xii, 78 leaves : ill., appendices / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry and General Physiology, 1973

Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry and General Physiology, 1973.

Outre son rôle dans le métabolisme du cycle de l’urée, la citrulline possède de nombreuses propriétés notamment celle de stimuler la synthèse protéique musculaire. Or, la synthèse protéique est un poste de dépense énergétique important de la cellule. Nos travaux ont ainsi exploré, à la fois in vivo et in vitro, les effets de la citrulline sur le métabolisme énergétique afin de comprendre comment l’activation de la synthèse protéique musculaire par cet acide aminé est coordonnée avec le métabolisme énergétique. Nos résultats ont permis de mettre en évidence que la citrulline module bien le métabolisme énergétique, notamment via une réorientation des flux énergétiques au profit de la synthèse protéique. De plus, nos travaux ont précisé les effets de la citrulline sur le métabolisme protéique avec notamment un effet synergique de la citrulline et de l’exercice sur la synthèse protéique et sur la performance. Enfin, ces travaux ont permis d’explorer pour la première fois in vitro les effets de la citrulline (et de la leucine) sur le sécrétome musculaire. Nous avons ainsi démontré que la citrulline module le sécrétome musculaire et mis en évidence la complexité de régulation des protéines sécrétées par les acides aminés. En conclusion, nos travaux sont une contribution à la meilleure compréhension de la régulation musculaire du métabolisme protéino-énergétique par la citrulline. / Besides his role in the metabolism of the urea cycle, citrulline has many properties including the ability to stimulate muscle protein synthesis. However, protein synthesis is an important item of cell energy expenditure. Our work has explored both the in vivo and in vitro effects of citrulline on energy metabolism in order to understand how the activation of muscle protein synthesis by this amino acid is coordinated with energy metabolism. Our results have shown that citrulline modulates energy metabolism via a reorientation of energy flux in favor of protein synthesis. Moreover, our work has clarified citrulline effects on protein metabolism with a synergistic effect of citrulline and exercise on protein synthesis and performance. Finally, this work allowed to explore for the first time citrulline (and leucine) effects on muscle secretome. We have thus demonstrated that citrulline modulates muscle secretome and highlighted how complex is the regulation of secreted proteins by amino acids. In conclusion, our work contributes to a better understanding on muscle regulation of protein-energy metabolism by citrulline.

La polyarthrite rhumatoïde (PR) est une maladie auto-immune (MAI) dans laquelle des anticorps anti-peptides citrullinés (ACPA) sont un outil diagnostique très spécifique. L’un des traitements clé de la PR est le méthotrexate (MTX). En plus de son action directe sur la maladie il renforce l’effet des anti-TNF alpha (aTNF). Ceci pourrait passer par une prévention de la formation d’anticorps anti-médicaments dirigés contre les aTNF ce qui leur fait perdre leur efficacité.Nous avons développé un modèle macaque pour reproduire la maladie humaine par immunisation avec des peptides citrullinés dans le contexte d’un facteur génétique favorisant la PR : l’épitope HLA partagé. L’immunisation de macaques avec divers peptides citrullinés en utilisant un boost intra-articulaire a déclenché une réponse T et B anti-citrulline et a entraîné une mono-arthrite chronique.Le rôle du MTX sur l’immunogénicité des aTNF a été étudié sur un modèle de souris autoimmunes, les souris BAFF transgéniques (tg) qui présentent une MAI. L’utilisation du MTX juste avant l’injection d’aTNF a permis de prévenir l’immunisation contre ce médicament uniquement chez ces souris BAFFtg et pas chez des souris sauvages ou chez des macaques. Nous avons démontré que ces souris BAFFtg surexprimaient CD73, ce qui permettait une sécrétion accrue d’adénosine et de cellules B régulatrices sous l’effet du MTX. L’interaction entre BAFF et le méthotrexate a été confirmée chez l’homme