Effects of the order of endurance and high-intensity interval exercise in combined training on mouse skeletal muscle metabolism.

Endurance exercise (EE) mainly improves oxidative capacity, whereas high-intensity interval exercise (HIIE) also improves glycolytic capacity. There is growing evidence that suggests that combining EE with HIIE can lead to improved athletic performance and fitness outcomes compared with either form of exercise alone. This study aimed to elucidate whether the order in which EE and HIIE are performed in combined training affects oxidative metabolism and glycolysis in mouse skeletal muscle. Male ICR mice at 7 wk of age were divided into three groups: control (CON), EE-HIIE, and HIIE-EE. The total training period was 3 wk (3 times/week). Mice performed running on a treadmill as endurance exercise and swimming with a weight load of 10% of body weight as high-intensity interval exercise. EE before HIIE (EE-HIIE) improved running performance in the maximal EE capacity test (all-out test) and partly enhanced the expression levels of molecular signals involved in glycolysis compared with HIIE before EE (HIIE-EE). The order of exercise did not, however, impact the expression of proteins related to mitochondrial dynamics, including those involved in the morphological changes of mitochondria through repeated fusion and fission, as well as oxidative energy metabolism. The findings suggest that the order of exercise has no significant impact on the expression of proteins associated with glycolytic and oxidative energy metabolism. Nevertheless, our results indicate that the order of EE-HIIE may enhance running performance.

Black Tea High-Molecular-Weight Polyphenol-Rich Fraction Promotes Hypertrophy during Functional Overload in Mice.

Mitochondria activation factor (MAF) is a high-molecular-weight polyphenol extracted from black tea that stimulates training-induced 5' adenosine monophosphate-activated protein kinase (AMPK) activation and improves endurance capacity. Originally, MAF was purified from black tea using butanol and acetone, making it unsuitable for food preparation. Hence, we extracted a MAF-rich sample "E80" from black tea, using ethanol and water only. Here, we examined the effects of E80 on resistance training. Eight-week old C57BL/6 mice were fed with a normal diet or a diet containing 0.5% E80 for 4, 7 and 14 days under conditions of functional overload. It was found that E80 administration promoted overload-induced hypertrophy and induced phosphorylation of the Akt/mammalian target of rapamycin (mTOR) pathway proteins, such as Akt, P70 ribosomal protein S6 kinase (p70S6K), and S6 in the plantaris muscle. Therefore, functional overload and E80 administration accelerated mTOR signaling and increased protein synthesis in the muscle, thereby inducing hypertrophy.

Wnt protein-mediated satellite cell conversion in adult and aged mice following voluntary wheel running.

Muscle represents an abundant, accessible, and replenishable source of adult stem cells. Skeletal muscle-derived stem cells, called satellite cells, play essential roles in regeneration after muscle injury in adult skeletal muscle. Although the molecular mechanism of muscle regeneration process after an injury has been extensively investigated, the regulation of satellite cells under steady state during the adult stage, including the reaction to exercise stimuli, is relatively unknown. Here, we show that voluntary wheel running exercise, which is a low stress exercise, converts satellite cells to the activated state due to accelerated Wnt signaling. Our analysis showed that up-regulated canonical Wnt/ß-catenin signaling directly modulated chromatin structures of both MyoD and Myf5 genes, resulting in increases in the mRNA expression of Myf5 and MyoD and the number of proliferative Pax7(+)Myf5(+) and Pax7(+) MyoD(+) cells in skeletal muscle. The effect of Wnt signaling on the activation of satellite cells, rather than Wnt-mediated fibrosis, was observed in both adult and aged mice. The association of ß-catenin, T-cell factor, and lymphoid enhancer transcription factors of multiple T-cell factor/lymphoid enhancer factor regulatory elements, conserved in mouse, rat, and human species, with the promoters of both the Myf5 and MyoD genes drives the de novo myogenesis in satellite cells even in aged muscle. These results indicate that exercise-stimulated extracellular Wnts play a critical role in the regulation of satellite cells in adult and aged skeletal muscle.

ES cell differentiation system recapitulates the establishment of imprinted gene expression in a cell-type-specific manner.

Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.

[Transdisciplinary Approach for Sarcopenia. The effects of exercise on skeletal muscle hypertrophy and satellite cells].

Skeletal muscle has a high degree of plasticity. The mass of skeletal muscle maintains owing to muscle protein synthesis and the regeneration by satellite cells. Skeletal muscle atrophy with aging (sarcopenia) is developed by decline of muscle protein synthesis and dysfunction of satellite cells. It is urgently necessary for today's highly aged society to elucidate the mechanism of sarcopenia and to establish prevention measure. This review shows that the positive effects of "exercise" on muscle protein synthesis and satellite cell function including their main molecular mechanism.

