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The Journal of Nutrition logoLink to The Journal of Nutrition
. 2014 Jul 23;144(9):1409–1414. doi: 10.3945/jn.114.196691

Skeletal Muscle Ras-Related GTP Binding B mRNA and Protein Expression Is Increased after Essential Amino Acid Ingestion in Healthy Humans1,2

Matthew B Carlin 3, Ruth E Tanner 3, Jakob Agergaard 6, Thunder Jalili 3, Donald A McClain 4, Micah J Drummond 5,*
PMCID: PMC4130827  PMID: 25056691

Abstract

Essential amino acids (EAAs) are potent stimulators of mechanistic target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis. However, regulators upstream of mTORC1 that are responsive to EAA availability are not well described, especially in human skeletal muscle. The purpose of this study was to determine changes in leucyl-tRNA synthetase (LARS/LARS) and Ras-related GTP binding B (RAGB/RAGB) mRNA and protein expression in healthy human skeletal muscle after acute EAA ingestion. Muscle biopsies sampled from the vastus lateralis were obtained from 13 young adults (7 males, 6 females; aged 22.9 ± 0.9 y; body mass index 21.7 ± 0.9 kg/m2) in the fasting state (baseline) and 1 and 3 h after EAA (13 g; 2.4 g of Leu) ingestion. Real-time quantitative polymerase chain reaction and Western blotting were used to determine changes in LARS/LARS and RAGB/RAGB mRNA and protein expression, respectively. Stable isotope tracers and gas chromatography mass spectrometry were used to determine Leu intracellular concentrations and muscle protein synthesis. EAA ingestion increased RAGB/RAGB mRNA (∼60%) and protein (∼100%) abundance in adult skeletal muscle (P ≤ 0.05). EAAs also increased muscle Leu concentrations (∼130%), mTOR phosphorylation (∼30%), and muscle protein synthesis (∼50%; P ≤ 0.05) but did not alter muscle LARS/LARS abundance (P > 0.05). We conclude that acute EAA ingestion is capable of increasing RAGB expression in human skeletal muscle. Future work is needed to determine whether this adaptive response is important to promote muscle protein anabolism in humans. This trial was registered at clinicaltrials.gov as NCT01669590.

Introduction

The mechanistic target of rapamycin complex 1 (mTORC1)7 is fundamental in stimulating human muscle protein synthesis (MPS) after essential amino acid (EAA) ingestion (1–4). However, less is known of the upstream molecular events that are responsible for mTORC1 activation in the presence of amino acid availability (5), especially in human skeletal muscle. An improved understanding of these regulators in human muscle will provide important information for future development of therapeutic approaches to regulate cell size in conditions of atrophy (e.g., sarcopenia, disuse).

Recently, leucyl-tRNA synthetase (LARS) was proposed to be critical for activating mTORC1 and protein synthesis (6, 7). In these studies, LARS, when bound to Leu, promoted the translocation of mTORC1 to the lysosome. Alternately, LARS knockdown cells prevented mTORC1 translocation to the lysosomal membrane (6, 7), whereas knockdown of isoleucyl-tRNA synthetase, methionyl-tRNA synthetase, or valyl-tRNA synthetase did not affect mTORC1 translocation. Together, these data suggest that LARS has a unique role compared with the other EAA amino acyl-tRNA synthetases in sensing Leu availability and regulating mTORC1 translocation.

The Ras-related GTP binding (RAG) GTPases (RAGA–RAGD) are also critical for amino acid–induced regulation of mTORC1 (8, 9) and were proposed to involve LARS (6, 7). The RAG proteins function as heterodimers such that RAGA or RAGB form a complex with RAGC or RAGD. In the presence of Leu, the Ragulator complex acts as a guanine–nucleotide exchange factor and facilitates RAGB into the GTP-bound state (10, 11), consequently activating the RAG heterodimeric complex. The Ragulator complex tethers the RAG proteins to the lysosomal membrane in which the RAGs physically interact with Raptor (10, 11). Interestingly, LARS bound with Leu interacted directly with RAGD and functioned as a GTPase activating protein to promote the transition from RAGD–GTP to RAGD–GDP, in turn activating mTORC1 (6, 7). Together, LARS and the RAG proteins (i.e., RAGB) are activated in the presence of increased amino acid availability and cooperate in the translocation and activation of mTORC1.

