Age-related Sarcopenia (ArS) is a phenomenon of skeletal muscle atrophy and functional decline with advancing age (Bauer et al., 2019). ArS increases the risk of falls, disability and mortality in the elderly, affects both physical and mental health and quality of life, and is a direct contributor to rising healthcare costs in the elderly population (Xia et al., 2017a; Seol et al., 2020). Skeletal muscle anabolic resistance, the dampened muscle protein synthesis response to protein ingestion and mechanical tension, has currently been identified as a major cause of ArS, to which blunted nutrition-sensing contributes (Zhao et al., 2021). Moreover, age-related anabolic resistance was considered to be exacerbated during musculoskeletal disuse and reduced physical activity (Marshall et al., 2020).

The impaired intracellular induction or propagation of mammalian target of rapamycin complex 1 (mTORC1) signaling is a hallmark of anabolic resistance, and thought to be partially a result of weakened anabolic nutrient sensing (Wilkinson et al., 2018). Rag GTPase is the mediator of the mTORC1 pathway in response to nutrient stimulation, specifically the proteogenic amino acid leucine. Four Rag GTPases (A, B, C, and D) are expressed in skeletal muscle. RagA-RagC and RagB-RagD, respectively, from heterodimers to regulate mTORC1 activation induced by different amino acids (Sancak et al., 2008; Lee et al., 2018). In response to leucine stimulation, mTORC1 translocates from the cytoplasm to the surface of lysosomes, interacting with the active heterodimer formed by GTP-loaded RagB and GDP-loaded RagD, and then activates its downstream molecular events (Sancak et al., 2008, 2010). Although mTORC1 signaling is highly sensitive to changes in leucine levels, mTOR itself is not a leucine sensor and cannot detect leucine levels in skeletal muscle cells. Thus, in the process of promoting skeletal muscle protein synthesis and inducing adaptive hypertrophy mediated by mTORC1 in response to anabolic stimulation, a leucine sensor must be involved as a rate-limiting factor. But which proteins are involved as leucine sensors in skeletal muscle, and how they participate in regulation is not fully understood (Lushchak et al., 2019).

To date, Leucyl-tRNA synthetase (LeuRS) and Sestrin2 have been identified as the leucine sensors, both of which are able to regulate mTORC1 signaling (Han et al., 2012; Peng et al., 2014). Among them, LeuRS directly combines with GTP bound RagD and stimulates the GTP hydrolysis of GTPase-activating protein (GAP), and the inactive RagD^GTP-RagB^GDP heterodimer is transformed to a pre-activated state (i.e., RagD^GDP-RagB^GDP). Further, the Ragulator complex mediates the nucleotide exchange of RagB as the guanine nucleotide exchange factors (GEF) and forms the active heterodimer RagD^GDP-RagB^GTP which induces the activation and lysosomal translocation of mTORC1. In contrast to LeuRS, Sestrin2 negatively regulates mTORC1 activity by controlling GTP hydrolysis of RagB. Since the Michaelis constant (Km value) of LeuRS for leucine in the amino acids activation reaction and the dissociation constant (Kd value) of leucine for Sestrin2 are similar, whether LeuRS and Sestrin2 co-regulate mTORC1 collectively or separately (Pang and Martinis, 2009; Wolfson et al., 2016) requires further research (Lee et al., 2018). We recently proposed that exercise may effectively enhance the sensitivity of skeletal muscle tissues to amino acid stimulation (Zhao et al., 2021). Resistance exercise has been widely recognized as a potent intervention to maintain or increase skeletal muscle protein synthesis and hypertrophy. Although not considered the most efficient intervention to stimulate a high magnitude of muscle hypertrophy, aerobic exercise can also attenuate the inhibition of muscle protein synthesis and deposition (Xia et al., 2016). However, to date there are no studies that have investigated the role of exercise on the LeuRS/Sestrin2-mTOR axis, and it's not clear which mode of training is most efficacious in regulating mTORC1 signaling and muscle hypertrophy mediated by LeuRS and/or Sestrin2 (Zhao et al., 2021). Thus, the preliminary evidence exploring these relationships are warranted, and the results will inspire more translational research on the topic.

