乳酸/GPR81信号通路对运动诱导肌细胞甘油三酯堆积的调节作用
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摘要
目的:旨在观察乳酸/GPR81信号通路是否参与调节电脉冲刺激(electricalpulsestimulation,EPS)诱发C2C12肌管细胞收缩诱导甘油三酯(triglyceride,TG)堆积的过程。方法:首先用乳酸处理C2C12肌管细胞,比色法检测细胞中TG含量,WesternBlot方法检测CREB、pCREB和β-actin蛋白含量;然后采用两种EPS方案(30 V 0.5 Hz 2 ms和30 V 1 Hz 2 ms)使C2C12肌管细胞收缩,比色法检测细胞中乳酸含量,化学发光法检测细胞中ATP含量,RT-PCR法检测MyHCI、MyHCIIa、MyHCIIb和MyHCIIx等m RNA含量;随后观察EPS处理后不同时间点C2C12肌管细胞中TG含量的变化,以建立EPS诱发的C2C12肌管细胞TG堆积模型;最后使用GPR81抑制剂3-OBA处理细胞,将C2C12肌管细胞分为6组(n=6):对照组、30 V 0.5 Hz 2 ms组、30 V 1 Hz 2 ms组、3-OBA组、3-OBA+30 V 0.5 Hz 2 ms组和3-OBA+30 V 1 Hz 2 ms组,比色法检测细胞内TG含量。结果:1)与对照组相比,乳酸显著增加C2C12肌管细胞中TG含量(P<0.05)并显著降低p CREB的蛋白含量(P<0.01);2)与未电刺激组相比,两种EPS方案均显著增加肌管细胞中乳酸含量(P<0.05),而显著降低细胞中ATP含量(P<0.01);同时,两组MyHCI和MyHCIIb mRNA含量均显著增加(P<0.05),而MyHCIIa和MyHCIIx mRNA含量变化不明显(P>0.05);在电刺激后36h,细胞中TG含量均显著增加(P<0.05),而电刺激后12h和24h肌管细胞中TG含量未出现显著变化(P>0.05);3)与未刺激组相比,30 V 0.5 Hz 2 ms组C2C12肌管细胞中TG含量出现增加趋势(P=0.052),当使用3-OBA处理细胞后,这一现象无显著变化(P>0.05);而30V1Hz2ms组TG含量出现非常显著增加(P<0.01),当使用3-OBA处理后,这一作用得到显著减弱(P<0.05)。结论:长时间的EPS能够诱导肌管细胞收缩,增加乳酸和TG含量。乳酸/GPR81信号通路参与调节EPS诱发C2C12肌管细胞收缩诱导TG堆积的过程是骨骼肌细胞收缩诱导能源物质脂滴增加的分子机制之一。
Objective: To investigate whether lactate/GPR81 signal pathway is involved in the regulation of skeletal muscle contraction-induced lipid droplets accumulation. Methods: C2C12 cells were treated with lactate firstly, then the cells' triglyceride(TG) content was determined by colorimetric method. The expression levels of CREB, pCREB and β-actin protein were detected by Western Blot. After that, C2C12 cells were treated by two kinds of electric pulse stimulation(EPS) protocol(i.e., 30 V 0.5 Hz 2 ms and 30 V 1 Hz 2 ms). The content of lactate was determined by colorimetric method, the level of ATP was detected by chemiluminescence method, and the mR NA level of MyHCI, MyHCIIa, MyHCIIb and MyHCIIx were detected by RT-PCR. Subsequently, the changes of TG content in C2C12 myotubes at different time points after EPS treatment were observed, in order to establish the model of EPS-induced TG accumulation in C2C12 myotubes. Finally, the cells were treated with GPR81 inhibitor 3-hydroxy-butyrate(3-OBA) and divided into six groups: control group, 30 V 0.5 Hz 2 ms group, 30 V 1 Hz 2 ms group, 3-OBA group, 3-OBA+30 V 0.5 Hz 2 ms group and 3-OBA+30 V 1 Hz 2 ms group. The content of TG was detected by chemiluminescence method. Results: 1) Compared with control group, the lactate increased the content of TG in C2 C12 myotubes(P<0.05) and decreased the pCREB signaling status(P<0.01) significantly; 2) Compared with control group, two EPS protocols increased the lactate concentration(P<0.05) and reduced the ATP content(P<0.01) in C2C12 cells significantly; the mRNA levels of MyHCI and MyHCIIb were significantly increased in these two groups, but the mRNA level of MyHCIIa and MyHCIIx were unchanged(P>0.05); the TG content was significantly increased at 36 h after EPS(P<0.05), but the TG content was unchanged at 12 h and 24 h(P>0.05); 3) Compared with non-EPS group, the TG content in 30 V 0.5 Hz 2 ms group was increased(P=0.052), but there was no significant change when the 3-OBA was applied(P>0.05); the TG content in 30 V 1 Hz 2 ms group was significantly increased(P<0.05), but the TG content was significantly reduced after the treatment of 3-OBA(P<0.05). Conclusions: Long-time EPS induced the contraction of the myotubes and increased the contents of lactate and TG. Lactate/GPR81 signaling pathway mediated the C2C12 myotubes cell contraction-induced TG accumulation, and it is one of the molecular mechanisms in skeletal muscle cell contraction-induced lipid droplets accumulation of energy substrate.
