Cellular fatty acid level regulates the effect of tolylfluanid on mitochondrial dysfunction and insulin sensitivity in C2C12 skeletal myotubes

Previous research suggests that the endocrine disrupting chemical tolylfluanid (TF) may promote metabolic dysfunction and insulin resistance in humans. The potential impact of TF on skeletal muscle metabolism has yet to be fully investigated. The purpose of this study was to determine whether TF can promote insulin resistance and metabolic dysfunction in mammalian skeletal muscle cells. C2C12 murine skeletal myotubes were exposed to 1 ppm TF for 24 h. To examine the potential effect of cellular fatty acid levels on TF-dependent regulation of mitochondrial metabolism and insulin signaling, we treated skeletal myotubes with 0.25 mM or 1.0 mM oleic acid (OA) during TF exposure trials. Tolylfluanid (1-10 ppm) reduced lipid accumulation by approximately 20% in 0.25 and 1.0 mM OA treated cells. The addition of 0.25 mM OA completely inhibited the TF-dependent reduction in maximal mitochondrial oxygen consumption rate (OCR) while 1.0 mM OA exacerbated the TF-dependent reduction in mitochondrial OCR. Exposing skeletal myotubes to 1 ppm TF promoted an 80% reduction in mitochondrial membrane potential, which was completely inhibited by 0.25 mM OA and partially inhibited by1.0 mM OA. The addition of 0.25 mM OA promoted a TF-dependent increase in insulin-dependent P-Akt (Ser473). In contrast, the addition of 1.0 mM OA promoted a significant reduction in insulin-dependent P-Akt (Ser473). Further, the addition of 1 ppm TF significantly reduced insulin-dependent mTORC1 activity regardless of OA concentration. Finally, TF significantly reduced insulin-dependent protein synthesis in the 1 mM OA treated cells only. Our results demonstrate that the effect of 1 ppm TF on mitochondrial function and insulin-dependent protein synthesis in skeletal myotubes was largely dependent upon cellular fatty acid levels.