Detection Method for Gene Doping in a Mouse Model Expressing Human Erythropoietin from Adeno-Associated Virus Vector-9.

With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.

Combined effects of functional overload and denervation on skeletal muscle mass and its regulatory proteins in mice.

Skeletal muscle is a highly pliable tissue and various adaptations such as muscle hypertrophy or atrophy are induced by overloading or disuse, respectively. However, the combined effect of overloading and disuse on the quantitative adaptation of skeletal muscle is unknown. Thus, the aim of this study was to investigate the effects of the combined stimuli of overloading and disuse on mouse skeletal muscle mass and the expression of regulatory factors for muscle protein anabolism or catabolism. Male mice from the Institute Cancer Research were subjected to denervation concomitant with unilateral functional overload or functional overload concomitant with unilateral denervation. Disuse and functional overload were induced by sciatic nerve transection (denervation) and synergist ablation, respectively, and the plantaris muscle was harvested 14 days after the operation. Our results showed that denervation attenuated functional overload-induced muscle hypertrophy and functional overload partially ameliorated the denervation-induced muscle atrophy. P70S6K phosphorylation, an indicator of mechanistic target of rapamycin complex 1 (mTORC1) activation, was not increased by unilateral functional overload in denervated muscles or by unilateral denervation in functional overloaded muscles. Denervation did not affect the increase of LC3-II, a marker of autophagy activation, and ubiquitinated protein expression upon unilateral functional overload. Also, functional overload did not affect ubiquitinated protein expression during unilateral denervation. Thus, our findings suggest that functional overload-induced muscle hypertrophy or denervation-induced muscle atrophy was attenuated by the combined stimuli of overload and denervation.

High Throughput Screening of Mitochondrial Bioenergetics in Myoblasts and Differentiated Myotubes.

Skeletal muscles contain stem cells called satellite cells, which are essential for muscle regeneration. The population of satellite cells declines with aging and the incidence of pathological conditions such as muscular dystrophy. There is increasing evidence that metabolic switches and mitochondrial function are critical regulators of cell fate decision (quiescence, activation, differentiation, and self-renewal) during myogenesis. Thus, monitoring and identifying the metabolic profile in live cells using the Seahorse XF Bioanalyzer could provide new insights on the molecular mechanisms governing stem cell dynamics during regeneration and tissue maintenance. Here we described a method to assess mitochondrial respiration (oxygen consumption rate) and glycolysis (ECAR) in primary murine satellite cells, multinucleated myotubes, and C2C12 myoblasts.

The effect of serum from calorie-restricted mouse on mTOR signaling in C2C12 myotubes.

Abstract: Calorie restriction (CR) is a widely recognized dietary approach with beneficial impacts on the entire body, including enhancements in oxidative metabolism and life span extension, while maintaining nutritional balance and calorie intake. However, CR leads to reductions in skeletal muscle and fat mass due to decreased food intake. Consequently, CR significantly modifies the metabolic profile of the entire body and its tissues. The observed benefits in skeletal muscle during CR may be attributed to CR-induced signaling mediators or significant changes in blood profiles associated with CR that regulate homeostasis maintenance. This study aimed to examine the mammalian target of rapamycin signaling and mitochondrial function of skeletal muscle from mice that undergone 8 weeks of CR and cells cultured in their serum to determine whether changes in blood secreted factors during CR affect skeletal muscle cells. C57BL6/J male mice were used. For 8 weeks, these were subjected to ad libitum (AL) or 40% CR. C2C12 myotubes were subsequently treated with media containing 10% mouse serum from AL or CR for 24 h. The results indicated that 8 weeks of CR decreased muscle mass and protein synthesis response compared with the AL group. Interestingly, myotubes conditioned with CR serum exhibited an elevation in the protein synthesis response compared with those treated with AL serum. Furthermore, mitochondrial function was enhanced in both CR mice and cells treated with CR serum. These findings suggest that while CR decreases the protein synthesis response, secretory factors present in the blood during CR can activate protein synthesis and promote mitochondrial function.

The role of the mechanistic target of rapamycin complex 1 in the regulation of mitochondrial adaptation during skeletal muscle atrophy under denervation or calorie restriction in mice.

