The benefits of endurance exercise on general health make it desirable to identify orally active agents that would mimic or potentiate the effects of exercise to treat metabolic diseases. Although certain natural compounds, such as resveratrol, have endurance-enhancing activities, their exact metabolic targets remain elusive. We therefore tested the effect of pathway-specific drugs on endurance capacities of mice in a treadmill running test. We found that PPARβ/δ agonist and exercise training synergistically increase oxidative myofibers and running endurance in adult mice. Because training activates AMPK and PGC1α, we then tested whether the orally active AMPK agonist AICAR might be sufficient to overcome the exercise requirement. Unexpectedly, even in sedentary mice, 4 weeks of AICAR treatment alone induced metabolic genes and enhanced running endurance by 44%. These results demonstrate that AMPK-PPARδ pathway can be targeted by orally active drugs to enhance training adaptation or even to increase endurance without exercise.
Skeletal muscle is an adaptive tissue composed of multiple myofibers that differ in their metabolic and contractile properties, including oxidative slow-twitch (type I), mixed oxidative-glycolytic fast-twitch (type IIa) and glycolytic fast-twitch (type IIb) myofibers.
Type I fibers preferentially express enzymes that oxidize fatty acids, contain slow isoforms of contractile proteins, and are more resistant to fatigue than are glycolytic fibers. Type II fibers preferentially metabolize glucose and express the fast isoforms of contractile proteins. Endurance exercise training triggers a remodeling program in skeletal muscle that progressively enhances performance in athletes such as marathon runners, mountain climbers, and cyclists. This involves change in metabolic programs and structural proteins within the myofiber that alter the energy substrate utilization and contractile properties that act to reduce muscle fatigue (
Training-based adaptations in the muscle are linked to increases in the expression of genes involved in the slow-twitch contractile apparatus, mitochondrial respiration, and fatty acid oxidation.
These adaptations that improve performance can also protect against obesity and related metabolic disorders.
Moreover, skeletal muscles rich in oxidative slow-twitch fibers are resistant to muscle wasting.
Given the numerous benefits of exercise on general health, identification of orally active agents that mimic or potentiate the genetic effects of endurance exercise is a long-standing, albeit elusive, medical goal. High doses of certain natural extracts such as resveratrol can improve endurance.
The aerobic effects of resveratrol are thought to dependent on activation of SIRT1-PGC1α coactivator complex in skeletal muscle. However, the downstream transcriptional factor(s) targeted by SIRT1/PGC1α in mediating these effects are not known. More importantly, both SIRT1/PGC1α 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.
We and others previously identified a critical role for PPARβ/δ (henceforth referred to as PPARδ) in transcriptional regulation of skeletal muscle metabolism.
Overexpression of a constitutively active PPARδ (VP16-PPARδ) in skeletal muscles of transgenic mice preprograms an increase in oxidative muscle fibers, enhancing running endurance by nearly 100% in untrained adult mice.
One of the best understood serine-threonine kinases is AMP-activated protein kinase (AMPK), a master regulator of cellular and organismal metabolism whose function is conserved in all eukaryotes.
In mammals, AMPK has been shown to contribute to glucose homeostasis, appetite, and exercise physiology.
These observations raise the question as to whether synthetic PPARδ or AMPK agonists can reprogram established fiber specification in adult muscle toward an overt endurance phenotype. We have found that the PPARδ agonist GW1516 (shown to be bioactive in humans; enables mice to run 60%–75% longer and further than the nontreated controls only when 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. A more critical role of AMPK in the super-endurance phenotype is revealed in our unexpected finding that the orally active AMPK agonist AICAR is sufficient as a single agent to improve running endurance by nearly 45% in nonexercised mice. Together, these results provide new insights into the pharmacological malleability of muscle performance.
