The Molecular Mechanism By Which Ursolic Acid Regulates Insulin-and Leucine-stimulated MTOR Signaling

Part1Effect of UA on insulin-and leucine-stimulated mTOR signalingObjective:(1)To explore the effect of UA on insulin-stimulated mTOR signaling in C2C12myoblasts and differentiated C2C12myotubes.(2)To explore the effect of UA on insulin-stimulated mTOR signaling in differentiated3T3-L1adipocytes.(3)To explore the effect of UA on leucine-stimulated mTOR signaling in C2C12myoblasts and differentiated C2C12myotubes.(4)To explore the effect of UA on leucine-stimulated mTOR signaling in differentiated3T3-L1adipocytes.Methods:(1)Serum-starved C2C12myoblasts and differentiated C2C12myotubes were pretreated with or without UA at the indicated concentrations for60min, followed with or without10nM insulin treatment for10min, the expression and phosphorylation of S6K and4E-BP1were determined by Western Blot using the indicated antibodies.(2)Serum-starved differentiated3T3-L1adipocytes were pretreated with or without UA at the indicated concentrations for60min, followed with or without10nM insulin treatment for10min, the expression and phosphorylation of S6K and4E-BP1were determined by Western Blot using the indicated antibodies.(3)Serum-starved C2C12myoblasts and differentiated C2C12myotubes were pretreated with or without UA at the indicated concentrations for60min, followed with or without10mM leucine treatment for60min, the expression and phosphorylation of S6K and4E-BP1were determined by Western Blot using the indicated antibodies.(4)Serum-starved differentiated3T3-L1adipocytes were pretreated with or without UA at the indicated concentrations for60min, followed with or without10mM leucine treatment for60min, the expression and phosphorylation of S6K and4E-BP1were determined by Western Blot using the indicated antibodies.Results:(1)When compared to the control group, the stimulatory effect of insulin on S6K at Thr389and4E-BP1at Thr37/46phosphorylation was significantly reduced by cotreating the C2C12myoblasts and myotubes with UA in a dose-dependent manner.(2)When compared to the control group, the stimulatory effect of insulin on S6K at Thr389and4E-BP1at Thr37/46phosphorylation was significantly reduced by cotreating the differentiated3T3-L1adipocytes with UA in a dose-dependent manner.(3)When compared to the control group, the stimulatory effect of leucine on S6K at Thr389and4E-BP1at Thr37/46phosphorylation was significantly reduced by cotreating the C2C12myoblasts and myotubes with UA in a dose-dependent manner.(4)When compared to the control group, the stimulatory effect of leucine on S6K at Thr389and4E-BP1at Thr37/46phosphorylation was significantly reduced by cotreating the differentiated3T3-L1adipocytes with UA in a dose-dependent manner.Conclusion:UA inhibits insulin-and leucine-stimulated phosphorylation of S6K and4E-BP1in C2C12cells and3T3-L1adipocytes. Part2Role of PI3K/Akt pathway in insulin-and leucine-stimulated mTOR signalingObjective:(1)To explore whether PI3K/Akt pathway is involved in the inhibition effect of UA on insulin-stimulated mTOR signaling.(2)To explore whether PI3K/Akt pathway is involved in the inhibition effect of UA on leucine-stimulated mTOR signaling.Methods:(1)Serum-starved C2C12cells and3T3-L1adipocytes were pretreated with or without UA at the indicated concentrations for60min, followed with or without10nM insulin for10min, the expression of Akt as well as the phosphorylation of Akt at Thr308and Ser473were determined by Western Blot using the indicated antibodies.(2)Serum-starved C2C12myoblasts were pretreated with or without100nM Wortmannin for60min, followed with or without50μM UA for60min, then cotreat with or without10nM insulin for10min, the expression of S6K,4E-BP1and Akt as well as the phosphorylation of S6K at Thr389,4E-BP1at Thr37/46and Akt at Thr308were determined by Western Blot using the indicated antibodies.