to, meniscal tears or broken bones or other cartilage injuries resulting from prolonged knee or limb immobility.
  • Multiple sclerosis is a degenerative disease that cause arm and leg atrophy as a result of chronic progressive weakness; spasticity and contractures may also develop. Osteoarthritis eventually causes atrophy proximal to involved joints as a result of progressive weakness and disuse. Parkinson's disease causes muscle rigidity, weakness, and disuse producing muscle atrophy. Peripheral nerve trauma or injury to or prolonged pressure on a peripheral nerve leads to muscle weakness and atrophy. Peripheral neuropathy can lead to muscle weakness that progresses slowly to flaccid paralysis and eventually atrophy. Distal extremity muscles are generally affected first.
  • Associated findings include loss of vibration sense; paresthesia, hyperesthesia, or anesthesia in the hands and feet; mild to sharp, burning pain; anhidrosis; glossy red skin; and diminished or absent deep tendon reflexes. Damaged spinal nerve roots can cause muscle atrophy as well as weakness. Rheumatoid arthritis causes muscle atrophy in the late stages of this disorder, as joint pain and stiffness decrease range of motion and discourage muscle use. Spinal cord injury or trauma can produce severe muscle weakness and flaccid, then spastic, paralysis, eventually leading to atrophy. Stroke may produce contralateral or bilateral weakness and eventually atrophy of the arms, legs, face, and tongue.
  • Associated signs and symptoms depend on the site and extent of vascular damage and may include dysarthria, aphasia, ataxia, apraxia, agnosia, and ipsilateral paresthesia or sensory loss.
  • Prolonged steroid therapy can interfere with muscle metabolism and can lead to atrophy, most prominently in the limbs.
  • prolonged immobilization from bed rest, casts, splints, or traction may cause muscle weakness and atrophy. Any of these diseases or disorder can be treated with a composition or combination of compositions of the disclosure.
  • Other muscle wasting disease or disorders are recognized in the art.
  • the disclosure provides compositions and methods useful for treating such disease and disorders by promoting or maintaining muscle mass or tone.
  • administering an AMPK agonist such as AICAR alone or in combination with a PPARd agonist can promote muscle tone or mass. In healthy subjects this can contribute to an enhanced exercise effect. In subjects with a potential for losing muscle mass or having a muscle wasting disease or disorder the compositions and methods of the disclosure can slow or eliminate the rate of such muscle atrophy.
  • Enhancing an exercise effect means that such effect is improved in a subject more than would have occurred by exercise alone.
  • an enhanced exercise effect is determined by discontinuing administration of an AMPK agonist or a PPAR ⁇ 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) .
  • an exercise effect of interest such as a AMPK-agonist induced effect or the PPAR ⁇ - enhanced portion of which is lost upon discontinuance of an AMPK agonist or PPAR ⁇ agonist 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.
  • a subject is a living multi-cellular vertebrate organism ⁇ e.g., human and/or non-human animals).
  • 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) .
  • a subject is an adult, an exercise-trained subject, or a healthy subject. Some representative adult, human subjects are 16 years old or older, 18 years old or older, or 21 years old or older. In some embodiment, the subject does not perform any routine exercise.
  • 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.
  • 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.
  • the AMPK agonist may be administered orally, parenterally, intramuscularly, intravascularly or by any appropriate route.
  • a subject can be any mammalian subject (e.g., equine, canine, or human).
  • An AMPK agonist is particularly useful in combination with agents that promote muscle fiber development and growth. Examples of such agents include agonists of the PPAR family of proteins.
  • 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.
  • 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.
  • PPAR isotypes exist in mammals: ⁇ (also known as NRlCl) , Y (also known as NR1C3) , and ⁇ (also known as ⁇ or NR1C2) .
  • PPAR ⁇ 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) .
  • PPAR ⁇ transgenic mouse models and discoveries aided by the development of high-affinity PPAR ⁇ agonists have revealed PPAR ⁇ 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) .
  • a subject comprising administering an AMPK agonist sufficient to produce an exercise effect, enhance mitochondrial expression or activity.
  • the subject is a an exercising subject.
  • the subject is a sedentary subject.
  • the subject is immobilized or has an immobilized limb.
  • the disclosure further includes administering to the subject an effective amount of a PPAR ⁇ 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) .
  • 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.
  • a PPAR ⁇ agonist is administered on the same day(s) on which the AMPK agonist is administered or on the same day upon which physical exertion will be performed.
  • the combination of an AMPK agonist and PPAR agonist are administered on the same day as a physical exertion will be performed.
  • administration of the AMPK agonist is by oral administration, intravenous injection, intramuscular injection, and/or subcutaneous injection.
  • the effective amount of the AMPK agonist is from about 1 mg per day to about 20 mg per day in a single dose or in divided doses.
  • administration of the PPAR ⁇ agonist is by oral administration, intravenous injection, intramuscular injection, and/or subcutaneous injection.
  • the effective amount of the PPAR ⁇ agonist is from about 1 mg per day to about 20 mg per day in a single dose or in divided doses.
  • PPAR ⁇ agonists Preferably such agonist would be non-toxic in the subject to which it is administered.
  • exemplary PPAR ⁇ 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/97/2784 ⁇ , 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) .
