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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 myostatinknockout 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 realtime 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. [ABSTRACT FROM AUTHOR]

TAKEMASA, T., ABE, Y., KUMAGAI, C. and KIYOSAWA, H. Possible Involvement of MicroRNA-21 in Skeletal Muscle Hypertrophy. Adv. Exerc. Sports Physiol., Vol.18, No.1 pp.5-15, 2012. In order to elucidate the function of microRNAs (miRNAs) in the plasticity of skeletal muscle, we analyzed global changes in miRNA expression using the microarray technique during muscle hypertrophy induced by compensatory overload following bilateral Achilles tenotomy in mice. The most prominent change observed by microarray analysis was that of miR-21 expression. The expression of myogenic genes, myogenin and MEF2D was increased, and that of MyoD and MHCIIb was decreased in overloaded muscle. We also investigated the changes in expression of PDCD4, PTEN, and SPRY2, which are potential targets of miR- 21, and reduction in the expression of all three genes was observed in hypertrophic muscle We next investigated the expression of miR-21 and its prospective target genes using an in vitro differentiation system with C2C12 cultured myocytes through overexpressed or suppressed miR-21 by introducing the miR-21 precursor or inhibitor. When the miR-21 precursor was transfected, miR-21 expression was significantly increased, but the genes controlling muscle differentiation (MyoD, myogenin, MHCIIb, MEF2D) and possible miR-21 target genes (PDCD4, PTEN, and SPRY2) were not significantly changed in myoblasts. Similary, when we applied the miR-12 inhibitor to C2C12, the expression of myogenic genes was not significantly changed, although possible miR-21 target genes (PDCD4, PTEN, and SPRY2) tended to be up-regulated. These results raise the possibility that microRNA may regulate muscle plasticity during exercise and training [ABSTRACT FROM AUTHOR]

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 () for 3 min, 5 days week. Thereafter, the horses either underwent detraining for 6 weeks by either 3 min of moderate-intensity training (MIT) at 70%, 5 days week (HIT-MIT group) or stall rest (HIT-SR group). The horses underwent an incremental exercise test, 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%, but the increase in the MCT4 protein content cannot be maintained by MIT. [ABSTRACT FROM AUTHOR]

Endurance exercise training pro- motes angiogenesis in skeletal muscle. In order to elucidate the mechanisms of exercise-induced angiogenesis in skeletal muscle, many researchers have focused on gene expression in skeletal muscle during and after an exercise, and it is well understood that acute endurance exercise induces temporarily increasing VEGF mRNA in skeletal muscle and secreting VEGF from active muscle. Because of these results, exercise-induced VEGF expression from skeletal muscle has been believed to be an early step of exercise-induced skeletal muscle angiogenesis. Recently it was revealed that inflammatory cells including macrophage secreted pro-angiogenesis cytokine including VEGF, which had an ability to facilitate angiogenesis in vivo. However the relationship between exercise-induced skeletal muscle angiogenesis and these inflammatory cells is not revealed. Therefore we had hypothesized that exercise-induced angiogenesis in skeletal muscle is facilitated by inflammatory cells secreting VEGF. To explore this hypothesis, we subjected ICR mice (male, 8 week-old) to acute endurance exercise (90 min, 20 m/min, 10° incline), sacrificed these mice by cervical dislocation at pre-exercise, 6 hour, 1 day, 2 day and 3 day after exercise, and harvested both gastrocnemius and plantaris muscle as hind-limb muscle. And, we used immunohistochemical method and observed significant increase of macrophage in hind-limb muscle at 1 day after exercise compared with pre exercise. Subsequently we observed that increment of percent- age of VEGF-secreting macrophage. These results suggest that migrating macrophages have a positive role in exercise-induced angiogenesis in skeletal muscle. [ABSTRACT FROM AUTHOR]