Objective: To investigate whether lactate/GPR81 signal pathway is involved in the regulation of skeletal muscle contraction-induced lipid droplets accumulation. Methods: C2C12 cells were treated with lactate firstly, then the cells' triglyceride(TG) content was determined by colorimetric method. The expression levels of CREB, pCREB and β-actin protein were detected by Western Blot. After that, C2C12 cells were treated by two kinds of electric pulse stimulation(EPS) protocol(i.e., 30 V 0.5 Hz 2 ms and 30 V 1 Hz 2 ms). The content of lactate was determined by colorimetric method, the level of ATP was detected by chemiluminescence method, and the mR NA level of MyHCI, MyHCIIa, MyHCIIb and MyHCIIx were detected by RT-PCR. Subsequently, the changes of TG content in C2C12 myotubes at different time points after EPS treatment were observed, in order to establish the model of EPS-induced TG accumulation in C2C12 myotubes. Finally, the cells were treated with GPR81 inhibitor 3-hydroxy-butyrate(3-OBA) and divided into six groups: control group, 30 V 0.5 Hz 2 ms group, 30 V 1 Hz 2 ms group, 3-OBA group, 3-OBA+30 V 0.5 Hz 2 ms group and 3-OBA+30 V 1 Hz 2 ms group. The content of TG was detected by chemiluminescence method. Results: 1) Compared with control group, the lactate increased the content of TG in C2 C12 myotubes(P<0.05) and decreased the pCREB signaling status(P<0.01) significantly; 2) Compared with control group, two EPS protocols increased the lactate concentration(P<0.05) and reduced the ATP content(P<0.01) in C2C12 cells significantly; the mRNA levels of MyHCI and MyHCIIb were significantly increased in these two groups, but the mRNA level of MyHCIIa and MyHCIIx were unchanged(P>0.05); the TG content was significantly increased at 36 h after EPS(P<0.05), but the TG content was unchanged at 12 h and 24 h(P>0.05); 3) Compared with non-EPS group, the TG content in 30 V 0.5 Hz 2 ms group was increased(P=0.052), but there was no significant change when the 3-OBA was applied(P>0.05); the TG content in 30 V 1 Hz 2 ms group was significantly increased(P<0.05), but the TG content was significantly reduced after the treatment of 3-OBA(P<0.05). Conclusions: Long-time EPS induced the contraction of the myotubes and increased the contents of lactate and TG. Lactate/GPR81 signaling pathway mediated the C2C12 myotubes cell contraction-induced TG accumulation, and it is one of the molecular mechanisms in skeletal muscle cell contraction-induced lipid droplets accumulation of energy substrate.
引文
AAS V, TORBLA S, ANDERSEN M H,et al., 2002. Electrical stimulation improves insulin responses in a human skeletalmuscle cell model of hyperglycemia[J]. Ann N Y Acad Sci, 967:506-515.
