No description available.

The resident microbes of human gastrointestinal tract cause both harmful and beneficial effects and these effects can be modulated by the administration of beneficial probiotic bacteria. Probiotics attribute several therapeutic and preventive beneficial effects, for both humans and animals. Despite the good effects of probiotic bacteria, the role of probiotic bacteria or their metabolites on the nutrient uptake by enterocytes is very less known. Most studies describe the genomic effects of probiotic bacteria on the transport properties. This thesis describes the short term (10 min or less) non-genomic effects of probiotic bacteria on the glucose uptake by human enterocytes like Caco-2 cells. The focus of the present study was to identify if metabolites of Lactobacilli sp. trigger a rapid non genomic regulation of glucose transporter proteins of enterocytes. The findings indicate that the regulatory molecules of bacterial metabolites can cause a rapid increase in glucose uptake by enterocytes.

La voie de signalisation Hedgehog (HH) est impliquée dans le développement du tractus gastro-intestinal chez de nombreuses espèces. Elle est importante chez ladulte en participant au maintien de lhoméostasie de lépithélium intestinal. Récemment la suractivation de la voie HH a été mise en évidence dans de nombreux cancers dont ceux du tractus gastro-intestinal tels que les cancers de lestomac et du pancréas. Au cours de ce travail, nous nous sommes intéressés à la voie de signalisation HH dans les cancers du côlon. Lexpression des membres principaux de la voie HH a dabord été caractérisée dans sept lignées cellulaires de cancers du côlon (Colo205, Colo320, HCT116, HT29, WiDr, SW480, Caco-2). Labsence dexpression de certains membres clés de la voie indique que celle-ci nest pas activée, de façon autocrine, dans les lignées cellulaires. Cette observation est également appuyée par des expériences réalisées en traitant les cellules à la cyclopamine, inhibiteur de la voie HH. Enfin GLI1, facteur de transcription mimant une activation de la voie HH, a été surexprimé stablement dans les cellules HCT116. Nous avons pu montrer que lactivation de la voie HH induisait lexpression du gène BMP4, gène cible de la voie HH lors de lembryogenèse intestinale, ainsi que lactivité phosphatase alcaline, marqueur de différenciation du côlon. Ces résultats suggèrent que la voie de signalisation HH nest pas activée de façon autocrine dans les cancers du côlon mais semble plutôt associée à la différenciation des cellules du côlon.

The small intestine generally transports dietary fats to circulation in triglyceride (TG)-rich lipoproteins. The two main intestinal lipoproteins are chylomicron (CM) and very low-density lipoprotein (VLDL). Unfortunately, studies on the CM biogenesis and intestinal transport of dietary fats have been hampered by the lack of an adequate in vitro model. In this study, we investigated the possible factors that might increase the efficiency of CM production by Caco-2 cells. We utilized sequential NaCl gradient ultracentrifugation to isolate the CMs that were secreted by the Caco-2 cells. To confirm the successful isolation of the CMs, we performed Fat Red 7B staining, TG reading, apolipoprotein B (ApoB) measurement, and transmission electron microcopy (TEM) analysis. We then tested the effects of cell differentiation, oleic acid, mono-olein, egg lecithin, incubation time, and collagen matrix on CM secretion. We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion. Using the Transwell system, we further confirmed that the CMs produced by our Caco-2 cells contained significant amount of TGs and ApoB-48 such that they could be detected without the use of isotope labeling. In conclusion, when fully differentiated Caco-2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80-200 nm.

Não consta resumo na publicação / Biological and immunological properties of phytohemagglutins from two brazilian varieties of beans (Phaseolus vulgaris) were studied. Through immunoprecipitation in agar gel, immuoenzymatic assays (ELISA) and eletrophoretic behavior after affinity chromatography purification it was shown that the lectins are similar. Both lectins when given to rats are toxic. They reduce growth and intestinal dissacharidase activity. They also able to smooth the \"microvillous\" of the enterocyte and to induce the appearance of autophagosomes in the enterocyte citoplasm. All this modification result in a reduced rate of carbohydrates absorption either in \"vitro\" or \"in vivo\".

