Leu promotes C2C12 cell differentiation by regulating the GSK3β/β‐catenin signaling pathway through facilitating the interaction between SESN2 and RPN2

BACKGROUND Leucine (Leu) is an essential amino acid that facilitates skeletal muscle satellite cell differentiation, yet its mechanism remains underexplored. Sestrin2 (SESN2) serves as a Leu sensor, binding directly to Leu, while ribophorin II (RPN2) acts as a signaling factor in multiple pathways. This study aimed to elucidate Leu's impact on mouse C2C12 cell differentiation and skeletal muscle injury repair by modulating RPN2 expression through SESN2, offering a theoretical foundation for clinical skeletal muscle injury prevention and treatment. RESULTS Leu addition promoted C2C12 cell differentiation compared to the control, enhancing early differentiation via myogenic determinant (MYOD) up‐regulation. Sequencing revealed SESN2 binding to and interacting with RPN2. RPN2 overexpression up‐regulated MYOD, myogenin and myosin heavy chain 2, concurrently decreased p‐GSK3 β and increased nuclear β ‐catenin. Conversely, RPN2 knockdown yielded opposite results. Combining RPN2 knockdown with Leu rescued increased p‐GSK3 β and decreased nuclear β ‐catenin compared to Leu absence. Hematoxylin and eosin staining results showed that Leu addition accelerated mouse muscle damage repair, up‐regulating Pax7, MYOD and RPN2 in the cytoplasm, and nuclear β ‐catenin, confirming that the role of Leu in muscle injury repair was consistent with the results for C2C12 cells. CONCLUSION Leu, bound with SESN2, up‐regulated RPN2 expression, activated the GSK3 β / β ‐catenin pathway, enhanced C2C12 differentiation and expedited skeletal muscle damage repair.