【作者】 漆正堂；

【导师】 丁树哲；

【作者基本信息】 华东师范大学 ， 运动人体科学， 2009， 博士

【摘要】 胰岛素抵抗是2型糖尿病、心血管疾病、肥胖症等多种代谢性疾病的前期表现，而线粒体损伤和功能异常又是导致外周组织发生胰岛素抵抗的重要原因。骨骼肌线粒体对长期运动的代谢适应被认为是运动预防冠心病、2型糖尿病和肥胖的关键因素。已有研究认为，骨骼肌线粒体的运动适应对运动方式、运动强度、运动频率和持续时间有着较高的特异性和依赖性。线粒体的运动适应包括线粒体生物发生、线粒体管网融裂、物质代谢与ATP合成等诸多变化。然而，骨骼肌运动适应，尤其是线粒体对具体运动方式发生特异性适应的基因表达机制还鲜为人知，与运动敏感性基因表达有关的调控因子还知之甚少。目的：本研究分别建立以长时间、低强度为特征的耐力训练模型和以短时间、大强度、间歇性为特征的冲刺训练模型，探讨、比较骨骼肌运动敏感性基因在长期训练后的累积性应答特征，期望从基因表达水平阐释骨骼肌线粒体适应不同训练方式的特异性，为运动预防代谢性疾病提供一些科学参考。此外，本研究也建立了一种“以骨骼肌负载降低为主要特征”的负对照生理模型——后肢悬吊，期望以此模型探讨骨骼肌废用性萎缩的有关机理，辅证运动适应的相关结论。方法：清洁级Sprague-Dawley雄性大鼠40只，随机分为4组，即安静组(C，n=10)，后肢悬吊组(D，n=8)，耐力训练组(E，n=10)，间歇性冲刺训练组(S，n=12)。间歇性冲刺训练：每天9-10次10s极量强度(≥42m／min)的跑台运动，间歇时间30-60s；耐力训练：每天30-60min低强度(≤16．7m／min)的持续跑台运动；每周训练6天，训练8周。后肢悬吊组在第1-5周与安静组饲养方式相同，在第6-8周实施悬吊，以悬尾方式保持后肢离地1-2cm。最后一次训练结束后24h，所有大鼠断颈处死。心脏取血，检测血糖、血浆胰岛素、瘦素、甘油三酯；取腓肠肌检测丙酮酸、乳酸的含量以及己糖激酶(HK)、丙酮酸激酶(PK)、苹果酸脱氢酶(MDH)的活性，分离腓肠肌线粒体，检测柠檬酸合成酶(CS)、羟脂酰CoA脱氢酶(HCD)的活性；用实时荧光定量PCR检测腓肠肌GLUT4、AMPKα2、PDK4、CPT-1β、PGC-1α、ERRα、NRF-2α、Mfn1、Mfn2、OPA1、Drp1、Fisl的基因转录水平；用Western blot测定腓肠肌线粒体PDK4、CPT-1β、Mfn2、Drp1的蛋白表达及细胞核PGC-1、ERRα的蛋白表达：用ELISA法(酶联免疫吸附试验)检测腓肠肌线粒体和胞浆的细胞色素c的含量。结果：(1)机体在两种运动方式和后肢悬吊的生理条件下，整体生理调节尚处于稳态范围内，正常生理稳态未受干扰。耐力训练能显著降低血浆甘油三酯，间歇性冲刺训练和后肢悬吊对血浆甘油三酯无显著影响，表明耐力训练与间歇性冲刺训练在此有着不同的运动适应效果。(2)耐力训练与间歇性冲刺训练对静息骨骼肌有着类似的丙酮酸提升效应，但间歇性冲刺训练能提高静息骨骼肌的乳酸水平，说明间歇性冲刺训练很可能使肌纤维在静息时的无氧代谢已经处于活跃状态。耐力训练、间歇性冲刺训练和后肢悬吊对静息骨骼肌HK、PK、MDH、HCD活性没有显著影响，但间歇性冲刺训练导致CS活性增加。(3)健康机体GLUT4 mRNA表达对于耐力训练并不表现敏感。间歇性冲刺训练能诱导GLUT4 mRNA表达上调。耐力训练后骨骼肌AMPKα2 mRNA表达下调，可能是AMPK信号通路活性对耐力训练适应性下调的一种表现。耐力训练对CPT-1的基因转录和蛋白表达均无显著作用，对PDK4也仅仅只在转录水平有下调作用。间歇性冲刺训练在基因转录和蛋白表达两个水平都导致PDK4下调。后肢悬吊与间歇性冲刺训练对PDK4、CPT-1基因表达的调控方向一致。(4)后肢悬吊、耐力训练、间歇性冲刺训练对NRF-2αmRNA表达均无显著作用。PGC-1、ERRα的基因转录和核蛋白表达并不具有同步性。耐力训练和间歇性冲刺训练都可能诱导了PGC-1α、ERRα基因的“转录节省化”效应。PGC-1与ERRα的核蛋白表达具备较高的线性正相关。后肢悬吊对PGC-1α、ERRα、NRF-2α基因转录的调控方向与耐力训练一致；后肢悬吊对PGC-1、ERRα蛋白表达的调控方向与间歇性冲刺训练一致。(5)耐力训练上调Mfn1 mRNA表达，但Mfn2 mRNA以及线粒体Mfn2蛋白表达对耐力训练均表现沉默。间歇性冲刺训练后Mfn2 mRNA转录下调，但线粒体Mfn2蛋白表达上调。间歇性冲刺训练可能增加Mfn2 mRNA及其蛋白的稳定性，导致Mfn2基因“转录节省化”。耐力训练或间歇性冲刺训练并不导致Fisl基因的转录适应性变化。间歇性冲刺训练后Drp1mRNA转录上调，线粒体Drp1蛋白表达下调，但Drp1的基因转录和蛋白表达对耐力训练均表现沉默。(6)后肢悬吊导致胞浆CytC显著增加，但线粒体CytC无显著增减。耐力训练使线粒体和胞浆CytC同步增加，间歇性冲刺训练导致线粒体CytC显著增加，但不导致胞浆CytC显著增加。结论：(1)血糖-胰岛素-瘦素三者之间可能存在相互依赖的稳定性，运动对健康机体三者之间的稳态联系可能有维持作用。间歇性冲刺训练能作为一种省时的运动方式提高骨骼肌有氧代谢能力，但在提高有氧代谢能力的同时也能促进丙酮酸向乳酸的转化。(2)间歇性冲刺训练导致PDK4酶蛋白的下调有可能削弱PDK4对糖氧化的抑制，从而有利于糖原的迅速动员，这对于间歇性冲刺训练的强度特点是一种有利的代谢适应。PGC-1与ERRα可能在细胞核中协同发挥转录激活作用，该激活作用在间歇性冲刺训练组比较显著，这也可能是PGC-1／ERRα转录激活通路对不同训练方式的差异性适应。(3)本实验耐力训练对健康大鼠骨骼肌糖代谢以及线粒体融合的作用并不显著。间歇性冲刺训练能诱导线粒体融合加强，分裂抑制，线粒体Drp1蛋白下调、Mfn2蛋白上调、PDK4蛋白下调可能协同促进线粒体的糖有氧氧化和结构融合。本实验的耐力训练未见此协同效果。间歇性冲刺训练诱导线粒体融合活性显著高于耐力训练。(4)骨骼肌废用性萎缩可能首先通过细胞凋亡减少肌纤维数量，单根肌纤维线粒体的相对密度则不会即刻下降。耐力训练可能使单根肌纤维的线粒体相对密度和氧化磷酸化能力有非常显著的增加。间歇性冲刺训练可能比耐力训练更有利于防止细胞凋亡和肌萎缩、维持肌肉体积，这可能是腓肠肌对间歇性冲刺训练的一种混合型、过渡性的适应特征。(5)面对骨骼肌负荷降低与负荷增加(运动)的不同生理刺激，骨骼肌细胞未必就存在不同的应答机制。本实验的后肢悬吊和间歇性冲刺训练对线粒体系统的影响比耐力训练要大得多，从维持机体的健康稳态看，耐力训练可能是的一种较好的运动方式；从提高线粒体机能、预防线粒体疾病的角度看，间歇性冲刺训练也不失为一种省时的运动方式。更多还原

