Edith Brot-Laroche

GLUT2 mutations, translocation, and receptor function in diet sugar managing.

Cloned 20 years ago, GLUT2 is a facilitative glucose transporter in the liver, pancreas, intestine, kidney, and brain. It ensures large bidirectional fluxes of glucose in and out the cell due to its low affinity and high capacity. It also transports other dietary sugars, such as fructose and galactose, within the range of physiological concentrations. Sugars and hormones regulate its gene expression. The contribution of GLUT2 to human metabolic diseases previously appeared modest. However, in the past decade, three major features of the GLUT2 protein have been revealed. First, GLUT2 mutations cause the severe but rare Fanconi-Bickel syndrome, mainly characterized by glycogenosis. Recently, a GLUT2 polymorphism has been associated with preferences for sugary food. Second, the GLUT2 location at the cell surface is regulated; this governs cellular activities dependent on glucose in the intestine and possibly those in the liver and pancreas. For instance, GLUT2 translocation from an intracellular pool to the apical membrane after a sugar meal transiently increases sugar uptake by enterocytes (reviewed in 32). Third, GLUT2 functions as a membrane receptor of sugar. Independently of glucose metabolism, GLUT2 detects the presence of extracellular sugar and transduces a signal to modulate cell functions, including beta-cell insulin secretion, renal reabsorption, and intestinal absorption according to the sugar environment. These recent developments are examined here in heath and metabolic disease, highlighting various unanswered questions.

Insulin Internalizes GLUT2 in the Enterocytes of Healthy but Not Insulin-Resistant Mice

OBJECTIVES—A physiological adaptation to a sugar-rich meal is achieved by increased sugar uptake to match dietary load, resulting from a rapid transient translocation of the fructose/glucose GLUT2 transporter to the brush border membrane (BBM) of enterocytes. The aim of this study was to define the contributors and physiological mechanisms controlling intestinal sugar absorption, focusing on the action of insulin and the contribution of GLUT2-mediated transport. RESEARCH DESIGN AND METHODS—The studies were performed in the human enterocytic colon carcinoma TC7 subclone (Caco-2/TC7) cells and in vivo during hyperinsulinemic-euglycemic clamp experiments in conscious mice. Chronic high-fructose or high-fat diets were used to induce glucose intolerance and insulin resistance in mice. RESULTS AND CONCLUSIONS—In Caco-2/TC7 cells, insulin action diminished the transepithelial transfer of sugar and reduced BBM and basolateral membrane (BLM) GLUT2 levels, demonstrating that insulin can target sugar absorption by controlling the membrane localization of GLUT2 in enterocytes. Similarly, in hyperinsulinemic-euglycemic clamp experiments in sensitive mice, insulin abolished GLUT2 (i.e., the cytochalasin B-sensitive component of fructose absorption), decreased BBM GLUT2, and concomitantly increased intracellular GLUT2. Acute insulin treatment before sugar intake prevented the insertion of GLUT2 into the BBM. Insulin resistance in mice provoked a loss of GLUT2 trafficking, and GLUT2 levels remained permanently high in the BBM and low in the BLM. We propose that, in addition to its peripheral effects, insulin inhibits intestinal sugar absorption to prevent excessive blood glucose excursion after a sugar meal. This protective mechanism is lost in the insulin-resistant state induced by high-fat or high-fructose feeding.

GLUT2 Accumulation in Enterocyte Apical and Intracellular Membranes: A Study in Morbidly Obese Human Subjects and ob/ob and High Fat–Fed Mice