(3)Serum-starved C2C12cells and3T3-L1adipocytes were pretreated with or without50μM UA for60min, followed with or without10mM leucine for60min, the expression of Akt and phosphorylation of Akt at Thr308and Ser473were determined by Western Blot using the indicated antibodies.(4)Serum-starved PDK1KO/WT MEFs were pretreated with or without50uM UA for60min, followed with or without10mM leucine for60min, the expression of S6K and4E-BP1as well as the phosphorylation of S6K at Thr389and4E-BP1at Thr37/46were determined by Western Blot using the indicated antibodies.Results:(1)UA treatment significantly reduced insulin-stimulated Akt phosphorylation at Thr308and Ser473in C2C12cells and3T3-L1adipocytes.(2)PI3K inhibitor Wortmannin significantly inhibited insulin-stimulated Akt phosphorylation at Thr308, S6K phosphorylation at Thr389and4E-BP1phosphorylation at Thr37/46in C2C12cells, but UA had no additive inhibitory effect on the phosphorylation of S6K and4E-BP1.(3)UA and leucine had no significant effect on Akt phosphorylation in C2C12cells and3T3-L1adipocytes.(4)Leucine markedly stimulated the phosphorylation of S6k at Thr389in PDK1KO/WT MEFs, UA treatment significantly inhibited S6K phosphorylation at Thr389in PDK1KO MEFs. Conclusion:UA inhibits insulin-stimulated mTOR activation mostly through inhibiting PI3K/Akt signaling; UA inhibits leucine-stimulated mTOR activation via a PI3K/Akt-independent manner. Part3Role of TSC1/2-Rheb signaling pathway as well as the interaction of mTOR and Raptor/Deptor in UA-mediated inhibition of leu cine-stimulated mTOR activationObjective:(1)To explore whether UA inhibits leucine-stimulated mTOR activation in a TSC1/2-dependent manner.(2)To explore whether UA inhibits leucine-stimulated mTOR activation in a Rheb-dependent manner.(3)To explore whether UA inhibits leucine-stimulated mTOR activation through inhibiting the expression of mTORC1components.(4)To explore whether UA inhibits leucine-stimulated mTOR activation through affecting the interaction between mTOR and Raptor/Deptor.Methods:(1)Serum-starved TSC1/2KO/WT MEFs were pretreated with or without50μM UA for60min, followed with or without lOmM leucine for60min, the expression of S6K and4E-BP1as well as the phosphorylation of S6K at Thr389and4E-BP1at Thr37/46were determined by Western Blot using the indicated antibodies.(2)Serum-starved Vector-, RhebWT-, and Rhebs16H-overexpressed C2C12cells were pretreated with or without50μM UA for60min, followed with or without10mM leucine for60min, the expression of S6K and4E-BP1as well as the phosphorylation of S6K at Thr389and4E-BP1at Thr37/46were determined by Western Blot using the indicated antibodies.(3)Serum-starved Deptor scrambled and suppressed C2C12myoblasts were pretreated with or without50μM UA for60min, followed with or without10mM leucine for60min, the expression of S6K and4E-BP1as well as the phosphorylation of S6K at Thr389and4E-BP1at Thr37/46was determined by Western Blot using the indicated antibodies.(4)Serum-starved C2C12myoblasts were pretreated with or without50uM UA for60min, followed with or without lOmM leucine for60min, the expression of mTOR, Raptor, Deptor and Tubulin in cell lysates as well as the immunoprecipitated mTOR and the co-immunoprecipitated Raptor/Deptor were determined by Western Blot using the specific antibodies indicated.Results:(1)The phosphorylation of S6K at Thr389was markedly increased in TSC1/2KO MEFs compared with WT MEFs, the phosphorylation of S6K at Thr389was further stimulated by leucine in TSC1/2KO MEFs, treating the cells with UA greatly inhibited the leucine-stimulated phosphorylation of S6K in these cells.(2)RhebWT and Rhebs16H overexpression in C2C12myoblasts increased significantly the phosphorylation of S6K at Thr389, leucine had the additive stimulatory effect on the phosphorylation of S6K, although UA didn’t inhibit the phosphorylation of S6K in RhebWT-and Rhebs16H-overexpressed C2C12myoblasts, it inhibited leucine-stimulated S6K phosphorylation.