有氧运动训练中补充Apelin对骨骼肌AMPK活化及线粒体能量代谢的影响
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摘要
目的:探讨有氧运动训练补充apelin对骨骼肌AMPK活化及线粒体能量代谢的影响。方法:1)细胞实验部分采用小鼠骨骼肌C2C12细胞系作为研究对象,将靶向AMPKα基因的小干扰RNA(AMPKαsi RNA)或阴性对照小干扰RNA(Control siRNA)通过脂质体介导转染细胞。转染48 h后,进行6 h的饥饿处理,而后在完全培养基中加入apelin-13(100nmol/L)或者同体积的PBS与细胞孵育6h。按转染si RNA和加apelin-13情况,实验分为Control si RNA+PBS组、ControlsiRNA+apelin组、AMPKsiRNA+PBS组与AMPKsi RNA+apelin组。完成干预后,采用Seahorse细胞能量代谢分析系统,测定细胞线粒体基础呼吸、ATP生成和呼吸功能变化。2)动物实验部分采用C57BL/6J小鼠做为研究对象,将40只小鼠随机分为安静未注射组、安静注射apelin组、运动未注射组和运动注射apelin组,每组10只。注射apelin组小鼠连续4周腹腔注射apelin-13(0.1μmol/kg体重/天)。运动组采用75%左右最大摄氧量强度(1～2周坡度5o,速度15m/min;3～4周坡度5o,速度20m/min)、1h/天、6天/周、持续4周的跑台运动。最后一次运动后休息48h,脱颈处死,取两侧股四头肌。WesternBlotting测定骨骼肌apelin、APJ、AMPKα、 p-AMPKα(Thr172)和COXⅣ蛋白表达。结果:1)细胞实验, ControlsiRNA+apelin组与Control si RNA+PBS组相比,细胞基础呼吸率、线粒体ATP生成和最大呼吸率均显著增加;而AMPKsi RNA+apelin组与ControlsiRNA+apelin组相比,线粒体ATP生成和最大呼吸率显著降低。2)动物实验,安静注射apelin组与安静未注射组相比、运动注射apelin组与运动未注射组相比,小鼠骨骼肌apelin、APJ、COXⅣ蛋白表达和pAMPKα/AMPKα比值均显著增加。结论:外源性补充apelin可显著增加C2C12细胞AMPK介导的线粒体呼吸功能,并提高有氧运动训练小鼠骨骼肌apelin/APJ、AMPKα磷酸化和COXIV蛋白表达,提示,有氧运动训练补充apelin可能对骨骼肌的apelin-AMPK通路及其介导的线粒体能量代谢有一定的积极促进作用。
Objective:To investigate the effects of apelin supplement during aerobic training on AMPK activation and mitochondrial energy metabolism in skeletal muscle. Methods: In the cell experiments, the AMPKα specific small interfering RNA(AMPKαsiRNA)or a negative control interfering RNA(Control siRNA) were transfected into mice skeletal muscle C2C12 cells by Lipofectamine. Starvation treatment was performed for 6 hours after 48 hours transfection, and then cells were incubated with apelin-13 or PBS for 6 hours in complete medium to observe the changes in mitochondrial basal respiration, ATP production, and respiratory function. In the animal experiments, forty 8-week-old C57BL/6J mice were randomly divided into four groups(n=10 in each group): sedentary without apelin treatment, sedentary with apelin treatment, exercise training without apelin treatment and exercise training with apelin treatment group. The apelin treatment groups were injected intraperitoneally with apelin-13 at 0.1 μmol/kg/day for 4 weeks. The exercise groups were trained 6 days/week, 1 hour/day for 4 weeks by running on a treadmill at 75% VO2 max. 48 hours after the last session of exercise training, the quadriceps were collected. The protein expressions of apelin, APJ, AMPKα, p-AMPKα(Thr172) and COX IV in skeletal muscles was measured by Western Blotting. Results: 1) In the cell experiments, the basal respiration rate, mitochondrial ATP production and maximum respiration rate were significantly increased in the Control siRNA+apelin group compared with the Control si RNA+PBS group, but the mitochondrial ATP production and maximum respiration rate were significantly decreased in the AMPK siRNA+apelin group compared with the Control siRNA+apelin group. 2) In the animal experiments, the protein expression of apelin, APJ, COX IV and the p-AMPKα/AMPKα ratio in skeletal muscles were significantly increased in the apelin treatment groups compared with no apelin treatment groups. Conclusion: The exogenous supplementation of apelin was significantly increased the AMPK-mediated mitochondrial respiratory function in C2C12 cells, and the apelin supplement during aerobic training was also significantly increased the apelin/APJ, COX IV protein expression and the p-AMPKα/AMPK ratio in skeletal muscles. The results suggested that the combination of aerobic exercise training with apelin supplement might activates the apelin-AMPK pathway and improves the mitochondrial energy metabolism in skeletal muscles.