Vitamin D is hydroxylated to form several active metabolites, of these, 1,25- dihydroxyvitamin D3 [1,25(OH)2D3] is the most studied stimulatory product. It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate homeostasis through its membrane receptor 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. Another metabolite, 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] has been reported to be inhibitory with respect to calcium and phosphate absorption in intestine. Previous work in this laboratory has indicated that 24,25(OH)2D3 inhibits phosphate uptake in isolated intestinal cells and perfused duodenal loops and in vivo. This thesis further tested the hypothesis that the actions of 24,25(OH)2D3 on phosphate homeostasis are physiologically important. Catalase has been identified as a binding protein for 24,25(OH)2D3. We determined the localization of catalase in the presence and absence of steroid, monitored catalase mRNA levels related to gene 24,25(OH)2D3 gene transcription regulation. We studied the effects of the two isomers of 24,25(OH)2D3 on localization of catalase in chicken enterocytes over a time course of 15 sec to 60 min. It was demonstrated that 24R,25(OH)2D3 is the effective metabolite for catalase redistribution in vitro. We also studied the effects of vitamin D on catalase and phosphate uptake in chicken intestinal cells. It was once again demonstrated that 24R,25(OH)2D3 is the effective metabolite for decreasing phosphate uptake and catalase gene expression. These combined results lead us to conclude that 24,25(OH)2D3 is an important hormone in phosphate homeostasis in chick intestinal epithelial cells.

Não consta resumo na publicação / Biological and immunological properties of phytohemagglutins from two brazilian varieties of beans (Phaseolus vulgaris) were studied. Through immunoprecipitation in agar gel, immuoenzymatic assays (ELISA) and eletrophoretic behavior after affinity chromatography purification it was shown that the lectins are similar. Both lectins when given to rats are toxic. They reduce growth and intestinal dissacharidase activity. They also able to smooth the \"microvillous\" of the enterocyte and to induce the appearance of autophagosomes in the enterocyte citoplasm. All this modification result in a reduced rate of carbohydrates absorption either in \"vitro\" or \"in vivo\".

25-Hydroxyvitamin D3[25(OH)D3] is a metabolite of vitamin D3 that has long been considered to be an inactive precursor of the hormonally active metabolite 1,25- dihydroxyvitamin D3[l,25(OH)2D3]; consequently very few studies have addressed the potential biological activity of 25(OH)2D3. However, it is known that 100 nM 25(OH)2D3 increases calcium transport in the perfused duodenal loop of the chicken to 200% of controls within 20 minutes. The hypothesis of the current study is that 25(OH)2D3 may be a hormonally active metabolite and its effects can be studied in isolated chick enterocytes. To begin testing this postulate, time course studies of 45Ca uptake were undertaken in isolated intestinal cells (from 7 wk chicks). After establishing the basal uptake of 45Ca for 5 minutes, cells were treated with vehicle(< 0.01% v/v ethanol, final concentration) or 25 nM, 50 nM, 100 nM, or 300 nM 25(OH)2D3 and samples were taken at T = 1, 3, 5, 7, and 10 min. With increasing concentrations of steroid, the enterocyte 45Ca decreased. The optimal concentration of 100 nM 25(OH)2D3 induced the most rapid response: within 1 min 45Ca decreased to 54% of controls (P < 0.001) and 70% of the controls at T= 3, 5, and 7 min (P < 0.01 to < 0.05, relative to controls). Comparison of the 7- min time points for 25 nM, 50 nM, 100 nM, and 300 nM 25(OH)2D3 appeared to yield a biphasic dose response curve with values of 45Ca observed at 99% (NS, not significant), 75% (P < 0.05), 70%(P < 0.01%), and 80% (NS) of corresponding controls, respectively. Physiological levels of 24,25(OH)2D3 (6.5 nM) inhibited the action of 100 nM 25(OH)2D3 in isolated chick enterocytes. Time course studies with isolated enterocytes from 14 wk and 28 wk chickens treated with 100 nM 25(OH)2D3 also showed decreased responsiveness: at T= 1 min 45Ca levels in 7 wk, 14 wk, and 28 wk were 54% (P < 0.01), 83% (NS), and 80% (NS) of corresponding controls, respectively. Experiments with the calcium channel activator BAY K8644 (2 μM) and protein kinase A (PKA) activator forskolin (20 μM) revealed enhanced levels of 45Ca at T= 10 min that were 132% and 140% of corresponding controls, respectively (each, P < 0.05). Phorbol ester treatment of the cells resulted in significant increases in the levels of 45Ca between the treated cells and corresponding controls at T=7 and 10 min. Cells treated with 100 nM 25(OH)2D3 revealed 89.8% and 78.4% increases above controls in PKA activity at T =1 min (P < 0.05) and T=3 min, relative to corresponding controls. However, there was no evidence for the activation of PKC by 25(OH)2D3 during the time period studied.