The objective of this study was to preliminarily investigate whether exercise stimulates skeletal muscle mTORC1 signaling via enhancing leucine-sensing (i.e., LeuRS/Sestrin2-mTOR axis), and if an increase in mTORC1 signaling results in skeletal muscle hypertrophy.

In the current study, we observed a significant increase in the CSA and diameter of gastrocnemius muscle fibers, gastrocnemius wet weight, muscle protein concentration, the phosphorylation state of mTOR signaling, and the relative expression of LeuRS in resistance exercise-trained rats. Aerobic exercise only resulted in a significant but modest increase of muscle protein concentration and p70S6K phosphorylation, and no differences in Sestrin2 expression level between groups was detected. We suggest that LeuRS increases in the RE group only, is part of a coordinated response of the resistance exercised muscle, which may contribute for muscle protein accretion.

We demonstrated for the first time that up-regulation of LeuRS expression and mTOR activation (in its two branches, namely 4EBP-1 and P70S6K) was induced by 4 weeks resistance exercise. In contrast, aerobic exercise training did not increase LeuRS levels, but P70S6K phosphorylation was increased compared to the control group (which suggests a moderate activation compatible with the smaller increases of muscle mass in this group). Since mTOR was not activated in this group, it is possible to suppose that P70S6K activation was caused by other pathways involved in muscle protein synthesis, like GSK3-β. However, the non-activation of mTOR or increased LeuRS in the AE group, suggests a role of LeuRS in activating the mTOR signaling in the RE group.

The effects of LeuRS on protein synthesis in skeletal muscle have not been widely reported, and only 4 studies provide data concerning the role of LeuRS in C2C12 myoblasts or muscle samples. Sato's work showed that LeuRS knockdown via siRNA did not decrease phosphorylated mTOR in differentiated myotubes, nor did it affect hypertrophy (Sato et al., 2018). Son et al. found that LeuRS negatively regulated myoblast differentiation in vitro, and this function was independent of its regulation of protein synthesis (Son et al., 2019). Carlin and colleagues found that acute essential amino acid ingestion did not alter the expression of LeuRS in skeletal muscle of young participants, but did up-regulate RagB expression (Carlin et al., 2014). While the consumption of a diet containing twice recommended daily allowance of protein intake for 10 weeks showed a tendency to decrease the mRNA expression of LeuRS, it did not affect the fasting mTORC1 signaling. However, increased total RPS6 might still suggest improved muscular translational capacity to maintain muscle mass (Zeng et al., 2019). Taking these clues into consideration, this might suggest a compensatory system for sensing leucine through Sestrin2. As the affinity of LeuRS for leucine is similar to that of Sestrin2, it remains elusive how these crucial sensors orchestrate leucine sensing to stimulate mTOR activation. In the present work, our results demonstrate the potential of LeuRS to modulate mTOR activity in skeletal muscle. It's reasonable to hypothesize that LeuRS may be a key sensor responding to anabolic signals, and capable of regulating protein synthesis via mTORC1 signaling in skeletal muscle. Moreover, increased muscle protein synthesis is always linked to increased intramuscular availability of amino acids (especially leucine). Thus, it's also reasonable to speculate that resistance exercise training may have resulted in an increased uptake of leucine, and then the increased intramuscular leucine level promotes muscular leucine-sensing by up-regulating of LeuRS expression. However, further studies exploring the relationships between different exercise protocols, age of subjects and LeuRS/Sestrin2-mTOR axis are warranted.

