Acetyl-CoA derived from mitochondrial fatty acid β oxidation triggers mTORC1 activation and insulin resistance under PA condition. (A) The levels of acyl-CoA and acylcarnitine in fish myocytes treated with 500 μM PA, 500 μM OA, or 500 μM LA for 12 h (n = 3 independent wells per treatment). (B) Measurement of CO₂ production and ASM in fish myocytes using [1-¹⁴C]-palmitic acid or [1-¹⁴C]-oleic acid (n = 3 independent wells per treatment). (C and D) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were measured by quantitative PCR in the muscle of fish fed CON, OO, or LO diet (n = 4 fish per group). (E and F) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were examined by quantitative PCR in C2C12 myotubes with control, 500 μM OA, or 500 μM LA treatment for 12 h (n = 3 independent wells per treatment). (G and H) Relative mRNA (G) and protein (H) levels of CPT1B were analyzed by quantitative PCR and immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT1B for 48 h (n = 3 independent wells per treatment). (I and J) Relative mRNA (I) and protein (J) levels of CPT2 were tested by quantitative PCR and immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT2 for 48 h (n = 3 independent wells per treatment). (K and L) Relative mRNA (K) and protein (L) levels of ACLY were analyzed by quantitative PCR and immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against ACLY for 48 h (n = 3 independent wells per treatment). The results are presented as the mean ± SEM and were analyzed using independent t tests (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are available for this figure: SourceData FS3.