RSV inhibits leucine-stimulated mTOR activation in a PI3K/Akt-independent manner. A, serum-starved C2C12 myoblasts were pretreated with 50 μm RSV for 20 min and then stimulated with or without 10 nm insulin (Ins) for 10 min. The tyrosine phosphorylation and protein levels of the immunoprecipitated insulin receptor were determined by Western blotting using the antibodies indicated. B, serum-starved C2C12 cells were pretreated with or without RSV at the indicated concentrations for 20 min, followed with or without 10 nm insulin for 10 min. The insulin-stimulated phosphorylation of Akt and ERK1/2 and the protein levels of these kinases in cell lysates were determined by Western blotting using the antibodies indicated. C, serum-starved C2C12 cells were pretreated with or without the Akt inhibitor III (Akti III) for 60 min, followed with 50 μm RSV for 20 min. Cells were then stimulated with or without 10 nm insulin for 10 min and lysed. The insulin-stimulated S6K and 4E-BP1 phosphorylation in cell lysates was determined by Western blotting using the antibodies indicated. D, serum-starved C2C12 cells were pretreated with or without 10 mm leucine for 60 min and then cotreated using different concentrations of RSV for 30 min. The phosphorylation and protein levels of S6K, Akt, and ERK1/2 were determined by Western blotting with the indicated antibodies. E, serum-starved wild-type and PDK1-null MEFs were pretreated with or without leucine for 60 min and then cotreated with or without 50 μm RSV for 30 min. The phosphorylation and protein levels of the interesting signaling molecules were determined by Western blotting with the indicated antibodies and were quantified using the NIH Scion Image program. KO, knock-out. Unless indicated otherwise, all data are representative of at least three independent experiments with similar results. Differences between groups were examined for statistical significance using ANOVA. *, p < 0.05; **, p < 0.01. N, no addition; IRb, insulin receptor β-subunit.

RSV enhances the association between mTOR and DEPTOR. A, serum-starved C2C12 cells were pretreated with or without leucine for 60 min and then cotreated with or without 50 μm RSV for 20 min, followed with or without insulin (Ins) stimulation for 10 min. Cells were lysed, and mTOR proteins were immunoprecipitated from the cell lysates and used for mTOR kinase activity assays. B, the quantification of S6K phosphorylation in A was performed using the NIH Scion Image program and normalized with S6K protein levels. Ctr, control. C, serum-starved C2C12 cells were pretreated with or without leucine for 60 min and then cotreated with 50 μm RSV for 30 min. The immunoprecipitated (IP) mTOR and the co-immunoprecipitated DEPTOR were determined by Western blotting using the specific antibodies indicated. Unless indicated otherwise, all data are representative of at least three independent experiments with similar results. Nlg, normal immunoglobulin. D, serum-starved DEPTOR-suppressed and scrambled C2C12 cells were pretreated with or without leucine for 60 min and then cotreated with or without 50 μm RSV for 30 min. The phosphorylation of S6K and the protein levels of S6K, DEPTOR, and tubulin were determined by Western blotting with specific antibodies. E, quantification of S6K phosphorylation in D was performed using the NIH Scion Image program and normalized with S6K protein levels. Differences between groups were examined for statistical significance using ANOVA. *, p < 0.05; **, p < 0.01.

RSV inhibits insulin- and leucine-stimulated mTOR activation via a Sirt1-independent mechanism. A, serum-starved C2C12 myoblasts were pretreated with or without RSV at the indicated concentrations for 20 min, followed with or without 10 nm insulin (Ins) for 10 min. B, serum-starved C2C12 cells were pretreated with or without 10 mm leucine for 60 min and then cotreated with RSV for 30 min. C, serum-starved C2C12 myoblasts transiently overexpressing (O/E) Sirt1 were pretreated with 50 μm RSV for 20 min, followed with or without 10 nm insulin for 10 min. D, serum-starved C2C12 cells transiently overexpressing Sirt1 were pretreated with 10 mm leucine for 60 min, followed with or without 50 μm RSV for 30 min. E, serum-starved Sirt1^+/+ and Sirt1^−/− MEFs were pretreated with 50 μm RSV for 20 min and then treated with or without 10 nm insulin for 10 min. KO, knock-out. F, serum-starved Sirt1^+/+ and Sirt1^−/− MEF cells were pretreated with 10 mm leucine for 60 min, followed with or without 50 μm RSV for 30 min. The expression and phosphorylation of proteins in cell lysates were determined by Western blotting using the indicated antibodies. Tubulin was used as a loading control (Ctr) for all experiments. All data (C–F) are representative of at least three independent experiments with similar results and were quantified using the NIH Scion Image program. Differences between groups were examined for statistical significance using ANOVA. *, p < 0.05; **, p < 0.01. N, no addition.

RSV-promoted Inhibition of mTOR activation is TSC1/2- and AMPK-independent. A, serum-starved TSC1/2^+/+ and TSC1/2^−/− MEF cells were pretreated with 10 mm leucine for 60 min and then cotreated with or without 50 μm RSV for 30 min. B, serum-starved TSC1/2^+/+ and TSC1/2^−/− MEFs were pretreated with 50 μm RSV for 20 min, followed with or without 10 nm insulin (Ins) for 10 min. KO, knock-out. C, serum-starved AMPKα2-suppressed C2C12 cells were pretreated with or without leucine for 60 min and then cotreated with or without 50 μm RSV for 30 min. For all experiments, the phosphorylation and protein levels of S6K, 4E-BP1, and AMPK and the protein levels of TSC1 and TSC2 in cell lysates were determined by Western blotting with the indicated antibodies. All data are representative of at least three independent experiments with similar results, and A–C were quantified using the NIH Scion Image program. Differences between groups were examined for statistical significance using ANOVA. *, p < 0.05; **, p < 0.01. N, no addition.