Role of PGC-1α during acute exercise-induced autophagy and mitophagy in skeletal muscle

Abstract

Regular exercise leads to systemic metabolic benefits, which require remodeling of energy resources in skeletal muscle. During acute exercise, the increase in energy demands initiate mitochondrial biogenesis, orchestrated by the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Much less is known about the degradation of mitochondria following exercise, although new evidence implicates a cellular recycling mechanism, autophagy/mitophagy, in exercise-induced adaptations. How mitophagy is activated and what role PGC-1α plays in this process during exercise have yet to be evaluated. Thus we investigated autophagy/mitophagy in muscle immediately following an acute bout of exercise or 90 min following exercise in wild-type (WT) and PGC-1α knockout (KO) animals. Deletion of PGC-1α resulted in a 40% decrease in mitochondrial content, as well as a 25% decline in running performance, which was accompanied by severe acidosis in KO animals, indicating metabolic distress. Exercise induced significant increases in gene transcripts of various mitochondrial (e.g., cytochrome oxidase subunit IV and mitochondrial transcription factor A) and autophagy-related (e.g., p62 and light chain 3) genes in WT, but not KO, animals. Exercise also resulted in enhanced targeting of mitochondria for mitophagy, as well as increased autophagy and mitophagy flux, in WT animals. This effect was attenuated in the absence of PGC-1α. We also identified Niemann-Pick C1, a transmembrane protein involved in lysosomal lipid trafficking, as a target of PGC-1α that is induced with exercise. These results suggest that mitochondrial turnover is increased following exercise and that this effect is at least in part coordinated by PGC-1α.

Keywords: Niemann-Pick C1; biogenesis; endurance; mitochondria; physical activity.

Fig. 1.

Lack of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) results in diminished mitochondrial content and reduced exercise performance. A: representative images of cytochrome oxidase (COX) and succinate dehydrogenase (SDH) staining of extensor digitorum longus muscle from control [wild-type (WT)] and PGC-1α knockout (KO) animals. Scale bars = 100 μm. B: COX activity as a surrogate measure of mitochondrial content in WT and KO animals. C: after 2 days of habituation to the treadmill, animals were run to failure utilizing an incremental exercise protocol on a 0% incline. Animals began with a warm-up period of 5 min at 5 m/min and 10 min at 10 m/min followed by 45 min of endurance running at 15 m/min. Finally, running speed was increased by 1 m/min every 2 min until the animals refused to continue. D: running performance (i.e., total distance run) in WT and KO animals injected with water [vehicle (Veh)] or 0.4 mg/kg colchicine (Col). E: blood lactate levels in WT and KO animals prior to exercise (Con), immediately following exercise (Ex), and following 90 min of recovery (Ex + R). Values are means ± SE; n = 4–12 for all groups. *P < 0.05, significant effect of exercise. †P < 0.05, significant effect of genotype.

Fig. 2.

Signaling kinases are activated with exercise. A–C: blots and quantification of signaling kinases in WT and KO animals in Con, Ex, and Ex + R groups. A: representative blots of signaling kinases in WT and KO animals in Con, Ex, and Ex + R groups. Total protein and GAPDH were used as loading controls. P-AMPK and T-AMPK, phosphorylated and total AMP-activated kinase; P-p38 and T-p38, phosphorylated and total p38. B and C: quantification of signaling kinases in WT and KO animals in Con, Ex, and Ex + R groups. AU, arbitrary units. Values are means ± SE; n = 5–9. *P < 0.05, significant effect of exercise. †P < 0.05, significant effect of genotype.

Fig. 3.

Exercise induces increased nuclear localization and expression of PGC-1α and mitochondrial biogenesis. A: blot and quantification of PGC-1α levels in the nuclear subfraction of WT animals in Con, Ex, and Ex + R groups. B: PGC-1α gene (Ppgargc1a) expression in WT animals in Ex and Ex + R groups compared with WT animals in Con group. C and D: COX subunit IV (Coxiv) and mitochondrial transcription factor A (Tfam) gene expression WT and KO animals in Ex and Ex + R groups compared with WT animals in Con group. GAPDH (Gapdh) and β-actin (Actb) were used as housekeeping genes. Values are means ± SE; n = 4–9. *P < 0.05, significant effect of exercise. †P < 0.05, significant effect of genotype.

Fig. 4.

Expression of autophagy genes and induction of autophagy with exercise are differentially regulated in PGC-1α KO animals. A and B: gene expression of autophagy factors, microtubule-associated protein 1 light chain 3 [Maplc3b (LC3)] and sequestosome 1 [Sqstm1 (p62)], in WT and KO animals in Ex and Ex + R groups compared with WT animals in Con group. Gapdh and Actb were used as housekeeping genes. C–E: blots and quantification of autophagic protein in whole muscle extracts from WT and KO animals in Con, Ex, and Ex + R groups treated with vehicle or colchicine (0.4 mg·kg⁻¹·day⁻¹) for 2 days. F and G: autophagy flux as determined by percent change in protein content from colchicine- and vehicle-treated WT and KO animals in Con, Ex, and Ex + R groups. GAPDH was used as loading control. Values are means ± SE; n = 5–9. *P < 0.05, significant effect of exercise. †P < 0.05, significant effect of genotype.

Fig. 5.

Exercise-induced mitophagy signaling and flux are attenuated in PGC-1α KO animals. A–E: blots and quantification of autophagic proteins and flux in isolated mitochondrial fractions from WT and KO animals in Con, Ex, and Ex + R groups treated with vehicle or colchicine (0.4 mg·kg⁻¹·day⁻¹) for 2 days. Voltage-dependent anion channel (VDAC) was used as loading control. Values are means ± SE; n = 7–9. *P < 0.05, significant effect of exercise.

Fig. 6.

Lack of PGC-1α results in attenuated exercise-mediated mitophagy signaling. A: Parkin (Park2) gene expression in WT and KO animals in Con, Ex, and Ex + R groups compared with WT animals in Con group. Gapdh and Actb were used as housekeeping genes. B–E: blots and quantification of proteins in isolated mitochondrial subfractions. Drp-1, dynamin-related protein 1; Ub, ubiquitin. VDAC was used as loading control. Values are means ± SE; n = 4–9. *P < 0.05, significant effect of exercise.

Fig. 7.

Transcriptional regulators of autophagy with exercise. A and B: gene expression of transcriptional regulators of autophagy [Forkhead box O3 (Foxo3) and transcription factor EB (TFEB)] in WT and KO animals in Con, Ex, and Ex + R groups compared with WT animals in Con group. Gapdh and Actb were used as housekeeping genes. Values are means ± SE; n = 4–9. *P < 0.05, significant effect of exercise. †P < 0.05, significant effect of genotype.

Fig. 8.

Identification of Niemann-Pick C1 (NPC1) as a PGC-1α-regulated autophagy factor through PCR-array analysis. A: heat map showing expression of 84 autophagy-related genes in WT and KO animals. Green indicates reduction, while red indicates increase, in gene expression; the brighter the color, the greater the change in gene expression. (For the full list of fold changes and statistical significance see Supplemental Table S1.) B: gene expression of NPC1 in WT and KO animals in Con, Ex, and Ex + R groups compared with WT animals in Con group. Gapdh and Actb were used as housekeeping genes. C: representative blot and quantification of NPC1 in tibialis anterior muscle extracts. GAPDH was used as loading control. Values are means ± SE; n = 3–4. *P < 0.05, significant effect of exercise. †P < 0.05, significant effect of genotype.