The Regulation Of Fibroblast Growth Factor 21(FGF21) Gene On Skeletal Muscle Differentiation And Muscle Fiber Type Transformation

Fibroblast growth factor 21(FGF21)belongs to fibroblast growth factor family,the protein encoded by this gene is a secretion protein,and liver is the major organ synthesizes this protein.Unlike canonical fibroblast growth factors,the protein encoded by FGF21 can act on various tissues through combination with its receptors and co-factors after secretion into the circulatory system.FGF21 plays important role in growth,development,energy metabolism and reproduction owning to its multiple target tissues.In the past decade,the role of FGF21 in fat metabolism,glucose uptake,insulin resistance and obesity were studied.FGF21 was considered as a therapeutic drug these years since its critical role in energy metabolism and non-mitosis character.Muscle and fat tissue are two most important tissues in energy homeostasis,they have critical role in glucose uptake and insulin resistance.Furhtermore,muscle is a very important economic trait of livestocks,the improvement of mass and quality of muscle is a goal of animal breeding.Even though much work has been done and great breakthrough was made about the function of FGF21,the emphasis of these studies was the role of FGF21 in obesity and type ? diabetes,whether FGF21 plays a role in muscle development remains unclear.This study focused on the role of FGF21 during myogenesis,and its regulation mechanism.The results were as follows:1.The mRNA and protein expression levels of FGF21 during myogenic differentiation were measured using quantitative PCR,Western Blot and ELISA.The results showed that mRNA level of FGF21 was increased during myogenic differentiation,it reached peak value in 2 days of differentiation and then decreased.Western Blot results indicated that the intracellular protein of FGF21 was increased in the whole differentiaton process,and the extracellular protein of FGF21 measured by ELISA showed that there was a dramatically increase of this protein after 2 days differentiation,and then it maintains at the high level through the process.2.Overexpression and knockdown of FGF21 gene were performed in mouse myoblasts C2C12 cells to investigate the role of FGF21 in myogenic differentiation,myofiber type transformation and energy metabolism.The results showed that overexpression of FGF21 increased the mRNA levels of key genes involved in myogenic differentiation(MyoD,MyoG),marker gene of aerobic myofiber(MyHC ?)and mitochondrial oxidative related genes(COX 6c?COX 8b),while knockdown of FGF21 decreased the mRNA levels of these genes.The key proteins of myogenic differentiation(MyoD and MyoG),aerobic myofiber abundant proteins(MyHC ? and Myoglobin),as well as mitochondrial oxidative related proteins(PGC1? and Cox4)were upregulated in FGF21 overexpression group while downregulated in FGF21 knockdown group.In addition,overexpression of FGF21 promotes cell fusion and myotubes formation,while FGF21 knockdown inhibits cell fusion and myotubes formation.3.In vivo study was performed to investigate the effects of FGF21 gene on muscle development by employing muscle specific transgenic mice,which overexpress mouse FGF21 gene.It was observed that the body mass and tissue mass of FGF21 transgenic mice was smaller than their control littermates and the gastrocnemius tissue has a redder color in transgenic mice.The mRNA levels of MyHC? and MyHC ?a were upregulated in transgenic mice,and the proportion of aerobic myofiber was higher in transgenic mice than control mice.4.Analyzing the promoter region of mouse FGF21 gene by using online prediction software,several potential bingding sites of transcription factor MyoD were predicted and finaly one binding site was verified,and the results showed that MyoD regulates the expression of FGF21 by binding to its promoter.5.The effect of FGF21 on cell cycle was also investigated in FGF21 overexpression and knockdown cells,the results showed that overexpression of FGF21 promoted the expression of cell cycle exit genes,cells were blocked in G0 phase and total number of cells in S phase was decreased,while knockdown of FGF21 decreased the expression of cell cycle exit genes and increased the expression of cell cycle genes.6.The regulatory mechanism of FGF21 affects myogenic differentiation and myofiber transformation was investigated,the results demonstrated that compared to control group,AMPK was activated by phosphorylation,protein levels of SIRT1 and PGC1? were increased in FGF21 overexpression group;after AMPK activator treatment,the expression levels of myogenic differentiation and aerobic myofiber abundant genes were upregulated,while in AMPK inhibitor treated cells,the expression levels of these genes were downregulated.Taking together,this study investigated the role of FGF21 in muscle development.The results demonstrated that FGF21 promotes myogenic differentiation through two ways,transcriptional activation by MyoD and promoting cell cycle exit.Furthermore,this study inllustrated that FGF21 enhances the formation of aerobic myofiber may through FGF21-SIRT1-AMPK-PGC1? pathway.This study provided evidence for understanding the role of FGF21 in myogenesis.