天麻素在L6肌管中促进葡萄糖消耗的作用和机制研究
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摘要
目的研究天麻素(GSTD)在L6肌管中促进葡萄糖消耗的作用和可能机制。方法体外培养大鼠L6骨骼肌细胞并诱导分化成肌管,血清饥饿后以GSTD单独处理或与浓度为0.05 nmol/L的胰岛素联合处理24 h,以二甲双胍(Met)为阳性对照药。部分实验中L6肌管先以浓度为100 nmol/L的胰岛素处理24 h以诱导胰岛素抵抗,再进行药物处理。以试剂盒测定细胞糖消耗、L-乳酸释放和ATP产生,以Western blot和实时荧光定量反转录PCR检测目标蛋白和基因的表达水平。结果 GSTD浓度依赖性地促进L6肌管的基础糖消耗[与对照细胞比较,差异有统计学意义(P <0.05或P <0.01)],并且显著增加胰岛素刺激的糖消耗[与单用胰岛素或GSTD处理的细胞比较,差异有统计学意义(P <0.05)]。在胰岛素抵抗状态下,浓度为500μmol/L的GSTD仍能有效促进细胞糖消耗(P <0.05)。Met处理后显著增加细胞的L-乳酸释放并抑制ATP产生[与对照细胞比较,差异有高度统计学意义(P <0.01)],但GSTD没有作用。GSTD显著上调L6肌管GLUT4蛋白和m RNA的表达,并且激活AMPK和Akt通路,表现为处理后p-AMPKα和p-Akt的水平显著增加[与对照细胞比较,差异有统计学意义(P <0.05或P <0.01)]。结论 GSTD作用于L6肌管显著增加其糖消耗并能克服胰岛素抵抗,其机制可能与GLUT4的上调以及AMPK和Akt通路的激活有关。
Objective To investigate the activities and possible mechanisms of gastrodin(GSTD) to promote glucose consumption in L6 myotubes. Methods Rat L6 skeletal muscle cells were cultured in vitro and differentiated into myotubes. After serum starvation, GSTD was used to treat the myotubes for 24 h, either alone or in combination with insulin at a concentration of 0.05 nmol/L. Metformin(Met) was used a positive control drug. In some experiments, L6 myotubes were treated with 100 nmol/L of insulin for 24 h to induce insulin resistance before drug treatment. Commercial kits were used to determine cell glucose consumption, L-lactate release, and ATP production. The expression levels of target proteins and genes were determined by Western blot and real-time quantitative reverse transcription-PCR(RT-PCR). Results GSTD promoted the basal glucose consumption of L6 myotubes in a concentration-dependent manner [comparing with control cells, the diferences were statistically significant(P < 0.05 or P < 0.01)]. In addition, GSTD greatly increased the insulin-stimulated glucose consumption [comparing with cells treated with insulin or GSTD alone,the diferences were statistically significant(P < 0.05)]. In a state of insulin resistance, GSTD at a concentration of 500μmol/L was still effective to promote cell glucose consumption(P < 0.05). After treatment, Met significantly increased cellular L-lactate release but decreased ATP production [comparing with control cells, the diferences were highly statistically significant(P < 0.01)]. However, GSTD did not have such effects. GSTD greatly up-regulated the protein and mRNA expression levels of GLUT4 in L6 myotubes. In addition, it activated the AMPK and Akt pathways, as indicatedby the significant increase of p-AMPKα and p-Akt levels after treatment [comparing with control cells, the diferences were statistically significant(P < 0.05 or P <0.01)]. Conclusion When used to treat L6 myotubes,GSTD greatly increases glucose consumption and is able to overcome insulin resistance. The mechanisms may be associated with the up-regulation of GLUT4 as well as the activation of AMPK and Akt pathways.
