Biology of the Nutraceutical L-Citrulline

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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.