PA-induced insulin resistance is dependent on the negative regulation of IRS1 by mTORC1. (A) Immunoblotting of IRS1 phosphorylation and protein levels in the muscle of fish fed CON or PO diet (n = 6 fish per group). (B) IRS1 phosphorylation and protein levels were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). (C) IRS1 phosphorylation and protein levels were measured by immunoblotting in fish myocytes and C2C12 myotubes treated with 500 nM rapamycin or 500 nM Torin1 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (D) IRS1 phosphorylation and protein levels were tested by immunoblotting in fish myocytes treated with control or 10 μM MHY1485 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (E) Relative mRNA levels of insa, insb, irs1, and irs2 were tested by quantitative PCR in the muscle of fish fed CON or PO diet (n = 4 fish per group). (F) Relative mRNA levels of insa, insb, irs1, and irs2 were analyzed by quantitative PCR in fish myocytes under control or PA treatments for 12 or 24 h (n = 3 independent wells per treatment). (G) Relative mRNA levels of insr, irs1, and irs2 were measured by quantitative PCR in C2C12 myotubes under control or 500 μM PA treatments for 12 or 24 h (n = 3 independent wells per treatment). (H) Relative mRNA levels of irs1 were analyzed by quantitative PCR in fish myocytes and C2C12 myotubes treated with control, 500 nM rapamycin or 500 nM Torin1 in the presence or absence of 500 μM PA for 12 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) and Tukey’s tests (bars bearing different letters are significantly different among treatments [P < 0.05]). Source data are available for this figure: SourceData F7.