dans la cohorte ABIRISK : le MTX prévient plus efficacement l’immunogénicité chez les patients avec des taux de BAFF élevés.En conclusion, nous avons mis au point deux nouveaux modèles animaux permettant de mieux comprendre la physiopathologie de la PR et d’optimiser l’utilisation des traitements biologiques qui s’étend dans tous les domaines de la médecine. / Title : Development of new animal models to study the pathophysiology of RA and the role of methotrexate-induced tolerance.Keywords : Rheumatoid arthritis, shared epitope, ACPA, immunogenicity, methotrexate, TNF inhibitorsAbstract: Rheumatoid arthritis (RA) is an autoimmune disease (AID) where antibodies directed against citrullinated peptides (ACPA) are highly specific for the diagnosis. One of the key treatments of RA is methotrexate. It has an action on both the disease and reinforces the effect of second line TNF inhibitors (TNFi). MTX might act via prevention of anti-drug antibodies (ADAb) directed against TNFi that are implicated in loss of efficacy of TNFi. We have developed a macaque model to recapitulate the human disease by immunization with citrullinated peptides in the context of a genetic factor favoring RA: the shared epitope on the HLA. Immunization of macaques with citrullinated peptides and intra-articular boost cause an anti-citrulline T and B cell response and a chronic monoarthritis.The role of MTX-induced tolerance against TNFI has been studied in autoimmune BAFF transgenic (tg) mice using MTX just before treatment with TNFi we were able to prevent ADAb formation in BAFFtg mice and not wild type mice or macaques. We identified that BAFFtg mice expressed elevated CD73 leading to more adenosine and regulatory B cells as actors in MTX-induced tolerance. This MTX-BAFF interaction was further confirmed in humans in the ABIRISK cohort where MTX was more efficient to prevent ADAb formation in RA patients with elevated BAFF levels.Setting up two new animal models allows better understanding of RA pathophysiology and better use of biologics that extend to other domains of medicine.

Ergogenic L-citrulline and malate are amino acids used in specific combination to effect muscular endurance during athletic performances. Purpose: The study aimed to investigate the ergogenic properties of citrulline-malate (CM) during a resistance training protocol. Methods: Utilizing a randomized, counterbalanced, double blind study, fifteen trained males completed a resistance training protocol once using placebo (PL) and once with CM (8.0g). Results: CM supplementation increased repetitions in chin-ups, reverse chin-ups, push-ups, and total trial repetitions. Blood lactate was significantly increased post-exercise compared to pre-exercise, but was not significantly different between CM and placebo. Further, a significant interaction effect was revealed for systolic blood pressure, a significant condition effect for diastolic blood pressure, and a significant time effect for HR. Post-hoc analysis revealed that SBP responses were more elevated in the placebo condition during recovery. Conclusion: Collectively, these novel findings suggest CM increases muscular endurance during upper body resistance exercise.

L-citrulline and malate are amino acids within the body that have the potential to affect muscular endurance during athletic performances. Purpose: This study intended to test the effectiveness of CM on muscular performance and fatigue during a lower body resistance protocol. Methods: Twelve trained males completed a lower body resistance training protocol for two sessions, one using CM (8.0g) and the other with placebo (PL), within a randomized, double blind, counterbalanced study. Results: CM supplementation increased repetitions in leg press, hack squat and leg extension. Blood lactate was significantly increased post-exercise compared to pre-exercise, but no significant difference was found between CM and placebo. In addition, no significant differences were found for systolic blood pressure, diastolic blood pressure, or HR between the CM and PL groups. Conclusion: These findings suggest CM attenuates muscular fatigue during a specific lower body exercise protocol.

