A complementary amino acid formulation enhances exercise-induced skeletal muscle remodeling during resistance training

Abstract

[Purpose]

This study investigated whether a five-amino-acid formulation integrating complementary anabolic, metabolic, and redox-supporting functions enhances skeletal muscle remodeling during resistance training.

[Methods]

Male C57BL/6N mice were assigned to eight groups receiving vehicle, leucine (300 mg/kg), or amino acid formulation (150-1,500 mg/kg). All mice completed four weeks of ladder resistance training, while the assigned supplements were administered orally once daily. Muscle function, body composition, gastrocnemius fiber cross-sectional area (CSA), anabolic and metabolic signaling proteins, the muscular NAD⁺/NADH ratio, and circulating markers of muscle damage, inflammation, and glutathione status were assessed.

[Results]

The formulation improved grip strength at doses of 600-1,500 mg/kg and increased gastrocnemius fiber CSA at 1,200 and 1,500 mg/kg compared with vehicle (p < 0.05), without affecting body weight, food intake, body composition, or absolute muscle mass. It also enhanced AKT-related signaling and increased the expression of PGC-1α, citrate synthase, and UCP3. Several anabolic and mitochondrial protein responses exceeded those observed with leucine alone. The NAD⁺/NADH ratio increased at doses of 150, 900, and 1,200 mg/kg, with the greatest response observed at 900 mg/kg. The formulation reduced circulating TNF-α at a dose of 1,200 mg/kg, produced a dose-related decrease in CKM that approached statistical significance at 1,500 mg/kg, and most prominently increased total and reduced glutathione levels at 900 mg/kg.

[Conclusion]

The complementary amino acid formulation enhanced functional, structural, anabolic, metabolic, and redox adaptations during resistance training. These coordinated effects support its potential as a nutritional strategy for improving skeletal muscle quality during chronic resistance exercise.

INTRODUCTION

Skeletal muscle is increasingly recognized as a pivotal tissue for maintaining and promoting overall health [1,2], owing to its diverse metabolic, endocrine, and functional roles. Metabolically, skeletal muscle plays a central role in regulating whole-body energy metabolism [1]. As an endocrine organ, it secretes signaling molecules, including irisin, cathepsin B, and lactate, which mediate physiological and functional adaptations in distant organs [3-6]. Functionally, skeletal muscle is the primary tissue responsible for generating movement and enabling physical activity. Consequently, declines in skeletal muscle quantity and quality are closely associated with impairments in physical performance and physiological function, thereby contributing to an increased risk of developing various noncommunicable diseases [2]. Therefore, strategies aimed at improving skeletal muscle mass and quality have emerged as a major research priority, not only for preventing and treating conditions, such as sarcopenia and cachexia [7], but also for promoting healthy aging and improving overall quality of life [8].

Regular exercise is widely recognized as the most effective strategy for preserving and improving skeletal muscle mass and quality. However, from the perspective of exercise nutrition, exercise alone represents only one component of the approach required to optimize skeletal muscle health [9]. Rather than directly constructing new muscle tissue, exercise serves as the primary stimulus that activates and suppresses molecular pathways governing skeletal muscle remodeling [10-12], thereby creating the physiological potential for skeletal muscle adaptation. The successful translation of this adaptive potential into quantitative and qualitative improvements in skeletal muscle ultimately depends on an adequate supply of nutrients that serve as the structural and metabolic building blocks required for muscle remodeling [13].

Among the nutrients involved in skeletal muscle remodeling, amino acids are unique because they not only serve as the principal structural building blocks of skeletal muscle but also regulate remodeling through distinct cellular signaling mechanisms [14]. Leucine is the best-characterized example. Unlike many other amino acids, leucine functions as a nutrient-sensing signaling molecule when its intracellular concentration increases. Leucine directly binds to intracellular leucine sensors, including Sestrin2 proteins and secretion-associated Ras-related GTPase 1B (SAR1B), thereby relieving their inhibitory effects on the GTPase-activating protein activity toward Rags 2 (GATOR2) complex and facilitating Rag GTPase-dependent activation of mechanistic target of rapamycin complex 1 (mTORC1) [15,16]. Through these nutrient-sensing mechanisms, leucine ultimately promotes skeletal muscle protein synthesis [15-17].

If leucine is widely recognized as the prototypical amino acid regulating the anabolic adaptation of skeletal muscle to exercise, glutamine has emerged as an important metabolic amino acid that supports exercise-induced skeletal muscle remodeling [18]. As the most abundant free amino acid in skeletal muscle, glutamine serves as a major precursor of glutamate, which is subsequently converted to α-ketoglutarate, thereby replenishing tricarboxylic acid (TCA) cycle intermediates through anaplerosis and sustaining oxidative ATP production during exercise [19]. In addition, through its close metabolic coupling with glutamate, glutamine contributes to amino group transfer and branched-chain amino acid (BCAA) metabolism, thereby supporting the elevated protein turnover and metabolic demands associated with skeletal muscle remodeling [20-23]. Despite its large endogenous pool, glutamine availability can be altered during strenuous exercise and heavy training; thus, supplementation may provide additional substrate support for the increased metabolic demands associated with exercise and recovery [24,25]. Together, these metabolic characteristics indicate that glutamine supplementation plays an important role in supporting the metabolic adaptation of skeletal muscle to exercise.

In addition to leucine and glutamine, cysteine, glycine, and taurine may provide complementary support for skeletal muscle remodeling by maintaining cellular redox and metabolic homeostasis. Cysteine and glycine serve as essential substrates for glutathione synthesis, with cysteine availability often representing a rate-limiting factor in cellular glutathione production, whereas glycine contributes to glutathione maintenance, protein synthesis, and skeletal muscle progenitor-cell function [26,27]. Taurine, a sulfur-containing amino acid metabolite derived from cysteine, further supports skeletal muscle homeostasis through its roles in mitochondrial function, membrane stabilization, and protection against metabolic stress [28].

Collectively, these findings suggest that individual amino acids exert distinct yet complementary roles during exercise-induced skeletal muscle remodeling. Because repeated exercise training increases protein and amino acid requirements to support skeletal muscle recovery and adaptation, targeted supplementation may provide a practical means of ensuring sufficient amino acid availability to support these increased demands [29,30]. Accordingly, combining amino acids with complementary physiological functions may represent a rational nutritional approach for simultaneously supporting the anabolic, metabolic, and redox adaptations required for optimal skeletal muscle remodeling. However, whether such an approach provides additive or synergistic benefits during exercise training remains unknown.

