【作者】 马慧娟；

【导师】 宋光耀；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 博士

【摘要】 近年来2型糖尿病的发病呈快速增长趋势,目前估算到2030年全球糖尿病患病人数将超过3.5亿。由糖尿病引起的死亡人数仅次于心脑血管疾病、恶性肿瘤,被称为“第三杀手”,我国现有糖尿病人数目前居世界第二位。流行病学资料表明,随年龄增加2型糖尿病患病率也明显增加,是影响中老年人健康的主要疾病之一。与糖尿病相关的慢性并发症如心脑血管疾病、糖尿病肾病、糖尿病视网膜病变以及糖尿病神经病变是致死、致残的主要原因,严重威胁人类健康。胰岛素抵抗(insulin resistanse, IR)是肥胖和2型糖尿病早期和重要的发病机制,其定义为由于肝脏、脂肪和肌肉等靶组织对胰岛素生物效应的反应性降低。导致胰岛素抵抗的具体机制尚不十分清楚。骨骼肌是机体葡萄糖、脂质摄取和利用的器官,是胰岛素发挥作用的重要组织。遗传因素和环境因素均可诱导IR的发生,动物和人的实验均表明无论是通过脂质灌注,还是慢性的高脂饮食,血中游离脂肪酸(free fatty acids,FFA)升高均可导致胰岛素抵抗,而且不同类型脂肪酸作用不同,而饱和脂肪酸尤为重要,高脂是诱发IR的独立危险因素之一。长期高脂饮食或短期脂肪酸输入导致人或动物骨骼肌线粒体相关蛋白表达变化和功能异常。老年人、2型糖尿病患者、2型糖尿病一级亲属的骨骼肌线粒体代谢能力下降,而且胰岛素抵抗个体线粒体代谢的标志物的表达已经发生改变、出现线粒体功能下降,导致肥胖和脂质堆积,所以线粒体代谢的标志物在胰岛素抵抗和2型糖尿病发病机制中扮演重要作用。由此可见,骨骼肌线粒体功能紊乱可能是导致IR的一个重要原因,但具体机制尚不清楚。老年人易发生的IR可能与肌肉细胞内的线粒体作用减退或缺陷有关。过氧化物酶体增生物激活受体γ共激活因子1α(peroxisome proliferator-activated receptorγcoactivator 1α,PGC-1α),是一种核转录共激活因子,可调节线粒体生物发生、糖代谢、脂肪酸氧化、胰岛素分泌等诸多生物过程。线粒体融合蛋白2(mitofusin 2,Mfn2),不仅促进线粒体的融合,维持线粒体结构完整性,而且还参与线粒体的代谢调节。核呼吸因子-1(nuclear respiratory factor-1,NRF-1)是一种由核基因组编码的,对线粒体基因组表达的复制转录和翻译过程中重要的酶及蛋白因子起调控作用,对维持线粒体结构与功能发挥重要作用。PGC-1α作为转录共激活因子可以调节Mfn2的表达和NRF-1的转录。因此,骨骼肌线粒体相关因子PGC-1α、Mfn2和NRF-1的表达异常可能导致骨骼肌线粒体形态和功能的改变,其中PGC-1α在线粒体功能和胰岛素抵抗中的作用成为近年来研究的热点。研究发现高脂引起骨骼肌线粒体相关蛋白PGC-1α,Mfn2、NRF-1表达下降,同时伴有胰岛素信号通路相关蛋白的表达异常及胰岛素抵抗,说明骨骼肌线粒体功能和胰岛素抵抗关系密切,而且PGC-1α可能在调控线粒体功能和胰岛素抵抗方面发挥重要作用。目前假说认为血中FFA升高或上述各种原因导致的线粒体功能异常均影响了脂肪酸进入线粒体的氧化过程,从而导致长链乙酰辅酶A(long-chain fatty acyl coenzyme A ,LCACoA)的堆积,甘油二酯(diacylglycerol ,DAG)生成增加,激活蛋白激酶C(protein kinase C ,PKC),进而抑制胰岛素受体底物-1(insulin receptor substrate-1 IRS-1)活性,干扰胰岛素信号传导系统,减低葡萄糖的转运,导致胰岛素抵抗的形成。PGC-1α作为重要的核转录共激活因子是否在高脂导致的线粒体功能变化、胰岛素抵抗中发挥重要作用,其机制是什么,目前国外尚无定论,国内鲜有报道。本课题通过高脂饮食喂养老年大鼠,造成胰岛素抵抗模型,分析胰岛素抵抗状态下和罗格列酮(Rosiglitazone,RSG)干预后骨骼肌线粒体相关蛋白PGC-1α,mfn2、NRF-1的表达变化及相关关系,分析高脂饮食喂养老年大鼠线粒体功能和胰岛素敏感性的变化。采用脂肪酸孵育C2C12肌细胞,了解不同类型脂肪酸对骨骼肌PGC-1α表达的影响,寻找细胞水平研究PGC-1α作用机制的模型,并通过药物和小干扰RNA(small interference RNA,siRNA)技术调节肌细胞PGC-1α的表达,分析线粒体相关蛋白和胰岛素信号通路相关蛋白的表达变化,探讨PGC-1α在高脂、线粒体功能和胰岛素抵抗中的作用机制及其上游调控因子,为研发防治胰岛素抵抗和2型糖尿病的药物提供新的作用靶点和理论依据。本实验内容主要包括以下四部分:第一部分高脂诱导老年胰岛素抵抗大鼠骨骼肌线粒体相关蛋白的表达目的:测定高脂饮食喂养及罗格列酮干预老年大鼠的胰岛素敏感性以及骨骼肌PGC-1α、Mfn2和NRF-1的表达变化,探讨高脂与线粒体功能、IR的关系。方法:22-24月龄老年雄性Wistar大鼠40只随机分为2组:老年对照(OC)组16只、老年高脂(OF)组24只, 4-5月龄青年大鼠16只,为青年对照组(YC)。对照组给予基础饲料,热量组成:碳水化合物65.5%,脂肪10.3%,蛋白质24.2%,总热量为348kcal/100g;OF组给予高脂饲料,其热量组成:碳水化合物20.1%,脂肪59.8 %,蛋白质20.1%,总热量为501kcal/100g;给予老年两组大鼠每日等热量投喂饲料,喂养八周。于喂养第四周末心脏采血测空腹血糖(FBG),胰岛素(INS),血清甘油三酯(TG),血清总胆固醇(TC),游离脂肪酸(FFA)和骨骼肌甘油三酯(TGm)的含量。喂养四周末,每组均随机选8只行高胰岛素-正葡萄糖钳夹试验判断胰岛素抵抗情况,判断造模成功后高脂组随机分为2组:高脂组和罗格列酮干预(OR)组,每组8只,除继续给予高脂饲料外OR组给予罗格列酮3mg/kg灌胃,高脂组给予等量生理盐水灌胃,继续喂养四周。实验第八周末,行高胰岛素-正葡萄糖钳夹试验评价各组胰岛素抵抗情况,实验前抽取血样用于测定血清各项指标;实验结束后颈动脉放血处死动物,留取股四头肌标本,-70℃保存。采用逆转录聚合酶链反应(RT-PCR)及Western-blot方法检测骨骼肌PGC-1α、Mfn2以及NRF-1的表达。结果:1各组一般项目的比较:与青年组相比,老年对照组空腹胰岛素、空腹血糖和游离脂肪酸在喂养第八周均升高;与老年对照组比较,老年高脂组空腹胰岛素、空腹血糖、游离脂肪酸、总胆固醇和甘油三酯明显升高;罗格列酮干预组空腹胰岛素、空腹血糖、游离脂肪酸、总胆固醇和甘油三酯与老年高脂组比较均下降,差异均有统计学意义(p<0.05或p<0.01)。2骨骼肌甘油三酯含量变化及葡萄糖输注率:与青年对照组相比,在喂养第四周和第八周,老年对照组骨骼肌甘油三酯含量升高,葡萄糖输注率明显降低,差异有统计学意义(P<0.05);与老年对照组相比,老年高脂组骨骼肌甘油三酯从喂养四周后开始升高,葡萄糖输注率下降,第八周骨骼肌甘油三酯含量明显高于第四周,葡萄糖输注率明显低于第四周,差异有统计学意义(P<0.01)。与老年高脂组相比,罗格列酮组骨骼肌甘油三酯含量下降,葡萄糖输注率升高,差异有统计学意义(P<0.01)。3各组大鼠骨骼肌PGC-1α、Mfn2的蛋白表达:在8周末,与青年对照组比较,老年对照组骨骼肌PGC-1α和Mfn2的蛋白表达均下降,差异有统计学意义(P<0.01);老年高脂组较老年对照组下降,差异有统计学意义(P<0.01);罗格列酮干预组PGC-1α和Mfn2的蛋白表达均较老年高脂组升高,但仍低于老年对照组,组间差异有统计学意义(P<0.01)。4各组大鼠骨骼肌PGC-1α、Mfn2以及NRF-1的mRNA表达:与青年对照组比较,老年对照组骨骼肌PGC-1α、Mfn2以及NRF-1的mRNA表达均下降,差异有统计学意义(P<0.01);高脂组较老年对照组下降,差异有统计学意义(P<0.01);罗格列酮干预组PGC-1α、Mfn2以及NRF-1 mRNA表达均较高脂组升高,但仍低于老年对照组,组间差异有统计学意义(P<0.01)。