REGULATION OF 15N UREA ISOTOPOMERS PRODUCTION

描述（由申请人提供）：在正常和病理状态下肝脏尿素生成的急性调节一直是广泛研究和许多争议的主题。最近，我们发现胍丁胺，通过精氨酸脱羧酶（ADC）反应的精氨酸代谢产物，刺激脂肪酸氧化（FAO），从而合成N-乙酰谷氨酸（NAG），氨甲酰磷酸合成酶-I（CPS-I）的激活剂。此外，胍丁胺可提高从婴儿高胰岛素血症/高氨血症（HI/HA）综合征转基因小鼠模型获得的肝脏中NAG水平和尿素合成。因此，胍丁胺可能被证明是一个有价值的治疗辅助HI/HA的情况下。因此，当前更新提案的总体目标是：（i）阐明胍丁胺调节FAO、NAG和尿素合成的机制;以及（ii）仔细研究先天性HI损害肝脏尿素生成并导致HA的机制。在这些目标中寻求的信息可能会推进胍丁胺在治疗尿素生成障碍中的潜在应用。基于我们的研究结果，我们提出探索两个主要假设：（i）胍丁胺对FAO的刺激触发代谢级联，导致用于合成NAG的乙酰辅酶A和谷氨酸的可用性增加，从而导致CPS-I的激活;和（ii）HI/HA患者中β细胞胰岛素的不受控制的释放增加导致FAO减少。FAO的减少，以及由GDH相关的功能获得性突变引起的肝谷氨酸氧化的增加，导致乙酰辅酶A和谷氨酸的消耗，从而减少NAG和尿素合成。然而，胍丁胺将通过刺激FAO和增加NAG合成来逆转这种代谢级联。 我们将使用在胰腺β细胞或肝脏中表达谷氨酸脱氢酶（GDH）功能获得性突变的野生型和转基因小鼠作为人HI/HA模型。同时，我们将使用最先进的方法，包括15 N和/或13 C标记的前体，气相色谱-质谱（GC-MS）和核磁共振（NMR），以确定胍丁胺对HI/HA小鼠模型中肝脏NAG和尿素生成的有益作用。所提出的研究具有临床和科学意义。通过推进改善受损尿素合成的方案的开发，待生成的数据可能具有巨大的临床影响。

DESCRIPTION (provided by applicant): The acute regulation of hepatic ureagenesis under normal and pathologic states has been the subject of extensive investigation and numerous controversies. Recently, we have discovered that agmatine, the product of arginine metabolism via the arginine decarboxylase (ADC) reaction, stimulates fatty acid oxidation (FAO), and thus synthesis of N-acetylglutamate (NAG), an activator of carbamoyl phosphate synthetase-I (CPS-I). In addition, agmatine elevates NAG levels and urea synthesis in livers obtained from the transgenic mouse model of infant hyperinsulinism/hyperammonemia (HI/HA) syndrome. Thus, agmatine might prove a valuable therapeutic adjunct in the case of HI/HA. Therefore, the overall aims of the current renewal proposal are: (i) To elucidate the mechanism(s) by which agmatine regulates FAO, NAG and urea synthesis; and (ii) To scrutinize the mechanism(s) by which congenital HI impairs hepatic ureagenesis and leads to HA. The information sought in these aims may advance the potential application of agmatine in the treatment of impaired ureagenesis. Based on our findings we propose to explore two main hypotheses: (i) The stimulation of FAO by agmatine, triggers a metabolic cascade that leads to an increased availability of acetyl-CoA and glutamate for the synthesis of NAG, thus resulting in the activation of CPS-I; and (ii) The increased uncontrolled release of insulin by beta-cells in HI/HA patients leads to decreased FAO. A decrease in FAO, together with increased hepatic glutamate oxidation, resulting from the GDH-linked gain-of-function mutation, leads to the depletion of acetyI-CoA and glutamate and thus, diminished NAG and urea synthesis. Agmatine, however, will reverse this metabolic cascade by stimulating FAO and increasing NAG synthesis. We will use wild type and transgenic mice expressing the glutamate dehydrogenase (GDH) gain-of-function mutation in pancreatic beta-cells or the liver as a model of human HI/HA. In conjunction, we will use state-of-the-art methodologies, including 15N and/or 13C labeled precursors, Gas Chromatography-Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance (NMR), to determine the beneficial effect of agmatine on hepatic NAG and ureagenesis in this mouse model of HI/HA. The proposed studies are of clinical as well as scientific significance. The data to be generated may have tremendous clinical impact by advancing the development of a protocol to ameliorate impaired urea synthesis.