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The importance of gut barrier integrity in intestinal homeostasis and the consequences of its alteration in the etiology of human pathologies have been subjects of exponentially growing interest during the last decade [...]

An increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls.

Paracellular permeability of the intestinal epithelium is a feature of the intestinal barrier, which plays an important role in the physiology of gut and the whole organism. Intestinal paracellular permeability is controlled by complex processes and is involved in the passage of ions and fluids (called pore pathway) and macromolecules (called leak pathway) through tight junctions, which seal the intercellular space. Impairment of intestinal paracellular permeability is associated with several diseases. The identification of a defect in intestinal paracellular permeability may help to understand the implication of gut barrier as a cause or a consequence in human pathology. Here we describe two complementary methods to evaluate alteration of paracellular permeability in cell culture, using the human intestinal cell line Caco-2 and its clone Caco-2/TC7.

International audience; An increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls.

Paracellular permeability of the intestinal epithelium is a feature of the intestinal barrier, which plays an important role in the physiology of gut and the whole organism. Intestinal paracellular permeability is controlled by complex processes and is involved in the passage of ions and fluids (called pore pathway) and macromolecules (called leak pathway) through tight junctions, which seal the intercellular space. Impairment of intestinal paracellular permeability is associated with several diseases. The identification of a defect in intestinal paracellular permeability may help to understand the implication of gut barrier as a cause or a consequence in human pathology. Here we describe two complementary methods to evaluate alteration of paracellular permeability in cell culture, using the human intestinal cell line Caco-2 and its clone Caco-2/TC7.

International audience; Bacteria are known to communicate with each other and regulate their activities in social networks by secreting and sensing signaling molecules called autoinducers, a process known as quorum sensing (QS). This is a growing area of research in which we are expanding our understanding of how bacteria collectively modify their behavior but are also involved in the crosstalk between the host and gut microbiome. This is particularly relevant in the case of pathologies associated with dysbiosis or disorders of the intestinal ecosystem. This review will examine the different QS systems and the evidence for their presence in the intestinal ecosystem. We will also provide clues on the role of QS molecules that may exert, directly or indirectly through their bacterial gossip, an influence on intestinal epithelial barrier function, intestinal inflammation, and intestinal carcinogenesis. This review aims to provide evidence on the role of QS molecules in gut physiology and the potential shared by this new player. Better understanding the impact of intestinal bacterial social networks and ultimately developing new therapeutic strategies to control intestinal disorders remains a challenge that needs to be addressed in the future.

Sporozoite forms of the malaria parasite Plasmodium are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytic cells. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection.IMPORTANCEMalaria is caused by Plasmodium parasites and remains one of the deadliest parasitic diseases worldwide. The parasite is transmitted by a blood feeding mosquito and first invades the liver for an initial, obligatory and silent round of replication. The liver infection is an attractive target for antimalarial vaccine strategies, however the molecular mechanisms of parasite invasion of hepatocytes remain to be fully elucidated. Two hepatocyte surface proteins are known to be important for parasite entry into hepatocytes, the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1). These receptors constitute independent gateways depending on the Plasmodium species. Here, we identified the structural determinants of SR-B1, an important hepatocyte entry factor for human-infecting P. vivax. This study paves the way toward a better characterization of the molecular interactions underlying the crucial early stages of infection, a pre-requisite for the development of novel malaria vaccine strategies.

International audience; he mechanisms leading to the low-grade inflammation observed during obesity are not fully understood. Seeking the initiating events, we tested the hypothesis that the intestine could be damaged by repeated lipid supply and therefore participate in inflammation. In mice, 1-5 palm oil gavages increased intestinal permeability via decreased expression and mislocalization of junctional proteins at the cell-cell contacts; altered the intestinal bacterial species by decreasing the abundance of Akkermansia muciniphila, segmented filamentous bacteria, and Clostridium leptum; and increased inflammatory cytokine expression. This was further studied in human intestinal epithelial Caco-2/TC7 cells using the two main components of palm oil, i.e., palmitic and oleic acid. Saturated palmitic acid impaired paracellular permeability and junctional protein localization, and induced inflammatory cytokine expression in the cells, but unsaturated oleic acid did not. Inhibiting de novo ceramide synthesis prevented part of these effects. Altogether, our data show that short exposure to palm oil or palmitic acid induces intestinal dysfunctions targeting barrier integrity and inflammation. Excessive palm oil consumption could be an early player in the gut alterations observed in metabolic diseases.

International audience; The worldwide prevalence of metabolic diseases is increasing, and there are global recommendations to limit consumption of certain nutrients, especially saturated lipids. Insulin resistance, a common trait occurring in obesity and type 2 diabetes, is associated with intestinal lipoprotein overproduction. However, the mechanisms by which the intestine develops insulin resistance in response to lipid overload remain unknown. Here, we show that insulin inhibits triglyceride secretion and intestinal microsomal triglyceride transfer protein expression in vivo in healthy mice force-fed monounsaturated fatty acid-rich olive oil but not in mice force-fed saturated fatty acid-rich palm oil. Moreover, when mouse intestine and human Caco-2/TC7 enterocytes were treated with the saturated fatty acid, palmitic acid, the insulin-signaling pathway was impaired. We show that palmitic acid or palm oil increases ceramide production in intestinal cells and that treatment with a ceramide analogue partially reproduces the effects of palmitic acid on insulin signaling. In Caco-2/TC7 enterocytes, ceramide effects on insulin-dependent AKT phosphorylation are mediated by protein kinase C but not by protein phosphatase 2A. Finally, inhibiting de novo ceramide synthesis improves the response of palmitic acid-treated Caco-2/TC7 enterocytes to insulin. These results demonstrate that a palmitic acid-ceramide pathway accounts for impaired intestinal insulin sensitivity, which occurs within several hours following initial lipid exposure.

