Figure 4.
Acetyl-CoA derived from mitochondrial fatty acid β oxidation triggers mTORC1 activation and insulin resistance under PA condition. (A) Acetyl-CoA levels were measured in the muscles of fish fed CON or PO diet (n = 6 fish per group). (B) Acetyl-CoA levels in fish myocytes were measured in the presence of the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). (C) Acetyl-CoA levels were measured in fish myocytes under control or 500 μM PA treatment with or without 25 μM perhexiline maleate for 12 h (n = 3 independent wells per treatment). (D) Acetyl-CoAs labeling pattern from fish myocytes treated with [U-13C16]-labeled palmitate (n = 5 independent wells per treatment). (E) Schematic representation of the main cellular pathways involved in ACLY-governed citrate transport and ACSS2-mediated acetyl-CoA production. (F) The activities of mTORC1 and AKT were assayed via immunoblotting in fish with intraperitoneal injection of control dsRNA or dsRNA targeting ACLY for 36 h (n = 6 fish per group). (G) The activity of mTORC1 was tested by immunoblotting in fish myocytes with control or 25 μM BMS-303141 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (H) The activity of mTORC1 signaling was analyzed by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against ACLY and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (I) The activity of mTORC1 signaling was measured by immunoblotting in fish myocytes and C2C12 myotubes with the indicated concentrations of sodium acetate under control or PA treatment for 12 h (n = 3 experimental replicates). (J) The activity of mTORC1 signaling was measured by immunoblotting in fish myocytes and C2C12 myotubes under control or 25 μM perhexiline maleate treatment with or without 25 mM sodium acetate addition in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (K) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or 25 μM perhexiline maleate treatment with or without 25 mM sodium acetate addition in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (L) AKT phosphorylation levels were tested by immunoblotting in fish myocytes (n = 3 experimental replicates). Cells were pretreated under control or 25 μM perhexiline maleate treatment with or without 25 mM sodium acetate addition in the presence or absence of 500 μM PA for 12 h, and then stimulated with 100 nM insulin for 5 min. (M) AKT phosphorylation levels were detected by immunoblotting in fish myocytes (n = 3 experimental replicates). Cells were pretreated with control or 25 μM BMS-303141 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 F4.

Acetyl-CoA derived from mitochondrial fatty acid β oxidation triggers mTORC1 activation and insulin resistance under PA condition. (A) Acetyl-CoA levels were measured in the muscles of fish fed CON or PO diet (n = 6 fish per group). (B) Acetyl-CoA levels in fish myocytes were measured in the presence of the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). (C) Acetyl-CoA levels were measured in fish myocytes under control or 500 μM PA treatment with or without 25 μM perhexiline maleate for 12 h (n = 3 independent wells per treatment). (D) Acetyl-CoAs labeling pattern from fish myocytes treated with [U-13C16]-labeled palmitate (n = 5 independent wells per treatment). (E) Schematic representation of the main cellular pathways involved in ACLY-governed citrate transport and ACSS2-mediated acetyl-CoA production. (F) The activities of mTORC1 and AKT were assayed via immunoblotting in fish with intraperitoneal injection of control dsRNA or dsRNA targeting ACLY for 36 h (n = 6 fish per group). (G) The activity of mTORC1 was tested by immunoblotting in fish myocytes with control or 25 μM BMS-303141 treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (H) The activity of mTORC1 signaling was analyzed by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against ACLY and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (I) The activity of mTORC1 signaling was measured by immunoblotting in fish myocytes and C2C12 myotubes with the indicated concentrations of sodium acetate under control or PA treatment for 12 h (n = 3 experimental replicates). (J) The activity of mTORC1 signaling was measured by immunoblotting in fish myocytes and C2C12 myotubes under control or 25 μM perhexiline maleate treatment with or without 25 mM sodium acetate addition in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (K) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or 25 μM perhexiline maleate treatment with or without 25 mM sodium acetate addition in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (L) AKT phosphorylation levels were tested by immunoblotting in fish myocytes (n = 3 experimental replicates). Cells were pretreated under control or 25 μM perhexiline maleate treatment with or without 25 mM sodium acetate addition in the presence or absence of 500 μM PA for 12 h, and then stimulated with 100 nM insulin for 5 min. (M) AKT phosphorylation levels were detected by immunoblotting in fish myocytes (n = 3 experimental replicates). Cells were pretreated with control or 25 μM BMS-303141 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 F4.

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