Objective:To investigate the effects of apelin supplement during aerobic training on AMPK activation and mitochondrial energy metabolism in skeletal muscle. Methods: In the cell experiments, the AMPKα specific small interfering RNA(AMPKαsiRNA)or a negative control interfering RNA(Control siRNA) were transfected into mice skeletal muscle C2C12 cells by Lipofectamine. Starvation treatment was performed for 6 hours after 48 hours transfection, and then cells were incubated with apelin-13 or PBS for 6 hours in complete medium to observe the changes in mitochondrial basal respiration, ATP production, and respiratory function. In the animal experiments, forty 8-week-old C57BL/6J mice were randomly divided into four groups(n=10 in each group): sedentary without apelin treatment, sedentary with apelin treatment, exercise training without apelin treatment and exercise training with apelin treatment group. The apelin treatment groups were injected intraperitoneally with apelin-13 at 0.1 μmol/kg/day for 4 weeks. The exercise groups were trained 6 days/week, 1 hour/day for 4 weeks by running on a treadmill at 75% VO2 max. 48 hours after the last session of exercise training, the quadriceps were collected. The protein expressions of apelin, APJ, AMPKα, p-AMPKα(Thr172) and COX IV in skeletal muscles was measured by Western Blotting. Results: 1) In the cell experiments, the basal respiration rate, mitochondrial ATP production and maximum respiration rate were significantly increased in the Control siRNA+apelin group compared with the Control si RNA+PBS group, but the mitochondrial ATP production and maximum respiration rate were significantly decreased in the AMPK siRNA+apelin group compared with the Control siRNA+apelin group. 2) In the animal experiments, the protein expression of apelin, APJ, COX IV and the p-AMPKα/AMPKα ratio in skeletal muscles were significantly increased in the apelin treatment groups compared with no apelin treatment groups. Conclusion: The exogenous supplementation of apelin was significantly increased the AMPK-mediated mitochondrial respiratory function in C2C12 cells, and the apelin supplement during aerobic training was also significantly increased the apelin/APJ, COX IV protein expression and the p-AMPKα/AMPK ratio in skeletal muscles. The results suggested that the combination of aerobic exercise training with apelin supplement might activates the apelin-AMPK pathway and improves the mitochondrial energy metabolism in skeletal muscles.
引文
ABBOTT M J,TURCOTTE L P,2014.AMPK-a2 is involved in exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following high-fat diet[J].J Appl Physiol,117(8):869-879.
ATTANE C,FOUSSAL C,LE GONIDEC S,et al.,2012.Apelin treatment increases complete Fatty Acid oxidation,mitochondrial oxidative capacity,and biogenesis in muscle of insulin-resistant mice[J].Diabetes,61(2):310-320.
BAJER B,VLCEK M,GALUSOVA A,et al.,2015.Exercise associated hormonal signals as powerful determinants of an effective fat mass loss[J].Endocr Regul,49(3):151-163.
BESSE P A,MONTASTIER E,Vinel C,et al.,2014.Effect of endurance training on skeletal muscle myokine expression in obese men:identification of apelin as a novel myokine[J].Int J Obes,38(5):707-713.
BERTRAND C,VALET P,CASTANLAURELL I,2015.Apelin and energy metabolism[J].Front Physiol,6:115.
