Study On The Effects Of Alanyl-Glutamine On Nitrogen Utilization And Its Regulation Mechanisms In Piglets

After ingested by animals,the dietary protein was degraded into free amino acids and peptides by the action of digestive enzymes in gastro-intestine.These amino acids and peptides entered into blood circulation through epithelial cells and participated in the metabolism of body.The digestion and metabolism of dietary nitrogen not only depends on the gastro-intestinal,but also depends on liver and skeletal muscle.The digestibility and metabolism of nitrogen will be directly or indirectly influenced by the actions of exogenous substances,such as anti-nutritional factors and bioactive peptides.Therefore,it will provide the theoretical reference for effective utilization of nitrogen and reducing nitrogen emission through investigating the effects of exogenous substance on nitrogen metabolism network.Alanyl-glutamine was approved to improve the growth performance of animals and enhanced the nutrients absorption and utilization in small intestine.Moreover,Ala-Gln could improve the utilization of net nitrogen and effectively alleviated the immune stress of animals.Until now,however,the mechanism of Ala-Gln on nitrogen metabolism is still unknown.Therefore,the study was to investigate the effects of Ala-Gln on the nitrogen metabolism of normal and LPS challenged piglet,and furtherly elucidate its related mechanism.1.The study was aimed to investigate the effects of alanyl-glutamine(Ala-Gln)and alanine and glutamine(Ala+Gln)combination supplementation on growth performance,plasma biochemical parameters and the contents of nitrogenous compounds in feces of piglet.A total of 180 piglets with initial weight 10.00 kg were randomly allocated to three treatments with 3 replicates of 20 piglets each,fed with diets containing 0.62%Ala,0.5%Ala-Gln,0.21%Ala + 0.34%Gln,respectively.The duration of the experiment was 28 days.The results indicated that Ala-Gln supplementation significantly increased the average daily gain(ADG)and decreased the ration of feed to gain(F/G,P<0.05)in comparison with Ala supplementation,but did not affect the average daily feed intake(ADFI,P>0.05);dietary supplemented with Ala+Gln did not affect the ADG,ADFI or F/G(P>0.05);Ala-Gln supplementation significantly incrased the contents of total protein(TP)and decreased the activity of lactate dehydrogenase(LDH,P<0.05)in plasma,but did not affect the activity of alkaline phosphatase in plasma(AKP,P>0.05);Ala+Gln supplementation increased the contents of TP(P<0.05),but did affect the activity of LDH or AKP in plasma(P>0.05);Ala-Gln supplementation decreased the contents of ammonium nitrogen,tryptamine,phenethylamine,histamine and tyramine in feces of piglet(P<0.05),but did not affect the contents of putrescine or cadaverine(P>0.05)in feces;Ala+Gln supplementation significantly decreased the contents of tryptamine and phenethylamine(P<0.05),but did not affect the contents ammonium nitrogen,histamine,tyramine,putrescine or cadaverine in feces of piglets(P>0.05).2.The study was to investigate the effects of Ala-Gln and Ala+Gln on nutrients apparent digestibility and the expression of intestinal amino acid and peptide sensors and transporters.Experiment design was the same with experiment 1.The results showed that,compared with Ala supplementation,Ala-Gln supplementation significantly increased the crude protein apparent digestibility(P<0.05);Ala-Gln supplementation increased the activity of trypsin in jejunum and pacrease(P<0.05);Ala-Gln supplementation increased the concentration of leucine(Leu),phenylalanine(Phe),histidine(His),arginine(Arg),alanine(Ala),cysteine(Cys),serine(Ser),glutamate(Glu),glutamine(Gln)and proline(Pro,P<0.05);Ala+Gln supplementation increased the concentrations of Leu,Phe,His,Ser and Glu in plasma(P<0.05);Ala-Gln supplementation partially increased the expression of jejunal mucosa amino acid and peptide sensors,such