The University Scholars Program

The non-essential amino acid Citrulline plays a critical role in the metabolism of ammonium in the urea cycle, and is a precursor of the amino acid Arginine. Recent studies have shown that Citrulline supplementation in rodents enhanced exercise performance by reducing exercise-related fatigue and energetic cost of contraction (Giannesini B., et al., 2011; Takeda K., et al., 2011). Although the exact mechanisms by which dietary Citrulline exerts these effects are not yet clear, it was suggested that the elimination of the metabolic by-products plasma lactate and ammonium, a known cause of fatigue, mediates this effect(Giannesini B., et al., 2011; Takeda K., et al., 2011). This project aims to evaluate the effect of Citrulline supplementation on mitochondrial function and biogenesis in horse skeletal muscle. We hypothesize that Citrulline supplementation will induce mitochondrial biogenesis and improve mitochondrial function. If Citrulline is found to stimulate mitochondrial biogenesis and enhance mitochondrial function the findings of this study will shed more light on how Citrulline supplementation enhances exercise performance. To test our hypothesis we will utilize muscle samples previously taken from twelve untrained mature horses (average age 10.8 years) enrolled in a Citrulline-Malate supplementation study. The animals’ diet was supplemented daily for a period of 15 days with either Citrulline-Malate (86 mg Citrulline/kg body weight; treatment group, n=6) or placebo (25 mg urea/kg body weight; control group, n=6). Muscle samples were collected prior to supplementation and again on Day 14 of supplementation. On Day 15, the horses were exercised for 2 hours on the free-stall exerciser (medium intensity) and muscle tissue was again collected 1 hour post-exercise. We will test the effects of Citrulline supplementation on mitochondrial function and biogenesis in these muscle samples using established biochemical methods. To study mitochondrial number, the amount of mitochondrial DNA will be assessed. In addition, protein expression of markers for mitochondrial biogenesis, PGC1α and TFAM, will be determined using Western Blot technique. To examine mitochondrial function, expression (by Western Blot) and activity (spectrophotometric enzyme assay) of the mitochondrial enzymes Cytochrome c oxidase and Citrate Synthase will be determined.