Icariin improves metabolic response to exercise by promoting TFEB-dependent mitochondrial clearance and metabolic reprogramming in C57BL/6 mice and C2C12 myotubes

Background: Fatigue during intensive exercise is closely associated with metabolic inefficiency and lactate accumulation. While Icariin, a natural flavonoid, has demonstrated potential in enhancing exercise performance, the precise cellular mechanisms governing its anti-fatigue effects remain incompletely elucidated. Methods: C2C12 myotube systems. Mice received Icariin supplementation (50 or 100 mg/kg) for 4 weeks before comprehensive physiological assessments. Cellular studies utilized caffeine stimulation, transcriptomic profiling, and metabolic analyses. Molecular mechanisms were investigated through western blotting, immunofluorescence, and genetic knockdown approaches. Results: max) and prolonged exhaustive running time. This improvement was accompanied by reduced blood lactate accumulation, skeletal muscle hypertrophy, and a shift toward oxidative fiber types. In C2C12 myotubes, Icariin directly attenuated lactate production by suppressing LDH activity and reprogramming cellular metabolism toward oxidative phosphorylation. Transcriptomic analysis revealed significant enrichment of mitophagy pathways, which was validated by enhanced mitophagic flux and improved mitochondrial membrane potential. Mechanistically, we identified TFEB as the key transcriptional regulator mediating Icariin's effects, evidenced by its dephosphorylation, nuclear translocation, and transactivation of mitophagic genes. Crucially, TFEB knockdown completely abolished Icariin-induced mitophagy, metabolic improvements, and lactate reduction. Conclusion: Our findings establish a comprehensive mechanistic pathway wherein Icariin activates TFEB to drive mitophagic clearance of dysfunctional mitochondria, thereby optimizing mitochondrial function and shifting energy metabolism toward oxidative phosphorylation. This TFEB-mitophagy axis represents the core mechanism through which Icariin enhances exercise performance and metabolic efficiency, providing novel insights into its anti-fatigue properties and potential therapeutic applications.