as calcium-sensing receptor(CaSR),taste receptor type 1 member 1(T1R1),taste receptor type 1 member 3(T1R3)and G protein-coupled receptor 93(GPR93);Ala-Gln supplementation also increased the expressions of solute carrier family 1 member 5,solute carrier family 6 member 19,solute carrier family 7 member 7,solute carrier family 15 member 1 of jejunal mucosa(P<0.05).3.The experiment was to investigate the effects of Ala-Gln and Ala+Gln supplementation on liver protein synthesis,muscle protein synthesis and degradation of piglets.The experiment design was same with experiment 1.The results showed that Ala-Gln supplementation significantly increased the concentrations of Gln and glutamate(Glu)in liver and skeletal muscle(P<0.05);Ala-Gln significantly increased the activity of aspartate aminotransferase and alanine aminotransferase in liver and skeletal muscle,and also increased the expression of glutamate dehydrogenase(GDH)and glutaminase(GA)in liver and muscle(P<0.05).Additionally,Ala-Gln supplementation increased the expression of carbamyl phosphate synthetase I(CPS-1)in liver(P<0.05).Ala-Gln supplementation increased the abundance of phosphorylation of mTOR,4EBP1 and S6K1 in liver and skeletal muscle and decreased the expression of MAFbx and MuRF1 in skeletal muscle(P<0.05).4.The study was to investigate the effect of Ala-Gln supplementation on growth performance,plasma biochemical parameters and the contents of nitrogenous compounds in feces of piglet challenged by LPS.Based on the results of study in piglets under normal condition,Ala-Gln was chosen as exogenous activity substance.The immune challenge model was established through the intraperitoneal injection of LPS into piglet.Piglets were arranged in a 2×2 factorial design and the main effects were LPS challenge(0 or 100 units)and diets(0.62%Ala or 0.5%Ala-Gln).After treatment with either Ala or Ala-Gln for 10days,piglets were injected twice with either saline or LPS on d 11 and d 15.During days11-15(post-challenge),LPS challenge affected the growth performance of piglets.Ala-Gln supplementation tended to alleviate the reduction of the ADG(P=0.071)and the ADFI(P=0.087)of the LPS-challenged piglets.LPS challenge increased the activity of LDH and decreased the activity of AKP(P<0.05);LPS challenge decreased the contents of IGF-1and FT4 in plasma,and increased the contents of plasma cortisol(P<0.05);Ala-Gln supplementation alleviated the increased cortisol induced by LPS challenge(P<0.05);LPS challenge increased the contents of tryptamine,phenethylamine,putrescine,cadaverine,histamine,tyramine and ammonium nitrogen in feces of piglet(P<0.05),however,Ala-Gin supplementation decreased the contents of tryptamine,tyramine and ammonium nitrogen(P<0.05).5.The study was to investigate the effects of Ala-Gln supplementation on nutrients apparent digestibility,and the expression of jejunal mucosa amino acid and peptide sensors and transporters of piglets challenged with LPS.The experiment design was the same with experiment 4.The results indicated that LPS challenge significantly decreased dry matter(DM),organic matter(OM),ether extract(EE)and crude protein(CP,P<0.05).Ala-Gln supplementation tended to increase the apparent digestibility of DM(P=0.0095),OM(P=0.071)and CP(P=0.083).LPS challenge decreased the activities of pepsin,trypsin in jejunum and pacrease of piglets(P<0.05);Ala-Gln increased the activity of trypsin in pacrease(P<0.05)and tended to increase the activity of jejunum(P=0.074).LPS challenge significantly decreased the concentrations of lysine(Lys),arginine(Arg),tyrosine(Tyr),phenylalanine(Phe),leucine(Leu)and isoleucine(Ile,P<0.05);Ala-Gln supplementation increased the concentratin of most amino acids,such as Gln,Ile,Leu,Lys and valine(P<0.05).Both LPS challenge and Ala-Gln increased the expressions of CaSR,T1R1,T1R3,GPRC6A and GPR93(P<0.05),and there was an interaction between LPS challenge and Ala-Gln supplementation for the