  • other PPAR ⁇ 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) .
  • the PPAR ⁇ 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) methyl) sulfanyl)phenoxy) acetic acid as has been sho ⁇ n to be is bioactive in humans (Sprecher et al., Arterioscler . Thromb. Vase. Biol. 27(2): 359-65, 200 7 ) .
  • GW1516 is administered orally, for example 1 mg - 20 mg/day, such as 2.5 mg or 10 mg per day.
  • the disclosed methods envision the use of any method of administration, dosage, and/or formulation of an AMPK agonist alone or in combination with a PPAR ⁇ 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.
  • AMPK agonist of the disclosure may be administered in the form of a drug to a human or an animal.
  • the AMPK agonist may be incorporated into a variety of foods and beverages or pet foods so as to be consumed by humans or animals.
  • the AMPK agonist may be applied to a common food or beverage; or may be applied to a functional food or beverage, a food for a subject suffering a disease, or a food for specified health use, the food (or beverage) bearing a label thereon indicating that it has a physiological function; for example, energy supplement, exercise enhancer or the like.
  • the AMPK agonist alone or in combination with a PPAR ⁇ agonist may be formulated into a drug product; for example, a peroral solid product such as a tablet or a granule, or a peroral liquid product such as a solution or a syrup.
  • Modes of administering an AMPK agonist (or a formulation including a PPAR ⁇ 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.
  • the AMPK agonist or AMPK agonist and PPAR ⁇ agonist is administered orally.
  • an AMPK agonist or a formulation including a PPAR ⁇ agonist
  • routes for administration of an AMPK 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.
  • an AMPK agonist or an AMPK agonist and PPAR ⁇ agonist may be desirable to administer an AMPK agonist or an AMPK agonist and PPAR ⁇ 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.
  • a pump such as a transplanted minipump
  • a pump may be used to deliver an AMPK agonist or a combination of an AMPK agonist and a PPAR ⁇ agonist (or a formulation including a PPAR ⁇ agonist)
  • a pump such as a transplanted minipump
  • a pump such as a transplanted minipump
  • an AMPK agonist (or a formulation including a PPAR ⁇ 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).
  • an AMPK agonist alone or in combination with a PPAR ⁇ 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.
  • polymeric materials useful in controlled- released formulations are known (see, e.g., Ranger et al., Macromol. ScL Rev. Macromol. Chem. 23, 61, 1983; Levy et al., Science 228, 190, 1985; During et al., Ann. Neurol. 25, 351, 1989; Howard efc al., J. Neurosurg. 71, 105, 1989).
  • an 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.
  • the disclosed methods contemplate the use of any dosage form of an AMPK agonist alone or in combination with a PPAR ⁇ agonist (or formulation containing the same) that delivers the agonist (s) 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 biologies).
  • 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 e.g., ointments, creams, and/or gels
  • 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, ul
  • 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 an AMPK agonist or PPAR ⁇ agonist) w/ith pharmaceutically acceptable excipients and/or other components (such as one or more other active ingredients) .
  • An aim of a drug formulation is to provide proper administration of an active ingredient (such as an AMPK agonist alone or in combination with a PPAR ⁇ agonist) to a subject.
  • a formulation should suit the mode of administration.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the U.S.
  • 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.
  • 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.
  • Excipients may also include, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodext ⁇ ns, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.
  • a dosage regimen utilizing a PPAR ⁇ 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 ⁇ agonist formulation employed. An ordinarily skilled physician or veterinarian can readily determine an effective amount of a PPAR ⁇ agonist (or formulation thereof) useful for enhancing an exercise effect in a subject .
  • oral dosages of an AMPK agonist alone or in combination with a PPAR ⁇ 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) .
  • a neutral molecule which may be a free acid or free base
  • 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.
  • formulation including an AMPK agonist alone or in combination with a PPAR ⁇ agonist may be administered in divided doses of two, three, or four times daily.
  • 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) .
  • 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.
  • 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.
  • 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.
  • an 80 kg subject receiving 8 ml trfice 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.
  • an AMPK 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) .
  • the dose is varied during the course of an AMPK agonist formulation 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.
  • long-term administration of an AMPK agonist or combination therapy (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).
  • 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 presence of ZMP or other non-naturally occurring AMP analog and/or 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.
  • a ZMP or AMP analog will be measured alone or in combination with whether (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, ClQ 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
  • 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. 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 ⁇ receptor or an AMPK-binding fragment thereof; (ii) providing a second component comprising an AMP- activated protein kinase (AMPK), AMPK ⁇ l, AMPK ⁇ 2, or a PPAR ⁇ -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.
  • AMPK AMP- activated protein kinase
  • 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.
  • PES performance-enhancing substances
  • 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 ⁇ agonist) that results in enhanced physical performance (see Table 2) .
  • the particular genes (and/or proteins encoded thereby) were up- or dorfn-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.
  • 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
  • AMPK agonist such as AICAR
  • LC-MS/MS assay utilizes a structurally related analog to the AMPK activator as an internal standard.
