How certain amino acids help muscle growth: roles, functions, and uses

BCAA serve dual metabolic roles as both direct substrates for protein synthesis and potent signaling molecules regulating anabolic processes. As structural precursors, they facilitate transcription and translation to support muscle protein accretion. Additionally, BCAA function as key nutrient signals that activate the mechanistic target of rapamycin (mTOR) pathway. The 280 kDa serine/threonine kinase mTOR belongs to the phosphatidylinositol kinase-related kinase family and exists in two functional distinct complexes with unique structures and functions [52]. mTORC1 primarily promotes protein synthesis through S6K1/4E-BP1 activation while suppressing autophagy. mTORC2 serves as downstream effector of insulin/PI3K signaling, primarily regulating glucose metabolism, cytoskeleton organization, cell migration, and adhesion [53].

mTORC1 activation at the lysosomal membrane is regulated by two small GTPases, Rheb and Rag-GTP [62]. Efficient mTORC1 signaling depends on the coordinated input of growth factors and AA, as maximal activation occurs when both Rheb and Rag-GTP are in their active states. At the cellular level, mTORC1 activity exhibits a graded response. Increasing concentrations of growth factors or AA elevate the proportion of active Rheb and Rag-GTP, respectively, leading to a higher probability of mTORC1 activation. Additionally, BCAA promote β-cell proliferation upon their transport into pancreatic cells by the SNAT2 and LAT1 transporters [63]. For this reason, BCAA and insulin/insulin-like growth factor 1 (IGF-1) signaling synergistically enhance mTOC1 activation [53]. Rag GTPases (Rag A/B and Rag C/D) are crucial for mTORC1 signaling by promoting its recruitment to the lysosomal membrane via the Ragulator complex [64, 65]. Under high AA conditions, Rag GTPases facilitate mTORC1 translocation to the lysosomal membrane, where its activation is further potentiated by Rheb-GTP [66] (Fig. 3).

Leucine is uniquely potent in stimulating mTORC1 signaling, with studies showing that Leu alone can activate mTORC1 as effectively as a complete AA mixture [74]. Ospina-Rojas et al. [75] found that supplementing broiler diets with Leu significantly increased the mRNA expression of mTOR and S6K1 genes in muscle tissue, whereas supplementing with Val had no effect on the mRNA expression of these genes, highlighting Leu's distinct role in mTOR pathway activation. Hao et al. [76] reported that Ile can stimulate milk protein and fat synthesis in mammary epithelial cells through the PI3K-BRG1-mTOR/SREBP-1c pathway in a dose-dependent manner. Similarly, Arriola Apelo et al. [77] demonstrated that Ile linearly increased mTOR and S6K1 phosphorylation in the mammary tissue of dairy cows, underscoring its role in lactation-related anabolic signaling.

Recent studies have uncovered novel mediators in mTOR signaling, revealing additional layers of complexity in its regulation. Jiang et al. [78] identified Wnt-1-induced secreted protein 3 (WISP3) as a potential new signaling factor linking mTOR with AA, making it a crucial regulator of AA-mediated milk protein synthesis and cell growth. Further expanding the regulatory landscape, Hayatine, a newly discovered mTOR-Rag A/C interaction inhibitor, was shown to suppress AA-induced mTORC1 activation [79], providing a potential therapeutic target for mTOR-related disorders. Luo et al. [80] found that Glycyl-tRNA synthase (GlyRS) is a key mediator in AA-induced mTOR expression and activation in bovine mammary epithelial cells, suggesting a direct connection between translational machinery and mTOR signaling. Beyond canonical pathways, Son et al. [81] demonstrated that downstream metabolites of Leu, such as acetyl-CoA, can independently activate mTORC1 by promoting EP-300-mediated acetylation of Raptor, bypassing traditional Leu-sensing mechanisms. As more key regulators of the mTOR signaling pathway are discovered, our understanding of this complex network will continue to expand, offering new insights into its role in metabolism, protein synthesis, and cell growth.

mTORC1 exerts indirect control over the UPS through its regulation of AKT and FOXO signaling cascades. As the primary effector of PI3K-mediated insulin signaling, AKT mediates cellular proliferation and metabolic reprogramming via FOXO1/3a and NAD kinase (NADK) [94, 95]. The regulatory network extends to autophagy control through serum and glucocorticoid-regulated kinase (SGK), where mTORC2-SGK-1 inhibition potently induces autophagic flux in Caenorhabditis elegans [96]. AKT further coordinates metabolic responses by inactivating glycogen synthase kinase 3β (GSK3β) to support cell survival and glucose metabolism [57]. Crucially, AKT serves as a signaling nexus between mTOR complexes. It can phosphorylate tuberous sclerosis complex (TSC2) to activate mTORC1 while modifying mSin1 to modulate mTORC2 assembly and function [97, 98].