BI F,XU Y,SUN Q,2018.Catalpol pretreatment attenuates cardiac dysfunction following myocardial infarction in rats[J].Anatol JCardiol,19(5):296-302.
BOOTH F W,ROBERTS C K,LAYE M J,2012.Lack of exercise is a major cause of chronic diseases[J].Compr Physiol,2(2):1143-1211.
FALCO M D,LUCA L D,ONORI N,et al.,2002.Apelin expression in normal human tissues[J].In vivo,16(5):333-336.
DRAY C,KNAUF C,DAVIAUD D,et al.,2008.Apelin stimulates glucose utilization in normal and obese insulin-resistant mice[J].Cell Metab,8(5):437-45.
FENTZ J,KJφBSTED R,KRISTENSEN C M,et al.,2015.AMPKαis essential for acute exercise-induced gene responses but not for exercise training-induced adaptations in mouse skeletal muscle[J].Am J Physiol Endocrinol Metab,309(11):E900-914.
FRIER B C,WILLIAMS D B,WRIGHT D C,2009.The effects of apelin treatment on skeletal muscle mitochondrial content[J].Am JPhysiol Regul Integr Comp Physiol,171(297):R1761-1768.
GILBERT J S,2017.From apelin to exercise:emerging therapies for management of hypertension in pregnancy[J].Hypertens Res,40(6):519-525.
INDRAKUSUMA I,SELL H,ECKEL J,2015.Novel Mediators of Adipose Tissue and Muscle Crosstalk[J].Curr Obes Rep,4(4):411-417.
ISABELLE C L,CAMILLE A,THIBAUT D,et al.,2011.Apelin,diabetes,and obesity[J].Endocrine,40(1):1-9.
KATUGAMPOLA S D,MAGUIRE J J,MATTHEWSON S R,et al.,2001.[(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man[J].British journal of pharmacology,132(6):1255-60.
LI J,LI S,WANG F,et al.,2017.Structural and biochemical insights into the allosteric activation mechanism of AMP-activated protein kinase[J].Chem Biol Drug Des,89(5):663-669.
MARCINKOarcinko K,STEINBERG G R,2014.The role of AMPK in controlling metabolism and mitochondrial biogenesis during exercise[J].Exp Physiol,99(12):1581-1585.
MASRI B,KNIBIEHLER B,AUDIGIER Y,2005.Apelin signalling:a promising pathway from cloning to pharmacology[J].Cellular signalling,17(4):415-426.
O'DOWD B F,HEIBEReiber M,Chan A,et al.,1993.A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11[J].Gene,136(1-2):355-360.
OLIVA C R,MARKERT T,GILLESPIE G Y,et al.,2015.Nuclearencoded cytochrome c oxidase subunit 4 regulates BMI1 expression anddetermines proliferative capacity of high-grade gliomas[J].Oncotarget,6(6):4330-4344.
RITCHIE I R,MACDONALDT L,WRIGHT D C,et al.,2014.Adiponectin is sufficient,but not required,for exercise-induced increases in the expression of skeletal muscle mitochondrial enzymes[J].J Physiol,592(12):2653-2665.
ROZYCKA M,KUROWSKA P,GRZESIAK M,et al.,2018.Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3β-hydroxysteroid dehydrogenase(3β-HSD)in pigs[J].Anim Reprod Sci,192:251-260.
SON J S,KIM H J,SON Y,et al.,2017.Effects of exercise-induced apelin levels on skeletal muscle and their capillarization in type 2diabetic rats[J].Muscle Nerve,56(6):1155-1163.
SON J S,SONG A C,TESTROET E D,et al.,2018.Exercise-induced myokines:a brief review of controversial issues of this decade[J].Expert Rev Endocrinol Metab,13(1):51-58.
STROHBACH A,PENNEWITZ M,GLAUBITZ M,et al.,2018.The apelin receptor influences biomechanical and morphological properties of endothelial cells[J].J Cell Physiol,233(8):6250-6261.