GW1516 Increases Muscle Gene Expression but Not Endurance in Sedentary Mice
To examine whether treatment with PPARδ ligands alone can reprogram the muscle transcriptome and endurance capacity, we treated wild-type C57Bl/6J age matched cohorts with vehicle or GW1516 for 4 weeks. QPCR analysis of selective target genes confirmed that drug treatment induced oxidative metabolic biomarkers such as uncoupling protein 3 (Ucp3), muscle carnitine palmitoyl transferase I (mCPT I, Cpt 1b), and pyruvate dehydrogenase kinase 4 (Pdk4) (Figure 1A). These changes in gene expression were detected as early as 4 days after treatment, as well as with drug concentrations ranging from 2–5 mg/kg/day. Moreover, in all our gene expression studies, maximal effects of PPARδ activation were detected in predominantly fast-twitch (quadricep and gastrocnemius) but not slow-twitch (soleus) muscles (data not shown). In primary muscle cells cultured from wild-type and PPARδ null mice we confirmed that the induction of oxidative genes by GW1516 is mediated via selective activation of PPARδ in skeletal muscles (Figures S1A–S1C available online). Moreover, this is similar to the expression changes found in the same genes in muscles expressing the constitutively active VP16-PPARδ transgene (Figure 1A), supporting the concept that pharmacological activation of PPARδ is sufficient to initiate an oxidative response in adult skeletal muscle. To determine the functional effects of ligand, age- and weight-matched cohorts of treated and control mice were subjected to an endurance treadmill performance test before (week 0) and after (week 5) treatment. Curiously, running performance was unchanged by GW1516 treatment (Figure 1B). Furthermore, long-term drug treatment of up to 5 months also did not change running endurance (data not shown). These results indicate that pharmacologic activation of the PPARδ genetic program in adult C57Bl/6J mice is insufficient to promote a measurable enhancement of treadmill endurance.
GW1516 Remodels Skeletal Muscle in Exercise-Trained Mice
Since endurance exercise remodels the skeletal muscle to progressively alter performance we speculated whether coadministration of GW1516 in the context of exercise training might enhance anticipated changes in fiber type composition and mitochondrial biogenesis. The effect of GW1516 and exercise on fiber type composition was determined via metachromatic staining of cryosections of the gastrocnemius.
As expected from the results of the running performance in Figure 1B, there was no significant difference in the proportion of type I fibers between vehicle- and GW1516-treated sedentary mice (Figure 1C). In contrast, in trained mice, GW1516 increased the proportion of type I fibers (by ∼38%) compared to the vehicle-treated sedentary mice (Figures 1C and 1D). In addition to its effects on the fiber type, exercise training increases skeletal muscle mitochondrial biogenesis, which was measured as a function of mitochondrial DNA expression levels via quantitative real-time PCR (QPCR). Similar to type I fiber changes, mitochondrial DNA expression was not changed by drug alone but was increased by approximately 50% with the combination of exercise and GW1516 treatment (Figure 1E).
The effects of GW1516 treatment and exercise, singly or in combination, on components of the oxidative metabolism of fatty acids were further analyzed by measurement of the gene expression levels of selective biomarkers for fatty acid β oxidation. As expected, we found that previously examined genes such as Ucp3, Cpt 1b, and Pdk4 were upregulated by GW1516 but showed no further induction with exercise (Figure 2A). Unexpectedly, we discovered a second set of genes that show no response to exercise or drug alone but are robustly induced by the combination. This intriguing response profile includes a series of genes involved in the regulation of fatty acid storage (such as steroyl-CoA-desaturase [Scd1], fatty acyl coenzyme A synthase [FAS, Fasn] and serum response element binding protein 1c [SREBP1c, Srebf1c]) and fatty acid uptake (such as the fatty acid transporter [FAT, Cd36] and lipoprotein lipase [Lpl]) (Figures 2B, 2C, and 3).
We also measured the protein levels of selective oxidative biomarkers including myoglobin, UCP3, cytochrome c (CYCS), and SCD1. In each case, a more robust upregulation of protein expression was found by combining exercise and GW1516 treatment relative to either drug or exercise alone (Figure 2D). Altered triglycerides are one way to assess changes in muscle oxidative capacity. Triglyceride levels were unchanged in vehicle- or GW1516-treated sedentary mice but showed a striking increase with exercise. In contrast, this increase was completely reversed by GW1516 treatment, presumably because of enhanced fat utilization (Figure S1D).
