Results Activation of PPARδ Leads to Muscle Fiber Transformation A role of PPARδ in muscle fiber was suggested by its enhanced expression—at levels 10-fold and 50-fold greater than PPARα and γ isoforms, respectively (unpublished data). An examination of PPARδ in different muscle fibers reveals a significantly higher level in type I muscle (soleus) relative to type II–rich muscle (extensor digitorum longus) or type I and type II mixed muscle (gastrocnemius) (Figure 1A); this expression pattern closely resembles that of PGC-1α (Lin et al. 2002). A similar pattern but with more pronounced differences was found at the protein level (Figure 1B). Figure 1 Expression of Endogenous PPARδ and VP16-PPARδ Transgene in Muscle (A) Pooled RNA isolated from various muscles of five wild-type male C57B6 mice was hybridized with indicated probes. EDL, extensor digitorum longus; Gastro, gastrocnemius. (B) Pooled nuclear proteins (15 μg/lane) isolated from muscles of five wild-type male C57B6 were probed with anti-PPARδ antibody. RNA polymerase II (Pol II) is shown as a loading control. (C) Expression of the VP16-PPARδ transgene in various tissues. 10 μg of total RNA from each tissue was hybridized with a VP16 cDNA probe. Gastrocnemius muscle was used here. (D) Nuclear proteins (15 μg/lane) isolated from gastrocnemius muscle of the transgenic mice (TG) and the wild-type littermates (WT) were probed with indicated antibodies. The upper, nonspecific band that cross-reacted with the anti-PPARδ antibody serves a loading control. To directly assess the role of activation of PPARδ in control of muscle fiber plasticity and mitochondrial biogenesis, we generated mice expressing a transgene in which the 78-amino-acid VP16 activation domain was fused to the N-terminus of full-length PPARδ, under control of the 2.2-kb human α-skeletal actin promoter. In agreement with the previous characterization of this promoter (Brennan and Hardeman 1993; Clapham et al. 2000), the VP16-PPARδ transgene was selectively expressed in skeletal muscle, with 10-fold less in the heart (Figure 1C). Among different types of muscle fibers, the levels of VP16-PPARδ expression appeared to be similar (unpublished data). As shown in Figure 1D for gastrocnemius muscle, VP16-PPARδ fusion protein was produced at a level similar to that of endogenous PPARδ in wild-type littermates. Interestingly, the level of endogenous muscle PPARδ protein in the transgenic mice was much higher than in the control littermates. The substantial increase of endogenous PPARδ may have been caused by a switch to type I fiber (see below), which intrinsically expresses higher levels of PPARδ (Figure 1A and 1B). Type I muscle can be readily distinguished from type II or mixed muscle by its red color, because of its high concentration of myoglobin, a protein typically expressed in oxidative muscle fibers. We found that muscles in the transgenic mice appeared redder (Figure 2A), which is particularly evident in the mixed type I/II fibers of the hindlimb (Figure 2B). Indeed, metachromatic staining revealed a substantial muscle fiber transformation (Figure 2C). In gastrocnemius muscle, we estimated that there was a 2-fold increase of type I fibers. A diagnostic component of oxidative fibers is their high myoglobin and mitochondrial content, which is supported by the mRNA analysis shown in Figure 3A. In addition to myoglobin, mitochondrial components for electron transfer (cytochrome c and cytochrome c oxidase [COX] II and IV) and fatty-acid β-oxidation enzymes were elevated (Figure 3A; unpublished data). These effects appear to be direct consequences of PPARδ activation, as levels of PGC-1α, a coactivator involved in muscle fiber switch and mitochondrial biogenesis (Wu et al. 1999; Lehman et al. 2000; Lin et al. 2002), remained unchanged. Southern blot analysis detected a substantially higher copy number of the mitochondrial genome–encoded COXII DNA in the transgenic mice (Figure 3B). Mitochondrial DNA was increased 2.3-fold in gastrocnemius muscle of the transgenic mice (Figure 3C). These results reveal a marked stimulation of mitochondrial biogenesis and further