Regulation of 15N Urea Isotopomers Production

描述（由申请人提供）：尿素合成受损和随后的高氨血症（HA）常见于尿素循环和脂肪酸氧化（FAO）先天性缺陷、非酒精性脂肪肝（NAFLD）和/或“代谢综合征”（MS）疾病。FAO缺陷和脂肪肝损害尿素生成的生化和代谢机制仍不清楚。也没有有效的治疗方法。在目前的资助期间，我们发现胍丁胺（AGM），精氨酸脱羧酶的产物，提高肝[cAMP]和刺激尿素生成和FAO。我们的初步数据表明，AGM或5-氨基咪唑-4-甲酰胺-1-<$-D-呋喃核糖苷（AICAR），AMP激活的蛋白激酶（AMPK）的激活剂，增强了NAFLD或MS大鼠模型中的尿素生成。总之，观察结果强烈表明，AGM具有许多预期的AMPK激活剂的作用。在这项更新提案中，我们的总体目标是阐明AGM或AICAR调节NAFLD或MS患者肝脏谷氨酰胺代谢和尿素合成的机制。长期目标是开发临床适用的药物干预，以改善NAFLD和/或MS患者的尿素生成。我们建议探索两个相关的特定目标/假设：（i）NAFLD中的尿素生成受损是线粒体功能障碍和由此导致的N-乙酰谷氨酸（NAG）合成减少的结果，NAG是氨甲酰磷酸合成酶-I（CPS-I）的强制性激活剂，是尿素生成的初始和限速步骤。AGM和AICAR增强FAO，从而触发代谢级联，减弱与NAFLD和MS相关的代谢紊乱。结果是尿素生成增强;和（ii）NAFLD降低肝摄取和谷氨酰胺代谢。这将限制线粒体[谷氨酸]和NAG的合成。最终结果是CPS-I活化失败。AGM和AICAR刺激FAO，改善谷氨酰胺摄取，并允许更多的谷氨酸可用于NAG合成，因此，更大的CPS-I活性。基于这些假设，需要解决的问题包括：（1）AGM对FAO和尿素合成的作用是否通过AMPK和/或cAMP-PKA的激活介导？(2)急性或慢性AGM或AICAR治疗如何影响NAFLD或MS患者肝脏FAO、TCA循环和尿素生成之间的代谢协调？和（3）用AGM或AICAR治疗以及随后AMPK和/或cAMP-PKA的活化如何影响NAFLD或MS中的肝谷氨酰胺摄取和代谢、全身蛋白质周转和尿素生成。将使用脂肪肝和/或MS的大鼠模型和各种系统进行实验，包括（a）分离的肝细胞;（B）肝灌注系统;（c）分离的线粒体;和（d）体内研究。我们将使用15 N和/或13 C标记的前体和气相色谱-质谱（GC-MS），MALDI-TOF-质谱，核磁共振（NMR）和分子生物学。结合这些方法提供了一个极好的工具，以查明年度股东大会或AICAR行动的主要机制。这些结果将为阐明AGM或AICAR在肝脏谷氨酰胺代谢和尿素生成调控中的作用提供新的和关键的信息。这一知识可能导致开发一种新的药物干预，以改善NAFLD或MS的尿素合成。 公共卫生相关性：在非酒精性脂肪肝（NAFLD）或代谢综合征（MS）的情况下，尿素合成缺陷和随后的毒性高氨血症（HA）是常见的，这是成人和儿童中日益认识到的医学问题。该提案的总体目标是：（i）阐明NAFLD或MS中调节肝脏尿素合成的机制;（ii）开发临床适用的药物干预，以改善NAFLD和/或MS患者的尿素生成。我们的初步结果表明，这种治疗是可行的。该研究结果可能为NAFLD或MS中尿素合成缺陷的评估、诊断和治疗提供新的指导。

DESCRIPTION (provided by applicant): Impaired urea synthesis and consequent hyperammonemia (HA) are common occurrences in disorders of congenital defects of the urea cycle and of fatty acid oxidation (FAO), nonalcoholic fatty liver disease (NAFLD) and/or "Metabolic Syndrome" (MS). Still unknown are the biochemical and metabolic mechanisms by which defective FAO and fatty liver impair ureagenesis. Nor is an effective treatment available. During the current funding period we found that agmatine (AGM), the product of arginine decarboxylase, elevates hepatic [cAMP] and stimulates both ureagenesis and FAO. Our preliminary data demonstrate that AGM or 5-aminoimidazole-4-carboxamide-1-¿-D-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), enhances ureagenesis in a rat model of NAFLD or MS. Together, the observations strongly suggest that AGM has many of the effects expected for an activator of AMPK. In this renewal proposal our overall aim is to elucidate the mechanisms by which AGM or AICAR regulates hepatic glutamine metabolism and urea synthesis in NAFLD or MS. A long-term objective is to develop a clinically applicable pharmacotherapeutic intervention to improve ureagenesis in patients with NAFLD and/or MS. We propose to explore two related Specific Aims/Hypotheses: (i) Impaired ureagenesis in NAFLD is a consequence of mitochondrial