OBJECTIVE In healthy rodents, intestinal sugar absorption in response to sugar-rich meals and insulin is regulated by GLUT2 in enterocyte plasma membranes. Loss of insulin action maintains apical GLUT2 location. In human enterocytes, apical GLUT2 location has not been reported but may be revealed under conditions of insulin resistance. RESEARCH DESIGN AND METHODS Subcellular location of GLUT2 in jejunal enterocytes was analyzed by confocal and electron microscopy imaging and Western blot in 62 well-phenotyped morbidly obese subjects and 7 lean human subjects. GLUT2 locations were assayed in ob/ob and ob/+ mice receiving oral metformin or in high-fat low-carbohydrate diet–fed C57Bl/6 mice. Glucose absorption and secretion were respectively estimated by oral glucose tolerance test and secretion of [U-14C]-3-O-methyl glucose into lumen. RESULTS In human enterocytes, GLUT2 was consistently located in basolateral membranes. Apical GLUT2 location was absent in lean subjects but was observed in 76% of obese subjects and correlated with insulin resistance and glycemia. In addition, intracellular accumulation of GLUT2 with early endosome antigen 1 (EEA1) was associated with reduced MGAT4a activity (glycosylation) in 39% of obese subjects on a low-carbohydrate/high-fat diet. Mice on a low-carbohydrate/high-fat diet for 12 months also exhibited endosomal GLUT2 accumulation and reduced glucose absorption. In ob/ob mice, metformin promoted apical GLUT2 and improved glucose homeostasis. Apical GLUT2 in fasting hyperglycemic ob/ob mice tripled glucose release into intestinal lumen. CONCLUSIONS In morbidly obese insulin-resistant subjects, GLUT2 was accumulated in apical and/or endosomal membranes of enterocytes. Functionally, apical GLUT2 favored and endosomal GLUT2 reduced glucose transepithelial exchanges. Thus, altered GLUT2 locations in enterocytes are a sign of intestinal adaptations to human metabolic pathology.

Sugar sensing by enterocytes combines polarity, membrane bound detectors and sugar metabolism

Sugar consumption and subsequent sugar metabolism are known to regulate the expression of genes involved in intestinal sugar absorption and delivery. Here we investigate the hypothesis that sugar‐sensing detectors in membranes facing the intestinal lumen or the bloodstream can also modulate intestinal sugar absorption. We used wild‐type and GLUT2‐null mice, to show that dietary sugars stimulate the expression of sucrase‐isomaltase (SI) and L‐pyruvate kinase (L‐PK) by GLUT2‐dependent mechanisms, whereas the expression of GLUT5 and SGLT1, did not rely on the presence of GLUT2. By providing sugar metabolites, sugar transporters, including GLUT2, fuelled a sensing pathway. In Caco2/TC7 enterocytes, we could disconnect the sensing triggered by detector from that produced by metabolism, and found that GLUT2 generated a metabolism‐independent pathway to stimulate the expression of SI and L‐PK. In cultured enterocytes, both apical and basolateral fructose could increase the expression of GLUT5, conversely, basolateral sugar administration could stimulate the expression of GLUT2. Finally, we located the sweet‐taste receptors T1R3 and T1R2 in plasma membranes, and we measured their cognate Galpha Gustducin mRNA levels. Furthermore, we showed that a T1R3 inhibitor altered the fructose‐induced expression of SGLT1, GLUT5, and L‐PK. Intestinal gene expression is thus controlled by a combination of at least three sugar‐signaling pathways triggered by sugar metabolites and membrane sugar receptors that, according to membrane location, determine sugar‐sensing polarity. This provides a rationale for how intestine adapts sugar delivery to blood and dietary sugar provision. J. Cell. Physiol. 213:834–843.

A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins

The bundled architecture of actin filaments is not needed for intestinal microvillar morphogenesis, as shown in knockout mice devoid of microvillar actin-bundling proteins. This architecture is essential for the apical anchorage of digestive proteins, probably via the recruitment of key players in apical retention, such as myosin-1a, and, as a result, for intestinal physiology.

Loss of Sugar Detection by GLUT2 Affects Glucose Homeostasis in Mice

Background Mammals must sense the amount of sugar available to them and respond appropriately. For many years attention has focused on intracellular glucose sensing derived from glucose metabolism. Here, we studied the detection of extracellular glucose concentrations in vivo by invalidating the transduction pathway downstream from the transporter-detector GLUT2 and measured the physiological impact of this pathway. Methodology/Principal Findings We produced mice that ubiquitously express the largest cytoplasmic loop of GLUT2, blocking glucose-mediated gene expression in vitro without affecting glucose metabolism. Impairment of GLUT2-mediated sugar detection transiently protected transgenic mice against starvation and streptozotocin-induced diabetes, suggesting that both low- and high-glucose concentrations were not detected. Transgenic mice favored lipid oxidation, and oral glucose was slowly cleared from blood due to low insulin production, despite massive urinary glucose excretion. Kidney adaptation was characterized by a lower rate of glucose reabsorption, whereas pancreatic adaptation was associated with a larger number of small islets. Conclusions/Significance Molecular invalidation of sugar sensing in GLUT2-loop transgenic mice changed multiple aspects of glucose homeostasis, highlighting by a top-down approach, the role of membrane glucose receptors as potential therapeutic targets.

