Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 91-100). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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Title: The effect of citrulline and citrulline malate supplementation on selected physiological indicators - review study. Objectives: Systematic findings overview of the effect of citrulline and citrulline malate on healthy individual's locomotor system and other physiological indicators. Methods: The study is designed as a findings overview. Electronic databases pubmed.com and ukaz.cuni.cz were used as an information source. "Citrulline" or "citrulline malate" and "exercise performance" and "randomized controlled trial" keywords were used for the information search. Results: Eighteen studies and 307 individuals are included in the overview. Studies are categorized according to duration and type of supplementation substance. Four studies agree on the strength improvement after acute use of citrulline malate. Four studies agree on endurance improvement in case of citrulline long term use. Three studies conclude on pain mitigation in case of substance use 24, 48 and 72 hours after training. Also, studies agree on increase of the levels of citrulline, arginine and ornithine plasmatic concentration after use of citrulline and citrulline malate. Three studies indicate possible fatigue decrease during the training in case of use of citrulline. Conclusion: Use of citrulline and citrulline malate can be...

A citrullinemic fibroblast cell line was used in this study to investigate two biochemical pathways involving citrulline. In the first section, the genetic mutation responsible for the argininosuccinate synthetase (-ASS) deficiency (1-5% activity) in this cell line was investigated. PCR analysis of the ASS cDNA revealed that the mRNA coding region (1236bp) was intact, showing no signs of major rearrangements. The ASS cDNA (1307bp) was cloned and sequenced and showed the presence of a single base mutation at position 1045bp, which represented a G->A transition. This mutation resulted in a glycine -> serine amino acid substitution at position 324 in the ASS subunit protein sequence. Although this glycine residue was not found to occur in any potential substrate binding sites, it was shown to be highly conserved among species, indicating a possible role of this amino acid in ASS catalytic activity. In the second section, the presence of the nitric oxide pathway in fibroblasts was investigated. Inducible nitric oxide synthase activity was assayed by measuring the production of ¹⁴C-citrulline from ¹⁴C-arginine after cytokine stimulation. By using the citrullinemic cell line (ASS deficient) any citrulline that may be produced by this pathway would accumulate, allowing detection. Under the assay conditions that were tested, no detectable ¹⁴C-citrulline was formed. Evidence suggests that human fibroblasts have the potential to synthesise nitric oxide, although a more sensitive assay system may need to be employed (longer cytokine activation, nitrite/nitrate detection).

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The interaction between the embryo and fetus with the maternal environment can have both short- and long-term consequences on health and development after birth. In some cases, these changes may be detrimental to the individual, but in other cases these developmental changes may be beneficial and manipulated to produce desired effects. Our interest is to use this concept of fetal programming to improve skeletal muscle development and meat production in ruminants. To achieve this, we targeted the period of gestation when fetal muscle fiber formation occurs. Primary muscle fibers form during embryonic development, and it is this small number of primary muscle fibers that will serve as templates for secondary fiber formation that occurs in the fetus during mid-gestation. Supplementing amino acids that influence blood flow within the reproductive tract is one potential way to provide fetuses with added nutrients during gestation, and this supplementation strategy may be especially useful when the maternal diet is compromised. L-citrulline was chosen for this work because of its long half-life in maternal circulation. This work utilized twin-bearing ewes with a moderate dietary energy restriction to assess the effects of mid-gestational L-citrulline supplementation on umbilical blood flow, placental function, neonatal lamb size, and lamb performance. We hypothesize that i.v. administration of L-citrulline will increase uterine and placental blood flow in gestating ewes and this will improve fetal growth, development, and overall postnatal performance. Blood flow parameters were not influenced by treatment (P>0.05). Circulating levels of progesterone and pregnancy-specific protein B (PSPB) were used as indicators of placental function and were unaffected by treatment administration (P>0.05). A treatment by time interaction was detected in both analyses, but no differences between treatments were detected within any time points. There was no effect of treatment on lamb weights or survival to weaning (P>0.05). Lamb sex effects are absent with the exception that body weights were greater in ewe lambs (P>0.05). There was no effect of treatment on any carcass traits or visceral organ weights assessed, though there was an effect of sex on dressing percentage and pancreas weight with wethers having a greater dressing percentage and heavier pancreases per kg body weight than that of ewes (P<0.05). In summary, contrary to our hypothesis L-citrulline supplementation to pregnant ewes under a minor to moderate metabolic challenge had no impact on blood flow and provided no programming benefit to the lambs. / Master of Science / The global population continues to grow, along with the consumption of animal protein. This can be met with increasing the numbers of animals within our food production systems, however, there is also increasing pressure for livestock production systems to produce more while utilizing less space and resources. And simultaneously, we face growing concerns about climate change, its impacts on agriculture, and the role of agriculture in both the cause and any future solution. To combat both these issues, the efficiency of our livestock systems needs to improve with each individual animal becoming much more efficient. This increase in efficiency can occur in many ways including reproductive efficiency, feed efficiency, and in overall producing more meat per individual. The improvement in efficiency of an animal can begin in the womb. Livestock in meat production spend 35-40% of their life within the uterus being nourished by their mother. The interactions the embryo and fetus have with the maternal environment during this time can have both short- and long-term impacts on health and development after birth. In some cases, these changes may be detrimental to the individual, but in other cases these developmental changes may be beneficial and manipulated to produce the desired effects. Thus, it is important to understand the impact of these fetal-maternal interactions as it directly affects both fetal growth and growth and development after birth. This concept is known as fetal programming. Our interest is to use this concept to improve skeletal muscle development and meat production in cattle and sheep. To achieve this, we targeted the period of pregnancy when fetal muscle formation occurs. Primary muscle fibers form early in pregnancy, and it is this small number of primary muscle fibers that will serve as templates for secondary fiber formation that occurs in the fetus during mid-pregnancy. Supplementing amino acids that influence blood flow within the reproductive tract is one potential way to provide fetuses with added nutrients during pregnancy, and this supplementation strategy may be especially useful when the maternal diet is compromised. L-citrulline was chosen for this work because of its long half-life in maternal circulation. This work utilized twin-bearing ewes with a moderate dietary energy restriction to assess the effects of L-citrulline supplementation on blood flow, placental function, newborn lamb size, and lamb performance. We hypothesize that intravenous administration of L-citrulline will increase uterine and placental blood flow in pregnant ewes and this will improve fetal growth, development, and overall postnatal performance. There was no beneficial effect on blood flow to the fetus and on placental function. Additionally, there were very minimal effects on carcass traits or internal organ weights assessed. In summary, contrary to our hypothesis L-citrulline supplementation to pregnant ewes under a moderate metabolic challenge had no impact on blood flow and provided no programming benefit to the lambs. We can conclude that the potential benefit of amino acid supplementation was not realized in our sheep model.