Under normal physiological conditions, skeletal muscle maintains proteostasis through balanced protein degradation and synthesis [99]. However, catabolic states such as fasting trigger accelerated proteolysis via the UPS and the autophagy-lysosomal system, leading to net muscle loss [100]. In response to diseases, inactivity, or acute endotoxemia, BCAA or Leu administration inhibits protein degradation through the UPS [101] and the autophagy-lysosomal system [102]. Zi et al. [103] revealed that mTORC1 phosphorylates serine/threonine kinase 11 interacting protein (STK11IP) to regulate lysosomal vacuolar-ATPase (V-ATPase) activity, providing a direct link between mTORC1 signaling and autophagy control. These findings establish BCAA as potent inhibitors of atrophy-induced proteolysis, suggesting their therapeutic potential for muscle preservation during energy deficit through dual modulation of major degradation pathways.

Leu and its metabolites play pivotal roles in regulating protein metabolism, energy homeostasis, and cellular signaling. As the most metabolically active BCAA, approximately 80% of Leu is directly utilized for protein synthesis in pigs, while the remaining 20% undergoes conversion to α-KIC and β-hydroxy-β-methylbutyrate (HMB) in skeletal muscle [104]. Notably, endogenous HMB production represents only 0.66% of total Leu turnover [105]. In the liver mitochondria, about 90% of α-KIC is oxidized to isovaleryl-CoA (IVA-CoA), forming β-hydroxy-β-methylglutaryl-CoA (HMG-CoA), which is further metabolized to acetoacetate and acetyl-CoA. The remaining α-KIC is oxidized to HMB in the cytoplasm via α-KIC dioxygenase (KICD). HMB is cleared by the kidneys or metabolized into HMG-CoA, a precursor for cholesterol synthesis [106, 107].

As a leucine metabolite, HMB activates mTORC1 signaling via AKT phosphorylation to promote protein synthesis while suppressing degradation pathways [108, 109], though this occurs independently of IRS-1 [110]. Given its mechanistic overlap with Leu, HMB effectively substitutes for Leu in enhancing muscle protein synthesis [107, 111]. HMB concurrently reduces muscle catabolism and apoptosis by inhibiting the UPS and autophagy-lysosomal pathways [112, 113]. Cohen-Or et al. [114] demonstrated that HMB could induce myogenesis in C2C12 cells independently of AKT and mTOR, via phospholipase D2 (PLD2) activation of p70 ribosomal protein S6 kinase and ribosomal protein S6. Furthermore, HMB promotes muscle growth by stimulating the IGF-1 axis [115], enhancing AKT phosphorylation, and inhibiting autophagy.

Growing evidence from poultry and swine indicates that HMB modulates lipid metabolism and intestinal health in a dose and tissue-dependent manner. In broilers, dietary supplementation of approximately 0.10% HMB reduced hepatic fat deposition in parallel with shifts in gut microbial communities, suggesting involvement of the gut–liver axis in lipid regulation [116]. Furthermore, HMB alters muscle fatty acid composition in a muscle-specific manner. In leg muscle, 0.10%–0.15% HMB supplementation increased monounsaturated fatty acids (MUFA) while decreasing polyunsaturated fatty acids (PUFA), associated with the downregulation of lipogenic genes like SREBP-1c and reduced activities of lipid-metabolizing enzymes [117]. In breast muscle, 0.10% HMB exhibited an even stronger suppressive effect on lipogenesis, indicating distinct tissue-specific responses [117]. The benefits for intestinal integrity are supported by studies in weaned piglets, where HMB supplementation enhanced tight junction function, increased digestive enzyme activity, and modulated gut microbiota and short-chain fatty acid production, particularly under an LPS-induced inflammatory challenge [118]. Collectively, these findings demonstrate HMB's potential to attenuate fat accumulation and maintain intestinal homeostasis.