Insulin regulates the expression of the GLUT5 transporter in L6 skeletal muscle cells

Skeletal muscle, a primary insulin target tissue, expresses the GLUT5 fructose transporter. Although insulin has no acute effect on GLUT5 expression and function in muscle, we show here that long‐term (24 h) insulin treatment of L6 muscle cells induces a dose‐dependent increase in GLUT5 protein (by up to two‐fold), leading to a concomitant increase in fructose uptake. The increase in GLUT5 expression and function was suppressed by inhibitors of gene transcription and protein synthesis, suggesting that insulin promotes de novo carrier synthesis. Transfection of the GLUT5 gene promoter fused to luciferase into L6 cells revealed that insulin induced a 1.8‐fold increase in GLUT5 promoter activity. Our findings indicate that insulin is capable of increasing the abundance and functional activity of GLUT5 in skeletal muscle cells and that this is most likely mediated via activation of the GLUT5 promoter.

PO18 Altération de la différenciation et de l’abondance des cellules entéroendocrines par l’obésité et le diabète chez l’Homme

Introduction Le bypass gastrique est actuellement le traitement le plus efficace de lobesite morbide qui conduit a une amelioration du diabete ou a sa resolution grâce a des mecanismes inconnus saccompagnant de changements de la secretion intestinale dhormones. La secretion de ces enterohormones est dependante du nombre de cellules, de leur fonctionnement en reponse a lenvironnement et de la balance proliferation/differenciation/apoptose. Cette etude evalue limpact de lobesite et du diabete sur labondance et la differenciation des cellules enteroendocrines, en particulier les cellules L productrices de lhormone incretine GLP-1 chez lHomme. Patients et methodes Des prelevements de jejunum proximal sont collectes lors de bypass gastriques de sujets obeses morbides diabetiques ou non. Des patients subissant une resection gastroduodenale suite a un cancer digestif constituent le groupe controle. Lexpression des facteurs de transcription impliques dans la differenciation du lignage enteroendocrine est etudiee par RTqPCR dans des cellules epitheliales isolees a partir des prelevements jejunaux. Labondance des cellules enteroendocrines est evaluee par immunohistochimie sur ces memes fragments jejunaux. Resultats Dans le jejunum des sujets obeses, le nombre de cellules L est trois fois plus faible que chez les sujets minces controles. Le diabete diminue encore le nombre de cellules GLP-1 positives/mm 2 . Ces resultats sont concordants avec les resultats dexpression des facteurs de transcriptions impliques dans la differentiation des cellules enteroendocrines tels que NGN3, NeuroD1, PAX6, ISL1, FOXA1 et FOXA2 chez les sujets obeses comparativement aux controles. Discussion Lobesite et le diabete affectent le lignage enteroendocrine chez lHomme en limitant labondance de ces cellules au niveau du jejunum, site majeur de labsorption des nutriments. Une deregulation de la differenciation enteroendocrine pourrait etre a lorigine des alterations de la secretion des enterohormones observees chez les sujets obeses et obeses diabetiques.

082 Des mutations identifiées dans le syndrome d’hyperinsulinisme congénital soulignent l’importance de GLUT2 dans le pancréas humain.