To test this hypothesis, we developed a novel formulation comprising five amino acids designed to integrate complementary anabolic, metabolic, and redox-supporting functions. Using a mouse model of resistance training, we investigated whether supplementation with this formulation enhances skeletal muscle quality in a dose-dependent manner by improving muscle function, muscle morphology, and molecular adaptations associated with skeletal muscle remodeling.

METHODS

Ethic Approval

The animal study was reviewed and approved by the Konkuk University Institutional Animal Care and Use Committee (No. KU25105). All methods were performed in accordance with the relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines: https://arriveguidelines.org/. Furthermore, efforts were made to minimize discomfort and exposure to stressful conditions throughout the study.

Animals

Male C57BL/6N mice, aged six weeks, were purchased from Orient Bio Inc. (Seongnam, Republic of Korea) and acclimated to the laboratory environment until 12 weeks of age before the start of the experiment. Mice were housed in standard transparent plastic cages under controlled environmental conditions (25-26 °C, 40-50% relative humidity) with a 12-h light/dark cycle (lights on from 07:00 to 19:00). Standard laboratory chow and water were provided ad libitum.

Experimental Design

Mice were randomly assigned to one of eight experimental groups: an exercise plus vehicle-treated group (Con), an exercise plus leucine-treated group (Leu, 300 mg/kg; nutritional reference group), and six exercise groups receiving the amino acid formulation at doses of 150 (A150), 300 (A300), 600 (A600), 900 (A900), 1200 (A1200), or 1500 mg/kg (A1500) body weight.

The intervention lasted for four weeks. Resistance exercise was performed five times per week, whereas the vehicle, leucine, or amino acid formulation was administered orally once daily, seven days per week. On exercise days, supplementation was administered immediately after each exercise session, whereas on non-exercise days, it was administered at approximately the same time of day.

To evaluate the effects of the intervention on skeletal muscle function and body composition, grip strength, maximal voluntary carrying capacity (MVCC), rotarod performance, hanging wire performance, and dual-energy X-ray absorptiometry (DXA) were assessed before and after the intervention. Body weight and food intake were recorded daily throughout the experimental period. Following completion of the intervention and all post-intervention assessments, mice were euthanized, and biological samples were collected for subsequent molecular analyses (Figure 1A).

Figure 1. Effects of Amino Acid Formulation on Serum ALT and AST Levels.

(A) Schematic overview of the 4-week experimental design. Following the intervention, serum (B) alanine aminotransferase (ALT) and (C) aspartate aminotransferase (AST) levels were measured to evaluate potential hepatic toxicity. Values are presented as the mean ± standard error of the mean (SEM) (n = 8-10 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the Con group; ##p < 0.01 versus the Leu group. DXA, dual-energy X-ray absorptiometry; MVCC, maximal voluntary carrying capacity.

Preparation and Administration of the Amino Acid Formulation

The amino acid formulation used in this study, designated as Pentamino™, was developed and supplied by Samyang Foods Co., Ltd. (Seoul, Republic of Korea). Pentamino™ is a patent-pending proprietary formulation consisting of five amino acids: leucine, glutamine, cysteine, glycine, and taurine.

All test substances were administered orally by gavage. In the exercise groups, the assigned treatment was administered immediately after each exercise session. A leucine-treated group was included as a nutritional reference group because leucine is a well-characterized amino acid that plays a key role in regulating anabolic signaling in skeletal muscle. Based on previous studies, an oral dose of 300 mg/kg leucine was selected as a biologically relevant reference dose for anabolic adaptation during resistance exercise [31,32]. The leucine used for this group was supplied by the same manufacturer and was identical to that incorporated into the amino acid formulation.

Before administration, the amino acid formulation and leucine powder were weighed according to the assigned dose using an analytical balance and dissolved in 0.5 N HCl to facilitate complete dissolution. The pH of each preparation was subsequently adjusted to 7.4 before oral administration. Mice in the control group received an equivalent volume of vehicle solution prepared using 0.5 N HCl and adjusted to pH 7.4.

Resistance Training Protocol

Resistance training was performed using a custom-built ladder-climbing apparatus consisting of a 1-m ladder with 3-mm diameter rungs spaced 10 mm apart. The ladder was positioned at an inclination of approximately 80° throughout the training period. To induce resistance exercise, mice climbed the ladder while carrying a weight attached to the base of the tail. Loaded ladder climbing is a well-established model of progressive resistance training in rodents and has been shown to induce skeletal muscle hypertrophy and improvements in maximal load-carrying capacity [33].

Before the training intervention, all mice underwent a 3-day familiarization period during which they voluntarily climbed the ladder 10 times per day without an external load. Following familiarization, MVCC, defined as the one-repetition maximum (1RM), was determined for each mouse to individually prescribe the training load. Based on the purpose of the study to induce skeletal muscle hypertrophy and functional adaptation, the training intensity was set at 75% of each animal’s 1RM during weeks 1-2 and increased to 80% during weeks 3-4 to maintain a comparable relative training stimulus throughout the intervention.

Resistance training was performed five days per week for four consecutive weeks using a 3-days-on/1-day-off/2-dayson/1-day-off schedule. Each training session consisted of 10 climbing repetitions. The first two climbs were performed without an external load as a warm-up, followed by eight loaded climbs at the prescribed training intensity. A 2-min recovery period was provided between consecutive climbing repetitions.

Dual-Energy X-Ray Absorptiometry

Body composition was assessed using a DXA system specifically designed for rodents (iNSiGHT, OsteoSys, Seoul, Republic of Korea). Before each scan, mice were anesthetized with isoflurane and positioned in the prone position to minimize movement during image acquisition. Whole-body scans were performed to determine fat mass, lean mass, fat mass percentage, and lean mass percentage. All measurements were conducted under identical conditions according to the manufacturer’s instructions. Each mouse was scanned twice, and the mean value of the two measurements was used for subsequent analyses. Body composition was analyzed using the manufacturer’s dedicated analysis software. DXA measurements were performed at two time points: immediately before the start of the intervention and after completion of the 4-week intervention period.

Grip Strength

Forelimb and hindlimb grip strength was assessed using a grip strength meter (JD-A-22, Jeongdo B&P, Seoul, Republic of Korea). Before testing, mice were allowed to acclimate to the testing environment, and all measurements were performed at the same time of day. For each assessment, mice were placed on the metal grid of the apparatus and allowed to grasp the grid using all four limbs (all-limb grip test). The mice were then gently pulled backward horizontally by the base of the tail at a constant speed until they released the grid. The peak grip force was automatically recorded by the instrument and expressed in gram-force (gf).

