PA inhibits the nuclear translocation of TFEB to impede IRS1 transcription. (A) Relative dual luciferase activity analysis was conducted to measure the effect of ATF4, PPARα, PPARγ, TFEB, and HNF1α on IRS1 promoter activity in HEK293T cells (n = 3 independent wells per treatment). (B) Relative dual luciferase activity analysis was conducted to measure the effect of TFEB at different concentration gradients on IRS1 promoter activity in HEK293T cells (n = 3 independent wells per treatment). (C) Relative dual luciferase activity analysis was performed to test the effect of TFEB on IRS1 promoter activity with mutation of the predicted binding sites in HEK293T cells (n = 3 independent wells per treatment). (D and E) The binding between TFEB and the predicted region of the IRS1 promoter was demonstrated by EMSA (D) and ChIP (E) in HEK293T cells. (F) TFEB and IRS1 protein levels were measured by immunoblotting in fish myocytes transfected with pCS2 (empty vector) or pCS2-TFEB plasmids (n = 3 independent wells per treatment). (G) TFEB nuclear translocation was assayed by immunoblotting nuclear fractions of fish myocytes and C2C12 myotubes treated with control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (H) TFEB nuclear translocation was tested by immunoblotting nuclear fractions of fish myocytes and C2C12 myotubes treated with control, 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). (I) Relative mRNA levels of tfeb and irs1 were analyzed by quantitative PCR in fish myocytes treated with control or TFEB activator 1 treatment in the presence or absence of PA for 12 h (n = 3 independent wells per treatment). (J) IRS1 protein levels and the phosphorylation of AKT were tested by immunoblotting in fish myocytes treated with control or 15 μM TFEB activator 1 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (K) AKT phosphorylation levels were measured by immunoblotting in fish myocytes and C2C12 myotubes (n = 3 experimental replicates). Cells were pretreated with control or 15 μM TFEB activator 1 treatment in the presence or absence of 500 μM PA for 12 h and then stimulated with 100 nM insulin for 5 min. 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 F8.