In terms of Sestrin2, studies that have compared untrained young and old mice have reported conflicting results, with one study showing lower levels (Lenhare et al., 2017), another showing increased levels in older mice (Xia et al., 2017b), and a third showing no difference between young and old human males (Zeng et al., 2018). Sestrin2 functions as a negative regulator of mTORC1 (Wolfson et al., 2016), but if Sestrin2 and LeuRS regulate the downstream mTORC1 signaling cooperatively in aging skeletal muscle, it is unclear how they affect mTORC1 activity. In light of these information, some scholars proposed that LeuRS and Sestrin2 may function at different developmental stages (Sato et al., 2018). Results concerning the effects of exercise on the expression level of Sestrin2 are unclear. In the research conducted by Crisol and colleagues, a decrease in basal Sestrin2 in the quadriceps was found in mice following a 4 week chronic exercise protocol on a treadmill running at 60% of peak workload (5 days/week, 60 min/day) (Crisol et al., 2018). However, a series of research from Fu's lab showed a significant increase in Sestrin2 expression in the quadriceps muscle of mice after 6 or 8 weeks treadmill running with progressively increasing workloads (up 75% VO2max) (Liu et al., 2015; Wang et al., 2018; Yu et al., 2019). As for the effects of resistance exercise, to our knowledge only one report exists. Zeng et al. showed no significant change in the expression of Sestrin2 in muscle biopsies from the vastus lateralis following a 12 week lower body strength training protocol (2 days/week, separated by 72 h) (Zeng et al., 2017). On the other hand, we found that 4 weeks of concurrent training (3 days of resistance exercise and 3 days of aerobic exercise weekly) in 2-year old mice lowered Sestrin2 compared to 2-year old controls, but was still significantly greater than young controls. Our results in the present study are in line with Zeng and colleagues' findings concerning the effects of resistance exercise, but being different from the reports of Crisol and colleagues as well as Fu's group concerning the effects of aerobic exercise. In our humble opinion, the differences observed between these reports could be explained by differences in exercise protocols, especially the intensity and duration of exercise sessions. Four weeks of lower intensity of aerobic exercise (<60% VO2max) might not been strong enough to result in an adaptive change of Sestrin2 expression, while higher intensity and longer duration might lead to possible discrepancies in the results (such as 60 vs. 75% VO2max; 4 weeks training vs. 6–8 weeks training).

Some limitations should be taken into consideration when interpreting the results of this study. First, we did not investigate the effects of acute exercise and time course changes on the expression of leucine-sensing related proteins (i.e., LeuRS, Sestrin2 and mTORC1 signaling). This information may help explain the seemingly contradictory results between gastrocnemius muscle protein concentration and the activity of mTOR signaling in aerobic exercise-trained rats. Second, we were not able to use a group with RagGTPases knockout mice, to fully establish the interaction between LeuRS, Sestrin2, RagGTPases, and downstream mTORC1 signaling.

In conclusion, 4 weeks resistance exercise training induced favorable adaptations in the gastrocnemius muscle of young rats, including increased wet weight, protein concentration, muscle diameter and CSA, while aerobic exercise only resulted in a significant increase of protein concentration. These positive results were likely the result of elevated leucine-sensing and subsequent protein deposition. From a molecular mechanistic standpoint, LeuRS/mTOR signaling axis may be involved in this adaptive process induced by exercise (especially resistance exercise), as no differences between groups in Sestrin2 were found. Further studies are needed to investigate these hypotheses via the use of bioinformatics or gene chip assay, and should be further verified by using targeted gene knockout/overexpression animal models or blockers/agonists (i.e., LeuRS and mTOR). Future studies are also needed to determine whether these adaptations translate into healthy benefits in human subjects when interventions are performed with different protocols concerning the exercise duration and the intensity.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Animal Ethics Committee of Jinggangshan University and Chengdu Sport University.

QW, NZ, and ZX designed the experiment. YZ, HS, YY, XD, and SL performed the experimental analyses and collected the data. YZ, JC, and HS performed the statistical analyses. YZ and HS wrote the manuscript. JC, QW, NZ, and ZX proofread the manuscript. All authors read and approved the final version.

This study was supported by grants from the National Natural Science Foundation of China (Nos. 31960192 and 31900842), the Jiangxi Provincial Science Fund for Distinguished Young Scholars (No. 20202ACBL216004), and the Jiangxi Provincial Natural Science Foundation (No. 20192BAB205081).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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