Le diabète est une maladie chronique avec une progression alarmante. L’insuline-résistance et la diminution de la masse fonctionnelle des cellules beta, associée à l'inflammation des îlots, sont les principaux défauts impliqués dans la pathogenèse du diabète de type 2 (DT2). La compréhension des mécanismes impliqués dans l'inflammation des îlots pancréatiques, et l'identification de cibles moléculaires à visée anti-inflammatoire, sont des approches intéressantes pour le traitement du diabète. La glycogène synthase kinase 3 (GSK3), est une sérine-thréonine kinase qui régule des fonctions cellulaires essentielles. Cette enzyme a été récemment décrite comme un régulateur important de l'inflammation dans différentes conditions pathologiques. Cependant, l'implication potentielle de GSK3beta dans l'inflammation des îlots au cours du diabète reste inexplorée. Le but de ce travail était d'étudier l'implication de GSK3beta dans l'inflammation des îlots pancréatiques et d'évaluer l'impact de l'inhibition de GSK3beta dans l’amélioration de l’hyperglycémie du rat diabétique Goto-Kakizaki. Le rat Goto-Kakizaki (GK) est un modèle spontané de DT2, avec une hyperglycémie chronique apparaissant au sevrage, une masse beta cellulaire réduite et une altération profonde de la sécrétion d'insuline en réponse au glucose. Peu après le sevrage, l'inflammation se développe dans les îlots du rat GK et participe au dysfonctionnement des cellules beta. Nous avons traité les rat GK mâles avec du chlorure de lithium (LiCl), un inhibiteur de GSK3. Le traitement chronique de jeunes rats GK a permis d’éviter l’installation de l’hyperglycémie chronique qui se développe normalement dans ce modèle chez les adultes. A la fin du traitement, la glycémie basale des rats GK traités par le LiCl était fortement réduite, en comparaison avec celle des rats GK non traités. Ces améliorations étaient associées à une réduction de l'expression des cytokines et des chimiokines pro-inflammatoires dans les îlots. L’inhibition de GSK3 a également diminué la fibrose des îlots et rétabli partiellement la sensibilité à l’insuline et la sécrétion d'insuline induite par le glucose chez les rats GK. De plus, des études ex vivo sur des îlots humains et des îlots de rats Wistar, exposés à un environnement inflammatoire en culture, ont révélé l'implication directe de GSK3 dans la réponse inflammatoire autonome des îlots. Ceci était entre autres associée à l’activation du facteur de transcription STAT3. En conclusion, nous montrons pour la première fois que GSK3beta est impliquée dans l’inflammation des îlots pancréatiques humains et de rongeurs. L’inhibition de GSK3beta atténue fortement l’inflammation insulaire, et prévient l’installation de l’hyperglycémie chronique chez le rat GK. L’ensemble des résultats de ce travail nous permet de proposer GSK3beta comme une cible potentielle pour le développement de traitements anti-inflammatoires dans le contexte du diabète de type 2 / Diabetes Mellitus (DM) is a chronic disabling disease with epidemic dimension. It is now established that islet inflammation is associated with defective functional beta cell mass in type 2 diabetes. The understanding of the mechanisms that govern diabetes-associated inflammation in pancreatic islets, and the identification of molecular targets to dampen inflammation are important steps to address this pathological condition. GK rat is a spontaneous model of type 2 diabetes with impaired beta cell function and mass, closely associated with islet inflammation. Glycogen Synthase Kinase 3 (GSK3) is a multi-tasking serine-threonine kinase which regulates crucial cellular functions. In recent years, GSK3beta has been found to be an important regulator of inflammation in different diseased conditions. However, the potential role of GSK3beta in the context of islet inflammation remains unexplored. In this study, we tested the potential of lithium, an inhibitor of GSK3, in improving islet inflammation and glucose metabolism in the GK rat. In vivo, treatment of young GK rats prevented the development of overt diabetes which normally occurs in adult individuals. Lithium improved the glycemic status of the GK rats after few weeks of treatment. At the end of the protocol, GK rats treated with lithium had a blood glucose levels that were significantly lower than that of age-matched untreated GK rats, which were overtly diabetic at this stage. Lithium treatment resulted in reduced expression of pro-inflammatory cytokines and chemokines, decreased fibrosis and