Contribution of proteomic and metalloproteomic analyses for the study of geophagia

Les travaux présentés dans ce manuscrit s'inscrivent dans un projet pluridisciplinaire portant sur l'étude de la géophagie. Parallèlement à des approches de physiologie et de morphologie intestinale, cette thèse a porté d'une part, sur l'identification de protéines de la muqueuse intestinale dont l'expression est modifiée par l'ingestion d'une argile, la kaolinite, et d'autre part, sur l'identification de protéines liant l'aluminium, élément présent en grande quantité dans cette argile. Des analyses protéomiques différentielles par électrophorèse bidimensionnelle, réalisées sur des échantillons de muqueuse intestinale de rat ayant reçu un apport en kaolinite au cours de différents régimes alimentaires, ont révélé une modification de l'expression de protéines du cytosquelette et de protéines participant au métabolisme des lipides. Parmi celles-ci, nous avons mis en évidence une augmentation de l'expression de l'apolipoprotéine A-IV, protéine impliquée dans le transport des triglycérides et jouant un rôle de facteur de satiété. L'étude des protéines liant l'aluminium a nécessité un développement méthodologique basé sur l'utilisation de l'électrophorèse en conditions natives pour la séparation des protéines, et l'utilisation de la spectrométrie de masse ICPMS pour la détection de l'aluminium et la localisation de métalloprotéines. Après une validation progressive de l'ensemble de la procédure à l'aide de plusieurs métalloprotéines modèles, la stratégie analytique a été appliquée à des échantillons de muqueuse intestinale dans lesquels des protéines pouvant lier l'aluminium ont été identifiées.The aim of this study was to investigate geophagia through a pluridisciplinar approach. Besides physiological and morphological studies, our work consisted (i) on the identification of intestinal mucosa proteins whose expression is modified by kaolinite ingestion, and (ii) on the identification of proteins bound to aluminium, a major element of this clay. Differential proteomic analyses have been carried out using two-dimensional electrophoresis, on intestinal mucosa samples from kaolinite complemented rats after various diets. These analyses revealed a modification of expression of cytoskeletal proteins or involved in lipid metabolism. In particular, the expression apolipoprotein A-IV involved in triglycerides transport and satiety was increased in kaolinite complemented rats. The study of aluminium-binding proteins has required a methodological development based on native electrophoreses for protein separation, and inductively coupled plasma mass spectrometry for aluminium detection and metalloproteins localisation. The whole procedure was validated using various model metalloproteins and the analytical strategy was then applied to intestinal mucosa samples. Potential aluminium-binding proteins were thus identified

Contribution of proteomic and metalloproteomic analyses for the study of geophagia

Les travaux présentés dans ce manuscrit s'inscrivent dans un projet pluridisciplinaire portant sur l'étude de la géophagie. Parallèlement à des approches de physiologie et de morphologie intestinale, cette thèse a porté d'une part, sur l'identification de protéines de la muqueuse intestinale dont l'expression est modifiée par l'ingestion d'une argile, la kaolinite, et d'autre part, sur l'identification de protéines liant l'aluminium, élément présent en grande quantité dans cette argile. Des analyses protéomiques différentielles par électrophorèse bidimensionnelle, réalisées sur des échantillons de muqueuse intestinale de rat ayant reçu un apport en kaolinite au cours de différents régimes alimentaires, ont révélé une modification de l'expression de protéines du cytosquelette et de protéines participant au métabolisme des lipides. Parmi celles-ci, nous avons mis en évidence une augmentation de l'expression de l'apolipoprotéine A-IV, protéine impliquée dans le transport des triglycérides et jouant un rôle de facteur de satiété. L'étude des protéines liant l'aluminium a nécessité un développement méthodologique basé sur l'utilisation de l'électrophorèse en conditions natives pour la séparation des protéines, et l'utilisation de la spectrométrie de masse ICPMS pour la détection de l'aluminium et la localisation de métalloprotéines. Après une validation progressive de l'ensemble de la procédure à l'aide de plusieurs métalloprotéines modèles, la stratégie analytique a été appliquée à des échantillons de muqueuse intestinale dans lesquels des protéines pouvant lier l'aluminium ont été identifiées.The aim of this study was to investigate geophagia through a pluridisciplinar approach. Besides physiological and morphological studies, our work consisted (i) on the identification of intestinal mucosa proteins whose expression is modified by kaolinite ingestion, and (ii) on the identification of proteins bound to aluminium, a major element of this clay. Differential proteomic analyses have been carried out using two-dimensional electrophoresis, on intestinal mucosa samples from kaolinite complemented rats after various diets. These analyses revealed a modification of expression of cytoskeletal proteins or involved in lipid metabolism. In particular, the expression apolipoprotein A-IV involved in triglycerides transport and satiety was increased in kaolinite complemented rats. The study of aluminium-binding proteins has required a methodological development based on native electrophoreses for protein separation, and inductively coupled plasma mass spectrometry for aluminium detection and metalloproteins localisation. The whole procedure was validated using various model metalloproteins and the analytical strategy was then applied to intestinal mucosa samples. Potential aluminium-binding proteins were thus identified

Contribution of proteomic and metalloproteomic analyses for the study of geophagia

Les travaux présentés dans ce manuscrit s'inscrivent dans un projet pluridisciplinaire portant sur l'étude de la géophagie. Parallèlement à des approches de physiologie et de morphologie intestinale, cette thèse a porté d'une part, sur l'identification deThe aim of this study was to investigate geophagia through a pluridisciplinar approach. Besides physiological and morphological studies, our work consisted (i) on the identification of intestinal mucosa proteins whose expression is modified by kaolinite

Interactions between ingested kaolinite and the intestinal mucosa in rat: proteomic and cellular evidences