(3)Knockdown of Deptor significantly increased the phosphorylation of S6K at Thr389, leucine had the additive stimulatory effect on the phosphorylation of S6K in Deptor-suppressed C2C12cells, treating the cells with UA greatly inhibited the leucine-stimulated phosphorylation of S6K.(4)UA didn’t affect the expression of mTOR, Raptor, and Deptor as well as the interaction between mTOR and Raptor/Deptor.Conclusion:UA inhibits leucine-stimulated mTOR activation in TSC1/2-and Rheb-independent manner; UA inhibits leucine-stimulated mTOR signaling not via affecting the expression of mTORC1proteins and the interaction between mTOR and Raptor/Deptor. Part4Role of RagB in leucine-stimulated mTOR signalingObjective:(1)To explore whether RagB is involved in UA-mediated inhibition of leucine-stimulated mTOR signaling.(2)To explore whether UA affects the interaction between RagB and Raptor.(3)To explore whether UA inhibits mTOR localization to lysosome.Methods:(1)Serum-starved Vector-, RagBWT-and RagBQ99L-overexpressed C2C12myoblasts were pretreated with or without50μM UA for60min, followed with or without10mM leucine for60min, the expression of S6K and4E-BP1and the phosphorylation of S6K at Thr389 and4E-BP1at Thr37/46were determined by Western Blot using the indicated antibodies.(2)Serum-starved RagB scrambled and suppressed C2C12myoblasts were pretreated with or without50μM UA for60min, followed with or without lOmM leucine for60min, the expression of S6K and the phosphorylation of S6K at Thr389were determined by Western Blot using the indicated antibodies.(3)Serum-starved RagB-overexpressed C2C12myoblasts were pretreated with or without50μM UA for60min, followed with or without lOmM leucine for60min, cells were then treated with the cross-linker chemical DSP and lysed. Raptor was immunoprecipitated from cell lysates, the immunoprecipitated Raptor and the co-immunoprecipitated RagB were detected by Western Blot.(4)Serum-starved RagB-overexpressed C2C12cells were pretreated with or without50μm UA for60min, followed with or without lOmM leucine for60min, cells were then treated with the cross-linker chemical DSP and lysed. Flag-RagB was immunoprecipitated from cell lysates, the immunoprecipitated Flag-RagB and the co-immunoprecipitated Raptor were detected by Western Blot.(5)Serum-starved C2C12cells were pretreated with or without50μM UA for60min, followed with or without lOmM leucine for60min, cells were stained using antibodies specific to mTOR (red) and LAMP1(green) and the lysosomal localization of mTOR was visualized by a confocal immunofluorescence microscope. Results:(1)Overexpression of RagB per se had no significant effect on S6K phosphorylation but potentiated leucine-stimulated S6K phosphorylation at Thr378, treatment of the cells with UA inhibited markedly leucine-stimulated mTORC1signaling in these cells; overexpression of RagBQ99L significantly promoted basal S6K phosphorylation, which was not further enhanced by leucine treatment, treatment of the cells with UA significantly inhibited mTORC1signaling in these cells.(2)UA has no effect on leucine-stimulated mTOR signaling in RagB-suppressed C2C12myoblasts.(3)Co-immunoprecipitation experiments showed that leucine treatment significantly stimulated the interaction between RagB and Raptor, the leucine-stimulated association of RagB with Raptor and mTOR was inhibited by UA.(4)Confocal immunofluorescence experiments showed that treatment of C2C12myoblasts with leucine promoted the co-localization of mTOR with the lysosomal marker LAMP1, the co-localization of mTOR with LAMP1was greatly suppressed by pretreating the cells with UA.Conclusion:RagB is involved in UA-mediated the inhibition of leucine-stimulated mTOR signaling; UA inhibits leucine-stimulated mTOR activation by interrupting the interaction between RagB and Raptor and inhibiting mTOR localization to lysosome.