La stéatose hépatique non alcoolique (NAFLD) est une manifestation du syndrome métabolique dont la prévalence est en constante évolution. Les stratégies thérapeutiques sont soit difficiles à mettre en œuvre soit d’une efficacité limitée. Nous avons étudié une approche nutritionnelle avec 3 acides aminés particuliers : la glutamine, l’arginine et la citrulline (Cit) pour leurs propriétés de pharmaconutriments azotés. Dans un modèle de NAFLD modérée induite par le fructose, seule la citrulline (1 g/kg/j) permettait une amélioration du métabolisme lipidique. Toutefois, l’étude de la cinétique de NAFLD suggérait un effet protecteur du simple apport azoté. L’effet spécifique de la Cit par rapport au simple apport azoté (AANE) a donc été déterminé dans un modèle de NAFLD induite par 8 semaines de régime enrichi en fructose. Ceci a permis de confirmer l’effet protecteur de la Cit et des AANE. Toutefois, la Cit exerce un effet plus spécifique sur l’expression de Srebp1c et de Fas et améliore la disponibilité périphérique en Arg, un élément important de l’insulino-sensibilité. La stéatose est associée à une perte de masse maigre, suggérant une oxydation des AA aux dépens de l’anabolisme musculaire, et une accumulation de lipides à l’origine de la stéatose et du gain de masse grasse viscérale ; la Cit et les AANE en agissant sur la NAFLD préviendraient cet effet du fructose. Nous avons ensuite évalué les effets de la Cit dans un modèle de stéatose plus sévère induite par le western diet. La Cit améliore la fonction hépatique (diminution des lipides et de l’inflammation hépatique) et préserve l’axe intestin-foie (restauration du groupe Bacteroides/Prevotella dans la muqueuse colique, diminution de l’inflammation intestinale et augmentation de l’expression de la Claudine 1) mais ne permet pas de prévenir l’ensemble des altérations liées au western diet. Il serait intéressant d’évaluer la relation effet/dose et l’efficacité de la Cit en association avec d’autres traitements. Par ailleurs, les mécanismes cellulaires restent à élucider. / Nonalcoholic fatty liver disease (NAFLD) is a manifestation of the metabolic syndrome whose prevalence is constantly growing. Therapeutic strategies are either difficult to implement or of limited effectiveness. We studied a nutritional approach with three specific amino acids: glutamine, arginine and citrulline (Cit) for their pharmaconutrient properties. In a model of moderate fructose-induced NAFLD, citrulline alone (1 g/kg/day) improved lipid metabolism. However, the study of the kinetics of NAFLD suggested a protective effect of nitrogen supply by itself. The specific effect of Cit compared to that of nitrogen (NEAAs) has been determined in a model of 8 week fructose diet-induced NAFLD. This has confirmed the protective effect of Cit and NEAAs. However, Cit exerted a specific effect on the expression of Fas and SREBP1c and improves peripheral Arg availability, an important component of insulin sensitivity. Steatosis was associated with loss of lean mass, suggesting AA oxidation at the expense of muscle anabolism, and lipid accumulation causing steatosis and visceral fat gain; Cit and NEAAs by acting on NAFLD would prevent this effect of fructose. We then evaluated the effects of Cit in a model of more severe steatosis induced by western diet. Cit improved liver function (reduced fat and liver inflammation) and protected the liver-gut axis (restoration of Bacteroides/Prevotella group in the colonic mucosa, decreased intestinal inflammation and increased expression of claudin 1) but did not prevent all western diet-induced alterations. It would be interesting to assess the dose/effect relationship and the effectiveness of Cit in combination with other treatments. Furthermore, the cellular mechanisms remain to be elucidated.