International audienceAlthough some of the effects of clay ingestion by humans and animals, such as gastrointestinal wellness and the increase in food efficiency are well known, the underlying mechanisms are not yet fully understood. Therefore, the interactions between the intestinal mucosa and kaolinite particles and their effects on mucosal morphology were observed using light microscopy (LM), transmission electron microscopy (TEM), conventional (CSEM) and environmental (ESEM) scanning electron microscopy combined with an EDX micro-analysis system. Kaolinite consumption, given with free access to rats, varied considerably from one animal to the other but was regular through time for each individual. Some kaolinite particles appeared chemically dissociated in the lumen and within the mucus barrier. Aluminium originating from ingested clay and present in the mucus layer, could directly cross the intestinal mucosa. A significant increase in the thickness of the villi with large vacuoles at the base of the mucosal cells and a decrease in the length of enterocyte microvilli characterized complemented animals. The proteomic analyses of the intestinal mucosa of complemented rats also revealed several modifications in the expression level of cytoskeleton proteins. In summary, kaolinite particles ingested as food complement interact with the intestinal mucosa and modify nutrient absorption. However, these data, together with the potential neurotoxicity of aluminium, need further investigation

Kaolinite ingestion facilitates restoration of body energy reserves during refeeding after prolonged fasting.

International audienceClay consumption is a spontaneous behavior currently observed in animals and humans, particularly during undernutrition. Often regarded as intestinal care products, ingested clays also enhance food efficiency, notably by increasing intestinal lipid uptake. Clay complementation could then optimize the reconstitution of energy reserves in animals with low lipid stocks consecutive to intensive fasting. The aim of this study was therefore to observe the effects of voluntarily kaolinite complementation during the refeeding of fasted rats to determine whether body mass, food uptake, lipid and mineral contents as intestinal morphology and protein profile were modified. This study examined two types of refeeding experiments after prolonged fasting. Firstly, rats with ad libitum access to food were compared to rats with ad libitum access to food and kaolinite pellets. Animals were randomly put into the different groups when the third phase of fasting (phase III) reached by each individual was detected. In a second set of experiments, rats starting phase III were refed with free access to food and kaolinite pellets. When animals had regained their body mass prior to fasting, they were euthanized for chemical, morphological, and proteomic analyses. Although kaolinite ingestion did not change the time needed for regaining prefasting body mass, daily food ingestion was seen to decrease by 6.8% compared with normally refed rats, without affecting lipid composition. Along the intestinal lining, enterocytes of complemented animals contained abundant lipid droplets and a structural modification of the brushborder was observed. Moreover, the expression of two apolipoproteins involved in lipid transport and satiety (ApoA-I and ApoA-IV) increased in complemented rats. These results suggest that kaolinite complementation favors intestinal nutrient absorption during refeeding despite reduced food uptake. Within the intestinal lumen, clay particles could increase the passive absorption capacity and/or nutrient availability that induce mucosal morphological changes. Therefore, clay ingestion appears to be beneficial for individuals undergoing extreme nutritional conditions such as refeeding and limited food supplies

Effects of controlled ingestion of kaolinite (5%) on food intake, gut morphology and in vitro motility in rats.

International audienceGeophagia is found in various animal species and in humans. We have previously shown that spontaneously ingested kaolinite interacts with the intestinal mucosa modifies nutrient absorption and slows down gastric emptying and intestinal transit in rats in vivo. However, the precise mechanisms involved are not elucidated. The aim of this work was to investigate the effects of controlled kaolinite ingestion on food intake, gut morphology and in vitro motility in rats. Male Wistar rats were fed with 5% kaolinite in standard food pellets during 7, 14 and 28 days. Body mass and food consumption were measured daily. Intestinal morphological and proteomic analyses were conducted. The length of mucosal lacteals was evaluated. Plasmatic levels of leptin and adiponectin were determined. Finally, organ bath studies were conducted to evaluate smooth muscle contractility. Food consumption was significantly increased during the first two weeks of kaolinite ingestion without any mass gain compared to controls. Kaolinite induced weak variations in proteins that are involved in various biological processes. Compared to control animals, the length of intestinal lacteals was significantly reduced in kaolinite group whatever the duration of the experiment. Leptin and adiponectin plasmatic levels were significantly increased after 14 days of kaolinite consumption. Changes in spontaneous motility and responses to electrical nerve stimulation of the jejunum and proximal colon were observed at day 14. Altogether, the present data give evidence for a modulation by kaolinite-controlled ingestion on satiety and anorexigenic signals as well as on intestinal and colonic motility

Characterization of the Active Bacterial Community Involved in Natural Attenuation Processes in Arsenic-Rich Creek Sediments

International audienceAcid mine drainage of the Carnoulès mine (France) is characterized by acid waters containing high concentrations of arsenic and iron. In the first 30 m along the Reigous, a small creek draining the site, more than 38% of the dissolved arsenic was removed by co-precipitation with Fe(III), in agreement with previous studies, which suggest a role of microbial activities in the co-precipitation of As(III) and As(V) with Fe(III) and sulfate. To investigate how this particular ecosystem functions, the bacterial community was characterized in water and sediments by 16S rRNA encoding gene library analysis. Based on the results obtained using a metaproteomic approach on sediments combined with high-sensitivity HPLC-chip spectrometry, several GroEL orthologs expressed by the community were characterized, and the active members of the prokaryotic community inhabiting the creek sediments were identified. Many of these bacteria are β-proteobacteria such as Gallionella and Thiomonas, but γ-proteobacteria such as Acidithiobacillus ferrooxidans and α-proteobacteria such as Acidiphilium, Actinobacteria, and Firmicutes were also detected