利拉鲁肽影响C2C12成肌细胞分化及合成Irisin的机制探讨

发布时间：2018-08-29 17:11

【摘要】：糖尿病是影响全身各个器官的内分泌系统疾病,以血液循环中葡萄糖浓度升高为特点,在肌肉、脂肪、肝脏组织的胰岛素抵抗为主要原因。骨骼肌是胰岛素作用的重要部位及人体调节血糖代谢的主要器官,可以消耗人体近80%的葡萄糖,但是高血糖环境也可以损害骨骼肌细胞,骨骼肌病变又使外周胰岛素抵抗加重,所以治疗糖尿病的药物对骨骼肌的生长发育及损伤后修复的作用受到越来越广泛的关注。利拉鲁肽(LG)是一种胰高血糖素样肽-1(GLP-1)类似物,与人体天然的GLP-1氨基酸序列具有97%的同源性,通过激活胰高血糖素样肽-1受体(GLP-1R)作用于胰岛β细胞产生葡萄糖依赖性的促进胰岛素分泌及其抑制胰岛a细胞分泌胰高血糖素的作用已应用于2型糖尿病的临床治疗。GLP-1还可作为促分化因子促进前脂细胞的分化以及胰岛细胞的增生。然而GLP-1在对骨骼肌细胞分化及功能的作用及调控机制尚未阐明,因此我们首先研究了 GLP-1R激动剂LG能否促进骨骼肌细胞的分化,以改善糖尿病病人肌细胞的萎缩与凋亡。另外骨骼肌细胞胰岛素抵抗是2型糖尿病发生的一个重要因素,研究报道LG可能通过促进糖原的合成及葡糖糖转运体4(GLUT4)的转运增强骨骼肌细胞对葡萄糖的利用,改善胰岛素抵抗,但其机制尚未完全清楚。鸢尾素(irisin)是一种新发现的由肌肉细胞分泌的细胞因子,其前体物质为III型纤连蛋白结构域(FNDC5)。有研究表明Irisin作为AMPK激活后PGC-1 a的表达产物,可能参与BCL-2所调控的骨骼肌细胞内自噬作用,从而改善骨骼肌的胰岛素的敏感性,所以一些临床研究已经证实的2型糖尿病病人的Irisin分泌会减少可能是肌肉组织发生胰岛素抵抗的一个原因,因此我们进一步研究了 LG是否通过调控Irisin的合成来发挥改善胰岛素抵抗的作用。第一部分利拉鲁肽对骨骼肌成肌细胞C2C12分化的影响研究目的:探讨LG对骨骼肌成肌细胞分化的影响研究内容:1.于C2C12细胞诱导分化过程中第0、2、4、6天收集样品,提取mRNA和蛋白,分别用荧光定量PCR和Western blot检测肌管细胞分化标志物以及GLP-1R的基因和蛋白水平的表达。2.于C2C12细胞诱导分化过程中,给予100nM浓度GLP-1受体激动剂LG处理,分别于第0、2、4、6天收集样品,提取mRNA和蛋白,分别用荧光定量PCR和Western blot检测肌管细胞分化标志物基因蛋白水平的表达。3.于C2C12细胞诱导分化过程中,给予100 nM LG和10 nM GLP-1R拮抗剂exendin(9-39)处理,于第6天收取样品,提取蛋白,用Western blot检测肌管细胞分化标志物蛋白水平的表达。研究结果:1.Western blot及荧光定量PCR的结果显示,GLP-1R在骨骼肌成肌细胞C2C12中表达,且在分化成熟肌管细胞中的表达水平明显高于未分化成肌细胞。2.C2C12细胞分化过程中,用LG进行处理,结果显示成肌细胞决定基因1(MYOD1)及肌球蛋白轻链3(MYL3)的蛋白表达水平明显高于对照组;同样MYOD1及肌球蛋白(Myosin)的基因表达水平明显高于对照组。3.C2C12细胞分化过程中,用LG及exendin(9-39)进行处理,结果显示分化第六天,LG组细胞MYOD1及MYL3蛋白表达显著高于对照组;与LG组细胞比较,Ex组细胞的MYOD1及MYL3蛋白表达受到抑制,显著降低,而LG + Ex组细胞的MYOD1及MYL3蛋白表达未受到Ex的抑制,与LG组持平。研究结论:1.GLP-1R存在于肌管细胞中2.在C2C12成肌细胞分化成熟为肌管细胞的过程中,LG发挥促进作用。第二部分利拉鲁肽对肌管细胞合成FNDC5的影响及其机制研究研究目的:探讨LG对肌管细胞合成Irisin前体物质FNDC5的影响及其机制研究内容:1.小鼠骨骼肌成肌细胞C2C12经诱导分化为肌管细胞后,给予梯度浓度LG(1-1000 nm/L)处理不同时间(0-24 h),观察LG对FNDC5表达及磷酸化腺苷酸活化蛋白激酶(cAMP-activated protein kinase,AMPK)和丝裂原活化激酶(mitogen-activated protein kinase,MAPK)信号通路活性的影响2.分别应用GLP-1R拮抗剂Exendin(9-39),AMPK的抑制剂Compound c及MAPK的抑制剂SCH772984预处理肌管细胞,应用Western blot方法观察FNDC5蛋白表达的改变,检测AMPK及MAPK信号通路的的活性。研究结果:1.LG可促进肌管细胞FNDC5的蛋白表达,并具有时间-剂量依赖性。2.LG可激活AMPK与MAPK信号通路,呈时间-剂量依赖性。3.Compound c抑制LG诱导的AMPK表达,显著降低LG诱导的FNDC5蛋白表达,SCH772984抑制LG诱导的MAPK表达,并显降低著LG诱导的FNDC5蛋白表达。4.Exendin(9-39),可抑制LG激活的MAPK及AMPK磷酸化水平信号通路;LG促进的FNDC5蛋白表达也受到抑制。研究结论:LG通过GLP-1R激活AMPK和MAPK信号通路促进肌管细胞FNDC5的表达
[Abstract]:Diabetes mellitus is an endocrine disorder affecting all organs of the body. It is characterized by elevated glucose concentration in the blood circulation. Insulin resistance is the main cause in muscle, fat and liver tissues. Skeletal muscle is an important part of insulin and the main organ of regulating blood glucose metabolism. It can consume nearly 80% of human glucose. Hyperglycemic environment can also damage skeletal muscle cells, skeletal muscle lesions and aggravate peripheral insulin resistance, so the role of diabetic drugs on skeletal muscle growth and development and repair after injury has received more and more attention. Liraglutide (LG) is a glucagon-like peptide-1 (GLP-1) analogue, and the human body's natural GLP-1. Glucagon-like peptide-1 receptor (GLP-1R) stimulates glucose-dependent insulin secretion by islet beta cells and inhibits glucagon secretion by islet a cells. GLP-1 can also be used as a differentiation-promoting factor to promote preadipheresis in type 2 diabetes mellitus. However, the role of GLP-1 in the differentiation and function of skeletal muscle cells has not been clarified. Therefore, we first studied whether GLP-1R agonist LG could promote the differentiation of skeletal muscle cells to improve the atrophy and apoptosis of skeletal muscle cells in diabetic patients. LG may enhance glucose utilization and improve insulin resistance by promoting glycogen synthesis and glucose transporter 4 (GLUT4) transport in skeletal