  • the adenosine analog tubercidin (4-amino-7-beta-D- ⁇ bufuranosyl-7H-pyrrolo [2, 3-d] py ⁇ mindine; 7-beta-D-riburuanosyl- 7H-pyrrolo [2, 3-d] pyrimindin-4-amine; 7-deazaadenosine) is structurally related to AICAR and can be used as the internal standard rfhen assaying for AICAR. Samples containing a known concentration of the internal standard can be directly analyzed by LC-MS/MS.
  • the substance of interest and the internal standard can be resolved on a LC-MS/MS (tandem mass spec) system, for example, suing a hydrophobic column with a solvent or acidified solvent gradient, and detected as positive ions using selective reaction monitoring.
  • Other analyzers and detection methods knouvn in the art can be used.
  • a standard curve over one two , three or four orders of magnitude constructed using agonist-spiked serum can be constructed to facilitate quantification of AICAR levels.
  • the biomarkers of substance-induced performance enhancement identified herein and useful in a disclosed method include one or more (or any combination of) of an AMP analog or the genes (and/or proteins encoded thereby) listed in Table 2, and in some examples listed in Table 4.
  • 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.
  • at least one gene (and/or protein encoded thereby) from each class listed in Table 2 e.g., cytokines, fat metabolism is analyzed.
  • 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, ClQ 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; lnsulin- like growth factor binding protein 5; protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), beta isoform;
  • 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.
  • 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 El 1.
  • 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.
  • 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).
  • Yet other method embodiments involve the detection in a sample of a combination of an AMP analog (e.g., ZMP) and one or more 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 ⁇ administration.
  • an AMP analog e.g., ZMP
  • a subject is a living multi-cellular vertebrate organism (e.g., human and/or non-human animals).
  • 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) .
  • 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.
  • 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) .
  • Any sample from a subject e.g., a biological sample
  • a subject in which can be detected an AMP analog and/or one or more genes and/or proteins uniquely regulated by exercise in combination with PPAR ⁇ 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.
  • Methods of detecting the expression of genes and/or proteins in a sample are very well known (see, e.g., U.S. Patent 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).
  • 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 ⁇ 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.
  • nucleic acid amplification such as, polymerase chain reaction (PCR) or any adaptation thereof, ligase chain reaction, transcription-based amplification systems, cycling probe reaction, Q ⁇ replicase amplification, strand displacement a
  • 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) .
  • 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
  • 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 ⁇ agonist (e.g., GW1516) .
  • a PES such as a PPAR ⁇ agonist
  • 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 disclosure identifies a previously unknown protein- protein interaction between PPAR ⁇ and particular exercise-induced kinases (e.g., AMPK, such as the AMPK ⁇ l and/or AMPK ⁇ 2 subunit(s) of AMPK) .
  • AMPK exercise-induced kinases
  • the interaction between PPAR ⁇ and AMPK may have important functional outcomes, such as enhancing exercise performance (e.g., aerobic exercise performance, such as running endurance) in a subject .
  • agents e.g., having potential to enhance exercise performance (e.g., aerobic exercise performance, such as running endurance) in a subject.
  • agents that affect e.g., enhance, weaken, or substantially disrupt
  • agents that affect e.g., increase, decrease, or substantially eliminate
  • AMPK-dependent phosphorylation of a PPAR ⁇ complex are identified.
  • 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 associated with AMPK activation cascade (e.g., AMPK-dependent phosphorylation of a PPAR ⁇ complex), or useful for modifying or affecting a protein-protein interaction (e.g., PPAR ⁇ - AMPK interaction) or ATP metabolism.
  • AMPK activation cascade e.g., AMPK-dependent phosphorylation of a PPAR ⁇ complex
  • PPAR ⁇ - AMPK interaction e.g., PPAR ⁇ - AMPK interaction
  • Any agent that has potential (whether or not ultimately realized) to modulate any aspect of the PPAR ⁇ - AMPK interaction disclosed herein is contemplated for use in the screening methods of this disclosure.
  • 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 .
  • 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, 12: '1 ⁇ l-118, 1993), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')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.
  • phosphopeptides including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang et al., Cell, 12: '1
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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 ⁇ protein-protein interaction) .
  • the compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • 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.
  • PPAR ⁇ forms a protein-protein interaction with AMPK or one or more of its subunits (such as AMPK ⁇ l and/or AMPKcc2) .
  • Agents that affect (e.g., increase or decrease) an AMPK- PPAR ⁇ interaction or AMP-dependent phosphorylation of a PPAR ⁇ 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.
  • 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.
  • 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.
  • PPAR ⁇ or AMPK such as AMPK ⁇ l or AMPKcc2
  • PPAR ⁇ ligand optionally is included (or is omitted) in disclosed methods.
  • a "direct association" between two or more polypeptides 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 ⁇ to AMPK (or AMPK ⁇ l and/or AMPK ⁇ 2) and vice versa) is considered “specific binding” .
  • Agents that affect an AMPK-PPAR ⁇ interaction can be identified by a variety of assays, including solid-phase or solution-based assays.
  • PPAR ⁇ or an AMPK-binding fragment thereof and AMPK or a subunit thereof are mixed under conditions in which PPAR ⁇ 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, fluorescein, 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.
  • 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.
  • 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.
  • 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).
  • each of two proteins that are capable of physical interaction are labeled with fluorescent dye molecule tags with different emission spectra and overlapping adsorption spectra.
