Pathological and physiological stimuli, including acute exercise, activate autophagy; however, it is unknown whether exercise training alters basal levels of autophagy and whether autophagy is required for skeletal muscle adaptation to training. We observed greater autophagy flux (i.e., a combination of increased LC3-II/LC3-I ratio and LC3-II levels and reduced p62 protein content indicating a higher rate of initiation and resolution of autophagic events), autophagy protein expression (i.e., Atg6/Beclin1, Atg7, and Atg8/LC3) and mitophagy protein Bnip3 expression in tonic, oxidative muscle compared to muscles of either mixed fiber types or of predominant glycolytic fibers in mice. Long-term voluntary running (4 wk) resulted in increased basal autophagy flux and expression of autophagy proteins and Bnip3 in parallel to mitochondrial biogenesis in plantaris muscle with mixed fiber types. Conversely, exercise training promoted autophagy protein expression with no significant increases of autophagy flux and mitochondrial biogenesis in the oxidative soleus muscle. We also observed increased basal autophagy flux and Bnip3 content without increases in autophagy protein expression in the plantaris muscle of sedentary muscle-specific Pgc-1α transgenic mice, a genetic model of augmented mitochondrial biogenesis. These findings reveal that endurance exercise training-induced increases in basal autophagy, including mitophagy, only take place if an enhanced oxidative phenotype is achieved. However, autophagy protein expression is mainly dictated by contractile activity independently of enhancements in oxidative phenotype. Exercise-trained mice heterozygous for the critical autophagy protein Atg6 showed attenuated increases of basal autophagy flux, mitochondrial content, and angiogenesis in skeletal muscle, along with impaired improvement of endurance capacity. These results demonstrate that increased basal autophagy is required for endurance exercise training-induced skeletal muscle adaptation and improvement of physical performance.

Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3–12 h and mitophagy at 6 h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer, that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle.

Background: Mitochondrial health is difficult to assess in vivo. Results: We have generated a reporter gene, MitoTimer, which targets mitochondria, and fluoresces green and shifts to red when oxidized, for assessment of mitochondrial content, structure, stress, and damage under physiological and pathological conditions. Conclusion: MitoTimer is useful for assessment of mitochondrial health in vivo. Significance: MitoTimer could advance mitochondrial research in multiple disciplines.

Abnormal conditions during early development adversely affect later health. We investigated whether maternal exercise could protect offspring from adverse effects of a maternal high-fat diet (HFD) with a focus on the metabolic outcomes and epigenetic regulation of the metabolic master regulator, peroxisome proliferator-activated receptor γ coactivator-1α (Pgc-1α). Female C57BL/6 mice were exposed to normal chow, an HFD, or an HFD with voluntary wheel exercise for 6 weeks before and throughout pregnancy. Methylation of the Pgc-1α promoter at CpG site −260 and expression of Pgc-1α mRNA were assessed in skeletal muscle from neonatal and 12-month-old offspring, and glucose and insulin tolerance tests were performed in the female offspring at 6, 9, and 12 months. Hypermethylation of the Pgc-1α promoter caused by a maternal HFD was detected at birth and was maintained until 12 months of age with a trend of reduced expression of Pgc-1α mRNA (P = 0.065) and its target genes. Maternal exercise prevented maternal HFD-induced Pgc-1α hypermethylation and enhanced Pgc-1α and its target gene expression, concurrent with amelioration of age-associated metabolic dysfunction at 9 months of age in the offspring. Therefore, maternal exercise is a powerful lifestyle intervention for preventing maternal HFD-induced epigenetic and metabolic dysregulation in the offspring.

While the timing of food intake is important, it is unclear whether the effects of exercise on energy metabolism are restricted to unique time windows. As circadian regulation is key to controlling metabolism, understanding the impact of exercise performed at different times of the day is relevant for physiology and homeostasis. Using high-throughput transcriptomic and metabolomic approaches, we identify distinct responses of metabolic oscillations that characterize exercise in either the early rest phase or the early active phase in mice. Notably, glycolytic activation is specific to exercise at the active phase. At the molecular level, HIF1a, a central regulator of glycolysis during hypoxia, is selectively activated in a time-dependent manner upon exercise, resulting in carbohydrate exhaustion, usage of alternative energy sources, and adaptation of systemic energy expenditure. Our findings demonstrate that the time of day is a critical factor to amplify the beneficial impact of exercise on both metabolic pathways within skeletal muscle and systemic energy homeostasis.