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The Role Of Glucocorticoid Receptor Pathway In Mice Lipopenia Induced By Conjuated Linoleic Acid And Erhualian Pigs Of High Cortisol Tolerance

Posted on:2014-08-12Degree:MasterType:Thesis
Country:ChinaCandidate:X L QuFull Text:PDF
GTID:2250330425952801Subject:Basic veterinary science
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As a food additive widely used, conjugated linoleic acid (CLA)can cause metabolic disorders like lipodystrophy in mice. We previously found thatCLA resulted in aberrant lipolysis though reducing plin1expression significantly inmouse adipose tissue. The purpose of this experiment was to further explore themechanism on CLA-induced fat loss by reducing plin1expression. We studed theglucocorticoid receptor(GR) which is the upstream of plin1mediating singal pathway.The specific methods are six weeks old kunming male mice were randomized intotwo groups (n=12) with one group receiving a diet supplemented with LA (1.5%w/w)and another receiving a diet with CLA (1.5%w/w) for13days and then mearued thecortisol concentration in the plasma, in addtion,we mearued the mRNA expression ofthe GR gene in epididymal fat tissue(EPI).We also cloned the GR and Plin1genespromoter and let these promoters control the luciferase activity and then structuredthe recombination plasmids pGL3-GR and pGL3-Plin1(GRE). In Hela cellstimulating dexamethasone(DEX) after the transient transfection the pGL3-GR,andthen mearued the GR promoter activity after add the LA/CLA(100uM) in the mediawhich transfected GR promoter of stimulation. We mearued the plin1promoteractivity after DEX stimulating the transfected pGL3-Plin1(GRE) cell. Compared tothe control group, CLA-fed mice showed43%loss of fat mass(P<0.01), significantdecrease of plasma cortisol(P<0.01). In adipose tissues of CLA-fed mice comparedto that in control mice, significant decrease of mRNA levels of GR was found (P<0.01).The result of luciferase assay showed that the transfection GR promoter of Hela, DEX can make significantly increased the activity of the GR promoter (P <0.05), but the CLA addition can inhibit this response. This expriment showedDEX induced the plin1(GRE) promoter activity.Through the above results can beconcluded that DEX as activator of Plin1gene can make Plin1higher expression, andcortisol levels drop, CLA decreased the expression of GR, in the process of signaltransduction of Plin1, GR as its upstream signaling molecules inhibit the expressionof Plin1in the process of CLA induced abnormal metabolism of fat, thus the decreaseof the epididymal adipose tissue in mice. The combination of glucocorticoid(GC) plays an important role inthe physiological metabolism bycombitation glucocorticoid receptor(GR).Accordingto some reports,erhualian(EHL) and pietrain(PIE) pigs have different stress responsesduring transport. PIE pigs have stronger responses,and EHL pigs don not exhibitapparent responses,and the plasma cortisol levels of EHL pigs is much higher thanPIE pigs by detecting plasma cortisol levels of this two breeds pigs. In normalcondition, the plasma cortislol levels of EHL pigs are about twice as much as LW pigsin other reports. This two different experiments show together the high cortisoltolerance in the GR signaling pathway. Therefore,the experiment is to study themechanism of EHL pig high cortisol tolerance. Methods: we can detect plasmacortisol levels after executing some4-5weeks EHL and PIE pigs. In the GR signalingpathway, P65gene can reduce the GR target gene expression though combining someprotein,as a subunit of NF-KB gene. So we detect the difference of mRNA level ofGR gene and P65gene by RT-qPCR with livers of EHL and large white(LW) pigs. Inorder to further research the mechanism of EHL pigs high cortisol.e had also clonedpromoter of plin1gene in the reverse of GR gene. This promoter contains GREsequence,and we structured the pGL3-pigplin1(GRE)recombinant plasmid. And letthe promoter control the luciferase expression. DEX stimulated the Hela cell aftertransfection the promoter and measured the luciferase activity. We cotransfected theP65and Plin1promoter in the Hela cell and then stimulate the cell with DEX,measured the luciferase activity.Results: The plasma cortisol level of EHL pigswas significantly higher than LW pigs, and the former was about as twice as thelater(P<0.05); On the mRNA level, GR gene and P65gene of EHL pigs’livers weresignificantly higer than LW pigs’.(P<0.05); The luciferase system found that on thecells level, P65gene lowered activity of promoter of GR gene (P<0.05). Results showthat EHL pigs high cortisol torlerance phenomenon may be associated with theincrease of P65gene.
Keywords/Search Tags:CLA, Plin1, GR, GREglucocorticoid, glucocorticoid receptor, high cortisol tolerance, P65, target gene
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