BACKGROUND & AIMS: Reducing postprandial triglyceridemia may be a promising strategy to lower the risk of cardiovascular disorders associated with obesity and type 2 diabetes. In enterocytes, scavenger receptor class B, type 1 (SR-B1, encoded by SCARB1) mediates lipid-micelle sensing to promote assembly and secretion of chylomicrons. The nuclear receptor subfamily 1, group H, members 2 and 3 (also known as liver X receptors [LXRs]) regulate genes involved in cholesterol and fatty acid metabolism. We aimed to determine whether intestinal LXRs regulate triglyceride absorption. METHODS: C57BL/6J mice were either fed a cholesterol-enriched diet or given synthetic LXR agonists (GW3965 or T0901317). We measured the production of chylomicrons and localized SR-B1 by immunohistochemistry. Mechanisms of postprandial triglyceridemia and SR-B1 regulation were studied in Caco-2/TC7 cells incubated with LXR agonists. RESULTS: In mice and in the Caco-2/TC7 cell line, LXR agonists caused localization of intestinal SR-B1 from apical membranes to intracellular organelles and reduced chylomicron secretion. In Caco-2/TC7 cells, LXR agonists reduced SR-B1-dependent lipidic-micelle-induced Erk phosphorylation. LXR agonists also reduced intracellular trafficking of the apical apolipoprotein B pool toward secretory compartments. LXR reduced levels of SR-B1 in Caco-2/TC7 cells via a post-transcriptional mechanism that involves microRNAs. CONCLUSION: In Caco-2/TC7 cells and mice, intestinal activation of LXR reduces the production of chylomicrons by a mechanism dependent on the apical localization of SR-B1.

The intestine is home to the largest microbiota community of the human body and strictly regulates its barrier function. Tight junctions (TJ) are major actors of the intestinal barrier, which is impaired in inflammatory bowel disease (IBD), along with an unbalanced microbiota composition. With the aim to identify new actors involved in host-microbiota interplay in IBD, we studied N-acyl homoserine lactones (AHL), molecules of the bacterial quorum sensing, which also impact the host. We previously identified in the gut a new and prominent AHL, 3-oxo-C12:2, which is lost in IBD. We investigated how 3-oxo-C12:2 impacts the intestinal barrier function, in comparison to 3-oxo-C12, a structurally close AHL produced by the opportunistic pathogen P. aeruginosa. Using Caco-2/TC7 cells as a model of polarized enterocytes, we compared the effects on paracellular permeability and TJ integrity of these two AHL, separately or combined with pro-inflammatory cytokines, Interferon-γ and Tumor Necrosis Factor-α, known to disrupt the barrier function during IBD. While 3-oxo-C12 increased paracellular permeability and decreased occludin and tricellulin signal at bicellular and tricellular TJ, respectively, 3-oxo-C12:2 modified neither permeability nor TJ integrity. Whereas 3-oxo-C12 potentiated the hyperpermeability induced by cytokines, 3-oxo-C12:2 attenuated their deleterious effects on occludin and tricellulin, and maintained their interaction with their partner ZO-1. In addition, 3-oxo-C12:2 limited the cytokine-induced ubiquitination of occludin and tricellulin, suggesting that this AHL prevented their endocytosis. In conclusion, the role of 3-oxo-C12:2 in maintaining TJ integrity under inflammatory conditions identifies this new AHL as a potential beneficial actor of host–microbiota interactions in IBD.

Intestine contributes to energy homeostasis through the absorption, metabolism, and transfer of nutrients to the organism. We demonstrated previously that hepatocyte nuclear receptor-4α (HNF-4α) controls intestinal epithelium homeostasis and intestinal absorption of dietary lipids. HNF-4γ, the other HNF-4 form highly expressed in intestine, is much less studied. In HNF-4γ knockout mice, we detect an exaggerated insulin peak and improvement in glucose tolerance during oral but not intraperitoneal glucose tolerance tests, highlighting the involvement of intestine. Moreover, the enteroendocrine L-type cell lineage is modified, as assessed by the increased expression of transcription factors Isl1, Foxa1/2, and Hnf4a, leading to an increase of both GLP-1–positive cell number and basal and stimulated GLP-1 plasma levels potentiating the glucose-stimulated insulin secretion. Using the GLP-1 antagonist exendin (9-39), we demonstrate a direct effect of GLP-1 on improved glucose tolerance. GLP-1 exerts a trophic effect on pancreatic β-cells, and we report an increase of the β-cell fraction correlated with an augmented number of proliferative islet cells and with resistance to streptozotocin-induced diabetes. In conclusion, the loss of HNF-4γ improves glucose homeostasis through a modulation of the enteroendocrine cell lineage.

