Fullerol Attenuates Diabetic Muscle Atrophy and Ferroptosis-Associated Injury: Intervention Timing Influences Myogenesis-Associated Responses In Vitro

Glucolipotoxic stress contributes to diabetic muscle atrophy by disrupting myoblast homeostasis and myogenesis-associated responses. Fullerol is a water-soluble polyhydroxylated fullerene derivative with potent free-radical-scavenging activity. We investigated the protective effects of fullerol and compared concurrent and post-HGPA intervention schedules in C2C12 myoblasts. We evaluated C2C12 myoblasts exposed to high glucose plus palmitic acid (HGPA), RAW264.7 macrophages, macrophage-conditioned medium, and male C57BL/6J mice subjected to a high-fat diet (HFD) and streptozotocin (STZ). In the animal experiment, fullerol was administered locally during diabetic model development, whereas intervention timing was compared only in the C2C12 model. In myoblasts, HGPA induced Fe2+ and superoxide anion accumulation, lipid peroxidation, GSH depletion, mitochondrial depolarization, increased ACSL4, and decreased GPX4, all of which were attenuated by fullerol. Fullerol also attenuated erastin-induced ferroptosis-associated injury. Concurrent treatment preserved proliferation-associated markers, reduced P21 and γ-H2AX, restored Myod1 and Myog expression and MYHC protein abundance, and suppressed Murf1 and Atrogin-1 expression. At the 48-h endpoint, both concurrent and 24-h post-HGPA fullerol treatment attenuated ferroptosis-associated changes. At the separate 96-h endpoint, post-HGPA fullerol treatment only partially restored proliferation- and myogenesis-associated marker profiles, even when fullerol treatment was continued for 72 h. In macrophages, fullerol reduced CCR7 and TNF-α and increased CD206 and SOD1. Conditioned medium from HGPA-plus-fullerol-treated macrophages induced less superoxide anion and Fe2+ accumulation in myoblasts than medium from HGPA-stimulated macrophages. In diabetic mice, fullerol preserved myofiber size and relative muscle weight, improved myogenesis-associated gene and protein expression, suppressed atrophy-associated gene expression, reduced 4-HNE accumulation and F4/80-positive macrophage abundance, and normalized GPX4 and ACSL4 protein expression toward control levels. These findings support the protective potential of fullerol during the development of diabetic muscle atrophy and indicate that intervention timing influences proliferation- and myogenesis-associated responses in vitro.