A Study On The Roles Of MTOR And AMPK Signalings In The Regulatory Effects Of Glutamine On Growth Of Intestinal Porcine Epithelial Cells

This stdudy was conducted to investigate the effect of L-glutamine (Gln) oncell proliferation and viability of intestinal porcine epithelial cells (IPEC-1), anddefine the roles of mTOR and AMPK signalings in the regulation of IPEC-1growthby Gln.1. Effects of amino acids and their derivatives on cellular vitality and cellproliferation of intestinal porcine epithelial cells.To investigate the effects of Gln, Nα-acetyl-L-glutamine (NAG), L-arginine(ARG), N-alpha-acetyl-L-arginine (NAA), L-citrulline (CIT), Alpha-ketoglutaricacid (AKG) and L-Proline (PRO) on the cellular vitality and proliferation ofintestinal porcine epithelial cells(IPEC-1), the IPEC-1cells were incubated with thecorresponding concentrations of amino acids or their derivatives for4days．Thecellular vitality was measured by CCK8(Cell counting kit8), and the cell numberswere recorded. The results showed that: the IPEC-1cell numbers and OD value inGln group were higher than the other groups (P<0.05). On day4of the trial, theIPEC-1cell numbers of NAG, AKG, CIT, NAA, and ARG groups were higher thanthat of the control group (P﹤0.05or0.01). There were no significant alterations inIPEC-1cell numbers by PRO supplementation. In conclusion, Gln is the mosteffective amino acid in increasing the IPEC-1cell numbers and viability; NAG, aswell as AKG, CIT, NAA, and ARG can also promote the cell growth on day4of thetrial.2. Effects of different levels of Gln on cell proliferation of IPEC-1.IPEC-1cells were incubated with the special DMEM medium containingdifferent levels of Gln (0,0.2,0.5,1,2, and5mM) for4days. Cell viability wasmeasured by CCK8kit, and the cell numbers were determined by automatic cellcounter. The results showed that: the optimum level of Gln in culture medium ofIPEC-1is2mM.3. Effects of Gln on mRNA expression of IPEC-1genes. To define the underlying mechanism of increased cell proliferation by Gln,IPEC-1cells were incubated with different levels (as mentioned in Exp.2) of Glnfor3days. mRNA abundance of IPEC-1were determined by RT-PCR. The resultsshowed that: Gln increased the relative mRNA expression of STAT3, CFTR,Na+/K+-ATPase, SGLT-1, ZO-1, pBD1, HSP70, and COX7C (P<0.05), butdown-regulated the gene expressions of Akt, Bcl-2, IGFBP-3, and Sirt1mRNA(P<0.05).4. The role of mTOR signaling in regulation of cell growth by Gln.RAP (rapamycin), an inhibitor of mTOR, was supplementated in the IPEC-1culture medium, and RAP inhibited the growth of IPEC-1. The optimum levels forinhibiting IPEC-1growth of RAP were10nM (3d). The cells were divided into4groups by using a2×2factorial design to define the role of mTOR signaling inregulation of cell growth by Gln. Four goups were:0mM Gln without RAP;2mMGln without RAP;0mM Gln with RAP;2mM Gln with RAP. The experiment lastedfor3days. Similarly, RT-PCR method used to determine mgenes expression ofIPEC-1cells. However, the relative protein expressions of key moleculars involvedin mTOR and AMPK signaling were measured by western blotting. The resultsshowed that:(1) supplementation of Gln alleviate the inhibition of RAP on cellproliferation on days3and4of the trial;(2) Gln upregulated the gene expressionsof AMPK, S6K1,4EBP1, ASCT2, Bax, CFTR, SGLT-1, ZO-1, ODC, eIF2B andCOX7C (P<0.05), whereas downregulated the gene expressions of Akt, eIF2α andMAPK6(P<0.05);(3) RAP treatment enhanced the mRNA expressions of AMPK,mTOR, S6K1,4EBP1, ASCT2, SGLT-1, HSP70, ZO-1, ODC, eIF2B, Bcl-xl, Bcl-2,COX7C and Sirt1(P<0.05), but reduced the mRNA expressions of eIF2α, MAPK6and CFTR (P<0.05);(4) Gln supplementation alleviated the upregulation of RAP onmRNA expressions of4EBP1, HSP70, ZO-1, Bcl-xl, eIF2B and Sirt1(P<0.05);(5)RAP treatment elevated the relative expression of p-mTOR, S6K1, and p-S6K1(P<0.05).5. The role of.AMPK signaling in regulation of cell growth by Gln P5499, a inhibitor of AMPK, was supplemented in IPEC-1culture medium andP5499inhibited the cell growth and vitality of IPEC-1. The optimum level forinhibiting cell growth of P5499was1μM (3d). The trial was divided into4groupsby using a2×2factorial design to ascertain the role of AMPK signaling inregulation of cell growth by Gln, Four goups were:0mM Gln without P5499;2mM Gln without P5499;0mM Gln with P5499;2mM Gln with P5499. The cellswere incubated with the corresponding concentrations of Gln and P5499for3days.Cells were collected to determine relative gene expressions of IPEC-1by RT-PCRand measure the related protein expression of mTOR and AMPK signaling bywestern blotting. The results showed that:(1) supplementation of Gln alleviate theinhibition of P5499on cell proliferation on days3and4of the trial;(2) Glnincreased the mRNA expressions of mTOR, S6K1,4EBP1, CFTR, SGLT-1, HSP70,ZO-1, ASCT2, ODC, Bax, Bcl-2, eIF2B and COX7C (P<0.05) and downregulatedthe expression of Akt and Sirt1mRNA (P<0.05);(3) P5499treatment upregulatedthe mRNA expression of Akt, AMPK, ASCT2and eIF2B (P<0.05), butdownregulatedthe expression of Bcl-2, SGLT-1, HSP70and Bcl-xl mRNA (P<0.05);(4) Gln mitgated the upregulation of P5499on the mRNA expressions of AMPK,CFTR, SGLT-1and ZO-1(P<0.05);(5) Gln enhanced the protein content of AMPK(P<0.05);(6) P5499treatment improved the protein expression of p-AMPK anddecreased the protein content of phosphorylated mTOR (P<0.05).In conclusion, the optimum level of Gln in IPEC-1culture medium is2mM;2mM Gln supplementation enhanced the mRNA expressions of4EBP1, CFTR,SGLT-1, Na+/K+-ATPase, ZO-1, HSP70, COX7C and Bax of IPEC-1, whereasdecreased the gene expressions of Akt, IGFBP3, MAPK6and Sirt1mRNA; Glnregulates the growth of IPEC-1may through the expression of4EBP1、S6K1andAMPK.