La dénutrition protéino-énergétique, qui touche une partie de la population âgée, accentue leur déclin musculaire (sarcopénie et dynapénie) et participe donc à l'augmentation de la morbi-mortalité. La prévention et le traitement de la dénutrition s'avèrent difficiles, lié au défaut de réponse à la renutrition observé chez les personnes âgées ; celui-ci est expliqué par une forte extraction splanchnique des acides aminés en période post-prandiale, responsable d'une moindre stimulation de la protéosynthèse musculaire. La citrulline, acide aminé non protéinogène qui échape à la séquestration splanchnique des acides aminés, est un facteur stimulant de la synthèse protéique musculaire. Nous avons montré qu'un apport en citrulline s'accompagne d'une amélioration de la masse et des capacités fonctionnelles musculaires (activité motrice, force musculaire maximale isométrique) chez le rat âgé dénutri. Cet effet bénéfique de la citrulline s'expliquerait, en partie, par une surexpression des protéines contractiles myofibrillaires et une diminution de l'expression d'éléments des voies de dégradation protéique. Son mécanisme d'action reste encore à préciser bien que nos données suggèrent également une réorientation du métabolisme énergétique. En complément de ces données expérimentales, nous développons une étude clinique afin de vérifier les effets bénéfiques de la citrulline sur le métabolisme protéique de la personne âgée dénutrie. Suite à cette étude, nous pourrons réaliser un essai thérapeutique de plus grande ampleur, afin de montrer l'efficacité de la citrulline sur l'amélioration de paramètres reflétant la fonction musculaire. Ainsi, l'administration de citrulline pourrait être une stratégie nutritionnelle efficace et innovante afin de limiter le déclin musculaire survenant au cours du vieillissement. En s'opposant à la dénutrition protéino-énergétique des personnes âgées, elle pourrait retarder l'entrée dans la dépendance, améliorer la qualité de vie et limiter la morbi-mortalité liée à la dynapénie.

The discovery of nitric oxide (NO) as the endothelial-derived relaxing factor has led to significant research on NO and the proteins involved in its function, generation, location and regulation. Synthesis of NO by blood vessel endothelial cells results from the enzymatic oxidation of arginine by endothelial nitric oxide synthase (eNOS) resulting in the formation of equimolar amounts of NO and citrulline. Citrulline is sequentially recycled to arginine by successive reactions involving the enzymes argininosuccinate synthase (AS) and argininosuccinate lyase (AL), respectively. eNOS activity has been shown to be regulated by post-translational modifications including dynamic phosphorylation on multiple serine/threonine and tyrosine residues and dynamic O-linked beta-N-acetylglucosamine (O-GlcNAc) modifications on serine/threonine residues.Previous studies showed that even though intracellular endothelial arginine levels range from 0.1 to 0.8 mM and the Km of eNOS for arginine is 3 uM, the addition of exogenous arginine caused an increase in NO production. To explain this "arginine paradox" we hypothesize that there is a separate and distinct cellular source of arginine substrate directed to NO production and that this source is maintained through the regeneration of arginine via a citrulline-NO cycle. The presented research has provided the following evidence in support of this hypothesis: Citrulline stimulates NO production in an arginine-rich medium, without an increase in intracellular arginine. The enzymes of the citrulline-NO cycle, eNOS, AS and AL, co-fractionate with caveolin-1 in an endothelial cell caveolar membrane fraction. In vitro interaction assays demonstrate protein-protein interactions between fusion tagged AS or AL with eNOS or caveolin-1. Simultaneous monitoring of apparent citrulline and NO production demonstrates an efficient and essential coupling of the reactions of the citrulline-NO cycle. Glucosamine treatment of endothelial cells results in increased NO production in the basal state and decreased NO production in the stimulated state.Our findings demonstrate the enzymes of the citrulline-NO cycle, eNOS, AS and AL, are functionally associated, the reactions are efficiently coupled and enzyme activities are changed by post-translational modifications based on nutrient levels. These alterations ensure a constant and distinct source of arginine which is available for NO production to ensure vascular health.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 130-144). Also available online.