  • fluorescent dye molecule tags with different emission spectra and overlapping adsorption spectra.
  • FRET fluorescence resonance energy transfer
  • FRET allows one to determine the kinetics of two interacting molecules based on the emission spectra of the sample.
  • two labeled protein components are added under conditions where their interaction resulting in FRET emission spectra.
  • 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.
  • Interactions between PPAR ⁇ (or AMPK-binding fragments thereof) and AMPK or AMPK ⁇ l or AMPK ⁇ 2 (or PPAR ⁇ -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 ⁇ (or AMPK-binding fragments thereof) and AMPK or AMPK ⁇ l or AMPK ⁇ 2 (or PPAR ⁇ -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.
  • one or more AMPK (such as AMPK ⁇ l and/or AMPKcc2) -binding fragments of PPAR ⁇ and/or one or more PPAR ⁇ -binding fragments of AMPK (such as AMPK ⁇ l and/or AMPK ⁇ 2) are used.
  • Polypeptide fragments having the desired binding activities can be identified by making a series of defined PPAR ⁇ fragments and/or AMPK (such as AMPK ⁇ l or AMPK ⁇ 2) fragments using methods standard in the art.
  • cDNA encoding the protein (s) of interest 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.
  • 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.
  • a PPAR ⁇ -AMPK such as AMPK ⁇ l and/or AMPKcc2
  • complexes including one or both of PPAR ⁇ -binding AMPK fragments and/or AMPK- binding PPAR ⁇ fragments or the affinity of PPAR ⁇ (or AMPK-binding fragments thereof) and AMPK (or PPAR ⁇ -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) .
  • 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
  • a PPAR ⁇ -AMPK such as AMPK ⁇ l and/or AMPKcc2
  • complexes including one or both of PPAR ⁇ -binding AMPK fragments and/or AMPK-binding PPAR ⁇ fragments or the affinity of PPAR ⁇ (or AMPK-binding fragments thereof) and AMPK (or PPAR ⁇ - 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) .
  • 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
  • AMPK e.g., AMPK ⁇ l and/or AMPK ⁇ 2
  • Agents that affect AMPK-dependent phosphorylation of the PPAR ⁇ 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 ⁇ complex .
  • 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.
  • a PPAR ⁇ complex (or a fragment thereof containing an AMPK phosphorylation site) and AMPK or one or more of it subunits (such as AMPK ⁇ l and/or AMPKcc2) or functional fragments thereof that are capable of phosphorylation are mixed under conditions whereby a PPAR ⁇ complex is phosphorylated by AMPK.
  • a PPAR ⁇ complex is adsorbed to a support, such as a microtiter well or beads.
  • AMPK or its one or more subunits (such as AMPK ⁇ l and/or AMPKcc2) 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 ⁇ complex.
  • phosphorylation of immobilized PPAR ⁇ 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.
  • such reactions can take place in solution (i.e., with no immobilized components)
  • PPAR ⁇ complex can be isolated from the solution (e.g., by immunoprecipitation with PPAR ⁇ - 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.
  • the phosphorylation of an AMPK is measured, wherein an agent that modulates AMPK activity is thus identified as an AMPK agonist.
  • the phosphorylation of a PPAR ⁇ 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) .
  • the phosphorylation of PPAR ⁇ 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) .
  • a PPAR ⁇ polypeptide useful in a disclosed screening method is any known PPAR ⁇ receptor. Also useful in the disclosed screening methods are homologs, functional fragments, or functional variants of a PPAR ⁇ that retains at least AMPK-bmding activity as described herein for a prototypical PPAR ⁇ polypeptide (see Example 6) .
  • amino acid sequences of prototypical PPAR ⁇ polypeptides are well known.
  • Exemplary PPAR ⁇ amino acid sequences and PPAR ⁇ -encoding nucleic acid sequences are described, for instance, in U.S. Patent 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_035275 (GI : 33859590) (Mus musculus amino acid sequence); NM_011145.3 (GI : 89001112) (Mus musculus nucleic acid sequence); NP_006229 (GI : 5453940) (Homo sapiens amino acid sequence); NM_006238.3 (GI : 89886454) (Homo sapiens nucleic acid sequence); NP_037273 (GI : 69S13S4) (Rattus norvegicus amino acid sequence); NM_013141.1 (GL6981383) (Rattus norvegicus nucleic acid sequence); NP_990059 (gi45382025) (Gallus gallus amino acid sequence) or NM_204728.1 (GI : 45382024) (Gallus gallus nucleic acid sequence) .
  • a PPAR ⁇ homolog or functional variant shares at least 60% amino acid sequence identity with a prototypical PPAR ⁇ 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. Patent No. 5,861,274, U.S. Pat. Appl. Pub. No. 20060154335, or GenBank Accession No.
  • a PPAR ⁇ homolog or functional variant has one or more conservative amino acid substitutions as compared to with a prototypical PPAR ⁇ 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. Patent No.
  • NP_035275 GI : 33859590
  • NP_006229 GI: 5453940
  • NP_037273 GL.69$13$4
  • NP_990059 gi45382025)
  • NP_990059 gi45382025)
  • a PPAR ⁇ functional fragment such as an AMPK-binding fragment
  • a PPAR ⁇ functional fragment can be any portion of a full-length known PPAR ⁇ 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 ⁇ function of interest (e.g., AMPK binding) .