TAHERI S,MURPHY K,COHEN M,et al.,2002.The effects of centrally administered apelin-13 on food intake,water intake and pituitary hormone release in rats[J].Biochem Biophys Res Commun,291(5):1208-1212.
THAN A,ZHANG X,LEOW M K,et al.,2014.Apelin attenuates oxidative stress in human adipocytes.[J].J Biol Chem,289(6):3763-3774.
WANG L,DI L,NOGUCHI CT,2014.AMPK is Involved in Mediation of Erythropoietin Influence on Metabolic Activity and Reactive Oxygen Species Production in White Adipocytes[J].Int J Biochem Cell Biol,54(8):1-9.
WU Z,PUIGSERVER P,ANDERSSON U,et al.,1999.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1[J].Cell,98(1):115-124.
YAMAMOTO T,HABATA Y,MATSUMOTO Y,et al.,2011.Apelintransgenic mice exhibit a resistance against diet-induced obesity by increasing vascular mass and mitochondrial biogenesis in skeletal muscle[J].Biochim Biophys Acta,1810(9):853-862.
YANG H,ZHAO L,ZHANG J,et al.,2015.Effect of Treadmill Running on Apelin and APJ Expression in Adipose Tissue and Skeletal Muscle in Rats Fed a High-fat Diet[J].Int J Sports Med,36(7):535-541.
YUE P,JIN H,AILLAUD M,et al.,2010.Apelin is necessary for the maintenance of insulin sensitivity[J].Am J Physiol-Endoc M,298(1):E59-67.
VINEL C,LUKJANENKO L,BATUT A,et al.,2018.The exerkine apelin reverses age-associated sarcopenia[J].Nat Med,24(9):1360-1371.
ATTANE C,FOUSSAL C,LE GONIDEC S,et al.,2012.Apelin treatment increases complete Fatty Acid oxidation,mitochondrial oxidative capacity,and biogenesis in muscle of insulin-resistant mice[J].Diabetes,61(2):310-320.
BAJER B,VLCEK M,GALUSOVA A,et al.,2015.Exercise associated hormonal signals as powerful determinants of an effective fat mass loss[J].Endocr Regul,49(3):151-163.
BESSE P A,MONTASTIER E,Vinel C,et al.,2014.Effect of endurance training on skeletal muscle myokine expression in obese men:identification of apelin as a novel myokine[J].Int J Obes,38(5):707-713.
BERTRAND C,VALET P,CASTANLAURELL I,2015.Apelin and energy metabolism[J].Front Physiol,6:115.
BI F,XU Y,SUN Q,2018.Catalpol pretreatment attenuates cardiac dysfunction following myocardial infarction in rats[J].Anatol JCardiol,19(5):296-302.
BOOTH F W,ROBERTS C K,LAYE M J,2012.Lack of exercise is a major cause of chronic diseases[J].Compr Physiol,2(2):1143-1211.
FALCO M D,LUCA L D,ONORI N,et al.,2002.Apelin expression in normal human tissues[J].In vivo,16(5):333-336.
DRAY C,KNAUF C,DAVIAUD D,et al.,2008.Apelin stimulates glucose utilization in normal and obese insulin-resistant mice[J].Cell Metab,8(5):437-45.
FENTZ J,KJφBSTED R,KRISTENSEN C M,et al.,2015.AMPKαis essential for acute exercise-induced gene responses but not for exercise training-induced adaptations in mouse skeletal muscle[J].Am J Physiol Endocrinol Metab,309(11):E900-914.
FRIER B C,WILLIAMS D B,WRIGHT D C,2009.The effects of apelin treatment on skeletal muscle mitochondrial content[J].Am JPhysiol Regul Integr Comp Physiol,171(297):R1761-1768.
GILBERT J S,2017.From apelin to exercise:emerging therapies for management of hypertension in pregnancy[J].Hypertens Res,40(6):519-525.
INDRAKUSUMA I,SELL H,ECKEL J,2015.Novel Mediators of Adipose Tissue and Muscle Crosstalk[J].Curr Obes Rep,4(4):411-417.