No description available.

L’intestin joue un rôle clé dans le contrôle de l’homéostasie énergétique. Les entérocytes sont des cellules polarisées qui permettent les échanges entre la lumière intestinale (membrane apicale) et le compartiment lymphatique et sanguin (membrane baso-latérale). Dans cette thèse, nous nous sommes particulièrement intéressés au contrôle par les entérocytes de deux processus liés au métabolisme des lipides et du cholestérol : l’excrétion trans-intestinale de cholestérol (TICE) et l’absorption des lipides alimentaires.Très récemment, il a été montré que l’intestin contribue à 20-30% de l’excrétion fécale du cholestérol chez la souris. Ce mécanisme, appelé TICE, implique le passage direct du cholestérol provenant de la circulation sanguine à travers les entérocytes vers les fèces. De par son caractère modulable par des substances pharmacologiques comme l’ézétimibe et les statines, le TICE représente une cible thérapeutique potentielle pour corriger les dyslipidémies athérogènes du diabétique. Cependant, les mécanismes moléculaires gouvernant le transport rétrograde du cholestérol (du pôle baso-latéral au pôle apical) dans l’entérocyte lors du TICE, sont complètement inconnus. Dans une première étude, nous avons mis en évidence la lignée entérocytaire humaine Caco-2/TC7 comme un modèle d’étude des processus trans-entérocytaires liés au TICE. Nous avons d’abord montré que suite à l’incubation avec du plasma humain dans le compartiment baso-latéral et des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 miment des caractéristiques du TICE in vivo. De plus, grâce à ce modèle in vitro, nous avons pu identifier les microtubules comme acteurs nécessaires au transport rétrograde du cholestérol dans l’entérocyte. Dans une seconde étude, nous nous sommes intéressés au contrôle par le récepteur nucléaire Rev-erbα de la production des chylomicrons (CM) par les entérocytes. En effet, bien qu’essentiellement vue comme la conséquence d’une clairance retardée, des données émergentes présentent la surproduction de CM par l’intestin comme un contributeur majeur de la dyslipidémie chez l’insulino-résistant. Il existe une balance, au sein de l’entérocyte, entre l’utilisation des lipides absorbés pour un stockage transitoire sous forme de gouttelettes lipidiques (GL) cytosoliques ou pour l’assemblage de lipoprotéines riches en triglycérides (LRT). Le récepteur nucléaire Rev-erbα est un répresseur transcriptionnel impliqué dans le métabolisme énergétique et le rythme circadien. Rev-erbα contrôle particulièrement le métabolisme lipidique au niveau du foie et le catabolisme des LRT. Pour cette seconde étude, une lignée Caco-2/TC7 invalidée pour Rev-erbα (sh Rev-erbα) a donc été développée par infection lentivirale et différenciée sur insert. Les résultats indiquent que suite à l’incubation avec des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 sh Rev-erbα sécrètent plus de LRT dans le milieu baso-latéral et stockent moins de lipides sous la forme de GL cytosoliques. De plus, la lignée Caco-2/TC7 sh Rev-erbα présente une activité lipophagique plus importante et l’inhibition de l’autophagie par la bafilomycine dans cette lignée restaure la sécrétion baso-latérale de LRT et le stockage intracellulaire de GL aux mêmes niveaux que ceux de la lignée sh control. Cette seconde étude montre donc que l’invalidation de Rev-erbα dans l’entérocyte entraîne une augmentation de la mobilisation des lipides des GL via le processus de la lipophagie résultant en une augmentation de la sécrétion de LRT. Notre hypothèse est que Rev-erbα joue un rôle clé dans le contrôle de la balance GL/LRT et donc de la triglycéridémie post-prandiale.Les deux études présentées dans cette thèse permettent une meilleure compréhension des mécanismes liés au contrôle du métabolisme lipidique par l’intestin et mettent ainsi en avant l’intestin comme une cible thérapeutique potentielle pour corriger les dyslipidémies du