La citrulline d'origine intestinale contrôle-t-elle la fonction musculaire?

Thèse en cours au laboratoireThe thesis target tends to better understand the key role of the citrullin (amino acid produces by the intestine)in musclar protein metabolism regultaion. On one hand, regarding the role of OTC enzyme through the development and use of a new mice line with a inducible KO for the OTC gene. On another hand, regarding the regulation of this enzyme thanks to a human duodenal explant model.Le sujet de thèse vise à mieux comprendre le rôle de la citrulline (acide aminé produit par l'intestin) dans la régulation du métabolisme protéique musculaire. D'une part, concernant le rôle de l'OTC par la mise au point et l'utilisation d'une lignée de souris KO inductible pour le gène de l'OTC,d'autre part, concernant la régulation de cet enzyme grâce à l'utilisation d'un modèle d'explant duodénal

Ornithine transcarbamylase (OTC) : Rôle et régulation dans l'homéostasie protéino-énergétique

Beyond its transitional role in the urea cycle, numerous works have evidenced pleïotropic effects of citrulline in the metabolism, especially in the proteino-energy metabolism. Indeed, its amino acid is able to stimulate muscle protein synthesis which is closely related to cellular energy metabolism. Nevertheless, studies conducted until now do not allow to conclude about the importance of the specific endogenous citrulline production by the enterocytes. Also, in the organim, citrulline is produced thanks to a key enzyme, the ornithine transcarbamylase (OTC), giving to its functionnality a huge implication in the regulation of the proteino-energy homeostasis. Works presented here set up an in vitro model of human duodenal explants, as well as, an in vivo murine model of conditional knock out for the OTC gene. Results obtained have allowed to evidence the importance of the citrulline metabolism since it remains stable over energy status deregulation. Moreover, they have allowed to specify that a healthy organism is able to compensate an OTC deficiency, however, the induction of an energy deregulation with a low protein diet bring to light a major role of the endogenous citrulline in these situations.Outre son rôle d’intermédiaire dans le cycle de l’urée, de nombreux travaux mettent en évidence les effets pléïotropiques de la citrulline dans le métabolisme et notamment dans le métabolisme protéino-énergétique. En effet, cet acide aminé est capable de stimuler la synthèse protéique musculaire étroitement liée avec le métabolisme énergétique cellulaire. Cependant les études menées jusqu’alors ne permettent pas de conclure quant à l’importance de la production endogène de citrulline par les entérocytes spécifiquement. Aussi, dans l’organisme, la citrulline est produite par une enzyme clé, l’ornithine transcarbamylase (OTC), donnant à la fonctionnalité de cette dernière une implication tout aussi importante dans le maintien de l’homéostasie protéino-énergétique. Les travaux présentés ici mettent en place un modèle d’étude in vitro d’explants duodénal humain ainsi qu’un nouveau modèle murin de knock out conditionnel du gène de l’OTC. Les résultats obtenus ont permis de mettre en évidence l’importance du métabolisme de la citrulline puisque celui-ci reste stable avec l’induction d’une altération du statut énergétique. De plus ils ont permis de spécifier qu’un la capacité d’un organisme sain à compenser une déficience en OTC, cependant, l’induction de dérégulation énergétique par un régime hypoprotéique met en avant un rôle majeur de la citrulline endogène dans ces situations

Ornithine transcarbamylase (OTC) : role and regulation in energy protein homeostasis

