Skeletal muscle, being the most abundant tissue in the body, is indispensable for movement as the body relies on the contraction and relaxation of skeletal muscle. Furthermore, a key predictor of mortality is muscle mass quality (Baskin et al., 2015). The skeletal muscle plays a crucial role in maintaining the balance of metabolic processes, making it one of the primary sites for glucose disposal (DeFronzo and Tripathy, 2009). Unhealthy lifestyles and habits have led to an alarming increase in the incidence of various chronic diseases, imposing a significant economic burden on healthcare systems worldwide (Kim et al., 2016; Farr and Khosla, 2019; Castellanos et al., 2020). It is well known that exercise, as a stressor, has a significant impact on physical function (Cook et al., 2012; Wang et al., 2021). Moreover, exercise is a major factor in the extensive metabolic and molecular remodeling of skeletal muscles (Egan and Zierath, 2013). The skeletal muscle undergoes various adaptations in response to exercise, including the synchronization of muscle contraction and ATP production, as well as the utilization of energy triggered by mechano- and other metabolic sensors (Egan and Zierath, 2013; Camera et al., 2016; Fan and Evans, 2017).

Studies have shown that regular physical activity enhances exercise performance, induces increased skeletal muscle protein synthesis, inhibits skeletal muscle protein degradation (Qiu et al., 2014), improves mitochondrial function, and subsequently promotes metabolic homeostasis in the body (Shen et al., 2021). However, intensive or prolonged exercise can trigger an oxidative imbalance, which can cause oxidative damage and lead to muscle fatigue (Reid et al., 1992; O'Neill et al., 1996). If the ability to remodel skeletal muscles decreases and their quality deteriorates, it can affect the bodyʼs motor function and increase the risk of falls and injuries. The enhancement of muscle function is achieved through improved the exercise capacity and the optimized metabolic functions. Despite significant efforts to uncover the comprehensive changes in muscle caused by exercise, there are still many regulators of skeletal muscle that have yet to be discovered.

Transcriptomic profiling is a powerful technique that allows for the identification of intricate gene expression patterns (Wang et al., 2009). On the other hand, metabolomics serves as a valuable tool in detecting and elucidating changes in metabolites within a biological system under varying conditions (Dettmer et al., 2007), but few studies have investigated the comprehensive transcriptomic and metabolomic profile of exercise muscular physiology. The objective of this study was to evaluate and analyze the transcriptional and metabolic networks of skeletal muscle that are regulated by exercise in mice. By doing so, we were able to gain insights into the mechanisms through which exercise intervention impacts the overall metabolic balance of skeletal muscle.

Skeletal muscle, a crucial component in controlling movement, plays a fundamental role as the primary regulator of metabolism in various body systems. Its susceptibility to the metabolic regulations imposed by exercise is particularly notable. To the best of our knowledge, this study represents the first investigation that employs transcriptomics and metabolomics to extensively explore the collective effects of acute exercise on skeletal muscle. This study identified a total of 34 metabolites that exhibited differential regulation. These metabolites are primarily associated with several metabolic pathways, including the citrate cycle (TCA cycle), oxidative phosphorylation, and Valine, leucine and isoleucine degradation. This transcriptomics analysis unveiled 245 differentially expressed genes. The KEGG pathways involved in this study include FoxO, ubiquitin mediated proteolysis signaling pathway. By conducting a joint analysis of transcriptomics and metabolomics, 20 enriched KEGG pathways were identified, including biosynthesis of amino acids, ferroptosis, protein digestion and absorption signaling pathway. Based on the aforementioned findings, we focus on a comprehensive analysis to investigate the potential mechanism by which exercise regulates skeletal muscle function.