Delivery of alimentary lipids induces immediate autophagic response in enterocytes. Forming autophagosomes are recruited to the ER membrane, where they capture nascent lipid droplets and later fuse with lysosomes, illustrating for the first time the role of autophagy in neutral-lipid distribution in enterocytes., Enterocytes, the intestinal absorptive cells, have to deal with massive alimentary lipids upon food consumption. They orchestrate complex lipid-trafficking events that lead to the secretion of triglyceride-rich lipoproteins and/or the intracellular transient storage of lipids as lipid droplets (LDs). LDs originate from the endoplasmic reticulum (ER) membrane and are mainly composed of a triglyceride (TG) and cholesterol-ester core surrounded by a phospholipid and cholesterol monolayer and specific coat proteins. The pivotal role of LDs in cellular lipid homeostasis is clearly established, but processes regulating LD dynamics in enterocytes are poorly understood. Here we show that delivery of alimentary lipid micelles to polarized human enterocytes induces an immediate autophagic response, accompanied by phosphatidylinositol-3-phosphate appearance at the ER membrane. We observe a specific and rapid capture of newly synthesized LD at the ER membrane by nascent autophagosomal structures. By combining pharmacological and genetic approaches, we demonstrate that autophagy is a key player in TG targeting to lysosomes. Our results highlight the yet-unraveled role of autophagy in the regulation of TG distribution, trafficking, and turnover in human enterocytes.

Cette etude s’interesse a l’humour en tant qu’indice de la resilience chez les etudiants deficients visuels. Une chute consequente de la vue peut etre vecue comme un evenement traumatique, qui peut etre la cause d’un frein a la communication, que ce soit avec les enseignants ou les pairs. Cette difficulte a communiquer restreint alors le soutien social dont pourraient beneficier les etudiants. Nous avons alors effectue une recherche des indices de l’humour produit par les etudiants deficients visuels lors d’activites universitaires enregistrees in situ ainsi qu’au cours d’entretiens semi-directifs. Cette analyse a permis de montrer que l’humour agit de differentes facons, comme mecanisme de defense mais aussi comme agent constructif de la resilience.

Rationale: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. Objective: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. Methods and Results: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-β-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. Conclusions: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.

Resume L’hypertriglyceridemie postprandiale est un facteur de risque des maladies cardiovasculaires. L’intestin de part son role dans l’absorption des lipides alimentaires participe a la secretion des lipoproteines riches en triglycerides (LRT) et contribue a l’augmentation du taux plasmatique de triglycerides en periode postprandiale. L’etude des mecanismes moleculaires impliques dans la secretion intestinale des LRT permettra l’identification de nouvelles cibles therapeutiques pour le traitement des maladies metaboliques. La detection des lipides alimentaires par les cellules intestinales constitue un nouveau mecanisme pouvant moduler la secretion des LRT. Alors que de nombreuses etudes relatent l’importance des cellules enteroendocrines dans la detection des lipides alimentaires, plusieurs donnees suggerent l’implication des enterocytes, les cellules absorbantes de l’intestin dans ce processus. Des travaux recents rapportant le role du recepteur scavenger SR-BI dans la detection des lipides alimentaires apportes sous leur forme physiologique de micelles lipidiques seront presentes.

Intestine contributes to lipid homeostasis through the absorption of dietary lipids, which reach the apical pole of enterocytes as micelles. The present study aimed to identify the specific impact of these dietary lipid-containing micelles on gene expression in enterocytes. We analyzed, by microarray, the modulation of gene expression in Caco-2/TC7 cells in response to different lipid supply conditions that reproduced either the permanent presence of albumin-bound lipids at the basal pole of enterocytes or the physiological delivery, at the apical pole, of lipid micelles, which differ in their composition during the interprandial (IPM) or the postprandial (PPM) state. These different conditions led to distinct gene expression profiles. We observed that, contrary to lipids supplied at the basal pole, apical lipid micelles modulated a large number of genes. Moreover, compared with the apical supply of IPM, PPM specifically impacted 46 genes from three major cell function categories: signal transduction, lipid metabolism, and cell adhesion/architecture. Results from this first large-scale analysis underline the importance of the mode and polarity of lipid delivery on enterocyte gene expression. They demonstrate specific and coordinated transcriptional effects of dietary lipid-containing micelles that could impact the structure and polarization of enterocytes and their functions in nutrient transfer.

Cytosolic lipid droplets (LDs) are observed in enterocytes of jejunum during lipid absorption. One important function of the intestine is to secrete chylomicrons, which provide dietary lipids throughout the body, from digested lipids in meals. The current hypothesis is that cytosolic LDs in enterocytes constitute a transient pool of stored lipids that provides lipids during interprandial period while lowering chylomicron production during the post-prandial phase. This smoothens the magnitude of peaks of hypertriglyceridemia. Here, we review the composition and functions of lipids and associated proteins of enterocyte LDs, the known physiological functions of LDs as well as the role of LDs in pathological processes in the context of the intestine.

Cette recherche s’intéresse à la mise en œuvre de l’humour dans un contexte spécifique : une salle de cours informatique où un enseignant enseigne l’utilisation d’un logiciel alors que ce dernier n’est pas adapté à l’un des étudiants du groupe qui est déficient visuel. L’enseignant n’est pas préparé à cette situation et doit alors improviser. L’observation directe de cette séance de cours nous permettra de mettre en exergue les fonctions du rire et de l’humour dans cette situation. Nous verrons comment l’enseignant y a recours pour dépasser son embarras et instaurer/conserver une atmosphère de travail agréable. This research focuses on using humor in a specific context: a classroom where a teacher teaches the use of software so that it is not suitable for one of the students who is visually impaired. The teacher is not prepared for this situation and must improvise. Direct observation during this session will highlight the functions of laugh and humor in this situation. We’ll see how teacher uses them to overcome his embarrassment and establish / maintain a pleasant working atmosphere.

