Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.

Exercise is among the most effective interventions for age-associated mobility decline and metabolic dysregulation. Although long-term endurance exercise promotes insulin sensitivity and expands respiratory capacity, genetic components and pathways mediating the metabolic benefits of exercise have remained elusive. Here, we show that Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits. In both fly and mouse models, genetic ablation of Sestrins prevents organisms from acquiring metabolic benefits of exercise and improving their endurance through training. Conversely, Sestrin upregulation mimics both molecular and physiological effects of exercise, suggesting that it could be a major effector of exercise metabolism. Among the various targets modulated by Sestrin in response to exercise, AKT and PGC1α are critical for the Sestrin effects in extending endurance. These results indicate that Sestrin is a key integrating factor that drives the benefits of chronic exercise to metabolism and physical endurance.

Endurance exercise is an effective therapeutic intervention with substantial pro-healthspan effects. Male Drosophila respond to a ramped daily program of exercise by inducing conserved physiological responses similar to those seen in mice and humans. Female flies respond to an exercise stimulus but do not experience the adaptive training response seen in males. Here, we use female flies as a model to demonstrate that differences in exercise response are mediated by differences in neuronal activity. The activity of octopaminergic neurons is specifically required to induce the conserved cellular and physiological changes seen following endurance training. Furthermore, either intermittent, scheduled activation of octopaminergic neurons or octopamine feeding is able to fully substitute for exercise, conferring a suite of pro-healthspan benefits to sedentary Drosophila. These experiments indicate that octopamine is a critical mediator of adaptation to endurance exercise in Drosophila.

Endurance exercise has emerged as a powerful intervention that promotes healthy aging by maintaining the functional capacity of critical organ systems. In addition, long-term exercise reduces the incidence of age-related diseases in humans and in model organisms. Despite these evident benefits, the genetic pathways required for exercise interventions to achieve these effects are still relatively poorly understood. Here, we compare gene expression changes during endurance training in Drosophila melanogaster to gene expression changes during selective breeding for longevity. Microarrays indicate that 65% of gene expression changes found in flies selectively bred for longevity are also found in flies subjected to three weeks of exercise training. We find that both selective breeding and endurance training increase endurance, cardiac performance, running speed, flying height, and levels of autophagy in adipose tissue. Both interventions generally upregulate stress defense, folate metabolism, and lipase activity, while downregulating carbohydrate metabolism and odorant receptor expression. Several members of the methuselah-like (mthl) gene family are downregulated by both interventions. Knockdown of mthl-3 was sufficient to provide extension of negative geotaxis behavior, endurance and cardiac stress resistance. These results provide support for endurance exercise as a broadly acting anti-aging intervention and confirm that exercise training acts in part by targeting longevity assurance pathways.

Endurance exercise is an inexpensive intervention that is thought to provide substantial protection against several age-related pathologies, as well as inducing acute changes to endurance capacity and metabolism. Recently, it has been established that endurance exercise induces conserved alterations in physiological capacity in the invertebrate Drosophila model. If the genetic factors underlying these exercise-induced physiological alterations are widely conserved, then invertebrate genetic model systems will become a valuable tool for testing of genetic and pharmacological mimetics for endurance training. Here, we assess whether the Drosophila homolog of the vertebrate exercise response gene PGC-1α spargel (srl) is necessary or sufficient to induce exercise-dependent phenotypes. We find that reduction of srl expression levels acutely compromises negative geotaxis ability and reduces exercise-induced improvement in both negative geotaxis and time to exhaustion. Conversely, muscle/heart specific srl overexpression improves negative geotaxis and cardiac performance in unexercised flies. In addition, we find that srl overexpression mimics some, but not all, exercise-induced phenotypes, suggesting that other factors also act in parallel to srl to regulate exercise-induced physiological changes in muscle and heart.

The development of endurance exercise paradigms in Drosophila has facilitated study of genetic factors that control individual response to exercise. Recent work in Drosophila has demonstrated that activation of octopaminergic neurons is alone sufficient to confer exercise adaptations to sedentary flies. These results suggest that adrenergic activity is both necessary and sufficient to promote endurance exercise adaptations.