To begin with, the results of metabolomics in this study demonstrated a significant increase in succinate levels within the TCA cycle following acute exercise, as illustrated in Figure 3. Succinate, being an integral component of the TCA cycle, can accumulate when the flow of succinic acid within the cycle is hindered. The metabolic dysfunction in the TCA cycle can lead to a deterioration in physiological function. Succinate is generated within the mitochondria and its main destiny upon transition to an aerobic environment is to undergo oxidation at the same location. Consequently, in a scenario where aerobic metabolism significantly outweighs anaerobic metabolism, succinate will not accumulate. However, the accumulation of succinate under aerobic conditions can occur if oxygen cost far exceeds the individual’s maximal oxygen uptake (VO₂max) and anaerobic energy production is significant (Hochachka and Dressendorfer, 1976). In the analysis of metabolic pathways, succinate is not only closely related to the TCA cycle, but is also involved in various other metabolic pathways, including oxidative phosphorylation (Zhao et al., 2017).

The TCA cycle is an important pathway for intracellular oxidative phosphorylation to produce ATP, and mitochondria are the main site where the TCA cycle takes place. Moderate-intensity exercise can improve mitochondrial function, whereas both high-intensity and exhaustive exercise may disrupt mitochondrial structure and cause mitochondrial disorders (Tondera et al., 2009; Schoepe et al., 2012). In particular, skeletal muscle is an important site for the uptake and utilization of fatty acids, and mitochondria play an important role in cellular energy metabolism, being the main site for energy conversion, the TCA cycle, and oxidative phosphorylation. The mitochondria undergo pathological changes following acute exercise, leading to impaired function (Magalhaes et al., 2013). As a result, the cellular capacity for oxidative phosphorylation is affected. This implies that the disruption of mitochondria by acute exercise may lead to an inadequate energy supply and adversely affect the organism.

Disruptions in mitochondrial function could be related to amino acid pool imbalances and altered amino acid metabolism (Naseer et al., 2020). The fascinating finding in this study was that strenuous exercise resulted in the degradation of amino acids such as valine, leucine, isoleucine, and so on (as depicted in Figure 3), while amino acids such as glycine and serine increased, demonstrating that the body undergoes degradation and production of proteins associated with the injury zone immediately after the onset of the injury. It is believed that succinate is produced as a result of the catabolism of amino acids. The findings of this study demonstrated that the levels of 15 amino acids and their derivatives, such as l-asparagine anhydrous, l-lysine, d-ornithine, pyroglutamic acid, and n, n-dimethylarginine and so on, were upregulated following an acute bout of intensive exercise. These changes in metabolites involve pathways such as d-amino acid metabolism, pyruvate metabolism, propanoate metabolism, and fatty acid degradation and so on. Besides, a source of anaerobic energy may be generated by amino acid catabolism (Hochachka and Dressendorfer, 1976). The noteworthy elevation of amino acids in vivo following acute exercise suggests that the rate of acid production in the skeletal muscle of exercising mice exceeds the rate of elimination, there is an increase in muscle protein degradation, which results in the release of amino acids from muscle tissue into the bloodstream. This phenomenon could be a compensatory mechanism to provide amino acids as a substrate for energy production and gluconeogenesis during exercise. Exercise could stimulate the activity of key enzymes involved in amino acid catabolism (Harris et al., 2005).

The general consensus is that protein synthesis in skeletal muscle is suppressed during exercise, as evidenced by findings in both humans and other species (Rennie et al., 1980; Dohm et al., 1985). The results of transcriptomics in this study show that acute exercise could induce an increase in KLF-15, FoxO1, MAFbx, and MuRF1 expression (Table 2). KLF-15, a key transcription factor, not only inhibits skeletal muscle protein synthesis but also plays a crucial role in enhancing skeletal muscle proteolysis by promoting MuRF1 and MAFbx transcription, and stimulates FoxO1 (Kuo et al., 2013). FoxO1, in particular, is the main regulatory factor among all FoxO subtypes, expressed significantly in skeletal muscle. This study demonstrates that the activation of the FoxO signaling pathway is significantly enhanced, as evidenced by the data presented in Figure 6. In the process of KLF-15 mediated skeletal muscle degradation, FoxO plays a pivotal role as a transcription factor and serves as a central component of multiple degradation pathways. Participating in moderate or vigorous physical activity leads to an intensified generation of reactive oxygen species (ROS) (Vezzoli et al., 2016). The FoxO signaling pathway may be activated as a result of increased generation of ROS (Dodd et al., 2010). The importance of this heightened FoxO signaling lies in its role in increasing FoxO nuclear translocation, therefore leading to an intensified transcription of FoxO target genes, such as MAFbx and MuRF-1, which are directly involved in muscle catabolism (Bodine et al., 2001; Gomes et al., 2001; Crow, 2002), and MAFbx and MuRF-1 play a crucial role in specifically targeting proteins for degradation by the proteasome (Bodine et al., 2001; Gomes et al., 2001; Puthanveetil et al., 2013). In addition, we found that the HSP70, UCP3 and SIRT6 genes were significantly upregulated in skeletal muscle after exercise in this study. The increase in expression of the differential gene HSP70 can induce a protective anti-apoptotic response, alleviate membrane protein denaturation caused by injury stimuli, and protect the protein synthesis pathway in cells from damage (Mosser et al., 2000; Mokranjac et al., 2006). This high expression of UCP3 can reduce the release of ROS and prevent damage to the body (Nabben et al., 2008; Toime and Brand, 2010), while SIRT6 overexpression inhibits the protein degradation pathway related to muscle atrophy (Samant et al., 2017). However, their protective effects may not be sufficient to counteract the physical damage caused by acute exercise.