reduced macrophage infiltration in the islets. Lithium partially restored the pancreatic insulin content, the insulin sensitivity and the glucose induced insulin secretion in the GK rats. Moreover, ex vivo studies in non-diabetic human and rat islets exposed to inflammatory environment in culture, revealed the direct implication of GSK3 in the islet autonomous inflammatory response. Moreover, we showed that GSK3 controls the islet inflammatory response at least in part by regulating the activity of the pro-inflammatory transcription factor STAT3. Taken together, our results identified GSK3 as a viable target to treat diabetes-associated inflammation, and could have potential clinical application in the treatment of diabetes and metabolic syndrome

京都大学 / 0048 / 新制・論文博士 / 博士(人間・環境学) / 乙第12951号 / 論人博第42号 / 新制||人||178(附属図書館) / 27||論人博||42(吉田南総合図書館) / 32250 / 三重大学大学院医学系研究科生命医科学専攻 / (主査)教授 石原 昭彦, 教授 神﨑 素樹, 准教授 久代 恵介, 准教授 月浦 崇, 教授 藤野 英己 / 学位規則第4条第2項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

La rétinopathie diabétique (RD) se compose d’une part d’une ischémie rétinienne périphérique et d’autre part d’une exsudation rétinienne responsable d’un œdème maculaire diabétique, première cause de cécité chez les moins 55 ans. Les traitements utilisés actuellement sont non spécifiques et traitent les complications tardives de la RD. Les phases précoces de la RD ne sont donc pas ciblées. L’hyperglycémie chronique entraine un stress oxydant et une activation des PKC qui participent à l’altération des BHR. L’objectif de ce travail a été 1°) d’étudier l’implication de la PKCζ et de la voie Rho/ROCK/Myosine II sur la physiopathogénie de la RD et 2°) de montrer l’effet bénéfique de leurs inhibiteur sur les BHR et sur la reperfusion des capillaires rétiniens. Nous avons confirmé l’hyperactivation de la PKCζ et de la voie Rho/ROCK/Myosine II chez les rats diabétiques et leur participation à la rupture de la BHR externe. Le traitement par leurs inhibiteurs respectifs normalise l’activation des deux enzymes et restaure l’intégrité anatomique et fonctionnelle de la BHR externe. De plus l'hyperactivation de ROCK altère la perfusion rétinienne par 1) constriction focale artériolaire, 2) protrusions membranaires endoluminales des cellules endothéliales (blebbing) et 3) vasoconstriction capillaire diffuse. Nous avons montré que l'ensemble de ces phénomènes étaient réversibles par traitement intravitréen de son inhibiteur le Fasudil. De manière importante le traitement par Fasudil induit également une diminution du VEGF rétinien responsable de la perméabilité des barrières et témoin indirect de l’ischémie rétinienne. Ces travaux éclairent la physiopathogénie de la RD et ouvre des perspectives thérapeutiques permettant de cibler les événements précoces de la RD. / Diabetic retinopathy (DR) mainly results from peripheral retinal ischemia and exudation leading to sight threatening complications such as retinal neovascularization or macular edema. This latter represents the main cause of visual loss among working age individuals. Current treatments address late complications of DR and are non-specific. Therefore, early events are currently not addressed. Chronic hyperglycemia increases oxidative stress and activates PKC leading to blood retinal barrier (BRB) breakdown. The aims of the present work were two fold. First, to assess the implication of PKCζ and the Rho/ROCK/Myosin II pathway on the pathogenesis of DR and second, to assess whether their specific inhibitors have the potential to restore the phenotype. Herein we have demonstrated the pathogenic role of PCKζ and ROCK hyperactivation on the development of diabetes induced external BRB breakdown. Furthermore their inhibitors restored the morphologic and functional aspect of the external BRB. We also found that ROCK hyperactivation was responsible for altered retinal perfusion through several mechanism namely 1) focal constriction of retinal arterioles; 2) endoluminal protrusions of the endothelial cell membrane (blebs) and 3) capillary diffuse vasoconstriction. We were able to demonstrate that all this aspects were reversible by Fasudil, a ROCK inhibitor, administrated into the vitreous. Of importance this treatment decreased also retinal VEGF that is a well-known factor responsible for barrier breakdown and a surrogate marker for retinal ischemia. To conclude the present findings not only shed light on the mechanisms of DR but also open new therapeutic avenues addressing the early events of DR a current unmet medical need.