The catabolism of Val generates 3-hydroxyisobutyrate (3-HIB) through the action of 3-hydroxyisobutyryl-CoA hydrolase on its precursor 3-hydroxyisobutyryl-CoA. This muscle-derived metabolite has emerged as a significant contributor to systemic insulin resistance through its effect on lipid metabolism [119]. 3-HIB stimulates fatty acid uptake in muscle tissue, leading to lipid accumulation and subsequent metabolic dysfunction. Jang et al. [120] indicated that 3-HIB could activate endothelial fatty acid transport, facilitating lipid uptake into muscle and promoting metabolic dysfunction in mice. Importantly, inhibition of 3-HIB synthesis has been shown to block peroxisome proliferators-activated receptor γ coactivator l α (PGC-1α) mediated endothelial fatty acid uptake. Recent studies further implicate HIBCH and 3-HIB in regulating lipid accumulation in the muscle and adipose tissues [121], with 3-HIB now recognized as a novel adipocyte-specific regulatory molecule. Its association with obesity, insulin resistance, and type 2 diabetes underscores the growing appreciation of BCAA metabolites as key modulators of meat quality. Che et al. [122] demonstrated that 3-HIB supplementation in late-gestation sows improved maternal-to-fetal fatty acid transport and neonatal oxidation, as evidenced by increased placental triglycerides and fatty acid transporters (SLC27A1 and FABP3), elevated piglet plasma MUFA and PUFA, and greater muscle CPT-1.

3-HIB is further metabolized to form methylmalonate semialdehyde, which interacts with 4-aminobutyrate aminotransferase to produce β-aminoisobutyric acid (BAIBA). This downstream metabolite exhibits significant metabolic benefits, particularly in enhancing muscle function through multiple mechanisms [123]. BAIBA exerts its effects primarily via the AMPK pathway. Jung et al. [124] found that moderate BAIBA levels not only increase AMPK phosphorylation but also reduce pro-inflammatory cytokines (TNFα and MCP-1) in LPS treated fibroblast cells, thereby mitigating inflammation associated insulin resistance. Beyond its anti-inflammatory properties, BAIBA improves lipid metabolism and induces white adipose tissue browning by upregulating the expression of peroxisome proliferator-activated receptor-α (PPARα) and uncoupling protein 1 (UCP-1) [125]. Minato et al. [126] further revealed that L-BAIBA treatment enhances AMPK and AKT phosphorylation, underscoring its beneficial effects on the liver, skeletal muscle, and adipose tissue. The beneficial effects of enhancing AKT and AMPK signaling are distinct yet complementary, with direct implications for body composition and muscle mass. However, their specific effects and mechanisms in livestock species require further investigation.

The liver is a central metabolic organ that regulates energy homeostasis by coordinating the metabolism of skeletal muscle, adipose tissue, and other organs. BCAA regulate glucose and lipid metabolism primarily through the PI3K-AKT-mTOR pathway [10]. In skeletal muscle, elevated BCAA levels activate mTORC1/S6K1 signaling axis, which inhibits the PI3K/AKT pathway, thereby impairing insulin signaling and sensitivity [70, 127]. Experimental evidence supports this mechanism across diverse models. Leal Yepes et al. [128] reported that adding 550 g/d of rumen-protected BCAA into diets significantly increased plasma insulin levels in lactating cows. Conversely, Rivera et al. [129] found that excess BCAA reduces insulin sensitivity in cultured myotubes. Lynch and Adams [130] proposed an alternative perspective, arguing that increased BCAA may not directly induce insulin resistance but instead serve as a biomarker of underlying metabolic dysfunction.

Beyond direct signaling, BCAA metabolites also contribute to metabolic regulation. The metabolite BCKA has been implicated in insulin resistance [131]. Furthermore, impaired catabolism of BCAA and BCKA in the liver may disrupt systemic glucose homeostasis. This is demonstrated by liver-specific knockout of Ppm1k, a positive regulator of BCAA metabolism. Nishi et al. [132] found that Ppm1k knockout in mice impaired BCKA catabolism, leading to metabolite accumulation. This buildup suppressed the conversion of glycolytic precursors (pyruvate and lactate) into glucose via gluconeogenic pathways. While these findings suggest a potential mechanistic link between BCAA metabolism and insulin resistance, further research is needed to clarify whether BCAA directly drive insulin resistance or if their metabolites primarily contribute to metabolic dysregulation, as well as to disentangle their roles as causative agents versus biomarkers of impaired insulin action.

