Excessive Food Intake, Obesity and Inflammation Process in Zucker fa/fa Rat Pancreatic Islets

Inappropriate food intake-related obesity and more importantly, visceral adiposity, are major risk factors for the onset of type 2 diabetes. Evidence is emerging that nutriment-induced β-cell dysfunction could be related to indirect induction of a state of low grade inflammation. Our aim was to study whether hyperphagia associated obesity could promote an inflammatory response in pancreatic islets leading to ß-cell dysfunction. In the hyperphagic obese insulin resistant male Zucker rat, we measured the level of circulating pro-inflammatory cytokines and estimated their production as well as the expression of their receptors in pancreatic tissue and β-cells. Our main findings concern intra-islet pro-inflammatory cytokines from fa/fa rats: IL-1β, IL-6 and TNFα expressions were increased; IL-1R1 was also over-expressed with a cellular redistribution also observed for IL-6R. To get insight into the mechanisms involved in phenotypic alterations, abArrays were used to determine the expression profile of proteins implicated in different membrane receptors signaling, apoptosis and cell cycle pathways. Despite JNK overexpression, cell viability was unaffected probably because of decreases in cleaved caspase3 as well as in SMAC/DIABLO and APP, involved in the induction and amplification of apoptosis. Concerning β-cell proliferation, decreases in important cell cycle regulators (Cyclin D1, p35) and increased expression of SMAD4 probably contribute to counteract and restrain hyperplasia in fa/fa rat islets. Finally and probably as a result of IL-1β and IL-1R1 increased expressions with sub-cellular redistribution of the receptor, islets from fa/fa rats were found more sensitive to both stimulating and inhibitory concentrations of the cytokine; this confers some physiopathological relevance to a possible autocrine regulation of β-cell function by IL-1β. These results support the hypothesis that pancreatic islets from prediabetic fa/fa rats undergo an inflammatory process. That the latter could contribute to β-cell hyperactivity/proliferation and possibly lead to progressive β-cell failure in these animals, deserves further investigations

Marqueurs de structure et de fonction dans les ependymocytes in vivo et in vitro: mise en evidence de l'expression des proteines G a l'aide d'anticorps polyclonaux et monoclonaux

SIGLEINIST T 76245 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Auto-anticorps recombinants humains anti-thyroperoxydase (de la caractérisation )

Les auto-anticorps (aAc) antithyroxydase (anti-TPO) constituent l'un des principaux marqueurs des maladies auto-immunes thyroïdiennes (MAIT). Ils sont présents, en forte concentration dans la plupart des sera de patients atteints de MAIT et reconnaissent des épitopes discontinus, localisés au niveau d'une région immunodominante (RID). Au cours des cinq dernières années, notre équipe a produit, grâce à la technologie des banques combinatoires de phages Ac, quarante quatre fragments humains d'Ac anti-TPO de type scFv (single chain fragment variable) dont une majorité est constituée d'une chaîne légère de type lambda. Ce travail de thèse s'appuyant sur ces outils Ac, s'est articulé principalement selon deux axes. 1. : Evaluer l'importance des aAcs anti-TPO de type de chaîne légère lambda dans les MAIT. En effet, jusqu'ici seuls quatre aAc anti-TPO recombinants humains possédant une chaîne légère de type lambda, avaient été décrits parmi près de 200 caractérisés. Dans cet objectoif, une étude comparative de deux aAc recombinants humains anti-TPO possédant une chaîne variable lourde très proche, mais des chaînes variables légères de type lambda ou kappa (T2/kappa et T13 /lambda), nous a permis de montrer, à la fois l'importance des aAc humains anti-TPO de chaînes légères lambda dans les MAIT, mais aussi de confirmer que l'épitope de l'aAc T13/lambda est contenu dans le région immunodominante (RID) de la TPO. 2. : Délimiter sur un modèle tridimensionnel la RID de la TPO. Grâce à la technologie des banques combinatoires de peptides exprimés à la surface de phages, nous avons sélectionné plusieurs mimes peptidiques (mimotopes) de l'épitope de l'aAc T13. L'alignement de ces mimotopes avec la séquence primaire de la TPO humaine a permis de définir des zones putatives d'interaction de l'aAc T13 sur la TPO. L'implication de quatre de ces zones (353-363, 377-386, 713-720 et 766-775) dans la formation de la RID a pu être confirmée par des expériences de mutagenèse dirigée sur la TPO. Nous avons pu ainsi définir, pour la première fois, l'épitope discontinu d'un aAc humain anti-TPO reconnaissant la RID. La caractérisation de la RID de la TPO devrait nous éclairer, à terme, sur le mode de présentation de cet auto-antigène au système immunitaire, lors de l'apparition et du développement des MAIT.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Les anticorps anti-thyroperoxydase (de l'identification de la région immuno-dominante de la thyroperoxydase à l'utilisation du potentiel cytotoxique des anticorps anti-thyropéroxydase dans les cancers thyroïdiens)