GW1516 and Exercise Training Synergistically Increase Running Endurance
As described above, although GW1516 treatment alone induces widespread genomic changes associated with oxidative metabolism, it fails to increase running endurance. On the other hand, drug treatment in conjunction with exercise produces an enriched remodeling program that includes a series of transcriptional and posttranslational adaptations in the skeletal muscle. This suggests that exercise training serves as a key trigger to unmask a cryptic set of PPARδ target genes, leading us to re-examine the ability of the drug to modulate endurance. Indeed, the same dose and duration of GW1516 treatment that previously failed to alter performance, when paired with 4 weeks of exercise training, increases running time by 68% and running distance by 70% over vehicle-treated trained mice (Figures 3A and 3B, compare week 5). It is also important to note that comparison of running time and distance before (week 0) and after (week 5) exercise and drug treatment revealed a 100% increment in endurance capacity for individual mice, underscoring the robustness of the combination paradigm (Figures 3A and 3B). Finally, it is noteworthy that the combined effects of GW1516 and exercise reduces the ratio of epididymal fat to body weight and fat cross-sectional area in these mice (Figures S1E and S1F), suggesting the broader systemic effects of this protocol.
PPARδ Agonist and Exercise Establish an Endurance Gene Signature
To dissect the mechanism underlying the super-endurance phenotype, we conducted a comprehensive study of the muscle transcriptome induced by ligand, exercise, or the combination, which produced three overlapping networks of 96, 113, and 130 genes, respectively (Figure 3C). Approximately 50% of the target genes were common between GW1516 and exercise, demonstrating that PPARδ activation partially mimics exercise. To our surprise, combined GW1516 treatment and exercise established a unique gene expression pattern that was neither an amalgamation nor a complete overlap of the individual interventions (Figure 3C). This signature included 48 new target genes (Table S1) not regulated by either GW1516 or exercise alone while excluding 74 genes regulated by GW1516 or exercise (selective genes are listed in Table S3). The majority of the genes in the GW1516-exercise signature were induced (108/130), the components of which are described in Figure 3D. Although the largest gene subclass (32% of genes) was linked to positive regulation of aerobic capacity, additional pathways implicated in muscle remodeling and endurance were also represented in the signature (see Table S2 for detailed description). It is noteworthy that comparative expression analysis of the 48 exclusive genes of the endurance signature (but not of either intervention alone) revealed a striking similarity to “untrained” VP16-PPARδ transgenic mice (Figure 3E). This observation confirms the primary dependence of the 48 genes on PPARδ and points to the possibility that exercise-generated signals may function to synergize PPARδ transcriptional activity to levels comparable to transgenic overexpression.
AMPK-PPARδ Interaction in Transcriptional Regulation
What might be the molecular interface between mechanical exercise and PPARδ transcription? Exercise training is known to activate multiple kinases, among which AMPK has profound effects on skeletal muscle gene expression and oxidative metabolism.
We found increased AMPK activation in the quadriceps of exercised mice relative to the sedentary controls (Figure 4A). Furthermore and unexpectedly, AMPK is constitutively activated in muscles of VP16-PPARδ transgenic mice in absence of exercise or drug (Figure 4B). In contrast, in our experiments, GW1516 treatment alone does not activate AMPK in either sedentary or exercise trained muscles, as previously suggested by some.
Taken together, these results strongly suggest that the ability to promote endurance in mice is associated with activation of both AMPK and PPARδ.
According to this hypothesis, selective coactivation of AMPK and PPARδ would induce gene expression changes that mimic those triggered by combined exercise and PPARδ as well as VP16-PPARδ overexpression. To investigate this possibility, we compared the transcriptional changes induced in skeletal muscle by combined exercise and GW1516 treatment with that of combined AMPK activator (the cell-permeable AMP analog AICAR) and GW1516 treatment. It is noteworthy that simultaneous GW1516 and AICAR treatment created a unique gene expression signature in the quadriceps of untrained C57Bl/6J mice (Figure S2) that shares 40% of the genes with that of combined GW1516 treatment and exercise (Figure 4C). Classification of the 52 genes common to the two signatures (Figure 4D, listed in Table S4) revealed that the majority of the targets were linked to oxidative metabolism. Quantitative expression analysis of selective oxidative genes by QPCR showed that several of these biomarkers, including Scd1, ATP citrate lyase (Acly), hormone sensitive lipase (HSL) (Lipe), muscle fatty acid binding protein (mFABP, Fabp3), Lpl, and Pdk4, were induced in a synergistic fashion by GW1516 and AICAR in the quadriceps (Figures 4E–4J). It is also noteworthy that all of the above genes were induced in quadriceps of untrained VP16-PPARδ mice, where AMPK is constitutively active (Figure S1G). Collectively, these results show that interaction between AMPK and PPARδ substantially contributes to reprogramming of the skeletal muscle transcriptome during exercise.