Outre son rôle d’intermédiaire dans le cycle de l’urée, de nombreux travaux mettent en évidence les effets pléïotropiques de la citrulline dans le métabolisme et notamment dans le métabolisme protéino-énergétique. En effet, cet acide aminé est capable de stimuler la synthèse protéique musculaire étroitement liée avec le métabolisme énergétique cellulaire. Cependant les études menées jusqu’alors ne permettent pas de conclure quant à l’importance de la production endogène de citrulline par les entérocytes spécifiquement. Aussi, dans l’organisme, la citrulline est produite par une enzyme clé, l’ornithine transcarbamylase (OTC), donnant à la fonctionnalité de cette dernière une implication tout aussi importante dans le maintien de l’homéostasie protéino-énergétique. Les travaux présentés ici mettent en place un modèle d’étude in vitro d’explants duodénal humain ainsi qu’un nouveau modèle murin de knock out conditionnel du gène de l’OTC. Les résultats obtenus ont permis de mettre en évidence l’importance du métabolisme de la citrulline puisque celui-ci reste stable avec l’induction d’une altération du statut énergétique. De plus ils ont permis de spécifier qu’un la capacité d’un organisme sain à compenser une déficience en OTC, cependant, l’induction de dérégulation énergétique par un régime hypoprotéique met en avant un rôle majeur de la citrulline endogène dans ces situations.Beyond its transitional role in the urea cycle, numerous works have evidenced pleïotropic effects of citrulline in the metabolism, especially in the proteino-energy metabolism. Indeed, its amino acid is able to stimulate muscle protein synthesis which is closely related to cellular energy metabolism. Nevertheless, studies conducted until now do not allow to conclude about the importance of the specific endogenous citrulline production by the enterocytes. Also, in the organim, citrulline is produced thanks to a key enzyme, the ornithine transcarbamylase (OTC), giving to its functionnality a huge implication in the regulation of the proteino-energy homeostasis. Works presented here set up an in vitro model of human duodenal explants, as well as, an in vivo murine model of conditional knock out for the OTC gene. Results obtained have allowed to evidence the importance of the citrulline metabolism since it remains stable over energy status deregulation. Moreover, they have allowed to specify that a healthy organism is able to compensate an OTC deficiency, however, the induction of an energy deregulation with a low protein diet bring to light a major role of the endogenous citrulline in these situations

Chronic intermittent hypoxia due to obstructive sleep apnea slightly alters nutritional status: a pre-clinical study

International audienceObstructive sleep apnea syndrome (OSAS) is associated with chronic intermittent hypoxia (cIH) that causes disturbances in glucose and lipid metabolism. Animals exposed to cIH show lower body weight and food intake, but the protein-energy metabolism has never been investigated. Here, to address the gap, we studied the impact of cIH on nutritional status in rats. A total of 24 male Wistar rats were randomized into 3 groups ( n = 8): a control group (Ctrl), a cIH group (cIH) exposed to cIH (30 s 21–30 s 5% fraction of inspired oxygen, 8 h per day, for 14 days), and a pair-fed group (PF) exposed to normoxia with food intake adjusted to the intake of the cIH group rats with anorexia. Body weight and food intake were measured throughout the study. After 14 days, the rats were euthanized, the organs were collected, weighed, and the liver, intestine mucosa, and muscles were snap-frozen to measure total protein content. Food intake was decreased in the cIH group. Body weight was significantly lower in the cIH group only (−11%, p < 0.05). Thymus and liver weight as well as EDL protein content tended to be lower in the cIH group than in the Ctrl and PF groups. Jejunum and ileum mucosa protein contents were lower in the cIH group compared to the PF group. cIH causes a slight impairment of nutritional status and immunity. This pre-clinical work argues for greater consideration of malnutrition in care for OSAS patients. Further studies are warranted to devise an adequate nutritional strategy

Citrulline protects human retinal pigment epithelium from hydrogen peroxide and iron/ascorbate induced damages

International audienceAbstract Oxidative stress plays an important role in the ageing of the retina and in the pathogenesis of retinal diseases such as age‐related macular degeneration (ARMD). Hydrogen peroxide is a reactive oxygen species generated by the photo‐excited lipofuscin that accumulates during ageing in the retinal pigment epithelium (RPE), and the age‐related accumulation of lipofuscin is associated with ARMD. Iron also accumulates with age in the RPE that may contribute to ARMD as an important source of oxidative stress. The aim of this work was to investigate the effects of L‐Citrulline (CIT), a naturally occurring amino acid with known antioxidant properties, on oxidative stressed cultured RPE cells. Human RPE (ARPE‐19) cells were exposed to hydrogen peroxide (H 2 O 2 ) or iron/ascorbate (I/A) for 4 h, either in the presence of CIT or after 24 h of pretreatment. Here, we show that supplementation with CIT protects ARPE‐19 cells against H 2 O 2 and I/A. CIT improves cell metabolic activity, decreases ROS production, limits lipid peroxidation, reduces cell death and attenuates IL‐8 secretion. Our study evidences that CIT is able to protect human RPE cells from oxidative damage and suggests potential protective effect for the treatment of retinal diseases associated with oxidative stress

Effets de l'hypoxie et de l'inflammation sur la synthèse de citrulline dans les entérocytes humains