Each mouse was tested three times with a 1-min recovery period between trials to minimize the effects of fatigue. Trials showing abnormal movements or measurement errors were excluded from the analysis. The mean value of the three valid trials was used for subsequent statistical analyses. All measurements were performed by the same investigator to minimize inter-observer variability. Grip strength was assessed at two time points: immediately before the start of the intervention and after completion of the 4-week intervention period.

Maximal Voluntary Carrying Capacity Test

MVCC was assessed using the same ladder-climbing apparatus described for the resistance training protocol. Following completion of the familiarization sessions, mice performed the MVCC test while carrying a weight attached to the base of the tail.

The initial load was set at 15 g and progressively increased following each successful climb. The load was increased by 4 g between successive trials up to the fourth attempt and by 2 g thereafter. A successful climb was defined as the ability of the mouse to reach the top of the ladder within 1 min without falling. Failure was defined as the inability to complete the climb within 1 min or falling from the ladder during the attempt. A recovery period of 2-3 min was provided between consecutive trials to minimize the effects of fatigue. When a mouse failed a trial, the same load was repeated after the recovery period. If the mouse successfully completed the repeated trial, that load was defined as the MVCC. If the repeated trial was also unsuccessful, the load completed in the previous successful trial was considered the MVCC. MVCC was assessed immediately before the start of the intervention and after completion of the 4-week intervention period.

Hanging Wire Test

Wire hanging performance was assessed using the alllimb hanging test. Each mouse was placed on a metal wire grid, which was then gently inverted, allowing the animal to hang using all four limbs. The latency to fall was recorded. The wire grid was positioned approximately 50 cm above a padded surface to prevent injury in the event of a fall. The maximum test duration (cut-off time) was set at 300 s. Mice that remained suspended for the entire testing period were assigned a score of 300 s. Each mouse was tested twice with a 5-min recovery period between trials to minimize the effects of fatigue. The mean value of the two trials was used for subsequent statistical analyses. The hanging wire test was performed immediately before the start of the intervention and after completion of the 4-week intervention period.

Rotarod Test

Motor coordination and balance were assessed using a rotarod apparatus for mice (JD-A-07TSMR, Jeongdo B&P, Seoul, Republic of Korea). Before testing, mice were allowed to acclimate to the testing environment. Each mouse was initially placed on a stationary rotating rod for 30 s, followed by a 30-s familiarization period at a constant rotational speed of 4 rpm. The test was then performed under an accelerating protocol, during which the rotational speed increased linearly from 4 to 44 rpm over a 2-min period. The latency to fall and the rotational speed (rpm) at the time of falling were automatically recorded. Each mouse completed two trials with a 1-min recovery period between trials to minimize the effects of fatigue. The mean value of the two trials was used for subsequent statistical analyses. The rotarod test was performed immediately before the start of the intervention and after completion of the 4-week intervention period.

Tissue Collection

All tissue collection procedures were performed under isoflurane anesthesia using a rodent inhalation anesthesia system. To minimize the influence of acute exercise-induced molecular responses and to evaluate chronic adaptations to the 4-week intervention, tissue collection was performed 48 h after the final exercise session. Following induction of deep anesthesia, whole blood was first collected from the inferior vena cava. Subsequently, mice were transcardially perfused with ice-cold 0.9% saline to remove residual blood from the tissues before tissue harvesting.

Skeletal muscles, including the soleus, plantaris, gastrocnemius, tibialis anterior, and extensor digitorum longus (EDL), as well as visceral adipose tissues (epididymal and perirenal adipose tissue) and subcutaneous adipose tissues (inguinal and abdominal adipose tissue), were rapidly excised. All tissues were weighed immediately after dissection, snap-frozen in liquid nitrogen, and stored at −80 °C until further analysis. Whole blood was allowed to clot at room temperature for 60-90 min and subsequently centrifuged at 1,500 × g for 15 min at 4 °C. The resulting serum was collected and stored at −80 °C until further analysis.

For the gastrocnemius muscles, the tissue was divided longitudinally through the mid-belly immediately after excision. One half was snap-frozen in liquid nitrogen and stored at −80 °C for western blot and NAD⁺/NADH analyses. The remaining half was post-fixed in 4% paraformaldehyde prepared in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 24 h and subsequently cryoprotected in cold 30% sucrose prepared in 0.1 M phosphate buffer (PB, pH 7.4) until the tissue had completely sunk in the solution prior to histological analysis.

Histological Analysis

To evaluate skeletal muscle morphology, the cross-sectional area (CSA) of gastrocnemius muscle fibers was analyzed following hematoxylin and eosin (H&E) staining (ab245880, Abcam, Cambridge, UK). Following cryoprotection in 30% sucrose, the gastrocnemius muscles were sectioned transversely at the mid-belly using a cryostat to obtain 5-μm-thick sections. During OCT embedding, each muscle was carefully positioned with its longitudinal axis parallel to the base of the embedding mold. The OCT-embedded tissue block was subsequently mounted and oriented in the cryostat with the longitudinal axis of the muscle fibers perpendicular to the sectioning plane, thereby ensuring transverse sectioning and minimizing the occurrence of oblique sections [34]. Tissue sections were mounted onto glass slides, air-dried at room temperature, and stained according to the manufacturer’s instructions.

For morphometric analysis, images were acquired from the deep region of the gastrocnemius muscle using an EVOS M5000 microscope (Thermo Fisher Scientific, Waltham, MA, USA) at ×20 magnification. Approximately 250-300 muscle fibers were analyzed per animal. Muscle fiber segmentation was performed using a Cellpose-based image analysis pipeline, followed by manual inspection to exclude fibers with indistinct borders, sectioning artifacts, overlapping fibers, or abnormal morphology. The CSA of each muscle fiber was subsequently quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the mean CSA from multiple fields of view was used for statistical analysis.

Western Blot Analysis

Western blot analysis was performed to investigate molecular mechanisms associated with skeletal muscle hypertrophy, function, and metabolic adaptation. Total protein was extracted from the left gastrocnemius muscle using an EzRIPA Lysis Kit (WSE07420, ATTO, Tokyo, Japan). Muscle tissues were homogenized in 600 μL of lysis buffer using a TissueRuptor (QIAGEN, Hilden, Germany), followed by centrifugation at 20,000 × g for 15 min at 4 °C. The resulting supernatant was collected for subsequent analysis.

Protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were denatured at 100 °C for 5 min, and equal amounts of protein (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were subsequently transferred onto polyvinylidene difluoride (PVDF) membranes under refrigerated conditions. For high-molecular-weight proteins, including mTOR, transfer was performed using a modified transfer buffer containing 0.03% SDS and 20% methanol at 30 V for 12 h at 4 °C.

Membranes were blocked with 5% non-fat dried milk in PBS containing 0.1% Tween-20 (PBS-T) for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 90 min at room temperature. Detailed information regarding the antibodies used in this study is provided in Supplementary Table S1.

Immunoreactive bands were visualized using ECL™ Prime Western Blotting Detection Reagent (Cytiva, Marlborough, MA, USA). Total protein staining using No-Stain™ Protein Labeling Reagent (Invitrogen, Carlsbad, CA, USA) was used for normalization. Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

NAD⁺/NADH Ratio Analysis

The NAD⁺/NADH ratio was determined in the remaining half of the right gastrocnemius muscle as an indicator of skeletal muscle metabolic status. Tissue NAD⁺ and NADH levels were measured using a commercial assay kit (NAD/NADH Assay Kit, ab65348, Abcam, Cambridge, UK) according to the manufacturer’s instructions.

Briefly, muscle samples were homogenized in the supplied extraction buffer, and the homogenates were centrifuged to collect the supernatant. Protein was subsequently removed using a 10-kDa molecular weight cutoff spin column (ab93349, Abcam) prior to analysis. The deproteinized samples were used for the determination of total NAD and NADH. For NADH measurement, samples were heated to selectively decompose NAD⁺. NAD⁺ concentrations were calculated by subtracting NADH from total NAD, and the NAD⁺/NADH ratio was subsequently calculated. All samples were analyzed in duplicate.

Serum Biochemical Analysis

Serum biochemical analyses were performed to assess systemic antioxidant status, inflammatory responses, muscle damage, and potential toxicity associated with the intervention. All analyses were conducted using commercially available assay kits according to the manufacturers’ instructions.

The glutathione/glutathione disulfide (GSH/GSSG) ratio was determined using a colorimetric assay kit (ab239709, Abcam, Cambridge, UK). Serum tumor necrosis factor-α (TNF-α) concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (EM0183-HS, FineTest, Wuhan, China). Serum muscle creatine kinase (CKM) concentrations were determined using an ELISA kit (SEA109Mu, Cloud-Clone Corp., Wuhan, China). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using ELISA kits (ab282882 and ab263882, respectively; Abcam, Cambridge, UK).

Statistical Analysis

All statistical analyses were performed using IBM SPSS Statistics version 29.0.2 (IBM Corp., Armonk, NY, USA). Graphs were generated using GraphPad Prism version 11.0.2 (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard error of the mean (S.E.M.).

The normality of data distribution was assessed using the Shapiro-Wilk test. All datasets satisfied the assumption of normality except for total food intake, which was analyzed using the Kruskal-Wallis test because of the small sample size. All other data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test for post hoc analysis. Statistical significance was set at p < 0.05.

RESULTS

Effects of Amino Acid Formulation on Serum ALT and AST Levels

After the 4-week intervention, we first measured serum ALT and AST levels to evaluate potential hepatic toxicity (Figure 1B, C). One-way ANOVA revealed a significant group effect for serum ALT levels (p < 0.001). Post hoc analysis demonstrated that serum ALT levels were significantly lower in the A600, A900, A1200, and A1500 groups than in the Con group (p < 0.05), with the greatest reduction observed in the A600 group (Figure 1B). In addition, the A600 group exhibited significantly lower ALT levels than the Leu group (p < 0.01; Figure 1B). Overall, ALT levels tended to be lower in the intermediate- to high-dose amino acid formulation groups than in the Con and Leu groups.

No significant group differences were observed in serum AST levels (p = 0.119); however, the amino acid formulation groups generally exhibited lower AST levels than the Con and Leu groups (Figure 1C).

Collectively, these findings indicate that supplementation with the amino acid formulation was not associated with adverse changes in serum ALT and AST levels during resistance training.

Amino Acid Formulation Does Not Alter Body Composition

To determine whether amino acid formulation supplementation influenced body composition and tissue mass, final body weight, total food intake, whole-body composition assessed by DXA, and tissue weights were evaluated (Tables 1 and 2). Final body weight and total food intake throughout the intervention did not differ significantly among the groups (Table 1). Likewise, DXA analysis revealed no significant differences in lean mass, lean mass percentage, fat mass, or fat mass percentage among the groups (Table 1).

Table 1.

Effects of amino acid formulation supplementation on body weight, food intake, and whole-body composition assessed by DXA

		Group								p-value
		Con	Leu	A150	A300	A600	A900	A1200	A1500
Final body weight (g)		26.73 ± 0.41	27.1 ± 0.39	27.3 ± 0.47	27.18 ± 0.35	26.69 ± 0.28	27.18 ± 0.58	27.14 ± 0.28	26.79 ± 0.48	0.942
Total food intake (g/mouse)		85.19 ± 2.08	81.12 ± 2.46	84.65 ± 3.02	87.86 ± 2.12	82.93 ± 0.41	85.84 ± 0.46	82.87 ± 1.63	88.96 ± 5.28	0.352
DXA	Lean mass (g)	22.98 ± 0.3	23.11 ± 0.32	23.38 ± 0.37	23.26 ± 0.27	22.93 ± 0.3	23.36 ± 0.48	23.48 ± 0.28	23.06 ± 0.4	0.924
	Lean mass percentage (%)	85.38 ± 0.46	84.62 ± 0.37	84.42 ± 0.34	84.75 ± 0.45	84.64 ± 0.27	84.46 ± 0.28	84.5 ± 0.23	84.72 ± 0.52	0.714
	Fat mass (g)	3.54 ± 0.16	3.8 ± 0.13	3.9 ± 0.14	3.8 ± 0.015	3.75 ± 0.07	3.88 ± 0.14	3.88 ± 0.08	3.76 ± 0.18	0.654
	Fat mass percentage (%)	13.31 ± 0.46	14.1 ± 0.37	14.28 ± 0.33	13.97 ± 0.45	14.06 ± 0.26	14.21 ± 0.29	14.17 ± 0.24	13.97 ± 0.52	0.719

Values are presented as mean ± standard error of the mean (SEM) (n = 10 per group). DXA, dual-energy X-ray absorptiometry.

Table 2.