La citrulline est un acide aminé dont les propriétés anaboliques n’ont été mises en évidence que très récemment. Ces propriétés pourraient relever en particulier de son rôle de précurseur direct de l’arginine. L’arginine est un acide aminé central du métabolisme des mammifères, notamment comme précurseur de nombreuses molécules exerçant des fonctions physiologiques essentielles, en particulier les polyamines, que ce soit les polyamines « classiques » putrescine, spermidine et spermine, dérivés de décarboxylation de l’ornithine impliquées entre autres dans la prolifération cellulaire, ou l’agmatine, dérivé de la décarboxylation de l’arginine. Toutefois, les interactions métaboliques entre citrulline et polyamines restent encore peu connues. Le but de ce travail a été d’explorer le lien métabolique entre citrulline et polyamines, selon deux démarches distinctes.La première a été d’évaluer l’influence d’une complémentation prolongée (3 mois) en citrulline sur le métabolisme tissulaire des polyamines « classiques » chez le rat âgé. La citrulline n’affecte que de manière modérée le métabolisme des polyamines, principalement en corrigeant les altérations liées au vieillissement.La seconde, par analogie avec la putrescine et l’agmatine, a exploré l’implication du dérivé décarboxylé de la citrulline, la N-carbamoyl-putrescine (NCP) dans le métabolisme des mammifères après avoir mis au point une technique de dosage de celle-ci dans les milieux biologiques. Nous n’avons pas pu mettre en évidence la présence de NCP dans des tissus de rats ayant reçu de la citrulline par voie orale ou par voie parentérale suggèrant soit une absence de synthèse soit un métabolisme extrêmement rapide de la NCP. Par ailleurs, la NCP administrée par voie orale à des doses de 5 et 50 mg/kg/j ne semble pas induire de toxicité ni d’effets biologiques majeurs mais la démonstration de son absorption digestive reste à faire. Cependant, la NCP exerce quelques effets modérés sur le métabolisme des acides aminés et des polyamines, l’évaluation de doses plus élevées ou l’administration par voie parentérale permettrait de confirmer ces observations. / The anabolic properties of the amino acid citrulline have only been recently demonstrated. These properties may result at least in part from its role as an arginine precursor. Arginine plays a central role in mammalian amino acid metabolism, notably as the precusor of many molecules involved in essential physiological functions, such as polyamines either “classical” ones, namely putrescine, spermidine, and spermine, derived from ornithine decarboxylation and notably involved in cell proliferation, or more recently-discovered molecules such as agmatine, the decarboxylation derivative of arginine. Metabolic interactions between citrulline and polyamines are still mostly unknown.The aim of this work was to explore the metabolic link between citrulline and polyamines using two different strategies.First, the influence of a long term citrulline supplementation on tissue “classical” polyamine metabolism in aged rats has been evaluated. Polyamine metabolism is only moderately affected by citrulline supplementation, mostly correcting aging-related alterations.Second, by analogy with putrescine and agmatine, the involvement of the decarboxylated derivative of citrulline, N-carbamoyl putrescine (NCP), in mammalian metabolism was investigated after having developped an HPLC assay for the determination of NCP in biological media. We have not been able to demonstrate the presence of NCP in tissues of rats having received citrulline orally or parenterally suggesting either a lack of synthesis or an extremely rapid metabolism of NCP. In addition, oral administration of NCP 5 or 50 mg/kg/d did not appear to induce toxicity or major biological effects but the demonstration of its gastrointestinal absorption remains to be done. However, NCP has some moderate effects on amino acid and polyamine metabolism. The evaluation of higher doses of NCP or of its parenteral administration is required to confirm these observations.