  • PPAR ⁇ encompasses known functional motifs (such as ligand-binding domain, a DNA-binding domain, and a transactivation domain) .
  • Mammalian AMP-activated kinase 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 ( ⁇ l and ⁇ 2) .
  • An AMPK (such as AMPK ⁇ l and/or AMPK ⁇ 2) polypeptide useful in a disclosed screening method is any known AMPK protein or subunit thereof (such as AMPK ⁇ l and/or AMPK ⁇ 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 ⁇ l and/or AMPK ⁇ 2) that retains at least PPAR ⁇ -binding activity as described herein (see Example 6) .
  • amino acid sequences of prototypical AMPK subunits (such as AMPK ⁇ l and/or AMPK ⁇ 2) (and nucleic acids sequences encoding prototypical AMPK subunits (such as AMPK ⁇ l and/or AMPK ⁇ 2) ) are well known.
  • Exemplary AMPK ⁇ l amino acid sequences and the corresponding nucleic acid sequences are described, for instance, in GenBank Accession Nos.
  • NM_206907.3 (GI : 94557298) (Homo sapiens transcript variant 2 REFSEQ including amino acid and nucleic acid sequences); NM 006251.5 (GI : 94557300) (Homo sapiens transcript variant 1 REFSEQ including amino acid and nucleic acid sequences); NM_001013367.3 (GI : 94681060) (Mus musculus REFSEQ including amino acid and nucleic acid sequences); NMJ) 01039603.1 (GI : 88853844) (Gallus gallus REFSEQ including amino acid and nucleic acid sequences); and NM_019142.1 (GI: 11862979XRaJfWS norvegicus REFSEQ including amino acid and nucleic acid sequences) .
  • Exemplary AMPK ⁇ 2 amino acid sequences and the corresponding nucleic acid sequences are described, for instance, in GenBank Accession Nos. NM_006252.2 (GI : 468 ⁇ 067) (Homo sapiens REFSEQ including amino acid and nucleic acid sequences) ; NM_178143.1 (GI : 54792085) (Mus musculus REFSEQ including amino acid and nucleic acid sequences); NM_001039605.1 (GI : 88853850) (Gallus gallus REFSEQ including amino acid and nucleic acid sequences) ; and NM_214266.1 (GI : 4 ⁇ 523597) (Mus musculus REFSEQ including amino acid and nucleic acid sequences) .
  • a homolog or functional variant of an AMPK subunit shares at least 60% amino acid sequence identity with a prototypical AMPK ⁇ l and/or AMPK ⁇ 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_206907.3; NM_006251.5; NMJ) 01013367.3; NM_001039603.1; NM_019142.1; NMJD06252.2; NM_178143.1; NM_001039605.1 ; or NM 214266.1.
  • a homolog or functional variant of an AMPK subunit has one or more conservative amino acid substitutions as compared to a prototypical AMPK ⁇ l and/or AMPK ⁇ 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_206907.3; NM_006251.5; NM_001013367.3; NM_001039603.1; NM_019142.1; NM_006252.2; NM_178143.1; NM_001039605.1 ; or NM 214266.1.
  • Exemplary conservative amino acid substitutions have been previously described herein.
  • Some method embodiments involve a functional fragment of AMPK or a subunit thereof (such as AMPK ⁇ l and/or AMPK ⁇ 2), including a PPAR ⁇ -binding fragment or a fragment with PPAR ⁇ phosphorylation activity.
  • Functional fragments of AMPK or a subunit thereof can be any portion of a full-length or intact AMPK polypeptide complex or subunit thereof (such as AMPK ⁇ l and/or AMPK ⁇ 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 ⁇ l and/or AMPK ⁇ 2) function of interest (e.g., PPAR ⁇ binding and/or PPAR ⁇ phosphorylation activity).
  • AMPK or AMPK ⁇ l and/or AMPK ⁇ 2
  • an AMPK PPAR ⁇ -binding fragment includes a functional fragment encompassing (or consisting of) the catalytic core domain of an alpha subunit of AMPK (such as AMPK ⁇ l and/or AMPK ⁇ 2) .
  • an "isolated" biological component such as a polynucleotide, polypeptide, or cell
  • a mixed sample such as a cell or tissue extract
  • 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) .
  • the term "purified" refers to the removal of one or more extraneous components from a sample.
  • 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.
  • 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.
  • 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.
  • NCBI National Center for Biotechnology Information
  • BLASTTM Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • NCBI Bethesda, MD
  • 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 BLASTTM.
  • binding partner such as a binding agent
  • 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) .
  • specific binding sites are saturable.
  • 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.
  • the disclosure also provides methods for identifying agents useful for effecting muscle tone or mass.
  • the disclosure provides endurance gene signatures (see, e.g., Table 2 and Table 4) comprising genes that are modulated in the presence of AICAR or GW1516, or a combination of AICAR and GW1516. Such gene signature are useful for identifying agents that provides an increase in muscle tone or mass and thereby can modulate physical endurance.
  • a GW-AI endurance gene signature refers to a set of genes described in Table 4 or a subset thereof.