In our previous work in human skeletal muscle, we observed repeatedly an acute upregulation of the mRNA and protein of select amino acid transporters (important regulators of mTORC1 activation) after EAA ingestion alone, after a bout of resistance exercise, and after the combination of EAA or protein ingestion immediately after resistance exercise (12–15). Interestingly, these nutrient regulators had an overlapping time course response with changes in skeletal mTORC1 signaling and MPS and thus may play a role in promoting protein anabolism. Therefore, we hypothesized that, in response to acute EAA ingestion, skeletal muscle LARS/LARS and RAGB/RAGB (mRNA and protein) would similarly increase and would have an overlapping time course with mTORC1 signaling and MPS. As a follow-up to our findings, we also hypothesized that EAAs would increase the binding of LARS and RAGB with mTORC1 in human skeletal muscle.

Materials and Methods

Participants.

A total of 13 healthy male and female young participants (aged 18–28 y) were recruited through poster advertisements on the University of Utah campus and in the surrounding Salt Lake City community. All participants gave their written informed consent before participating in the study. Participant characteristics are listed in Table 1. The participants were recreationally active but were not engaged in any regular exercise training program. All women were studied in the follicular phase of their menstrual cycle. Exclusion criteria included, but were not limited to, the following diseases: 1) heart; 2) lung; 3) blood; 4) vascular; 5) liver; 6) kidney; 7) infectious; 8) oncologic; and 9) neurologic. This study was approved by the University of Utah Institutional Review Board and conformed to the Declaration of Helsinki and Title 45, U.S. Code of Federal Regulations, Part 46, Protection of Human Subjects.

TABLE 1.

Participant characteristics of young healthy adults1

Gender, M/F 7/6
Age, y 22.9 ± 0.9
Height, m 1.7 ± 0.1
Weight, kg 65.9 ± 3.8
BMI, kg/m2 21.7 ± 0.9
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1

Values are means ± SEMs.

Experimental protocol.

The evening before the experiment, participants ingested a standardized evening meal (30% fat, 15% protein, 55% carbohydrate). The next morning, after an overnight fast and refrainment of intense physical activity over a 48-h period, participants arrived at 0600 by transportation to the Center for Clinical and Translational Sciences at the University of Utah. After arriving, an 18-gauge polyethylene catheter was inserted into the antecubital vein for the primed, constant infusion of l-[ring-13C6]Phe (Cambridge Isotopes Laboratories) to determine MPS and l-[1-13C]Leu for determination of intracellular Leu concentrations. The priming dose for the labeled Phe was 2 μmol/kg, and the infusion rate was 0.05 μmol ⋅ kg−1 ⋅ min−1. The priming dose for the labeled Leu was 4.8 μmol/kg, and the infusion rate was 0.08 μmol ⋅ kg−1 ⋅ min−1. Two and 4 h after the start of the infusion (0700), a muscle biopsy was taken from the vastus lateralis for determination of baseline MPS rates and usage for Western blotting and qPCR. Immediately after biopsy 2, participants ingested ∼13 g of crystalline EAAs (Glanbia Nutritionals) mixed in a 400-mL flavored low-calorie, noncaffeinated beverage. The EAA beverage contained 7.7% l-[ring-13C6]Phe and 5.2% l-[1-13C]Leu to minimize tracer dilution. Additional muscle biopsies were taken 1 and 3 h after EAA ingestion. The composition of EAA mixture was the following: l-His (1.6 g), l-Ile (1.0 g), l-Leu (2.4 g), l-Lys (3.1 g), l-Met (0.8 g), l-Phe (1.2 g), l-Thr (1.2 g), and l-Val (1.5 g). The dosage of EAAs used in this study, as determined by the quantity of Leu, is effective to maximally stimulate MPS in young adults (1, 4, 16).

Muscle biopsy.

Muscle biopsies were sampled from the vastus lateralis using aseptic technique, local anesthesia (1% lidocaine), and a 5-mm Bergström biopsy needle with manual suction (17). Muscle biopsies 1 and 2 were taken from a single incision. Biopsies 3 and 4 were taken from a separate incision, ∼7 cm proximal from the first incision. Biopsies taken from the same incision (biopsies 1 vs. 2, 3 vs. 4) were separated by ≥5 cm by angling the biopsy needle. All muscle tissue was immediately blotted and dissected of visible nonmuscle tissue, flash frozen in liquid nitrogen, and stored at −80°C for later analysis.