support the idea that there is a muscle fiber switch. This conclusion was also confirmed by Western blot analysis. As shown in Figure 3D, the characteristic type I fiber proteins, such as myoglobin and cytochrome c and b, were significantly increased. More importantly, the specialized contraction protein troponin I (slow) of type I fiber was robustly induced; this was accompanied by a marked reduction of the specialized contraction protein troponin I (fast) of type II fiber, indicating a high degree of fiber transformation. We next examined whether acute activation of endogenous PPARδ would induce similar target genes. In agreement with the chronic effects in the transgenic mice, we found that, after treatment of wild-type C57B6J mice with the PPARδ-specific agonist GW501516 for only 10 d, genes for slow fiber contractile proteins, mitochondrial biogenesis, and β-oxidation were all upregulated (Figure 3E). This indicates that rapid, systematic, and coordinated changes of muscle fiber properties toward type I can be achieved by activation of the endogenous PPARδ pathway. Figure 2 Increased Oxidative Type I Fibers in the Transgenic Mice (A and B) Muscles in transgenic mice (TG) are redder than those in wild-type mice (WT). (C) Metachromatic staining of the type II plantaris muscle. Type I fibers are stained dark blue. Figure 3 Activation of PPARδ Induces Genes Typical for Type I Fibers and Promotes Mitochondrial Biogenesis (A) Total RNA (10 μg/lane) prepared from gastrocnemius muscle of transgenic (TG) and wild-type (WT) littermates was probed with indicated probes. The fold increase of induction of each gene is shown. (B) Total genomic DNA (10 μg/lane) prepared from gastrocnemius muscle was digested with Nco1 and subjected to Southern analysis with COXII (mitochondrial genome–encoded) and MCIP1 (nuclear genome–encoded) DNA probes. (C) Equal amounts of gastrocnemius muscle were collected from both transgenic mice and control littermates. Total mitochondrial DNA was isolated and separated on 1% agarose gel. The relative abundance of mitochondrial DNA in transgenic and wild-type mice is presented. (D) Western blot analysis of muscle fiber markers and mitochondrial components. Each lane was loaded with 80 μg of total gastrocnemius muscle extracts. (E) Wild-type C57B6 mice were treated with vehicle or PPARδ agonist GW501516 for 10 d. Total RNA (10 μg/lane) prepared from the gastrocnemius muscle was probed with indicated probes. Muscle Fiber Switch by PPARδ Protects Against Obesity A number of previous studies have shown that obese individuals have fewer oxidative fibers, implying that the presence of oxidative fibers alone may play a part in obesity resistance. To test this possibility, we fed the transgenic mice and their wild-type littermates with a high-fat diet for 97 d. Although the initial body weights of the two groups were very similar, the transgenic mice had gained less than 50% at day 47, and only one-third at day 97, of the weight gained by the wild-type animals (Figure 4A). The transgenic mice displayed significantly higher oxygen consumption on the high-fat diet than the control littermates (unpublished data). By the end of this experiment, the control littermates became obese, whereas the transgenic mice still maintained a normal body weight and fat mass composition (Figure 4A). A histological analysis of inguinal fat pad revealed a much smaller cell size in the transgenic mice (Figure 4B), due to the increased muscle oxidative capacity. While there was no significant difference in intramuscular glycogen content, the triglyceride content was much less in the transgenic mice (Figure 4C and 4D), which may explain their improved glucose tolerance (Figure 4E). We also placed wild-type C57BJ6 mice on the high-fat diet and treated them with either vehicle or the PPARδ agonist GW501516 for 2 mo. GW501516 produced a sustained induction of genes for type I muscle fibers; this, at least in part, resulted in an only 30% gain in body weight, a dramatically reduced fat mass accumulation, and improved glucose tolerance, compared to the vehicle-treated group (Figure 5). Thus, muscle