Mechanistic target of rapamycin complex 1 (mTORC1) is a protein complex that regulates skeletal muscle protein synthesis and hypertrophy. mTORC1-mediated signaling activities are activated during denervation-induced skeletal muscle atrophy and suppressed during calorie restriction-induced atrophy. Mitochondria control the qualitative plasticity of skeletal muscles primarily through biogenesis, fusion, and fission. We recently showed that mTORC1 activation contributes toward mitochondrial homeostasis. In this study, we examined the role of mTORC1 in mitochondrial adaptation during denervation- or calorie restriction-induced skeletal muscle atrophy. Seven-week-old Institute of Cancer Research mice were subjected to 14 days of denervation or calorie restriction combined with the administration of the mTORC1 inhibitor-"rapamycin". Our results showed that although mTORC1 inhibition did not alter mitochondrial biogenesis, content and enzyme activity, it suppressed the activation of dynamin-related protein 1 (DRP1), a mitochondrial fission-related protein in denervated muscle, and reduced DRP1 expression in calorie-restricted muscle. Furthermore, calorie restriction-induced mitochondrial fragmentation was partially suppressed by mTORC1 inhibition. Taken together, our results indicate that mTORC1 activation upon denervation and inhibition upon calorie restriction contributes to qualitative changes in muscle plasticity by at least partially regulating the mitochondrial fission response.

Effects of maslinic acid supplementation on exercise-induced inflammation and oxidative stress in water polo athletes: A randomized, double-blind, crossover, and placebo-controlled trial.

BACKGROUND: Olive fruit is rich in bioactive pentacyclic triterpenoids, primarily maslinic acid (MA). Previous studies have demonstrated that MA exhibits anti-inflammatory and anti-oxidative effects; however, it is unclear whether MA intake during training inhibits perceptual fatigue and muscle soreness in athletes. This study analyzed the effects of MA supplementation during athletic training on perceptual fatigue and muscle soreness. METHODS: This randomized, double-blind, cross-over, and placebo-controlled trial involved 12 young, healthy male water polo athletes. After daily training for seven days, they ingested either olive fruit extract, containing 60 mg/day MA, or a placebo. We measured perceptual fatigue and muscle soreness during the intervention using a visual analog scale and inflammatory and oxidative stress-related proteins. RESULTS: Perceptual fatigue and muscle soreness and the area under the curve during the training period were significantly lower (main effect of MA; P < 0.05) following MA supplementation than those for the placebo. MA supplementation during training lowered perceptual fatigue and muscle soreness by decreasing inflammatory factors in water polo athletes. Additionally, we examined the detailed mechanism of MA, added the participant's serum to the culture medium at a 10% concentration to determine inflammation- and oxidative stress-related intracellular signals. Skeletal muscle cells (C2C12) cultured with MA-conditioned serum before and after intervention also suppressed expression of inflammation and oxidative stress-related proteins. CONCLUSION: These findings suggest that MA intake not only reduces perceptual fatigue and muscle soreness but also decreases inflammation and oxidative stress in the blood and skeletal muscle.

Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs.

In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog (Canis lupus familiaris), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare.

Reduction of ribosome biogenesis with activation of the mTOR pathway in denervated atrophic muscle.

Mammalian target of rapamycin (mTOR) pathway positively regulates the cell growth through ribosome biogenesis in many cell type. In general, myostatin is understood to repress skeletal muscle hypertrophy through inhibition of mTOR pathway and myogenesis. However, these relationships have not been clarified in skeletal muscle undergoing atrophy. Here, we observed a significant decrease of skeletal muscle mass at 2 weeks after denervation. Unexpectedly, however, mTOR pathway and the expression of genes related to myogenesis were markedly increased, and that of myostatin was decreased. However, de novo ribosomal RNA synthesis and the levels of ribosomal RNAs were dramatically decreased in denervated muscle. These results indicate that ribosome biogenesis is strongly controlled by factors other than the mTOR pathway in denervated atrophic muscle. Finally, we assessed rRNA transcription factors expression and observed that TAFIa was the only factor decreased. TAFIa might be a one of the limiting factor for rRNA synthesis in denervated muscle.

Fundamental study of detection of muscle hypertrophy-oriented gene doping by myostatin knock down using RNA interference.

To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control. Key pointsUsing a myostatin knockdown plasmid, we have succeeded in creating a model system for gene doping using mice that resulted in muscle hypertrophy greater than that reported previously.We confirmed that there was a limit of gene doping detection using real-time PCR, either from serum or muscle smple.This model experimental system can be utilized for examining indirect methods of gene doping detection such as immune responses to gene transfer or a profiling approach using DNA microarray.

Effects of exercise intensity on white adipose tissue browning and its regulatory signals in mice.