Citrullinemia and argininosuccinic aciduria are two disorders resulting from defects in two consecutive enzymes of the urea cycle, argininosuccinate synthetase and argininosuccinate lyase. Fibroblast cell lines were derived from patients with these disorders and the diagnoses, which had been made on the basis of amino acid levels in plasma and urine, were confirmed by demonstrating that the cell lines were unable to incorporate 14 c-citrulline into protein. DNA from the argininosuccinate synthetase-deficient (ASS⁻) cells was analysed by restriction enzyme digestion and hybridisation to a cDNA probe which had been cloned from human argininosuccinate synthetase mRNA. No defect in the patient's DNA could be demonstrated, indicating that no major deletions in the argininosuccinate synthetase genes were present in this patient. Co-cultures of the ASS⁻ and argininosuccinate lyase-deficient (ASL⁻) fibroblasts were able to incorporate 14 citrulline into protein at rates comparable to normal fibroblasts. This complementation did not require cell fusion, was dependent on cell contact, and was not the result of exchange of metabolites or enzymes via the culture medium. These results indicated that complementation occurred by the exchange of metabolites via intercellular junctions between the two cell types. Co-cultures of ASS⁻ and ASL⁻ cells were used as an assay system for measuring intercellular junctional communication. This allowed quantitation of the effects of pH and extracellular divalent cations on junctional communication. Tumor promoters such as phorbol esters and organochlorine pesticides have been reported to inhibit intercellular junctional communication in other systems, and this inhibitory activity may be related to the mechanism of tumor promotion. The organochlorine pesticide 1,1,1-trichloro- 2,2-bis(p-chlorophenyl)ethane (DDT) was shown to be an inhibitor of junctional communication in ASS⁻/ASL⁻ cocultures. This inhibition was reversible, of rapid onset, and independent of extracellular calcium. The tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13- acetate (TPA) also rapidly induced inhibition of junctional communication. However, co-cultures between Chinese hamster V79 cells, which are deficient in ASS⁻, and ASL⁻ human fibroblasts were more sensitive to inhibition by TPA than the original ASS⁻/ASL⁻ co-cultures. Refractoriness to TPA occurred following prolonged treatment with high concentrations of TPA. Retinoic acid and other retinoids also inhibited junctional communication, and the inhibitory effects of retinoic acid and TPA were additive. The significance of these results in relation to the anti-tumor-promoting activity of retinoic acid is discussed. It is concluded that co-cultures of ASS⁻ and ASL⁻ cells constitute a useful system for providing quantitative measurements of intercellular junctional communication under a wide range of experimental conditions.

Aims There is a high prevalence of hypertension in the African population and it is known that vascular dysfunction (including nitric oxide (NO) bio-availability markers) play an important role in the development of cardiovascular diseases. Since very little is known regarding the role of markers of NO bio-availability in Africans, the aim of this study was to compare markers of NO bio-availability (namely L-arginine, L-citrulline, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA)), ambulatory blood pressure (BP) and markers of end organ damage between African and Caucasian school teachers. Additionally, we also aimed to determine whether these markers of NO bio-availability are associated with ambulatory BP and markers of end organ damage in both ethnic groups. Methods The SABPA (Sympathetic activity and Ambulatory Blood Pressure in Africans) study was a cross-sectional study, including urbanised African (N=181) and Caucasian (N=209) men and women, between the ages of 25 and 65 years. Cardiovascular measurements included ambulatory blood pressure, pulse wave velocity (PWV), electrocardiographic Cornell product and carotid intima media thickness (cIMT). Anthropometric measurements included height, weight and waist circumference. Various bio-markers were analysed, including glucose, L-arginine, ADMA, SDMA, Lcitrulline, reactive oxygen species, albumin-to-creatinine ratio (ACR) and estimated creatinine clearance (eCCR). Characteristics of groups were compared with independent T-tests and Chi-square tests. Single and partial analyses were used to investigate associations between NO bioavailability markers with ambulatory BP measurements and markers of end organ damage. Analyses of covariance (ANCOVA) were used for comparison of variables between groups