Induces Pig Adipocyte Browning Through Overexpressing Mice UCP1 And Pig PGC-1α Gene,and Explore The Mechanism

Due to the poor thermogenic ability,piglets need electric heating to survive,which lead to enormous energy and economic loss.The brown fat non-shivering thermogenesis is necessary for most mammals to survive the cold environment.UCP1 is the functional marker of the brown adipose tissue(BAT).The essence of non-shivering thermogenesis in BAT is the adipocytes uncoupling respiration thermogenesis mediated by UCP1 protein.PGC-1α is a key co-transcription factor for mitochondrial biogenesis and mitochondrial energy metabolism.PGC-1 also plays a key role in mammal UCP1 transcription and adaptive thermogenesis.Mammals,such as humans and mice,have brown adipose tissue,but it is not clear if brown adipose tissue exists in swine.The project intends to explore the mechanism of weak thermogenic ability in piglet and repair the non-shivering thermogenesis in piglet.The results will not only resolve the controversy of the existence of porcine brown adipose,but also have implication for analysis obesity related diseases such as diabetes in human.To obtain the monoclonal fetal fibroblasts with mice UCP1 and porcine PGC-1α genes inserted in the H11 locus of its genome used for generating the transgenic pig.These pigs with non-shivering thermogenesis ability will be the important experimental material for the future to cultivate new varieties of energy-saving emission reduction pig.In this study,we first prepare pig UCP1 specific antibody,to examine porcine UCP1 protein expression.And then,induces pig adipocyte browning through overexpressing mouse UCP1 and porcine PGC-1α gene,and explore the mechanism.Finally,construct the homologous recombination vector,obtain the monoclonal fetal fibroblasts with mice UCP1 and porcine PGC-1α genes inserted in the H11 locus of its genome used for generating the transgenic pig,the main results are as follows:1.We found only exons 1 and 2 of the UCP1 gene are transcribed in pig by 3’RACE.We also found UCP1 protein was not translated in various pig tissues in 1-day or 7-day old pig,that means pig have no brown adipose tissue.2.We detected the mitochondrial biogenesis,cell uncoupling respiration rate and mitochondrial energy metabolism in preadipocytes transfected with pc DNA3.1(+),pc DNA3.1(+)-pig UCP1,pc DNA3.1(+)-mice UCP1,pc DNA3.1(+)-PGC-1α,pc DNA3.1(+)-PGC-1α + pc DNA3.1(+)-pig UCP1,pc DNA3.1(+)-PGC-1α + pc DNA3.1(+)-mice UCP1,respectively.Through the transmission electron microscopy observation and Q-PCR analysis,we found mitochondrial biogenesis was increased(P<0.01)in preadipocytes transfected with,pc DNA3.1(+)-PGC-1α,pc DNA3.1(+)-PGC-1α + pc DNA3.1(+)-pig UCP1,pc DNA3.1(+)-PGC-1α + pc DNA3.1(+)-mice UCP1,respectively.Through the WB and Q-PCR analysis,we found PGC-1α increased mitochondrial energy metabolism related gene FABP1,CPT1β,ACOX1,MCAD,Cyt C,ATP Synthase and COX III expression(P<0.01).Through detecting the cell oxygen consumption,we found PGC-1α increased mitochondrial energy metabolism.And after transfection with pc DNA3.1(+)-PGC-1α + pc DNA3.1(+)-mice UCP1,preadipocytes not only obtain uncoupling respiration ability only in preadipocytes,but also the brown/beige adipocyte marker gene Lhx8,Zic1,Cidea,PDK4,CD137 and SLC27a1 expression was increased(P<0.05).In addition,compared with transfected with pc DNA3.1(+)-PGC-1α,preadipocytes transfected with pc DNA3.1(+)-PGC-1α + pc DNA3.1(+)-mice UCP1,the brown/beige adipocyte marker genes PDK4,CD137 and SLC27a1 and mitochondrial energy metabolism related gene CPT-1βand ATP synthase were increased(P<0.05),and Lhx8 was decreased(P<0.05).3.Overexpressing pig PGC-1α promotes mitochondrial biogenesis in pig preadipocytes.In this process,mice UCP1 assembled into the mitochondrial inner membrane exerting function.The uncoupled respiration of functional UCP1 increased p AMPK and SIRT1 protein level.The PGC-1α activity was increased by SIRT1 deacetylation effects.The active transcriptional co-activator PGC-1α interacted with transcription factors Lhx8,Zic1,REEα and PPARα regulated mitochondrial energy metabolism and preadipocytes browning related gene expression.Thus,co-overexpressing mice UCP1 and pig PGC-1α repaired uncoupling respiration in pig preadipocytes.4.Obtain positive monoclonal porcine fetal fibroblasts of porcine PGC-1α and mouse UCP1 gene homologous recombinant in pig genome H11 locus.We first construct the adipose tissue-specific expressing porcine PGC-1α and mouse UCP1 gene homologous recombinant vector,expressing sg RNA and Cas9 protein p X330 vector.The homologous recombination fragment was inserted into porcine adipocyte genome H11 locus by CRISPR/Cas9.We found the homologous recombinant vector could not only express porcine PGC-1α and mouse UCP1 protein,but also the preadipocytes obtain uncoupled respiration ability.The homologous recombination fragments were inserted into the pig fetal fibroblasts genome H11 locus by CRISPR/Cas9,and the positive monoclonal cells were prepared for transgenic pigs.