Biology of the Nutraceutical L-Citrulline

背景：L瓜氨酸是L-鸟氨酸和氨基甲酰磷酸在尿素循环中通过鸟氨酸氨基甲酰转移酶合成的a-氨基酸，同时也是一氧化氮合酶反应的副产物，将L-精氨酸转化为精氨酸和一氧化氮。有趣的是，LC可以作为膳食补充剂在柜台上购买。在人体上的研究表明，LC可能是一种性能增强剂，然而，LC的生物学作用以及LC提高性能的机制还相对研究较少。我的目标是描述LC在骨骼肌和心肌中的生物学作用。*假设：我假设LC通过增加过氧化物酶体增殖物激活受体γ辅活化子1a的活性来增强运动肌肉的代谢，从而增强线粒体的功能。*方法学：我的实验室将利用一些体外/体内方法来阐明LC在骨骼肌和心肌中的生物学作用。这包括培养新生大鼠心肌细胞(NRCM)、H9c2心室肌细胞和C2C12骨骼肌管。此外，小鼠将接受一系列的程序(即高脂饮食、运动)，以确定LC治疗是否影响线粒体功能和氧化代谢，通过间接量热法和肌肉/心脏提取物的mRNA/蛋白质表达谱来确定。最后，我们将评估分离的肌肉/心脏线粒体的呼吸。*目标：这项研究计划包括三个特定的目标：(1)确定LC治疗后肌肉和心脏中激活的信号通路，(2)确定LC处理是否增强肌肉和心脏的线粒体功能，以及(3)确定LC和NO在一氧化氮合酶(NOS)介导的线粒体功能增加中的相对重要性。在目标1中，我们将用LC和采集细胞提取液处理培养的C2C12肌管、H9c2肌细胞和NRCM，以评估mRNA/蛋白质的表达谱。在目标2中，我们将分离小鼠心脏和骨骼肌的线粒体，并确定LC治疗是否增强呼吸。此外，我们将使用间接量热法来测量瘦小和肥胖小鼠在LC处理后的饮用水中的O2消耗。在目标3中，我们将用LC或NO供体补充瘦小和肥胖的内皮一氧化氮合酶缺陷小鼠，并评估线粒体功能。*影响：我们的发现将深入了解LC在肌肉和心脏中的生物学作用。此外，这项研究计划的结果应该会加强我们对驱动线粒体呼吸的细胞过程的理解。最后，我们的发现还应该为那些将LC作为非处方药营养膳食补充剂来增强运动性能的制造商提供重要的见解。

Biology of the Nutraceutical L-Citrulline******Background: L-citrulline (LC) is an a-amino acid synthesized from L-ornithine and carbamoyl phosphate in the Urea Cycle via ornithine carbamoyltransferase, while it also serves as a by-product of the nitric oxide (NO) synthase (NOS) reaction, which converts L-arginine into LC and NO. Of interest, LC can be purchased over the counter as a dietary supplement. Studies in humans have shown that LC may act as a performance enhancer, however, the biological actions of LC, and the mechanisms by which LC may enhance performance have been relatively understudied. My goal is to delineate the biological actions of LC in skeletal and cardiac muscle.******Hypothesis: I hypothesize that LC enhances metabolism in exercising muscle via increasing peroxisome proliferator-activated receptor gamma coactivator 1a activity, which enhances mitochondrial function. ******Methodology: My laboratory will utilize a number of in vitro/in vivo approaches to elucidate the biological actions of LC in skeletal and cardiac muscle. This includes culturing of neonatal rat cardiac myocytes (NRCMs), H9c2 ventricular myocytes, and C2C12 skeletal myotubes. Furthermore, mice will be subjected to a number of procedures (i.e. high fat diet, exercise), to determine whether LC treatment influences mitochondrial function and oxidative metabolism via indirect calorimetry and mRNA/protein expression profiling of muscle/cardiac extracts. Last, we will assess respiration in isolated muscle/heart mitochondria.******Aims: This research proposal will encompass 3 specific aims; (1) to determine the signaling pathways activated in muscle and heart following treatment with LC, (2) to determine whether LC treatment enhances mitochondrial function in the muscle and heart, and (3) to determine the relative importance of LC versus NO formation in NOS-mediated increases in mitochondrial function. In aim 1, we will treat cultured C2C12 myotubes, H9c2 myocytes, and NRCMs with LC and harvest cell extracts to assess mRNA/protein expression profiles. In aim 2, we will isolate mitochondria from both mouse heart and skeletal muscle and determine whether LC treatment enhances respiration. Furthermore, we will use indirect calorimetry to measure O2 consumption in lean and obese mice treated with LC in their drinking water. In aim 3, we will supplement lean and obese endothelial NOS deficient mice with either LC or a NO donor and assess mitochondrial function.******Impact: Our findings will yield insight into the biological actions of LC in muscle and heart. Moreover, the results of this research program should enhance our understanding of the cellular processes that drive mitochondrial respiration. Last, our findings should also provide significant insights into what "claims" should be allowed for manufacturers who market LC as an over-the-counter nutraceutical dietary supplement to augment exercise performance.