Management of energy stores is critical during endurance exercise; a shift in substrate utilization from glucose toward fat is a hallmark of trained muscle. Here we show that this key metabolic adaptation is both dependent on muscle PPARδ and stimulated by PPARδ ligand. Furthermore, we find that muscle PPARδ expression positively correlates with endurance performance in BXD mouse reference populations. In addition to stimulating fatty acid metabolism in sedentary mice, PPARδ activation potently suppresses glucose catabolism and does so without affecting either muscle fiber type or mitochondrial content. By preserving systemic glucose levels, PPARδ acts to delay the onset of hypoglycemia and extends running time by ∼100 min in treated mice. Collectively, these results identify a bifurcated PPARδ program that underlies glucose sparing and highlight the potential of PPARδ-targeted exercise mimetics in the treatment of metabolic disease, dystrophies, and, unavoidably, the enhancement of athletic performance.

Despite the above five collectanea, the minimal components needed to enhanced running performance have eluded description. Here we show that activation of muscle PPARδ not only increases fat oxidation, but also coordinately decreases glucose catabolism to forestall hypoglycemia and facilitate progressively longer running time. Unexpectedly, this substrate prioritization does not depend on either oxidative fiber-type transformation or mitochondrial biogenesis. Mechanistically, we identify a PPARδ-induced genomic signature in muscle as the transcriptional basis of glucose conservation and ultimately enhanced stamina. These findings identify muscle PPARδ as a key regulator of energetic resilience and offer a quantitative molecular approach in the development of new, safe, and effective therapeutics for the treatment of metabolic disease and the promotion of metabolic health.

To address the above questions, we focused on the peroxisome proliferator-activated receptor delta (PPARδ), a nuclear receptor that serves as a key regulator of FA metabolism in muscle (). Muscle-specific overexpression of PPARδ not only induces an oxidative fiber-type transformation but also increases FA oxidation in skeletal muscle through the induction of two mitochondrial gatekeeper proteins, carnitine palmitoyl-transferase 1b (Cpt1b), the rate-limiting enzyme in the transport of FAs into the mitochondria (), and pyruvate dehydrogenase kinase isozyme 4 (Pdk4), which negatively regulates the influx of glucose-derived pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle (). Consequently, PPARδ transgenic mice run twice as long as the controls and represent a permanent strain of “marathon mice” (). Conversely, muscle-specific PPARδ knockout (PDmKO) mice show defects in fiber-type determination and mitochondrial biogenesis ().

In endurance sports such as marathon running and cycling, carbohydrate depletion, commonly known as “hitting the wall,” is a significant determinant of performance. Exercise training enhances endurance, in part, by delaying the depletion of carbohydrate stores (mainly glycogen in liver and muscle). The adaptive benefits of exercise training are commonly attributed to the glycolytic-to-oxidative fiber-type transformation and increased mitochondrial energetic capacity (), programs in which the AMPK-PGC1α signaling pathway is now known to play a major role. At the same time, exercise also enhances muscle fatty acid (FA) oxidation (), theoretically providing extra energy substrates for extended performance and reducing the dependence on glucose. This readily observed glucose sparing leads to the assumption that increased FA oxidation extends performance. However, the contribution of altered fat and glucose metabolism to endurance and the molecular mechanism underlying this metabolic shift are simply not known.

Previously, a functional interaction between PPARδ and AMPK was suggested based on a synergistic induction of FA metabolism genes by co-treatment with GW and the AMPK activator AICAR, an effect that was dependent on PPARδ (). To explore the regulatory hierarchy controlling metabolic substrate utilization, we determined the effects of AICAR treatment on PPARδ target genes ( Figures 3 A and 3B). AICAR alone did not significantly activate PPARδ target genes involved in FA metabolism, except for the induction of Pparγ, in agreement with an earlier study ( Figures S4 A and S4B). Furthermore, AICAR failed to repress glucose catabolism genes ( Figure S4 A). Consistent with this lack of transcriptional changes, 8 weeks of AICAR treatment did not alter substrate utilization in mice ( Figure S4 C). AICAR treatment did increase energy expenditure in mice, presumably through mitochondrial biogenesis ( Figures S4 C and S4D). Notably, the ability of AICAR to induce mitochondrial biogenesis is independent of PPARδ ( Figure S4 D), in line with the data that PPARδ activation or depletion did not affect mitochondrial biogenesis ( Figure S2 G).