【Abstract】 The insulin resistance syndrome (IRS)is a trait cluster composed of risk factors forthe future development of cardiovascular disease,obesity and/or type 2 diabetes.Impaired mitochondrial function is related to common insulin resistance in skeletalmuscle.Mitochondrial adaptation to long-term exercise training is redarded as a keyfactor for exercise to prevent cardiovascular disease,obesity and/or type 2 diabetes.Exercise-induced mitochondrial adaptations in muscle are highly specific and aredependent upon the type of exercise (i.e.resistance vs endurance)as well as itsintensity,frequency and duration.These adaptations include changes in mito-biogenesis,mito-fusion/fission,mitochondrial metabolism and ATP production.However,at the present time,most of the mechanisms underlying the mitochondrialadaptation of skeletal muscle to specific exercise still remain to be unclear.Little isknown about the regulatory factors directly modulating the expression ofexercise-responsive genes.Purpose:Exercise programs of endurance training and sprint interval training werefounded in the study,one of the purposes was to determine the molecular difference ofmitochondrial adaptations in muscle between the two training programs and providesome evidence for execise to prevent metabolic disease.Muscle disuse modelcharacterized by muscle unloading was also selected as a negative control group,another purpose was to investigate mechanisms underlying muscle disuse-inducedatrophy,and to support some conclusions about exercise adaptation.Methods:40 male Sprague-Dawley rats were distributed into four groups:sedentary(C,n=10),hindlimb suspension(D,n=8),sprint interval training(S,n=12)orendurance training(E,n= 10).Sprint interval training consisted of 9-10 repeats of a 10s“all out”treadmill test((?)42m/min)with 30-60s recovery between repeats,6 days/wk.Endurance training consisted of 30-60min of continuous treadmill exercise at a lowerintensity((?)16.7m/min)per day,6 days/wk.Group D were administered similarly toGroup C in the 1～5wk,hindlimb suspension was implemented in the 6～8wk bykeeping the rat’s hindlimb 1～2cm away from the ground.After 8 weeks of eithersprint interval or endurance training,all rats were decapitated 24h after the lasttreadmill test.Blood was collected from heart,blood glucose,insulin,leptin andtriglyceride were detected.The content ofpyruvate and lactate,the enzyme activity ofhexokinase(HK),pyruvate kinase(PK)and malate dehydrogenase(MDH)ingastrocnemius homogenate were measured by spectrophotometric assays.Thecapacity of citrate synthase(CS)andβ-hydroxyacyl-CoA dehydrogenase(HCD)wasdetermined in mitochondrial extact from gastrocnemius.Real-time