diabétique. / The intestine plays a key role in the control of energy homeostasis. Enterocytes, which constitute the main cellular type of intestinal epithelium (> 90%), are polarized cells allowing exchanges between intestinal lumen (apical membrane) and lymph/blood compartment (basolateral membrane). In this thesis, cholesterol and lipid metabolism control by enterocytes was studied and particularly, trans intestinal cholesterol excretion (TICE) and dietary lipid absorption.Recently, it has been estimated that intestine contributes 20-30% of fecal neutral sterol excretion in chow-fed mice. This pathway called TICE involves the direct luminal secretion of plasma-derived cholesterol by enterocytes. Moreover, TICE can be pharmacologically modulated, for instance by ezetimibe and statins and so, represents a new therapeutic target in order to prevent atherosclerosis in type 2 diabetic patients. However, at present, the molecular mechanisms behind the trans-enterocytic process of TICE are still unknown, especially the steps sustaining cholesterol entry, trafficking and efflux in enterocytes. In the first study of this thesis, we highlighted the human enterocytic Caco-2/TC7 cell line as a suitable model to study the enterocyte-related processes of TICE. We have first shown that upon basolateral incubation with human plasma and apical incubation with lipid micelles, differentiated Caco-2/TC7 cells mimic some of the in vivo TICE features. Moreover, using this model, we have identified a key role of the microtubule network in the process.In the second study of this thesis, chylomicron secretion by enterocytes and its control by the nuclear receptor Rev-erbα were investigated. Indeed, although chylomicron remnant accumulation has been associated to a delayed clearance by the liver, some recent studies show that chylomicron overproduction by the intestine is a major contributor to dyslipidemia in insulin resistant patients. Dietary lipid absorption results from a balance between transient storage in enterocytes as cytosolic lipid droplets (LD) and secretion as triglyceride-rich lipoproteins (TRL). The nuclear receptor Rev-erbα is a transcriptional repressor involved in the energy metabolism and the circadian rhythm. Particularly, Rev-erbα controls lipid metabolism in the liver and thus the catabolism of TRL. The aim of this second study was to investigate the role of Rev-erbα in intestinal lipid metabolism and particularly in TRL secretion. To study that, Caco-2/TC7 cells infected with lentivirus encoding or not a shRNA targeting Rev-erbα (sh Rev-erbα) were grown on transwells. Compared to sh control, sh Rev-erbα Caco-2/TC7 cells secrete higher amounts of micelle-derived LRT in the basolateral medium and exhibit lower quantity of neutral lipids stored as cytosolic LD, whereas the apical uptake is not different. Activation of lipophagy in sh Rev-erbα compared to sh control cells was evidenced by a higher autophagic flux and an increased colocalization of the autophagy marker LC3 with LD. Finally, autophagy inhibition with bafilomycin in sh Rev-erbα cells restores lipid secretion to the same level as in sh control cells. This second study show that Rev-erbα knock-down in enterocytes leads to a higher lipophagy-mediated remobilization of intracellular lipids and an increased TRL secretion. Our hypothesis is that Rev-erbα may be a molecular gear in the control of chylomicron secretion and a major regulator of post-prandial triglyceridemia.In conclusion, these two studies allow to better understand lipid metabolism control by the intestine: the first one by identifying the contribution of the microtubule network in enterocytes for trans-enterocytic retrograde cholesterol transport; the second one by highlighting the nuclear receptor Rev-erbα as a regulator of TRL secretion by enterocytes. These two studies point out the intestine as a potential therapeutic target to treat dyslipidemia in type 2 diabetic patients.