BCAA, particularly Leu, stimulate insulin secretion by allosterically activating glutamate dehydrogenase (GDH) [133]. In pancreatic β-cells, Leu uptake is mediated primarily by the LAT1 transporter, which is essential for Leu-stimulated insulin release. While Leu enhances insulin secretion, Ile and Val regulate glucose homeostasis through distinct mechanisms. Isabel et al. found that Ile and Val inhibit hepatic glucose production, though this regulatory effect diminishes following high-fat diet treatment [134]. A study in pigs fed low-protein diets demonstrated that supplementing BCAA increases uncoupling protein 3 (UCP3) expression in glycolytic skeletal muscle fibers, likely through activation of the AMPK-SIRT1-PGC-1α pathway [135]. Since UCP3 facilitates glucose transporter 4 (GLUT4) translocation, this mechanism links BCAA metabolism to improved glucose uptake in the skeletal muscle [135]. The metabolic impact of BCAA depends on their availability. On one hand, BCAA deficiency can disrupt glucose metabolism [136]. On the other hand, chronic BCAA accumulation downregulates the hexosamine biosynthetic pathway (HBP), leading to pyruvate dehydrogenase complex (PDH) inactivation and reduced glucose oxidation [137].

Additionally, mTORC1 enhances glycolysis by upregulating glycolytic enzymes via hypoxia-inducible factor 1α (HIF1α) activation [138, 139]. Conversely, AMPK plays a key role in maintaining systemic energy balance [140]. When cellular energy levels are low, AMPK is phosphorylated and activated by liver kinase B1 (LKB1) [141, 142]. Once activated, AMPK improves insulin sensitivity and promotes adipose tissue mobilization. Meanwhile, it also upregulates BCAA catabolic mechanisms in the skeletal muscle [143] and shifts metabolism toward increased catabolism and reduced anabolism by phosphorylating key proteins involved in mTORC1, lipid homeostasis, glycolysis, and mitochondrial function [144]. Collectively, these findings emphasize the significant role of BCAA and their metabolites in insulin sensitivity through its interactions with both mTORC1 and AMPK pathways. This understanding underscores the potential for targeted nutritional interventions to modulate these pathways for improved metabolic health in farm animals.

BCAA catabolism influences lipid synthesis by contributing to the synthesis of N-acyl amino acids, branched-chain fatty acids, and odd-chain fatty acids [8]. Both white and brown adipocytes are functionally connected to circulating BCAA levels and systemic insulin resistance regulation [119]. Specifically, during adipogenesis, enzymes involved in BCAA catabolism increase, and BCAA oxidation supplies intermediates for cholesterol and odd-chain fatty acid synthesis. Elevated BCAA levels increase lipid accumulation during adipocyte differentiation [145]. Sterol-regulatory element binding proteins (SREBP) regulate lipogenesis [146], and BCAA activate mTORC1, which promotes SREBP1/2 nuclear translocation and processing via the S6K1-dependent mechanism, thereby stimulating lipogenesis. Additionally, mTORC1 phosphorylates Lipin-1, inhibiting its nuclear translocation and consequently relieving its suppression of SREBP-mediated lipogenic activity [147]. While these findings establish a clear connection between BCAA metabolism and lipid synthesis, it should be noted that current evidence primarily comes from studies on brown adipose tissue or in vitro models. Further research is needed to determine whether these mechanisms similarly operate in white adipose tissue of livestock.

While BCAA promote lipogenesis through mTORC1-SREBP signaling in adipocytes, emerging evidence reveals contrasting effects in other tissues and physiological contexts. Dietary BCAA supplementation paradoxically reduces lipid accumulation in the liver of largemouth bass (Micropterus salmoides) [148], suggesting tissue-specific regulatory mechanisms. This dichotomy extends to skeletal muscle, where Leu enhances fatty acid oxidation and mitochondrial biogenesis via AMPK and SIRT1 activation, as demonstrated in C2C12 myotubes in mice [149]. Similarly, dietary BCAA supplementation in laying hens suppressed de novo lipogenesis [150]. The isolated administration of Ile alone to obese mice further illustrates this complexity, showing selective reduction in white adipose tissue mass [151].