5相关结果表明:相关结果表明,葡萄糖输注率与胰岛素、游离脂肪酸和骨骼肌甘油三酯明显负相关(γ值分别为-0.4931、-0.5825、-0.4270,P值分别为0.0376,0.0112,0.0398。骨骼肌PGC-1α与Mfn2、NRF-1呈正相关,γ值分别为0.4931、0.532,P值为0.0076、0.002。结论:1.高脂饮食喂养老年大鼠,血FFA升高,骨骼肌甘油三酯升高,葡萄糖输注率下降,成功造成胰岛素抵抗模型。2.高脂饮食喂养老年大鼠骨骼肌PGC-1α以及线粒体蛋白Mfn2和NRF-1表达下降。3.高脂饮食喂养的老年大鼠,其骨骼肌PGC-1α与Mfn2、NRF-1之间存在正相关关系。4.罗格列酮干预后,大鼠胰岛素敏感性增强,骨骼肌PGC-1α、Mfn2和NRF-1表达升高,验证了PGC-1α与线粒体功能、IR的密切关系。第二部分不同脂肪酸对C2C12肌细胞PGC-1α表达的影响目的:采用不同类型长链脂肪酸分别培养小鼠C2C12肌细胞,测定其PGC-1αmRNA和蛋白的表达,在细胞水平探讨脂肪酸类型对骨骼肌PGC-1α的影响,为进一步研究PGC-1α与高脂、线粒体功能、胰岛素抵抗的关系打基础。方法:用含10 %新生小牛血清的DMEM培养C2C12肌细胞,接种于6孔板中,培养24h后换为无血清培养基,分别加入软脂酸组(C16:0)、硬脂酸组(C18:0)、棕榈酸组(C16:1)、油酸组(C18:1)以及亚油酸组(C18:2)孵育,均设置0.25mM、0.5 mM和0.75 mM三个浓度梯度,继续培养24h。采用实时荧光定量PCR及Western-blot方法检测细胞中PGC-1α的表达。结果:1不同浓度长链脂肪酸对C2C12细胞PGC-1α表达的影响:在0.25mM浓度时,软脂酸组、硬脂酸组、棕榈酸组、油酸组以及亚油酸组肌细胞PGC-1αmRNA和蛋白表达较对照组差异无统计学意义(P> 0.05);在0.5 mM和0.75 mM浓度时,软脂酸组肌细胞PGC-1αmRNA和蛋白表达较对照组降低(P <0.05);硬脂酸组、棕榈酸组、油酸组、亚油酸组PGC-1αmRNA和蛋白表达与对照组相比差异无统计学意义(P>0.05)。2软脂酸培养的C2C12细胞不同时间PGC-1α表达变化:PGC-1αmRNA和蛋白表达在1h与0h比较差异无统计学意义(P>0.05),而在培养6h、12h和24h的PGC-1αmRNA和蛋白表达较0h降低,其中24h降低最明显,差异有统计学意义(P <0.01)。结论:1软脂酸孵育的C2C12肌细胞PGC-1α的mRNA和蛋白表达下降,并且有浓度和时间依赖性。2单不饱和或多不饱和脂肪酸培养的C2C12肌细胞对PGC-1α表达无明显影响。第三部分C2C12肌细胞PGC-1α表达对线粒体相关蛋白和胰岛素信号通路蛋白的影响目的:通过二甲双胍、罗格列酮和AMPK激动剂(AICAR)孵育C2C12细胞,筛选上调PGC-1α效率最高的药物,分别采用药物上调和siRNA抑制PGC-1α的表达,分析PGC-1α表达变化对粒体功能相关蛋白和胰岛素信号通路蛋白的影响,探讨PGC-1α在线粒体功能紊乱、IR中的作用。方法:细胞培养与传代同第二部分。C2C12细胞分别以2mmol/l二甲双胍、2mmol/l罗格列酮及10μmmol/l AMPK激动剂(AICAR)孵育30 min,然后加入0.5 mM的C16:0软脂酸孵育24h,收取各组细胞并测定PGC-1α的表达。通过上述方法筛选出上调PGC-1α效率最高的药物,采用此药物上调PGC-1α(分对照组、软脂酸组和药物干预组)测定PGC-1α、Mfn2、NRF-1、IRS-1以及GLUT4的蛋白表达。将C2C12细胞转染PGC1α的特异性siRNA(PGC1α-siRNA组)或无关序列对照siRNA(NS-siRNA组)6小时后以10μmmol/l AICAR孵育细胞48h,并设阴性对照组、AICAR组做为比较,分别测定PGC-1α、Mfn2、NRF-1、IRS-1以及GLUT4的蛋白表达。结果:1药物对软脂酸孵育的C2C12细胞PGC-1α表达的影响:二甲双胍、罗格列酮和AICAR组与对照组比较均使肌细胞PGC-1α蛋白表达升高,但以AICAR组最明显,差异有统计学意义(P <0.01),所以接下来选用AICAR作为上调药物研究其它蛋白的表达。2应用AICAR上调C2C12细胞PGC-1α表达后线粒体相关蛋白的变化:软脂酸组PGC-1α蛋白表达较对照组低,AICAR组较软脂酸组、对照组升高;软脂酸组Mfn2、NRF-1蛋白的表达与对照组相比均降低,差异有统计学意义(P <0.01);AICAR干预组Mfn2蛋白表达较软脂酸组升高(P <0.01),较对照组无统计学差异(P >0.05),NRF-1蛋白表达较软脂酸组升高(P <0.01),但较对照组仍低(P <0.05)。3应用AICAR上调C2C12细胞PGC-1α后胰岛素信号通路相关蛋白表达的变化:软脂酸组IRS-1、GLUT4的蛋白表达较对照组降低,差异有统计学意义(P <0.01),AICAR干预组IRS-1和GLUT4的蛋白表达较软脂酸组升高,但较对照组低,差异有统计学意义(P <0.01)。4应用siRNA下调C2C12细胞PGC-1α后线粒体相关蛋白的表达变化:AICAR组和NS-siRNA组PGC-1α表达较对照组升高,差异有统计学意义(P <0.01),AICAR组和NS-siRNA组相比表达无明显变化,PGC-1α-siRNA组PGC-1α表达较其余三组均下降(P <0.01);AICAR组和NS-siRNA组Mfn2和NRF-1的蛋白表达均较对照组增加,差异有统计学意义(P <0.01),NS-siRNA组与AICAR组比较无统计学差异(P >0.05),PGC-1α-siRNA组Mfn2、NRF-1蛋白表达与对照组无差异,较AICAR组和NS-siRNA组明显下降,差异均有统计学意义(P <0.05或P <0.01)。5应用siRNA下调C2C12细胞PGC-1α后胰岛素信号通路蛋白的表达变化:IRS-1蛋白表达在PGC-1α-siRNA组较对照组、AICAR组和NS-siRNA组降低,差异均有统计学意义(P <0.05),在对照组、AICAR组和NS-siRNA组比较无统计学差异(P >0.05);GLUT4的蛋白表达在AICAR组和NS-siRNA组均较对照组增加(P <0.01),PGC-1α-siRNA组与对照组比较无明显变化(P >0.05),与AICAR组和NS-siRNA组比较明显下降(P <0.01)。结论:1.二甲双胍、罗格列酮以及AICAR干预均可以使软脂酸孵育的C2C12细胞PGC-1α表达上调,而AICAR的上调作用最明显,二甲双胍、罗格列酮以及AICAR可能逆转了软脂酸对C2C12的作用。2.药物上调或siRNA下调C2C12细胞PGC-1α的表达同时伴有线粒体相关蛋白以及胰岛素信号通路蛋白的变化,表明PGC-1α表达下降与线粒体功能异常及IR密切相关。3.高浓度软脂酸诱导的线粒体功能下降由PGC-1α介导。第四部分MAPKs信号通路对C2C12肌细胞PGC-1α表达的调控作用目的:测定软脂酸培养的C2C12细胞ERK,p38MAPK(p38)和JNK的表达水平,并用p38MAPK抑制剂(SB203580)干预软脂酸培养的C2C12细胞,测定PGC-1α的表达变化,揭示MAPKs信号通路与PGC-1α表达的关系,寻找软脂酸诱导C2C12细胞PGC-1α表达变化的上游调节通路。方法:细胞培养与传代同第二部分。以0.5 mM的C16:0软脂酸孵育C2C12细胞,分别于0h、0.5h、1 h、6 h和12 h收取细胞检测PGC-1α和ERK、JNK、p38及其磷酸化蛋白的表达水平。用p38MAPK抑制剂干预后测定PGC-1α、P38及p-P38的表达(分为对照组,软脂酸组,p38MAPK抑制剂组,软脂酸+p38MAPK抑制剂组)。肌细胞ERK、JNK、p38和PGC-1α蛋白表达用Western-blot方法检测。结果:1软脂酸培养的C2C12细胞ERK和JNK的表达:ERK和P-ERK以及JNK和P-JNK的表达在1h、6h、12h以及24h较0h无明显变化,其差异均无统计学意义(P >0.05)。2软脂酸培养的肌细胞p38MAPK的表达:p38MAPK的表达在1h、6h、12h以及24h较0h无明显变化,其差异无统计学意义(P >0.05)。P-p38MAPK的表达在1h、6h、12h以及24h逐渐升高,组间两两比较差异均有统计学意义(P <0.05)。3用p38MAPK抑制剂干预C2C12细胞后PGC-1α的表达:PGC-1α的表达在软脂酸组与其它三组比较最低(P <0.01),p38MAPK抑制剂组和软脂酸+p38MAPK抑制剂组较软脂酸组升高(P <0.01),p38MAPK抑制剂组最高(P <0.01)。结论:1.在软脂酸培养的不同时间(0-12h)的C2C12细胞的p38MAPK表达无变化,而P- p38MAPK表达逐渐升高;2.在软脂酸培养的不同时间(0-12h)的C2C12细胞ERK、P-ERK以及JNK、P-JNK表达无明显变化;3.用p38MAPK抑制剂干预软脂酸培养的C2C12细胞,PGC-1α表达较加入抑制剂前明显升高,表明软脂酸诱导的的PGC-1α表达下降可能由p38MAPK调控。更多还原