It has long been known that acute cold stress suppresses immune responses, and that swimming training increases tolerance to cold. The aim of the current study was to investigate whether the improved cold tolerance occasioned by swimming training prevented the suppression of immune responses as a result of acute cold stress, focusing in particular on the generation of suppressor macrophages and glucocorticoid hormones. This is because acute cold stress induces the generation of suppressor macrophages that express large numbers of receptors to the Fc portion of immunoglobulin G (MAC-1+Fc γ RII/IIIbright cells), leading to the suppression of splenocyte mitogenesis; moreover, the generation of MAC-1+Fc γγ RII/IIIbright cells is partly mediated by increased glucocorticoid levels during acute cold stress. The trained mice underwent a 6-week endurance swimming training (5 times/week) in water at 30γ for 90min. The swimming training remarkably increased brown adipose tissue mass, suggesting an improved cold tolerance. Indeed, when the swimming-trained mice were exposed to 5γ for 3h (acute cold stress), the rectal temperature was not substantially decreased, unlike the case for control mice. The proportion of MAC-1+Fc γ RII/IIIbright cells in peritoneal exudate cell population from the trained mice was unaffected by the acute cold stress. The swimming training significantly attenuated the increases in serum corticosterone levels in response to acute cold stress. Taken together, the results obtained suggegst that swimming training not only improves cold tolerance but also inhibits the generation of suppressor macrophages under acute cold stress. [ABSTRACT FROM AUTHOR]

Oligonol is a new lychee fruit-derived polyphenol converted into a low-molecular-form, which has improved absorption and strong antioxidative activity. The aim of the current study was to investigate the effects of Oligonol supplementation on perceived subjective mood states in addition to oxidative stress in 47 undergraduate athletes during 52 days of track-and-field training. This was a prospective single blind crossover study. The ratings of perceived exertion (RPE) responses were significantly (P<0.05) lower following Oligonol supplementation, suggesting that Oligonol intake caused the subjects to feel less fatigued during regular training. The results of our own questionnaire on fatigue and pain were as follows: Oligonol supplementation significantly (P<0.05)improved all the fatigue scores and tended to attenuate the feeling of three kinds of pains(muscular/anticular pain, lumbago, and menstrual pain), followed by a change fot the worse after the discontinuance of Oligonol intake. Unexpectedly, however, there were no definite changes in the Profile of Moods States (POMS) scores or oxidative stress markers (8-hydroxy-2'-deoxyguanosine and hexanoyllysine) in urine after Oligonol supplementation. The results obtained suggest that Oligonol supplementation in young athletes shows significant subjective positive effect particularly on the feeling of fatigue during regular track and-field training, possibly contributing to the maintenance of good conditioning. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to elucidate the effect of early onset of exercise on muscle injury. Methods: Mice were divided into 3 groups, control group, injury group and exercise group. For injury group and exercise group, we forced micc to get muscle injury (eccentric-stimulation induced muscle injury). Then, for only exercise group, we let mice exercise (60 minutes swimming for 3 in every 4 days) after 7 days of muscle injury. All groups were sacrificed 21 days after muscle injury. Result and Discussion: we observed that the cross sectional area of the regenerating myofibers in exercise group was larger than that in injury group and the level of fibrosis in the exercise group was lower than that in injury group. Then, we measured mRNA level of collagenlα2 and collagen5α3, which are known to be fibrosis structural component. Collagen1α2 mRNA level are almost the same among 3 groups. On the other hand, in collagen5α3 mRNA level, exercise group showed less collagen5α3 mRNA level than injury group. Then, in order to reveal how exercise inhibits fibrosis, we measured mRNA level of TGF-β 1, which is a cytokine for promoting fibrosis. We observed that TGF-β 1 mRNA level of exercise group was less than that of injury group. Taken together, our data suggests that exercise improves healing process and exercise inhibit fibrosis via TGF-β 1 down regulation. [ABSTRACT FROM AUTHOR]

Purpose: Ammonia, one of the factors inducing fatigue, is transported by Rh b glycoprotein (Rhbg) and Rh c glycoprotein (Rhcg) in liver, kidney and other organs. The purpose of this study is examination of expression of Rhbg and Rhcg in skeletal muscle. Methods: Skeletal muscle samples (Soleus: Sol, Plantaris: Pia, Gastrocremius: Gas) were dissected from ICR mice (male, 8weeks old). Rhbg and Rhcg protein contents were analyzed by Western blot. Localization of Rhbg and Rhcg were analyzed by immunofluorescent microscopy. Myosin Heavy Chain (MHC) type composition of each muscles were measured by SDS-PAGE. Results: Rhbg and Rhcg protein much expressed in Sol than Pia and Gas. A positive correlation was found between MHC type I + II a/ II b and Rhbg/Rhcg expression. Immnofluorescent microscopy identified that Rhbg was colocalized with dystrophin, plasma membrane protein marker. On the other hand, staining for Rhcg showed colocalization with CD31 (vascular endothelial cell marker). Discussion: Ammonia transporters, both Rhbg and Rhcg, were rich in slow type fiber predominance muscle. Ammonia transporters may contribute in slow fiber than fast fiber. Rhbg and Rhcg localizations were different in types ofskeletal muscle. This result indicates that Rhbg and Rhbg have different role in skeletal muscle. [ABSTRACT FROM AUTHOR]