Introduction Limplication du transporteur des sucres GLUT2 dans la secretion dinsuline par les cellules beta pancreatiques est bien caracterisee chez les rongeurs mais reste controversee chez lHomme. Le syndrome de Fanconi-Bickel (FBS), caracterise par des desordres de lhomeostasie glucidique sans diabete, est associe a des mutations inactivantes des fonctions de hGLUT2. Certains jeunes patients FBS peuvent souffrir dhyperglycemie et dhypoinsulinemie en reponse a une charge de glucose; cependant ces symptomes disparaissent avec lâge. Reciproquement dans le syndrome dhyperinsulinisme congenital (CHI), des mutations activantes de hGLUT2 pourraient etre a lorigine de lhyperinsulinemie et de lhypoglycemie severe des nouveau-nes. Le but de cette etude est devaluer les consequences fonctionnelles sur le transport de sucre et la secretion dinsuline de mutations de hGLUT2 identifiees chez des patients FBS et CHI. Materiels et methodes Neuf mutations faux-sens de hGLUT2 (4 identifiees chez des patients FBS, 3 chez des patients CHI et 2 dans la population generale) ont ete introduites dans des vecteurs dexpression de hGLUT2 par mutagenese dirigee. Lexpression proteique de hGLUT2 a ete analysee par immunohistochimie et western blot. Les parametres cinetiques de transport ont ete mesures par le captage de 2-deoxy-D-glucose radiomarque dans des ovocytes de xenope exprimant les variants hGLUT2. La secretion dinsuline en reponse au glucose a ete mesuree dans les cellules insulino-secretrice MIN6 infectees avec les variants CHI de hGLUT2. Resultats Nous montrons que hGLUT2 est exprime dans les cellules insulino-positives du pancreas de patients CHI. De plus, deux mutations identifiees chez les patients CHI provoquent une secretion dinsuline exacerbee meme en absence de glucose dans le milieu de culture des cellules. En revanche, les mutations associees au FBS, inhibent lexpression proteique ou les capacites de transport de hGLUT2. Conclusion Dans le pancreas de nouveau-ne humain, hGLUT2 jouerait un role essentiel dans le controle de la secretion dinsuline. Nous proposons donc de sequencer hGLUT2 chez les patients CHI.

O86 GLUT2 devient un transporteur de sucre constitutif de la membrane apicale des entérocytes chez les souris rendues résistantes à l’insuline par un régime hyperlipidique

Introduction Nous avons montre que la consommation d’un repas de sucres simples recrute de facon transitoire la proteine de transport du glucose et du fructose, GLUT2, dans la membrane en bordure en brosse des enterocytes de souris. Il en resulte une augmentation de la capacite d’absorption intestinale de sucre. Nous avons donc montre que les sucres alimentaires favorisent l’insertion de GLUT2 dans la bordure en brosse, alors que l’insuline la previent par son internalisation. Cette internalisation de GLUT2 par l’insuline est perdue chez les souris rendues resistantes a l’insuline par la consommation d’un regime riche en fructose (65 %) pendant un mois. Materiels et methodes Le but de ce travail etait de connaitre les contributions respectives de la resistance a l’insuline et du fructose sur le defaut d’internalisation de GLUT2. Nous avons nourri des souris avec un regime, riche en graisse saturees (72 %) et pauvre en sucre ( Resultats Les souris developpent une intolerance au glucose et une resistance a l’insuline a partir de 2 mois de ce regime hyperlipidique. Le taux de proteine GLUT2 est diminue de 70 % par rapport a celui de souris nourries avec un regime riche en fructose. Comme chez les souris rendues resistantes a l’insuline par le regime riche en fructose, GLUT2 est majoritairement localise dans la bordure en brosse des enterocytes des souris soumises a un regime hyperlipidique en condition de clamp euglycemique et hyperinsulinemique. La localisation de GLUT2 est insensible a l’insuline. De plus, nous avons observe que la proportion de GLUT2 dans les membranes basolaterales etait reduite chez les souris resistantes a l’insuline par rapport aux souris nourries avec un regime standard (standard > fructose > graisse saturees). Conclusion Ces observations indiquent que l’etat de resistance a l’insuline est associe a une localisation apicale permanente de GLUT2 dans les membranes en bordure en brosse independamment de la presence de sucre dans le regime des souris. Cette distribution pathologique de GLUT2 dans les membranes plasmiques des enterocytes est un facteur aggravant de la resistance a l’insuline parce qu’il augmente l’absorption intestinale des sucres.