【Abstract】 In the recent years, the prevalence of type 2 diabetes is increasing quickly, estimated at 3 5000 billion suffers worldwide by the year 2030.The death toll of diabetes just follows cardiovascular disease and malignant tumor, thus called“the third killer”. The number of diabetics in China ranks No. 2 in the world. Epidemiologic data indicate that the rate of type-2 diabetes increases sharply with aging, being one of the main diseases affecting the health of the middle-aged and the old. Some chronic complications related to diabetes, such as cardiovascular disease, diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy are main reasons causing death or deformity, seriously affecting the health of mankind. Insulin resistance (IR), defined as the decreased reactivity of the target tissues such as liver, fat, and muscles to the bio-effect of insulin, is the early-stage and important mechanism of obesity and type 2 diabetes. However, the specific mechanism of IR is not clear. Yet we do know that skeletal muscles (SM) are the place where glucose and lipid are taken in and utilized, and where insulin plays its important role.Genetic and environmental factors both can induce IR. The human and animal experiments all indicate that the increased free fatty acids (FFA), caused either by lipid perfusion or by chronic high-lipid diet, can result in IR, the functions of the fatty acids differ, with the saturated fatty acids the most important, and high lipid is the independent risk factor triggering IR. Long time high-lipid diet and transient fatty acids input both can cause the changed expression and dysfunction of the mitochondria-related proteins of the skeletal muscles in human and animals. In the old, type 2 diabetes, and their first-degree relatives, the metabolic capacity of the SM mitochondria declines, the expression of the landmark, indicating insulin resisting individual mitochondria metabolism, also has changed, with decreased mitochondria function, which leads to obesity and fat accumulating. Accordingly, the landmark of mitochondria metabolism makes a decisive difference in IR and in the pathogenesis of type-2 diabetes. Considering this, the dysfunction of SM mitochondria is the main reason of IR, but the mechanism is not clear yet. In the old, IR is probably associated with decreased function or defect of the mitochondria in the muscle cells.Peroxisome proliferator-activated receptorγco-activator 1α(PGC-1α), a nuclear transcription co-activator, may regulate many bioprocesses, such as mitochondria biogenesis, glycometabolism, fatty acid oxidation, and insulin secretion etc. Mitofusin 2 (Mfn2) can not only promote mitochondria fusion, sustain the completeness of mitochondria,but also be involved in the metabolic regulation. Nuclear respiratory factor-1 (NRF-1), a nuclear transcription factor coded by nuclear genome, regulating the expression of respiratory chain subunits and the transcription and duplication of mitochondria DNA, serves to regulate the key enzymes and protein factors in the