Purpose: We investigated the effects of exercise on adult satellite cell conversion focusing at Wnt signaling, which plays important roles in lineage control during embryonic myogenesis and postnatal development. Methods: Male C57BL/6J mice ages 8 weeks (adult) and 24 months (aged) were used and divided into control group and runner group. Runner group mice were housed individually in cages equipped with a running wheel and performed voluntary wheel running for 4 weeks. After 4 weeks of exercise, mice were sacrificed and the gastrocnemius muscles were dissected out quickly from each mouse. Results: We demonstrated that voluntary wheel running exercise, which was a low-stress exercise, converted satellite cells to activated state due to accelerated Wnt signaling. Our analyses showed that upregulated canonical Wnt/b-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. Discussion: These results indicate that exercise-stimulated extracellular Wnts play a critical role in the regulation of satellite cells in adult and aged skeletal muscle. [ABSTRACT FROM AUTHOR]

Purpose: A number of studies have shown that changes in muscle contractile activity regulate the expression of monocarboxylate transporters (MCTs) in the skeletal muscle. The aim of this study was to investigate the effect offunctionaloverload on MCTI and MCT4 protein expression. Method: Plantaris muscles. from male ICR mice (8-week-old) were functionally overloaded for 15 days by ablation of the synergistic muscles. Blood samples are collected for measurirrg plasma testosterone and lactate·middot; ·middot;concentration MCT1, MCT4 ·middot;and AMPK protein expression was determined by Western blotting. Results and Discussion: The body weight was not altered thorough the experimental period. The ptanmris muscle weight increased after 1 day of overload (p<0.05). MCTl and MCT4 protein expression increased after 12 days after·middot; functional overload (p<0.05}. AMP-activated protein kinase (AMPK) phosphorylation status [phospho-AMPK (Thr172) tota TMPK] was elevated after 3-9 days of' functional overload (p<0.05). Plasma testosterone concentration was elevated after 12 days of functional overload (p<0.0-5), while lactate concentration was not altered. Thus, the current study demonstrated that heavy mechanical loading induces increase in MCTl and MCT4 protein expression in the muscles with increase in AMPK phosphorylation status and plasma testosterone concentration. [ABSTRACT FROM AUTHOR]

Purpose: Ibuprofen is a non-steroidal antiinflammatory drug that is often used as an analgesic, but its effect on skeletal muscle adaptation during endurance training is unclear Methods: In the present study, we administered ibuprofen to mice during running wheel exercise for four weeks, and examined its effects on the increase in the I and Ua myofibre and the C/F in skeletal muscle. Results and Discussion: We observed a significant increase of the I and Ila myofibre and C/F even in the presence of ibuprofen. Moreover, in untreated mice, there was a significant positive and strong correlation between these parameters and running distance. These results indicate that the increase in the I and lla myofibre and the C/F in skeletal muscle usually depend on running distance. Interestingly, we observed no significant correlation between these parameters and running distance in ibuprofen-administered mice. Moreover, we found no significant increase of these parameters when the running distance was significantly increased, in comparison with untreated mice. These results indicate that ibuprofen administration during endurance training cancels running-distance-dependent adaptations in skeletal muscle. This suggests that even if ibuprofen administration facilitates longer-distance running, no further effects of training on skeletal muscle can be expected. [ABSTRACT FROM AUTHOR]

Purpose: Muscle damage is occurred by excessive exercise training. Recently, it has been suggested that anti-oxidants and/or anti-inflammatory agents supplementation are effective for suppressing muscle damage and inducing muscle hypertrophy. The purpose of this study was to examine the effect of EMR supplementation on muscular hypertrophy model mice. Methods: Male ICR mice underwent ablation of the gastrocnemius and the soleus muscle to induce compensatory hypertrophy of the plantaris muscle. EMR and EMR-mixed whey protein were administered to half of each group for 3weeks, after which the cross-sectional area and minimum fiber diameter of the plantaris muscle fiber were measured Results: The muscle cross-sectional area and minimum fiber diameter of the EMR group and the EMR-mixed whey protein group significantly increased compared to their respective control groups. Discussion: EMR and EMR-mixed whey protein were effective for muscle hypertrophy in compensatory overload. [ABSTRACT FROM AUTHOR]

An abstract of the article "Expression of monocarboxylate transporter (MCT)1 and MCT4 in overloaded mouse plantaris muscle," by Yu Kitaoka and colleagues is presented.