Glucose-6-phosphatase (G6Pase) catalyzes the release of glucose from glucose 6-phosphate. This enzyme was mainly studied in the liver, but while detected in the small intestine little is known about the regulation of its intestinal expression. This study describes the mechanisms of the glucose-dependent regulation of G6Pase expression in intestinal cells. Results obtained in vivo and in Caco-2/TC7 enterocytes showed that glucose increases the G6Pase mRNA level. In Caco-2/TC7 cells, glucose stabilized G6Pase mRNA and activated the transcription of the gene, meaning that glucose-dependent G6Pase expression involved both transcriptional and post-transcriptional mechanisms. Reporter-gene studies showed that, although the -299/+57 region of the human G6Pase promoter was sufficient to trigger the glucose response in the hepatoma cell line HepG2, the -1157/-1133 fragment was required for maximal activation of glucose-6-phosphatase gene transcription in Caco-2/TC7 cells. This fragment binds the aryl receptor nuclear translocator (ARNT), cAMP-responsive element-binding protein, and upstream stimulatory factor transcription factors. The DNA binding activity of these transcription factors was increased in nuclear extracts of differentiated cells from the intestinal villus of mice fed sugar-rich diets as compared with mice fed a no-sugar diet. A direct implication of ARNT in the activation of G6Pase gene transcription by glucose has been observed in Caco-2/TC7 cells using RNA interference experiments. These results support a physiological role for G6Pase in the control of nutrient absorption in the small intestine.

Apolipoprotein (apo) A-IV, a component of triglyceride-rich lipoproteins secreted by the small intestine, has been shown to play an important role in the control of lipid homeostasis. Numerous studies have described the induction of apoA-IV gene expression by lipids, but the molecular mechanisms involved in this process remain unknown. In this study, we have demonstrated that a lipid bolus induced transcription of the apoA-IV gene in transgenic mice and that the regulatory region of the apoA-IV gene, composed of the apoC-III enhancer and the apoA-IV promoter (eC3-A4), was responsible for this induction. In enterocyte Caco-2/TC7 cells, a permanent supply of lipids at the basal pole induced expression of the apoA-IV gene both at the transcriptional level and through mRNA stabilization. ApoA-IV gene transcription and protein secretion were further induced by an apical supply of complex lipid micelles mimicking the composition of duodenal micelles, and this effect was not reproduced by apical delivery of different combinations of micelle components. Only induction of the apoA-IV gene by lipid micelles involved the participation of hepatic nuclear factor (HNF)-4, as demonstrated using a dominant negative form of this transcription factor. Accordingly, lipid micelles increased the DNA binding activity of HNF-4 on the eC3-A4 region. These results emphasize the importance of physiological delivery of dietary lipids on apoA-IV gene expression and the implication of HNF-4 in this regulation.

Aminopeptidase B (Ap-B), a ubiquitous enzyme, catalyses the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. The physiological function of Ap-B still remains an open question, even though its activity suggests that it could be involved in inflammatory processes and proliferation of tumor cells. This study was conducted to determine the expression of Ap-B in the developing and adult retina as a path to envisage physiological roles of Ap-B. RT-PCR and in situ hybridization were used to detect expression of Ap-B mRNA and activity tests, Western blotting and immunofluorescence microscopy were performed to identify and localize the enzyme in the rat retina. These biochemical and morphological methods show that Ap-B is expressed in the retina from embryo to adult. Expression level is restricted to specific layers (pigmented epithelium, outer and inner plexiform layers and ganglion cell layer) and is developmentally regulated. Moreover, a preliminary analysis indicates that Ap-B, the glucose transporter GLUT3 and choline acetyltransferase (ChAT) share a similar expression pattern in retina. Altogether, Ap-B appears predominantly expressed in neuronal cells lying in retinal layers containing neuritic extensions and synaptic junctions. Such expression is up-regulated during ontogenesis allowing to hypothesized that Ap-B participates in processes accompanying retinal neuronal cell differentiation.

The gastrointestinal tract represents the first barrier met by the exogenous compounds of food or orally delivered drugs. To be transferred to the whole body, drugs and xenobiotics have first to pass through the intestinal epithelium, where detoxification systems have to minimize the potential of damage from toxic xenobiotics. However, most studies on xenobiotic-metabolizing enzymes have focused on liver enzymes. Such a situation may be explained by the fact that this organ is the site of toxification/detoxification for both endogenous and exogenous compounds, and also because adequate in vitro hepatocytes models have been available for a long time. By contrast, normal cellular models for the in vitro study of the intestinal processes of biotransformation still remain difficult to obtain. In the present report we will thus focus on the most commonly used models, which are Caco-2 cells and their derivative clones, and we will report recent procedures that allow the isolation of normal enterocytes which maintain their functions and integrity for several hours or even several days. Their respective performance and advantages for the study of the induction of the drug-metabolizing enzymes will be discussed.