  • a GW-TR endurance gene signature refers to a set of genes described in Table 2 or a subset thereof.
  • An agent to be tested can be administered to a subject and the gene expression profile measure in a muscle sample (e.g., a biopsy) or other biological sample.
  • a muscle sample e.g., a biopsy
  • the gene expression profile comprises a set of the endurance gene signatures (e.g., the overlapping 52 genes associated with GW1516 and AICAR administration, see Table 4) or a subset thereof such an agent can be identified as an agent or drug useful for treating or modifying muscle activity.
  • Example 1 [00135] Administration of PPAR ⁇ agonist does not enhance physical performance in non-exercised subjects.
  • Wang et a previously demonstrated that skeletal muscle-specific expression of a constitutively active form of PPAR ⁇ 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 . , PLoC Biol., 2:e294, 2004).
  • This Example demonstrates that administration of a PPAR ⁇ agonist (G ⁇ / ⁇ 1516) to non-transgenic mice also results in the expression in skeletal muscle of some biomarkers of oxidative metabolism.
  • PPAR ⁇ 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.
  • Male C57B/6J mice (8 wks old) were obtained from Jackson Laboratory and housed in the SaIk 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.
  • 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) .
  • mice then were treated once per day for 4 weeks with vehicle or the PPAR ⁇ agonist, GW 1516 (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 .
  • QPCR Real time quantitative PCR
  • PPAR ⁇ agonist remodels skeletal muscle in exercised-trained subjects. 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.
  • 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) .
  • muscle fibers with high ATPase activity ⁇ e.g., type I (slow oxidative) muscle fibers) are darkly stained.
  • a PPAR ⁇ agonist e.g., GW1516
  • GW1516 a PPAR ⁇ agonist
  • administration of a PPAR ⁇ agonist does not significantly affect the number of type I (slow-twitch, oxidative) muscle fibers in hind limb muscles, but can increase the number of type I muscle fibers in hind limb muscles of trained subjects .
  • 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.
  • MHC I myosin heavy chain
  • MHC lib a marker of fast-twitch, glycolytic muscle fibers
  • GW1516 treatment did not alter the expression of MHC Ha (a marker of fast-twitch oxidative/glycolytic muscle fibers) in sedentary mice. Therefore, at least at the transcriptional level, the PPAR ⁇ agonist was capable of inducing some proteins characteristic of a slow-twitch muscle fiber phenotype .
  • This example illustrates that the genetic or pharmacologic activation of the PPAR ⁇ 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 ⁇ 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 ⁇ program in the sedentary, normal adult.
  • the cellular "template" for PPAR ⁇ effects on skeletal muscle is very different in a normal subject as compared to a genetically engineered transgenic subject.
  • PPAR ⁇ -regulated program 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 ⁇ -regulated program.
  • the constitutively active PPAR ⁇ transgene is active all the while muscle fiber specification is being determined and connections between muscle fibers and motor neurons are being made.
  • the effects of activation of endogenous PPAR ⁇ receptor by a single daily dose of a PPAR ⁇ 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 VPl ⁇ -PPAR ⁇ transgene.
  • PPAR ⁇ agonist treatment significantly affected fatty acid metabolism and markers of fatty acid oxidation.
  • exercise training also increases skeletal muscle mitochondrial density (e.g., Freyssenet et al., Arch. Physiol. Biochem., 104(2): 129-141, 1996).
  • PPAR ⁇ agonist treatment e.g., GW1516
  • 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 ⁇ -oxidation (FAO) .
  • mice 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 ⁇ ere gradually increased.
  • mice 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.
  • This interesting response profile includes a series of genes involved in the regulation of fatty acid storage (such as steroyl-CoA-desaturase (SCDl), fatty acyl coenzyme A synthase (FAS) and serum response element binding protein Ic (SREBPIc) ) 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) .
  • protein expression was determined for selective oxidative biomarkers including myoglobin, UCP3, cytochrome c (CYCS) and SCDl, using Western blotting.
  • Protein homogenates were prepared from guadriceps 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) SCDl (Santacruz), and, as a loading control, tubulin (Sigma).
  • myoglobin Dako
  • UCP3 Affinity Bioreagents
  • cytochrome c Santacruz
  • SCDl SCDl
  • tubulin tubulin
  • mTG Muscle triglyceride
  • FIG. 4 Muscle triglyceride (mTG) content was measured as previously described (Wang et al., PLoC 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) .
  • Gene and/or protein expression that is induced by a combination of exercise and drug treatment (e.g., PPAR ⁇ 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.
  • one or more genes and/or proteins uniquely regulated by one or more drugs ⁇ e.g., PPAR ⁇ agonists) and exercise can be used as markers, for instance, of illicitly boosting performance in professional and/or amateur athletes .
  • PPAR ⁇ agonist enhances the physical performance of exercise-trained subjects.
  • 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 ⁇ gene network (in the VPl ⁇ -PPAR ⁇ transgenic mouse) lead to a distance-running phenotype (familiarly, a "marathon mouse") .
  • PPAR ⁇ agonist e.g., GW1516 treatment in conjunction with exercise produced an enriched remodeling program that included a series of transcriptional and post-translational adaptations in the skeletal muscle.
