Effects And Molecular Mechanism Of Daidzein On Fat Deposition In Mouse C2C12 Myoblast Cells

Intramuscular fat(IMF)plays an important role in meat quality.Proper increase of the content of intramuscular fat plays an important role in improving the flavor,tenderness and acceptability of meat.As research continues,it has been discovered that intramuscular fat is mainly stored in intramuscular adipocytes.At least 80% of triglycerides(TGs)are stored in intramuscular adipocytes.However,there are still 5%-20% of TGs exist in the cytoplasm of skeletal muscle cells in lipid droplet(LD)form,which are equally crucial for the fat content in muscles.Daidzein(7,4'-dihydroxy isoflavone)is one of the main forms of soy isoflavones(SIF).Daidzein is a natural phytoestrogen which can bind to intracellular estrogen receptors or directly stimulate the activation of related gene expression in cells,plays an effect on anti-cancer,anti-hypertensive and anti-neuronal disease.Evidences have proved that soy isoflavones can regulate anabolic metabolism in animals,but whether and how they regulate fat deposition in muscle cells are still unknown.In this study,mouse C2C12 myoblast cells was used as the research materials.Different concentrations of daidzein(0,2.5,5,10,20 and 40 ?M)were administrated to C2C12 cells which were cultured in OPTI-MEM ? medium without FBS.Western blotting detected that the expression levels of f SREBP-1c and n SREBP-1c proteins were increased with the increase of daidzein concentration,in a dose-dependent manner,and reached a peak at the daidzein concentration of 10 ?M,then gradually decreased.These data reveal that daidzein can increase SREBP-1c expression and maturation.However,TGs detection and BODIPY staining indi cated that daidzein did not affect TGs synthesis and LDs formation in C2C12 cells.Subsequently,different concentrations of FAs(0,0.3,0.6,1.2,2.4,and 4.8 ?M)were administrated to C2C12 cells.Western blotting detected that,with the FAs concentration increasing,the expression protein levels of f SREBP-1c and n SREBP-1c were gradually increased,and reached a peak at the FAs concentration of 4.8 ?M,then decreased.TGs detection and BODIPY staining also show ed the same trend with the FAs concentration increased: TGs synthesis and LDs formation were increased with the FAs concentration increasing,and reached a peak at the FAs concentration of 2.4 ?M,then decreased.These data confirm that FAs induce fat deposition in C2C12 myoblast cells.The optimal concentration of daidzein and FAs were administrated to C2C12 cells which were cultured in OPTI-MEM ? medium without FBS.The experimental results showed that,the expression of f SREBP-1c and n SREBP-1c,TGs synthesis and LDs formation in C2C12 cells were significantly increased when daidzein and FAs were added simultaneously,compared with the cell group that only treated with daidzein or FAs.These data validate that FAs are substrate for fat synthesis in C2C12 cells,and reveal that daidzein can enhance FAs-induced fat deposition in C2C12 cells.To investigate the molecular mechanism via which daidzein promotes fat deposition in C2C12 cells,PI3K-AKT signaling pathway inhibitor LY294002 was administrated together with daidzein.Western blotting detected that daidzein significantly raised the protein levels of p-AKT,p-NF?B1,f SREBP-1c and n SREBP-1c in C2C12 cells.However,the stimulatory effects of daidzein on the protein expression of p-PI3 K,p-AKT,p-NF?B1,f SREBP-1c and n SREBP-1c were greatly reduced after inhibition of the PI3 K signaling pathway.These data reveal that daidzein enhances the SREBP-1c expression and maturation through the PI3K-AKT-NF?B1 signaling pathway in C2C12 cells.Real-time quantitative PCR showed that daidzein significantly increased SREBP-1c m RNA level;Ch IP-q PCR detected that daidzein accelerated the binding of p-NF?B1 to the SREBP-1c promoter,thereby promoting the transcription of SREBP-1c gene.These data suggest that daidzein promotes fat deposition in C2C12 cells by upregulating the transcription of SREBP-1c through the PI3K-AKT-NF?B1 signaling pathway.In order to further investigate whether the promotion of daidzein on fat deposition in C2C12 cells is dependent on estrogen receptors(ERs),daidzein was administrated to C2C12 cells transfected with interfering RNA against estrogen receptor ?(ER?).Western blotting detected that the expression of ER? did not change when daidzein was added.ER? knockdown did not affect the stimulation of daidzein on the protein levels of f SREBP-1c and n SREBP-1c.These data reveal that,in C2C12 myoblast cells,ER? does not mediate the signaling of daidzein to SREBP-1c.Similarly,daidzein was administrated to C2C12 cells transfected with interfering RNA against G protein-coupled receptor 30(GPR30).Western blotting data showed that the expression of GPR30 was significantly increased when daidzein was added.GPR30 knockdown totally abrogated daidzein-stimulated PI3 K activation,NF?B1 phosphorylation,and protein levels of f SREBP-1c and n SREBP-1c.Western blotting and immunofluorescence found that daidzein promoted GPR30 protein expression and cytoplasmic localization in a dose-dependent manner,and reached a peak at the daidzein concentration of 10 ?M.These data reveal that,in C2C12 myoblast cells,GPR30 is a key mediator of the signaling of daidzein to the PI3K-NF?B1-SREBP-1c pathway.In summary,our findings uncover that daidzein can dose-dependently promote the SREBP-1c signaling and FAs-induced fat deposition in C2C12 myoblast cells.Daidzein stimulates the m RNA expression of SREBP-1c via the GPR30-PI3K-NF?B1 signaling pathway...