Résumé L’augmentation de l’espérance de vie chez les patients atteints de fibrose kystique (FK) entraine une augmentation de la prévalence des anomalies de tolérance au glucose, soit l’intolérance au glucose (IGT) et le diabète (DAFK). En dépit du fait, que les anomalies de la tolérance au glucose soient associées à un mauvais prognostic, l’origine de ces troubles n’est pas encore clairement établie. Notre objectif était d’examiner le rôle de l’insulinopénie et de la résistance à l’insuline dans la détérioration de la tolérance au glucose dans une cohorte prospective observationelle patients FK non diabétique. Nos résultats démontrent une réduction marquée de la phase précoce de la sécrétion de l’insuline ainsi qu’une augmentation de l’aire sous la courbe de la glycémie (AUC) chez tous les groupes de patients. Nous avons aussi démontré qu’une variation de la sensibilité à l’insuline joue un rôle prépondérant dans les changements de catégorie de tolérance au glucose dans cette population. Finalement, il semblerait que l’augmentation de l’AUC du glucose et la réduction de la phase précose de la sécrétion d’insuline sont des meileurs prédicateurs du status clinique que les catégories de tolérance au glucose. Il existe plusieurs indices pour évaluer la sécrétion d’insuline et pourtant aucun n’a été validé chez les patients FK. Nous avons examiné la validité des index de la sécrétion de l’insuline dérivés de l’hyperglycémie provoquée par voie orale (HGPO) ainsi que des valeurs à jeun par rapport au test de référence. Alors que la plupart des index calculés à partir de l’HGPO corrèlent significativement avec les valeurs du test de référence, cela n’est pas le cas pour les index calculés à partir des valeurs à jeun. La validation de ces index nous permet d’évaluer la sécrétion de l’insuline prospectivement dans notre cohorte de FK à partir de l’HGPO, est un test recommandé pour le dépistage du DAFK. Les recommandations nutritionnelles suggèrent aux patients FK une diète comprenant environ 40% de lipides afin d’éviter la malnutrition. Nous avons examiné le profil lipidique des patients FK diabétiques et non diabétiques après une HGPO et un repas hyperlipidique (RT). Nos résultats démontrent que la FK est associée à des augmentations de l’excursion glycémique lors de l’HGPO ou d’un RT comparativement aux témoins. Cependant, le RT provoque 1) une plus faible excursion glycémique comparativement à l’HGPO ; 2) une meilleure suppression de la production hépatique de glucose et 3) l’excursion lipidique postprandiale des patients FK reste normale. Il est donc peu probable que les anomalies de la sécrétion de l’insuline par les cellules  soient secondaires à une lipotoxicité. Nous avons aussi examiné les taux d’adiponectine, une hormone sécrétée par le tissu adipeux et pouvant moduler l’action de l’insuline. Les niveaux d’adiponectine corrèlent négativement avec plusieurs facteurs présents chez les patients FK incluant l’IGT, l’inflammation et une adiposité centrale en dépit d'un faible poids corporel. Les patients FK ne présentaient aucune altération des taux d’adiponectine malgré la présence d’une résistance à l’insuline, une inflammation sub-clinique et de l’IGT. La FK apparaît donc comme une condition où il existe une discordance entre les taux d’adiponectine et la résistance à l’insuline ou l’inflammation. En conclusion la prévalence de l’IGT est élevée dans cette population caractérisée par une excursion glycémique anormalement plus élevée, due principalement à une altération de la sécrétion de l’insuline exacerbée par une résistance à l’insuline. Mots clés : Adiponectine, Hyperglycémie par voie oral (HGPO), intolérance au glucose, fibrose kystique, diabète, résistance à l’insuline, sécrétion de l’insuline. / Abstract Abnormal glucose tolerance is a frequent co-morbidity in cystic fibrosis patients (CF), and is associated with a worse prognosis. However, the most pertinent factors to predict the clinical status and the physiopathology of glucose intolerance remain unclear. The aim of this study was to investigate the roles of impaired insulin secretion and insulin resistance in the progression of glucose intolerance from the normal state up to diabetes (CFRD) using an ongoing prospective observational cohort of non-diabetic CF patients. We demonstrated that CF patients displayed a reduced first phase insulin secretion across glucose tolerance categories (normal; intolerance and CFRD). Moreover, variation in insulin resistance has a significant impact on glucose tolerance in this population. Finally, early in the course of the disease, increased glucose area under the curve (AUC) and reduced first phase insulin secretion are better predictors of clinical status than conventional glucose tolerance categories. Numerous indices to evaluate insulin secretion have been proposed, but their validity has not been explored in cystic fibrosis (CF). The aim of this study was to validate surrogate indices of insulin secretion calculated from fasting values or the oral glucose tolerance test (OGTT) in CF patients against the gold standard, the intravenous glucose tolerance test (IVGTT). This is an essential step to study the physiopathological role of defective insulin secretion in the development of CFDR in large cohort. We showed that all insulin secretion indices correlated significantly with the IGVTT in control individuals. However, while OGTT-derived indices correlated significantly with the gold standard test in all CF