Branched-Chain Amino Acids Modulate Amino Acid And Glucose Transport In Intestine And Muscle Of Piglets

Four experiments were conducted in this study to investigate the effects of branched-chain amino acid (BCAA) on intestinal amino acid transporters, and intestinal and muscular glucose transporters in weanling piglets. The mechanism of amino acid transporter expression in intestinal epithelial cells regulated by BCAA was studied in vitro. (1) In Exp.1, one hundred and eight Large White x Landrace ± Duroc piglets (7.97±0.11 kg) were randomly assigned to 1 of 3 treatments and fed a normal protein diet (NP,20.9% crude protein), a low protein diet (LP,17.1% crude protein), or a low protein diet supplemented with BCAA (LP+BCAA,17.9%crude protein), respectively. The results showed that dietary protein restriction reduced growth performance and small intestinal villus height in piglets, while the performance and villus height were restored in pigs fed the NP diet or the LP+BCAA diet (P < 0.05). There was no significant difference in the performance and villus height between the NP treatment and the LP+BCAA treatment (P>0.05). mRNA levels for ASCT2, CAT1, b0,+ AT and 4F2hc were more abundant in piglets fed the LP+BCAA diet than those in LP diet (P< 0.05). However, mRNA and protein levels for PepT1 were lower in piglets fed the LP+BCAA diet as compared to those fed the LP diet (P< 0.05). (2) In Exp.2, we studied the transcriptional and translational regulation of BCAA on amino acid transporter production in IPEC-J2 cells and the signaling pathways involved. Treatment of IPEC-J2 cells with 7.5 mM leucine enhanced mRNA expression of ASCT2 and 4F2hc and caused an increase in ASCT2 protein expression (P< 0.05). Leucine also activated phosphorylation of 4E-BP1 and eIF4E through the mTOR, Akt and ERK signaling pathways in IPEC-J2 cells (P< 0.05). Pre-treatment of IPEC-J2 cells with inhibitors of mTOR and Akt (rapamycin and wortmannin) or an inhibitor of ERK (PD098059) for 30 min before leucine treatment attenuated the positive effect of leucine in enhancing the protein abundance of ASCT2 (P< 0.05). In Exp.3, twelve Large White× Landrace× Duroc weanling piglets (5.53±0.65 kg) were randomly divided into 2 treatments and fed a low protein diet supplemented with alanine (LP+Ala,17.4% crude protein) or a low protein diet supplemented with isoleucine (LP+Ile,17.5% crude protein) for 14 days. For the intestinal glucose transporters, the expression of SGLTl was up-regulated in the duodenum, jejunum and ileum of piglets in the LP+Ile treatment, compared with piglets in the LP+Ala treatment (P< 0.05). For the muscular glucose transporters, the expression of GLUT1 in red muscle was higher in piglets fed the LP+Ile diet compared with piglets fed the LP+Ala diet (P< 0.05), but not in white muscle and intermediate muscle (P> 0.05). The expression of GLUT4 in red muscle, white muscle and intermediate muscle was higher in the LP+Ile treatment compared with the LP+Ala treatment (P< 0.05). In Exp.4, we studied the effects of BCAA on expression of intestinal and muscular glucose transporters in IPEC-J2 cells and C2C12 myotubes respectively. Culture of C2C12 myotubes at both 7 or 10 mM isoleucine for 3,5,7 or 10 h significantly increased cellular 2-deoxyglucose uptake (P< 0.05), and similar results were observed when treated C2C12 myotubes with 5 mM isoleucine for either 3 or 5 h (P< 0.05).7 mM leucine treatment for 5 or 7 h, and 10 mM leucine treatment for 3,5 or 7 h also significantly enhanced the 2-deoxyglucose uptake in C2C12 myotubes (P< 0.05). For GLUT1, supplemented with 3,5 or 7 mM isoleucine, and 7 or 10 mM leucine increased the expression of glucose transporter GLUT1 in C2C12 myotubes (P< 0.05). For GLUT4, only supplemented with 3,5 or 7 mM isoleucine (not leucine or valine) increased the expression of glucose transporter GLUT4 in C2C12 myotubes (P< 0.05). No significant difference of 2-deoxyglucose uptake from medium was found when IPEC-J2 cells were treated with any concentration of leucine, isoleucine or valine at any time (P> 0.05). In conclusion, leucine up-regulate the expression of amino acid transporters through transcriptional and translational regulation by PI3K/Akt/mTOR and ERK activation in epithelial intestinal cells. Isoleucine supplementation enhances the expression of glucose transporters in intestine and muscle. Therefore, BCAA has an important implication for amino acid and glucose transportation and utilization.