ISABELLE C L,CAMILLE A,THIBAUT D,et al.,2011.Apelin,diabetes,and obesity[J].Endocrine,40(1):1-9.
KATUGAMPOLA S D,MAGUIRE J J,MATTHEWSON S R,et al.,2001.[(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man[J].British journal of pharmacology,132(6):1255-60.
LI J,LI S,WANG F,et al.,2017.Structural and biochemical insights into the allosteric activation mechanism of AMP-activated protein kinase[J].Chem Biol Drug Des,89(5):663-669.
MARCINKOarcinko K,STEINBERG G R,2014.The role of AMPK in controlling metabolism and mitochondrial biogenesis during exercise[J].Exp Physiol,99(12):1581-1585.
MASRI B,KNIBIEHLER B,AUDIGIER Y,2005.Apelin signalling:a promising pathway from cloning to pharmacology[J].Cellular signalling,17(4):415-426.
O'DOWD B F,HEIBEReiber M,Chan A,et al.,1993.A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11[J].Gene,136(1-2):355-360.
OLIVA C R,MARKERT T,GILLESPIE G Y,et al.,2015.Nuclearencoded cytochrome c oxidase subunit 4 regulates BMI1 expression anddetermines proliferative capacity of high-grade gliomas[J].Oncotarget,6(6):4330-4344.
RITCHIE I R,MACDONALDT L,WRIGHT D C,et al.,2014.Adiponectin is sufficient,but not required,for exercise-induced increases in the expression of skeletal muscle mitochondrial enzymes[J].J Physiol,592(12):2653-2665.
ROZYCKA M,KUROWSKA P,GRZESIAK M,et al.,2018.Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3β-hydroxysteroid dehydrogenase(3β-HSD)in pigs[J].Anim Reprod Sci,192:251-260.
SON J S,KIM H J,SON Y,et al.,2017.Effects of exercise-induced apelin levels on skeletal muscle and their capillarization in type 2diabetic rats[J].Muscle Nerve,56(6):1155-1163.
SON J S,SONG A C,TESTROET E D,et al.,2018.Exercise-induced myokines:a brief review of controversial issues of this decade[J].Expert Rev Endocrinol Metab,13(1):51-58.
STROHBACH A,PENNEWITZ M,GLAUBITZ M,et al.,2018.The apelin receptor influences biomechanical and morphological properties of endothelial cells[J].J Cell Physiol,233(8):6250-6261.
TAHERI S,MURPHY K,COHEN M,et al.,2002.The effects of centrally administered apelin-13 on food intake,water intake and pituitary hormone release in rats[J].Biochem Biophys Res Commun,291(5):1208-1212.
THAN A,ZHANG X,LEOW M K,et al.,2014.Apelin attenuates oxidative stress in human adipocytes.[J].J Biol Chem,289(6):3763-3774.
WANG L,DI L,NOGUCHI CT,2014.AMPK is Involved in Mediation of Erythropoietin Influence on Metabolic Activity and Reactive Oxygen Species Production in White Adipocytes[J].Int J Biochem Cell Biol,54(8):1-9.
WU Z,PUIGSERVER P,ANDERSSON U,et al.,1999.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1[J].Cell,98(1):115-124.
YAMAMOTO T,HABATA Y,MATSUMOTO Y,et al.,2011.Apelintransgenic mice exhibit a resistance against diet-induced obesity by increasing vascular mass and mitochondrial biogenesis in skeletal muscle[J].Biochim Biophys Acta,1810(9):853-862.
YANG H,ZHAO L,ZHANG J,et al.,2015.Effect of Treadmill Running on Apelin and APJ Expression in Adipose Tissue and Skeletal Muscle in Rats Fed a High-fat Diet[J].Int J Sports Med,36(7):535-541.
YUE P,JIN H,AILLAUD M,et al.,2010.Apelin is necessary for the maintenance of insulin sensitivity[J].Am J Physiol-Endoc M,298(1):E59-67.
VINEL C,LUKJANENKO L,BATUT A,et al.,2018.The exerkine apelin reverses age-associated sarcopenia[J].Nat Med,24(9):1360-1371.