groups, this was not the case for fasting-derived indices. Since the OGTT is required on a regular basis in CF patients to screen for CFRD, OGTT-derived indices should be used to evaluate insulin secretion. Abnormal insulin secretion combined with recommended high fat intake could be associated with dysregulation of glucose and lipid metabolisms in CF patients. Thus, the second objective was to examine postprandial glucose and lipid profiles during an OGTT and a standardized high-fat test meal (TM) in CF patients. CF patients presented higher glucose excursion compared to controls after the OGTT and TM. This excursion was significantly reduced in both amplitude and length during the TM for CF patients. In addition, control and CF patients presented similar profiles for both triglycerides and fatty acids. These results exclude lipotoxicity as a major player in ß cell defect for these patients. Circulating adiponectin levels are negatively associated with glucose intolerance, inflammation and central adiposity. Since these conditions are common in CF patients, we examined whether adiponectin values are altered in this population. CF patients did not show any changes in adiponectin levels despite insulin resistance, glucose intolerance and sub-clinical inflammation. Thus, CF appears to be one of the rare conditions in which discordance between adiponectin values and insulin resistance or inflammation is evident. In conclusion, CF patients are characterized by a high prevalence of glucose abnormalities due to an insulin secretion defect but also the contribution of insulin resistance. Key words: Adiponectin, Cystic fibrosis, diabetes, glucose intolerance, Oral glucose tolerance test, insulin resistance, insulin secretion.

Le diabète est une maladie chronique de l’homéostasie du glucose caractérisée par une hyperglycémie non contrôlée qui est le résultat d’une défaillance de la sécrétion d’insuline en combinaison ou non avec une altération de l’action de l’insuline. La surnutrition et le manque d’activité physique chez des individus qui ont des prédispositions génétiques donnent lieu à la résistance à l’insuline. Pendant cette période dite de compensation où la concentration d’acides gras plasmatiques est élevée, l’hyperinsulinémie compense pleinement pour la résistance à l’insuline des tissus cibles et la glycémie est normale. Le métabolisme du glucose par la cellule pancréatique bêta entraîne la sécrétion d’insuline. Selon le modèle classique de la sécrétion d’insuline induite par le glucose, l’augmentation du ratio ATP/ADP résultant de la glycolyse et de l’oxydation du glucose, induit la fermeture des canaux KATP-dépendant modifiant ainsi le potentiel membranaire suivi d’un influx de Ca2+. Cet influx de Ca2+ permet l’exocytose des granules de sécrétion contenant l’insuline. Plusieurs nutriments comme les acides gras sont capables de potentialiser la sécrétion d’insuline. Cependant, le modèle classique ne permet pas d’expliquer cette potentialisation de la sécrétion d’insuline par les acides gras. Pour expliquer l’effet potentialisateur des acides gras, notre laboratoire a proposé un modèle complémentaire où le malonyl-CoA dérivé du métabolisme anaplérotique du glucose inhibe la carnitine palmitoyltransférase-1, l’enzyme qui constitue l’étape limitante de l’oxydation des acides gras favorisant ainsi leur estérification et donc la formation de dérivés lipidiques signalétiques. Le modèle anaplérotique/lipidique de la sécrétion d'insuline induite par le glucose prédit que le malonyl-CoA dérivé du métabolisme du glucose inhibe la bêta-oxydation des acides gras et augmente la disponibilité des acyl-CoA ou des acides gras non-estérifiés. Les molécules lipidiques agissant comme facteurs de couplage du métabolisme des acides gras à l'exocytose d'insuline sont encore inconnus. Des travaux réalisés par notre laboratoire ont démontré qu’en augmentant la répartition des acides gras vers la bêta-oxydation, la sécrétion d’insuline induite par le glucose était réduite suggérant qu’un des dérivés de l’estérification des acides gras est important pour la potentialisation sur la sécrétion d’insuline. En effet, à des concentrations élevées de glucose, les acides gras peuvent être estérifiés d’abord en acide lysophosphatidique (LPA), en acide phosphatidique (PA) et en diacylglycérol (DAG) et subséquemment en triglycérides (TG). La présente étude a établi l’importance relative du processus d’estérification des acides gras dans la production de facteurs potentialisant la sécrétion d’insuline. Nous avions émis l’hypothèse que des molécules dérivées des processus d’estérification des acides gras (ex : l’acide lysophosphatidique (LPA) et le diacylglycerol (DAG)) agissent comme signaux métaboliques et sont responsables de la modulation de la sécrétion d’insuline en présence d’acides gras. Afin de vérifier celle-ci, nous avons modifié le niveau d’expression des enzymes clés contrôlant le processus d’estérification par des approches de biologie moléculaire afin de changer la répartition des acides gras dans la cellule bêta. L’expression des différents isoformes de la glycérol-3-phosphate acyltransférase (GPAT), qui catalyse la première étape d’estérification des acides gras a été augmenté et inhibé. Les effets de la modulation de l’expression des isoenzymes de GPAT sur les processus d’estérifications, sur la bêta-oxydation et sur la sécrétion d’insuline induite par le glucose ont été étudiés. Les différentes approches que nous avons