An abstract of the article "Ribosome biogenesis is reduced under activated mTOR-pathway condition in denervated atrophying muscle," by Masanao Machida and colleagues is presented.

An abstract of the article "Assumption of influential molecular events for skeletal muscle hypertrophy from analysis of global gene expression during tenotomy-induced hypertrophy," by Masanao Machida and Tohru Takemasa is presented.

An abstract of the study "Expression of miRNA-21 during overload induced skeletal muscle hypertrophy," by Chiaki Kumagai and Tohru Takemasa is presented.

An abstract of the article "Effects of High-molecular-weight Polyphenol "MAF" on Gene Expression Are Comparable to That of Exercise in Skeletal Muscle Cells," by Chiaki Kumagai and Tohru Takemasa is presented.

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping. [ABSTRACT FROM AUTHOR]

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-h EPO ), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-h EPO 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.

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.

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.

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.

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.

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., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

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.

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., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

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., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Calorie restriction (CR) involves a reductions of calorie intake without altering the nutritional balance, and has many beneficial effects, such as improving oxidative metabolism and extending lifespan. However, CR decreases in skeletal muscle mass and fat mass in correlation with the reduction in food intake. Lactate is known to have potential as a signaling molecule rather than a metabolite during exercise. In this study, we examined the effects of the combination of caloric restriction and lactate administration on skeletal muscle adaptation in order to elucidate a novel role of lactate. We first demonstrated that daily lactate administration (equivalent to 1 g/kg of body weight) for 2 weeks suppressed CR-induced muscle atrophy by activating mammalian/mechanistic target of rapamycin (mTOR) signaling, a muscle protein synthesis pathway, and inhibited autophagy-induced muscle degradation. Next, we found that lactate administration under calorie restriction enhanced mitochondrial enzyme activity (citrate synthase and succinate dehydrogenase) and the expression of oxidative phosphorylation (OXPHOS) protein expression. Our results suggest that lactate administration under caloric restriction not only suppresses muscle atrophy but also improves mitochondrial function., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

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-3β) 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., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

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., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

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., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Nutritional supplements are sometimes important for athletes to improve their sports performance and maintain their condition. Maslinic acid (MA) is a type of compound with a pentacyclic triterpene structure extracted from olives, and has a strong anti-inflammatory effect and improves metabolic function. This study aimed to investigate the effects of MA on muscle hypertrophy by functional overload using an animal model. Mice plantaris muscles were overloaded by synergist ablation surgery with/without MA and they were sampled at 4, 7, and 14 d after the operation. We demonstrated that MA significantly increased plantaris' cross-sectional area and activated the mechanistic target of rapamycin (mTOR) signaling compared with the non-supplemented group (main effect of MA, p<0.05). In addition, MA also significantly reduced catabolic proteins compared with the non-supplemented group. MA supplementation increased muscle fiber size and promoted muscle hypertrophy via mTOR signaling. Our results indicate that MA supplementation may be useful for promoting hypertrophy of skeletal muscle.

The enhanced rate of protein synthesis in skeletal muscle cells results in a net increase in total protein content that leads to skeletal muscle growth/hypertrophy. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent regulation of the activity of mechanistic target of rapamycin (mTOR) and subsequent protein synthesis has been suggested as a regulatory mechanism; however, the exact molecular processes underlying such a regulation are poorly defined. The purpose of this study was to investigate regulatory mechanisms involved in the MEK/ERK-dependent pathway leading to mTORC1 activation in skeletal muscle cells. Treatment with phorbol-12-myristate-13-acetate (PMA), a potent agonist of protein kinase C (PKC) and its downstream effector in the MEK/ERK-dependent pathway, resulted in the activation of mTORC1 signaling and phosphorylation of the upstream regulator tuberous sclerosis 2 (TSC2) in C2C12 myoblasts. PMA-induced activation of mTORC1 signaling was partially prevented by treatment with U0126 (a selective inhibitor of MEK1/2) or BIX-02189 (a selective inhibitor of MEK5) and completely blocked with BIM-I (a selective inhibitor of upstream PKC). TSC2 phosphorylation at Ser664 (an ERK-dependent phosphorylation site) was prevented with U0126, and BIM-I treatment blocked PMA-induced phosphorylation of TSC2 at multiple residues (Ser664, Ser939, and Thr1462). Overexpression of Ras homolog enriched in brain (Rheb), a downstream target of TSC2, and an mTORC1 activator, was sufficient to activate mTORC1 signaling. We also identified that PMA-induced activation of mTORC1 signaling was significantly inhibited in the absence of Rheb with siRNA knockdown. These observations demonstrate that the PKC/MEK/ERK-dependent activation of mTORC1 is mediated through TSC2 phosphorylation and its downstream target Rheb in C2C12 myoblasts.