AHMED K, TUNARU S, TANG C, et al., 2010. An autocrine lactate loop mediates insulin-dependent inhibition of lipolysis through GPR81[J]. Cell Metab, 11(4):311-319.
AM ATI F, DUBE J J, ALVAREZ-CARNERO E, et al., 2011. Skeletal muscle triglycerides, diacylglycerols, and ceramides in insulin resistance:Another paradox in endurance-trained athletes?[J].Diabetes, 60(10):2588-2597.
BJORNTORP P, 1965. The effect of lactic acid on adipose tissue metabolism in vitro[J]. Acta Med Scand, 178(2):253-255.
BOZZO L, PUYAL J, CHATTON J Y, 2013. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway[J]. PLoS One, 8(8):e71721.
BURCH N, ARNOLD A S, ITEM F, et al., 2010. Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle[J]. PLoS One, 5(6):e10970.
CAI T Q, REN N, JIN L, et al., 2008. Role of GPR81 in lactatemediated reduction of adipose lipolysis[J]. Biochem Biophys Res Commun, 377(3):987-991.
CHEETHAM M E, BOOBIS L H, BROOKS S, et al., 1986. Human muscle metabolism during sprint running[J]. J Appl Physiol(1985),61(1):54-60.
CORI C F, SHINE W M, 1935. The formation of carbohydrate from glycerophosphate in the liver of the rat[J]. Science, 82(2119):134-135.
FEINGOLD K R, MOSER A, SHIGENAGA J K,et al.,2011.Inflammation inhibits GPR81 expression in adipose tissue[J].Inflamm Res, 60(10):991-995.
FUJITA H, NEDACHI T, KANZAKI M, 2007. Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12myotubes[J]. Exp Cell Res, 313(9):1853-1865.
GOODPASTER B H, HE J, WATKINS S, et al., 2001. Skeletal muscle lipid content and insulin resistance:Evidence for a paradox in endurance-trained athletes[J]. J Clin Endocrinol Metab, 86(12):5755-5761.
HOPPELER H, HOWALD H, CONLEY K, et al., 1985. Endurance training in humans:Aerobic capacity and structure of skeletal muscle[J]. J Appl Physiol(1985), 59(2):320-327.
HUGHSON R L, WEISIGER K H, SWANSON G D, 1987. Blood lactate concentration increases as a continuous function in progressive exercise[J]. J Appl Physiol(1985), 62(5):1975-1981.
HWANG K A, HWANG Y J, KIM G R, et al., 2015. Extracts from Aralia elata(Miq)Seem alleviate hepatosteatosis via improving hepatic insulin sensitivity[J]. BMC Complement Altern Med, 15:347.
ISSEKUTZ B J, MILLER H I, PAUL P, et al., 1965. Effect of lactic acid on free fatty acids and glucose oxidation in dogs[J]. Am J Physiol, 209(6):1 137-1144.
KAMIJO Y, TAKENO Y, SAKAI A, et al., 2000. Plasma lactate concentration and muscle blood flow during dynamic exercise withnegative-pressure breathing[J]. J Appl Physiol(1985), 89(6):2196-2205.
KUEI C, YU J,ZHU J,et al.,2011. Study of GPR81, the lactate receptor, from distant species identifies residues and motifs critical for GPR81 functions[J]. Mol Pharmacol, 80(5):848-858.
LAM T K, GUTIERREZ-JUAREZ R, POCAI A, et al., 2005.Regulation of blood glucose by hypothalamic pyruvate metabolism[J]. Science, 309(5736):943-947.
LANGIN D, 2010. Adipose tissue lipolysis revisited(again!):Lactate involvement in insulin antilipolytic action[J]. Cell Metab, 11(4):242-243.
LAURITZEN F, EID T, BERGERSEN L H, 2015. Monocarboxylate transporters in temporal lobe epilepsy:Roles of lactate and ketogenic diet[J]. Brain Struct Funct, 220(1):1-12.
LAURITZEN K H, MORLAND C, PUCHADES M, et al., 2014.Lactate receptor sites link neurotransmission, neurovascular coupling, and brain energy metabolism[J]. Cereb Cortex, 24(10):2784-2795.