utilisées ont changé les niveaux de DAG et de TG sans toutefois altérer la sécrétion d’insuline induite par le glucose. Ainsi, les résultats de cette étude n’ont pas associé de rôle pour l’estérification de novo des acides gras dans leur potentialisation de la sécrétion d’insuline. Cependant, l’estérification des acides gras fait partie intégrante d’un cycle de TG/acides gras avec sa contrepartie lipolytique. D’ailleurs, des études parallèles à la mienne menées par des collègues du laboratoire ont démontré un rôle pour la lipolyse et un cycle TG/acides gras dans la potentialisation de la sécrétion d’insuline par les acides gras. Parallèlement à nos études des mécanismes de la sécrétion d’insuline impliquant les acides gras, notre laboratoire s’intéresse aussi aux effets négatifs des acides gras sur la cellule bêta. La glucolipotoxicité, résultant d’une exposition chronique aux acides gras saturés en présence d’une concentration élevée de glucose, est d’un intérêt particulier vu la prépondérance de l’obésité. L’isoforme microsomal de GPAT a aussi utilisé comme outil moléculaire dans le contexte de la glucolipotoxicité afin d’étudier le rôle de la synthèse de novo de lipides complexes dans le contexte de décompensation où la fonction des cellules bêta diminue. La surexpression de l’isoforme microsomal de la GPAT, menant à l’augmentation de l’estérification des acides gras et à une diminution de la bêta-oxydation, nous permet de conclure que cette modification métabolique est instrumentale dans la glucolipotoxicité. / Diabetes is a chronic disease of glucose homeostasis characterized by hyperglycemia and the result of a failure of insulin secretion in combination or not with impaired insulin action. Overnutrition and lack of physical activity in individuals who have acquired or inherited genetic predispositions lead to insulin resistance. During the period of compensation where the concentration of plasma fatty acids is high, hyperinsulinemia fully compensates for the insulin resistance of target tissues and blood sugar is normal. Glucose promotes insulin secretion through its metabolism by the pancreatic β cell. According to the classical model of glucose-induced insulin secretion, the increase in the ATP/ADP ratio resulting from glycolysis and glucose oxidation induces the closure of KATP channels thus changing membrane potential followed by an influx of Ca2+. This influx of Ca2+ allows the exocytosis of secretory granules containing insulin. Several nutrients like fatty acids are capable of potentiating insulin secretion. However, the classical model does not explain the potentiation of insulin secretion by fatty acids. To explain the potentiating effect of fatty acids, our laboratory has proposed a complementary model in which malonyl-CoA derived from glucose anaplerotic metabolism inhibits carnitine palmitoyltransferase 1, the enzyme catalyzing the limiting step of fatty acid oxidation, thereby promoting their esterification and thus the formation signaling derivatives. The anaplerotic model of insulin secretion predicts that malonyl-CoA derived from glucose metabolism inhibits β-oxidation of fatty acids and increases the availability of acyl-CoA or non esterified fatty acids. Thus, lipid molecules can act as coupling factors for insulin exocytosis. Fatty acid-derived signalling molecules that are active remain to be identified. Work performed by our laboratory has shown that increasing the partition of fatty acids toward β-oxidation reduced glucose-induced insulin secretion, suggesting that derivatives of fatty acid esterification are important for the potentiation of insulin secretion. Indeed, at high concentrations of glucose, fatty acids are esterified into lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG) and subsequently in triglycerides (TG). The present study established the relative importance fatty acid esterification in the production of factors potentiating insulin secretion. We hypothesized that molecules derived from the process of esterification of fatty acid (eg lysophosphatidic acid (LPA) and diacylglycerol (DAG)) act as metabolic signals and are responsible for the modulation of the secretion of insulin in the presence of fatty acids. Thus, the level of expression of key enzymes controlling the process of esterification has been altered by molecular biology approaches to increase distribution of fatty acids toward esterification in the β cell. The expression of various isoforms of glycerol-3-phosphate acyltransferase (GPAT), which catalyzes the first step of esterification of fatty acids was increased and inhibited. The effects of GPAT isoenzyme modulation on the esterification process, on β-oxidation and on glucose-induced insulin secretion were investigated. The various approaches we used have changed the levels of DAG and TG without altering insulin secretion induced by glucose in the presence or absence of fatty acids. Thus, the results of this study do not suggest a role for de novo synthesis of glycerolipid intermidiates via esterification of fatty acids in the potentiation of insulin secretion. However, the esterification of fatty acids is an integral part of a TG/fatty acid cycle with its counterpart lipolysis. Moreover, parallel studies conducted by colleagues of the laboratory have demonstrated a role for lipolysis and a cycle TG/fatty acid in the potentiation of insulin secretion by fatty acids. In parallel with our studies of the mechanisms of insulin secretion