Effects of amino acid formulation supplementation on skeletal muscle and adipose tissue weights

		Group								p-value
		Con	Leu	A150	A300	A600	A900	A1200	A1500
Skeletal muscle (mg)	Soleus	9.08 ± 0.17	9.04 ± 0.13	8.83 ± 0.17	9.04 ± 0.14	9.04 ± 0.21	9.42 ± 0.22	8.64 ± 0.22	8.96 ± 0.24	0.251
	Plantaris	16.79 ± 0.36	16.79 ± 0.46	16.79 ± 0.33	16.67 ± 0.33	16.54 ± 0.26	17.21 ± 0.42	17.5 ± 0.24	17.08 ± 0.38	0.619
	Gastrocnemius	135.33 ± 2.29	135 ± 1.67	132.5 ± 2.04	133.04 ± 1.58	132.25 ± 1.48	136 ± 1.8	135.14 ± 1.69	134.83 ± 2.54	0.786
	Tibialis anterior	45.17 ± 0.91	45.33 ± 1.01	45.08 ± 0.58	45.17 ± 0.66	45.29 ± 0.68	43.88 ± 1.2	44.45 ± 1	43.63 ± 0.8	0.752
	EDL	9.67 ± 0.37	9.42 ± 0.32	9.83 ± 0.26	9.96 ± 0.27	9.75 ± 0.26	10 ± 0.28	10.41 ± 0.23	10.04 ± 0.26	0.420
Skeletal muscle/Body weight (mg/g)	Soleus	0.34 ± 0.01	0.33 ± 0.01	0.32 ± 0.0	0.33 ± 0.01	0.34 ± 0.01	0.35 ± 0.01	0.32 ± 0.01	0.33 ± 0.01	0.164
	Plantaris	0.63 ± 0.01	0.62 ± 0.02	0.62 ± 0.01	0.61 ± 0.01	0.62 ± 0.01	0.63 ± 0.01	0.65 ± 0.01	0.64 ± 0.01	0.714
	Gastrocnemius	5.07 ± 0.09	4.99 ± 0.07	4.86 ± 0.09	4.9 ± 0.08	4.96 ± 0.05	5.02 ± 0.07	4.98 ± 0.05	5.05 ± 0.11	0.610
	Tibialis anterior	1.69 ± 0.04	1.67 ± 0.04	1.66 ± 0.03	1.66 ± 0.03	1.7 ± 0.02	1.62 ± 0.04	1.64 ± 0.04	1.63 ± 0.03	0.701
	EDL	0.36 ± 0.02	0.35 ± 0.01	0.36 ± 0.01	0.37 ± 0.01	0.37 ± 0.01	0.37 ± 0.01	0.38 ± 0.01	0.38 ± 0.03	0.514
Adipose tissue (mg)	Epididymal	556.75 ± 41.83	681.42 ± 44.67	512.33 ± 81.57	607.58 ± 50.26	626.92 ± 24.48	655.92 ± 45.84	599.45 ± 52.14	619 ± 57.48	0.425
	Perirenal	228.17 ± 32.51	274.67 ± 22.07	261.08 ± 20.66	260.75 ± 32.36	233.17 ± 13.81	241.58 ± 17.73	250 ± 13.59	232 ± 26.15	0.842
	Inguinal	192.17 ± 22.16	218.33 ± 19.29	199.75 ± 14.95	205 ± 18.93	233.17 ± 13.81	209.92 ± 17.49	223 ± 22.91	232.92 ± 28.34	0.969
	Abdominal	264.75 ± 22.16	317.42 ± 15.47	314.33 ± 19.75	309.25 ± 22.69	278.33 ± 12.6	297.08 ± 24.2	286.45 ± 24.89	202.67 ± 24.79	0.760
Adipose tissue/Body weight (mg/g)	Epididymal	21.11 ± 1.34	25.02 ± 1.41	18.75 ± 2.92	22.21 ± 1.65	23.49 ± 0.88	23.98 ± 1.35	22.04 ± 1.88	22.96 ± 1.93	0.300
	Perirenal	8.43 ± 1.09	10.06 ± 0.7	9.49 ± 0.64	9.5 ± 1.09	8.74 ± 0.51	8.86 ± 0.59	9.2 ± 0.47	8.62 ± 0.97	0.850
	Inguinal	7.12 ± 0.77	7.99 ± 0.62	7.27 ± 0.47	7.49 ± 0.63	7.78 ± 0.39	7.67 ± 0.57	8.2 ± 0.81	7.51 ± 0.83	0.953
	Abdominal	9.85 ± 0.75	11.68 ± 0.47	11.5 ± 0.66	11.33 ± 0.76	10.45 ± 0.51	10.83 ± 0.75	10.56 ± 0.92	10.93 ± 1.05	0.711

Values are presented as mean ± standard error of the mean (SEM) (n = 10 per group). EDL, extensor digitorum longus.

Similarly, neither the absolute nor the relative weights (normalized to body weight) of the five hindlimb skeletal muscles (soleus, plantaris, gastrocnemius, tibialis anterior, and EDL) differed significantly among the groups. No significant differences were observed in either the absolute or relative weights of the epididymal, perirenal, inguinal, and abdominal adipose tissues (Table 2).

Collectively, these findings indicate that amino acid formulation supplementation did not alter body composition, skeletal muscle mass, or adipose tissue mass during the 4-week intervention.

Overall Beneficial Effects of Amino Acid Formulation Supplementation on Functional Adaptations to Resistance Exercise

To evaluate functional adaptations of skeletal muscle, changes in grip strength, MVCC, hanging wire performance, and rotarod performance from pre- to post-intervention were analyzed (Figure 2). One-way ANOVA revealed significant group effects for changes in grip strength (p = 0.011; Figure 2A) and MVCC (p = 0.015; Figure 2B). The increase in grip strength was significantly greater in the A600, A900, A1200, and A1500 groups than in the Con group (p < 0.05; Figure 2A). For MVCC, the A150, A300, and A600 groups exhibited greater improvements than the Con group, although these differences did not reach statistical significance. However, the A1500 group showed a significantly smaller improvement than the Leu group (p < 0.05; Figure 2B).

Figure 2. Overall beneficial effects of amino acid formulation supplementation on functional adaptations to resistance exercise.

Pre- to post-intervention changes in (A) grip strength, (B) MVCC, (C) hanging wire performance, and (D) rotarod performance were assessed after the 4-week intervention. Values are presented as the mean ± SEM (n = 10 per group). *p < 0.05 versus the Con group; #p < 0.05 versus the Leu group. MVCC, maximal voluntary carrying capacity; bw, body weight.