Emerging evidence demonstrates that BCAA deficiency substantially impacts systemic lipid metabolism. A Leu-deficient diet significantly suppresses hepatic lipogenesis, promotes lipolysis in white adipose tissue, and increases UCP1 expression in brown adipose tissue, collectively enhancing fat mobilization [152]. This phenomenon extends to other BCAA, as Ile or Val deficient diets similarly promote lipid catabolism while inhibiting hepatic lipid synthesis, reinforcing the crucial role of BCAA homeostasis in regulating adiposity [153]. These findings reveal a biphasic relationship between BCAA availability and lipid metabolism. While adequate BCAA levels support metabolic homeostasis, both deficiency and excess induce significant alterations in fat deposition and mobilization patterns. This dose-dependent response suggests an optimal range of BCAA intake for metabolic health. The regulation window likely varies by species, metabolic state, and individual BCAA composition, warranting further investigation to establish precise nutritional guidelines.

Dietary protein content is a key determinant of growth performance. Habibi et al. [154] reported that supplementing high levels of BCAA in protein-restricted diets partially mitigated the negative effects of protein restriction on growth performance in swine. Piglets fed a low-protein diet (LP, 17.1% crude protein) had lower serum BCAA levels, whereas piglets fed an LP + BCAA diet had BCAA concentrations comparable to those fed a normal-protein diet (20.9% crude protein). Supplementing BCAA in the LP diet improved growth performance, gut development, and intestinal AA transport protein expression in weaned piglets [155]. BCAA supplementation increased average daily gain (ADG) and feed intake in piglets, likely by modulating the hypothalamic general AA control or mTOR pathways, which could enhance feed intake and promote growth [156]. Tian et al. [157] demonstrated that BCAA deficiency may suppress feed intake by regulating AA T1R1/T1R3 receptors in the intestine or activating the hypothalamic general control nonderepressible 2 (GCN2)/eIF2α pathway. The intestinal T1R1/T1R3 receptors regulate food intake by modulating the release of gut hormones such as GLP-1, PYY, and CCK [158]. Specifically, BCAA deficiency upregulates intestinal T1R1/T3R3 expression, stimulating CCK secretion and subsequent feed intake reduction. At the central level, BCAA deficiency leads to accumulation of uncharged tRNAs that activate the GCN2 pathway, increasing eIF2α phosphorylation in the brain [159]. This hypothalamic eIF2α signaling is a well-established anorexigenic pathway [160], as evidenced by significantly elevated eIF2α phosphorylation in piglets fed BCAA-deficient diets.

Beyond appetite regulation, BCAA supplementation significantly impacts swine production. Zhang et al. [161] found that dietary supplementation of 0.4% Leu can prevent the reduction of loin eye area caused by low crude protein diets in fattening pigs. Luo et al. [162] reported that adding extra 35% of Ile improved pork quality by increasing intramuscular fat, reducing drip loss and shear force without affecting growth performance in crossbred barrows. Xu et al. [163] reported that the combination of low Val (SID Val = 0.31%) and high Ile (SID Ile = 0.53%) minimized drip loss. Conversely, a high Val diet is detrimental to meat quality, particularly reducing water holding capacity.