involving fatty acids, our laboratory is also interested in the negative effects of fatty acids on the β cell. The glucolipotoxicity resulting from chronic exposure to saturated fatty acids in the presence of high glucose concentrations is of particular interest in the context of obesity rates. The microsomal isoform of GPAT was also used as a molecular tool under glucolipotoxicity conditions to study the role of de novo synthesis of complex lipids in the context of decompensation when β-cell function decreases. Increased esterification of fatty acids by the overexpression of microsomal isoform of GPAT has increased the toxic effects of fatty acids in the context of glucolipotoxicity. Thus, our results allow us to conclude that the distribution of lipids toward esterification and a decrease in β-oxidation is instrumental in glucolipotoxicity.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Le diabète de type 2 (DT2) apparaît lorsque la sécrétion d’insuline par les cellules β des îlots du pancréas ne parvient plus à compenser la résistance à l’insuline des organes cibles. Parmi les médicaments disponibles pour traiter le DT2, deux classes agissent en améliorant la sensibilité à l’insuline : les biguanides (metformine) et les thiazolidinediones (pioglitazone et rosiglitazone). Des études suggèrent que ces médicaments protègent également la fonction des cellules β. Dans le but d’identifier des mécanismes par lesquels les médicaments insulinosensibilisateurs protègent les cellules β, nous avons étudié les effets aigus de la metformine et de la pioglitazone sur le métabolisme et la fonction des cellules INS 832/13, sécrétrices d’insuline et des îlots pancréatiques isolés de rats. Nous avons aussi validé in vivo avec des rats Wistar les principales observations obtenues en présence de pioglitazone grâce à des clamps glucidiques et par calorimétrie indirecte. Le traitement aigu des cellules β avec de la pioglitazone ou de la metformine inhibe la sécrétion d’insuline induite par le glucose en diminuant la sensibilité des cellules au glucose (inhibition en présence de concentrations intermédiaires de glucose seulement). Dans les mêmes conditions, les traitements inhibent aussi plusieurs paramètres du métabolisme mitochondrial des nutriments et, pour la pioglitazone, du métabolisme des lipides. Les composés affectent le métabolisme en suivant un patron d’inhibition similaire à celui observé pour la sécrétion d’insuline, que nous avons nommé « décélération métabolique ». La capacité de la pioglitazone à inhiber la sécrétion d’insuline et à ralentir le métabolisme mitochondrial de façon aigüe se confirme in vivo. En conclusion, nous avons identifié la décélération métabolique de la cellule β comme nouveau mode d’action pour les médicaments insulinosensibilisateurs. La décélération métabolique causée par les agents insulinosensibilisateurs les plus utilisés semble provenir d’une inhibition du métabolisme mitochondrial et pourrait être impliquée dans les bienfaits de ceux-ci dans un contexte de stress métabolique. Le fait que les deux agents insulinosensibilisateurs étudiés agissent à la fois sur la sensibilité à l’insuline et sur la sécrétion d’insuline, les deux composantes majeures du DT2, pourrait expliquer pourquoi ils sont parmi les agents antidiabétiques les plus efficaces. La décélération métabolique est une approche thérapeutique à considérer pour le traitement du DT2 et d’autres maladies métaboliques. / Type 2 diabetes (T2D) appears when insulin secretion by pancreatic β-cells fails to compensate for insulin resistance. Two classes of anti-diabetic drugs have been used to target insulin resistance: biguanides (metformin) and thiazolidinediones (pioglitazone and rosiglitazone). Some studies suggest that these compounds also protect β-cell function. In order to identify the mechanisms whereby insulin-sensitizing agents protect β-cell function, we used INS 832/13 insulin secreting cells and isolated pancreatic rat islets to study the acute effects of pioglitazone and metformin on β-cell metabolism and function. Key observations obtained with pioglitazone were also validated in vivo in Wistar rats with the use of glucose clamps and indirect calorimetry. In vitro, acute pioglitazone or metformin treatment inhibits glucose-induced insulin secretion by lowering β-cell sensitivity to glucose (inhibition only at sub-maximal glucose concentrations). The same treatments also inhibit parameters of nutrient mitochondrial metabolism and, in the case of pioglitazone, parameters of lipid metabolism. Both compounds alter metabolism following a pattern similar to that observed with insulin secretion, a pattern that we label “metabolic deceleration”. Pioglitazone also acutely inhibits insulin secretion and slows down mitochondrial metabolism in vivo. In conclusion, we identified metabolic deceleration of the pancreatic β-cell as a new mode of action for insulin-sensitizing agents. Pioglitazone and metformin both seem to cause metabolic deceleration of the β-cell via inhibition of mitochondrial metabolism. This mode of action could participate in the beneficial effects of these compounds in the context of metabolic stress. The fact that these drugs affect both insulin sensitivity and insulin secretion, the two major components of T2D, may explain why they are among the most powerful anti-diabetic agents. Metabolic deceleration is a new therapeutic approach worth considering for the treatment of T2D and other metabolic diseases.

