Cattle SHH Gene Regulating Adipogenesis Through PPARg Pathway

Quantitative PCR, gene clone, prokaryotic expression, protein purification, cell culture andeukaryotic expression were used to analyze the regulatory mechanism of SHH gene andPPARg pathway comprehensively. The research contents includes expression patterns ofcandidate genes in fat tissue from different parts of cattle, effecting of cattle SHH inadipogenesis, relationship between SHH gene and PPARg pathway, effecting of PPARg inadipogenesis of intramuscular fat, identification the promoter of PPARg gene and the geneticvariation analysis of PPARg gene in cattle herds. The main results including:1. The formation and differentiation of preadipocytes affect the fat deposition from differentparts of body.Quantitative PCR were used to test the expression patterns of candidate genes in thesubcutaneous fat, abdominal fat, pleural fat and intermuscular fat. The results showed that theexpressions of triglyceride metabolism-related genes including HSL, CIDEC, Perilipin, Leptinand Glut4were higher in the abdominal fat and intermuscular fat than the pleural fat andsubcutaneous fat, and the expressions of adipose differentiation-related genes includingFABP4, PPARg and DLK1are lower in the subcutaneous than the intermuscular fat,abdominal fat and pleural fat,suggesting that the adipose metabolism level, differentiationstatus and differentiation potential are different from different parts of cattle, and alsosuggenting that the formation and differentiation of preadipocytes affect the fat depositionfrom different parts of body.2. Recombinant bovine SHH protein can inhibit the differentiation of preadipocytesOverlap PCR was used to clone the SHH gene of Qinchuan cattle, and the specificity ofrecombinant SHH protein purified from prokaryotic expression was tested by western blotting.Recombinant SHH protein could inhibit the differentiation of3T3-L1cells at theconcentration of1μg/mL, indicating that the recombinanat bovine SHH protein processes theinhibiting activity on adipocyte differentiation.3. Cattle SHH inhibits adipocyte differentiation through PPARg pathwayCattle PPARg gene was cloned. Overexpression of PPARg could induce the adipocytedifferentiation, and co-overexpression of SHH and PPARg could rescue the inhibition of SHHon adipocyte differentiation, suggesting that SHH inhibits adipocyte differentiation throughPPARg pathway. Quantitative PCR analysis showed that overexpression of SHH could induce the expression of Nr2f2, while overexpression of Nr2f2could inhibit the expression of PPARgduring adipocyte differentiation. Co-overexpression of Nr2f2and PPARg could rescue theinhibition of Nr2f2on the adipocyte differentiation, suggesting that SHH is able to inhibit theexpression of PPARg by increasing the expression of Nr2f2, thereby inhibiting adipocytedifferentiation.4. Cloning and activity identification of PPARg promoterA novel dual color fluorescent reporter system was established by cloning a Discosoma redfluorescent protein gene and a green fluorescent protein gene into a single vector. This dualcolor fluorescent reporter system allows real time quantitative monitoring of promoter activity.Transcriptional activity of PPARg promoter was identified, the results showed that the PPARgpromoter located at the-446bp to-196bp from the transcriptional start site. This promoterdrives weak transcriptional activity in preadipocyte, and its transcriptional activity increasessignificantly during the adipocyte differentiation.5. Overexpression of PPARg in the bovine primary muscle cells contributes to the formationof intramuscular fatAdenovirus vector of PPARg (Ad-PPARg) was packed successfully, and infection with theAd-PPARg in the cattle primary muscle cells showed that, the expressions of adipogenesisup-regulating genes including FABP4, Glut4and AdPLA increased1.9,1.5and1.6times,respectively, while the expression of adipogenesis down-regulating genes including GATA2,HSL and Nr2f2decreased0.5,0.3and0.6times, suggesting that overexpression of PPARg inthe bovine primary muscle cells contributes to the formation of intramuscular fat.6. Asp7Gly mutation damages the inducing adipogenesis of PPARgPCR-SSCP, PCR-RFLP and sequencing were used to detect the genetic variations of PPARggene in Qinchuan cattle, Nanyang cattle, Jiaxian Red cattle and China Holstein cattle, and theassociations between the PPARg variations and cattle body measurements were analyzed. Theresults showed that four SNPs (-110G>C,-27C>T,+20A>G and+1344G>T) were detected inthe PPARg gene in Qinchuan cattle, Nanyang cattle and Jiaxian Red cattle rather than ChinaHolstein cattle. The mutations-110G>C,-27C>T and+20A>G under linkage disequilibriumwere associated with smaller body weight, heart girth and body length of cattle. The mutation+20A>G changed the7thamino acid of PPARg from Asp to Gly, so called PPARg Asp7GLy.Overexpression of the two haplotypes of PPARg in preadipocytes showed that, Asp7Glydamages the transcriptional activity of PPARg, thereby decreases its inducing adipogenesis.These findings may explains the associations results of the mutations. PPARg Asp7Gly can beused as a molecular marker in cattle breeding practices.