Phosphorylation/dephosphorylation processes are known to control the activity of several transcription factors. The nutrition-dependent expression of sucrase–isomaltase and Na+/glucose co-transporter 1, two proteins implicated in the intestinal absorption of glucose, has been shown to be closely related to modifications of hepatocyte nuclear factor 1 (HNF1) activity. This study was conducted to determine whether phosphorylation/dephosphorylation processes could control HNF1 activity. We show that expression of the gene encoding sucrase–isomaltase is inhibited in the enterocytic Caco-2 clone TC7 by okadaic acid at a concentration that is known to inhibit protein phosphatases 1/2A and that does not affect cell viability. At the same concentration, phosphorylation of the HNF1α and HNF1β isoforms is greatly enhanced and their DNA-binding capacity is decreased. The phosphorylation state of HNF1β isoforms directly affects their DNA-binding capacity. In contrast, the decreased DNA-binding activity of the HNF1α isoforms, which was observed after the inhibition of protein phosphatases 1/2A, is due to a net decrease in their total cellular and nuclear amounts. Such an effect results from a decrease in both the HNF1α mRNA levels and the half-life of the protein. This is the first evidence for the implication of protein phosphatases 1/2A in the control of the activity of HNF1 isoforms. Moreover, these results emphasize a physiological role for the balance between phosphatases and kinases in the nutrition-dependent regulation of HNF1-controlled genes.

Our purpose was to investigate whether inducers of cytochrome P450 1A1 (CYP1A1), which cause a decreased expression in Caco-2 cells, at both the mRNA and protein levels, of membrane proteins associated with the uptake and transport of hexoses, would also affect the expression of gamma-glutamyltranspeptidase (gammaGT) (EC 2.3.2.2). In Caco-2 clonal TC7 cells grown under standard conditions (25 mM glucose), exposure to beta-naphthoflavone (beta-NF), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene resulted in increased glucose consumption and decreased gammaGT activity in cells grown to confluence, i.e. when the differentiation is optimum. GammaGT activity was further analyzed during the time course of differentiation of TC7 cells treated or not with beta-naphthoflavone: while gammaGT activity in untreated cells showed a 10-fold increase from the exponential phase of growth until late postconfluence, gammaGT activity in beta-NF-treated cells, although increasing by 4-fold, remained at a much lower level (25%). This decreased activity of gammaGT was associated with a decreased level of gammaGT mRNA. This inhibiting effect was not dependent on the CYP1A1 activity, as it also occurred in the presence of CYP1A1 inhibitors such as alpha-naphthoflavone, 8-methoxypsoralen or ellipticin. It was however dependent on glucose supply as it was not observed when the cells were cultured in low glucose (1 mM). These results raise the question of whether, in Caco-2 cells, CYP1A1 inducers or the signal transduction system which controls CYP1A1 are involved in the regulation of the expression of gammaGT through a mechanism involving glucose metabolism.

Although induction of cytochrome P-450 1A1 (CYP1A1) in the Caco-2 clone TC7 alters glucose utilization and modifies the expression of sucrase-isomaltase (SI) and hexose transporters, nothing is known of the events that control these effects. In this study, we analyzed the effects of β-naphthoflavone (β-NF) and hypoxia on these parameters and expression of key enzymes of glucose metabolism. Both β-NF and hypoxia induce similar changes: 1) induction of CYP1A1 mRNA; 2) increased glucose consumption and lactic acid production and lower glycogen content; 3) downregulation of SI and upregulation of GLUT1 mRNAs; 4) downregulation of fructose-1,6-bisphosphatase and pyruvate kinase mRNAs and upregulation of phospho enolpyruvate carboxykinase, pyruvate dehydrogenase, lactate dehydrogenase, and phosphofructokinase mRNAs; and 5) upregulation of c- fos and c- jun mRNAs. Although addition of inhibitors of CYP1A1 catalytic activity to β-NF-treated cells totally inhibits the enzyme activity, it does not modify CYP1A1 mRNA response and associated effects, thus excluding a direct role for the enzyme per se. These results point to a possible physiological implication of the signal-transduction pathway responsible for CYP1A1 induction.

We have previously shown that the transcription of the human sucrase-isomaltase (SI) gene was negatively regulated by glucose. Using two clonal metabolic variants of the human colon adenocarcinoma cell line Caco-2 we demonstrate here that: 1) although similar growth-related variations of phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-diphosphatase (F1,6-dPase), pyruvate kinase (PK) and SI mRNA levels are observed, only F1,6-dPase, PK and SI mRNA levels vary in the same way in response to modifications of glucose utilization; and 2) regulatory elements responsible for the glucose-dependent transcription of the SI gene are located within the −370/+30 region of the promoter.

Cette recherche s'intéresse à la mise en œuvre d'une pédagogie différenciée dans le cadre d'un cursus de licence sur les nouvelles technologies de l’information et de la communication suivi par un étudiant non-voyant. Comment les enseignants qui ne sont pas informés des adaptations nécessaires à ce profil étudiant font-ils face à cette situation inédite ? Nous avons mis en place une méthodologie d'observation participante en tant que tuteur pédagogique auprès d'un étudiant non-voyant afin de recueillir des données nous permettant d'analyser les stratégies adaptatives des enseignants. Les premiers résultats nous permettent d'envisager la coopération enseignant/étudiant non-voyant comme principal facteur de réussite d'une pédagogie différenciée.

CYP3A is known to be expressed in liver, small intestine and colon. However, its isoform distribution (CYP3A4, 3A5 and 3A7) and inducibility have not been clearly elucidated in the colon. Therefore, we analyzed CYP3A in human colon and compared its expression and inducibility to the human colonic cell lines HT29 and Caco2, which were used as models. Patients suffered either from sigmoiditis or colonic adenocarcinoma. Patients as well as HT29 and Caco2 cells were treated with rifampicin. CYP3A protein expression was analyzed in the colon of patients and in the cells by immunoblot and by isoelectric focusing enabling separation of CYP3A isoforms, while mRNA expression was determined using specific reverse transcription-polymerase chain reaction. In both human colon and cells, CYP3A5 was the main isoform expressed at the protein and mRNA levels. Rifampicin treatment had no effect on CYP3A expression. HT29 and Caco2 cells exhibiting the same CYP3A expression and inducibility might therefore be useful in vitro models for studying xenobiotic metabolism in human colon.

