低氧耐力运动对营养性肥胖大鼠骨骼肌自噬蛋白表达的影响
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摘要
目的:通过对高脂膳食诱导的营养性肥胖大鼠模型进行低氧耐力训练,探讨不同低氧浓度的耐力运动对营养性肥胖大鼠骨骼肌自噬相关蛋白LC3、Beclin1、AMPKα2、Sestrin2表达的影响。方法:80只建模成功的营养性肥胖大鼠随机分为8组(常氧安静组、常氧运动组、11.3%组、13.3%组分别在11.3%、13.3%、16.3%静组不进行任何干预,运动组按照20 m/min、40 min/d、5次/周的安排训练8周,末次训练24 h后处死大鼠取腓肠肌,采用Western Blot方法测定腓肠肌自噬相关蛋白LC3(微管相关蛋白1轻链3)、Beclin1(酵母ATG6同源物)、AMPKα2、Sestrin2的表达水平。结果:(1)实施低氧暴露干预后,11.3%低氧组、13.3%低氧组大鼠腓肠肌自噬相关蛋白Beclin1、AMPKα2、Sestrin2表达及LC3Ⅱ/LC3Ⅰ比值显著高于常氧组(P<0.05)。(2)实施耐力运动干预后,与对应安静组相比,运动组Beclin1、AMPKα2表达水平及LC3Ⅱ/LC3Ⅰ比值显著性升高(P<0.05);常氧运动组、16.3%低氧运动组、11.3%表达水平显著性升高(P<0.05)。(3)低氧结合耐力运动干预后,与常氧安静组相比,常氧运动组、16.3%低低氧运动组Sestrin2、Beclin1、AMPKα2蛋白表达显著性升高(P<0.低氧运动组LC3Ⅱ/LC3Ⅰ比值显著性升高(P<0.05),低氧运动组LC3Ⅱ/LC3Ⅰ比值显著性升高(P<0.05);且随氧浓度降低,Beclin1、AMPKα2、Sestrin2表达水平及LC3Ⅱ/LC3Ⅰ比值呈现上升趋势。结论:运动和低氧都能显著激活骨骼肌细胞自噬,上调骨骼肌自噬相关蛋白的表达水平;与单纯运动和单纯低氧相比,低氧耐力训练对自噬的调控作用更加明显。
Objective To explore the effect of hypoxia exercise on the expression of autophagy-related proteins,including the microtubule-associated protein 1 light chain 3(LC3),yeast ATG6 homolog Beclin1,adenosine monophosphate activated protein kinase alpha 2(AMPKα2),and Sestrin2 in skeletal muscles of rats with alimentary obesity.Methods The alimentary obesity was successfully induced in 80 rats. Then they were randomly divided into a normoxic group,a normoxic group with endurance exercise,an 11.3% hypoxic group,an 11.3% hypoxic group with endurance exercise,a 13.3% hypoxic group,a 13.3% hypoxic group with endurance exercise,a 16.3% hypoxic group and a 16.3% hypoxic group with endurance exercise,each of 10. The hypoxic groups were exposed in the environment with the oxygen concentration of what their group names implied12 hours per day,five days a week for 8 weeks. The groups with exercise underwent treading running for 8 weeks,going with 20 m/min,40 min/d,and 5 times/week. Twenty-four hours after the last training,the rats were sacrificed and their gastrocnemius muscles were collected. The expression levels of LC3,Beclin1,AMPKα2 and Sestrin2 in the gastrocnemius muscle were determined using the Western Blotting.Results (1)After the hypoxic exposure,the expressions of Beclin1,AMPKɑ2,Sestrin2 and LC3Ⅱ/LC3Ⅰ of the 11.3% and 13.3%hypoxia groups were significantly higher than the normoxic groups(P<0.05).(2)The expression levels of Beclin1 and AMPKα2 and the ratio of LC3Ⅱ/LC3Ⅰ of the exercising groups were significantly higher than the corresponding resting groups(P<0.05). Moreover,a significant increase was observed in the expression level of Sestrin2 of the nomoxic,16.3% hypoxic and 11.3% hypoxic groups with endurance exercise compared with their corresponding resting groups(P<0.05).(3)After hypoxia combined with endurance exercise intervention,compared with the normoxic group,the expression of Sestrin2,Beclin1 and AMPKα2 protein of the normoxic group with endurance exercise group,as well as the 16.3%,13.3% and 11.3% hypoxic group with endurance exercise increased significantly(P<0.05). Compared with the normoxic group with endurance exercise,a significant increase was observed in the ratio of LC3Ⅱ/LC3Ⅰ of the 13.3% and 11.3% hypoxic group with endurance exercise(P<0.05),while compared with the 16.3% hypoxic group with endurance exercise,a significant increase was found in the ratio of LC3Ⅱ/LC3Ⅰ only of the 11.3% hypoxic group with endurance exercise(P<0.05). The expression level of Beclin1,AMPKα2 and Sestrin2 and the ratio of LC3Ⅱ/LC3Ⅰ increased with the decrease of the oxygen concentration.Conclusion Both exercise and hypoxia can significantly activate autophagy in skeletal muscles and up-regulate the expression levels of proteins associated with the skeletal muscle autophagy. Compared with exercise and hypoxia solely,their combination has a more pronounced regulatory effect on the autophagy.