  • This Example provides methods used to demonstrate that administration of a PPAR ⁇ agonist (e.g., GW1516) surprisingly improves physical performance in exercised (trained) subjects .
  • mice 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) GWl516-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.
  • V vehicle-treated and sedentary
  • GW1516-treated and sedentary GW1516-treated and sedentary
  • Tr vehicle-treated and exercise trained
  • GW+Tr GWl516-treated and exercise trained
  • 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).
  • 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.
  • PPAR ⁇ 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 :E497-50 ⁇ , 1997; Mader et al., Int. J. Sports Med., 22:344-9, 2001; Schmitt et al., Physiol.
  • PPAR ⁇ agonist and exercise can co-operatively re -program the muscle genome and raise endurance limits.
  • 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) .
  • 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) .
  • 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.
  • Lamp2 0.608 neighbor of P ⁇ nc Ell Nope 0.452 thyroid hormone responsive SP0T14 homolog (Rattus ⁇ Thrsp 2.685 cytochrome P450. family 2, subfamily e, polypeptide 1 Cyp2el 2.941 complement factor D (adipsm) Cfd 2.828 transietolase Tkt 2 256 OXYGEN CARRIERS
  • C/EBP CCAAT/enliancer binding protem
  • alpha Cebpa 2 168 nuclear receptor subfamily 1 group D 7 member 2(Reverb-b) Nrld2 1 794
  • 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 (PEPCKl) , 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 .
  • FBP2 fructose bisphosphate 2
  • PEPCKl phosphoenolpyruvate carboxykinase 1
  • lactate dehydrogenase B lactate dehydrogenase B
  • fatty acyl-CoA oxidase and medium chain acyl-CoA dehydrogenase 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.
  • angiogenesis e.g., angiopoietin-like 4 protein/also a known regulator of lipid metabolism
  • angiogenesis e.g., angiopoietin-like 4 protein/also a known regulator of lipid metabolism
  • transcription e.g., C/EBP ⁇ , Reverb ⁇ , NURRl
  • substrate transport e.g., transferrin, chloride channel 5
  • 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 ⁇ 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.
  • PPAR ⁇ agonists e.g., PPAR ⁇ agonists
  • PPAR ⁇ directly interacts with exercise-activated kinases, p44/42 MAPK and AMPK.
  • 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. 5 7 4: 33-9, 2006).
  • the interaction between exercise- regulated kinases and PPAR ⁇ signaling is described in this Example.
  • the levels of phospho-p44/42 MAPK and phospho-AMPK ⁇ 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 ⁇ l antibodies were obtained from Cell Signaling. The phospho- specific AMPK ⁇ l antibody recognizes the key activating threonine in the activation loop.
  • 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 ⁇ activation and exercise or PPAR ⁇ and AMPK co-activation) (listed in Table 4) revealed that the majority of the targets were linked to oxidative metabolism.
  • TRANSCRIPTION nuclear receptor subfamily 4 group A, member 2 Nr4a2 1.776 0.437
  • TRANSPORT solute earner family 1 neutral amino acid transporter
  • Slcla5 1.939 1.511 member 5 two pore channel 1
  • Tpcnl 2.842 1.487 seminal vesicle secretion 5
  • AMPK increases transcription activation by PPAR ⁇ .
  • the genetic synergism described in Example 6 indicates that AMPK directly regulates the transcriptional activity of PPAR ⁇ in skeletal muscles.
  • An analysis of the effects of GW1516 and AICAR on gene expression in primary muscle cells isolated from wild type and PPAR ⁇ null mice was performed. Primary muscle cells were isolated from wild type and PPAR ⁇ 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.
  • RNA expression of UCP3, PDK4, LPL, and HSL was determined using real time quantitative PCR as described in Example 1. As shown in FIGS. 10A-D, synergism is dependent on PPAR ⁇ and lost in the null cells. Similar synergistic regulation of gene expression by GW1516 and AICAR was also observed in differentiated C2C12 cells.
  • 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 ⁇ , CMX-Tk-PPRE, or CMX- ⁇ GAL, or an hAMPK ( ⁇ l and ⁇ 2 subunits, Origene) expression vector using LipofectamineTM 2000 in accordance with the manufacturer's instructions. Anti-Flag antibody-conjugated beads were incubated overnight at 4OC 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 ⁇ subunit(s).
  • AD 293 cells were transfected with PPAR ⁇ and hAMPk ( ⁇ l or ⁇ 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 32P-ortho ⁇ hos ⁇ hate in phosphate-free DMEM for 20 hours (100 ⁇ Ci/5 ml) . Cells were washed three times with ice-cold phosphate-free DMEM and lysed in ice-cold lysis buffer.
  • AMPK may be present in a transcriptional complex with PPAR ⁇ where it can potentiate receptor activity via direct protein-protein interaction and/or by phosphorylating co-activators such as PGCIo.
  • PGCIo phosphorylating co-activators
  • AMPK can integrate multiple transcriptional programs by interacting not only with PPAR ⁇ but also other transcriptional regulators of metabolism (e.g., PGCIo, PPAR ⁇ ) (Hong et al., 2003; Leff, 2003; Bronner et al., 2004; Jaager et al . , 2007) . This raises the interesting question as to whether chemical activation of AMPK is sufficient to increase running endurance without exercise.