In the hanging wire test, all amino acid formulation groups exhibited greater improvements than the Con and Leu groups; however, these differences were not statistically significant (Figure 2C). Likewise, no significant group differences were observed in changes in rotarod performance (Figure 2D).

Collectively, these findings support an overall beneficial effect of amino acid formulation supplementation on func-tional adaptations to resistance exercise.

Amino Acid Formulation Supplementation Promotes Skeletal Muscle Hypertrophy

To investigate the structural adaptations of skeletal muscle, gastrocnemius muscle fiber CSA was evaluated by H&E staining (Figure 3). Histological analysis revealed an overall dose-dependent increase in muscle fiber CSA following amino acid formulation supplementation. Although modest increases were observed in the A300-A900 groups, the A1200 and A1500 groups exhibited significantly greater muscle fiber CSA than the Con group (p < 0.05; Figure 3B).

Figure 3. Amino acid formulation supplementation promotes skeletal muscle hypertrophy.

Following the 4-week intervention, the gastrocnemius muscle tissues were harvested for histological analysis. (A) Representative images of hematoxylin and eosin (H&E)-stained gastrocnemius muscle cross-sections. (B) Quantitative analysis of the mean CSA of gastrocnemius muscle fibers. Values are presented as the mean ± SEM (n = 6-8 per group). *p < 0.05 versus the Con group. CSA, cross-sectional area; H&E, hematoxylin and eosin.

Collectively, these findings indicate that amino acid formulation supplementation promotes skeletal muscle hypertrophy during resistance exercise.

Amino Acid Formulation Supplementation Modulates Signaling Proteins Associated with Skeletal Muscle Hypertrophy

To investigate the molecular mechanisms underlying the hypertrophic response, the expression and phosphorylation of proteins involved in the AKT/mTOR and MEK/ERK signaling pathways were analyzed by western blotting (Figure 4). To comprehensively evaluate the effects of amino acid formulation supplementation on anabolic signaling, total protein expression, phosphorylated protein expression, and phosphorylation-to-total protein ratios were analyzed.

Figure 4. Amino acid formulation supplementation modulates signaling proteins associated with skeletal muscle hypertrophy.

Following the 4-week intervention, the gastrocnemius muscle tissues were harvested for western blot analysis. (M) Representative western blot bands. Quantitative analyses of protein expression and phosphorylation ratios are shown for (A) t-AKT, (B) p-AKT, (C) p-AKT/t-AKT ratio, (D) t-mTOR, (E) p-mTOR, (F) p-mTOR/t-mTOR ratio, (G) t-MEK, (H) p-MEK, (I) p-MEK/t-MEK ratio, (J) t-ERK, (K) p-ERK, and (L) p-ERK/t-ERK ratio. Values are presented as the mean ± SEM (n = 6-8 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the Con group; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus the Leu group.

Within the AKT/mTOR signaling pathway, amino acid formulation supplementation induced marked alterations in AKT-related signaling. Total AKT expression was significantly higher in the A300, A600, and A900 groups than in the Con group (p < 0.05); moreover, the A300 and A600 groups also exhibited significantly greater expression than the Leu group (p < 0.05; Figure 4A). Likewise, p-AKT expression showed an overall dose-dependent increase, with significantly greater levels observed in the A300-A1500 groups than in the Con group (p < 0.05). Furthermore, the A1200 and A1500 groups also exhibited significantly higher p-AKT expression than the Leu group (p < 0.05; Figure 4B). Consistent with these findings, the p-AKT/t-AKT ratio was significantly higher in the A600-A1500 groups than in the Con group (p < 0.05), whereas the A1200 group also showed a significantly greater ratio than the Leu group (p < 0.05; Figure 4C).

For mTOR, significant differences were observed in total protein expression. Total mTOR expression was significantly higher in the A300, A600, and A1200 groups than in the Con group (p < 0.05), whereas all groups from A300 to A1500 exhibited significantly greater expression than the Leu group (p < 0.05; Figure 4D). Although neither p-mTOR expression nor the p-mTOR/t-mTOR ratio differed significantly among the groups, relatively higher values were observed in several amino acid formulation groups, particularly A150, A300, A600, and A1200 (Figure 4E, F).

Within the MEK/ERK signaling pathway, total MEK expression also exhibited a dose-dependent increase following amino acid formulation supplementation. Total MEK expression was significantly greater in the A600, A900, and A1200 groups than in the Con group (p < 0.05), whereas all amino acid formulation groups showed significantly higher expression than the Leu group (p < 0.05; Figure 4G). Although p-MEK expression showed an overall decreasing tendency following amino acid formulation supplementation, no significant pairwise differences were detected despite a significant overall group effect (p = 0.035; Figure 4H). Consequently, the p-MEK/t-MEK ratio progressively decreased with increasing supplementation dose and was significantly lower in the Leu, A150, A300, A1200, and A1500 groups than in the Con group (p < 0.05; Figure 4I). No significant differences were observed in total ERK, p-ERK, or the p-ERK/t-ERK ratio among the groups (Figure 4J-L).

Collectively, these findings indicate that amino acid formulation supplementation modulates signaling proteins associated with skeletal muscle hypertrophy, with the most pronounced changes observed in AKT-related signaling.

Amino Acid Formulation Supplementation Enhances Metabolic Adaptations Associated with Mitochondrial Function

To investigate the metabolic adaptations induced by amino acid formulation supplementation, the expression of proteins involved in skeletal muscle energy metabolism and mitochondrial function was assessed by western blotting, followed by determination of the NAD⁺/NADH ratio in the gastrocnemius muscle (Figure 5).

Figure 5. Amino acid formulation supplementation enhances metabolic adaptations associated with mitochondrial function.

Following the 4-week intervention, the gastrocnemius muscle tissues were harvested for molecular and redox analyses. Quantitative analyses of protein expression and phosphorylation ratios are shown for (A) t-AMPK, (B) p-AMPK, (C) p-AMPK/ t-AMPK ratio, (D) PGC-1α, (E) CS, and (F) UCP3. (G) Representative western blot bands. (H) Quantitative analysis of the NAD⁺/NADH ratio. Values are presented as the mean ± SEM (n = 7-8 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the Con group; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus the Leu group; +++p < 0.001 versus all groups except the A1200 group.

Among the metabolic regulators, total AMPK expression exhibited a significant overall group effect (p = 0.021); however, no significant pairwise differences were observed among the groups (Figure 5A). Likewise, phosphorylated AMPK expression showed a significant overall group effect (p = 0.042), with the amino acid formulation groups generally exhibiting higher expression levels than the Con and Leu groups despite the absence of significant pairwise differences (Figure 5B). No significant differences were observed in the p-AMPK/t-AMPK ratio among the groups (Figure 5C).