process of duplication, transcription, and translation of the mitochondria genome expression; meanwhile, NRF-1 plays an important role in sustaining mitochondria morphologically and functionally. As the transcription co-activator, PGC-1αcan regulate Mfn2 expression and NRF-1 transcription. Therefore, the abnormal expression of the landmarks of mitochondria metabolism such as SM PGC-1α, Mfn2 and NRF-1 can cause the change of SM mitochondria morphologically and functionally, of which the roles of PGC-1αin the mitochondria function and IR has become the focus of study in the recent years.Some researches found out that high lipid can induce decreased expression of SM mitochondria-related proteins, such as PGC-1α, Mfn2, and NRF-1, meanwhile accompanied by abnormal expression of the insulin signal pathway-related proteins and IR, demonstrating that SM mitochondria function and IR are closely related and PGC-1αmay play an important role in regulating the functions of mitochondria and IR. The current hypothesis is that the increased FFAs in the blood or the dysfunctions of mitochondria, caused by any of the above mentioned reasons, affect the oxidation process of fatty acid entering mitochondria, and as a result, cause accumulated long-chain fatty acyl coenzyme A (LCACoA), increased genesis of diacylglycerol (DAG), and inactivated protein kinase C(PKC),and then restrain the activity of insulin receptor substrate-1 (IRS-1), finally interfere with the insulin signal conduction system, reduce the transportation of glucose, and lastly lead to IR. As to the functions of PGC-1α, the important nuclear transcription co-activator, in the changed function of mitochondria caused by high lipid and in IR, and as to the mechanism, there is no definite answer and the relative reports are rarely seen.After establishing IR models by feeding the old-aged rats with high lipid diet, we aim to analyze the expression changes of SM mitochondria-related proteins, such as PGC-1α, Mfn2, and NRF-1, and the correlation in the condition of IR and after being interfered with Rosiglitazone(RSG), and to analyze the changes of mitochondria functions and insulin sensitivity in the old-aged rats fed with high lipid diet. At the same time, muscle cells C2C12 were incubated with fatty acids to comprehend the effects of different types of acids on the expression of PGC-1αin skeletal muscles, with the purpose of finding the model of studying PGC-1αmechanism on cellular level. Meanwhile, after regulating the expression of PGC-1αthrough drugs and small interference RNA (siRNA) technique, we are to analyze the expression changes of mitochondria-related proteins and insulin signal pathway-related proteins, with the aim to explore the action mechanism of PGC-1αin the condition of high lipid, mitochondria function, and IR, and finally to provide novel target in developing drugs used to prevent IR and type 2 diabetes and ultimately to offer theoretical foundation. This experiment consists of the following 4 parts.Part I The expression of mitochondria-related proteins in skeletal muscles and of in the old-aged insulin resistant rats with high-fat-fedObjectives: To assay insulin insensitivity and the expression changes of PGC-1α, Mfn2 and NRF-1 in the skeletal muscles of the rats fed with high lipid and interfered with RSG, and to explore the correlation among high lipid, mitochondria function, and IR.Methods: Forty Wistar rats, male, ages 22-24 months, were divided into 2 groups: Old