The Effect Of ASIP Gene On Bovine Lipid Metabolism Related Traits

ASIP(Agouti signaling protein),as a secreted protein,can interact with receptors including MC1 R,MC4R and ATRN to influence the coat color and lipid metabolism in mice by regulating cellular cAMP content.So far,there are rare reports related to the effect of ASIP gene on the lipid metabolism of ruminant annimals.One study showed that the L1-BT element inserted at upstream of ASIP 5' UTR can increase the mRNA level of ASIP in cattle.But the influence of L1-BT element on animal meat and milk quality is still unknown.In the previous study,we found the mRNA level of ASIP is significantly different between Japanese Black and Red Step cattle.Hence,in this study,we mainly focused on the effect of ASIP gene on bovine lipid metabolism.To analyze the association between bovine lipid metabolism and ASIP gene and L1-BT element inserted upstream of ASIP,ASIP and its receptor genes,MC4 R,MC1R and ATRN were detected and analyzed with the transcription,translation level and cellular localization.RT-qPCR results indicated that a heterozygous L1-BT element inserted upstream ASIP 5' UTR in 17 bulls can lead to an ecotopic overexpression of ASIP mRNA compared to other individuals(P < 0.05).The mRNA level of individuals with L1-BT element did not show the significant associations with bovine carcass traits.However,the ASIP mRNA level in subcutaneous fat of bulls without L1-BT element had significantly positive correlations with fat deposition related traits.We selected 20 bulls with high carcass fat(HCF)and low carcass fat(LCF)and assigned them to HCF and LCF groups.In different adipose tissues,subcutaneous fat had a higher ASIP mRNA amount(P < 0.05).And the mRNA level of ASIP in subcutaneous fat of HCF bull is significantly higher than that in LCF bulls(P < 0.05).The protein of ASIP could not be detected in tissues of three groups of bulls by western.Immunohistochemistry result suggested that ASIP protein located inside or outside of adipocyte.No ASIP signal could be found in muscle tissue.In liver,ASIP protein could be observed in stellate cells.These results indicated that the insertion of L1-BT element can cause for the ASIP mRNA overexpression,but not ASIP protein.And there is no association between heterozygous L1-BT element and bovine carcass traits.Moreover,ASIP mRNA amount in subcutaneous tissue was significantly associated with the fat deposition related traits.To illustrate the effect of ASIP on lipid metabolism in bovine adipocyte,we used the optimized serum-free culture medium contained 10 nmol/L recombinant ASIP protein to culture the bovine preadipocyte,and then induced and differentiated the preadipocyte to mature adipocyte.Results showed that after two days treatment with rec.ASIP protein,the ASIP mRNA level and cellular triglyseride content were significantly increased compared to control group(P < 0.05).8 induction days later,in the rec.ASIP treatment group mRNA level of the adipogenesis related genes,PPAR?,CEBP?,ADIPOQ,LEPTIN,and STAT3 and the fatty acide metabolism related gene SCL27A6 were increased(P < 0.05).These results suggested that ASIP protein can improve the accumulation of triglyceride in bovine preadipocyte and influence the expression level of lipid metabolim related genes in bovine adipocyte.To demonstrate whether ASIP affected the bovine milk fat metabolism,we treated bovine mammary epithelial cells(bMEC)with 10 nmol/L rec.ASIP protein and established knock-out ASIP bMEC.The expression level of related genes and cellular content of triglyseride and cholesterol were detected and analyzed.Results showed that ASIP mRNA level did not alter after rec.ASIP protein treatment,but MC1 R and ATRN mRNA amount were elevated(P < 0.05).In the rec.ASIP treatment group mRNA level of the adipogenesis related genes,CEBP?,STAT3,PPARGC1?,and LPL and the fatty acide metabolism related gene ACOT6 b,SCL27A6,PRKACB,FABP4 were significantly higher than control group(P < 0.05).Moreover,the cellular content of triglyseride and cholesterol were raised compared to control group.In the KO-ASIP bMEC,ASIP mRNA was downregulated and the cellular cholesterol was decreased(P < 0.05).Results above indicated that ASIP protein could also promote the lipid accumulation and related genes expression in bovine mammary epithelial cells.To apply ASIP gene in the further breeding procedures,we sreened and analyzed the ASIP SNPs and L1-BT element with fat deposition related traits and milk quality traits in Chinese Simmental steers and Chinese Holstein population,respectively.In Chinese Simmental steers,L1-BT indel(g.-14643 indel L1-BT element)and three SNP(g.-568A>G,g.-554A>T,g.-4805A>T)located in intron of ASIP gene were detected.Among them,g.-568A>G,g.4805A>T and L1-BT indel were significantly associated with bovine carcass and fat-relate traits.e.g.live weight and back fat thickness(P < 0.05).Individuals with homozygous L1-BT element insertion had thinner back fat thickness and higher fat coverage rate(P < 0.05).The linkage disequilibrium analysis of g.-568A>G and g.4805A>T indicated 3 haplotypes(H1: AT,H2: AA,H3: GT)which formed 6 haplotype combinations.Individuals with H1H2 genotype had a higher back fat thickness and carcass fat coverage rate,buta lower content of linoleic acid and ?-linolenic acid(P < 0.05)in M.longissimus dorsi.H3H3 genotype steers possessed with a lower marbling score,perirenal fat weight and carcass weight.In 88 Chinese Holstein cattle,L1-BT element upsteam ASIP gene could not be detected and only one SNP,g.4805A>T,was found.However,there is no association between g.4805A>T and milk quality traits.In conclusion,ASIP could regulate adipogenesis and fatty acid metabolism genes expression and influence the lipid metabolism in bovine preadipocyte and mammary epithelial cell.The SNPs and their haplotype combinations associated with meat quality traits could provide the molecular theory for the breeding of cattle with high quality beef in the future.