The ubiquitin-proteasome system (UPS) is the primary pathway for intracellular protein degradation (Meng et al., 2014). The importance of the ubiquitination level of proteins for quality control is further highlighted by the fact that approximately 80% of proteins in the body are degraded by the ubiquitin-proteasome system (Cromm and Crews, 2017). There is evidence that abnormal protein metabolism can lead to excessive activation of the ubiquitin-proteasome system, resulting in protein degradation (Cunha et al., 2012). MAFbx and MuRF-1, which are both downstream signaling proteins of FoxO, are widely recognized as crucial skeletal muscle-specific F-box-type ubiquitin ligases involved in protein ubiquitination (Bodine and Baehr, 2014). This study demonstrates an enhanced process of ubiquitin mediated proteolysis, as depicted in Figure 6. Previous studies have examined the expression of MAFbx and MuRF1 genes in skeletal muscle during exercise. Mascher et al. (2008) found that after one session of knee joint resistance exercise in sedentary young men, the mRNA content of MuRF1 increased by 4.8 times. Louis et al. (2007) had subjects perform running and resistance exercises and found increased expression of MuRF1 and MAFbx through a muscle biopsy.

UPS could regulate the degradation of proteins involved in iron metabolism, thereby modulating ferroptosis (Zhang et al., 2010; Horie et al., 2017). In this study, Figure 7 vividly depicts the crucial discovery that ferroptosis serves as a pivotal biological process, identified through KEGG pathway enrichment analysis of differential genes and metabolites. Ferroptosis, a novel type of cell death has been reported (Dixon et al., 2012). It is a specific mechanism characterized by lipid peroxidation due to iron overload, which leads to cell death (Conrad et al., 2016). Studies have shown that ferroptosis is associated with various cellular processes, such as iron homeostasis, redox homeostasis, and lipid metabolism (Yang et al., 2014; Ingold et al., 2018). UPS, as a key regulatory system for homeostasis of eukaryotic cells (Glickman and Ciechanover, 2002). Ferroptosis impacted muscle fiber differentiation and promoted muscle protein degradation. Interestingly, some ubiquitin ligases are also involved in ferroptosis occurrence (Meng et al., 2022), while myasthenia-specific MAFbx and MuRF1 expression increases with iron accumulation in the skeletal muscle of aging individuals (Bodine and Baehr, 2014; Huang et al., 2021).

Thus, take all together, this study identified multiple metabolic pathways and signaling pathways that were involved in the regulation of skeletal muscle, such as the TCA cycle, oxidative phosphorylation, protein digestion and absorption, the FoxO signaling pathway, ubiquitin mediated proteolysis, and ferroptosis signaling pathway. Additionally, the study demonstrated that acute exercise led to the upregulation of KLF-15, FoxO1, MAFbx, and MuRF1 expression, which could play a critical role in enhancing skeletal muscle proteolysis. However, the study mainly focused on the metabolic and transcription profiling of mouse muscle, and did not involve other potential biomarkers or mechanisms, such as protein spectrometry or post-transcriptional modifications of gene expression.