Effect Of Leucine On Nutritional Transport Of Pig Placenta And Mammary Epithelial Cells And Regulation Mechanism

In this study,sow placenta and mammary epithelial cells were used as in vivo model and in vitro model,respectively,to explore the effect of leucine(Leu)on nutrient transport of sow-pigs and its regulation mechanism.The placenta samples of this test were taken from 200 sows on the 70 th day of pregnancy,randomly divided into 4 groups,and leucine containing 0,0.4%,0.8% and 1.2% proportions were added to their feed;pMEC is selected from the porcine mammary epithelial cell line isolated and cultured in our laboratory in the early stage,divided into 4 treatment groups,add Leu separately to achieve final concentrations of 0,1,5,and 10 mmol/L.MTT method was used to detect changes in pMEC cell viability;oil red O staining was used to detect pMEC lipid droplet synthesis;Detection of milk protein related genes,amino acid,glucose and fatty acid transporter and key protein genes of mTOR signaling pathway in pMEC by real-time fluorescence quantitative PCR.The main findings are as follows:1.The effect of diet supplemented with leucine in late pregnancy on sow placenta nutrient transport(1)0.4% and 0.8% Leu group significantly increased the mRNA expression of SLC7A1,SLC7A2,SLC7A8 and SLC7A11 in the sow placenta amino acid transporter(P<0.05);and0.8% Leu group significantly increased the mRNA expression level of SLC15A1,SNAT1 and SNAT2(P<0.05);the 1.2% Leu group significantly increased the mRNA expression levels of SLC7A8,SLC15A1,LAT1 and r BAT(P<0.05).(2)0.8% Leu group significantly increased the expression of glucose transporter GLUT1 and SGLT3 mRNA(P<0.05);1.2% Leu group significantly increased the expression of SGLT5 mRNA(P<0.05).(3)0.4% Leu group significantly increased the expression of fatty acid transporter FATP2,FATP4,FABP2,FABP4 and FABP5 mRNA(P<0.05);0.8% Leu group significantly increased the expression of CD36,FATP1 and FATP2 mRNA(P<0.05);1.2% Leu group significantly increased the expression of FATP2 mRNA(P<0.05).(4)Compared with the control group,the 0.4% and 0.8% leucine groups significantly increased the protein phosphorylation of mTOR in the placenta(P<0.05);and the protein phosphorylation of S6K1 in each treatment group was significantly increased(P<0.05).2.The effect of different concentrations of leucine on pMEC nutrient transport(1)After 48 hours of treatment,the pMEC cell viability of each treatment group was significantly increased(P<0.05),(2)The expression of CSN1S2 and CSN3 gene mRNA in the 1 mmol/L group was significantly increased(P<0.05);the expression of CSN1S2 gene mRNA in the 5 mmol/L group was significantly increased(P<0.05).(3)The 1 mmol/L group significantly increased the expression levels of amino acid transporters SLC1A4,SLC1A5,SNAT1,SLC7A11,LAT1,SLC7A2,and SLC7A7 mRNA(P<0.05);the 5 mmol/L group significantly increased SLC1A4,SNAT1,SLC7A11,LAT1,SLC7A2,SLC7A7,4F2 hc and r BAT mRNA expression(P<0.05);10 mmol/L group significantly increased SLC1A4,SLC1A5,SLC7A11,LAT1,SLC7A7,4F2 hc and r BAT mRNA expression(P<0.05).(4)The 5mmol/L and 10mmol/L groups significantly reduced the expression of GLUT8 mRNA(P<0.05).(5)The 1 mmol/L group significantly increased FATP1,FATP2,FABP3,FABP5,FABP7,and ACBP mRNA expression(P<0.05);the 5 mmol/L group significantly increased CD36,FATP1,FATP2 and FABP7 mRNA expression(P<0.05);10 mmol/L group significantly increased the expression of CD36 and FATP1 mRNA(P<0.05).(6)Treatment of pMEC with 1 mmol / L Leu significantly increased the phosphorylation of S6K1 protein(P<0.05);however,the phosphorylation expression of mTOR protein in each group was not significantly different from that of the control group(P>0.05).Based on the above results,supplementing 0.8% Leu in the diet of sows in late pregnancy can promote the expression of mRNA associated with placental amino acid transporter and glucose transporter through the mTOR signaling pathway,and promote the placenta’s intake of fatty acids.The addition of Leu to pMEC could significantly increase cell viability,expression of milk protein genes CSN1S2 and CSN3 mRNA,and promote the expression of amino acid transporter and fatty acid transmembrane transporter mRNA.