4周择时有氧运动对小鼠骨骼...和糖原代谢及运动耐力的影响_徐磊.pdf

第 45 卷 第 12 期2022 年 12 月北 京 体 育 大 学 学 报Journal of Beijing Sport UniversityVol.45 No.12Dec.20224周择时有氧运动对小鼠骨骼肌CHRONO-BMAL1通路和糖原代谢及运动耐力的影响徐磊1，2，贾杰2，于晶晶1，张缨2（1.北京体育大学运动与体质健康教育部重点实验室，北京 100084；2.北京体育大学运动人体科学学院，北京 100084）摘 要：目的：探讨择时有氧运动对小鼠骨骼肌CHRONO-BMAL1通路和糖原代谢及运动耐力的影响。方法：33只健康雄性C57BL/6J小鼠随机分为3组：安静对照组（Con），ZT12运动组（ZT12），ZT0运动组（ZT0）。在明暗循环中的时间用授时因子时间（zeitgeber time，ZT）表示，ZT0（7 00）为光照开始，ZT12（19 00）为光照结束。Con组不做干预，运动组进行4周的运动强度为75%最大摄氧量的跑台训练。干预结束48 h后，各组进行运动能力测试，再休息48 h后取材。取腓肠肌并称重，Western blot检测骨骼肌CHRONO和BMAL1的蛋白表达；免疫共沉淀检测CHRONO-BMAL1蛋白结合量；免疫荧光检测CHRONO-BMAL1共定位；试剂盒检测糖原含量、GYS活性、GPa活性；荧光实时定量PCR检测Gys1、Gbe1、Phka1 mRNA转录水平。结果：1）与Con和ZT0组相比，ZT12组小鼠的运动至力竭的时间和距离均显著提高。2）与Con组相比，ZT12组小鼠骨骼肌CHRONO蛋白表达显著降低；且与Con和ZT0组相比，ZT12组小鼠骨骼肌BMAL1蛋白表达显著增加。3）与Con和ZT0组相比，ZT12组小鼠骨骼肌CHRONO-BMAL1的结合量和共定位系数显著降低。4）与Con和ZT0组相比，ZT12组小鼠骨骼肌Gys1和Gbe1 mRNA水平、GYS活性及糖原含量显著提高。结论：与ZT0（7 00）相比，在ZT12（19 00）进行4周跑台运动训练，可更好地降低骨骼肌CHRONO对BMAL1转录活性的抑制作用，增加 BMAL1介导的骨骼肌糖原合成代谢调控以及肌糖原含量，进而提高小鼠的有氧运动耐力。关键词：择时有氧运动；CHRONO-BMAL1通路；骨骼肌；糖原代谢；有氧运动耐力Effects of 4-Week Aerobic Training at Different Times During the Day on CHRONO-BMAL1 Pathway，Glycogen Metabolism and Endurance Performance of Skeletal Muscle in MiceXU Lei1，2，JIA Jie2，YU Jingjing1，ZHANG Ying2（1.Key Laboratory of Sports and Physical Health of the Ministry of Education，Beijing Sport University，Beijing 100084，China；2.School of Kinesiology，Beijing Sport University，Beijing 100084，China）Abstract:This research aims to investigate the effects of aerobic training at two selected times during the day on CHRONO-BMAL1 pathway，glycogen metabolism and endurance performance of skeletal muscle in mice.Methods：33 male C57BL/6J mice were randomly divided into three groups：control group（Con），ZT12 exercise 徐磊，贾杰，于晶晶，等.4周择时有氧运动对小鼠骨骼肌CHRONO-BMAL1通路和糖原代谢及运动耐力的影响 J.北京体育大学学报，2022，45（12）：109-119.doi：10.19582/ki.11-3785/g8.2022.12.010投稿日期：2022-03-24 修稿日期：2022-08-28基金项目：国家自然科学基金项目“CHRONO-BMAL1通路调节骨骼肌糖代谢及其影响有氧运动能力的分子机制”（项目编号：32071168）；中央高校基本科研业务费专项基金“CHRONO-BMAL1通路在有氧运动预防高脂喂养小鼠骨骼肌糖代谢紊乱中的作用和机制研究”（项目编号：20221015）。作者简介：徐磊，博士研究生，研究方向运动与骨骼肌代谢适应。通信作者：张缨，教授，博士，博士研究生导师，研究方向运动与骨骼肌代谢适应。109北京体育大学学报第 45 卷第 12 期group（ZT12），ZT0 exercise group（ZT0）.The time in the light dark cycle is represented by zeitgeber time（ZT），ZT0 corresponds to the time when light was turned on at 7：00 and ZT12 corresponds to the time when light was turned off at 19：00.The Con group was not intervened，and the exercise group received treadmill training with exercise intensity of 75%maximum oxygen uptake for 4 weeks.48 