LIU C, WU J, ZHU J, et al., 2009. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor,GPR81[J]. J Biol Chem, 284(5):2811-2822.
MILLER H I,ISSEKUTZ B J,RODAHL K,et al.,1964. Effect of lactic acid on plasma free fatty acids in pancreatectomized dogs[J].Am J Physiol, 207:1226-1230.
MORGAN T E, SHORT F A,COBB L A, 1969. Effect of long-term exercise on skeletal muscle lipid composition[J]. Am J Physiol,216(1):82-86.
NEDACHI T, FUJITA H, KANZAKI M, 2008. Contractile C2C12myotube model for studying exercise-inducible responses in skeletal muscle[J]. Am J Physiol Endocrinol Metab, 295(5):E1191-E1204.
NEDACHI T, HATAKEYAMA H,KONG T, et al.,2009.Characterization of contraction-inducible CXC chemokines and their roles in C2C12 myocytes[J]. Am J Physiol Endocrinol Metab, 297(4):E866-E878.
NIKOLIC N,BAKKE S S,KASE E T,et al.,2012. Electrical pulse stimulation of cultured human skeletal muscle cells as an in vitro model of exercise[J]. PLoS One, 7(3):e33203.
NIKOOIE R, SAMANEH S. 2016.Exercise-induced lactate accumulation regulates intramuscular triglyceride metabolism via transforming growth factor-beta 1 mediated pathways[J]. Mol Cell Endocrinol, 419:244-251.
OHKUWA T, KATO Y, KATSUMATA K, et al., 1984. Blood lactate and glycerol after 400-m and 3,000-m runs in sprint and long distance runners[J]. Eur J Appl Physiol Occup Physiol, 53(3):213-218.
PARK H, BHALLA R, SAIGAL R, et al., 2008. Effects of electrical stimulation in C2C12 muscle constructs[J]. J Tissue Eng Regen Med,2(5):279-287.
PETTE D, DUSTERHOFT S, 1992. Altered gene expression in fasttwitch muscle induced by chronic low-frequency stimulation[J]. Am J Physiol, 262(3 Pt 2):R333-R338.
PETTE D, STARON R S, 2000. Myosin isoforms, muscle fiber types,and transitions[J]. Microsc Res Tech, 50(6):500-509.
PETTE D, STARON R S, 2001. Transitions of muscle fiber phenotypic profiles[J]. Histochem Cell Biol, 115(5):359-372.
PRUCHNIC R, KATSIARAS A, HE J, et al., 2004. Exercise training increases intramyocellular lipid and oxidative capacity in older adults[J]. Am J Physiol Endocrinol Metab, 287(5):E857-E862.
SAKURAI T, DAVENPORT R, STAFFORD S, et al., 2014.Identification of a novel GPR81-selective agonist that suppresses lipolysis in mice without cutaneous flushing[J]. Eur J Pharmacol,727:1-7.
SHEN Z, JIANG L, YUAN Y, et al., 2015. Inhibition of G proteincoupled receptor 81(GPR81)protects against ischemic brain injury[J]. CNS Neurosci Ther, 21(3):271-279.
SILVEIRA L R, PILEGAARD H, KUSUHARA K, et al., 2006. The contraction induced increase in gene expression of peroxisome proliferator-activated receptor(PPAR)-gamma coactivator 1 alpha(PGC-1 alpha), mitochondrial uncoupling protein 3(UCP3)and hexokinaseⅡ(HKII)in primary rat skeletal muscle cells is dependent on reactive oxygen species[J]. Biochim Biophys Acta,1763(9):969-976.
SPANGENBURG E E, BOOTH F W, 2003. Molecular regulation of individual skeletal muscle fibre types[J]. Acta Physiol Scand, 178(4):413-424.
THELEN M H, SIMONIDES W S, Van HARDEVELD C, 1997.Electrical stimulation of C2C12 myotubes induces contractions and represses thyroid-hormone-dependent transcription of the fast-type sarcoplasmic-reticulum Ca2+-ATPase gene[J]. Biochem J, 321(Pt 3):845-848.