Adipose tissue has been classified into white adipose tissue (WAT), brown adipose tissue (BAT), and beige adipose tissue the latter of which is produced as WAT changes into BAT due to exposure to cold temperature or exercise. In response to these stimulations, WAT produces heat by increasing mitochondrial contents and the expression of uncoupling protein 1 (UCP1), thus facilitating browning. Exercise is known to be one of the triggers for WAT browning, but the effects of exercise intensity on the browning of WAT remain to be unclear. Therefore, in this study, we aimed to examine the effects of high- or low-intensity exercises on the browning of WAT. Mice performed high- or low-intensity running on a treadmill running 3 days a week for four weeks. As per our findings, it was determined that four weeks of running did not significantly reduce inguinal WAT (iWAT) wet weight but did significantly reduce adipocytes size, regardless of exercise intensity. The protein expression level of UCP1 was significantly increased in iWAT by high-intensity running. In addition, the expression of oxidative phosphorylation proteins (OXPHOS) in iWAT was significantly increased by high-intensity running. These results demonstrated that high-intensity exercise might be effective for increasing mitochondrial contents and heat production capacity in iWAT. Furthermore, we found that high-intensity running increased the protein expression level of fibroblast growth factor 21 (FGF21) in skeletal muscle compared with that in low intensity running. We have also examined the relationship between browning of WAT and the expression of FGF21 in skeletal muscle and found a positive correlation between the protein expression of UCP1 in iWAT and the protein expression of FGF21 in gastrocnemius muscle. In conclusion, we suggest that high-intensity exercise is effective for the browning of WAT and the increase of FGF21 in skeletal muscle.

Effects of lactate administration on hypertrophy and mTOR signaling activation in mouse skeletal muscle.

Lactate is a metabolic product of glycolysis and has recently been shown to act as a signaling molecule that induces adaptations in oxidative metabolism. In this study, we investigated whether lactate administration enhanced muscle hypertrophy and protein synthesis responses during resistance exercise in animal models. We used male ICR mice (7-8 weeks old) were used for chronic (mechanical overload induced by synergist ablation: [OL]) and acute (high-intensity muscle contraction by electrical stimulation: [ES]) resistance exercise models. The animals were intraperitoneally administrated a single dose of sodium lactate (1 g/kg of body weight) in the ES study, and once a day for 14 consecutive days in the OL study. Two weeks of mechanical overload increased plantaris muscle wet weight (main effect of OL: p < 0.05) and fiber cross-sectional area (main effect of OL: p < 0.05), but those were not affected by lactate administration. Following the acute resistance exercise by ES, protein synthesis and phosphorylation of p70 S6 kinase and ribosomal protein S6, which are downstream molecules in the anabolic signaling cascade, were increased (main effect of ES: p < 0.05), but lactate administration had no effect. This study demonstrated that exogenous lactate administration has little effect on the muscle hypertrophic response during resistance exercise using acute ES and chronic OL models. Our results do not support the hypothesis that elevated blood lactate concentration induces protein synthesis responses in skeletal muscle.

Effect of training and detraining on monocarboxylate transporter (MCT) 1 and MCT4 in Thoroughbred horses.

The aim of this study was to investigate the effects of training and detraining on the monocarboxylate transporter (MCT) 1 and MCT4 levels in the gluteus medius muscle of Thoroughbred horses. Twelve Thoroughbred horses were used for the analysis. For 18 weeks, all the horses underwent high-intensity training (HIT), with running at 90-110% maximal oxygen consumption (VO2 max ) for 3 min, 5 days week(-1). Thereafter, the horses either underwent detraining for 6 weeks by either 3 min of moderate-intensity training (MIT) at 70% VO2 max, 5 days week(-1) (HIT-MIT group) or stall rest (HIT-SR group). The horses underwent an incremental exercise test, VO2 max was measured and resting muscle samples were obtained from the middle gluteus muscle at 0, 18 and 24 weeks. The content of MCT1 and MCT4 proteins increased after 18 weeks of HIT. At the end of this period, an increase was noted in the citrate synthase activity, while phosphofructokinase activity remained unchanged. After 6 weeks of detraining, all these indexes returned to the pretraining levels in the HIT-SR group. However, in the HIT-MIT group, the increase in the MCT1 protein content and citrate synthase activity was maintained after 6 weeks of MIT, while the MCT4 protein content decreased to the pretraining value. These results suggest that the content of MCT1 and MCT4 proteins increases after HIT in Thoroughbred horses. In addition, the increase in the MCT1 protein content and oxidative capacity induced by HIT can be maintained by MIT of 70% VO2 max, but the increase in the MCT4 protein content cannot be maintained by MIT.