fiber conversion by stimulation with the PPARδ agonist or the activated transgene has a protective role against obesity. Figure 4 Resistance to High-Fat-Induced Obesity in the Transgenic Mice (A) Seven-week-old transgenic (TG) and wild-type (WT) littermates (n = 5–6 for each group) were fed with a high-fat diet for 97 d. Left panel shows net body weight gain, which was calculated for individual mice and then averaged. Right panel shows the body weights before (Day 0) and after (Day 97) high-fat feeding. (B) Histology of inguinal fat pad in the transgenic and wild-type littermates under a high-fat diet for 2 mo. (C and D) Intramuscular glycogen content (C) and triglyceride content (D) of mice in (A) after high-fat feeding (n = 6). (E) Glucose tolerance test. Mice in (A) after high-fat feeding were fasted for 6 h and then injected with glucose at a concentration of 1g/kg body weight. Then blood glucose levels were measured periodically over 2 h (n = 6). Figure 5 PPARδ Agonists Counteract Obesity Induced by High-Fat Diet (A) Eleven-week-old wild-type C57B6 mice were fed a high-fat diet in combination with vehicle or GW501516 for 57 d. Total RNA (10 μg/lane) prepared from the gastrocnemius muscle was probed with indicated probes. (B) Net body weight gain for mice in (A) after treatment was calculated for individual mice and averaged. Initial body weights were 28.54 ± 1.04 g for vehicle group (n = 5) and 28.86 ± 0.80 g for GW501516 group (n = 5). (C) Various tissue weights for mice in (A) after treatment. ifat, inguinal fat; rdfat, reproductive fat; retrofat, retroperitoneal fat. (D) Glucose tolerance test. Mice in (A) after treatment were fasted for 6 h and then injected with glucose at a concentration of 1g/kg body weight. Blood glucose levels were then measured periodically over 2 h. Activation of PPARδ Enhances Physical Performance Muscle oxidative capacity is a crucial factor for determining endurance and fatigue. Indeed, type I fibers adaptively generated through exercise training are considered to be fatigue resistant. However, whether the type I fibers generated molecularly via PPARδ expression can contribute to enhanced performance in the absence of previous training is unclear. In fact, the consequence of genetically induced fiber switch on running capacity has to our knowledge never been evaluated. We thus compared exercise performance between untrained, body-weight-matched transgenic and wild-type littermates. Mice were run on oxygen-infused, enclosed treadmills until exhaustion. Strikingly, the running time and distance the transgenic mice were able to sustain were increased by 67% and 92%, respectively (Figure 6A; also see Videos S1 and S2). The transgenic mice ran about 1 h longer than the controls, which translates to nearly a kilometer further. No significant differences in muscle mass (unpublished data) and daily activity (total counts of activity per hour: 1618 ± 209 for transgenic versus 1987 ± 301 for wild-type, p > 0.35, n = 4) were observed between the transgenic and control mice. Thus, the remarkable increase in endurance is the physiologic manifestation of muscle fiber transformation. This suggests that genetically directed muscle fiber switch is physiologically and functionally relevant. In addition, we looked at what effect the absence of PPARδ function has on exercise endurance. In the treadmill test, the PPARδ-null mice could sustain only 38% of the running time and 34% of the distance of their age- and weight-matched wild-type counterparts (Figure 6B). These results further support a role for PPARδ in enhancement of physical performance. Figure 6 PPARδ Regulates Exercise Endurance (A) Enhanced exercise performance in the transgenic mice. Fourteen-week-old male transgenic and wild-type littermates with similar body weights (n = 4 for each group) were subjected to a forced treadmill exercise test. (B) Compromised exercise performance in PPARδ-null mice. Two-month-old male PPARδ-null mice and wild-type controls with similar body weights (n = 6 for each group) were subjected to a forced treadmill exercise test. (C) Functions of PPARδ in skeletal muscle.