AHMED K, TUNARU S, TANG C, et al., 2010. An autocrine lactate loop mediates insulin-dependent inhibition of lipolysis through GPR81[J]. Cell Metab, 11(4):311-319.
AM ATI F, DUBE J J, ALVAREZ-CARNERO E, et al., 2011. Skeletal muscle triglycerides, diacylglycerols, and ceramides in insulin resistance:Another paradox in endurance-trained athletes?[J].Diabetes, 60(10):2588-2597.
BJORNTORP P, 1965. The effect of lactic acid on adipose tissue metabolism in vitro[J]. Acta Med Scand, 178(2):253-255.
BOZZO L, PUYAL J, CHATTON J Y, 2013. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway[J]. PLoS One, 8(8):e71721.
BURCH N, ARNOLD A S, ITEM F, et al., 2010. Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle[J]. PLoS One, 5(6):e10970.
CAI T Q, REN N, JIN L, et al., 2008. Role of GPR81 in lactatemediated reduction of adipose lipolysis[J]. Biochem Biophys Res Commun, 377(3):987-991.
CHEETHAM M E, BOOBIS L H, BROOKS S, et al., 1986. Human muscle metabolism during sprint running[J]. J Appl Physiol(1985),61(1):54-60.
CORI C F, SHINE W M, 1935. The formation of carbohydrate from glycerophosphate in the liver of the rat[J]. Science, 82(2119):134-135.
FEINGOLD K R, MOSER A, SHIGENAGA J K,et al.,2011.Inflammation inhibits GPR81 expression in adipose tissue[J].Inflamm Res, 60(10):991-995.
FUJITA H, NEDACHI T, KANZAKI M, 2007. Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12myotubes[J]. Exp Cell Res, 313(9):1853-1865.
GOODPASTER B H, HE J, WATKINS S, et al., 2001. Skeletal muscle lipid content and insulin resistance:Evidence for a paradox in endurance-trained athletes[J]. J Clin Endocrinol Metab, 86(12):5755-5761.
HOPPELER H, HOWALD H, CONLEY K, et al., 1985. Endurance training in humans:Aerobic capacity and structure of skeletal muscle[J]. J Appl Physiol(1985), 59(2):320-327.
HUGHSON R L, WEISIGER K H, SWANSON G D, 1987. Blood lactate concentration increases as a continuous function in progressive exercise[J]. J Appl Physiol(1985), 62(5):1975-1981.
HWANG K A, HWANG Y J, KIM G R, et al., 2015. Extracts from Aralia elata(Miq)Seem alleviate hepatosteatosis via improving hepatic insulin sensitivity[J]. BMC Complement Altern Med, 15:347.
ISSEKUTZ B J, MILLER H I, PAUL P, et al., 1965. Effect of lactic acid on free fatty acids and glucose oxidation in dogs[J]. Am J Physiol, 209(6):1 137-1144.
KAMIJO Y, TAKENO Y, SAKAI A, et al., 2000. Plasma lactate concentration and muscle blood flow during dynamic exercise withnegative-pressure breathing[J]. J Appl Physiol(1985), 89(6):2196-2205.
KUEI C, YU J,ZHU J,et al.,2011. Study of GPR81, the lactate receptor, from distant species identifies residues and motifs critical for GPR81 functions[J]. Mol Pharmacol, 80(5):848-858.
LAM T K, GUTIERREZ-JUAREZ R, POCAI A, et al., 2005.Regulation of blood glucose by hypothalamic pyruvate metabolism[J]. Science, 309(5736):943-947.
LANGIN D, 2010. Adipose tissue lipolysis revisited(again!):Lactate involvement in insulin antilipolytic action[J]. Cell Metab, 11(4):242-243.
LAURITZEN F, EID T, BERGERSEN L H, 2015. Monocarboxylate transporters in temporal lobe epilepsy:Roles of lactate and ketogenic diet[J]. Brain Struct Funct, 220(1):1-12.
LAURITZEN K H, MORLAND C, PUCHADES M, et al., 2014.Lactate receptor sites link neurotransmission, neurovascular coupling, and brain energy metabolism[J]. Cereb Cortex, 24(10):2784-2795.
