Study On Lysine Transports Response Law By Different Types Of Protein In The Small Intestines Of Weaned Piglets And MTOR Signaling Pathway Regulation In IEPC Cells

Dietary protein is the maximum number of nutrient substance required by animals playing an important role in their metabolism and life. For non-ruminant animals, the key to protein nutrition is to study the absorption and metabolism of amino acid transport. As the first limiting amino acid to pigs, Lysine adequate intake can regulate the utilization of other amino acids to increase protein synthesis to promote the growth of piglets. Thus, the transportation and absorption of Lysine is particularly important.With people’s higher awareness of energy conservation and emission reduction, the application value research on low-protein diet has become a focus. However, the reasonable distribution of low-protein diet has many difficulties at the same time, which not only considers the mutual effect between amino acid and other substances to confirm the amount of needs of amino acid but also considers the balance of amino acid in the ideal protein model. Three experiments were carried out as follows.Experiment 1: Effect of low protein diet with different protein sources on the expression of major lysine transporters in the small intestine of weaned piglets. The diets were formulated by net energy, and 9 kinds of crystalline amino acids were added to balance essential amino acids in low protein diets(CP14%). Protein sources included soybean meal, cottonseed meal, soybean-cottonseed meal and fish meal, and then soybean meal group was a control group. The adjustment period lasted for 7 days, and the experiment period for 28 days. The CAT-1, b0,+AT and y+LAT1 mRNA expression abundances were investigated by real-time PCR using tissue samples of duodenum, anterior jejunum, Post-jejunum and ileum collected from piglets fed 4 treatment diets. The results showed that the mRNA abundance values of CAT-1, b0,+AT and y+LAT1 in all segments of cottonseed meal group were lower than those of other treatment groups. The mRNA abundance of CAT-1(in the Post-jejunum and ileum), b0,+AT(in the jejunum and ileum), y+LAT1(in the anterior-jejunum and ileum) in the soybean-cottonseed meal group showed no difference from that in the soybean meal group and fish meal group(P > 0.05). Although in low protein diety( CP14%), piglets also could gain better lysine transport in the jejunum and ileum when 50% of the SBM in their diet is replaced by CSM and 9 kinds of crystalline amino acids were added to the diets to balance essential amino acids.Experiment 2: Effect of lysine levels on the abundance expression of major lysine transporters in porcine epithelial cell line. Cells were cultured in six-well plates in DMEM/F12 medium supplemented with 8%(vol/vol) fetal bovine serum, 1% Penicilin-Streptomycin Solution at 37°C with humidified conditions with 5%CO2. After reaching 98% confluence, cells were starved for 2 h in an lysine-deprived medium with Earle’s Balanced Salt Solution(EBSS) and other amino acids mixture. After starvation, cells were cultured in the presence of 0, 0.5, 1.5, 4.5 or 13.5 mM lysine, respectively. After 6 or 12 h, cells were collected to examine the mRNA expression of CAT-1, b0,+AT and y+LAT1 with real-time PCR. The results showed that the addition of lysine could increase the mRNA expression of CAT-1, y+LAT1 and b0,+AT, and they were the highest in 1.5mM. These data showed that the mRNA expression of CAT-1, y+LAT1 and b0,+AT were regulated in the range of animal needs.Experiment 3: Mechanism on mTOR upstream signaling transduction pathway medicated by amino acid levels to select suitable Lysine level.Cells from experiment 2 were used to extract total RNA and total protein to detect mTOR, Rag, Rheb or TSC1/2 mRNA and mTOR, Rag or Rheb protein expression by real-time PCR and Western Blot. The results show: high concentrations of lysine had significant up-regulation effect on the mTOR, Rag, Rheb, TSC1/2 mRNA expression and there was an obvious dose-dependent effect(P < 0.05), but mTOR, Rheb, TSC1/2 mRNA expression had significant down-regulation effect in the 4.5mM lysine. Simultaneously, with the prolonging of treatment time, all target genes mRNA expression of 12 h group had significant down-regulation effect except Rheb than that of 6h group. Furthermore, in the 6h group, mTOR, Rag and Rheb protein expression had resembled their mRNA expression trends. Partial correlation analysis showed that there was significant positive correlation between Rheb and mTOR, RagC and Rheb; RagA and Rheb had significant negative correlation. However, RagA and mTOR did not show significant correlation. The ratio of phosphorylated mTOR and total mTOR results showed an obvious dose-dependent effect from 0 mM to 4.5 mM lysine in the 6h culture, and there were no difference between 1.5 and 4.5mM lysine(P > 0.05). However, the phosphorylation of mTOR can not be found at 13.5mM lysine. These data showed that Rags needed to cooperate with Rheb to regulate the growth of cells though lysine activating mTOR complex 1, and 1.5mM lysine was the most suitable concentration that Rags and Rheb activated mTOR complex 1 to regulate protein synthesis by lysine.