Outre leur rôle de marqueur des maladies auto-immunes thyroïdiennes (MAIT), les auto-anticorps (aAc) anti-thyroperoxydase (TPO) pourraient être impliqués dans la destruction de la glande thyroïde et/ou dans l emballement du processus auto-immuns. Ils reconnaissent à la surface de la TPO des épitopes discontinus et majoritairement conformationnels regroupés en deux domaines restreints A et B, définissant la région immuno-dominante (RID). Notre équipe a participé à la caractérisation de cette RID, tout d abord en produisant des aAc recombinants humains anti-TPO spécifiques des aAc de patients atteints de MAIT. Ces aAc recombinants ont été caractérisés au niveau génétique, épitopique et terme d affinité vis-à-vis de la TPO. L utilisation de l un de ces aAc recombinant produit sous forme d Immunoglobuline entière et nommé T13 a permis de définir à la surface de la TPO quatre régions (353-363, 377-386, 713-720 et 766-775) impliquées dans la reconnaissance de la TPO par cet aAc recombinant humain anti-TPO mais également des aAc de patients.Notre étude de la région 353-363, par mutagénèse dirigée où chaque acide aminé est muté ponctuellement en Alanine, a permis de déterminer les résidus impliqués dans l interaction aAc/TPO. Après production des protéines TPO mutées, l étude de la liaison de trois aAc recombinants humains anti-TPO (T13, B4 et ICA1) et des aAc de patients atteints de MAIT a permis d identifier les résidus critiques de cette région : Histidine en position 353 (H353), Acide Aspartique 358 (D358), Arginine 361 (R361). La caractérisation de la RID devrait nous permettre d expliquer le mode de présentation de la TPO au système immunitaire lors de l apparition des MAIT. Le potentiel destructeur des aAc anti-TPO sur la glande thyroïde passe par des mécanismes de cytotoxicité à médiation cellulaire (ADCC) et/ou dépendante du Complément (CDC). Les aAc anti-TPO sont capable de lyser de cellules thyroïdiennes issues de cultures primaires en présence d une source de Complément via la voie classique (après fixation de la fraction C1q du Complément) ou via l activation de cellules effectrices, les monocytes/macrophages capables d infiltrer la thyroïde. Ce mécanisme d ADCC passerait par l activation des récepteurs Fc RI (CD64) et/ou Fc RII (CD32) exprimés par les monocytes/macrophages. Puisque les aAc de patients atteints de MAIT sont capables d exercer une activité cytotoxique, il serait intéressant de pouvoir utiliser ce potentiel destructeur afin de cibler et détruire spécifiquement les cellules cancéreuses thyroïdiennes exprimant la thyroperoxydase (tumeur primaire ou métastases). Afin de pouvoir être utilisé pour la mise en place d une immunothérapie anti-cancéreuse, un aAc recombinant humain anti-TPO produit dans le système baculovirus/cellule d insecte a été sous-cloné et exprimé dans un autre système d expression (cellules CHO). Ces aAc recombinants anti-TPO ainsi que des aAc de patients ont été utilisés dans des tests de cytotoxicité. Ils ont la capacité de lyser les cellules d une lignée de cancer papillaire thyroïdien par des mécanismes de cytotoxicité cellulaire et/ou dépendant du complément. Ces résultats encourageants pourraient permettre l émergence d une thérapie complémentaire pour traiter les cancers thyroïdiens.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

IL-1β and TSH disturb thyroid epithelium integrity in autoimmune thyroid diseases

International audiencePro-inflammatory cytokines such as IL-1β and TNFα are known to affect thyroid function. They stimulate IL-6 secretion and modify epithelium integrity by altering junction proteins. To study the role of cytokines on thyroid epithelia tightness in autoimmune thyroid diseases (AITD), we analyzed the expression profiles of junction proteins (ZO-1, Claudin, JAM-A) and cytokines in human thyroid slices and also investigated the effect of IL-1β on the epithelium integrity in primary cultures of human thyrocytes. Junction proteins expression (ZO-1, Claudin, JAM-A) has been analyzed by immunohistochemistry on thyroid slices and by Western blot on membrane proteins extracted from thyrocytes of patients suffering from Graves and Hashimoto diseases. The high expression of junction proteins we found on Graves' disease thyroid slices as well as in cell membrane extracts acknowledges the tightness of thyroid follicular cells in this AITD. In contrast, the reduced expression of JAM and ZO-1 in thyroid cells from patients suffering from Hashimoto thyroiditis is in agreement with the loss of thyroid follicular cell integrity that occurs in this pathology. Concerning the effects on epithelium integrity of TSH and of the pro-inflammatory cytokine IL-1β in primary cultures of human thyroid cells, TSH appeared able to modify JAM-A localization but without any change in the expression levels of JAM-A, Claudin and ZO-1. Inversely, IL-1β provoked a decrease in the expression of- and a redistribution of both, Claudin and ZO-1 without modifying the expression and sub-cellular distribution patterns of JAM-A in thyroid cells. These results demonstrate (i) that Hashimoto's- and Graves' diseases display different junction proteins expression patterns with a loss of epithelium integrity in the former and (ii) that IL-1β modifies thyroid epithelial tightness of human thyrocytes by altering the expression and localization of junction proteins. Therefore, IL-1β could play a role in the pathogenesis of thyroid autoimmunity

Possible protective effect of membrane lipid rafts against interleukin-1β-mediated anti-proliferative effect in INS-1 cells.

