Mitochondrial Adaptations in Skeletal Muscle Following Incretin‐Based Therapies: In Vitro

BACKGROUND: Incretin-based therapies such as glucagon-like peptide-1 receptor agonists (GLP-1Ras), dual GLP-1/GIP agonists and amylin analogues have demonstrated significant weight loss benefits. However, their impact on skeletal muscle mitochondrial function, particularly under metabolic stress, remains unclear. This study aimed to investigate the effects of semaglutide (GLP-1RA), tirzepatide (dual GLP-1/GIP agonist) and cagrilintide (amylin analogue) on mitochondrial function in C2C12 skeletal muscle myotubes under both healthy and lipotoxic (palmitic acid-treated) conditions. METHODS: Differentiated C2C12 myotubes were treated with doses of each drug for 48 h and 5 days. Mitochondrial respiration was assessed using the Seahorse XFp analyser, mitochondrial DNA (mtDNA) copy number and oxidative phosphorylation (OXPHOS) complex protein expression were measured by qPCR and western blotting. Key findings were repeated in primary human skeletal muscle cells. RESULTS: Palmitic acid (PA) significantly impaired mitochondrial function, reducing basal oxygen consumption rate (OCR) by 22% (p = 0.0056) and ATP production by 25% (p = 0.0022). In healthy myotubes, semaglutide and cagrilintide transiently reduced basal respiration (↓21%-28%, p < 0.05) and ATP production (↓24%-31%, p < 0.01) at 48 h, along with reductions in Complexes I, III and IV protein expression, all of which resolved by 5 days. Tirzepatide significantly increased maximal respiration (↑20%-25%, p < 0.005) and spare respiratory capacity (↑22%-30%, p < 0.005) after 5 days. In PA-treated myotubes, semaglutide and cagrilintide acutely worsened mitochondrial impairment (↓ATP production by ~20%-25%, p < 0.01), but these effects resolved by Day 5. Tirzepatide initially suppressed mitochondrial function (↓ATP production, p = 0.0087) but reversed these effects by Day 5, significantly improving ATP production (↑27%-30%, p < 0.005), basal respiration (↑20%, p = 0.0152), and coupling efficiency. mtDNA content remained unchanged across all conditions. Similar responses were noted in human myotubes, with a transient reduction in respiration for semaglutide and cagrilintide (↓30%-62%, p < 0.05) at 48 h and a significant improvement in maximal respiration for tirzepatide at 5 days (↑42%-52%, p = 0.0022). CONCLUSION: Incretin-based therapies exert distinct, time and dose-dependent effects on skeletal muscle mitochondrial function. Tirzepatide promoted sustained improvements in mitochondrial respiration under both healthy and lipotoxic conditions, indicating potential benefits for maintaining skeletal muscle bioenergetic function. These findings underscore the need for further mechanistic studies and suggest that tirzepatide may have the potential to support skeletal muscle health in metabolic disease.