With an excessive postprandial accumulation of intestine-derived, triglyceride-rich lipoproteins being a risk factor of cardiovascular diseases, it is essential to characterize the mechanisms controlling the intestinal absorption of dietary lipids. Our aim was to investigate the role of the transcription factor hepatocyte nuclear factor (HNF)-4α in this process. We used transgenic mice with a specific and inducible intestinal knockout of Hnf-4α gene. One hour after a lipid bolus, in the presence of the lipase inhibitor tyloxapol, lower amounts of triglycerides were found in both plasma and intestinal epithelium of the intestine-specific Hnf-4α knockout (Hnf-4αintΔ) mice compared with the Hnf-4αloxP/loxPcontrol mice. These discrepancies were due to a net decrease of the intestinal uptake of fatty acid in Hnf-4αintΔmice compared with Hnf-4αloxP/loxPmice, as assessed by the amount of radioactivity that was recovered in intestine and plasma after gavage with labeled triolein or oleic acid, or in intestinal epithelial cells isolated from jejunum after a supply of labeled oleic acid-containing micelles. This decreased fatty acid uptake was associated with significant lower levels of the fatty acid transport protein-4 mRNA and protein along the intestinal tract and with a lower acyl-CoA synthetase activity in Hnf-4αintΔmice compared with the control mice. We conclude that the transcription factor HNF-4α is a key factor of the intestinal absorption of dietary lipids, which controls this process as early as in the initial step of fatty acid uptake by enterocytes.

The aim of our study was to establish a relationship between the ability of rat liver and gut to activate 2-aminoanthracene (2AA) into mutagens and their P450 enzyme composition. Rats were orally pretreated with beta-naphthoflavone (beta NF), phenobarbital (PB), dexamethasone (DEX) or acetone (AT). Mutagenic activation of 2AA was detected in the Ames test. P450IA1, IA2, IIB1/B2 and IIE1 were immunochemically quantified by Western blots. All the results were compared to those obtained in untreated rats. In all tissues, beta NF treatment considerably increased the mutagenicity of 2AA. PB treatment significantly reduced the mutagenicity of 2AA in the liver but not in the intestine. By contrast, AT treatment significantly decreased the number of revertants in the duodenum but not in the liver whereas DEX treatment significantly decreased the number of revertants in both tissues. 2AA appears to be metabolized by various P450s in both organs. In the liver, reactive metabolites may be produced after metabolism by the P450IA subfamily. The other P450 enzyme seems to play a part in the metabolism of 2AA leading to formation of either mutagenic or non-mutagenic metabolites.

Background The intestine is responsible for absorbing dietary lipids and delivering them to the organism as triglyceride-rich lipoproteins (TRL). It is important to determine how this process is regulated in enterocytes, the absorptive cells of the intestine, as prolonged postprandial hypertriglyceridemia is a known risk factor for atherosclerosis. During the postprandial period, dietary lipids, mostly triglycerides (TG) hydrolyzed by pancreatic enzymes, are combined with bile products and reach the apical membrane of enterocytes as postprandial micelles (PPM). Our aim was to determine whether these micelles induce, in enterocytes, specific early cell signaling events that could control the processes leading to TRL secretion. Methodology/Principal Findings The effects of supplying PPM to the apex of Caco-2/TC7 enterocytes were analyzed. Micelles devoid of TG hydrolysis products, like those present in the intestinal lumen in the interprandial period, were used as controls. The apical delivery of PPM specifically induced a number of cellular events that are not induced by interprandial micelles. These early events included the trafficking of apolipoprotein B, a structural component of TRL, from apical towards secretory domains, and the rapid, dose-dependent activation of ERK and p38MAPK. PPM supply induced the scavenger receptor SR-BI/CLA-1 to cluster at the apical brush border membrane and to move from non-raft to raft domains. Competition, inhibition or knockdown of SR-BI/CLA-1 impaired the PPM-dependent apoB trafficking and ERK activation. Conclusions/Significance These results are the first evidence that enterocytes specifically sense postprandial dietary lipid-containing micelles. SR-BI/CLA-1 is involved in this process and could be a target for further study with a view to modifying intestinal TRL secretion early in the control pathway.