Objective To explore the effect of hypoxia exercise on the expression of autophagy-related proteins,including the microtubule-associated protein 1 light chain 3(LC3),yeast ATG6 homolog Beclin1,adenosine monophosphate activated protein kinase alpha 2(AMPKα2),and Sestrin2 in skeletal muscles of rats with alimentary obesity.Methods The alimentary obesity was successfully induced in 80 rats. Then they were randomly divided into a normoxic group,a normoxic group with endurance exercise,an 11.3% hypoxic group,an 11.3% hypoxic group with endurance exercise,a 13.3% hypoxic group,a 13.3% hypoxic group with endurance exercise,a 16.3% hypoxic group and a 16.3% hypoxic group with endurance exercise,each of 10. The hypoxic groups were exposed in the environment with the oxygen concentration of what their group names implied12 hours per day,five days a week for 8 weeks. The groups with exercise underwent treading running for 8 weeks,going with 20 m/min,40 min/d,and 5 times/week. Twenty-four hours after the last training,the rats were sacrificed and their gastrocnemius muscles were collected. The expression levels of LC3,Beclin1,AMPKα2 and Sestrin2 in the gastrocnemius muscle were determined using the Western Blotting.Results (1)After the hypoxic exposure,the expressions of Beclin1,AMPKɑ2,Sestrin2 and LC3Ⅱ/LC3Ⅰ of the 11.3% and 13.3%hypoxia groups were significantly higher than the normoxic groups(P<0.05).(2)The expression levels of Beclin1 and AMPKα2 and the ratio of LC3Ⅱ/LC3Ⅰ of the exercising groups were significantly higher than the corresponding resting groups(P<0.05). Moreover,a significant increase was observed in the expression level of Sestrin2 of the nomoxic,16.3% hypoxic and 11.3% hypoxic groups with endurance exercise compared with their corresponding resting groups(P<0.05).(3)After hypoxia combined with endurance exercise intervention,compared with the normoxic group,the expression of Sestrin2,Beclin1 and AMPKα2 protein of the normoxic group with endurance exercise group,as well as the 16.3%,13.3% and 11.3% hypoxic group with endurance exercise increased significantly(P<0.05). Compared with the normoxic group with endurance exercise,a significant increase was observed in the ratio of LC3Ⅱ/LC3Ⅰ of the 13.3% and 11.3% hypoxic group with endurance exercise(P<0.05),while compared with the 16.3% hypoxic group with endurance exercise,a significant increase was found in the ratio of LC3Ⅱ/LC3Ⅰ only of the 11.3% hypoxic group with endurance exercise(P<0.05). The expression level of Beclin1,AMPKα2 and Sestrin2 and the ratio of LC3Ⅱ/LC3Ⅰ increased with the decrease of the oxygen concentration.Conclusion Both exercise and hypoxia can significantly activate autophagy in skeletal muscles and up-regulate the expression levels of proteins associated with the skeletal muscle autophagy. Compared with exercise and hypoxia solely,their combination has a more pronounced regulatory effect on the autophagy.
