| The study was to investigate how the methionine sources regulate the protein synthesis of porcine mammary gland and the related mechanism through culture of mammary cell and mammary tissue in vitro,and measurement of the enzymes'activities special for the conversion of methionine sources among tissues to further discuss the potential utilization by mammary gland of sows.The study designed four experiments:Exp 1 Primary culture of porcine mammary epithelial cellTwo methods including tissue explants and enzyme digestion were conducted in the primary culture of porcine mammary epithelial cell.The cells which were got from the two methods both had the characteristic keratin and the characteristic morphology of mammary cells,and were generated to the 10th generation whose morphology was not different from 1st generation.The above results showed that the method of primary culture of porcine mammary epithelial cells was successfully established.Exp 2 Different methionine sources on the regulation of signaling pathway which is responsible for protein synthesis of porcine mammary epithelial cellsThe mammary epithelial cells which is in the state of methionine deprivation were cultured for 24h with following medium:Negative control(with no methionine sources)or 0.6mM of any methionine source[L-methionine(L-MET),D-methionine(D-MET),DL-2-hydroxy-4-(methylthio)butyric acid(HMTBA),L-MET+D-MET(70:30),L-MET+HMTBA(70:30)].The cells were harvested to measure mRNA abundance of the CSN1S1(?s1 casein)gene with Real-time PCR method,and protein abundance of mechanistic Target of Rapamycin(mTOR),Ribosome protein S6 kinase 1(S6K1),Eukaryotic translation initiation factor 4E-binding protein 1(4EBP1)and their corresponding phosphorylated protein with Western Blotting.The results are as follows:1)Compared with the NC group,L-MET,HMTBA,L-MET+D-MET andL-MET+HMTBA group's CSN1S1 mRNA abundance significantly upregulated(P<0.05),but the D-MET group showed no significant change(P>0.05).2)Compared with NC group,pmTOR and mTOR protein of L-MET+HMTBA group were significantly higher(P<0.05),and mTOR protein of L-MET+D-MET group was significantly higher(P<0.05)while the pmTOR protein has a tendency to improve(P<0.10).However,the L-MET and D-MET group showed no significant changes(P>0.10).3)Compared with NC group,pS6K1 protein abundance of L-MET,HMTBA,L-MET+D-MET and L-MET+HMTBA group were significantly higher(P<0.05);p4EBP1 protein abundance was higher in the L-MET,HMTBA,L-MET+D-MET and L-MET+HMTBA group(P<0.05).However,D-MET had no influence on the protein abundance of pS6Kl and p4EBP1(P>0.10).The above results showed that,adequate L-MET and its precursor HMTBA can activate the mTOR signaling of porcine mammary epithelial cells and may promote the synthesis of mammary protein.Exp 3 Different methionine sources on the regulation of protein synthesis of porcine mammary gland tissueBased on Exp 2,mammary tissue slices which were taken from lactating sows were cultured for 2h in vitro with the following medium:no methionine sources(Negative Control)or 0.1 mM methionine sources(L-MET,D-MET,HMTBA and KMB).Fractional protein synthesis rate were measured with the stable isotype(1-13C Phe)enrichment,and the related protein(mTOR,S6K1 and 4EBP1)abundance of signaling pathways that regulate protein synthesis were measured with Western Blotting.The results were as follows:1)Compared with NC group,the FSR of L-MET and HMTBA group were significantly higher(P<0.05)while the D-MET and KMB group showed no significant changes(P>0.05);2)Compared with NC group,the mTOR,pmTOR,pS6K1 and p4EBPl protein of L-MET and HMTBA group were significantly higher(P<0.05)while these protein of D-MET group showed no significant changes(P>0.05).3)In the mammary tissue slices after culture,compared with NC group,the free methionine concentration of L-MET,HMTBA and KMB group were both significantly higher(P<0.05),and D-MET didn't change(P>0.05).The above results showed that increasing the addition of L-MET and its precursor HMTBA in mammary tissue can both improve the mammary protein synthesis rate and the improved rate may be related to the activation of mTOR signaling pathway induced by L-MET and HMTBA.Exp 4 Distribution of enzymes' activities special for the conversion of methionine sources among sow's tissuesThe experiment aimed to determine the conversion ability and potential utilization of methionine sources by the mammary gland,used D-MET and HMTBA as substrate,and evaluated the enzymes' activities special for the conversion of methionine sources by the generated amount of KMB.The main resuls are as follows:1)For the D-MET conversion,Kidney had the highest enzyme's activity,followed by liver and duodenum,and the mammary gland had the lowest enzyme's activity,10%of the liver's.2)For the HMTBA conversion,liver had the highest enzymes'activity,followed by muscle and fat tissue,and the enzymes' activity of mammary gland were 50%of liver's.The above results indicated that,the sow's tissues have the basic biochemistry condition to utilize different methionine sources and the mammary tissue have a relatively higher enzymes' activity to convert HMTBA and a relatively lower enzymes' activity to convert D-MET,which can further explain the difference between mTOR signaling activation and protein synthesis rate caused by different methionine sources.In conclusion,the research established the method of primary culture of porcine mammary epithelial cell,found the different protein synthesis rates caused by different methionine sources,proved that increasing the addition of L-MET and its precursor HMTBA can both improve the protein synthesis rate of mammary gland,and revealed that the mechanism may be related to the activation of mTOR signaling pathway and the enzymes' activity special for conversion of methionine sources. |
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