Figure 2.
Hyperactivation of mTORC1 is associated with PA-induced insulin resistance. (A) mTORC1 pathway activity was measured by immunoblotting for the phosphorylation of S6K and S6 in the skeletal muscle of fish fed CON or PO diet (n = 6 fish per group). (B) mTORC1 pathway activity was tested by immunoblotting in fish myocytes under control or 500 μM PA treatment for 8, 12, and 24 h (n = 3 independent wells per treatment). (C) mTORC1 pathway activity was assayed by immunoblotting in fish myocytes and C2C12 myotubes in the presence of the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). (D) mTORC1 pathway activity was analyzed by immunoblotting in fish myocytes and C2C12 myotubes under control or 500 μM PA treatment in the presence or absence of 500 nM rapamycin for 12 h (n = 3 independent wells per treatment). (E) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or 500 μM PA treatment with or without 500 nM rapamycin for 12 h (n = 3 independent wells per treatment). (F) Phosphorylation levels of AKT in fish myocytes and C2C12 myotubes were detected by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 500 μM PA treatment in the presence or absence of 500 nM rapamycin for 12 h and then stimulated with 100 nM insulin for 5 min. (G) mTORC1 pathway activity was measured by immunoblotting in fish myocytes under control or 500 μM PA treatment with or without 10 μM MHY1485 for 12 h (n = 3 independent wells per treatment). (H) Phosphorylation levels of AKT in fish myocytes were tested by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 500 μM PA treatment in the presence or absence of 10 μM MHY1485 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 F2.

Hyperactivation of mTORC1 is associated with PA-induced insulin resistance. (A) mTORC1 pathway activity was measured by immunoblotting for the phosphorylation of S6K and S6 in the skeletal muscle of fish fed CON or PO diet (n = 6 fish per group). (B) mTORC1 pathway activity was tested by immunoblotting in fish myocytes under control or 500 μM PA treatment for 8, 12, and 24 h (n = 3 independent wells per treatment). (C) mTORC1 pathway activity was assayed by immunoblotting in fish myocytes and C2C12 myotubes in the presence of the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). (D) mTORC1 pathway activity was analyzed by immunoblotting in fish myocytes and C2C12 myotubes under control or 500 μM PA treatment in the presence or absence of 500 nM rapamycin for 12 h (n = 3 independent wells per treatment). (E) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or 500 μM PA treatment with or without 500 nM rapamycin for 12 h (n = 3 independent wells per treatment). (F) Phosphorylation levels of AKT in fish myocytes and C2C12 myotubes were detected by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 500 μM PA treatment in the presence or absence of 500 nM rapamycin for 12 h and then stimulated with 100 nM insulin for 5 min. (G) mTORC1 pathway activity was measured by immunoblotting in fish myocytes under control or 500 μM PA treatment with or without 10 μM MHY1485 for 12 h (n = 3 independent wells per treatment). (H) Phosphorylation levels of AKT in fish myocytes were tested by immunoblotting (n = 3 experimental replicates). Cells were pretreated with control or 500 μM PA treatment in the presence or absence of 10 μM MHY1485 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 F2.

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