International audienc

Obstructive sleep apnea and malnutrition: a preliminary study

International audienc

Ornithine Transcarbamylase – From Structure to Metabolism: An Update

Ornithine transcarbamylase (OTC; EC 2.1.3.3) is a ubiquitous enzyme found in almost all organisms, including vertebrates, microorganisms, and plants. Anabolic, mostly trimeric OTCs catalyze the production of L-citrulline from L-ornithine which is a part of the urea cycle. In eukaryotes, such OTC localizes to the mitochondrial matrix, partially bound to the mitochondrial inner membrane and part of channeling multi-enzyme assemblies. In mammals, mainly two organs express OTC: the liver, where it is an integral part of the urea cycle, and the intestine, where it synthesizes citrulline for export and plays a major role in amino acid homeostasis, particularly of L-glutamine and L-arginine. Here, we give an overview on OTC genes and proteins, their tissue distribution, regulation, and physiological function, emphasizing the importance of OTC and urea cycle enzymes for metabolic regulation in human health and disease. Finally, we summarize the current knowledge of OTC deficiency, a rare X-linked human genetic disorder, and its emerging role in various chronic pathologies

An in vitro explant model for studies of intestinal amino acid metabolism

International audienceBackground & aimsThe main role of the small intestine to absorb the bulk of dietary nutrients, including proteins and amino acids (AA). Enterocytes have long been considered a major player in amino acid homeostasis, especially the glutamine–citrulline–arginine crossroads. In vivo intestinal epithelium cell lines for studies of nitrogen metabolism have now reached their limitations. To push research forward, an interesting approach could be to use the explant incubation model of intestinal tissue, involving in vitro incubation of duodenal tissue biopsy specimens taken from human subjects.MethodsEight duodenal biopsies were taken during endoscopy from patients without proven intestinal disease. Each biopsy was incubated in complete culture medium for 23 h then weighed, and cell viability was evaluated after 6 and 18 h. We then explored L-citrulline (L-CIT) production (reflecting metabolic activity) and the presence of the main enzymes involved in AA metabolism in the intestine (arginase II, glutaminase I, ornithine aminotransferase, and ornithine carbamoyltransferase).ResultsMean weight of biopsies was 12.36 ± 1.00 mg. Mortality was around 20% after 6 h and 50% after 18 h of incubation. CIT was amply produced by the biopsies, and enzymes implicated in intestinal AA metabolism were expressed in this model.ConclusionsThis in vitro explant model of intestinal tissue emerges as a reliable model for conducting ex vivo investigations on AA metabolism

J Cachexia Sarcopenia Muscle

Combating malnutrition and cachexia is a core challenge in oncology. To limit muscle mass loss, the use of proteins in cancer is encouraged by experts in the field, but it is still debated due to their antagonist effects. Indeed, a high protein intake could preserve lean body mass but may promote tumour growth, whereas a low-protein diet could reduce tumour size but without addressing cachexia. Here we used a realistic rodent model of cancer and chemotherapy to evaluate the influence of different protein intakes on cachexia, tumour response to chemotherapy and immune system response. The goal is to gain a closer understanding of the effect of protein intake in cancer patients undergoing chemotherapy. Female Fischer 344 rats were divided into six groups: five groups (n = 14 per group) with cancer (Ward colon tumour) and chemotherapy were fed with isocaloric diets with 8%, 12%, 16%, 24% or 32% of caloric intake from protein and one healthy control group (n = 8) fed a 16% protein diet, considered as a standard diet. Chemotherapy included two cycles, 1 week apart, each consisting of an injection of CPT-11 (50 mg/kg) followed by 5-fluorouracil (50 mg/kg) the day after. Food intake, body weight, and tumour size were measured daily. On day 9, the rats were euthanized and organs were weighed. Body composition was determined and protein content and protein synthesis (SUnSET method) were measured in the muscle, liver, intestine, and tumour. Immune function was explored by flow cytometry. Cancer and chemotherapy led to a decrease in body weight characterized by a decrease of both fat mass (-56 ± 3%, P < 0.05) and fat-free mass (-8 ± 1%, P < 0.05). Surprisingly, there was no effect of protein diet on body composition, muscle or tumour parameters (weight, protein content, or protein synthesis) but a high cumulative protein intake was positively associated with a high relative body weight and high fat-free mass. The immune system was impacted by cancer and chemotherapy but not by the different amount of protein intake. Using a realistic model of cancer and chemotherapy, we demonstrated for the first time that protein intake did not positively or negatively modulate tumour growth. Moreover, our results suggested that a high cumulative protein intake was able to improve moderately nutritional status in chemotherapy treated cancer rodents. Although this work cannot be evaluated clinically for ethical reasons, it nevertheless brings an essential contribution to nutrition management for cancer patients