Western blotting.

We reported details of Western blotting previously (18). Membranes were incubated overnight in LARS (1:1000; Abcam; catalog #ab31534), RAGB (1:2000; Cell Signaling Technology; catalog #8150), phosphorylated mTOR (Ser2448; 1:1000; Cell Signaling Technology; catalog #2971), or phosphorylated mTOR (Ser2481; 1:1000; Cell Signaling Technology; catalog #2974). The next morning, secondary antibody was added to the membrane. Chemiluminescence reagent (ECL Plus; GE Healthcare) was applied to each blot and then assessed with a digital imager (ChemiDoc XRS+ Bio-Rad). Membranes were stripped of primary and secondary antibodies and then reprobed for total mTOR (1:1000; Cell Signaling Technology; catalog #2972) and GAPDH (Cell Signaling Technology; catalog #2118) on a separate day. Densitometric analysis was performed using Lab version 4.1 software (Bio-Rad). Whole-muscle homogenate data were normalized to the internal control (loaded in duplicate on each gel), and replicate samples were averaged and reported as fold change from baseline. Phosphorylated mTOR (Ser2448 and Ser2481) was reported as phosphorylated mTOR/total mTOR (fold change). GAPDH was used to verify equal sample loading.

Coimmunoprecipitation.

To further describe whether the binding of LARS and RAGB to mTORC1 was altered after EAA ingestion, we evaluated a subset of muscle samples. We were restricted to n = 8 (4 males and 4 females; aged 22.8 ± 1.3 y; 22.8 ± 1.2 kg/m2) for this experiment because of limited available muscle tissue. Details of the coimmunoprecipitation for mTORC1 were reported by us and others previously (19, 20). Six microliters of mTOR antibody (Cell Signaling Technology; catalog #2972) was added to 700 μg of protein and then rotated overnight at 4°C. The mTOR protein–antibody complex was isolated by adding BioMag goat anti-rabbit IgG (Qiagen) beads, and then the immunoprecipitated sample (25 μL) was subjected to Western blotting as described above. Membranes were incubated in LARS or RAGB antibody overnight and then on another day reprobed for total mTOR. Coimmunoprecipitation data were normalized to the internal control, and replicate samples were averaged. Coimmunoprecipitation data were reported as target protein relative to total mTOR. No signal was present when using an IgG control antibody (1:1000; Cell Signaling Technology; catalog #2729) for precipitation.

RNA extraction, cDNA synthesis, and semiquantitative real-time PCR.

Total RNA, cDNA synthesis, and real-time qPCR were conducted as reported previously by our team (14). RNA integrity was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies). The mean RNA integrity number was 9.5 ± 0.5 (1–10 scale, in which 1 is low and 10 is high), and the 28S:18S ratio was 1.53 ± 0.03, indicating high RNA integrity. Real-time qPCR was performed with a CFX Connect real-time PCR cycler (Bio-Rad). Primers [LARS, RAGB, and hydroxymethybilane synthase (HMBS)] were custom designed (Beacon Designer), purchased from Life Sciences, and carefully optimized (i.e., efficiency, DNA gel). The primer sequences and GenBank accession numbers were the following: 1) LARS (NM_020117): forward, ATGATTGACGCTGGAGAT and reverse, CAGAGCCACAACACATTC; 2) RAGB (NM_016656): forward, AAGGCGGAGGCAAGAATA and reverse, CGCAACAAGTCCTTTCCA; and 3) HMBS (NM_000190): forward, CACAGTTGGTAGGCATCA and reverse, AGTTAATGGGCATCGTTA. RAGD (NM_021244) primers were generated from PrimerBank (21) and also purchased from Life Sciences. The cycle threshold values for HMBS were similar across time points; therefore, HMBS was used to normalize the endpoints of interest. Fold change values were calculated using the 2−∆∆Ct method (22).

Intracellular Leu concentrations and MPS.

Bound muscle proteins and muscle intracellular free amino acids were extracted from biopsy samples (biopsies 1–4) as described previously (23). GC-MS (6890 Plus CG, 5973N MSD, 7683 autosampler; Agilent Technologies) was used to determine muscle intracellular free concentrations of Leu via the internal standard method as described previously (24), which included measurement of tracer enrichments for l-[1-13C]Leu and the internal standard, [5,5,5-2H3]Leu. However, because of technical issues with the intracellular Leu enrichments, only 12 of the 13 participants were included in the final analysis.