  • AMPK- PPAR ⁇ interaction comes from the observation that GW1516 and AICAR (AMPK activator) synergistically induce several endurance-related genes in wild-type but not in PPAR ⁇ null primary muscle cells. More importantly, treatment of animals with AICAR and GW1516 creates a gene signature in skeletal muscle that replicates up to 40% of the genetic effects of combined exercise and GW1516 treatment. Notably, the shared genes between the two profiles are linked to oxidative metabolism, angiogenesis, and glucose sparing, pathways that are directly relevant to muscle performance .
  • PPAR ⁇ is important for normal cardiac contractility, as well as for the endocrine function of adipose tissue (Wang et al., 2003; Cheng et al., 2004).
  • AMPK is thought to mediate its ability to lower blood glucose levels (Shaw et al., 2005).
  • exercise has beneficial effects in a wide range of pathophysiological conditions, such as respiratory disorders, cardiovascular abnormalities, type 2 diabetes, and cancer risk.
  • the disclosure demonstrates that synthetic PPAR ⁇ activation and exercise—and, more importantly, AMPK activation alone-provide a robust transcriptional cue that reprograms the skeletal muscle genome and dramatically enhances endurance.
  • the disclosure provides a strategy for reorganizing the preset genetic imprint of muscle (as ⁇ eIl as other tissues) rfith exercise mimetic drugs has therapeutic potential in treating certain muscle diseases such as wasting and frailty as well as obesity inhere exercise is known to be beneficial.
  • Enhancing exercise effect in a subject 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.
  • 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) .
  • the subjects are orally administered GW1516 [ (2-methyl- 4 ( ( (4-methyl-2- (4- trifluoromethylphenyl) -1, 3-thiazol-5- yl) methyl) sulfanyl) ⁇ henoxy) 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.
  • the exercise effect achieved in the treated subjects e.g., running endurance
  • 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) .
  • the exercise effect of interest is increased in treated subjects by at least 5%, such as at least 10% as compared to untreated subjects.
  • Identifying performance enhancing substances in an exercise-trained subject This example describes methods that can be used to identify performance- enhancing substances in an exercised-trained subject.
  • 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.
  • PES e.g., GW1516
  • 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.
  • 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.
  • a change in expression such as an increase or decrease
  • 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.
  • 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.
  • nucleic acids need not be isolated prior to analysis.
  • Nucleic acids can be contacted vjith 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 guantified.
  • the seguence of the oligonucleotide probe can bind specifically to a nucleic acid molecule represented by the sequences listed in Table 2 or 4.
  • 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 ⁇ eIl 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, NY, 1989.
  • Oligonucleotides specific to sequences listed in Table 2 or 4 can be chemically synthesized using commercially available machines.
  • oligonucleotides can then be labeled, for example with radioactive isotopes (such as 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).
  • radioactive isotopes such as 32P
  • non-radioactive labels such as biotin (Ward and Langer et al., Proc. Natl. Aca
  • Proteins in the biological sample can also be analyzed.
  • proteins are isolated using routine methods prior to analysis.
  • SELDI-TOF surface-enhanced laser desorption- iomzation time-of-flight
  • 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 chromato graphic 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.
  • 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.
  • 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) .
  • any standard detection system such as one that includes a secondary antibody conjugated to horseradish peroxidase.
  • Bancroft and Stevens Theory and Practice of Histological Techniques, Churchill Livingstone, 1982
  • Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998) .
  • expression in the test sample can be compared to levels found in cells from a subject who has not taken a PES.
  • the pattern of expression identified in the test subject can be compared to that shown in Table 2 or 4.
  • 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)
  • a PES such as a PPAR ⁇ agonist (e.g., GW1516) .
  • 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 ⁇ agonist (e.g., GW1516) .
  • a PES such as a PPAR ⁇ agonist
  • 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.
  • 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.

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Abstract

L'invention porte sur l'utilisation d'agonistes de l'AMPK (protéine kinase activée par l'AMP) pour améliorer les effets de l'exercice et modifier le métabolisme énergétique chez un sujet. L'invention porte également sur une combinaison d'agonistes de l'AMPK et de PPARδ (récepteur activé par les proliférateurs des peroxisomes), permettant d'améliorer les performances lors de l'exercice chez un sujet, sur des méthodes d'identification de performances lors de l'exercice, améliorées au moyen de substances chez un sujet, et sur des méthodes d'identification de composés modifiant l'interaction entre le PPARδ et les kinases induites par l'exercice.
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US8084108P 2008-07-15 2008-07-15
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CA2809958A1 (fr) 2010-08-31 2012-03-08 Snu R & Db Foundation Utilisation de la reprogrammation fƒtale d'un agoniste des ppar ?
US20140147431A1 (en) * 2010-09-20 2014-05-29 The Regents Of The University Of California Compositions and Methods for Modulating Desnutrin-Mediated Adipocyte Lipolysis
EP2554182A1 (fr) * 2011-08-03 2013-02-06 Universitat Pompeu-Fabra Sestrines de mammifères pour le traitement de l'amyotrophie
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CN108349904B (zh) 2015-10-07 2021-08-31 米托布里奇公司 Ppar激动剂、化合物、药物组合物及其使用方法
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