Introduction Les desordres metaboliques comme le diabete, l’obesite et le syndrome metabolique sont des preoccupations majeures de sante publique. Malgre sa contribution a l’homeostasie energetique par sa fonction d’absorption des nutriments, l’intestin a recu peu d’attention concernant son role potentiel dans ces pathologies compare au foie, muscles et tissu adipeux. Deux formes du recepteur nucleaire HNF-4, alpha et gamma, sont fortement exprimes dans l’epithelium intestinal. Nous avons demontre precedemment que HNF-4alpha controle l’homeostasie de l’epithelium intestinal et l’absorption des lipides alimentaires par l’intestin. Contrairement a la forme alpha, HNF-4gamma a ete peu etudie. Notre objectif est de determiner son role physiologique. Materiels et methodes Nous avons utilise un modele murin d’invalidation totale et constitutive de HNF-4gamma presentant un gain de poids significatif des 4 mois suggerant une perturbation metabolique. Resultats La perte d’expression de HNF-4gamma entraine une augmentation de la tolerance au glucose et une hyperinsulinemie lors d’un test oral au glucose mais pas lors d’un test intraperitoneal, demontrant la part intestinale du phenotype observe. Grâce a un antagoniste du GLP-1, l’exendine 9, nous avons montre que le GLP-1, une enterohormone incretine, est directement impliquee dans cet effet. De plus, l’invalidation de HNF-4gamma entraine une augmentation du nombre de cellules exprimant le GLP-1 dans le jejunum et le colon (×1,7) ainsi que la quantite plasmatique basal et en reponse au glucose de GLP-1. L’analyse morphologique du pancreas et fonctionnelle sur ilots de Langherans isoles montre que la perte d’expression de HNF-4gamma entraine une augmentation du nombre d’ilots moyens et larges (respectivement ×1,5 et ×1,8) ainsi que de leurs contenus en insuline (×1,7) sans que leurs capacites secretoires soit affectees. Conclusion Nous demontrons pour la premiere fois un role specifique de HNF-4gamma dans le controle de l’homeostasie glucidique soulignant l’implication de l’intestin dans le controle de la balance energetique.

Our purpose was to analyze whether postmitotic Caco-2 colon cancer cells, although they express most of the differentiation characteristics of terminally differentiated intestinal epithelial cells, still maintain, unlike normal cells, a proliferation potential. Experiments were performed with clone TC7 of the Caco-2 cell line. Dividing TC7 cells are undifferentiated and express detectable levels of thymidylate synthase (TS) and cytochrome P450 1A1 (CYP1A1) mRNAs. When reaching confluence TS and CYP1A1 are downregulated, mitosis is no longer detectable, and differentiation takes place, as demonstrated by appearance and increasing levels of differentiation-associated marker mRNAs (e.g., sucrase-isomaltase (SI), dipeptidylpeptidase-IV (DPP-IV) or GLUT5), increasing activities of sucrase and DPP-IV, and increasing expression, on immunofluorescence analysis, of SI on the surface of the cell layer. Trypsinization and seeding of late postconfluent cells (day 30) expressing complete differentiation results within 24 h in upregulation of TS and CYP1A1, a concomitant and dramatic disappearance of differentiation marker mRNAs associated with a decrease in sucrase and DPP-IV activities, and delayed resumption of cell division. This is followed, after the cells have reached confluence again, by downregulation of TS and CYP1A1 and resumption of cell differentiation. The ability of differentiated cells to dedifferentiate was further confirmed by wounding the cell layer of late postconfluent differentiated cultures: within 24 h following the wound, cells migrate from the wound edge and dedifferentiate, as demonstrated by transmission electron microscopy and disappearance of SI from the cell surface of migrating cells. Late postconfluent differentiated cells were tumorigenic in nude mice. These results raise the question of the validity of the concept of differentiation therapy when applied to colon cancer cells.

Introduction et but de l’etude Le flux portal d’Arginine (Arg) est un element cle de la regulation de l’ureogenese hepatique. L’intestin est l’organe qui controle la disponibilite de l’Arg apportee par voie digestive. Cela dependrait d’une modulation de l’expression de differentes enzymes (arginase, glutaminase, ornithine ami-notransferase (OAT) et argininosuccinate lyase (ASL) et synthetase (ASS)) par les apports en proteines de la ration alimentaire. Le but de ce travail a ete d’etudier l’influence d’apports croissants en acides amines (AA) sur l’expression de ces enzymes dans un modele de cellules intestinales, les cellules Caco-2/TC7. Materiel et methodes Des cellules Caco-2/TC7 ont ete cultivees sur filtre semi-permeable pendant 15 jours en milieu standard puis ont ete reparties en 4 groupes (n=3). Les cellules ont alors ete exposees pendant 3 jours a differents milieux depourvu d’AA (0), ou contenant des concentrations croissantes en AA, correspondant a une (1X), deux (2X) et quatre (4X) fois les concentrations physiologiques plasmatiques post-prandiales. Les ARNm codant pour les enzymes d’interet ont ete quantifies par PCR en temps reel. Les resultats ont ete analyses par regression lineaire (test de Student sur la pente). Resultats Il existe une relation inverse entre les apports en AA et l’expression de la glutaminase (p= 0,028), de l’OAT (p= 0,035), de l’ASS (p= 0,028) et de l’arginase (p= 0,058). Conclusions Cette etude montre pour la premiere fois un effet direct des AA sur la regulation au niveau transcriptionnel des enzymes du carrefour intestinal de l’arginine en fonction de l’apport Tableau 1 [AA] Glutaminase Arginase OAT ASS O 1,03 ± 0,34 2,85 ± 1,81 ,33 ± 1,44 1,49±0,48 1X 1,05 ± 0,19 0,96 ± 0,01 1,02 ± 0,01 0,91 ± 0,10 2X 0,34 ± 0,05 1,11 ± 0,15 0,42 ± 0,07 0,55 ± 0,02 4X 0,37 ± 0,10 0,27 ± 0,07 0,29 ± 0,04 0,52 ± 0,11 Les niveaux d’expression des differentes enzymes, rapportees a la quantite d’ARNr 18S azote. Cette regulation permettrait ainsi, dans des situations de faibles apports azotes, de favoriser la synthese intestinale de citrulline et par consequent la neosynthese renale d’Arg. Lors d’apports azotes plus importants, cela favoriserait le passage de l’Arg absorbee dans le systeme porte et donc une stimulation adaptee de l’ureoge-nese hepatique.

