Mouse models for studying citrulline recycling for sustaining NO production

描述（由申请人提供）：该提案描述了一个五年的职业发展计划，以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理解复杂的代谢途径。Brendan Lee博士是遗传学领域公认的领导者，他将指导PI的科学发展。一个由备受尊敬的科学家组成的咨询委员会将为该项目提供指导和职业建议。在遗传学领域的培训将补充候选人之前的培训和经验，包括兽医学位，动物营养学硕士和博士学位以及营养生理学和代谢的博士后培训。这些研究和培训将为PI的独立研究计划提供基础，并产生将在一氧化氮（NO）生物学和氮代谢领域广泛应用的资源。一氧化氮是参与许多生理和病理生理过程的生物信使分子。l -精氨酸（Arg）是一氧化氮合成的前体，在某些情况下（生长、妊娠、败血症），它的可用性似乎限制了一氧化氮的产生。作为NO合成的副产物，瓜氨酸的细胞内循环可以提供精氨酸来维持NO的生成。尽管这一途径很重要，但尚未对瓜氨酸在体内的细胞内循环进行测定。鉴于脓毒症期间NO生成的重要性，脓毒症的内毒素模型将利用稳定同位素和已建立的组织特异性条件突变体来研究Arg可用性、细胞内瓜氨酸循环和NO生成。本研究的具体目的是：1)量化Arg缺乏和高精氨酸血症小鼠在禁食和喂养条件下一氧化氮的产生；2)量化内毒素挑战对Arg缺乏和高精氨酸血症小鼠产生NO的影响，并评估Arg和瓜氨酸的补充；3)开发一种新的条件小鼠突变体，在这种突变体中，瓜氨酸的细胞内循环受到阻碍，并量化其NO的产生。总之，这些研究将阐明细胞内瓜氨酸循环在维持NO生产中的作用，无论是在基础条件下还是当NO生产增加时。确定在哪些生理和病理生理条件下Arg会限制NO的合成，可以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.