Regulation of 15N Urea Isotopomers Production

15N尿素同位素生产监管

• 批准号: 8068083
• 负责人: ITZHAK NISSIM
• 金额: $ 10万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-18 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8068083
• 关键词: 5' -AMP-activated protein kinase; Acetyl Coenzyme A; Acetyl-CoA Carboxylase; Acute; Address; Adult; Affect; Agmatine; Amination; Arginine; Arginine decarboxylase; Aspartate; Attenuated; Biochemical; Carbamoyl-Phosphate Synthase (Ammonia); Carbamyl Phosphate; Child; Chronic; Citric Acid Cycle; Citrulline; Congenital Abnormality; Cyclic AMP; Data; Development; Disease; Down-Regulation; Face; Failure; Fatty Liver; Funding; Glutamate Dehydrogenase; Glutamates; Glutaminase; Glutamine; Guidelines; Hepatic; Hepatocyte; Hyperammonemia; Hyperinsulinism; Intervention; Kinetics; Knowledge; Label; Lead; Lipids; Liver; Liver diseases; MALDI-TOF Mass Spectrometry; Malonyl Coenzyme A; Mass Fragmentography; Mediating; Medical; Metabolic; Metabolic syndrome; Metabolism; Methodology; Mitochondria; Modeling; Molecular Biology; N acetyl L glutamate; NADH; Nuclear Magnetic Resonance; Outcome; Oxaloacetates; Patients; Perfusion; Production; Progress Reports; Protein Isoforms; Publications; Pyruvate Carboxylase; Rattus; Regulation; Role; Secondary to; Signal Pathway; Supplementation; Synthase I; System; Testing; Up-Regulation; Urea; Zucker Rats; base; diagnosis evaluation; effective therapy; enzyme activity; fatty acid oxidation; hepatic ureagenesis; improved; in vivo; inorganic phosphate; lipid biosynthesis; mitochondrial dysfunction; nitrogen metabolism; non-alcoholic fatty liver; novel; oxidation; protein degradation; public health relevance; research study; tool; uptake; urea cycle

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.

描述（由申请人提供）：尿素合成受损和随之而来的高氨血症（HA）常见于先天性尿素循环缺陷和脂肪酸氧化（FAO）、非酒精性脂肪性肝病（NAFLD）和/或“代谢综合征”（MS）。目前尚不清楚有缺陷的FAO和脂肪肝是如何影响尿变性的生化和代谢机制。目前也没有有效的治疗方法。在目前的资助期内，我们发现精氨酸脱羧酶的产物agmatine （AGM）可升高肝脏cAMP，并刺激尿原性和FAO。我们的初步数据表明，AGM或5-氨基咪唑-4-carboxamide-1-¿- d -核糖呋喃苷（AICAR），一种amp活化的蛋白激酶（AMPK）的激活剂，可以增强NAFLD或ms大鼠模型中的尿素生成。总之，这些观察结果强烈表明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中肝脏谷氨酰胺代谢和尿素合成的机制。长期目标是开发一种临床适用的药物治疗干预措施，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具体目标/假设：(i) NAFLD的尿原功能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是氨基甲酰磷酸合成酶- i （CPS-I）的强制性激活剂，是尿原作用的初始和限速步骤。AGM和AICAR增强了FAO，从而引发了代谢级联反应，从而减弱了与NAFLD和ms相关的代谢紊乱。NAFLD降低肝脏对谷氨酰胺的摄取和代谢。这将限制线粒体[谷氨酸]和NAG的合成。最终的结果是CPS-I的激活失败。AGM和AICAR刺激了粮农组织，改善了谷氨酰胺的吸收，并允许更多的谷氨酸可用于NAG的合成，从而提高了CPS-I的活性。基于这些假设，需要解决的问题包括：(1)AGM对FAO和尿素发生的作用是否通过激活AMPK和/或cAMP-PKA介导？(2)急性或慢性AGM或AICAR治疗如何影响NAFLD或MS中肝脏FAO、TCA循环和尿素发生之间的代谢协调？(3) AGM或AICAR治疗以及随后AMPK和/或cAMP-PKA的激活如何影响NAFLD或MS的肝脏谷氨酰胺摄取和代谢、全身蛋白质转换和尿素生成，实验将使用脂肪肝和/或MS大鼠模型和各种系统进行，包括(a)分离的肝细胞；(b)肝脏灌注系统；(c)分离线粒体；(d)体内研究。我们将使用15N和/或13C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来查明AGM或AICAR作用的主要机制。所获得的数据将为阐明AGM或AICAR在调节肝脏谷氨酰胺代谢和尿素生成中的作用提供新的关键信息。这一知识可能会导致一种新的药物治疗干预措施的发展，以改善NAFLD或MS的尿素合成。