We have previously shown, using the Caco-2 clone PF11, that glucose represses transcription of the human sucrase-isomaltase (SI) gene and that the -370/+30 fragment of the SI gene conferred glucose-regulated expression on a heterologous gene. Different fragments beginning at the already characterized SI footprint (SIF) 1 (-53/-37), SIFR (-153/-129) or SIF3 (-176/-156) elements [Wu, Chen, Forslund and Traber (1994) J. Biol. Chem. 269, 17080–17085] were tested, in comparison with the -370/+30 fragment, for their capacity to inhibit reporter gene expression under high-glucose (25 mM) conditions. Unlike SIF1 and SIFR, the addition of the HNF (hepatocyte nuclear factor)-1-binding element SIF3 to the promoter fragment was required for repression under high-glucose conditions. This effect was enhanced when the SI promoter was extended to position -370, indicating that the -370/-176 region contains elements that may co-operate with SIF3 to increase the metabolic control of the SI promoter. We have characterized an additional HNF-1-binding site near to and upstream from SIF3; SIF4. By mutagenesis of the three HNF-1-binding elements we show that the two distal HNF-1-recognition sites are the most important for the glucose regulation of the SI gene. Moreover, this glucose regulation was abolished in PF11 cells overexpressing vHNF-1C (variant HNF, an isoform of the HNF-1 family). We thus propose that the differential binding of HNF-1-family proteins to their DNA targets on the SI promoter constitutes the molecular mechanism that controls the glucose regulation of the SI gene transcription.

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc- a - O -benzyl results in inhibition of Gal b 1-3GalNAc: a 2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secre- tion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hemon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To de- termine the effects of prolonged inhibition of sialyla- tion, differentiated HT-29 populations were grown un- der permanent exposure to GalNAc- a - O -benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumu- lation in intracytoplasmic vesicles of brush border-asso- ciated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis -Golgi as substantiated by endoglycosidase treatment and bio- synthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that ( a ) like in mu- cins, NeuAc a 2-3Gal-R is expressed in the terminal po- sition of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and ( b ) treatment with GalNAc- a - O -benzyl results in an impairment of their sialylation. These effects are re- versible upon removal of the drug. It is suggested that a 2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.

Involvement of cytochrome P-4501A1 (CYP1A1) in the regulation of sucrase-isomaltase and hexose transporters was analyzed in low (TC7)- and high (PF11)-glucose-consuming Caco-2 clones. CYP1A1 mRNA is elevated in exponentially growing cells concomitantly with high rates of glucose consumption and high levels of GLUT-1 and GLUT-3 mRNA. After confluency, CYP1A1 is not detectable in TC7 cells; this is associated with a decreased glucose consumption, a downregulation of GLUT-1 and GLUT-3, and an upregulation of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. In PF11 cells CYP1A1 mRNA remains elevated, along with high glucose consumption, high levels of GLUT-1 and GLUT-3, and minimal expression of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. Exposure of TC7 cells to inducers of CYP1A1 results in high levels of CYP1A1 mRNA, a 10-fold increase of glucose consumption after confluency, an upregulation of GLUT-1 and GLUT-3, and a downregulation of sucrase-isomaltase, GLUT-2, and, to a lesser extent, SGLT-1 and GLUT-5. These results suggest that activation of CYP1A1, whether spontaneous or drug induced, is involved in the variations of glucose utilization and in the associated modifications of expression of sucrase-isomaltase and hexose transporters.

HT-29 sublines and Caco-2 clones were analyzed for the expression of cytochrome P-450 3A. The enzyme was found to be expressed in differentiated HT-29 cells selected by resistance to methotrexate and in one of seven Caco-2 clones, TC7. Its expression parallels the differentiation process, with highest levels being observed at late confluency. P-450 3A mRNA and protein patterns, as well as subcellular distribution, are intermediate between those observed in human adult intestine and fetal liver.

Chlorzoxazone, a centrally acting muscle relaxant, was previously shown to be hydroxylated on carbon 6 specifically by cytochrome P450 2E1. Accordingly, this drug has been proposed as a potential noninvasive in-vivo probe for screening the hepatic P450 2E1 activity. This study was carried out to test the specificity of such a substrate when first experiments conducted by using human hepatocyte cultures showed that the chlorzoxazone 6-hydroxylation activity increased after 3-methylcholanthrene treatment of cells. Indeed, the ability of both rat and human hepatocytes to metabolize chlorzoxazone significantly increased after treatments by 3-methylcholanthrene alone or plus ethanol, suggesting the involvement of P450 1A enzymes in this oxidative reaction. Identical results were obtained by in-vivo treatment of rats with four inducers of P450 1A enzymes, namely, beta-naphthoflavone, isosafrole, Arochlor 1254, and 3-methylcholanthrene. Furthermore, the chlorzoxazone 6-hydroxylation activity was inhibited by both alpha-naphthoflavone and dimethyl sulfoxide, both known to inhibit P450 1A and P450 2E1 activities, respectively. Finally, the use of yeasts genetically engineered for expression of human P450 1A1, 1A2, 2C9, and 3A4 demonstrated that P450 1A1 was significantly involved in this catalytic activity. In conclusion, these results taken together suggest that chlorzoxazone should be used with precaution as in-vivo tool for evaluating P450 2E1. However, the relative Km of P450 1A1 and 2E1 for chlorzoxazone and, on the other hand, the relative levels of these two enzymes in the human liver suggest that P450 2E1 would generally be the major form metabolizing chlorzoxazone in-vivo.