引文
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[14]Chai D,Wang G,Zhou Z,et al.Insulin increases Sestrin 2content by reducing its degradation through the PI3K/mTORsignaling pathway[J].Int J Endocrinol,2015;2015:505849.
[15]Lee J H,Budanov A V,Talukdar S,et al.Maintenance of metabolic homeostasis by Sestrin2 and Sestrin3[J].Cell Metab,2012,16(3):311-321.
[16]Zhang XY,Wu XQ,Deng R,et al.Upregulation of sestrin 2expression via JNK pathway activation contributes to autophagy induction in cancer cells[J].Cell Signal,2013,25(1):150-158.
[17]刘效磊.AMPK/mTOR介导有氧运动提高骨骼肌胰岛素敏感性的机制研究[D].天津医科大学,2015.
[18]Budanov AV,Karin M.p53 target genes sestrin1 and sestrin2connect genotoxic stress and mTOR signaling[J].Cell,2008,134(3):451-460.
[19]Borger DR,Gavrilescu LC,Bucur MC,et al.AMP-activated protein kinase is essential for survival in chronic hypoxia[J].Biochem Biophys Res Commun,2008,370(2):230-234.
[20]Liu H,Qiu H,Xiao Q,et al.Chronic hypoxia-induced autophagy aggravates the neuropathology of alzheimer’s disease through AMPK-mTOR signaling in the APP Swe/PS1 dE9mouse model[J].J Alzheimers Dis,2015,48(4):1019-1032.
[21]吴菊花.低氧运动对肥胖大鼠骨骼肌AMPK-PGC-1α及其下游分子介导脂代谢的影响[D].上海体育学院,2016.
[22]牛燕媚,张珊,王天怡,等.Sestrins与骨骼肌细胞自噬--有氧运动改善胰岛素抵抗机制研究新进展[J].中国运动医学杂志,2016,35(02):181-183.
[23]Lo Verso F,Carnio S,Vainshtein A,et al.Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity[J].Autophagy,2014,10(11):1883-1894.
[24]Maiuri MC,Malik SA,Morselli E,et al.Stimulation of autophagy by the p53 target gene Sestrin2[J].Cell Cycle,2009,8(10):1571-1576.
[25]陈福刁,林文弢,黄丽英.低氧运动干预对大鼠心肌细胞自噬作用的探讨[J].沈阳体育学院学报,2012,31(01):70-72.
[26]Lira VA,Okutsu M,Zhang M,et al.Increased contractile acitivity induces autophagy in skeletal muscle[J].FASEB J,2010,24(1 Supplement):lb646-lb646..
[27]姚璐,谢谨,李松波,等.低氧和低氧训练对AMPKα2转基因小鼠骨骼肌CPT-1表达的影响[J].北京体育大学学报,2011,34(01):51-53,57.
[2]崔迪,贺杰,孙婧瑜,等.耐力训练与p53抑制剂PFT-ɑ对小鼠骨骼肌线粒体自噬相关基因表达的影响[J].天津体育学院学报,2013,28(5):422-426.
[3]Jamart C,Benoit N,Raymackers J M,et al.Autophagy-related and autophagy-regulatory genes are induced in human muscle after ultraendurance exercise[J].Eur J Appl Physiol,2012,112(8):3173-3177.
[4]薄海,李玲,段富强,等.低氧联合运动对大鼠骨骼肌线粒体自噬的影响[J].中国康复医学杂志,2014,29(10):908-912.
[5]张素娴,朱磊,冯连世,等.4周低氧运动对营养肥胖型大鼠骨骼肌自噬相关因子时序性变化的影响[J].中国运动医学杂志,2017,36(12):1059-1065,1051.
[6]钱帅伟,罗艳蕊,漆正堂,等.细胞自噬的分子学机制及运动训练的调控作用[J].体育科学,2012,32(01):64-70.
