The Function And Mechanism Of Liraglutide On The Differentiation Of C2C12 Myoblasts And The Expression Of FNDC5 In Myotubes

Diabetes is a kind of chronic disease that can affect lots of organs in our body.An increase of glucose in circulation is a characteristic of diabetes and the major reason of insulin resistant that occur in skeletal muscle,fat and liver tissues.Skeletal muscle is one of the important target organs that take up approximately 80%of dietary glucose,and play primary role in insulin action and glucose metabolism.Hyperglycemic conditions lead to the injury of skeletal muscle and then aggravate insulin resistance.The proliferation and modification of skeletal muscle has attracted more attention.Liraglutide is a kind of GLP-1 analogue expressed 97%homology for amino acid sequences with GLP-1 that has been approved for the treatment of type 2 diabetes by the means of its related mechanisms include glucose-dependent insulin secretion in pancreatic ?-cells,the inhibition of glucagon secretion in pancreatic a-cells by means of GLP-1 receptor agonist.In addition,LG acts as differentiation factors that can regulate the differentiation of islet and pre-adipocytes.But the effect and mechanisms of GLP-1 on the differentiation and function of C2C12 myoblasts has not been fully elucidated.Firstly,we studied that the effect of LG on differentiation of C2C12 myoblasts.Skeletal insulin resistance is a characteristic of T2DM.Studies reported that the improvement of insulin resistance and the potential enhancement of glucose use in skeletal muscle by LG is associated with increasing in glycogen synthesis and the GLUT4 translocation.Irisin is a cytokine secreted by skeletal muscle,whose precursor substance is fibronectin type III domain-containing protein 5(FNDC5).Irisin is believed to a potential therapeutic agent for T2DM because irisin could promote metabolic rate and improve insulin resistance.Studies showed that irisin is the production of PGC-1 a that is activated by AMPK,and irisin may take part in BCL-2-induced autophagy of skeletal muscle.Several clinical studies have demonstrated that patients with type 2 diabetes have decreased serum irisin.Irisin might be a novel target for insulin resistance and Type 2 diabetes.So we further studied that mechanism of LG on synthesis of irisin.Part I:The effect of LG on the differentiation of C2C12 skeletal myoblasts Aim:To investigate the effect of LG on the differentiation of C2C12 skeletal myoblastsMethods:1.In the process of C2C12 skeletal myoblasts differentiation,cells were harvested for western blot or fluorescence quantitative PCR at day 0,2,4 and 6.The mRNA and protein levels of molecular markers of differentiation and GLP-IR were detected by fluorescence quantitative PCR and western blot.2.In the process of C2C12 skeletal myoblasts differentiation,cells were treated with LG and collected at day 0,2,4 and 6.mRNA and protein levels of MYOD1 and MYL3 were detected by fluorescence quantitative PCR and western blot.3.In the process of C2C12 skeletal myoblasts differentiation,cells were treated with GLP-1R agonist LG and antagonist Exendin(9-39).Proteins were collected at sixth day and were detected by western blot.Results:1.Western blot and fluorescence quantitative PCR results showed that GLP-1R existed in C2C12 skeletal myoblasts and myotubes,and the expression level of GLP-1R in myotubes was significantly higher than in C2C12 skeletal myoblasts.2.qPCR and Western blot showed that compared with control cells,the mRNA and protein levels of MYOD1 and MY03 were significantly increased in LG-treated C2C12 cells.Similarly,3.At D6,protein expression of MYOD1 and MYL3 were markedly increased in LG-induced cells,and Exendin(9-39)inhibited the protein levels.Conclusions:1.GLP-1R exist in the myotubes.2.LG induce the differentiation of C2C12 skeletal myoblasts.Part II the function of LG on the expression of FNDC5 and the underlying machanisms Aim:To investigate the effect of liraglutide on the expression of fibronectine type III domain-containing protein 5(FNDC5)in C2C12 myotubes and the underlying mechanisms.Methods:1.The C2C12 skeletal myoblasts was induced to differentiate.Differentiated cells were then stimulated with LG(1-1000 nmol/1).The effects of LG on the expression of FNDC5 and the activation of cAMP-activated protein kinase(AMPK)and mitogen-activated protein kinase(MAPK)signaling pathways were determined.2.The myotubes were pretreated with Exendin(9-39)the GLP-1R antagonist,Compound or SCH772984 the inhibitors of AMPK or MAPK,the changes of LG induced FNDC5 expression were then examined.The expression of FNDC5 and the activation of AMPK and MAPK were determined by western blot.Results:1.In myotubes,LG promoted the expression of FNDC5 in a dose-and time-dependent manners and activated AMPK and MAPK signaling pathways.2.These effects of LG on FNDC5,AMPK and MAPK were partly abolished by Exendin(9-39)and STO609.3.These effects of LG on FNDC5 were partly abolished by Compound c.Conclusions:LG promotes the expression of FNDC5 via Glucagon like peptide-1 receptor(GLP-1R)in myotubes possibly through activation of the AMPK and MAPK signaling pathways.