Objective To investigate the activities and possible mechanisms of gastrodin(GSTD) to promote glucose consumption in L6 myotubes. Methods Rat L6 skeletal muscle cells were cultured in vitro and differentiated into myotubes. After serum starvation, GSTD was used to treat the myotubes for 24 h, either alone or in combination with insulin at a concentration of 0.05 nmol/L. Metformin(Met) was used a positive control drug. In some experiments, L6 myotubes were treated with 100 nmol/L of insulin for 24 h to induce insulin resistance before drug treatment. Commercial kits were used to determine cell glucose consumption, L-lactate release, and ATP production. The expression levels of target proteins and genes were determined by Western blot and real-time quantitative reverse transcription-PCR(RT-PCR). Results GSTD promoted the basal glucose consumption of L6 myotubes in a concentration-dependent manner [comparing with control cells, the diferences were statistically significant(P < 0.05 or P < 0.01)]. In addition, GSTD greatly increased the insulin-stimulated glucose consumption [comparing with cells treated with insulin or GSTD alone,the diferences were statistically significant(P < 0.05)]. In a state of insulin resistance, GSTD at a concentration of 500μmol/L was still effective to promote cell glucose consumption(P < 0.05). After treatment, Met significantly increased cellular L-lactate release but decreased ATP production [comparing with control cells, the diferences were highly statistically significant(P < 0.01)]. However, GSTD did not have such effects. GSTD greatly up-regulated the protein and mRNA expression levels of GLUT4 in L6 myotubes. In addition, it activated the AMPK and Akt pathways, as indicatedby the significant increase of p-AMPKα and p-Akt levels after treatment [comparing with control cells, the diferences were statistically significant(P < 0.05 or P <0.01)]. Conclusion When used to treat L6 myotubes,GSTD greatly increases glucose consumption and is able to overcome insulin resistance. The mechanisms may be associated with the up-regulation of GLUT4 as well as the activation of AMPK and Akt pathways.
引文
[1]韩磊,乔爱敏,刘青.天麻素的抗糖尿病作用实验[J].华侨大学学报:自然科学版,2013,34(6):682-686.
[2]张勇,武燕彬,孔维佳.天麻素在KK-Ay小鼠中抗糖尿病作用的实验研究[J].中国药理学通报,2018,34(7):917-924.
[3]Shan YQ,Ren G,Wang YX,et al.Berberine analogue IMB-Y53 improves glucose-lowering efficacy by averting cellular efflux especially P-glycoprotein efflux[J].Metabolism,2013,62(3):446-456.
[4]Xu M,Xiao Y,Yin J,et al.Berberine promotes glucose consumption independently of AMP-activated protein kinase activation[J].PLoS One,2014,9(7):e103702.
[5]Liu LZ,Cheung SC,Lan LL,et al.Berberine modulates insulin signaling transduction in insulin-resistant cells[J].Mol Cell Endocrinol,2010,317(1/2):148-153.
[6]蒋根灵,李庆林.天麻素通过下调ERK1/2-p38信号通路保护谷氨酸损伤的PC12细胞[J].安徽中医学院学报,2013,32(3):71-75.
[7]Qu LL,Yu B,Li Z,et al.Gastrodin ameliorates oxidative stress and proinflammatory response in nonalcoholic fatty liver disease through the AMPK/Nrf2 pathway[J].Phytother Res,2016,30(3):402-411.
[8]Yang P,Han Y,Gui L,et al.Gastrodin attenuation of the inflammatory response in H9c2 cardiomyocytes involves inhibition of NF-κB and MAPKs activation via the phosphatidylinositol 3-kinase signaling[J].Biochem Pharmacol,2013,85(8):1124-1133.
[9]Richter EA,Hargreaves M.Exercise,GLUT4,and skeletal muscle glucose uptake[J].Physiol Rev,2013,93(3):993-1017.
[10]Hajiaghaalipour F,Khalilpourfarshbafi M,Arya A.Modulation of glucose transporter protein by dietary flavonoids in type 2 diabetes mellitus[J].Int J Biol Sci,2015,11(5):508-524.
[11]Day EA,Ford RJ,Steinberg GR.AMPK as a therapeutic target for treating metabolic diseases[J].Trends Endocrinol Metab,2017,28(8):545-560.
[12]Lee JO,Lee SK,Kim JH,et al.Metformin regulates glucose transporter 4(GLUT4)translocation through AMP-activated protein kinase(AMPK)-mediated Cbl/CAP signaling in 3T3-L1 preadipocyte cells[J].J Biol Chem,2012,287(53):44121-44129.