expression of T1R3(P<0.05);LPS challenge significantly decreased the expressions of amino acid and peptide transporters of jejunal mucosal,such as SLC1A5,SLC7A9,SLC6A19,SLC7A7,SLC1A1 and SLC15A5(P<0.05);Ala-Gln supplementation increased the expression of SLC1A5,SLC7A9,SLC6A19,SLC7A7 and SLC15A5(p<0.05),but did not affect the expression of SLC1A1(P>0.05).6.The study was conducted to investigate Ala-Gln supplementation on liver protein synthesis,muscle protein synthesis and degradation of piglets challenged with LPS.The experiment design was the same with experiment 4.The results showed that LPS challenge increased the contents of TNF-?,IL-1?and IL-6 in plasma(P<0.05),however,Ala-Gln supplementation decreased the contents of cytokines mentioned above(P<0.05);LPS challenge increased the concentration of Gln in liver and decreased it in muscle(P<0.05).Ala-Gln increased the activity of GS and the expression of GA in liver(P<0.05);LPS challenge increased the expression of C-reactive protein(CRP),serum amyloid(SAA)and ceruloplasmin(Cp)in liver,in contrast,Ala-Gln supplementation decreased the expression of acute-phase proteins mentioned above(P<0.05);Ala-Gln supplementation increased the expression of IGF-1 signaling and Akt in skeletal muscle,and increased the abundance of phosphorylation of mTOR,4EBP1 and S6K1 in liver and skeletal muscle(P<0.05).Additionally,Ala-Gln supplementation decreased the expression of Toll-like receptor 4(TLR-4),MAFbx and MuRF1(P<0.05),and suppressed the degradation of skeletal muscle.As mentioned above,the conclusions are as follows:(1)Dietary supplementation with 0.5%Ala-Gln significantly increased the growth performance of normal piglets and decreased the emission of nitrogenous compounds.Ala-Gln also improved the nitrogen apparent digestibility,the expression of jejunal mucosa sensors and transporters and improved the digestibility and absorption of nutrients.Additionally,the effects of Ala-Gln dipeptide was better than those of free Ala and Gln combination supplementation.(2)Dietary supplementation with Ala-Gln improved Gln utilization thourgh the increased expression of GA and GDH in liver and skeletal muscle.Ala-Gln supplementation might increase the protein synthesis through upregulated the mTOR signaling in liver and skeletal muscle.And Ala-Gln might suppress the degradation of skeletal muscle of piglet through decreasing the expression of MAFbx and MuRF1.Morover,the effect of Gin on protein synthesis and protein degradation may be associated with the increased Gin metabolism.(3)LPS challenge significantly decreased the growth performace and nutrients apparent digestibility of piglets challenged by LPS.Meanwhile,LPS challenge might inhibit the nutrient utilization through decreasing the expression of jejunal mucosa amino acid and peptide sensors and transporters.Ala-Gln supplemented alleviated the immune stress induced by LPS challenge,increased the expression of amino acid and peptide sensors and transporters in jejunal mucosa.Ala-Gln supplementation tended to increase the nutrient apparent digestibility and might increase the nutrient digestion and absorption induced by LPS challenge.(4)LPS challenge increased the contents of Gin,the expression of acute-phase proteins and mTOR signaling in liver.However,Ala-Gln supplementation decreased the expression of acute-phase protein in liver and effectively alleviated the liver stress induced by LPS challenge.LPS challenge decreased the contents of Gin and the abundance of molecular in mTOR signaling pathway and increased the degradation of skeletal muscle through increasing the expression of MAFbx and MuRF1.Ala-Gin supplementation increased the contents of Gin and the abundance of molecules in mTOR signaing pathway.Meanwhile,Ala-Gln supplementation effectively inhibited the protein degradation in skeletal muscle of LPS-challenged piglets through inhibiting the expression of signaling molecules in TLR4 and UPP pathways.