| Ammonia emissions exacerbate the protein resource scarcity and nitrogen emission pollution. Ammonia is a major product of nitrogen turnover in mammals. Ammonia can convert to urea which cannot be metabolized or convert to glutamine that can be further utilized. O ur previous results show that hepatic leucine metabolism rate is only 4% to 13%. It is now well recognized that leucine plays an important function in the regulation of nitrogen metabolism. The aim of the present study was to explore the regulatory role and mechanism of leucine on ammonia metabolism from different source using 15 N tracer and portal infusion technology. 1. Portal ammonia(NH4+) is the main hepatic ammonia source, 15NH4 Cl or leucine+15NH4Cl was infused to explore the regulation of leucine on hepatic ammonia(from portal vein) metabolism. 2. Alanine is an important transporter of ammonia, 15N-alanine or leucine+15N-alanine was infused to explore the regulation of leucine on hepatic ammonia(from alanine) metabolism. 3. The catabolism of mixed amino acid is also an important ammonia source in the liver, mixed amino acids or leucine+mixed amino acids was infused to explore the regulation of leucine on hepatic ammonia(from mixed amino acids) metabolism. The main findings are as follows:(1) The regulation of leucine on hepatic ammonia(from NH4Cl) metabolism. Compared with 15NH4 Cl perfusion group, leucine+15NH4Cl group decreased hepatic vein urea nitrogen concentration(P<0.05) in 40~80 min post- infusion, increased 15 Nglutamate and 15N-glutamine concentrations in both hepatic vein(40~80 min)(P<0.05) and liver tissue(P<0.01). Morever, the addition of leucine improved the glutamate dehydrogenase(GDH) activity(about 65%) and gene expression(1.8- fold)(P<0.01). Taken together, when NH4 Cl served as hepatic ammonia source, the addition of leucine could effectively inhibit the generation of urea and promote the generation of glutamate and glutamine by regulating GDH expression and activity.(2) The regulation of leucine on hepatic ammonia(from alanine) metabolism. Compared with the 15N-alanine perfusion group, leucine+15N-alanine group decreased hepatic vein urea nitrogen(P<0.05) and glucose(P<0.05) concentrations in 40~80 min post-infusion, increased 15N-glutamate and 15N-glutamine concentrations in both hepatic vein(40~80 min)(P<0.05) and liver tissue(P<0.01). Morever, the addition of leucine improved GDH activity(about 50%) and gene expression(about 1.6-fold)(P<0.01). Taken together, when alanine served as hepatic ammonia source, the addition of leucine could not noly effectively inhibit the generation of urea and promote the generation of glutamate and glutamine by regulating GDH expression and activity, but also inhibit hepatic gluconeogenesis.(3) The regulation of leucine on hepatic ammonia(from mixed amino acids) metabolism. Compared with the mixed amino acids perfusion group, leucine+mixed amino acids group decreased hepatic vein urea nitrogen(P<0.05) concentration in 40~80 min post- infusion, reduced most NEAA concentrations both in hepatic vein(P<0.05) and liver tissue(P<0.05) NEAA. Morever, the addition of leucine improved GDH activity(about 45%) and gene expression levels(about 1.5-fold)(P<0.01). Taken together, when mixed amino acids as hepatic ammonia source, the addition of leucine could not noly effectively inhibit the generation of urea by regulating GDH expression and activity, but also reduce hepatic NEAA contents.(4) The regulatory mechanis m of leucine on GDH activity. The addition of leucine can effectively improve the GDH activity(P<0.01) from different ammonia sources. Therefore, the expression of mitochondrial SIRT4, the inhibitory factor of GDH, was futher analyzed. With NH4 Cl, alanine and mixed amino acid as ammonia sources, the SIRT4 m RNA level were down by 50%, 41%, 45%, the SIRT4 protein were down by 55%, 51%, 59% in the presence of leucine(P<0.01), respectively.These results showed that:1. With NH4 Cl as ammonia source, the addition of leucine could effectively inhibit ureagenesis and promote the generation of glutamate and glutamine. With alanine as ammonia source, the addition of leucine could not noly inhibit ureagenesis and promote the generation of glutamate and glutamine, but also inhibit hepatic gluconeogenesis. With mixed amino acids as ammonia source, the addition of leucine could not noly inhibit ureagenesis, but also reduce hepatic NEAA contents.2. The effect of leucine on hepatic ammonia metabolism might involve in the regulation of epigenetic factor SIRT4: the addition of leucine could inhibit mitochondrial SIRT4 expression, then increased GDH activity and promoted ammonia escaping urea cycle and into glutamine circulation.This study clarified the role and mechanism of the regulation of leucine on hepatic ammonia metabolism, and provide the theoretical basis and practical guidance for the development of pig production. |
