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. ? ? ?
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