Interestingly, PPARδ employs distinct mechanisms to regulate target genes. Only a subset of genes showed increased PPARδ chromatin binding upon ligand treatment (e.g., Pdk4 and Mlycd, in contrast to Cpt1a, Slc25a20, Angptl4, and Ucp3, where PPARδ binding was independent of ligand) ( Figures 4 D–4F). Further investigation of the latter gene set revealed both ligand-induced loss of co-repressor binding (loss of NCoR binding on Angptl4 and Ucp3) and ligand-induced recruitment of co-activator (increased Pgc1α binding on Cpt1a and Slc25a20) ( Figures 4 G and 4H).

Correlating the PPARδ cistrome with the ligand-induced transcriptional changes ( Figures 3 and S3 ) revealed that ∼50% of GW-regulated genes (457 of 975 genes) were direct PPARδ target genes (based on proximity of binding sites to the closest transcription start site), with roughly equivalent numbers of these genes up- and downregulated (223 and 234, respectively; Figure 4 C). Furthermore, GO analysis of the 457 directly regulated genes showed enrichment of the same categories as seen in the entire GW transcriptome, including FA metabolism in upregulated and glucose catabolism in downregulated gene sets ( Figures 3 A, S3 , and 4 C). In contrast, the 518 indirect PPARδ target genes failed to show any significant GO enrichment. The marked overlap between the transcriptomic and cistromic findings supports a direct role for PPARδ-regulated transcriptional changes in both exercise- and ligand-induced adaptations in energy substrate utilization.

Chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) was used to establish the PPARδ cistrome in both the presence and absence of ligand. In C2C12 cells, 8,474 and 10,482 PPARδ binding sites were identified in vehicle- and GW-treated cells, respectively ( Figure 4 A). Motif analysis identified a consensus PPAR response element significantly enriched in both conditions ( Figure 4 A), confirming the quality of the ChIP. A total of 5,586 binding sites were common to both vehicle- and GW-treated cells, with GW recruiting an additional 4,896 unique sites and dismissing 2,888 pre-ligand sites. PPARδ binding sites were predominantly located in the intergenic and intronic regions, with only 4% in gene promoter regions ( Figure 4 B), as seen with many transcription factors.

(C) Pie chart showing the number of PPARδ-bound or non-bound genes from all 975 genes that are changed by GW treatment. GO and pathway analysis of up- and downregulated genes is listed.

Conversely, pathways of insulin signaling, glycolysis, and carbohydrate catabolism were significantly enriched in the downregulated gene set (including Irs2, Slc2a3, and Hk2) ( Figures 3 A, 3C, and S3 B). Notably, these transcriptional changes, combined with the suppression of the recently identified mitochondrial pyruvate carrier Mpc1, coordinately reduce muscle glucose catabolism ( Figures 3 A, 3C, and S3 B). These studies reveal that PPARδ reprograms muscle metabolism for endurance by reciprocal regulation of gene programs promoting FA oxidation and suppressing glucose metabolism ( Figure 3 D).

Global transcriptional analyses in the glycolytic white quadriceps muscle (WQ) identified 975 genes with altered expression upon GW treatment, with 492 up- and 483 downregulated. In addition to the key mitochondrial genes Pdk4 and Cpt1b described above ( Figures 2 F and 2G), gene ontology (GO) analysis of upregulated genes revealed significant enrichment in the PPAR signaling pathway as well as lipid and FA catabolism (including Lpl, Lipe, Acadl, Acads, and Acaa2) ( Figures 3 A, 3B, and S3 A). Interestingly, lipogenic genes including PPARγ (master adipogenic regulator) and FA synthase (Fasn) were also induced ( Figures 3 A, 3B, and S3 A), which would theoretically lead to a futile cycle of lipid catabolism and anabolism. Additionally, genes involved in antioxidant defense and glutathione synthesis (including Cat, Sod3, and Gpx1) were highly upregulated ( Figures 3 A, 3B, and S3 A). Counterintuitively, pathways in carbohydrate metabolism, including the hexose metabolic process, pentose-phosphate shunt, and insulin signaling, were also significantly enriched ( Figures 3 A, 3B, and S3 A). However, the induction of Fbp2, Pck1, and Pcx ( Figure 3 B) is consistent with the induction of an anabolic program, suggesting a possible role in muscle repair.

(D) Diagram showing the metabolism of glucose and FAs, the two major cellular energy substrates, as well as GW-induced gene expression changes that regulate their metabolism. Red and blue labels represent up- and downregulation induced by GW treatment, respectively.

