Figure 5.
PA enhances acetylation of Rheb at lysine 8 (K8) to induce mTORC1 activation and insulin resistance. (A) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). IP was performed with anti-AcK beads. (B) Immunoblotting of Rheb acetylation in C2C12 myotubes treated with the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). IP was performed with an antibody to Rheb. (C) Immunoblotting of Rheb acetylation in the muscle of fish fed CON or PO diet (n = 5 fish per group). IP was performed with anti-AcK beads. (D) Immunoblotting of Rheb acetylation and S6K phosphorylation 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). IP was performed with anti-AcK beads. (E) Immunoblotting of Rheb acetylation in 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). IP was performed with an antibody to Rheb. (F) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with 25 mM sodium acetate in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). IP was performed with anti-AcK beads. (G and H) Representative MS/MS spectrum of acetylated K8 peptides of Rheb in croaker (G) or mouse (H). (I) The activity of mTORC1 signaling was analyzed by immunoblotting in fish myocytes transfected with the indicated plasmids. (J) The activity of mTORC1 signaling was measured by immunoblotting in HEK293T cells transfected with the indicated plasmids. (K) The activity of mTORC1 signaling was measured by immunoblotting in Rheb KO HEK293T cells transfected with the indicated plasmids (n = 3 independent wells per treatment). (L) The activity of mTORC1 signaling was measured by immunoblotting in Rheb KO HEK293T cells transfected with the indicated plasmids and then treated with or without 500 μM PA for 12 h (n = 3 experimental replicates). (M) Immunoblotting of Rheb acetylation in HEK293T cells transfected with the indicated plasmids. IP was performed with anti-Flag beads. (N) HEK293T cells were transfected with plasmids as indicated and the protein was extracted for Co-IP to assay the interaction between Rheb and FKBP12. IP was performed with anti-HA beads. (O) HEK293T cells were transfected with plasmids as indicated, and the protein was extracted for Co-IP to assay the interaction between Rheb and FKBP38. IP was performed with anti-Flag beads. (P) HEK293T cells were transfected with plasmids as indicated, and the protein was extracted for Co-IP to assay the interaction between mTOR and FKBP38. IP was performed with anti-Flag beads. (Q) Insulin-stimulated glucose uptake was measured by 2-DG uptake assays in fish myocytes transfected with the indicated plasmids (n = 3 independent wells per treatment). (R) AKT phosphorylation levels were assayed by immunoblotting in fish myocytes (n = 3 experimental replicates). Cells were transfected with the indicated plasmids 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 F5.

PA enhances acetylation of Rheb at lysine 8 (K8) to induce mTORC1 activation and insulin resistance. (A) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). IP was performed with anti-AcK beads. (B) Immunoblotting of Rheb acetylation in C2C12 myotubes treated with the indicated concentrations of PA for 12 h (n = 3 independent wells per treatment). IP was performed with an antibody to Rheb. (C) Immunoblotting of Rheb acetylation in the muscle of fish fed CON or PO diet (n = 5 fish per group). IP was performed with anti-AcK beads. (D) Immunoblotting of Rheb acetylation and S6K phosphorylation 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). IP was performed with anti-AcK beads. (E) Immunoblotting of Rheb acetylation in 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). IP was performed with an antibody to Rheb. (F) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with 25 mM sodium acetate in the presence or absence of 500 μM PA for 12 h (n = 3 independent wells per treatment). IP was performed with anti-AcK beads. (G and H) Representative MS/MS spectrum of acetylated K8 peptides of Rheb in croaker (G) or mouse (H). (I) The activity of mTORC1 signaling was analyzed by immunoblotting in fish myocytes transfected with the indicated plasmids. (J) The activity of mTORC1 signaling was measured by immunoblotting in HEK293T cells transfected with the indicated plasmids. (K) The activity of mTORC1 signaling was measured by immunoblotting in Rheb KO HEK293T cells transfected with the indicated plasmids (n = 3 independent wells per treatment). (L) The activity of mTORC1 signaling was measured by immunoblotting in Rheb KO HEK293T cells transfected with the indicated plasmids and then treated with or without 500 μM PA for 12 h (n = 3 experimental replicates). (M) Immunoblotting of Rheb acetylation in HEK293T cells transfected with the indicated plasmids. IP was performed with anti-Flag beads. (N) HEK293T cells were transfected with plasmids as indicated and the protein was extracted for Co-IP to assay the interaction between Rheb and FKBP12. IP was performed with anti-HA beads. (O) HEK293T cells were transfected with plasmids as indicated, and the protein was extracted for Co-IP to assay the interaction between Rheb and FKBP38. IP was performed with anti-Flag beads. (P) HEK293T cells were transfected with plasmids as indicated, and the protein was extracted for Co-IP to assay the interaction between mTOR and FKBP38. IP was performed with anti-Flag beads. (Q) Insulin-stimulated glucose uptake was measured by 2-DG uptake assays in fish myocytes transfected with the indicated plasmids (n = 3 independent wells per treatment). (R) AKT phosphorylation levels were assayed by immunoblotting in fish myocytes (n = 3 experimental replicates). Cells were transfected with the indicated plasmids 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 F5.

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