Mixed-muscle protein-bound Phe enrichment for l-[ring-13C6]Phe was analyzed by GC-MS after protein hydrolysis and amino acid extraction (23, 25), using the external standard curve approach (26). MPS was calculated by measuring the incorporation rate of the Phe tracer into the proteins and using the precursor-product model to calculate the synthesis rate. Data are expressed as percentage per hour.

Statistical methods.

A repeated-measures ANOVA followed by pairwise comparisons (Fisher’s least significant differences) were used to assess specific differences between baseline vs. 1-h EAA and baseline vs. 3-h EAA. Significance was set at P < 0.05. All values are presented as mean ± SEM. All analyses were performed with SigmaPlot (version 12.0).

Results

mRNA and protein abundance.

We found that LARS/LARS mRNA and protein abundance (Fig. 1A, B) were not different from baseline at either 1 h (mRNA, P = 0.45; protein, P = 0.32) or 3 h (mRNA, P = 0.46; protein, P = 0.34) after EAA ingestion. RAGB/RAGB mRNA and protein abundance (Fig. 1C, D) were not different from baseline at 1 h (mRNA, P = 0.98; protein, P = 0.095) after EAA ingestion but increased at 3 h at both the mRNA and protein levels (mRNA, P = 0.039; protein, P = 0.025) after EAA ingestion. RAGD mRNA abundance was not different from baseline at 1 h (0.97 ± 0.17-fold, P = 0.85) or 3 h (0.85 ± 0.22-fold, P = 0.76) after EAA ingestion (data not shown).

FIGURE 1.

FIGURE 1

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LARS/LARS mRNA (A) and protein (B) expression and RAGB/RAGB mRNA (C) and protein (D) expression at baseline and 1 and 3 h after EAA ingestion in the skeletal muscle of young, healthy adults. Insets above panels B and D are Western blot images representing protein expression (in duplicate) for LARS, RAGB, and GAPDH at baseline and 1 and 3 h after EAA ingestion. Western blot images of GAPDH were used to verify equal protein across sample time points. Data are means ± SEMs (n = 13) and are reported as fold changes from baseline. *Different from baseline, P ≤ 0.05. B, baseline; EAA, essential amino acid; LARS/LARS, leucyl-tRNA synthetase; RAGB/RAGB, Ras-related GTP binding B.

Coimmunoprecipitation.

We found that the LARS–mTORC1 association was not different from baseline at 1 h (P = 0.28) or 3 h (P = 0.33) after EAA ingestion (Fig. 2A). Similarly, the RAGB–mTORC1 association was not different from baseline at 1 h (P = 0.87) or 3 h (P = 0.61) after EAA ingestion (Fig. 2B).

FIGURE 2.

FIGURE 2

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LARS–mTORC1 (A) and RAGB–mTORC1 (B) associations (relative to total mTOR) at baseline and 1 and 3 h after EAA ingestion in the skeletal muscle of young, healthy adults. Insets above figures are Western blot images representing protein expression (in duplicate) for LARS, RAGB, and total mTOR at baseline and 1 and 3 h after EAA ingestion. Data are means ± SEMs (n = 8). LARS and RAGB data are reported as fold change from baseline. B, baseline; EAA, essential amino acid; LARS, leucyl-tRNA synthetase; mTOR, mechanistic target of rapamycin; mTORC1, mechanistic target of rapamycin complex 1; RAGB, Ras-related GTP binding B.

mTOR, Leu concentrations, and MPS.

As expected, mTOR phosphorylation at Ser2448 was elevated above baseline at 1 h (P < 0.01) and returned to baseline at 3 h (P = 0.97) after EAA ingestion (Fig. 3A). Similarly, mTOR phosphorylation at Ser2481 increased at 1 h (1.29 ± 0.12-fold, P = 0.026) and returned to baseline 3 h (1.01 ± 0.12-fold, P = 0.95) after EAA ingestion (data not shown). Leu intracellular concentrations increased from baseline ∼2.3-fold at 1 h (P < 0.01) and ∼1.5-fold 3 h (P < 0.01) after EAA ingestion (Fig. 3B). Finally, mixed MPS increased from baseline ∼1.5-fold at 1 h (P = 0.036) but returned to baseline at 3 h (P = 0.76) after EAA ingestion (Fig. 3C).