to determine significant differences, while adjusting for age, body mass index and antihypertensive medication. Forward stepwise multiple regression analyses were performed to determine if independent associations exist between ambulatory BP measurements or markers of end organ damage with either- L-arginine, L-citrulline, ADMA or SDMA as the main independent variable. Results and conclusion The Africans and Caucasians were of similar ages. However, the Africans had higher blood pressure therefore their cardiovascular profile was unfavourable compared to that of the Caucasians. The inhibitors of NO biosynthesis, ADMA and SDMA, were significantly lower in the Africans (p=0.046; p<0.001, respectively). However, the NO bio-availability markers, L-arginine and L-citrulline, were higher in the African compared to the Caucasian participants (all p values <0.05) regarded as significant. When performing unadjusted analyses, we found significant negative associations between eCCR and L-citrulline in all four subgroups: African men (r=-0.27; p=0.013), African women (r=-0.24; p=0.021), Caucasian men (r=-0.21; p=0.044) and Caucasian women (r=-0.28; p=0.003). The association of eCCR with L-citrulline was confirmed to be independent of confounders in all groups: African men (R2=0.46; β=-0.23; p=0.006), African women (R2=0.68; β= -0.12; p=0.046), Caucasian men (R2=0.62; β= -0.24; p<0.001) and Caucasian women (R2=0.72; β= -0.13; p=0.029). This implicates that renal function may be detrimentally affected by L-citrulline concentrations. In the Caucasian men and women negative correlations between eCCR and SDMA were found before adjustments (r=-0.33; p=0.003 and r=-0.26; p=0.006, respectively). This phenomenon was confirmed in the forward stepwise multiple regression analysis in Caucasian men (R2=0.75; β= -0.27; p<0.001) and women (R2=0.73; β= -0.21; p<0.001), while no associations were found in the Africans. This result is not unexpected, since SDMA can only be eliminated by the kidneys and is therefore an important risk marker for the early detection of renal dysfunction. In Caucasian men we found that ADMA correlated with ACR (r=0.36; p=0.001), night-time SBP (r=0.34; p=0.002) and night-time DBP (r=0.25; p=0.023) with single linear regression analyses. A similar trend was shown in African men with night-time SBP (r= 0.20; p=0.089) and night-time DBP (r= 0.21; p=0.078) respectively, but this association was absent in the Caucasian and African women. After adjustments for age and body mass index, the associations with ADMA, ACR and SBP in the Caucasian men remained. However, a negative association between eCCR and ADMA also became evident in the African men (r=- 0.24; p=0.025) and remained significant in the forward stepwise multiple regression analysis (R2=0.44; β= -0.18; p=0.034). It is, however, not clear why our results were gender specific, but we could speculate that the female sex hormones may play a part in protecting the vascular endothelium. Apart from the associations described above, there were no significant independent associations between the markers of the NO cycle (such as L-arginine) and PWV, cIMT, eCCR, ACR or Cornell product. In conclusion, although Africans presented a more vulnerable cardiovascular profile, we found a consistent negative association between renal function and L-citrulline in all participants, which has only been reported previously in patients with chronic renal disease. Additionally we found a gender-specific link between renal function and ADMA in African and Caucasian men. Our results may indicate that in the general population, markers of NO bioavailability may be associated with early changes in renal function, accompanying elevated blood pressure. / Thesis (MSc (Physiology))--North-West University, Potchefstroom Campus, 2013

Aims There is a high prevalence of hypertension in the African population and it is known that vascular dysfunction (including nitric oxide (NO) bio-availability markers) play an important role in the development of cardiovascular diseases. Since very little is known regarding the role of markers of NO bio-availability in Africans, the aim of this study was to compare markers of NO bio-availability (namely L-arginine, L-citrulline, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA)), ambulatory blood pressure (BP) and markers of end organ damage between African and Caucasian school teachers. Additionally, we also aimed to determine whether these markers of NO bio-availability are associated with ambulatory BP and markers of end organ damage in both ethnic groups. Methods The SABPA (Sympathetic activity and Ambulatory Blood Pressure in Africans) study was a cross-sectional