Control (OC) with 16 and Old Fat (OF) with 24; another 16 young rats ages 4-5 months were used as Young Controls (YC). The controls were given basal feed, with the calories composition of carbohydrate 65.5%, fat 10.3%, and protein 24.2%, totaling 348kcal/100g. OF members were given high-lipid feed, with the calories composition of carbohydrate 20.1%, fat 59.8 %, and protein 20.1%, totaling 501kcal/100g. The two old groups were given the feed with the same amount of calories daily lasting 8 weeks, and weighed once per week. At the end of week 4, blood samples from heart were collected to determine the amount of fasting blood-glucose (FBG), insulin (INS), serum triglyceride (TG), serum total cholesterol (TC), and FFA. Meanwhile, 8 rats were randomly selected from each group to be given the oral glucose tolerance test(OGTT)to evaluate the glucose tolerance, and then to be administered hyperinsulinemic euglycemic clamp technique (HGCT) to evaluate the condition of IR. After the models were established, HF members were divided into 2 subgroups: high fat and Rosiglitazone interference( OR group), with 8 in each. In addition to high lipid feed, Rosiglitazone interference members were given RSG 3mg/kg through intragastric administration, while OF done with the same amount of physiological saline. After another 4 weeks’feeding, at the end of week 8, blood samples were collected again to determine the serum indexes, and OGTT and HGCT were done to evaluate IR in each group. Afterwards, the animals were executed at the carotid. Quadriceps femoris and fatty tissues were taken out to be frozen in the liquid nitrogen and preserved in the cryogenic refrigerator of -70℃. The expression of PGC-1α, Mfn2 and NRF-1 mRNA and protein was measured by semiquantitative PCR and Western blot method.Results: 1. The comparison of the general items in each group. Compared with those in YC, fasting insulin (FINS), FBG, and FFA were higher in the old groups. Compared with OC group, FINS, FBG, FFA, TG, and TG in OF group increased obviously, and those in OR group decreased compared with those in OF, with statistical significance in both (p<0.05 or p<0.01).2. The amount changes of SM TG and the glucose infusion rate. Compared with YC, at week 4 and 8, the amount of SM TG in OC increased and the glucose infusion rate decreased, with statistical significance (P<0.05). Compared with OC, in OF group, SM TG began to increase after 4 week feeding, and that in week 8 was apparently higher than that at week 4, and the infusion rate decreased markedly compared with that in week 4, with statistical significance (P<0.01). Compared with OF, SM TG in OR decreased and infusion rate increased, with statistical significance (P<0.01).3. The expression of SM PGC-1αand Mfn2. Compared with YC, the expression of SM PGC-1αand Mfn2 in OC decreased, with statistical significance (P<0.01), and those in OF decreased than those in OC, with statistical significance (P<0.01). The expression in OR was higher than that in OF, but still lower than that in OC, with statistical significance between groups (P<0.01)4. The expression of SM PGC-1α, Mfn2 and NRF-1 mRNA of Compared with YC, the expression of SM PGC-1α, Mfn2 and NRF-1 mRNA in OC decreased, with statistical significance (P<0.01); and those in OF decreased than those in OC, with statistical significance (P<0.01). The expressions in OR were higher than those in OF, but still lower than those in OC, with statistical significance between groups (P<0.01).5. Results indication: The results demonstrated that glucose infusion rate showed obvious negative correlation with INS, FFA, and SMTG (γ=-0.4931, -0.5825, -0.4270, respectively; and P=0.0376, 0.0112, 0.0398, respectively) SM PGC-1αshowed positive correlation with Mfn2 and NRF-1 (γ=0.4931,0.532; P=0.0076, 0.002 respectively).Conclusions: 1. IR animal models are successfully made after feeding the old-aged rats with high-lipid diet, followed by FFA increase in the blood, SM TG increase, and glucose infusion rate decrease. 2. The expressions of SM PGC-1αand mitochondria-related factors Mfn2 and NRF-1 decrease after the old-aged rats being fed with high-lipid diet. 3. There exists positive correlation between SM PGC-1αand mitochondria-related factors Mfn2 and NRF-1. 4. The INS sensitivity heightens in the rats interfered with RSG, and the expression of SM PGC-1αand mitochondria-related factors Mfn2 and NRF-1, shows up-regulation, testifying the close relationship between PGC-1αand mitochondria function and IR.Part II The effects of fatty adics on the expression of PGC-1αin muscle cell C2C12Objectives: To explore on cellular level the effects of different FAs on the expression SM PGC-1α, after the muscle cells C2C12 of the mice were incubated with different types of fatty adics and the expressions of PGC-1αmRNA and proteins assayed; and ultimately to lay groundwork for studying the relation between PGC-1αand high lipid, mitochondria function and IR.Methods: DMEM in the New-born calf’s serum was used to incubate muscle cell C2C12 in the 6-well plates. After 24 hours, the cells were incubated in the serum free medium, and grouped into palmitic acid (C16:0), stearic acid (C18:0), palmic acid(C16:1), oleic acid(C18:1), and linoleic acid (C18:2) after the related acids were added in, with 3 concentration gradients of 0.25mM,0.5mM and 0.75mM. After another 24 hours’incubation, the expression of PGC-1αwas determined by real-time fluorescent quantitative PCR and Western-blot.Results: 1. The effects of fatty adics with different concentrations on the expression of muscle cell PGC-1αmRNA and proteins. At concentration 0.25mM of FA, compared with the control group, there showed no statistical significance of the expression of muscle cell PGC-1αmRNA and proteins in the groups of palmitic acid, stearic acid, palmic acid, oleic acid, and linoleic acid (P> 0.05). At concentration 0.5mM and 0.75mM, the expressions in the palmitic acid group were lower than those in the control group(P <0.05), while the expressions in stearic acid ,palmic acid, oleic acid, and linoleic acid groups and those in the control group showed no statistical significance (P>0.05). 2. The expressions PGC-1αmRNA and proteins at different times in the muscle cells incubated with palmitic acid. As to the expressions, there showed no statistical significance in 0h and in 1h(P>0.05), while the expressions were lower at 6h, 12h, and 24h, compared with those in 0h and 1h, with the expression decreased most apparently in 24h, with statistical significance(P <0.01).Conclusions: The expression level of PGC-1αin the muscle cell C2C12 incubated with saturate fatty acids decreases, the level depending on concentration and time. 2. The muscle cell C2C12 incubated with monounsaturated and polyunsaturated fatty acids has no distinct effect on the expression level of PGC-1α.Part III The effects of SM PGC-1αexpression on mitochondria protein and insulin signal pathway in C2C12 cellObjectives: To analyze the functions of PGC-1αin mitochondria dysfunction and in IR induced by high lipid, using metformin, RSG, and agonist AMPK to up-regulate the expression level of PGC-1αin muscle cell C2C12, and using siRNA to restrain.Methods: Culturing and sub-culturing methods are the same as used in Part II. After C2C12 was incubated in the culture of C16:0 palmitic acid, with the concentration of 0.5 mM, 2mmol/l metformin, 2mmol/l RSG, and 10μmmol/l AMPK agonist (AICAR) were administered respectively to incubate the cells for another 24 hours, then PGC-1αexpression level was assayed. Then C2C12 was transfected with PGC1-α-siRNA or NS-siRNA. Six hours later, the cells were incubated with 10μmmol/l AICAR for 48 hours, followed by assay of the expression levels of PGC-1α, Mfn2, NFR-1, IRS-1 and GLUT4 mRNA and proteins. The cells interfered by AICAR were divided into groups of control, palmitic acid, and AICAR; and the siRNA group was subdivided into groups of control, AICAR (AICAR), AICAR+NS-siRNA (NS-siRNA), and AICAR+PGC1α-siRNA (PGC1α-siRNA).Results: 1. The effects of drugs on the expression of PGC-1αin muscle cell C2C12 incubated with palmitic acid. Metformin, RSG and AICAR all up-regulated the expressions of PGC-1αprotein and mRNA, compared with the condition in control group, with statistical significance (P <0.01).2. The effects of muscle cells incubated with palmitic acid and interfered with AICAR on the expression of mitochondria-related proteins. The expression levels of PGC-1α, Mfn2, NRF-1 mRNA and proteins decreased in the palmitic acid group compared with those in the controls, the difference with statistical significance (P <0.01). In addition, the above mentioned factors’mRNA and proteins in the AICAR group showed higher levels than those in the control and palmitic acid groups, also with statistical significance (P <0.01).3. The effects of muscle cells incubated with palmitic acid and interfered with AICAR on the expression of insulin signal pathway-related proteins. The expression levels of IRS-1 and GLUT4 mRNA and proteins in the palmitic acid group decreased compared with those in the control group, with statistical significance (P <0.01). Furthermore, IRS-1 and GLUT4 mRNA and proteins in the AICAR group showed higher expression levels than those in the control and palmitic acid groups, also with statistical significance (P <0.01).4. The effects of down-regulating PGC-1αexpression level in muscle cell C2C12 by using siRNA on the expression of mitochondria–related proteins. The expression levels of PGC-1α, Mfn2, NRF-1 mRNA and proteins increased in the AICAR group compared with those in the controls, the difference with statistical significance (P <0.01). The above mentioned factors’mRNA and proteins in the NS-siRNA group showed higher levels than those in the controls, also with statistical significance (P <0.01), no obvious difference with those in AICAR group (P >0.05). The indicators used above in PGC-1α-siRNA group showed distinct decrease than those in the other 3 groups, with statistical significance (P <0.01).5. The effects of down-regulating PGC-1αexpression level in muscle cell C2C12 by using siRNA on the expression of insulin signal pathway–related proteins. The expression levels of IRS-1 and GLUT4 mRNA and proteins increased in the AICAR group compared with those in the controls, the difference with statistical significance (P <0.01). Meanwhile, the expression levels of IRS-1 mRNA and proteins increased