hours after the final training session，the endurance performance of each group was tested，and the mice were sacrificed after 48 hours.The protein expression of CHRONO and BMAL1 in skeletal muscle were detected by Western blot；the binding amount of CHRONO-BMAL1 protein was detected by co-immunoprecipitation；the colocalization of CHRONO-BMAL1 was detected by immunofluorescence；the glycogen content，GYS activity and GPa activity were detected by kit；the mRNA transcription level of Gys1，Gbe1 and Phka1 were detected by RT-qPCR.Results：1）compared with the Con and ZT0 groups，the duration and distance to exhaustion of the ZT12 group were significantly longer.2）Compared with the Con group，the protein expression of CHRONO in skeletal muscle of the ZT12 group was significantly lower；compared with the Con and ZT0 groups，the protein expression of BMAL1 in skeletal muscle of the ZT12 group was significantly higher.3）Compared with the Con and ZT0 groups，the binding amount and colocalization coefficient of CHRONO-BMAL1 in skeletal muscle of the ZT12 group were significantly lower.4）Compared with the Con and ZT0 groups，the mRNA level of Gys1 and Gbe1，GYS activity and glycogen content of skeletal muscle in the ZT12 group were significantly higher.Conclusion：compared with ZT0（7 00），the 4-week treadmill training at ZT12（19 00）can better reduce the inhibitory effect of CHRONO on BMAL1 transcriptional activity，increase the glycogen anabolism and glycogen content of skeletal muscle，which is mediated by BMAL1，and then improve the endurance performance of mice.Keywords:timing aerobic training；CHRONO-BMAL1 pathway；skeletal muscle；glycogen metabolism；endurance performance生物钟是生物体内的一种无形的“时钟”，使人体睡眠觉醒循环、体温和能量代谢等许多生理活动均呈现近24 h昼夜节律变化1。哺乳动物的生物节律性变化受下丘脑视交叉上核的主生物钟和骨骼肌、肝脏等外周组织的生物钟调节2-3。脑肌类芳香烃受体核转位样蛋白1（Brain and Muscle Arnt-Like protein，BMAL1）是骨骼肌生物钟的核心组件，可与时钟节律调节因子（Circadian Locomotor Output Cycles Kaput，CLOCK）结合形成异二聚体，它们作为转录因子与生物钟基因Per1/2、Cry1/2启动子上E-box区域结合，促进其转录。Per1/2、Cry1/2转录后在细胞质被翻译成周期蛋白（Period，PER）、隐花色素蛋白（Cryptochrome，CRY），随后可转位再进入细胞核，抑制BMAL1-CLOCK转录活性。这种转录翻译反馈调节近24 h重复一次，使BMAL1-CLOCK 转录活性呈现节律性变化4-5。计算机模拟的生物节律抑制因子（Computationally Highlighted Repressor Of the Network Oscillator，Chrono）是近年来在哺乳动物中发现的生物钟基因，其编码的CHRONO蛋白能够直接与转录因子 BMAL1 结合，反馈性抑制 BMAL1-CLOCK异二聚体的转录活性6-8。因此，CHRONO-BMAL1 是调控骨骼肌生物节律的重要通路9。此外，BMAL1也是调控骨骼肌糖代谢的重要转录因子10-11。研究表明，骨骼肌特异性敲除Bmal1基因的小鼠因葡萄糖转运、糖酵解以及葡萄糖有氧氧化降低，可引起糖耐量受损和胰岛素抵抗12-14。因此，可以推测 CHRONO-BMAL1作为调控骨骼肌生物节律的重要通路，可能也参与调控骨骼肌的糖代谢，从而影响有氧运动耐力。人们在研究生物节律时，将白天/黑夜（光/暗）循环的时间用授时因子时间（zeitgeber time，ZT）表示。例