<p>The timing of cell differentiation is important for development and renewal of well functioning organs and tissues. One protein involved in this process is Preadipocyte factor 1 (Pref-1). Most likely, the role of this protein is to maintain cells in an undifferentiated state. </p><p>The work presented in this thesis, has employed the rat as an animal model for the studies of Pref-1. Rat models of obesity (Zucker, ZO) and type II diabetes (Goto-Kakizaki, GK) were used to determine metabolic influence on Pref-1 and adipokine mRNA expression in adipose tissues.</p><p>The Pref-1 cleavage product was purified from rat amniotic fluid and physicochemically characterised. Concentration of Pref-1 in serum, amniotic fluid and urine was determined by ELISA. Soluble Pref-1 and the compartmentalisation of the protein were highly similar to what had previously been demonstrated in mice and humans.</p><p>Immunohistochemistry studies displayed similar staining patterns of Pref-1 in adrenal glands, ovaries and pituitary glands of non-pregnant and pregnant rats. This suggests that pregnancy do not influence the protein expression of Pref-1 in these organs.</p><p>In the GK rats, Pref-1 mRNA was altered and a decrease in the visceral compared to subcutaneous adipose depots was demonstrated, in contrast to the ZO rats. Additionally, adiponectin, leptin, IL-6 and TNF-α mRNA levels were altered in the diabetic strain, indicating that this animal model expresses many of the typical features of type II diabetes.</p><p>In conclusion, the rat is an appropriate model for studies of FA1/Pref-1/dlk. Pref-1 is highly elevated in fetal and maternal serum during pregnancy. However, the expression of Pref-1 in some endocrine tissues did not alter due to pregnancy. The mRNA expression of Pref-1 was altered between adipose depots and demonstrated to be affected by metabolic disturbances in the animals.</p>

The timing of cell differentiation is important for development and renewal of well functioning organs and tissues. One protein involved in this process is Preadipocyte factor 1 (Pref-1). Most likely, the role of this protein is to maintain cells in an undifferentiated state. The work presented in this thesis, has employed the rat as an animal model for the studies of Pref-1. Rat models of obesity (Zucker, ZO) and type II diabetes (Goto-Kakizaki, GK) were used to determine metabolic influence on Pref-1 and adipokine mRNA expression in adipose tissues. The Pref-1 cleavage product was purified from rat amniotic fluid and physicochemically characterised. Concentration of Pref-1 in serum, amniotic fluid and urine was determined by ELISA. Soluble Pref-1 and the compartmentalisation of the protein were highly similar to what had previously been demonstrated in mice and humans. Immunohistochemistry studies displayed similar staining patterns of Pref-1 in adrenal glands, ovaries and pituitary glands of non-pregnant and pregnant rats. This suggests that pregnancy do not influence the protein expression of Pref-1 in these organs. In the GK rats, Pref-1 mRNA was altered and a decrease in the visceral compared to subcutaneous adipose depots was demonstrated, in contrast to the ZO rats. Additionally, adiponectin, leptin, IL-6 and TNF-α mRNA levels were altered in the diabetic strain, indicating that this animal model expresses many of the typical features of type II diabetes. In conclusion, the rat is an appropriate model for studies of FA1/Pref-1/dlk. Pref-1 is highly elevated in fetal and maternal serum during pregnancy. However, the expression of Pref-1 in some endocrine tissues did not alter due to pregnancy. The mRNA expression of Pref-1 was altered between adipose depots and demonstrated to be affected by metabolic disturbances in the animals.