【作者】 逄金柱；

【导师】 杨则宜； Dawei Gong；

【作者基本信息】 北京体育大学 ， 运动人体科学， 2011， 博士

【摘要】 目的：研究C2C12细胞超表达UCP3及共超表达CPT1对脂肪酸氧化、活性氧产生和胰岛素敏感性的影响,以及脂肪酸、WY14643、亮氨酸和AICAR对UCP3表达的影响。方法：(1)UCP3和CPT1重组腺病毒感染C2C12细胞后测试油酸氧化速率和2-脱氧葡萄糖摄取速率及活性氧产生水平；(2)不同物质干预C2C12细胞后,实时荧光定量PCR测试UCP3mRNA表达水平。结果：(1)与GFP相比,感染UCP3组、CPTla组及共感染UCP3和CPTla组,脂肪酸氧化增加62.8%(P<0.01),80.2%(P<0.001)、168.4%(P<0.001),与感染UCP3组、CPTla组和共感染UCP3和GFP组比,共感染UCP3和CPTla组脂肪酸氧化增加64.8%(P<0.001)、48.9%(P<0.001)、63.0%(P<0.001)；(2)在没有肉碱或含有爱克莫舍时,感染UCP3组脂肪氧化下降39.6%(P<0.05)、43.8%(P<0.01)；(3)感染UCP3组、CPTla组及共感染UCP3和CPTla组在施加胰岛素后比GFP组2-脱氧葡萄糖摄取速率增加25.8%(P<0.01)、13.3%(P<0.05)及21.8%(P<0.05)；(4)感染UCP3组、CPTla组及共感染UCP3和CPTla组比感染GFP组活性氧生成增加39.3%(P<0.01),54.1%(P<0.001)及53.7%(P<0.001)；(5)与0.75mM辛酸相比,0.75mM油酸组UCP3 mRNA增加117.9%(P<0.001)；(6)与丙氨酸相比,WY14643、亮氨酸以及亮氨酸和WY14643共同干预组,UCP3 mRNA表达分别增加63.2%(P<0.001)、41.0%(P<0.01)以及66.7%(P<0.001)。(7)AICAR干预2小时后,UCP3 mRNA表达水平增加(P<0.001)。结论：超表达UCP3能增加脂肪酸氧化水平和胰岛素敏感性,但没有减少活性氧产生；CPT1具有协同促进UCP3增加脂肪酸氧化水平的作用；长链脂肪酸能增加UCP3表达但中链脂肪酸对其没有影响；WY14643、亮氨酸和短时间AICAR干预能增加UCP3表达。更多还原

【Abstract】 Purpose:To study the effect of overexpression of UCP3 in C2C12 myotubes on fatty acid oxidation and the production of reactive oxygen species, especially under the condition of co-overexpression of CPT1. And to study the effect of fatty acids, WY14643, leucine and AICAR on the expression of UCP3.Methods:(1) To examine oleic acid oxidation rate and 2-deoxyglucose uptake rate by isotope method and to examine the production of superoxide by DHE fluorescence probe in C2C12 myotubes which were infected with UCP3 recombinant adenovirus and CPTla recombinant adenovirus which were prepared by cloning technique. (2) To examine the expression of UCP3 mRNA by real time RT-PCR in in C2C12 myotubes which are treated with fatty acids, WY14643, leucine and AICAR.Results:(1) Fatty acid oxidation was significantly increased by 62.8%(P< 0.01),80.2%(P< 0.001) respectively after infected with Adv-UCP3, Adv-CPTla, and increased by 168.4%(P<0.001) when co-infected cells with Adv-UCP3 and Adv-CPTla compared with corresponding Adv-GFP-infected myotubes. And more interestingly, fatty acid oxidation was markedly increased by 64.8%(P<0.001),48.9%(P<0.001),63.0%(P <0.001) when co-infected with Adv-UCP3 and Adv-CPTla compared with Adv-UCP3, Adv-CPTla, co-Adv-UCP3-Adv-GFP infected myotubes respectively. (2) Furthermore, fatty acid oxidation was reduced by 39.6%(P<0.05) and 43.8%(P<0.01) respectively in the absence of carnitine (activator of CPT1) and in the presence of etomoxir (inhibitor of CPT1) even upregulated expression of UCP3. (3) 2-deoxyglucose uptake was significantly increased by 25.8%(P<0.01),13.3%(P<0.05) and 21.8% (P<0.05) respectively after infected with Adv-UCP3, Adv-CPTla, and co-infected with Adv-UCP3 and Adv-CPTla compared with corresponding Adv-GFP infected myotubes under the treatment of insulin. (4) On the other hand, over-expression of UCP3 and CPTla in C2C12 myotubes also increased the production of reactive oxygen species by 39.3%(P<0.01),54.1%(P < 0.001)after infected with Adv-UCP3, Adv-CPTla, and increased by 53.7%(P<0.001) when co-infected Adv-UCP3 with Adv-CPTla. (5) UCP3 mRNA and CPT1a mRNA were increased by 117.9% (P<0.001) and 117.2%(P<0.001) respectively compared with the treatment of 0.75mM caprylic acid. (6) Compared with the treatment of alanine, UCP3 mRNA was increased by 63.2%(P <0.001),41.0%(P<0.01) and 66.7%(P<0.001) and fatty acid oxidation was increased by 23.2%(P<0.01),21.2%(P<0.001) and 35.7%(P<0.001) under the treatment of WY14643 (agonist of PPAR a), leucine and co-treatment of WY14643 and leucine respectively. (7) UCP3 mRNA and fatty acid oxidation were increased significantly (P<0.001) after 2 hours under the treatment of AICAR, but UCP3 mRNA was not changeable after 6 hours and 24 hours treatment of AICAR.Conclusion:Overexpression of UCP3 may significantly increase fatty acid oxidation and insulin sensitivity, but don’t decrease the production of reactive oxygen species. CPT1 promote the effect of UCP3 on increasing fatty acid oxidation.long-chain fatty acid but not medium-chain fatty acid increased the expression of UCP3. WY14643 and leucine increased the expression of UCP3 and the capacity of fatty acid oxidation while AICAR involve in this role at the early stage of its treatment.更多还原