Figure 3.
Mitochondrial fatty acid β oxidation is required for PA-induced mTORC1 activation and insulin resistance. (A) Heat map of the contents of acyl-CoAs and acyl-carnitines in fish myocytes treated with 500 μM PA, OA, or LA for 12 h. The relative fold change for each factor in each sample is represented as the relative average increase (red) or decrease (blue) (n = 3 independent wells per treatment). (B) Oxygen consumption rate (OCR) traces as measured by seahorse XF 24 Flux Analyzer in fish myocytes treated with 500 μM PA, OA or LA for 12 h (n = 3 independent wells per treatment). (C) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were analyzed by quantitative PCR in the muscle of fish fed CON or PO diet (n = 4 fish per group). (D and E) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were measured by quantitative PCR in fish myocytes (D) and C2C12 myotubes (E) with control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (F) Schematic representation of the main cellular pathways involved in mitochondrial fatty acid β oxidation. (G) The activities of mTORC1 and AKT were analyzed via immunoblotting in fish with intraperitoneal injection of control dsRNA or dsRNA targeting CPT1B for 36 h (n = 6 fish per group). (H) The activities of mTORC1 and AKT were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with control or 50 μM etomoxir treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (I) The activities of mTORC1 and AKT were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with control or 25 μM perhexiline maleate treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (J) The activity of mTORC1 signaling was examined by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT1B and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (K) The activity of mTORC1 signaling was measured by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT2 and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (L) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or 25 μM perhexiline maleate in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (M) AKT phosphorylation levels in fish myocytes and C2C12 myotubes were tested by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 25 μM perhexiline maleate treatment in the presence or absence of 500 μM PA for 12 h and then stimulated with 100 nM insulin for 5 min. (N) AKT phosphorylation levels in fish myocytes were tested by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 50 μM etomoxir treatment in the presence or absence of 500 μM PA for 12 h and then stimulated with 100 nM insulin for 5 min. (O) AKT phosphorylation levels in C2C12 myotubes were measured by immunoblotting (n = 3 experimental replicates). Cells were transfected with control siRNA or siRNA against CPT1B and pretreated with control or 500 μM PA treatments 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). Source data are available for this figure: SourceData F3.

Mitochondrial fatty acid β oxidation is required for PA-induced mTORC1 activation and insulin resistance. (A) Heat map of the contents of acyl-CoAs and acyl-carnitines in fish myocytes treated with 500 μM PA, OA, or LA for 12 h. The relative fold change for each factor in each sample is represented as the relative average increase (red) or decrease (blue) (n = 3 independent wells per treatment). (B) Oxygen consumption rate (OCR) traces as measured by seahorse XF 24 Flux Analyzer in fish myocytes treated with 500 μM PA, OA or LA for 12 h (n = 3 independent wells per treatment). (C) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were analyzed by quantitative PCR in the muscle of fish fed CON or PO diet (n = 4 fish per group). (D and E) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were measured by quantitative PCR in fish myocytes (D) and C2C12 myotubes (E) with control or 500 μM PA treatment for 12 h (n = 3 independent wells per treatment). (F) Schematic representation of the main cellular pathways involved in mitochondrial fatty acid β oxidation. (G) The activities of mTORC1 and AKT were analyzed via immunoblotting in fish with intraperitoneal injection of control dsRNA or dsRNA targeting CPT1B for 36 h (n = 6 fish per group). (H) The activities of mTORC1 and AKT were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with control or 50 μM etomoxir treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (I) The activities of mTORC1 and AKT were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with control or 25 μM perhexiline maleate treatment in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (J) The activity of mTORC1 signaling was examined by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT1B and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (K) The activity of mTORC1 signaling was measured by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT2 and then treated with or without 500 μM PA for 12 h (n = 3 independent wells per treatment). (L) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or 25 μM perhexiline maleate in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). (M) AKT phosphorylation levels in fish myocytes and C2C12 myotubes were tested by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 25 μM perhexiline maleate treatment in the presence or absence of 500 μM PA for 12 h and then stimulated with 100 nM insulin for 5 min. (N) AKT phosphorylation levels in fish myocytes were tested by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 50 μM etomoxir treatment in the presence or absence of 500 μM PA for 12 h and then stimulated with 100 nM insulin for 5 min. (O) AKT phosphorylation levels in C2C12 myotubes were measured by immunoblotting (n = 3 experimental replicates). Cells were transfected with control siRNA or siRNA against CPT1B and pretreated with control or 500 μM PA treatments 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). Source data are available for this figure: SourceData F3.

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