[7]李海英,苗卫国,马静芬,等.线粒体自噬在运动预适应抗急性低压低氧大鼠脑海马损伤中的作用[J].中国运动医学杂志,2014,33(06):524-529.
[8]崔迪,邱守涛,王海燕,等.耐力运动对营养性肥胖小鼠骨骼肌细胞自噬及线粒体自噬的影响[J].体育科学,2014,34(12):63-71.
[9]Lee JH,Budanov AV,Karin M.Sestrins orchestrate cellular metabolism to attenuate aging[J].Cell Metab,2013,18(6):792-801.
[10]Tsilioni I,Filippidis AS,Kerenidi T,et al.Sestrin-2 is significantly increased in malignant pleural effusions due to lung cancer and is potentially secreted by pleural mesothelial cells[J].Clin Biochem,2016,49(9):726-728.
[11]Budanov AV.Stress-responsive sestrins link p53 with redox regulation and mammalian target of rapamycin signaling[J].Antioxid Redox Signal,2011,15(6):1679-1690.
[12]Lee JH,Budanov AV,Talukdar S,et al.Maintenance of metabolic homeostasis by Sestrin2 and Sestrin3[J].Cell Metab,2012,16(3):311-321.
[13]Lee JH,Budanov AV,Park EJ,et al.Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies[J].Science,2010,327(5970):1223-1228.
[14]Chai D,Wang G,Zhou Z,et al.Insulin increases Sestrin 2content by reducing its degradation through the PI3K/mTORsignaling pathway[J].Int J Endocrinol,2015;2015:505849.
[15]Lee J H,Budanov A V,Talukdar S,et al.Maintenance of metabolic homeostasis by Sestrin2 and Sestrin3[J].Cell Metab,2012,16(3):311-321.
[16]Zhang XY,Wu XQ,Deng R,et al.Upregulation of sestrin 2expression via JNK pathway activation contributes to autophagy induction in cancer cells[J].Cell Signal,2013,25(1):150-158.
[17]刘效磊.AMPK/mTOR介导有氧运动提高骨骼肌胰岛素敏感性的机制研究[D].天津医科大学,2015.
[18]Budanov AV,Karin M.p53 target genes sestrin1 and sestrin2connect genotoxic stress and mTOR signaling[J].Cell,2008,134(3):451-460.
[19]Borger DR,Gavrilescu LC,Bucur MC,et al.AMP-activated protein kinase is essential for survival in chronic hypoxia[J].Biochem Biophys Res Commun,2008,370(2):230-234.
[20]Liu H,Qiu H,Xiao Q,et al.Chronic hypoxia-induced autophagy aggravates the neuropathology of alzheimer’s disease through AMPK-mTOR signaling in the APP Swe/PS1 dE9mouse model[J].J Alzheimers Dis,2015,48(4):1019-1032.
[21]吴菊花.低氧运动对肥胖大鼠骨骼肌AMPK-PGC-1α及其下游分子介导脂代谢的影响[D].上海体育学院,2016.
[22]牛燕媚,张珊,王天怡,等.Sestrins与骨骼肌细胞自噬--有氧运动改善胰岛素抵抗机制研究新进展[J].中国运动医学杂志,2016,35(02):181-183.
[23]Lo Verso F,Carnio S,Vainshtein A,et al.Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity[J].Autophagy,2014,10(11):1883-1894.
[24]Maiuri MC,Malik SA,Morselli E,et al.Stimulation of autophagy by the p53 target gene Sestrin2[J].Cell Cycle,2009,8(10):1571-1576.
[25]陈福刁,林文弢,黄丽英.低氧运动干预对大鼠心肌细胞自噬作用的探讨[J].沈阳体育学院学报,2012,31(01):70-72.
[26]Lira VA,Okutsu M,Zhang M,et al.Increased contractile acitivity induces autophagy in skeletal muscle[J].FASEB J,2010,24(1 Supplement):lb646-lb646..
[27]姚璐,谢谨,李松波,等.低氧和低氧训练对AMPKα2转基因小鼠骨骼肌CPT-1表达的影响[J].北京体育大学学报,2011,34(01):51-53,57.