Mouse models for studying citrulline recycling for sustaining NO production

描述(由申请人提供)：这份建议书描述了一项为期五年的职业发展计划，该计划旨在获得开发小鼠遗传模型的关键技能，并应用最先进的稳定同位素技术来了解复杂的代谢途径。遗传学领域公认的领导者Brendan Lee博士将指导PI的科学发展。一个由备受尊敬的科学家组成的顾问委员会将为该项目提供指导以及职业建议。拟议的遗传学培训将补充候选人以前的培训和经验，包括动物营养学的兽医学位、硕士和博士学位以及营养生理学和新陈代谢的博士后培训。研究和培训将为PI的独立研究计划提供基础，并产生将广泛用于一氧化氮(NO)生物学和氮代谢领域的资源。NO是一种生物信使分子，参与多种生理和病理生理过程。L-精氨酸(Arg)是NO合成的前体，在某些条件下(生长、妊娠、败血症)，其供应似乎限制了NO的产生。合成NO的副产物瓜氨酸的胞内循环可以为Arg提供维持NO产生的能力。尽管这一途径很重要，但目前还没有关于瓜氨酸在体内细胞内循环的测定。鉴于脓毒症中NO产生的重要性，将使用脓毒症的内毒素模型来研究Arg的可利用性、细胞内瓜氨酸的循环以及利用稳定的同位素和已建立的组织特异性条件突变体来研究NO的产生。这项研究的具体目的是：1)定量测定禁食和喂养条件下Arg缺乏和高精氨酸血症小鼠一氧化氮的产生；2)量化内毒素刺激对Arg缺乏和高精氨酸血症小鼠产生NO的影响，并评价Arg和瓜氨酸的补充作用；3)开发一种新的阻碍瓜氨酸细胞内循环的小鼠条件突变体，并量化其NO产生。总之，这些研究将阐明细胞内瓜氨酸循环在维持NO产生中的作用，无论是在基础条件下还是在NO产生增加的情况下。确定在哪些生理和病理生理条件下Arg对NO合成具有限制作用，可能会导致更好地确定治疗靶点和更有用的补充策略。

DESCRIPTION (provided by applicant): This proposal describes a five-year career development plan for acquiring critical skills for developing mouse genetic models and for applying state of the art stable isotope techniques to understand complex metabolic pathways. Dr. Brendan Lee, a recognized leader in the field of genetics, will mentor the PI's scientific development. An advisory committee of highly regarded scientists will provide guidance for this project as well as career advice. The proposed training in the field of genetics will complement the candidate's previous training and experience which includes a veterinary degree, masters and doctoral degrees in animal nutrition and post doctoral training in nutritional physiology and metabolism. The research and training will provide the basis of the PI's independent research program as well as generate resources that will be widely used in the field of nitric oxide (NO) biology and nitrogen metabolism. NO is a biological messenger molecule involved in many physiological and pathophysiological processes. L-arginine (Arg) is the precursor for NO synthesis and, in certain conditions (growth, pregnancy, sepsis) its availability seems to limit NO production. The intracellular recycling of citrulline, a co-product in the synthesis of NO, can provide Arg to sustain NO production. Despite the importance of this pathway, no determinations of intracellular recycling of citrulline in vivo have been conducted. In light of the importance of NO production during sepsis, an endotoxin model of sepsis will be used to investigate Arg availability, intracellular citrulline recycling and NO production utilizing stable isotopes and established and tissue-specific conditional mutants. The specific aims of the proposed research are 1) To quantify nitric oxide production in Arg deficient and hyperargininemic mice in fasted and fed condition, 2) To quantify the effect of endotoxin challenge in the production of NO by Arg deficient and hyperargininemic mice and to evaluate Arg and citrulline supplementation and 3) To develop a novel conditional mouse mutant in which intracellular recycling of citrulline is impeded and to quantify its NO production. Together these studies will elucidate the role of intracellular citrulline recycling in sustaining NO production, both during basal conditions and when NO production is increased. Identifying in which physiological and pathophysiological conditions Arg becomes limiting for NO synthesis, could lead to better defined therapeutic targets and more useful supplementation strategies.