PCR was used todetermine the mRNA content of GLUT4,AMPKα2,PDK4,CPT-1β,PGC-1α,ERRα,NRF-2α,Mfnl,Mfn2,OPAl,Drpl,Fisl in gastrocnemius.Western blot was used todetermine the protein content of PDK4,CPT-1β,Mfn2,Drpl in mitochondria andPGC-1,ERRαin nuclear extract.ELISA was used to determine the content ofcytochrome c in mitonchondria and cytoplasm. Results:(1)Hindlimb suspension and exercise program in the study had no negative effects onphysiological homeostasis in an organism.However,endurance training decreasedblood triglyceride,sprint interval training and hindlimb suspension had no markedeffects on blood triglyceride,suggesting that endurance training differed in bloodtriglyceride from sprint interval training.(2)Both endurance training and sprint interval training increased pyruvate level inresting muscle,but it was only sprint interval training that increased lactate level inresting muscle,suggesting that sprint interval training activated anerobic metabolismin resting muscle.Hindlimb suspension and the two exercise programs in the studyhad no marked effects on the activities of HK、PK、MDH、mito-HCD,but sprintinterval training elevated the capacity ofmito-CS.(3)Endurance training in the study had no marked effect on mRNA content ofGLUT4,sprint interval training induced the upregulation of GLUT4mRNA.Endurance training in the study induced a downregulation of AMPKα2 mRNA,suggesting that AMPK signalling in resting muscle may be attenuated after endurancetraining.Endurance training in the study had no marked effect on mRNA and proteincontent of CPT-1,and induced a decrease only in PDK4mRNA.Sprint intervaltraining in the study induced a marked decrease in mRNA and protein content ofPDK4.Hindlimb suspension had a very similar effect to sprint interval training onregulating gene expression of PDK4、CPT-1.(4)Hindlimb suspension and the two exercise programs in the study had no markedeffects on NRF-2αmRNA.The synchronization of mRNA and protein content wasvery poor in PGC-1\ERRαgene expression.The two exercise programs in the studywere likely to induce a“labor-saving”transcription of PGC-1αand ERRor.PGC-1αprotein was in linear correlation with ERRαin nuclear extract.Hindlimb suspensionhad a very similar effect to endurance training on regulating mRNA transcription ofPGC-1/ERRα/NRF-2,and had a very similar effect to sprint interval training onregulating protein expression of PGC-1/ERRα.(5)Endurance training in the study upregulated Mfn1 mRNA,but Mfn2 kept silenceto endurance training in transcriptional level and mitochondrial localization.Sprintinterval training induced a decrease in Mfn2 mRNA and an increase in mito-Mfn2protein.Sprint interval training maybe increased the stability of Mfn2 mRNA andprotein,and induced a“labor-saving”transcription of Mfn2.The two exerciseprograms in the study had no marked effects on Fisl mRNA.Sprint interval traininginduced an increase in Drpl mRNA and a decrease in mito-Drpl protein,but DrplmRNA and protein expression kept silence to endurance training.