FIGURE 3.

FIGURE 3

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Intracellular Leu concentrations (μmol/L) (A), mTOR phosphorylation (Ser2448) relative to total mTOR (B), and mixed muscle protein synthesis rates (percentage per hour) (C) at baseline and 1 and 3 h after EAA ingestion in skeletal muscle of young, healthy adults. Inset above panel B is a Western blot image representing samples (in duplicate) at baseline and 1 and 3 h after EAA ingestion. Western blot images of GAPDH were used to verify equal protein across sample time points. Data are means ± SEMs (n = 13). Data for panel B are reported as fold change from baseline. Panel A only contains data from 12 participants. *Different from baseline, P < 0.05. B, baseline; EAA, essential amino acid; mTOR, mechanistic target of rapamycin.

Discussion

The purpose of this study was to characterize the acute expression of 2 important molecular regulators of amino acid–mediated mTORC1 activation in human skeletal muscle. Our primary finding was that skeletal muscle RAGB/RAGB mRNA and protein expression (but not LARS/LARS) was acutely increased after EAA ingestion. However, despite an increase in mTOR phosphorylation, intracellular Leu concentrations, and MPS, we did not detect a change in LARS–mTORC1 and RAGB–mTORC1 associations from baseline at either 1 or 3 h after EAA ingestion in healthy human skeletal muscle. We conclude that an increase in amino acid availability is capable of acutely increasing RAGB/RAGB mRNA and protein abundance in human skeletal muscle. Future investigations are needed to determine whether this adaptive response is an important mechanism to improve sensitivity to a subsequent anabolic stimulus (e.g., meal, exercise).

The novel finding from our study was that RAGB/RAGB mRNA and protein abundance increased after EAA ingestion in human skeletal muscle. We observed previously acute increases in both protein and mRNA abundance of other nutrient regulators of mTORC1 after EAA ingestion in humans (18), suggesting that an adaptive upregulation of select amino acid–sensitive regulators of mTORC1 may be a common response in the presence of amino acid availability in humans. We speculate that an acute increase in RAGB/RAGB mRNA and protein may be an adaptive response important to amplify a subsequent anabolic stimulus. These findings, coupled with our previous findings (12–15), support conducting a future clinical trial to test whether a previous protein-enriched meal (i.e., breakfast) may serve as the basis for producing a magnified protein anabolic response to a second meal (i.e., lunch). Additionally, an acute upregulation of RAGB with EAA ingestion may be partly related to the additive anabolic response that is observed when EAAs are ingested in close proximity to a bout of resistance exercise (27–29).

We are unsure of the mechanism responsible for the nearly 100% increase in skeletal muscle RAGB protein abundance that occurs within hours of EAA ingestion. However, it is not unreasonable to suspect that the upregulation of RAGB mRNA observed in this study may have contributed to the accumulation of RAGB protein. Moreover, it is possible that the transcriptional upregulation of RAGB mRNA may have been detected earlier if more sampling time points were feasible because RAGB protein tended to increase at 1 h after EAA ingestion (P = 0.095). However, we cannot rule out the possibility that post-translational mechanisms may contribute to RAGB protein accumulation. Finally, although out of the scope of our study, it is possible that endogenous insulin release could have had a role in the induction of RAGB. This is based on previous lines of evidence that circulatory insulin concentrations increase after a bolus of EAAs (i.e., 10 g) (14) and an understanding that insulin is capable of upregulating transcription factors (30) and participates in the assembly of an active mTORC1 complex (31).

To the best of our knowledge, a transcriptional regulator has not been identified for RAGB. We predict that activating transcription factor 4 (ATF4) may be a candidate regulator in the acute induction of RAGB mRNA in response to amino acid availability. Given the role of ATF4 as an important transcriptional mediator of many amino acid transporters and amino acyl-tRNA synthetases (30, 32, 33) coupled with our previous observation of increased ATF4 after EAA ingestion in human muscle (14), we suggest that this might be a possibility. Therefore, we examined known amino acid response element sites (32) within a proximal region of the transcriptional start site of the RAGB promotor (data not shown), and, as a result, we could not verify the existence of such a sequence. Although these known composite sites are not enriched in RAGB and given the complexity of ATF binding and enhancers, we cannot conclude that this is evidence against ATF4 regulating RAGB but simply that experiments need to be conducted to rule against or in favor of ATF4 as an activator of RAGB transcription.