Effect of the order of concurrent training combined with resistance and high-intensity interval exercise on mTOR signaling and glycolytic metabolism in mouse skeletal muscle.

Athletes train to improve strength and endurance to demonstrate maximum performance during competitions. Training methods vary but most focus on strength, endurance, or both. Concurrent training is a combination of two different modes of training. In this study, we combined resistance exercise (RE) and high-intensity interval exercise (HIIE) to investigate the influence of the order of the concurrent training on signal molecules on hypertrophy and glycolysis in the skeletal muscle. The phosphorylation levels of mechanistic target of rapamycin (mTOR) signals, p70 S6 kinase (p70S6 K), ribosomal protein S6 (S6), and glycogen synthase kinase beta (GSK-3ß) were significantly increased in the HIIE first group compared with the control group. The combined training course did not affect the glycogen content and expression levels of proteins concerning glycolytic and metabolic capacity, suggesting that a combination of HIIE and RE on the same day, with HIIE prior to RE, improves hypertrophy response and glycolysis enhancement.

Effect of endurance exercise duration on muscle hypertrophy induced by functional overload.

For many ball games, both resistance and endurance training are necessary to improve muscle strength and endurance capacity. Endurance training has been reported to inhibit muscle strength and hypertrophy, but some studies have reported that endurance exercise (EE) does not inhibit the effects of resistance exercise. Here, we examined the effect of short- or long-duration EE on mouse skeletal muscle hypertrophy induced by functional overload (OL) at the molecular level. Plantaris muscle hypertrophy was induced by OL with synergist ablation in mice. Body mass was reduced with endurance training, but EE duration (30 or 90 min) had no effect. The ratio of plantaris muscle weight to body weight was higher in the OL and EE for 30 min (OL+EE30) and OL and EE for 90 min (OL+EE90) groups compared with the OL group. Expression of mechanistic target of rapamycin signaling proteins, which is related to protein synthesis and hypertrophy, was increased in the OL+EE30 group. Expression of Forkhead box-containing protein O1, which is related to protein breakdown and atrophy, remained unchanged. However, microtubule-associated protein 1 light chain 3, a known marker of autophagy, and MAFbx, which is related to protein breakdown, were significantly increased in the OL+EE90 group. Furthermore, markers of oxidative stress, ubiquitin and 4-hydroxynonenal were also significantly increased in the OL+EE90 group compared with other groups. In conclusion, EE duration did not affect body mass and plantaris mass and did not interfere with mechanistic target of rapamycin signaling, but it did increase ubiquitinated proteins and oxidative stress. It is therefore necessary to consider training durations for EE when combining endurance and resistance training.

Effect of mechanistic/mammalian target of rapamycin complex 1 on mitochondrial dynamics during skeletal muscle hypertrophy.

Mechanistic/mammalian target of rapamycin (mTOR) is a central factor of protein synthesis signaling and plays an important role in the resistance training-induced increase in skeletal muscle mass and subsequent skeletal muscle hypertrophy response. In particular, mTOR complex 1 (mTORC1) promotes protein synthesis in ribosomes by activating the downstream effectors, p70S6K and 4EBP1, in skeletal muscle and is highly sensitive to rapamycin, an mTOR inhibitor. Recently, resistance training has also been shown to affect mitochondrial dynamics, which is coupled with mitochondrial function. In skeletal muscle, mitochondria dynamically change their morphology through repeated fusion and fission, which may be key for controlling the quality of skeletal muscle. However, how the mechanisms of mitochondrial dynamics function during hypertrophy in skeletal muscle remains unclear. The aim of this study was to examine the impact of mTOR inhibition on mitochondrial dynamics during skeletal muscle hypertrophy. Consistent with previous studies, functional overload by synergist (gastrocnemius and soleus) ablation-induced progressive hypertrophy (increase in protein synthesis and fiber cross-sectional area) of the plantaris muscle was observed in mice. Moreover, these hypertrophic responses were significantly inhibited by rapamycin administration. Fourteen days of functional overload increased levels of MFN2 and OPA1, which regulate mitochondrial fusion, whereas this enhancement was inhibited by rapamycin administration. Additionally, overload decreased the levels of DRP1, which regulates mitochondrial fission and oxidative phosphorylation, regardless of rapamycin administration. These observations suggest that the relative reduction in mitochondrial function or content is complemented by enhancement of mitochondrial fusion and that this complementary response may be regulated by mTORC1.