LIU C, WU J, ZHU J, et al., 2009. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor,GPR81[J]. J Biol Chem, 284(5):2811-2822.
MILLER H I,ISSEKUTZ B J,RODAHL K,et al.,1964. Effect of lactic acid on plasma free fatty acids in pancreatectomized dogs[J].Am J Physiol, 207:1226-1230.
MORGAN T E, SHORT F A,COBB L A, 1969. Effect of long-term exercise on skeletal muscle lipid composition[J]. Am J Physiol,216(1):82-86.
NEDACHI T, FUJITA H, KANZAKI M, 2008. Contractile C2C12myotube model for studying exercise-inducible responses in skeletal muscle[J]. Am J Physiol Endocrinol Metab, 295(5):E1191-E1204.
NEDACHI T, HATAKEYAMA H,KONG T, et al.,2009.Characterization of contraction-inducible CXC chemokines and their roles in C2C12 myocytes[J]. Am J Physiol Endocrinol Metab, 297(4):E866-E878.
NIKOLIC N,BAKKE S S,KASE E T,et al.,2012. Electrical pulse stimulation of cultured human skeletal muscle cells as an in vitro model of exercise[J]. PLoS One, 7(3):e33203.
NIKOOIE R, SAMANEH S. 2016.Exercise-induced lactate accumulation regulates intramuscular triglyceride metabolism via transforming growth factor-beta 1 mediated pathways[J]. Mol Cell Endocrinol, 419:244-251.
OHKUWA T, KATO Y, KATSUMATA K, et al., 1984. Blood lactate and glycerol after 400-m and 3,000-m runs in sprint and long distance runners[J]. Eur J Appl Physiol Occup Physiol, 53(3):213-218.
PARK H, BHALLA R, SAIGAL R, et al., 2008. Effects of electrical stimulation in C2C12 muscle constructs[J]. J Tissue Eng Regen Med,2(5):279-287.
PETTE D, DUSTERHOFT S, 1992. Altered gene expression in fasttwitch muscle induced by chronic low-frequency stimulation[J]. Am J Physiol, 262(3 Pt 2):R333-R338.
PETTE D, STARON R S, 2000. Myosin isoforms, muscle fiber types,and transitions[J]. Microsc Res Tech, 50(6):500-509.
PETTE D, STARON R S, 2001. Transitions of muscle fiber phenotypic profiles[J]. Histochem Cell Biol, 115(5):359-372.
PRUCHNIC R, KATSIARAS A, HE J, et al., 2004. Exercise training increases intramyocellular lipid and oxidative capacity in older adults[J]. Am J Physiol Endocrinol Metab, 287(5):E857-E862.
SAKURAI T, DAVENPORT R, STAFFORD S, et al., 2014.Identification of a novel GPR81-selective agonist that suppresses lipolysis in mice without cutaneous flushing[J]. Eur J Pharmacol,727:1-7.
SHEN Z, JIANG L, YUAN Y, et al., 2015. Inhibition of G proteincoupled receptor 81(GPR81)protects against ischemic brain injury[J]. CNS Neurosci Ther, 21(3):271-279.
SILVEIRA L R, PILEGAARD H, KUSUHARA K, et al., 2006. The contraction induced increase in gene expression of peroxisome proliferator-activated receptor(PPAR)-gamma coactivator 1 alpha(PGC-1 alpha), mitochondrial uncoupling protein 3(UCP3)and hexokinaseⅡ(HKII)in primary rat skeletal muscle cells is dependent on reactive oxygen species[J]. Biochim Biophys Acta,1763(9):969-976.
SPANGENBURG E E, BOOTH F W, 2003. Molecular regulation of individual skeletal muscle fibre types[J]. Acta Physiol Scand, 178(4):413-424.
THELEN M H, SIMONIDES W S, Van HARDEVELD C, 1997.Electrical stimulation of C2C12 myotubes induces contractions and represses thyroid-hormone-dependent transcription of the fast-type sarcoplasmic-reticulum Ca2+-ATPase gene[J]. Biochem J, 321(Pt 3):845-848.