La greffe d’îlots pancréatiques est caractérisée par une réponse inflammatoire et procoagulante précoce, connue sous le nom d’IBMIR (Instant Blood Mediated Inflammatory Reaction). Les microparticules (MPs) porteuses de facteur tissulaire (TF) sont le témoin d’un important remodelage membranaire et constituent des acteurs centraux dans la dissémination du stress de l’IBMIR. Nous avons exploré l’effet d’un stress inflammatoire sur la survie et la fonction de la cellule β dans un modèle de communication cellulaire médiée par les MPs. La modulation pharmacologique par les analogues du GLP-1 a été évaluée, la par la mesure de la sécrétion d’insuline, de l’activité TF et l'analyse du remodelage de la membrane plasmique. Nos résultats décrivent les MPs comme des effecteurs autocrines et indiquent que les MPs sont des cibles potentielles pour les analogues du GLP-1 au cours de l'IBMIR. Les données apportent de nouvelles pistes sur les mécanismes cellulaires mis en jeu lors des phénomènes d’ischémie reperfusion durant l’IBMIR. / Pancreatic islets graft is characterized by early inflammatory and procoagulant events known as Instant Blood Mediated Inflammatory Reaction (IBMIR). Tissue factor (TF) bearing microparticles (MPs) are surrogates of important membrane remodeling and key players in the systemic and local dissemination of such stress.We investigated the effect of inflammatory stress on β cell survival and function in a MP-mediated cell crosstalk model. Pharmacological modulation by GLP-1 analogues was evaluated by measurement of insulin secretion, TF activity and assessment of plasma membrane remodeling. Our data evidenced MPs as autocrine effectors and possible new target for GLP-1 analogues. They bring new hints on the cellular mechanisms prompted by ischemia reperfusion during IBMIR.

Orientador: Carla Beatriz Collares Buzato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T15:01:37Z (GMT). No. of bitstreams: 1 Oliveira_RicardoBeltramede_M.pdf: 4343326 bytes, checksum: c45de34d36b3510fa7bea078864506dc (MD5) Previous issue date: 2011 / Resumo: Tem havido um grande interesse na determinação das vias envolvidas na proliferação e função/disfunção da célula beta e a aplicação deste conhecimento em terapias moleculares e celulares da diabetes. A patogênese da diabetes melito tipo 2 (T2DM) é complexa, mas frequentemente está associada com obesidade e distúrbios do metabolismo de lipídios (hipercolesterolemia e hipertrigliceridemia). A T2DM envolve o desenvolvimento de um quadro de resistência periférica à insulina parcialmente compensada por hiperinsulinemia e hiperplasia da célula beta pancreática, resultando em intolerância à glicose e hiperglicemia. Os mecanismos interligando os estados de obesidade/hipercolesterolemia e resistência à insulina ao fenômeno da hiperplasia da célula beta não são completamente conhecidos. A presente dissertação teve como objetivos: 1) caracterizar um modelo animal adequado para se estudar a proliferação e disfunção da célula beta pancreática, e 2) avaliar, no pâncreas endócrino desses animais, a possível ativação da via de sinalização Wnt/beta-catenina, conhecida por estar envolvida no processo de proliferação celular em outros tecidos/órgãos. Para tal, foram empregados camundongos C57BL/6, wild-type (WT) e knockout para receptor de lipoproteína LDL (LDLr-/-), os quais foram submetidos à dieta hiperlipídica (HF) por 60 dias. Após a dieta HF, os animais WT tornaram-se obesos e hipercolesterolêmicos, bem como moderadamente hiperglicêmicos, hiperinsulinêmicos, intolerantes à glicose e resistentes à insulina, caracterizando-os como pré-diabéticos. Além disso, os animais alimentados com dieta HF apresentaram uma diminuição significativa na resposta secretora das células beta à glicose. De modo geral, os animais LDLr-/- apresentaram uma susceptibilidade relativamente mais alta à dieta HF, sugerida pela acentuada hipercolesterolemia, intolerância à glicose, e reduzida secreção de insulina estimulada por glicose observadas nestes animais. No entanto, a dieta HF induziu, de forma semelhante em animais WT e LDLr-/-, uma diminuição significativa no conteúdo celular de Cx36, uma proteína associada à junção comunicante e um marcador de diferenciação terminal da célula beta. Ambos os grupos WT e LDLr-/- alimentados com dieta HF mostraram aumento na proliferação de células beta, como avaliada pela imunomarcação das ilhotas para a proteína Ki67, mas apenas os animais WT exibiram alterações morfométricas indicativas de hiperplasia do pâncreas endócrino, tais como aumento na massa total de ilhotas e de células beta. Uma vez estabelecido que camundongos WT alimentados com dieta HF por 60 dias consistiam em um modelo adequado para a segunda etapa deste estudo, fomos investigar a possível ativação da via Wnt/beta-catenina nas ilhotas pancreáticas desses animais, avaliando-se a distribuição e expressão celular das proteínas beta-catenina total, beta-catenina ativada, c-Myc e ciclina D. A análise por imunofluorescência para beta-catenina não mostrou acúmulo citoplasmático ou translocação para o núcleo desta proteína em ilhotas pancreáticas, que poderia indicar ativação da via Wnt/beta-catenina no nosso modelo de hiperplasia do