muscle cells, but the mechanism is not fully understood. Iris is a newly discovered cytokine secreted by muscle cells. Irisin, as an expression product of PGC-1a activated by AMPK, may be involved in the autophagy of skeletal muscle cells regulated by BCL-2, thus improving the insulin sensitivity of skeletal muscle. Therefore, Irisin secretion in type 2 diabetic patients has been confirmed by some clinical studies. Reduction may be one of the causes of insulin resistance in muscle tissue, so we further investigated whether LG can improve insulin resistance by regulating the synthesis of Irisin. Part I Effect of liraglutide on the differentiation of skeletal myoblasts C2C12 Objective: To investigate the effect of LG on the differentiation of skeletal myoblasts. Contents: 1. Samples were collected on the 0th, 2nd, 4th and 6th day of the induction of differentiation of C2C12 cells, and the mRNA and protein were extracted. The expression of differentiation markers and GLP-1R gene and protein were detected by fluorescence quantitative PCR and Western blot respectively. 2. During the induction of differentiation of C2C12 cells, 100nM concentration of GLP-1 receptor agonist LG was given. Samples were collected on the 0th, 2nd, 4th and 6th day, and mRNA and protein were extracted. The expression of gene protein, a marker of myotube differentiation, was detected by fluorescence quantitative PCR and Western blot respectively. Western blot and fluorescence quantitative PCR showed that GLP-1R was expressed in C2C12 of skeletal myoblasts, and the expression level of GLP-1R in differentiated mature myoblasts was significantly higher than that in undifferentiated myoblasts. The expression of MYOD1 and myosin light chain 3 (MYL3) in myoblasts was significantly higher than that in control group. The expression of MYOD1 and myosin was also significantly higher than that in control group. Compared with LG group, MYOD1 and MYL3 protein expression in Ex group was inhibited and significantly decreased, while MYOD1 and MYL3 protein expression in LG + Ex group was not inhibited by Ex, which was the same as that in LG group. Conclusion: 1. GLP-1R was present in myotube cells 2. MYOD1 and MYL3 protein expression in C2C12 myoblast differentiated into myotube cells. In the second part, the effect of liraglutide on the synthesis of FNDC5 by myotube cells and its mechanism were studied. Objective: To investigate the effect of LG on the synthesis of Irisin precursor FNDC5 by myotube cells and its mechanism: 1. After induction and differentiation of mouse skeletal muscle myoblast C2C12 into myotube cells, the gradient concentration of LG (LG) was given. Effects of LG on FNDC5 expression and signal pathway activity of cAMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) were observed at 1-1000 nm/L for different time (0-24 h). 2. GLP-1R antagonist Exendin (9-39), AMPK inhibitors UNDC and M PK inhibitors Exendin (9-39), respectively. APK inhibitor SCH772984 was used to pretreat myotubal cells. Western blot was used to observe the expression of FNDC5 protein and detect the activity of AMPK and MAPK signaling pathway. 3. Compound C inhibited LG-induced AMPK expression, significantly decreased LG-induced FNDC5 protein expression, SCH772984 inhibited LG-induced MAPK expression, and significantly decreased LG-induced FNDC5 protein expression. 4. Exendin (9-39) inhibited LG-activated MAPK and AMPK phosphorylation signaling pathway; LG-promoted FNDC5 protein expression was also inhibited. Activation of AMPK and MAPK signaling pathway through GLP-1R promotes FNDC5 expression in myotube cells
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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