The above data suggested a PPARδ-controlled metabolic shift that preserves systemic glucose contributing to a GW-induced endurance enhancement. To determine the magnitude and timing of these changes, we monitored blood glucose in mice treated with or without GW during a run-to-exhaustion test. As expected, both control and GW-treated mice showed time-dependent reductions in blood glucose to <70 mg/dL, at which point the mice stopped running and often lost consciousness ( Figures 2 J and S2 L). By increasing blood glucose to >120 mg/dL via intraperitoneal (i.p.) injection (250 mg/kg), exhausted mice were able to resume running for another ∼20 min, confirming that blood glucose is the major determinant of endurance. Interestingly, while the blood glucose in the control mice started dropping after 90–120 min of running, GW-treated mice were able to maintain normal glycemic levels for extended periods and delay the onset of blood glucose reduction even after 180 min of running ( Figure 2 J). It is important to note that the glucose-sparing effects of GW treatment parallel those seen with exercise training, suggesting a common underlying mechanism ( Figure S2 M). Blood lactate was also monitored during our run-to-exhaustion tests, which showed minimal fluctuation in both control and GW-treated mice ( Figure 2 J, dashed lines; Figure S2 N), indicating that the endurance regimen did not exceed the aerobic threshold of the tested mice. In combination, our data describe a PPARδ-controlled muscle reprogramming that boosts exercise endurance by inversely regulating fat and glucose metabolism, thereby preserving circulating glucose to support other tissues such as the brain ( Figure 2 K).

Consistent with an energy substrate shift, the longer 8 week GW treatment of sedentary mice was sufficient to confer ∼1.5 hr longer running time than untreated controls ( Figure 2 I). This endurance benefit is lost in PDmKO mice and thus dependent on muscle PPARδ activation ( Figure 2 I). Unexpectedly, muscular glycogen content ( Figures S2 E and S2F), mitochondrial quantity and OXPHOS activity ( Figures S2 C and S2G–S2I), and muscle fiber-type composition ( Figures S2 J and S2K)—changes commonly associated with endurance enhancement ()—were not affected by GW treatment. Thus, while establishing that ligand activation of PPARδ can enhance endurance in sedentary mice, these findings implicate a novel mechanism of action.

Previous studies showed that treatment of mice with the PPARδ agonist GW dramatically increased running endurance, but only when combined with daily exercise (). Based on the above, we re-examined the impact of GW on muscle energy substrate usage and endurance in fully sedentary mice. Unexpectedly, treatment of WT mice with GW (40 mg/kg in food) for a longer time (8 weeks compared to 4 weeks) reduced RER to a level similar to exercise training, indicative of increased FA metabolism ( Figures 2 A, 2B, S2 A, and S2B). Consistent with this, palmitate-fueled respiration was ∼50% higher in muscle mitochondria isolated from GW-treated mice, while succinate-fueled respiration was unchanged ( Figures 2 C and S2 C). GW treatment also more than doubled the palmitate-induced increase of OCR in cultured C2C12 myotubes ( Figures 2 D and 2E). In addition, GW strongly induced the expression of the mitochondrial gatekeeper genes Pdk4 and Cpt1a/b both in vivo and in vitro ( Figures 2 F–2H) while serum lactate, an indicator of anaerobic glycolysis, was reduced ∼40% in GW-treated mice ( Figure S2 D). Notably, GW treatment of PDmKO mice failed to alter RER ( Figures 2 A, 2B, S2 A, and S2B), FA oxidation in muscle mitochondria ( Figure 2 C), expression of muscle Pdk4 and Cpt1b ( Figures 2 F and 2G), or circulating lactate levels ( Figure S2 D), establishing that the above effects are dependent on muscle PPARδ.

Mouse experiments were performed in the same set of 4-month-old WT or PDmKO mice with or without 8 weeks of oral GW treatment (n = 8).

Despite the requirement for muscle PPARδ in exercise-induced metabolic adaptations and endurance enhancement, the glycolytic-to-oxidative fiber-type switch and mitochondrial biogenesis are still achieved in PDmKO mice ( Figures S1 A and S1E–S1J). Furthermore, sedentary PDmKO mice are indistinguishable from WT mice in terms of mitochondrial content and oxidative phosphorylation (OXPHOS) capacity ( Figures S1 A, S1E, and S1F), as well as muscle fiber-type composition ( Figures S1 E and S1G–S1J). In addition, whole-body energy expenditure (measured as oxygen consumption rate [VO]; Figures 1 A, 1B, S1 B, and S1C), energy substrate utilization (RER; Figures 1 A, 1B, S1 B, and S1C), and mitochondrial FA oxidation ( Figure 1 C) are all independent of muscle PPARδ expression. This indicates a role for PPARδ in adaptive, but not innate, muscle activity and stands in contrast to previous reports ().