pâncreas endócrino. No entanto, a análise por Western Blot revelou um aumento significativo na expressão de beta-catenina ativada e ciclina D em ilhotas de animais alimentados com dieta HF em relação ao grupo controle. Concluindo, a dieta HF por 60 dias induz alterações metabólicas típicas da pré-diabetes em animais WT e LDLr-/-. O estado de pré-diabetes está associado a uma diminuição da expressão de Cx36 nas células beta pancreáticas, sugerindo um possível papel da comunicação intercelular mediada pelas junções comunicantes na patogênese da T2DM. A maior susceptibilidade metabólica à dieta HF apresentada por camundongos LDLr-/-, em relação aos WT, pode ser explicada pela maior deficiência na secreção de insulina em resposta à glicose e ausência de hiperplasia compensatória do pâncreas endócrino. Ainda, a análise preliminar de expressão protéica de algumas proteínas da via Wnt/beta-catenina sugere que esta via parece estar ativada durante o processo de hiperplasia do pâncreas endócrino observada no nosso modelo animal / Abstract: The pathogenesis of type 2 diabetes mellitus (T2DM) is often associated with obesity and dyslipidemia (hypercholesterolemia and hypertriglyceridemia). T2DM involves intolerance to glucose and insulin resistance partially compensated by hyperinsulinemia and pancreatic beta cell hyperplasia. The mechanisms linking obesity/hypercholesterolemia and insulin resistance to beta cell hyperplasia are not fully known. The Wnt/beta-catenin signaling pathway has been reported to be involved in cell growth and differentiation in several tissues/organs but its role in endocrine pancreas development and function is still unclear. This work aimed at: 1) establishing an appropriate animal model of T2DM to study pancreatic beta cell proliferation and dysfunction and, 2) investigating a putative involvement of the Wnt/beta-catenin signaling pathway in the beta cell hyperplasia in this model. To this end, we employed C57BL/6 wild-type (WT) and LDL lipoprotein receptor knockout (LDLr-/-) mice, fed a high fat (HF) diet for 60 days. After feeding a HF diet, WT mice became obese, hypercholesterolemic and moderately hyperglycemic, hyperinsulinemic, glucose intolerant and insulin resistant, characterizing them as pre-diabetics. Moreover, animals fed a HF diet showed a significant decrease in beta-cell secretory response to glucose. In general, LDLr-/- animals showed a relatively higher susceptibility to HF diet, as suggested by a marked hypercholesterolemia, glucose intolerance and reduced insulin secretion stimulated by glucose observed in these animals as compared to the control ones. However, HF diet induced similarly in both WT and LDLr-/- mice a significant decrease in cellular content of Cx36, a gap junctional protein and marker of terminally differentiated beta cell. Both WT and LDLr-/- fed a HF diet showed increased proliferation of beta cells, as assessed by Ki67 immunostaining, but only WT mice exhibited morphometric changes indicative of endocrine pancreas hyperplasia, such as increased total islet and beta cell masses. After we investigated a possible activation of Wnt/beta-catenin signaling pathway in these hyperplasic pancreatic islets of WT animals fed a HF diet. This was done by assessing the distribution and cellular protein expression of some proteins associated to this pathway (i.e., total and activated beta-catenin, c-Myc and cyclin D) in islets of our animal model. Beta-catenin immunofluorescence showed no cytoplasmic accumulation or translocation into the nucleus of beta cells in HF-fed mice. However, immunoblotting revealed a significant increase of unphosphorylated beta-catenin (activated) and cyclin D expression in islets of HF diet-fed animals when compared to its control group. In conclusion, a HF diet for 60d induced pre-diabetes state in both WT and LDLr-/- mice. The pre-diabetes state is associated with a decreased expression of Cx36 in pancreatic beta cells, suggesting a possible role of intercellular communication mediated by gap junctions in the pathogenesis of T2DM. The relatively high metabolic susceptibility to the HF diet showed by LDLr-/- mice, as compared to WT, may be explained by a marked impairment of glucosestimulated insulin secretion and a lack of compensatory hyperplasia of the endocrine pancreas. In addition, the protein expression analysis suggests that the Wnt/beta-catenin pathway may be activated during the islet hyperplasia process in our animal model / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Indiana University-Purdue University Indianapolis (IUPUI) / In response to glucose, pancreatic islet beta cells secrete insulin in a biphasic manner, and both phases are diminished in type 2 diabetes. In beta cells, cortical F-actin beneath the plasma membrane (PM) prevents insulin granule access to the PM and glucose stimulates remodeling of this cortical F-actin to allow trafficking of insulin granules to the PM. Glucose stimulation activates the small GTPase Cdc42, which then activates p21-activated kinase 1 (PAK1); both Cdc42 and PAK1 are required for insulin secretion. In conjunction with Cdc42-PAK1 signaling, the SNARE protein Syntaxin 4 dissociates from F-actin to allow SNARE