AMPK and PPARdelta agonists are exercise mimetics

Abstract

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 reseveratrol, 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 PPARbeta/delta agonist and exercise training synergistically increase oxidative myofibers and running endurance in adult mice. Because training activates AMPK and PGC1alpha, 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-PPARdelta pathway can be targeted by orally active drugs to enhance training adaptation or even to increase endurance without exercise.

Figures

Figure 1. Synthetic PPARδ activation in mice

(A) Relative gene expression levels of Ucp3, Cpt 1 and Pdk4 in quadriceps isolated from vehicle (V) and GW1516 (GW)-treated wild type mice as well as from muscle VP16-PPARδ transgenic (Wang et al., 2004) (TG) and non-transgenic (WT) littermates. Data is presented as mean±SEM (N=4-9). * Indicates statistically significant differences between GW and V groups or TG and WT groups (p<0.05, Unpaired student’s t test). (B) Running endurance in sedentary mice. Endurance was tested in V (open bars) and GW (black bars)-treated wild type mice before (Week 0) and after (Week 5) treatment. Data is represented as mean±SD (N=6). (C) Representative meta-chromatically stained frozen gastrocnemius cross-sections from vehicle-treated sedentary (V), GW1516-treated sedentary (GW), vehicle-treated exercised (Tr) and GW-treated exercised (Tr+GW) mice. Type I fibers are stained dark blue. (D) Type I fiber quantification (N=3). (E) Fold change in mitochondrial DNA to nuclear DNA ratio (N=9). Data in (D) and (E) are presented as mean±SEM. * Indicates statistical differences between V and indicated groups (p<0.05, One Way ANOVA; post hoc: Dunnett’s Multiple Comparison Test).

Figure 2. Gene and protein expression in quadriceps

Relative gene expression levels of (A) FAO (Ucp3, Cpt 1b, Pdk4), (B) fatty acid storage (Scd1, Fasn, srebf1c) and (C) fatty acid uptake (Cd36, Lpl) biomarkers in quadriceps from V, GW, Tr and Tr+GW groups. Data is presented as mean±SEM (N=9) mice. * Indicates statistically significant difference between V and indicated groups (p<0.05, One Way ANOVA; post hoc: Dunnett’s Multiple Comparison Test). (D) Protein expression levels of oxidative biomarkers (myoglobin, UCP3, CYCS, SCD1) and loading control (tubulin) in quadriceps (N=3).

Figure 3. Running endurance and gene signature in exercise trained mice

Running endurance was tested in V (open bars) and GW- (black bars) treated mice before (Week 0) and after (Week 5) exercise training. Running endurance is depicted as time (A) and distance (B) that animals in each group ran. Data is represented as mean±SD (N=6). ***Indicates statistically significant difference between V and GW-treated exercised mice (p<0.001) (One Way ANOVA; post hoc: Tukey’s Multiple Comparison Test). (C) Venn diagram comparing GW, Tr and Tr+GW target genes identified in microarray analysis of quadriceps (N=3). The selection criteria used a p<0.05 on Bonferroni’s multiple comparison test. (D) Classification of target genes in Tr+GW mice. (E) Relative expression of 48 unique TR+GW target genes in GW, TR, TR+GW and VP16-PPARδ muscles. Each condition is represented by data from two samples (each sample is pooled from three mice). (Color scheme for fold change is provided).

Figure 4. Synergistic regulation of muscle gene expression by PPARδ and AMPK

(A) and (B) represent AMPK activation by exercise and VP16-PPARδ over-expression, respectively, in skeletal muscle. (C) Comparison of Tr+GW and AI+GW dependent gene signatures in quadriceps (N=3). The selection criteria used is similar to one used in Figure 3C. (D) Classification of 52 targets that were common to Tr+GW and AI+GW gene signatures. Expression of Scd1 (E), ATP citrate lyase (Acly) (F), HSL (Lipe) (G), Fabp3 (H), Lpl (I) and Pdk4 (J) transcripts in quadriceps of mice treated with vehicle (V), GW1516 (GW, 5mg/kg/day), AICAR (AI, 250 mg/kg/day) and the combination of the two drugs (GW+AI) for 6 days. Data is presented as mean±SEM (N=6). * Indicates statistically significant difference between V and indicated groups (p<0.05, One Way ANOVA; post hoc: Dunnett’s Multiple Comparison Test).

Figure 5. AMPK-PPARδ interaction

(A-D) Expression of metabolic genes in wild type and PPARδ null primary muscle cells treated with V, GW, AI and GW+AI for 24 hr. In (E-F, J), AD293 cells were transfected with PPARδ+RXRα+Tk-PPRE along with control vector, AMPK α1, α2 and/or PGC1α as indicated. (E) Induction of basal PPARδ transcriptional activity by AMPK α1 or α2. (F) Dose-dependent induction of PPARδ transcriptional activity is enhanced by AMPKα1 (closed circle) or AMPK α2 (closed square) compared to control (open triangle). In (G-I, K), AD293 cells were transfected and processed as indicated. (G-H) Representative blot showing co-immunoprecipitation of transfected (G) or endogenous (H) AMPK with Flag-PPARδ. (I) Metabolic p32 labeling of PPARδ in AD293 cells transfected as described. (J) Synergistic regulation of basal (V) and ligand (GW) dependent PPARδ transcriptional activity by AMPK α2 subunit and PGC1α. (K) Co-immunoprecipitation of PPARδ but not AMPK α2 subunit with Flag-PGC1α. Data in (A)-(D) (n = 6), (E), and (J) (n = 3-4) are presented as mean ± SEM, and * indicates statistical significance (p < 0.05, one-way ANOVA; post hoc: Dunnett’s multiple comparison test).

Figure 6. AICAR increases running endurance

In A-F, C57Bl/6J mice were treated with vehicle (open bars/thin lines) or AICAR (500mg/kg/day, 4 weeks) (closed bar/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. (C) Percent epididymal fat mass to body weight ratio. (D) Oxygen consumption rates (mg/kg/hr) measured over 12 hr period. (E) Data in (D) is represented as AUC. (F) Running endurance measured as a function of time (upper panel) and distance (lower panel). (G) Representative oxidative genes induced by AICAR treatment (250 mg/kg/day, 6 days). (H) Expression of oxidative biomarkers (Scd1, Fasn, Ppargc1a, Pdk4) in wild type and PPARδ null primary myoblast treated with vehicle (open bar) or AICAR (closed bar) for 72 hr. (I) Model depicting exercise/AMPK-PPARδ interaction in re-programming muscle genome. Data in (B) and (C) (n = 10), (D) and (E) (n = 4), (F) (n = 15-20), and (H) (n = 9) are presented as mean ± SEM, and * indicates statistical significance (p < 0.05, unpaired student’s t test).