study, including urbanised African (N=181) and Caucasian (N=209) men and women, between the ages of 25 and 65 years. Cardiovascular measurements included ambulatory blood pressure, pulse wave velocity (PWV), electrocardiographic Cornell product and carotid intima media thickness (cIMT). Anthropometric measurements included height, weight and waist circumference. Various bio-markers were analysed, including glucose, L-arginine, ADMA, SDMA, Lcitrulline, reactive oxygen species, albumin-to-creatinine ratio (ACR) and estimated creatinine clearance (eCCR). Characteristics of groups were compared with independent T-tests and Chi-square tests. Single and partial analyses were used to investigate associations between NO bioavailability markers with ambulatory BP measurements and markers of end organ damage. Analyses of covariance (ANCOVA) were used for comparison of variables between groups to determine significant differences, while adjusting for age, body mass index and antihypertensive medication. Forward stepwise multiple regression analyses were performed to determine if independent associations exist between ambulatory BP measurements or markers of end organ damage with either- L-arginine, L-citrulline, ADMA or SDMA as the main independent variable. Results and conclusion The Africans and Caucasians were of similar ages. However, the Africans had higher blood pressure therefore their cardiovascular profile was unfavourable compared to that of the Caucasians. The inhibitors of NO biosynthesis, ADMA and SDMA, were significantly lower in the Africans (p=0.046; p<0.001, respectively). However, the NO bio-availability markers, L-arginine and L-citrulline, were higher in the African compared to the Caucasian participants (all p values <0.05) regarded as significant. When performing unadjusted analyses, we found significant negative associations between eCCR and L-citrulline in all four subgroups: African men (r=-0.27; p=0.013), African women (r=-0.24; p=0.021), Caucasian men (r=-0.21; p=0.044) and Caucasian women (r=-0.28; p=0.003). The association of eCCR with L-citrulline was confirmed to be independent of confounders in all groups: African men (R2=0.46; β=-0.23; p=0.006), African women (R2=0.68; β= -0.12; p=0.046), Caucasian men (R2=0.62; β= -0.24; p<0.001) and Caucasian women (R2=0.72; β= -0.13; p=0.029). This implicates that renal function may be detrimentally affected by L-citrulline concentrations. In the Caucasian men and women negative correlations between eCCR and SDMA were found before adjustments (r=-0.33; p=0.003 and r=-0.26; p=0.006, respectively). This phenomenon was confirmed in the forward stepwise multiple regression analysis in Caucasian men (R2=0.75; β= -0.27; p<0.001) and women (R2=0.73; β= -0.21; p<0.001), while no associations were found in the Africans. This result is not unexpected, since SDMA can only be eliminated by the kidneys and is therefore an important risk marker for the early detection of renal dysfunction. In Caucasian men we found that ADMA correlated with ACR (r=0.36; p=0.001), night-time SBP (r=0.34; p=0.002) and night-time DBP (r=0.25; p=0.023) with single linear regression analyses. A similar trend was shown in African men with night-time SBP (r= 0.20; p=0.089) and night-time DBP (r= 0.21; p=0.078) respectively, but this association was absent in the Caucasian and African women. After adjustments for age and body mass index, the associations with ADMA, ACR and SBP in the Caucasian men remained. However, a negative association between eCCR and ADMA also became evident in the African men (r=- 0.24; p=0.025) and remained significant in the forward stepwise multiple regression analysis (R2=0.44; β= -0.18; p=0.034). It is, however, not clear why our results were gender specific, but we could speculate that the female sex hormones may play a part in protecting the vascular endothelium. Apart from the associations described above, there were no significant independent associations between the markers of the NO cycle (such as L-arginine) and PWV, cIMT, eCCR, ACR or Cornell product. In conclusion, although Africans presented a more vulnerable cardiovascular profile, we found a consistent negative association between renal function and L-citrulline in all participants, which has only been reported previously in patients with chronic renal disease. Additionally we found a gender-specific link between renal function and ADMA in African and Caucasian men. Our results may indicate that in the general population, markers of NO bioavailability may be associated with early changes in renal function, accompanying elevated blood pressure. / Thesis (MSc (Physiology))--North-West University, Potchefstroom Campus, 2013