Leucine promotes energy metabolism and stimulates slow-twitch muscle fibers expression through AMPK/mTOR signaling in equine skeletal muscle satellite cells.

Overview

• This study investigates how the amino acid leucine (Leu) influences energy metabolism and the expression of slow-twitch muscle fibers in horse skeletal muscle satellite cells (SCs).
• It demonstrates that leucine enhances mitochondrial function and promotes slow-MyHC expression through AMPK/mTOR signaling pathways, providing insight into muscle fiber regulation mechanisms.

Background

• Leucine is an essential branched-chain amino acid known to stimulate muscle cell proliferation.
• Previous research established that leucine can promote proliferation of equine skeletal muscle satellite cells and affect their gene expression profile.
• However, its role in regulating slow-twitch muscle fiber markers (slow-MyHC) and mitochondrial function in equine SCs was not fully understood.
• AMP-activated protein kinase (AMPK) and mammalian target of rapamycin complex (mTOR) are key signaling pathways involved in muscle metabolism and growth.

Methodology

• Equine skeletal muscle satellite cells were cultured in differentiation medium.
• Cells were treated with varying concentrations of leucine (0, 0.5, 1, 2, 5, and 10 mM) for 3 days to assess dose-dependent effects.
• To investigate mechanisms, AMPK inhibitor Compound C and mTOR inhibitor Rapamycin were applied to cells treated with leucine.
• Measurements included gene and protein expression of slow-twitch muscle fiber marker slow-MyHC, mitochondrial function genes (PGC-1α, Cytc, ND1, TFAM, COX1), and key markers in AMPK/mTOR pathways (e.g., LKB1, AMPK, S6, 4EBP1, mTOR and their phosphorylated forms).
• Respiration parameters such as basal, maximum, and standby respiration were assessed to evaluate mitochondrial function.

Key Findings

• Slow-MyHC expression was significantly higher at 2 mM leucine compared to 0, 0.5, and 10 mM doses, indicating an optimal concentration for promoting slow-twitch muscle fiber phenotype.
• Leucine supplementation significantly increased mitochondrial function, evidenced by enhanced basal, maximum, and standby respiration.
• Expression of mitochondrial biogenesis and function-related genes (PGC-1α, Cytc, ND1, TFAM, COX1) was elevated with leucine treatment.
• Leucine upregulated key proteins in both AMPK and mTOR signaling pathways, including their phosphorylated active forms, suggesting activation of these pathways.
• Inhibition of AMPK with Compound C or mTOR with Rapamycin reduced leucine-induced increases in slow-MyHC expression and signaling proteins, demonstrating reliance on these pathways for leucine’s effects.

Implications and Conclusion

• This study identifies the AMPK/mTOR signaling pathways as critical mediators of leucine’s effects on slow-twitch muscle fiber expression and mitochondrial function in equine skeletal muscle satellite cells.
• It highlights leucine’s role not only in muscle cell proliferation but also in promoting the energy-efficient slow-twitch muscle phenotype important for endurance and sustained muscle activity.
• The findings provide a molecular basis for nutritional strategies to enhance muscle metabolism and phenotype in horses, which may be relevant for training and recovery in equine athletes.
• This work also contributes novel mechanistic insights into how amino acid signaling can regulate muscle fiber type and mitochondria through AMPK and mTOR pathways.