PGC-1α, a master regulator of mitochondrial biogenesis, exhibited significantly greater expression in the A300, A1200, and A1500 groups than in the Con group (p < 0.05; Figure 5D). Although not strictly dose-dependent, PGC-1α expression generally increased following amino acid formulation supplementation, reaching the highest level in the A1500 group. Similarly, citrate synthase (CS), a key enzyme of the TCA cycle, was significantly upregulated in the A600-A1500 groups compared with the Con group (p < 0.05), whereas the A900 and A1200 groups also exhibited significantly greater expression than the Leu group (p < 0.05; Figure 5E). Similarly, UCP3 expression was significantly higher in the A300, A600, A1200, and A1500 groups than in the Con group (p < 0.05). Furthermore, all amino acid formulation groups from A300 to A1500 exhibited significantly greater UCP3 expression than the Leu group (p < 0.05; Figure 5F).

Consistent with these molecular changes, the NAD⁺/NADH ratio exhibited an overall increase following amino acid formulation supplementation, reaching the highest level in the A900 group before declining at higher supplementation doses (Figure 5H). The A150, A900, and A1200 groups exhibited significantly greater NAD⁺/NADH ratios than the Con group (p < 0.05). Notably, the A900 group also exhibited significantly higher NAD⁺/NADH ratios than all other groups except the A1200 group (p < 0.001), indicating that the maximal improvement in skeletal muscle redox status was achieved with the A900 supplementation dose.

Collectively, these findings demonstrate that amino acid formulation supplementation enhances mitochondrial metabolic adaptations and improves skeletal muscle redox status during resistance exercise.

Amino Acid Formulation Supplementation Attenuates Muscle Damage and Enhances Systemic Antioxidant Capacity

To evaluate the systemic effects of amino acid formulation supplementation following resistance exercise, circulating biomarkers associated with muscle damage, inflamma-tion, and antioxidant status were analyzed (Figure 6).

Figure 6. Amino acid formulation supplementation attenuates muscle damage and enhances systemic antioxidant capacity.

Following the 4-week intervention, circulating biomarkers associated with muscle damage and inflammation, including (A) muscle-type creatine kinase (CKM) and (B) TNF-α, were analyzed. Systemic antioxidant status was evaluated by measuring (C) the GSH/GSSG ratio, (D) total GSH, (E) reduced GSH, and (F) GSSG levels. Values are presented as the mean ± SEM (n = 8-10 per group). Gray text indicates a statistical trend (0.05 < p < 0.10) that did not reach the threshold for statistical significance. *p < 0.05 versus the Con group; #p < 0.05 versus the Leu group; $$p < 0.01 versus the A150 group; @p < 0.05 versus the A1500 group; &&p < 0.01 versus all groups except the A600 and A1200 groups.

As an indicator of exercise-induced muscle damage, circulating CKM concentrations generally decreased with increasing amino acid formulation dose (Figure 6A). Although no significant pairwise differences were detected, the A1500 group exhibited the lowest CKM concentration and showed a strong tendency toward a lower level than the Con group (p = 0.054). Likewise, circulating TNF-α concentrations differed significantly among the groups, with the A1200 group exhibiting significantly lower levels than the Con group (p < 0.05; Figure 6B).

To further investigate systemic antioxidant responses, the GSH/GSSG ratio, total GSH, reduced GSH, and GSSG concentrations were determined. No significant differences were observed in the GSH/GSSG ratio among the groups (Figure 6C). In contrast, total GSH, reduced GSH, and GSSG concentrations displayed a similar response pattern, increasing progressively with amino acid formulation supplementation, reaching peak levels in the A900 group, and declining at higher supplementation doses.

Specifically, total GSH concentrations were significantly higher in the A900 group than in all other groups except the A600 and A1200 groups (p < 0.01; Figure 6D). Reduced GSH concentrations were significantly greater in the A900 group than in the Con, Leu, and A150 groups (p < 0.05 or p < 0.01; Figure 6E). Likewise, GSSG concentrations were significantly higher in the A900 group than in the Con and A1500 groups (p < 0.05; Figure 6F).

Collectively, these findings demonstrate that amino acid formulation supplementation attenuates exercise-induced muscle damage while enhancing systemic antioxidant capacity during resistance exercise.

DISCUSSION

The present study investigated whether a novel formulation comprising five amino acids designed to integrate complementary anabolic, metabolic, and redox-supporting functions could enhance skeletal muscle remodeling during resistance training. The principal finding of this study is that supplementation with the formulation improved multiple aspects of skeletal muscle quality, including muscle function, muscle hypertrophy, molecular adaptations associated with skeletal muscle remodeling, and systemic antioxidant capacity. Furthermore, these beneficial effects were observed across a broad range of physiological outcomes without affecting body weight, food intake, or whole-body composition, suggesting that the observed adaptations were attributable to qualitative improvements in skeletal muscle rather than nonspecific changes in whole-body metabolism. Collectively, these findings support the concept that combining amino acids with complementary physiological functions represents an effective nutritional strategy for promoting exercise-induced skeletal muscle remodeling. To better interpret these findings, the following discussion focuses on two key aspects: the dose-response characteristics of the amino acid formulation and the potential mechanisms underlying the complementary actions of its constituent amino acids.

A central finding of the present study was that the amino acid formulation produced an outcome-specific rather than uniformly linear dose-response pattern. Functional and molecular adaptations emerged at relatively lower doses, whereas clear structural changes required higher exposure. Grip strength and AKT-related signaling showed improvements at doses of approximately 600 mg/kg and above (Figure 2A and Figure 4A-C), whereas significant increases in gastrocnemius muscle fiber CSA were observed at doses of 1,200 and 1,500 mg/kg (Figure 3). Notably, the increases in muscle fiber CSA following amino acid formulation supplementation were not accompanied by significant changes in whole-muscle mass (Tables 1 and 2). Although these measures are related, they represent distinct aspects of skeletal muscle morphology. Whole-muscle mass reflects the composite mass of the entire tissue, including muscle fibers as well as extracellular, connective, vascular, and fluid components, whereas fiber CSA provides a histological measure of remodeling at the individual myofiber level. Therefore, changes in myofiber size may not necessarily translate proportionally into detectable changes in whole-muscle mass, as previous studies have demonstrated that muscle mass and fiber CSA do not necessarily change in parallel [33,35]. Thus, the present findings may reflect myofiber-level structural remodeling that was detectable histologically without a corresponding measurable change in gross muscle mass. Together, these findings suggest that the effects of the formulation were initially manifested as functional, molecular, and histological adaptations rather than as detectable alterations in whole-body composition.