complex formation and insulin exocytosis. My central hypothesis is that, in the pancreatic beta cell, glucose signals through a Cdc42-PAK1-mediated pathway to remodel the F-actin cytoskeleton to mobilize insulin granules to SNARE docking sites at the PM to evoke glucose stimulated second phase insulin secretion. To investigate this, PAK1 was inhibited in MIN6 beta cells with IPA3 followed by live-cell imaging of F-actin remodeling using the F-actin probe, Lifeact-GFP. PAK1 inhibition prevented normal glucose-induced F-actin remodeling. PAK1 inhibition also prevented insulin granule accumulation at the PM in response to glucose. The ERK pathway was implicated, as glucose-stimulated ERK activation was decreased under PAK1-depleted conditions. Further study showed that inhibition of ERK impaired insulin secretion and cortical F-actin remodeling. One of the final steps of insulin secretion is the fusion of insulin granules with the PM which is facilitated by the SNARE proteins Syntaxin 4 on the PM and VAMP2 on the insulin granule. PAK1 activation was also found to be critical for Syntaxin 4-F-actin complex dynamics in beta cells, linking the Cdc42-PAK1 signaling pathway to SNARE-mediated exocytosis. Syntaxin 4 interacts with the F-actin severing protein Gelsolin, and in response to glucose Gelsolin dissociates from Syntaxin 4 in a calcium-dependent manner to allow Syntaxin 4 activation. Disrupting the interaction between Syntaxin 4 and Gelsolin aberrantly activates endogenous Syntaxin 4, elevating basal insulin secretion. Taken together, these results illustrate that signaling to F-actin remodeling is important for insulin secretion and that F-actin and its binding proteins can impact the final steps of insulin secretion.

<p>  </p> <p>Diabetes, a chronic condition characterized by elevated blood glucose levels, arises when pancreatic β-cells lose capacity to produce a robust, dynamic glucose-stimulated insulin secretion (GSIS) response. Accurate measurement of β-cell health and function <em>ex vivo</em> is thus fundamental to diabetes research, including studies evaluating disease mechanisms, novel drug candidates, and replacement β-cell populations. However, present-day dynamic GSIS assays typically represent end-point measurements, involve expensive commercial perifusion machines, and require time-consuming enzyme-linked immunosorbent assays (ELISA) for insulin detection. Microfluidic devices developed as accessible, low-cost alternatives still rely on secondary ELISAs and suspend islets in liquid medium, limiting their survival <em>in vitro</em>. Here, we present a novel, 3D-printed microphysiological system (MPS) designed to recreate components of <em>in-vivo</em> microenvironments through encapsulation in fibrillar type I collagen and restoration of favorable molecular transport conditions. Following computational-informed design and rapid prototyping, the MPS platform sustained collagen-encapsulated mouse islet viability and cytoarchitecture for 5 days and supported <em>in-situ</em> measurements of dynamic β-cell function. To rapidly detect insulin secretion from β-cells in the MPS, we then developed a highly sensitive electrochemical sensor for zinc (Zn2+), co-released with insulin, based on glassy carbon electrodes modified with bismuth and indium and coated with Nafion. Finally, we validated sensor detection of Zn2+ released from glucose-stimulated INS-1 β-cells and primary mouse islets, finding high correlation with insulin as measured by standard ELISA. Together, the 3D MPS and Zn2+ sensor developed in this dissertation represent novel platforms for evaluating β-cell health and function in a low-cost, user-friendly, and physiologically-relevant manner. </p>

Elevated serum levels of trimethylamine N-oxide (TMAO) were first associated with increased risk of cardiovascular disease (CVD) 10 years ago. Research has since defined that serum TMAO accumulation is controlled by the diet-microbiome-liver-kidney axis. Choline related nutrients are consumed in excess during over-nutrition from a Western diet. The resultant elevated serum TMAO is investigated across various chronic metabolic diseases and many tissue types. While TMAO is most clearly linked to CVD mechanisms in vascular tissue, its molecular effects on metabolic tissues are unclear. Here we report the current standing of TMAO research in metabolic disease context across relevant metabolic tissues including liver, kidney, brain, adipose, and muscle tissues. This review explores the variable TMAO effects in healthy and diseased conditions. Since impaired pancreatic β-cell function is a hallmark of metabolic disease pathogenesis which are largely unexplored in TMAO research, the following primary research results investigate TMAO effects on in vitro functional β-cell mass in relation to healthy and type 2 diabetes (T2D) conditions. Although we hypothesized that TMAO would aggravate functional β-cell mass, the data demonstrate that TMAO improves the T2D phenotype by increasing insulin secretion and production and reducing oxidative stress. Therefore, this work provides crucial support for the emerging context dependent molecular effects of TMAO during metabolic disease progression.