in the NS-siRNA group compared with those in the controls, the difference with statistical significance (P <0.01), no obvious difference with those in AICAR group (P >0.05). Additionally, the expression levels of IRS-1 and GLUT4 mRNA and proteins in PGC-1α-siRNA group showed apparent decrease compared with the other 3 groups, with statistical significance (P <0.01).Conclusions: 1. Metformin, RSG, and AICAR interfering can all up-regulate PGC-1αexpressions in C2C12 incubated with high-concentrated palmitic acid , indicating all the three drugs can reverse the function of palmitic acid on C2C12. 2. Up-regulating PGC-1αexpression level in C2C12 with drugs or down-regulating with siRNA will be accompanied by the changes of mitochondria–related and insulin signal pathway–related proteins, suggesting PGC-1αdecrease can cause mitochondria dysfunction and IR. 3. Mitochondria dysfunction and IR induced by high-concentrated palmitic acid are mediated by PGC-1α.Part IV The regulation of MAPKs pathway on PGC-1αexpression in C2C12 cellObjectives: To reveal the relation between MAPKs signal transduction pathway and PGC-1αexpression, and to investigate the up-stream regulatory pathway of the changed PGC-1αexpression in muscle cells induced by palmitic acid, by assaying the expression levels of Erk, p38, and JNK in palmitic acid-incubated C2C12.Methods: Culturing and sub-culturing methods were the same as used in Part II. After C2C12 was incubated in the culture of palmitic acid, with the concentration of 0.5 mM, the cells were collected at 0h, 0.5h, 1h, 6h, and 12h to determine the expression levels of PGC-1α, ERK, JNK, p38, and the phosphorylased proteins. After being interfered with inhibitor p38MAPK, the expression levels of PGC-1α, P38, and p-P38 were assayed, and the cells were divided into groups of control, palmitic acid, inhibitor p38MAPK, and palmitic acid + inhibitor p38MAPK.Results: 1. The expressions of ERK and JNK in muscle cells C2C12 incubated with palmitic acid. There showed no obvious change of ERK, P-ERK, JNK, and P-JNK at 0h, 1h, 6h, 12h, and 24h, with no statistical significance (P >0.05). 2. The p38MAPK expression in muscle cells incubated with palmitic acid. There showed no marked change of p38MAPK at 0h, 1h, 6h, 12h, and 24h, with no statistical significance (P >0.05). However, the expression of p38MAPK increased gradually at 0h, 1h, 6h, 12h, and 24h, all with statistical significance in any two groups (P <0.051). 3. The PGC-1αexpression in muscle cells C2C12 interfered with inhibitor p38MAPK. The expression level in the p38MAPK group was the highest, with statistical significance compared with the other 3 groups (P <0.05 or P <0.01). There showed increased expression level in group palmitic acid + inhibitor p38MAPK than those in control and palmitic acid groups, with statistical significance (P <0.05), with higher level in control group than that in palmitic acid group, but without statistical significance (P>0.05).Conclusions: 1. The expression levels of p38MAPK and P- p38MAPK in muscle cells increase gradually in the different time span incubated with palmitic acid. 2. There show no obvious changes of the expression of ERK, P-ERK, JNK, and P-JNK. 3. After the muscle cells incubated with palmitic acid are interfered with inhibitor p38MAPK, the expression level increases markedly compared with that before inhibitor adding, indicating the changed expression of PGC-1αinduced by palmitic acid may be regulated by p38MAPK.更多还原