[13]Waldhart AN,Dykstra H,Peck AS,et al.Phosphorylation of TXNIP by AKT mediates acute influx of glucose in response to insulin[J].Cell Rep,2017,19(10):2005-2013.
[14]Rowland AF,Fazakerley DJ,James DE.Mapping insulin/GLUT4 circuitry[J].Traffic,2011,12(6):672-681.
[15]Kim MJ,Yang HJ,Moon BR,et al.Gastrodia elata Blume rhizome aqueous extract improves arterial thrombosis,dyslipidemia,and insulin response in testosterone-deficient rats[J].Evid Based Complement Alternat Med,2017,2017:2848570.
[16]白永,范晓明,郭家智,等.天麻素对胰岛素抵抗模型中CBS表达的影响[J].昆明医科大学学报,2014,35(4):6-9.
[2]张勇,武燕彬,孔维佳.天麻素在KK-Ay小鼠中抗糖尿病作用的实验研究[J].中国药理学通报,2018,34(7):917-924.
[3]Shan YQ,Ren G,Wang YX,et al.Berberine analogue IMB-Y53 improves glucose-lowering efficacy by averting cellular efflux especially P-glycoprotein efflux[J].Metabolism,2013,62(3):446-456.
[4]Xu M,Xiao Y,Yin J,et al.Berberine promotes glucose consumption independently of AMP-activated protein kinase activation[J].PLoS One,2014,9(7):e103702.
[5]Liu LZ,Cheung SC,Lan LL,et al.Berberine modulates insulin signaling transduction in insulin-resistant cells[J].Mol Cell Endocrinol,2010,317(1/2):148-153.
[6]蒋根灵,李庆林.天麻素通过下调ERK1/2-p38信号通路保护谷氨酸损伤的PC12细胞[J].安徽中医学院学报,2013,32(3):71-75.
[7]Qu LL,Yu B,Li Z,et al.Gastrodin ameliorates oxidative stress and proinflammatory response in nonalcoholic fatty liver disease through the AMPK/Nrf2 pathway[J].Phytother Res,2016,30(3):402-411.
[8]Yang P,Han Y,Gui L,et al.Gastrodin attenuation of the inflammatory response in H9c2 cardiomyocytes involves inhibition of NF-κB and MAPKs activation via the phosphatidylinositol 3-kinase signaling[J].Biochem Pharmacol,2013,85(8):1124-1133.
[9]Richter EA,Hargreaves M.Exercise,GLUT4,and skeletal muscle glucose uptake[J].Physiol Rev,2013,93(3):993-1017.
[10]Hajiaghaalipour F,Khalilpourfarshbafi M,Arya A.Modulation of glucose transporter protein by dietary flavonoids in type 2 diabetes mellitus[J].Int J Biol Sci,2015,11(5):508-524.
[11]Day EA,Ford RJ,Steinberg GR.AMPK as a therapeutic target for treating metabolic diseases[J].Trends Endocrinol Metab,2017,28(8):545-560.
[12]Lee JO,Lee SK,Kim JH,et al.Metformin regulates glucose transporter 4(GLUT4)translocation through AMP-activated protein kinase(AMPK)-mediated Cbl/CAP signaling in 3T3-L1 preadipocyte cells[J].J Biol Chem,2012,287(53):44121-44129.
[13]Waldhart AN,Dykstra H,Peck AS,et al.Phosphorylation of TXNIP by AKT mediates acute influx of glucose in response to insulin[J].Cell Rep,2017,19(10):2005-2013.
[14]Rowland AF,Fazakerley DJ,James DE.Mapping insulin/GLUT4 circuitry[J].Traffic,2011,12(6):672-681.
[15]Kim MJ,Yang HJ,Moon BR,et al.Gastrodia elata Blume rhizome aqueous extract improves arterial thrombosis,dyslipidemia,and insulin response in testosterone-deficient rats[J].Evid Based Complement Alternat Med,2017,2017:2848570.
[16]白永,范晓明,郭家智,等.天麻素对胰岛素抵抗模型中CBS表达的影响[J].昆明医科大学学报,2014,35(4):6-9.