Aging is an inevitable physiological process that leads to the dysfunction of various tissues, and these changes may contribute to certain diseases, and ultimately death. Recent research has discovered biological pathways that promote aging. This review focuses on Wnt signaling, Wnt is a highly conserved secreted signaling molecule that plays an essential role in the development and function of various tissues, and is a notable factor that regulates aging. Although Wnt signaling influences aging in various tissues, its effects are particularly prominent in neuronal tissue and skeletal muscle. In neuronal tissue, neurogenesis is attenuated by the downregulation of Wnt signaling with aging. Skeletal muscle can also become weaker with aging, in a process known as sarcopenia. A notable cause of sarcopenia is the myogenic-to-fibrogenic trans-differentiation of satellite cells by excessive upregulation of Wnt signaling with aging, resulting in the impaired regenerative capacity of aged skeletal muscle. However, exercise is very useful for preventing the age-related alterations in neuronal tissue and skeletal muscle. Upregulation of Wnt signaling is implicated in the positive effects of exercise, resulting in the activation of neurogenesis in adult neuronal tissue and myogenesis in mature skeletal muscle. Although more investigations are required to thoroughly understand age-related changes and their biological mechanisms in a variety of tissues, this review proposes exercise as a useful therapy for the elderly, to prevent the negative effects of aging and maintain their quality of life.

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.

It is now evident that exercise training leads to increases in monocarboxylate transporter (MCT)1 and MCT4, but little is known about the mechanisms of coupling muscle contraction with these changes. The aim of this study was to investigate the effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) induced activation of AMP-activated protein kinase (AMPK) on MCT1, MCT4, and GLUT4 in denervated muscle. Protein levels of MCT4 and GLUT4 after 10 days of denervation were significantly decreased in mice gastrocnemius muscle, while MCT1 protein levels were not altered. AICAR treatment for 10 days significantly increased MCT4, and GLUT4 protein levels in innervated muscle as shown in previous studies. We found that the MCT1 protein level was also increased in AICAR treated innervated muscle. AICAR treatment prevented the decline in MCT4 and GLUT4 protein levels in denervated muscle. Thus, the current study suggests that MCT1 and MCT4 protein expression in muscles, as well as GLUT4, may be regulated by AMPK-mediated signal pathways, and AMPK activation can prevent denervation-induced decline in MCT4 protein.

The molecular mechanisms mediating the cellular adaptations to exercise training in human skeletal muscle are very poorly understood. To investigate the effect of endurance training on the expression of various genes at the mRNA levels in human skeletal muscle, focusing on angiogeneic factors, antioxidant enzymes, and uncoupling proteins (UCPs), seven untrained male students underwent an intensive swimming training five times a week for 3 months and two male students an intensive running training, respectively. Muscle biopsies were taken before training and about 48 h after the last session. All the subjects markedly increased their maximal oxygen uptake levels due to training (P < 0.001), indicating an improvement in aerobic capacity. After training, there were significant (P < 0.04) decreases in the expression of mRNAs for heat shock protein 70, Cu,Zn-superoxide dismutase (Cu,Zn-SOD), and Mn-SOD but a significant (P < 0.02) increase in UCP2 mRNA expression, whereas no definite changes were observed in the levels of mRNAs for vascular endothelial growth factor (VEGF), basic fibroblast growth factor, hypoxia-inducible factor-1alpha (HIF1alpha), myoglobin, or UCP3. The changes in HIF1alpha mRNA expression correlated well with those in VEGF mRNA expression after training (r=0.875, P < 0.01), suggesting that HIF1alpha influences the training-induced VEGF gene expression or alternatively that VEGF and HIF1alpha expressions are coregulated at the transcriptional level in human skeletal muscle. Taken together, it is envisioned that cumulative effects of transient changes in transcription during recovery from successive bouts of exercise may represent the underlying kinetic basis for the cellular adaptations associated with endurance training.