目的:运用蛋白组学技术研究有氧运动对胰岛素抵抗(Insulin Resistance,IR)小鼠骨骼肌蛋白表达的影响,初步探究骨骼肌蛋白质折叠/降解通路相关蛋白在有氧运动改善IR过程中的作用机制。方法:选取80只4周龄C57BL/6雄性小鼠,随机分为正常饮食组(NC,n=20)和IR模型组(n=60)并分别饲以基础和高脂饲料10周。10周后通过检测空腹血清胰岛素水平(Fasting Insulin,FIN)及口服葡萄糖耐量试验(Oral Glucose Tolerance Test,OGTT)鉴定IR模型是否成功。选取40只成模小鼠,再次随机分为高脂饮食运动组(HE)和高脂饮食安静组(HC)。HE组小鼠采用6周75%VO2 max有氧跑台运动训练。运动结束后处死动物、提取各组小鼠股四头肌总蛋白,经Bradford法检测蛋白浓度后进行蛋白质双向电泳(2-DE),采用ImageMaster 2D Platinum V5.0软件分析2-DE电泳图谱,并对所选取的差异蛋白点用基质辅助激光解吸附离子化飞行时间质谱(Matrix-assisted Laser Desorption/Ionization Time-of-Flight MassSpectrometry,MALDI-TOF-MS)和高效液相色谱-串联谱法(Liquid-Chromatography–tandemMass Spectrometry,LC-MS/MS)进行分析,经Mascot检索软件结合NCBI nr数据库鉴定差异蛋白后使用相关软件对所得数据进行统计分析。结果:10周高脂饮食后,IR模型组小鼠FIN水平比NC组增加50%,OGTT曲线峰值时间点显著后移并出现平台期,提示10周高脂饮食可诱导小鼠产生IR。6周有氧跑台运动后,HE组小鼠FIN水平较HC组下降17.2%,OGTT曲线峰值下降且时间点前移,平台期消失,表明6周有氧运动可显著改善IR小鼠症状。经双向电泳显影,设定1.5倍为筛选差异表达蛋白的标准,比较HC组与HE组蛋白,共发现差异表达蛋白22个,其中运动后表达下调蛋白14个,表达上调蛋白8个。差异表达蛋白中有5个蛋白与细胞蛋白折叠/降解通路密切相关。其变化倍数分别为:纤维蛋白原β链前体(Fibrinogen
Objective The aim of this study was to explore the role of fold / degradation proteins involved in aerobic exercise ameliorating insulin resistance(IR)by identifying the differentially expressed proteins in the skeletal muscle of mice after exercise.Methods Eighty male C57BL/6 mice were randomly divided into IR model group(IR,n = 60)and normal diet group(NC,n = 60).Rats in IR group were fed with high-fat diet for 10 weeks.The IR was confirmed by fasting serum insulin level(FIN)and oral glucose tolerance test(OGTT).Then the IR group was randomly subdivided into a high-fat-diet exercise group(HE)and a high-fat-diet control group(HC).The HE group was required to run on a treadmill for 6 weeks with the intensity of 75%VO2 max.After the completion of the 6-week exercise,the proteins extracted from the quadriceps femoris were quantified with Bradford and then separated by two-dimensional gel electrophoresis(2-DE).The result was analyzed by ImageMaster 2D Platinum V5.0.The selected differentially expressed proteins were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)and Tandem Mass Spectrometry(LC/LC-MS/MS).The differentially expressed proteins were identified using Mascot software and NCBI nr database.Results Compared with the NC group,FIN of 10-week high-fat-diet group increased by 50%,and time point of OGTT peak shifted backward obviously with a platform stage.After 6-week aerobic exercise,the FIN in HE group decreased by 17.2% as compared with the HC group;the time point of OGTT peak shifted forward,the peak value of the OGTT curve decreased significantly,and the platform stage was eliminated.The protein was considered to be significantly differentially expressed(over-expressed or under-expressed),if the ratio of intensity between two groups was greater than 1.5-fold.21 protein spots were found in the HE group as compared with the HC group using proteome analysis.After 6-week aerobic exercise,8 proteins were up-regu