Regulation of muscle fiber type and running endurance by PPARdelta

Abstract

Endurance exercise training can promote an adaptive muscle fiber transformation and an increase of mitochondrial biogenesis by triggering scripted changes in gene expression. However, no transcription factor has yet been identified that can direct this process. We describe the engineering of a mouse capable of continuous running of up to twice the distance of a wild-type littermate. This was achieved by targeted expression of an activated form of peroxisome proliferator-activated receptor delta (PPARdelta) in skeletal muscle, which induces a switch to form increased numbers of type I muscle fibers. Treatment of wild-type mice with PPARdelta agonist elicits a similar type I fiber gene expression profile in muscle. Moreover, these genetically generated fibers confer resistance to obesity with improved metabolic profiles, even in the absence of exercise. These results demonstrate that complex physiologic properties such as fatigue, endurance, and running capacity can be molecularly analyzed and manipulated.

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.

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.

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.

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.