While endurance exercise shifts muscle energy substrate usage from glucose to FAs (), the dependence on PPARδ for this shift is not known. Accordingly, we compared the benefits of treadmill training in wild-type (WT) and PDmKO mice ( Figure S1 A). After 4 weeks of daily running, WT mice show a clear shift in energy substrate usage from glucose to FA oxidation, as evidenced by the reduced respiratory exchange ratio (RER) ( Figures 1 A, 1B, S1 B, and S1C) and increased palmitate-fueled mitochondrial oxygen consumption rate (OCR) ( Figure 1 C). Notably, these metabolic changes are mostly abolished in PDmKO mice ( Figures 1 A–1C, S1 B, and S1C), demonstrating the dependence of exercise-induced metabolic adaptations on PPARδ. Mechanistically, we show that the exercise-induced upregulation of mitochondrial gatekeeper genes ( Figure 1 D) is heavily compromised (Pdk4 induction reduced by ∼50%; Figure 1 E) or completely absent (Cpt1b; Figure 1 F) in PDmKO mice. In terms of running endurance, the increase in performance of PDmKO mice after exercise training was only half that seen in WT controls (run-to-exhaustion treadmill test; Figure 1 G). Consistent with these results, PPARδ expression in skeletal muscle positively correlates with running distance, activity, and muscle mass in the BXD mouse genetic reference population (GRP) ( Figure 1 H) (). Additional negative correlations between Cpt1b and Pdk4 expression in muscle and plasma lactate (indicator for muscle glycolysis) and RER, respectively, further implicate PPARδ in the adaptive regulation of energy substrate utilization ( Figure S1 D).

Experiments were performed in the same set of 4-month-old WT or PDmKO mice with or without 4 weeks of exercise training (n = 5).

Discussion

In endurance sport competitions such cycling, marathon runs, race walking, and cross-country skiing, “hitting the wall” is a dramatic demonstration of sudden and complete exhaustion. It is thought to be due to the depletion of liver and muscle glycogen and can be averted by training that promotes mitochondrial biogenesis, increased type I fibers, and enhanced FA burning. In this study, we show that PPARδ expression correlates with endurance, and its activation by exercise mimetics, such as GW, is sufficient to increase running time by ∼100 min without changes in either muscle fiber type or mitochondrial biogenesis. Thus, the foundational core of endurance enhancement appears to be purely metabolic. Furthermore, even though the GW impact appears to be achieved via increased FA metabolism, the strongest correlation to endurance is maintenance of blood glucose above 70 mg/dL. Indeed, expression of key glycolytic genes such as Hk2, Gck, and the recently described mitochondrial pyruvate carrier (Mcp1) is dramatically reduced.

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This PPARδ-directed metabolic shift is driven by the induction of a metabolic gene signature change, which includes not only the induction of the mitochondrial gatekeeper genes Cpt1b and Pdk4, but also the upregulation of genes in FA transport, FA oxidation, lipogenesis, and gluconeogenesis, and the downregulation of genes in glucose uptake, glycolysis, and mitochondrial pyruvate entry. Overall, these gene expression changes promoted the metabolic shift in energy substrate from glucose to FA in the muscle. Antioxidant genes, as well as genes in glutathione synthesis, were also significantly induced by GW treatment, which could protect against increased reactive oxidative species (ROS) due to elevated mitochondrial energy metabolism.

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Mortensen R.M. Peroxisome proliferator-activated receptor-gamma-mediated effects in the vasculature. Our study reveals the molecular mechanism underlying muscle PPARδ regulation of its target genes. We show that ligand activation recruits PPARδ to new cis regulatory elements on its target genes. In addition, at sites where PPARδ is naturally bound, ligand binding modifies its interaction with co-activators or co-repressors for target gene regulation (). Our data also suggested PPARδ as the direct executor in GW-induced gene expression changes, since ∼50% of the GW-induced genes contain PPARδ binding sites. In those PPARδ bound genes, about half were upregulated and half downregulated upon GW activation, suggesting PPARδ could both directly activate and suppress downstream genes in a ligand-dependent manner, a circumstance observed for other nuclear receptor members such as PPARγ ().

The importance of muscle PPARδ in regulating energy metabolism has been further confirmed by the correlation study in 38 BXD mouse lines. We observed strong correlations between PPARδ target gene expression and metabolic fitness. As a nuclear receptor, the transcription activity of PPARδ is regulated by its endogenous ligands. Its activity, rather than its own expression level, controls its target gene expression. In support of this, exercise training highly induced the expression of PPARδ target genes such as Cpt1b and Pdk4 but had no effect on PPARδ itself in skeletal muscle.