Metabolic and redox-related responses showed a similarly nonuniform pattern. CS, UCP3, AMPK-related signaling, and the NAD⁺/NADH ratio generally improved within the intermediate dose range (600-1200 mg/kg; Figure 5), whereas glutathione-related indices were most favorable at a dose of approximately 900 mg/kg (Figure 6C-F). However, the highest dose did not consistently produce the greatest response across MVCC, NAD⁺/NADH, or glutathione-related outcomes. These findings suggest that the formulation reached a threshold at which multiple adaptive processes were recruited, followed by a plateau or attenuation in selected domains rather than a continuous linear benefit.

Taken together, these findings suggest that 600 mg/kg represents the lowest dose at which functional, anabolic, and metabolic responses converged, whereas doses between 900 and 1,200 mg/kg produced more pronounced metabolic, redox, and structural adaptations. Thus, the present findings support 600 mg/kg as the minimum coordinated effective dose and 600-1,200 mg/kg as a broader biologically effective dose range, rather than identifying a single dose that maximized all outcomes.

The broad adaptive profile of the formulation may reflect the complementary functions of its constituent amino acids. Skeletal muscle remodeling during resistance training requires not only anabolic signaling but also adequate substrate availability, metabolic support, redox buffering, and recovery from repeated contractile stress [10,36,37]. Accordingly, the concurrent improvements in AKT-related signaling, muscle fiber CSA, grip strength, mitochondrial markers (CS and UCP3), the NAD⁺/NADH ratio, glutathione-related indices, and circulating CKM in the present study are more consistent with coordinated skeletal muscle remodeling than with activation of a single pathway.

Leucine likely served primarily as an anabolic signaling molecule [15,17], whereas glutamine may have supported carbon and nitrogen metabolism through its conversion to glutamate and α-ketoglutarate [23,38]. These actions provide a plausible metabolic context for the observed increases in CS, UCP3, PGC-1α-related responses, and the NAD⁺/NADH ratio (Figure 5D-H). At the same time, cysteine and glycine provide direct substrates for glutathione synthesis [39], whereas glutamine may contribute indirectly by replenishing the glutamate pool. The increase in reduced and total glutathione without a change in the GSH/GSSG ratio in the present study (Figure 6C-F) is therefore more consistent with the expansion of antioxidant buffering capacity than with a simple reduction in oxidative stress. Taurine may have further complemented these actions through its roles in membrane stability and cytoprotection [28]. Consistent with these functions, the formulation lowered circulating CKM and TNF-α concentrations (Figure 6A, B) despite repeated moderate-to-high intensity resistance exercise, suggesting improved preservation of muscle membrane integrity and reduced low-grade inflammatory stress during chronic training adaptation.

This interpretation is also consistent with the reduction in the p-MEK/t-MEK ratio in the present study (Figure 4I). Although MEK-related signaling is rapidly activated by acute mechanical loading and cellular stress, accumulating evidence indicates that this response becomes progressively attenuated as skeletal muscle adapts to repeated exercise stimuli rather than remaining chronically elevated [40]. Previous studies have demonstrated that exercise-induced MAPK signaling is blunted following chronic training, reflecting a reduced requirement for stress-responsive MAPK activation in adapted skeletal muscle [40,41], while recent reviews have likewise emphasized that MEK/ERK primarily functions as an acute mechanosensitive signaling pathway rather than a constitutively activated mediator of skeletal muscle remodeling [42]. Given that skeletal muscle samples were collected 48 h after the final exercise bout, the lower p-MEK/t-MEK ratio observed in the present study is therefore unlikely to indicate impaired anabolic signaling; instead, it may reflect the resolution of transient stress-responsive MAPK activation accompanying physiological adaptation to repeated mechanical loading.

The multidimensional responses observed in the present study are consistent with previous supplementation studies investigating individual amino acids or related amino acid combinations. Leucine and leucine-enriched essential amino acids have been associated with enhanced anabolic responses to resistance exercise [43], whereas glutamine has been investigated for its potential roles in metabolic support and exercise recovery [44]. Similarly, supplementation strategies involving glycine and cysteine availability or taurine have been associated with improvements in glutathione metabolism, oxidative stress, mitochondrial function, and muscle recovery [45,46]. Although the contribution of each amino acid cannot be distinguished in the present study, these findings provide biological support for combining amino acids with complementary physiological functions to target multiple aspects of skeletal muscle adaptation.

Collectively, these findings suggest that the formulation supported skeletal muscle adaptation not through amplification of a single anabolic pathway, but through coordinated modulation of AKT-related signaling, metabolic capacity, antioxidant buffering, and recovery from repeated contractile stress. This integrated physiological response may explain the broader adaptive profile observed with the formulation compared with leucine alone.

Several limitations should be acknowledged. First, because the present study evaluated a multi-component amino acid formulation, individual contribution of each constituent amino acid and potential interactions among the components could not be determined. Future studies employing individual-component and combination groups will be required to clarify the relative contribution of each amino acid and whether their combined effects are additive or synergistic. Second, although several anabolic, metabolic, redox-related, and stress-responsive pathways were examined, direct measures of muscle protein synthesis and degradation, as well as functional assessments of mitochondrial capacity, were not performed. Such analyses would provide a more comprehensive understanding of the mechanisms underlying the observed skeletal muscle adaptations.

Despite these limitations, the present findings demonstrate that supplementation with a complementary five-amino-acid formulation enhances multiple aspects of skeletal muscle remodeling during resistance training. Rather than acting through a single anabolic mechanism, the formulation promoted coordinated functional, structural, metabolic, redox, and cytoprotective adaptations, supporting its potential as a nutritional strategy for improving skeletal muscle quality during chronic resistance exercise. From a practical sports nutrition perspective, this multi-component approach may be particularly relevant during periods of frequent or intensified resistance training, when athletes are required to repeatedly recover from and adapt to successive training sessions. In such settings, a formulation designed to concurrently support anabolic, metabolic, and redox-related processes may potentially complement conventional protein-centered nutritional strategies by targeting multiple aspects of skeletal muscle remodeling. Nevertheless, given the preclinical nature of the present study, human studies are warranted to establish its efficacy, determine the optimal dosing regimen, and evaluate its practical utility in athletic populations.

Supplementary Material

Supplemental Table S1.

The list of antibodies used in the current study