(6)Hindlimb suspension induced an increase in cytoplasmic CytC and had no markedeffect on mito-CytC.Endurance training induced an increase in cytoplasmic CytC andmito-CytC,Sprint interval training induced an increase in mito-CytC and had nomarked effect on cytoplasmic CytC. Conlusions:(1)There may be an interdependent stability among blood glucose,insulin and leptin,exercise training maybe have positive effects on maintaining the interdependence.Ourdata indicated that sprint interval training might be used as an efficient program toenhance oxidative capacity and increase the conversion of pyruvate to lactate inresting muscle.(2)The downregulation of PDK4 protein maybe attenuate the inhibitory effect ofPDK4 on mitochondrial oxidation of carbohydrate and contribute to glycogenoxidation.These changes were a favorable metabolic adaptation to sprint intervaltraining.PGC-1 coordinated likely the activation of metabolic genes with ERRαinnuclei of muscle,sprint interval training in the study probably induced more intensivetranscriptional activation than endurance training.(3)Endurance training in the study had less effect on muscle glycogen oxidation andmito-fusion than sprint interval training.The latter exercise program could inducemito-fusin and decrease mito-fission.Elevation of mito-Mfn2 protein with decrease inmito-PDK4 and Drpl protein maybe enhance mito-fusion and glycogen oxidation inmuscle.The present data suggested that sprint interval training was more likely toincrease mito-fusion than endurance training.(4)Muscle disuse in the study might decrease myofiber number by apoptosis,notdecrease mitochondrial density in muscle fiber.Mitochondrial density and oxidativecapacity in muscle fiber maybe increased dramatically after endurance training.Sprintinterval training was more likely to prevent muscular apoptosis and atrophy andmaintain muscle mass than endurance training,gastrocnemius adaptation to sprintinterval training showed mixed and transitive.(5)The similarity between exercise-and disuse-induced muscle suggested that musclecells were not necessary to response dissimilarly to muscle overloading or unloading.Hindlimb suspension and sprint interval training in the study had much rnore intensiveeffects on mitochondrial homeostasis than endurance training.Endurance trainingcould be used as a better program to keep healthy homeostasis.Sprint interval trainingcould be used as a timesaving program to enhance mitochondrial function and preventmitochondrial related diseases.更多还原