dysfunction and a resultant decrease in synthesis of N-acetylglutamate (NAG), an obligatory activator of carbamoyl phosphate synthetase-I (CPS-I), the initial and the rate-limiting step of ureagenesis. AGM and AICAR augment FAO, thereby triggering a metabolic cascade that attenuates the metabolic derangements associated with NAFLD and MS. The result is an augmentation of ureagenesis; and (ii) NAFLD decreases hepatic uptake and metabolism of glutamine. This would limit mitochondrial [glutamate] and NAG synthesis. The net result is a failure of activation of CPS-I. AGM and AICAR stimulate FAO, improve glutamine uptake and permit more glutamate to be available for NAG synthesis, and thus, greater CPS-I activity. Based on these hypotheses, questions to be addressed include: (1) Is the action of AGM on FAO and ureagenesis mediated via activation of AMPK and/or cAMP-PKA? (2) How does acute or chronic treatment with AGM or AICAR affect metabolic coordination between hepatic FAO, the TCA cycle and ureagenesis in NAFLD or MS?; and (3) How does treatment with AGM or AICAR and subsequent activation of AMPK and/or cAMP-PKA affect hepatic glutamine uptake and metabolism, whole-body protein turnover and ureagenesis in NAFLD or MS. Experiments will be performed using a rat model of fatty liver and/or MS and various systems, including (a) isolated hepatocytes; (b) a liver perfusion system; (c) isolated mitochondria; and (d) in vivo study. We will use 15N and/or 13C labeled precursors and gas chromatography-mass spectrometry (GC-MS), MALDI-TOF-mass spectrometry, nuclear magnetic resonance (NMR) and molecular biology. Combining these methodologies provides a superb tool to pinpoint the primary mechanism(s) of AGM or AICAR action. Data obtained will provide new and pivotal information to elucidate the role of AGM or AICAR in the regulation of hepatic glutamine metabolism and ureagenesis. This knowledge may lead to development of a novel pharmacotherapeutic intervention to improve urea synthesis in NAFLD or MS. PUBLIC HEALTH RELEVANCE: Defective urea synthesis and consequent toxic hyperammonemia (HA) are common occurrences in case of nonalcoholic fatty liver disease (NAFLD) or Metabolic Syndrome (MS), an increasingly recognized medical problem in adults and children. The overall aims of this proposal are: (i) to elucidate the mechanism(s) regulating hepatic urea synthesis in NAFLD or MS; and (ii) to develop a clinically applicable pharmacotherapeutic intervention to improve ureagenesis in patients with NAFLD and/or MS. Our preliminary results suggest that such treatment is feasible. The outcome of the propose study may provide new guidelines for evaluation, diagnosis and treatment of defective urea synthesis in NAFLD or MS.