AMPK Increases Transcriptional Activation by PPARδ
The above described pathway crosstalk raised the possibility that AMPK directly regulates the transcriptional activity of PPARδ in skeletal muscles. An analysis of the effects of GW1516 and AICAR on gene expression in primary muscle cells isolated from wild-type and PPARδ null mice revealed that synergism is completely dependent on PPARδ and lost in the null cells (Figures 5A–5D). These observations show that AMPK enhances a subset of ligand-dependent PPARδ transcriptional targets in a cell-autonomous fashion.
In this study, we show that the AMP-mimetic AICAR can increase endurance in sedentary mice by genetically reprogramming muscle metabolism in a PPARδ-dependent manner. We also found that a PPARδ agonist in combination with exercise synergistically induces fatigue-resistant type I fiber specification and mitochondrial biogenesis, ultimately enhancing physical performance. These changes correlate with an unexpected but interesting establishment of a muscle endurance gene signature that is unique to the drug-exercise paradigm. Such a signature is an outcome of molecular crosstalk and perhaps a physical association between exercise-activated AMPK and PPARδ. These findings identify a novel pharmacologic strategy to reprogram muscle endurance by targeting AMPK-PPARδ signaling axis with orally active ligands.
Transgenic overexpression as well as knockout studies have identified PPARδ and AMPK as key regulators of type I fiber specification and endurance adaptations during exercise.
Whether and how these endogenously expressed regulators can be targeted to reprogram adult muscle without exercise has been a subject of unresolved speculation. We found that the AMPK activator AICAR increased oxygen consumption and endurance in untrained adult mice in part by stimulating PPARδ-dependent oxidative genes. Despite a demonstrated role for PPARδ in endurance, 4 week treatment with a potent and selective agonist failed to alter either fiber type composition or endurance, revealing that direct and pharmacologic activation of PPARδ is insufficient to enhance running performance. In contrast, transgenic overexpression of activated PPARδ at birth preprograms the nascent myofibers to transdifferentiate into slow-twitch fibers, thus imparting a high basal endurance capacity to adult transgenic mice. Apparently, once fiber type specification is complete in adults, the potential plasticity of muscle to synthetic activation of a single transcriptional pathway is constrained. Along these lines, the unexpected yet successful reprogramming of endurance in untrained adults with synthetic AMP-mimetic might be linked to the ability of AMPK to simultaneously target multiple transcriptional programs governed by its substrates such as PGC1α, PPARα and PPARδ, triggering a genetic effect akin to exercise.
Interestingly, the recalcitrance of adult skeletal muscle endurance to manipulation by PPARδ agonist alone is relieved by combining drug treatment with exercise. Indeed, this strategy generates an endurance gene signature that is unique from either paradigm alone, reflecting a crosstalk between exercise and PPARδ signaling (Table S2). Although exercise activates a cascade of signaling events, we feel AMPK is central to this genetic adaptation for several reasons. First, AMPK is a metabolic sensor that detects low ATP levels (such as occur during exercise) and in turn increases oxidative metabolism.
Second, long-term effects of AMPK are in part mediated via regulation of gene expression (
Third, exercise induces activation and nuclear import of AMPK, where it can potentially interact with transcription factors (this study and
And finally, transgenic mice defective for AMPK activation exhibit reduced voluntary exercise (
The notion that exercise-activated AMPK interacts with PPARδ in regulating gene expression is supported by our demonstration that AMPK associates with PPARδ and dramatically increases basal and ligand-dependent transcription via the receptor. Despite physical interaction, we found that AMPK does not induce PPARδ phosphorylation in metabolic labeling studies. Interestingly, AMPK and its previously reported substrate PGC1α synergistically increased PPARδ transcription, suggesting indirect regulation of receptor function by AMPK via coregulator modification. Nevertheless, we cannot rule out the possible regulation of PPARδ by AMPK via direct protein-protein interaction. Indeed, regulation of other transcription factors by AMPK via similar mechanisms has been previously demonstrated (
A physiological validation of AMPK-PPARδ interaction comes from our observation that GW1516 and AICAR (AMPK activator) synergistically induce several endurance-related genes in wild-type but not in PPARδ 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 (Figure 4D, listed in Table S4).