We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1β takes part and controls β-cell function. In the present study, using the INS-1 rat pancreatic β-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1β signaling and biological effects on insulin secretion, β-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1β leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic β-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for β-cell secretory function. In the same conditions, IL-1β treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1β inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1β concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1β anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1β deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis

Adipose tissue derived-factors impaired pancreatic β-cell function in diabetes

International audienceInflammatory factors produced and secreted by adipose tissue, in particular peri-pancreatic adipose tissue (P-WAT), may influence pancreatic β-cell dysfunction. Using the ZDF Rat model of diabetes, we show the presence of infiltrating macrophage (ED1 staining) on pancreatic tissue and P-WAT in the pre-diabetes stage of the disease. Then, when the T2D is installed, infiltrating cells decreased. Meanwhile, the P-WAT conditioned-medium composition, in terms of inflammatory factors, varies during the onset of the T2D. Using chemiarray technology, we observed an over expression of CXCL-1, -2, -3, CCL-3/MIP-1α and CXCL-5/LIX and TIMP-1 in the 9 weeks old obese ZDF pre-diabetic rat model. Surprisingly, the expression profile of these factors decreased when animals become diabetic (12 weeks obese ZDF rats). The expression of these inflammatory proteins is highly associated with inflammatory infiltrate. P-WAT conditioned-medium from pre-diabetes rats stimulates insulin secretion, cellular proliferation and apoptosis of INS-1 cells. However, inhibition of conditioned-medium chemokines acting via CXCR2 receptor do not change cellular proliferation apoptosis and insulin secretion of INS-1 cells induced by P-WAT conditioned-medium. Taken together, these results show that among the secreted chemokines, increased expression of CXCL-1, -2, -3 and CXCL-5/LIX in P-WAT conditioned-medium is concomitant with the onset of the T2D but do not exerted a direct effect on pancreatic β-cell dysfunction

Adipose tissue derived-factors impaired pancreatic β-cell function in diabetes

International audienc

Effect of IL-1β on β-cell function: insulin secretion, apoptose and proliferation.

<p>(A) Insulin secretion assay on INS-1 cells cultured in the presence of increasing IL-1β concentrations for 2 days. At the end of the exposure time, cells were washed, deprived in glucose during 1 hour and incubated in the presence of 0, 2.8, 5.6, 8.3 and 16 mM glucose. Cells supernatant fractions were collected and insulin content extracted with acid/alcohol mixture. Insulin present in culture supernatants and insulin content was quantified using HTRF assay. (B) Insulin secretion with IL-1β treatment (White column), and after pre-treatment of INS-1 cells with 500 ng/ml of IL1 receptor 1 antagonist (IL1-RA) to inhibit IL1-β effect on secretion (Black column). Insulin has been quantified by HTRF. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102889#pone-0102889-g002" target="_blank">Fig (2A-2B</a>): All data represent insulin release normalized for insulin contents and are expressed as percentages of insulin secretion recorded in the presence of 16 mM glucose alone; they are all the result of 3 independent experiments, with each experimental condition performed in triplicate. (C) Annexin V labeling of apoptotic INS-1 cells after treatment by increased concentrations of IL-1β: Ins-1 cells were analyzed by confocal microscopy (pictures are representative of 3 independent experiments) and flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102889#pone-0102889-g003" target="_blank">Fig 3B</a>). (D) Cell proliferation test by BrdU incorporation in INS-1 cells after IL-1β treatment; controls are cells not exposed to the cytokine. Data represent means of 3 independent experiments with each experimental condition performed in triplicates. *: p<0.05 and **: p<0.01 vs cells incubated in the absence of IL-1 β; a: p<0.05 and b: p<0.01 vs in the absence of antagonist.</p

INS-1 cell line expresses IL-1R1 in lipid rafts.

<p>(A) Immunofluorescence studies in the INS-1 cell line; Immunostaining of receptor IL1-R1 and cytokine IL1-β. Irrelevant rabbit IgG and secondary antibody were used as negative controls. (B) Characterization of lipid raft fractions by Ganglioside M1 detection after isolation on sucrose gradient; Dot Blot characterization of Brij 98-extracted fractions. INS-1 cells were lysed with Brij 98 detergent at 37°C and separated into fractions by sucrose density gradient. Membrane rafts are present in fractions 3 and 4, as indicated by GM1 expression. Membrane lipid rafts localization in the INS-1 cell line by GM1 detection using immunofluorescence, without and with MβCD treatment. Results are representative of three independent experiments. (C) Western blots of IL-1R1 expression in lipid-rafts and non-rafts fractions. Forty micrograms of protein extracts from membrane lipid rafts- and non-lipid rafts were run on SDS-PAGE and blotted with anti Il-1R1 antibody.</p