Recent cell studies showed that a critical step in amino acid–induced mTORC1 activation is the recruitment of mTORC1 to the lysosome by LARS and RAG proteins (6, 9). Therefore, a follow-up experiment was to evaluate the mTORC1 association with LARS and RAGB. Contrary to our hypothesis, LARS–mTORC1 and RAGB–mTORC1 association was not altered after EAA ingestion. Certainly, for ethical considerations, we were limited to few sampling time points and number of participants (n = 8) to accurately detect differences. Nonetheless, we were surprised by our findings because EAA ingestion created a robust anabolic environment in skeletal muscle as evidenced by increased mTOR phosphorylation (Ser2448 and Ser2481), intracellular Leu concentrations, and MPS. The EAA dose used in the current study (13 g of EAAs; 2.4 g of Leu) was designed to elicit a maximal MPS response in young human skeletal muscle (4, 16). Furthermore, the time points we chose were in accordance with maximal amino acid–induced mTORC1 signaling and MPS in human muscle (2, 4, 14, 34, 35). Nonetheless, our findings are consistent with those of Suryawan and Davis (36), who found no change in RAGB–Raptor association after a 2-h amino acid infusion in skeletal muscle of neonatal pigs aged 6 and 28 d. Although we initially predicted that the LARS–mTORC1 and RAGB–mTORC1 associations might occur earlier than 2 h in human muscle (i.e., 1 h) and may be highly responsive to rapid increases in amino acid concentrations with a bolus (37), our chosen postprandial time points may have been too late to detect maximal LARS–mTORC1 or RAGB–mTORC1 associations. This notion is not unreasonable because an increase in plasma EAA/Leu concentrations and skeletal muscle ribosomal S6 kinase 1 and 4E-binding protein 1 phosphorylation (effectors of mTORC1) are detected in as little as 15–30 min after amino acid/protein ingestion in healthy young adults (4, 35, 38, 39). Additionally, changes in the LARS–mTORC1 and RAGB–mTORC1 associations may have been at a level undetectable by Western blotting methods yet still capable of maintaining an increase in mTORC1 signaling and MPS.

The lack of a change in the LARS–mTORC1 association after EAA ingestion is also difficult to explain beyond the limited sampling time points. A possible explanation is that other GTPase-activating proteins may exist in human muscle, such as folliculin tumor suppressor, to coordinate the nucleotide exchange of RAGD (40). Tsun et al. (40) noted that folliculin tumor suppressor and its binding partner, folliculin interacting protein 1, regulated RAG–mTORC1 binding by acting as a GTPase activating protein for RAGC/D, whereas LARS did not express GTPase activating activity. Therefore, the role of LARS in amino acid sensing should be investigated further in humans.

In conclusion, to our knowledge, this is the first time the integral nutrient signaling molecules LARS and RAGB, responsible for mTORC1 activation, were investigated in human skeletal muscle. The novel finding of this study was that skeletal muscle RAGB/RAGB mRNA and protein increased in response to amino acid availability in healthy humans. Additional work is needed to determine whether this adaptive response has a role in promoting protein anabolism.

Acknowledgments

The authors thank Ming Zheng, Shelley Medina, and Susan Wilson at the University of Texas Medical Branch for their assistance with the GC-MS analysis and Glanbia Nutritionals for providing the premixed EAAs. M.J.D. developed the research proposal and design; M.J.D. and M.B.C. wrote the manuscript; M.J.D. and M.B.C. analyzed the data; M.J.D., M.B.C., R.E.T., and J.A. collected the data; and M.J.D., M.B.C., R.E.T., J.A., T.J., and D.A.M. reviewed the manuscript. All authors read and approved the final manuscript.

Footnotes

7

Abbreviations used: ATF4, activating transcription factor 4; EAA, essential amino acid; HMBS, hydroxymethybilane synthase; LARS, leucyl-tRNA synthetase; MPS, muscle protein synthesis; mTORC1, mechanistic target of rapamycin complex 1; RAG, Ras-related GTP binding.

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