Balanced BCAA ratios can enhance growth and muscle synthesis while reducing lipid deposition in poultry. Wu [164] reported that the ideal BCAA profile in 10-day-old chicks follows a Leu:Val:Ile to Lys ratio of 1.13:0.68:0.58. Strategic reduction of crude protein with balanced BCAA supplementation has been shown to improve growth performance, meat tenderness, and carcass yield in broilers [165]. The anabolic effects of BCAA are primarily mediated through leucine's ability to stimulate muscle protein synthesis via mTOR and S6K1 gene expressions in the muscle tissue signaling [166]. However, excessive Leu supplementation negatively affected overall growth performance, carcass weight, breast muscle weight, and body mineral content [167]. These negative effects stem from BCAA antagonism, where high Leu levels competitively inhibit the transport and functions of Val and Ile through shares AA transporters. This antagonism can be effectively counterbalanced by maintaining appropriate Val and Ile supplementation levels [168]. Ma et al. [169] found that dietary Ile supplementation improved growth performance and meat quality in broilers fed a low protein diet. Moreover, Ile reduced lipid deposition by activating the AMPK/mTOR and Leptin/JAK2/STAT3 pathways, which suppressed fat synthesis, promoted hepatic fat breakdown, and inhibited abdominal fat cell differentiation and proliferation [169]. Similarly, Jian et al. [150] found that high BCAA diets could inhibit de novo lipogenesis in laying hens by suppressing the tryptophan-ILA-AHR axis and MAPK9 signaling, while promoting fatty acid β-oxidation through PPAR-RXR activation and autophagy. These findings highlight the importance of maintaining precise BCAA levels in poultry nutrition to simultaneously optimize protein accretion and lipid metabolism while avoiding the detrimental effects of BCAA imbalance.

The effects of BCAA supplementation on ruminant growth and production performance varies across species and dietary conditions. Crucially, because free BCAA are quickly degraded in the rumen, dietary supplements must be rumen-protected to increase arterial BCAA concentrations and exert systemic effects. In goats, a low-protein diets (8.27% CP) reduced ADG, whereas supplementation with rumen-protected Leu significantly increased ADG [170, 171]. Similarly, supplementing 6 g/100 kg BW of Leu daily improved ADG in beef cattle, with altered plasma AA profiles, and increased free Leu bioavailability [3]. Dietary Leu supplementation also improved meat quality by reducing subcutaneous fat thickness and increasing marbling score and crude protein content in the longissimus pectoral muscle. However, supplementing 7.1 g/kg of Leu in 5-day-old lambs' milk replacer had no effect on feed intake or ADG but lowered plasma urea nitrogen levels [172]. This suggests that young ruminants may have different Leu utilization patterns compared to growing or finishing animals.

In dairy cows, BCAA constitute approximately 40% of EAA supplied to the mammary glands [173]. Abomasal infusion of EAA without BCAA suppressed mTOR-mediated activation of eIF2Bε and eIF2α in the mammary glands, leading to decreased milk protein production in dairy cows [174]. However, studies suggest that BCAA supplementation may not directly enhance milk protein production when methionine and lysine are adequately supplied. Appuhamy et al. [175] found that, in the presence of adequate methionine and lysine, BCAA supplementation did not significantly enhance milk protein synthesis but reduced milk urea nitrogen levels, likely due to the activation of alternative protein synthesis pathways. Chen et al. [176] observed that adding 10.6 g/d rumen-protected Leu to diets significantly increased ADG without affecting milk yield in lactating goats. Similarly, Leal Yepes et al. [177] reported that postpartum supplementation with 550 g/d of BCAA only marginally increased milk yield and protein content, possibly because BCAA are preferentially metabolized for energy rather than protein synthesis in the mammary glands. In a parallel study, Leal Yepes et al. [128] demonstrated that supplementing 550 g/d of rumen-protected BCAA from calving to day 35 of lactation increased plasma insulin concentrations and reduced hepatic lipidosis. Overall, BCAA serve as critical substrates for transamination and nitrogen donation, supporting the de novo synthesis of non-EAA required for milk protein production [28].

Recent studies highlight the complex relationship between BCAA metabolism, residual feed intake (RFI), and metabolic disorders in cattle. Jorge-Smeding et al. [178] found that high-RFI cattle fed a high-starch diet exhibited BCAA accumulation, insulin resistance, and obesity, whereas these traits were not observed with high-fiber diets. In a follow-up work, Jorge-Smeding et al. [179] reported that elevated plasma BCAA in low-efficiency animals were associated with enhanced lipogenesis and fat deposition, possibly due to the ketogenic effects of Leu and Ile. Supporting these findings, a meta-analysis conducted by Duarte et al. [180] identified BCAA degradation as the sole metabolic pathway significantly correlated with RFI in beef cattle. These findings collectively position BCAA as key metabolic regulators of feed efficiency. Optimizing BCAA supplementation strategies could help mitigate metabolic inefficiencies and improve sustainability in ruminant production systems.