Although not all genes regulated by either exercise (data not shown) or exercise-PPARδ interaction (nonoverlapping signature, Figure 4D) are AMPK dependent, two key findings assign a critical role for the kinase in promoting endurance compared to other known exercise signals (
First, AMPK is constitutively active in VP16-PPARδ transgenic muscles that exhibit endurance without exercise. Second, AMPK activation by AICAR was sufficient to increase running endurance without additional exercise signals. Strikingly, the majority of the oxidative genes (30 out of 32) upregulated by AICAR are active in super-endurance VP16-PPARδ mice and perhaps are the core set of genes required to improve muscle performance. Interestingly, AICAR failed to induce oxidative gene expression in PPARδ null muscle cells, indicting the requirement of PPARδ, at least for regulation of oxidative metabolism by AMPK. Collectively, these findings demonstrate a molecular partnership between AMPK and PPARδ in reprogramming skeletal muscle transcriptome and endurance (Figure 6I) that can be readily exploited by orally active AMPK drugs to replace exercise.
In humans, endurance exercise leads to physiological adaptations in the cardiopulmonary, endocrine, and neuromuscular systems.
Although our current investigation focused on skeletal muscle, extramuscular effects of PPARδ, AMPK, and exercise may also contribute to increased endurance. Although potentiation of extramuscular adaptations by PPARδ and AMPK agonists remains to be studied, we found that drug treatment can reduce epididymal fat mass, possibly conferring additional systemic benefits. It is noteworthy that PPARδ is important for normal cardiac contractility, as well as for the endocrine function of adipose tissue.
Similarly, the activation of AMPK by metformin is thought to mediate its ability to lower blood glucose levels.
In addition to increasing performance in athletes, exercise has beneficial effects in a wide range of pathophysiological conditions, such as respiratory disorders, cardiovascular abnormalities, type 2 diabetes, and cancer risk. Therefore, understanding the effects of exercise on normal physiology and identifying pharmaceutically targetable pathways that can boost these effects is crucial. In this study, we revealed that synthetic PPARδ activation and exercise—and, more importantly, AMPK activation alone—provide a robust transcriptional cue that reprograms the skeletal muscle genome and dramatically enhances endurance. We believe that the strategy of reorganizing the preset genetic imprint of muscle (as well as other tissues) with exercise mimetic drugs has therapeutic potential in treating certain muscle diseases such as wasting and frailty as well as obesity where exercise is known to be beneficial.
Exercise Training and Drug Treatment
Male C57B/6J mice (8 weeks old) were randomly divided into four cohorts comprising (1) vehicle-treated and sedentary (V), (2) GW1516-treated and sedentary (GW), (3) vehicle-treated and exercise-trained (Tr), and (4) GW1516-treated and exercise-trained (Tr+GW) mice (n = 9). Mice in all groups were acclimated to moderate treadmill running (10 m/min for 15 min) every other day for 1 week. After acclimation, basal running endurances for the four groups were determined via a treadmill running test, where the speed was gradually increased from 0 to 15 m/min and then maintained constant until exhaustion (week 0). After the initial test, the mice in the exercise groups were subjected to 4 weeks (5 days/week) of exercise training. The mice were trained on a treadmill inclined at 5°, with progressively increasing intensity and time. At the end of 4 weeks, all exercise-trained mice were running for 50 min/day at 18 m/min. During the 4 weeks, mice from both the sedentary and trained groups were treated with either vehicle or GW1516 (5 mg/kg/day). At the end of the drug treatment and/or training protocol (week 5), six mice per group were subjected to the running test. Three mice in each group were not subjected to treadmill test to confirm that changes observed in the skeletal muscle were not due to the acute run, but related to the exercise training. It should be noted that the above interventions do not affect body weight and food intake in mice (data not shown).
In another study, male C57B/6J mice (8 weeks old) were treated with GW1516 (5 mg/kg/day, oral gavage), AICAR (250 mg/kg/day, i.p.), or the combination of the two drugs for 6 days for gene expression analysis. Additionally, C57B/6J mice (8 weeks old) were also treated with AICAR (500 mg/kg/day, i.p.) for 4 weeks for treadmill running tests.