【作者】 乔海荣；

【导师】 张勇；

【作者基本信息】 天津体育学院 ， 运动人体科学， 2010， 硕士

【摘要】 目的:通过高脂饲料喂养C57BL/6小鼠以建立胰岛素抵抗(IR)动物模型,再进行有氧运动干预,观察骨骼肌线粒体解偶联蛋白3(UCP3)、线粒体呼吸功能和游离脂肪酸(FFA)含量等,进而探讨UCP3在IR发生中的作用,与线粒体功能的关系,有氧运动对骨骼肌线粒体UCP3的表达和线粒体功能的影响,并探究其机制。方法:1)IR动物模型建立:8周龄雄性C57BL/6小鼠80只,经过适应性喂养一周,随机分为正常膳食和高脂膳食组,分别饲以正常饲料和高脂饲料。喂养8周后,检测相关指标以鉴定IR模型是否成功。待模型建立后,再次将正常膳食组随机分为正常膳食安静组(NC)和运动组(NE);高脂膳食组随机分为高脂膳食安静组(HC)和高脂膳食运动组(HE);2)运动方案:各运动组小鼠进行为期8周的12米/分(75% VO2max)强度的跑台训练,1次/天,60分钟/次,5天/周。3)指标检测:8周运动后检测空腹血糖(FBG)、空腹血清胰岛素(FINs)、态3、态4呼吸速率、ATP酶合成活力,骨骼肌游离脂肪酸(FFA)、线粒体UCP3和GLUT4表达以及骨骼肌油红O染色观察脂质沉积。结果:1)FBG,FINs和HOMA-IR的变化:HC组FBG、FINS和HOMA-IR显著高于NC组和NE组(p<0.01);HE组FBG、FINS和HOMA-IR显著低于HC组(p<0.01),但仍显著高于NC组和NE组(p<0.01),高脂膳食和运动均对FBG、FINS和HOMA-IR有影响,并存在交互作用。2)线粒体呼吸功能的变化:HC与NC组相比小鼠骨骼肌线粒体态4呼吸显著升高(p<0.05),态3呼吸和RCR显著降低(p<0.01);NE较NC组线粒体态3呼吸和呼吸控制比(RCR)显著升高(p<0.01),态4呼吸有下降趋势但无显著性;HE线粒体态3呼吸和RCR显著高于HC组(p<0.01),态4呼吸有下降趋势但无显著性。3)线粒体ATP酶合成活力: NE组小鼠骨骼肌线粒体ATP酶合成活力显著高于NC组(p<0.05);HC组显著低于NC组(p<0.01);HE组显著高于HC组(p<0.01),与正常膳食NC组差异无显著性。4)骨骼肌线粒体UCP3 mRNA和蛋白的表达:HC组小鼠骨骼肌线粒体蛋白UCP3 mRNA的表达NC组比显著升高了41.78%(p<0.01)UCP3蛋白表达显升高了69.47%;NE组与NC组相比mRNA和蛋白分别降低了25.37%和25.03%(p<0.01);HE组UCP3 mRNA比HC降低了将近100%(p<0.01),蛋白降低56.64%(p<0.01)。5)骨骼肌GLUT4 mRNA和蛋白的表达:HC组骨骼肌GLUT4 mRNA表达比NC组低49%(p<0.01),蛋白表达降低42.71%(p<0.01);NE组骨骼肌细胞GLUT4 mRNA的表达比NC组高8%(p<0.05),蛋白表达却比NC组高73.81%(p<0.01);HE组骨骼肌GLUT4 mRNA表达比HC组高40%(p<0.01),蛋白表达高36.9%(p<0.01)。6)骨骼肌游离脂肪酸: HC组骨骼肌游离脂肪酸显著高于NC组(p<0.01);各运动组显著低于对照组(p<0.01);7)UCP3蛋白与RCR、态4、骨骼肌FFA的相关性分析:RCR与UCP3蛋白水平呈显著负相关;UCP3与态4和骨骼肌FFA水平分别呈显著正相关。8)形态学检测:油红O染色发现HC组的脂质沉积较多。结论:1)长期高脂膳食可诱导小鼠IR,表现为HOMA-IR升高并伴有骨骼肌脂质堆积。2)长期高脂负荷所致IR小鼠骨骼肌线粒体态4呼吸增加,诱导呼吸链解偶联,下调RCR,呼吸功能降低;高脂状态下UCP3表达的上调,可能与代偿性增加FFA代谢,对抗IR的发生与发展有关。3)长期有氧耐力训练可减少IR小鼠骨骼肌线粒体态4呼吸,增加线粒体能量转换偶联,提高线粒体呼吸功能;下调的骨骼肌UCP3表达,反映骨骼肌线粒体功能的适应能力增加,加速高脂状态下糖脂代谢速率,延缓IR的发生与发展。更多还原

