The Study Of Physiological Function Of Mitochondria Uncoupling Protein 3 And The Regulation Of Its Expression

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.