Type I diabetes (T1D) is an autoimmune disorder affecting the insulin-producing beta cells of the islets of Langerhans, disrupting the glucose homeostasis regulatory system. Nowadays, islet transplantation is one of the anticipated treatments to revive the endocrinal function by injecting isolated pancreatic islets from a deceased donor into the patient’s liver’s portal vein. Regardless of the promising aspect, the main issue prior to transplantation is the inconsistent quality and low percentage of functioning islets post-transplantation. Therefore, a rapid islet functionality test with minimal complicated operation becomes necessary to tackle the pre-transplantation issue. This project revolves around the end-to-end development of an electrophysiology recording platform to monitor extracellular activities in murine pancreatic islets. A microfluidic perfusion system with hydrodynamic trapping is integrated with planar gold microelectrode arrays (MEA) as the preliminary device directly interfacing the islets. The design and fabrication of both the microfluidics and electrode devices, as well as in-house front-end electronics with analog filters and amplifiers tailored to capture the microvolt-scale signals, covered most of the project. Offline digital processing was performed in Python to analyse the recorded signals further. As a result, the complete platform and recording setup have been fully integrated, with successful islet trapping on top of electrodes and front-end electronics with 220x voltage gain and 0.1-3000 Hz bandwidth to record extracellular electrophysiology signals from intact pancreatic islets. While the current preliminary electrophysiology recordings are still quite inconclusive and require further validation, the project serves as a starting point in developing devices for extracellular electrophysiology measurement, which has not commonly been investigated specifically in pancreatic islets, and enables further exploration in the field. / Typ I-diabetes (T1D) är en autoimmun sjukdom som påverkar de insulinproducerande betacellerna på de Langerhanska öarna och stör det reglerande systemet för glukoshomeostas. Nuförtiden är ötransplantation en av de förväntade behandlingarna för att återuppliva den endokrina funktionen genom att injicera isolerade pankreasöar från en avliden donator i patientens levers portven. Oavsett den lovande aspekten är huvudfrågan före transplantation den inkonsekventa kvaliteten och låga andelen fungerande öar efter transplantationen. Därför blir ett snabbt funktionstest av öar med minimalt komplicerad operation nödvändigt för att ta itu med problemet före transplantation. Detta projekt kretsar kring end-to-end utveckling av en elektrofysiologisk inspelningsplattform för att övervaka extracellulära aktiviteter i murina pankreatiska öar. Ett mikrofluidiskt perfusionssystem med hydrodynamisk infångning är integrerat med plana guldmikroelektrodarrayer (MEA) som den preliminära enheten som direkt gränsar till öarna. Designen och tillverkningen av både mikrofluidik och elektrodenheter, såväl som intern front-end-elektronik med analoga filter och förstärkare skräddarsydda för att fånga signalerna i mikrovoltskala, täckte större delen av projektet. Offline digital bearbetning utfördes i Python för att analysera de inspelade signalerna ytterligare. Som ett resultat har den kompletta plattformen och inspelningsuppsättningen integrerats helt, med lyckad ö-infångning ovanpå elektroder och front-end-elektronik med 220x spänningsförstärkning och 0,1-3000 Hz för att registrera extracellulära elektrofysiologiska signaler från intakta pankreatiska öar. Medan de nuvarande preliminära elektrofysiologiska inspelningarna fortfarande är ganska ofullständiga och kräver ytterligare validering, fungerar projektet som en utgångspunkt för att utveckla enheter för extracellulär elektrofysiologisk mätning, som inte vanligtvis har undersökts specifikt i pankreasöar, och möjliggör ytterligare utforskning inom området.