【Abstract】 Objective: High-fat feed C57BL/6 mice to induce animal model of insulin resistance, then carried out program of aerobic exercise intervention, observe the changes of mitochondrial uncoupling protien 3 expression ,mitochondrial respiratory function, free fatty acid content in skeleton muscle. Explore role of UCP3 in mechanism of IR occurance and in the aerobic exercise prevent IR.Methods: 1)Creating mouse model of insulin resistance: First, 80 male, 8-week old C57BL/6 mice were divide into normal chow group and high-fat diet group randomly, fed by normal and high fat diet respectively. 8 weeks later, The normal chow group was randomly divided into normal chow diet contorl (NC) and normal chow exercise group (NE), meanwhile mice from high fat diet groups which have insulin resistance continued to be fed with high-fat diet and randomly divided into high-fat diet control (HC) and high-fat diet exercise training group (HE) during the subsequent 8-week period. 2) Exercise protocol: Exercise training consisted of motorized treadmill running for 1 hour/day, 5days/week at an intensity of 75%VO2max. 3) Measurement of Indicators: after 8-week exercise, measured fasting blood glucose concentration, fasting serum insulin level, mitochondrial respiratory state 3 and state 4 and ATP synthesis enzyme activity. Detected content of FFA, UCP3 and GLUT4 expression in skeletal muscle. observe lipid desposition with Oil red O stain skeletal muscle. Results: 1) Changes of FBG and FINs:The FBG and FINs were altered significantly by either high fat diet or aerobic exercise. 2) Changes in mitochondrial respiratory function: HC compared with NC, mitochondrial state 4 respiration of mice skeletal muscle was significantly higher (p<0.05), state 3 respiration and respiratory control ratio (RCR) were significantly lower (p<0.01). NE and HE compared with respective control groups, state 3 and RCR were significantly increased (p<0.01). 3) Mitochondrial ATP synthesis enzyme activity: ATP synthesis enzyme activity was increased by high-fat feeding (p<0.01) but were decreased by exercise training (p<0.05). 4) The expression of UCP3 mRNA and protien: we found UCP3 mRNA level of HC group was increased by 41.78% than NC group (p<0.01), similarly UCP3 protien level was increased by 69.47% (p<0.01). Two exercise groups were decreased significantly in mRNA and protien. 5) The expression of GLUT4 mRNA and protien: The expression of GLUT4 mRNA and protien were significantly decreased in HC group compared to NC group (espectively decreased 49% and 42.71%, p<0.01). The expression of GLUT4 mRNA and protien of HE group were espectively increased by 40% and 36.9% than HC group. 6) Free fatty acid content: HC group were significantly higher than in skeletal muscle free fatty acid NC group (p<0.01). The exercise group was significantly lower than the control group (p<0.01). 7)Correlation analysis: RCR and UCP3 protien were significantly negative correlation, UCP3 protien and FFA and State 4 were significantly positive correlation. 8) Lipid desposition : Oil red staining, we found lipid desposition in HC was more than NC. Conclusion:1) High-fat can induce IR accomponied higher HOMA-IR and lipid accumulation. 2) Long term high-fat load increase state 4 respiration, induce uncoupling of respiratory chain, reduced RCR, respiratory function was reduced in the mitochondria of mice skeletal muscle; Up-regulation of UCP3 is maybe induced by elevation of FFA, be related to compensatory increase in FFA metabolism, fight IR occurrence and development.3)Long-term aerobic training can reduce skeletal muscle mitochondria state 4 respiration in the IR mice, increased coupling of mitochondrial energy conversion and improve mitochondrial respiratory function; Down-regulation of UCP3 expression reflect the adaptability of skeletal muscle mitochondrial function to accelerate the glucose and lipid metabolism rate under high-fat, delay the occurrence and development of IR.更多还原