The progressive decline in metabolic homeostasis is a hallmark of aging, leading to impaired organ function and reduced physical capacity. Mitochondria, the powerhouses of cells, play a critical role in this process, not only producing energy but also coordinating cellular homeostasis through communication with the nucleus and other cellular compartments. While mitochondria are traditionally known for their 13 protein-coding genes, recent discoveries have revealed short open reading frames (sORFs) that produce bioactive peptides, including mitochondrial-derived peptides (MDPs). MOTS-c (mitochondrial ORF of the 12S rRNA type-c) is one such MDP that has shown promise in regulating metabolic homeostasis, partly through AMPK activation and direct regulation of nuclear gene expression. Given its age-dependent expression in various tissues, including skeletal muscle and circulation, MOTS-c has been proposed as a "mitochondrial hormone" or "mitokine". Previous research showed that MOTS-c treatment reversed diet-induced obesity and insulin resistance in mice. This study aimed to investigate whether MOTS-c functions as a mitochondrial-encoded regulator of physical capacity and performance.

The paper references extensive literature on the hallmarks of aging, the role of mitochondria in aging, and the importance of mitonuclear communication in cellular homeostasis. Previous research on MOTS-c's role in metabolic homeostasis, particularly its ability to reverse diet-induced obesity and insulin resistance, is also highlighted. The literature review sets the stage for exploring MOTS-c's impact on physical performance and lifespan.

The study employed a multi-faceted approach, including human studies and experiments with mice and C2C12 myoblast cells. Human participants (sedentary healthy young males) underwent a stationary bicycle exercise regimen, with blood and skeletal muscle samples collected at various time points (before, during, and after exercise, and after a 4-hour rest) to assess endogenous MOTS-c levels. In mice, the researchers used different strains (CD-1 and C57BL/6J/N) and ages (young, middle-aged, and old) to investigate the effects of MOTS-c treatment (daily or intermittent) on physical performance (treadmill running, rotarod, grip strength, walking test, gait analysis), metabolic parameters (body composition, respiratory exchange ratio, blood glucose, metabolomics), and cognitive function (Barnes maze and Y-maze tests). C2C12 myoblast cells were used to study the *in vitro* effects of MOTS-c on metabolic stress resistance, cell viability, proliferation, and gene expression. Various techniques were employed, including Western blotting, ELISA, time-domain NMR, metabolic cages, RNA sequencing, GSEA, and confocal microscopy.

The study revealed several key findings: 1. Exercise induced significant increases in MOTS-c expression in both skeletal muscle and plasma in humans. 2. MOTS-c treatment significantly improved physical performance in young, middle-aged, and old mice, regardless of diet (normal or high-fat diet). This improvement was observed in treadmill running tests, where treated mice ran significantly longer and farther, demonstrating enhanced endurance and speed. 3. In old mice, MOTS-c treatment not only improved physical performance but also outperformed untreated middle-aged mice, suggesting a potential for physical reprogramming beyond simple rejuvenation. 4. Late-life intermittent MOTS-c treatment significantly improved several aspects of healthspan in old mice, including grip strength, gait, and walking capacity. 5. Metabolomics analysis showed that MOTS-c treatment regulated skeletal muscle metabolism, impacting glycolysis, pentose phosphate pathway, and amino acid metabolism. In old mice, MOTS-c improved metabolic flexibility, shifting fuel preference towards fat utilization. 6. *In vitro* studies showed that MOTS-c protected C2C12 myoblasts from metabolic stress by enhancing cell viability and proliferation. 7. MOTS-c treatment regulated gene expression in C2C12 cells, particularly those involved in heat shock response and proteostasis, with HSF1 identified as a potential key transcription factor mediating these effects. The RNA-seq data revealed common pathways affected by MOTS-c in both *in vivo* and *in vitro* models, including metabolism and proteostasis.

The findings strongly support the hypothesis that MOTS-c acts as a mitochondrial-encoded regulator of physical capacity and aging. The improvement in physical performance in mice of different ages and the induction of MOTS-c expression by exercise in humans suggest a crucial role for this peptide in the adaptive response to physical activity. The impact on healthspan, even when treatment was initiated late in life, is particularly significant, suggesting the potential of MOTS-c as a therapeutic target to extend healthy lifespan. The observed metabolic reprogramming, including enhanced metabolic flexibility and improved proteostasis, further underscores the peptide's multifaceted effects. The study provides strong evidence for a bi-genomic basis of aging, implying that both the mitochondrial and nuclear genomes collaborate in the aging process.

This study demonstrates that MOTS-c, a mitochondrial-encoded peptide, plays a significant role in regulating age-dependent physical decline and muscle homeostasis. Exercise induces its expression, and its administration improves physical performance and healthspan in mice, even when initiated late in life. The mechanisms appear to involve regulation of skeletal muscle metabolism and proteostasis, potentially through HSF1. Future research could focus on optimizing MOTS-c treatment strategies, investigating its cellular uptake mechanisms, and exploring its therapeutic potential in humans.

The study primarily focused on mice, and further research is needed to confirm the findings and investigate the potential therapeutic applications in humans. The sample sizes in some experiments were relatively small. Also, while the study demonstrated a trend towards increased lifespan with late-life intermittent MOTS-c treatment, a larger cohort would be needed to confirm this effect conclusively. The precise mechanisms underlying MOTS-c's action remain to be fully elucidated.