Human pancreatic beta cells are susceptible to infection by enteroviruses, especially Coxsackie B viruses, and such infections could contribute to the development of Type 1 diabetes. Enteroviruses gain entry via cell surface receptors, one of which, the Coxsackie and Adenovirus receptor (CAR), is a transmembrane cell adhesion protein which serves as a key entry receptor for Coxsackie B viruses and is thought to be localised mainly within regions where contacts are formed between adjacent cells. CAR exists as at least 5 isoforms and this study has examined their expression profile and distribution in the human pancreas utilising; formalin-fixed paraffin-embedded pancreatic sections from non-diabetic individuals, type 1 diabetes patients and a human tissue microarray. Isolated human islets, human pancreatic beta and ductal cell lines were also studied. Immunological and molecular approaches were employed to examine the expression and cellular localisation of the known CAR isoforms in human pancreas. One specific isoform of CAR (CAR-SIV) with a unique C terminal PDZ binding domain, was highly expressed in human beta cells at the protein level. Surprisingly, it was distributed in a punctate manner mainly within the cytoplasm of the cells, rather than at the cell surface. In human beta cells, within the cytoplasm CAR-SIV co-localised with ZnT8, PC1/3 and insulin but less so with proinsulin suggesting that CAR-SIV is associated with insulin secretory granules. Immunogold labelling and electron microscopic analysis revealed that CAR-SIV is localised both to maturing insulin secretory granules and to fully mature, dense-core (insulin) secretory granules. Intriguingly, CAR-SIV colocalises and interacts with a cytosolic protein, PICK1, which plays a role in the budding, maturation and trafficking of insulin secretory granules. On this basis, a model is proposed whereby CAR-SIV and PICK1 interact to regulate the maturation and trafficking of insulin secretory granules. Overall, this study suggests that the specialised role and subcellular localisation of CAR-SIV in human beta cells may contribute to their sensitivity to enteroviral infection following externalisation of the protein at the cell surface, during insulin exocytosis.

胰臟內分泌細胞分化的調控事件的研究揭示了胰島素分泌細胞的形成。這一原理既有利於體外誘導用於移植的胰島素分泌細胞，又可應用于糖尿病病人自體胰島素分泌細胞的再生。正在發育的組織和器官中，發現了腎素血管緊張素（RAS）成員，揭示了他們在發育過程中的潛在調控作用。另外，對 RAS 信號系統做出應答的活性氧化物質（ROS），被認為是第二信使，通過對轉錄調控因子的氧化還原的修飾促進分化。作為 ROS 的主要來源，NADPH 已被證實在各類細胞和組織中參與了祖細胞的分化。儘管如此，依賴於 NADPH 氧化酶的 ROS對于胰腺內分泌細胞分化的調控作用仍不清楚。基於這個背景，本研究致力於揭示 RAS 和 NADPH 氧化酶依賴性 ROS 在胰腺內分泌細胞分化中的作用。本實驗將在小鼠胰臟原基培養物和尿鏈黴素（STZ）誘導的新生大鼠上進行。 結果顯示，經典 RAS 成員中的血管緊張素 2 型受體（AT₂R）分佈於內分泌祖細胞的細胞核，之後穿梭定位於胰島素分泌細胞的細胞質。阻斷 AT₂R 功能抑制了Ngn3，胰島素的表達以及 β 細胞的增值。在不同的胚胎期 ROS 的水平發生了改變。對于培養的胰臟原基施加適當的外源 ROS，刺激了內分泌細胞的分化。同時，ROS 清除劑減弱了胰島細胞分化和成熟的標記基因的表達。NOX4 以及其相關的亞基 p22phox 是 NADPH 氧化酶成員，其在胰臟發育過程中的變化同 ROS 水平的變化相似，並且持續表達與內分泌細胞系統。在 NGN3 高表達的胚胎期15.5 天，它們定位于表達 NGN3 的細胞；在 NGN3 表達下調，且胰島素表達升高的胚胎期 17.5 天，它們分佈於胰島素表達細胞。而且，NADPH 氧化酶的抑製劑 DPI 削弱了胰臟培養物中的內分泌祖細胞的分化, 外源 H₂O₂ 的加入扭轉了這一現象。 / 另一方面，在 STZ 誘導的新生大鼠的研究中，DPI 負調節 β 細胞的再生。血糖失調，胰島結構毀壞以及血清胰島素匱乏的現象發生在了 DPI 處理組。另外，DPI 減弱了 NGN3 的表達而並非 Ki67, 顯示 β 細胞的分化而並非增值對於 ROS 的刺激進行了應答。在體內和體外的實驗中，DPI 也抑制了 NGN3 的轉綠調控因子 SOX9 在胰腺祖細胞中的表達。有趣的是，過表達 SOX9 可以恢復 DPI 引發的對於 NGN3 的抑制 。結合以上數據，本研究顯示 NADPH 氧化酶依賴性ROS 誘導的信號通路參與了胰腺祖細胞到胰島素分泌細胞的分化。 / Investigations into the regulatory events that modulate pancreatic endocrine cell differentiation shed light on the generation of sufficient insulin-producing cells in vitro for transplantation or regeneration of β cells in patients with diabetes. The expression of renin-angiotensin system (RAS) components has been detected in development tissue and organs, implicating their regulatory role in developmental processes. On the other hand, reactive oxygen species (ROS) are responsive to RAS signaling pathways and act as second messengers to promote differentiation through redox modification of transcriptional factors essential for differentiation. As a major source of ROS, NADPH oxidase has been shown to participate in the progenitor differentiation in a variety of cells and tissues. Despite this finding, the role of NADPH oxidase-dependent ROS in regulating pancreatic endocrine cell differentiation remains ambiguous. Against this background, the study was aimed at elucidating the roles of RAS components and NADPH oxidase-derived ROS during differentiation of pancreatic endocrine cells using mouse pancreatic rudiments and streptozotocin-treated neonatal rats. / Results showed that angiotensin II type 2 receptor (AT₂R), a major component of the classical RAS, was localized within the nuclei of endocrine progenitors in the cultured pancreatic rudiments; following the differentiation of endocrine progenitors into insulin producing cells, it translocated to cytoplasm. Blockade of AT₂R impeded the expression of Ngn3 and insulin as well as proliferation of β-cells. In addition, the dynamic changes of ROS levels were found in mouse pancreata at different embryonic days, concomitant with induction of endocrine cell differentiation induced by modest exogenous ROS in pancreatic rudiment cultures. Moreover, scavenger of ROS diminished the expression of islet cell markers for differentiation and maturation. NOX4 and its associated subunit p22phox, which are the member of NADPH oxidase, exhibited similar changes of expression to that of ROS levels during pancreas development and persisted in the endocrine lineage; they were located in NGN3⁺ cells at E15.5 during the burst of NGN3 expression and then distributed in insulin⁺ cell at E17.5, the latter being the phase that has a decline in NGN3 expression with an increase of insulin. Furthermore, administration of NADPH oxidase inhibitor, diphenylene iodonium (DPI) attenuated the differentiation of endocrine progenitors in rudiment cultures, while exogenous ROS reversed this effect. / On the other hand, studies performed in streptozotocin-induced neonatal rats showed that β cell regeneration was negatively affected by DPI treatments; consistently, impaired blood glucose control, disturbed islet architecture and deficient serum insulin were observed in DPI-treated groups. In addition, DPI treatments blunted NGN3 expression, but not Ki67-labeling beta-cells, suggesting that differentiation beyond proliferation of β-cells was accountable in response to ROS stimulation. Administration of DPI also suppressed the levels of SOX9, a transcriptional regulator of NGN3, in pancreatic progenitor cells, as evidenced by both in vivo and in vitro studies. Interestingly, over-expression of SOX9 could restore the repression of NGN3 induced by DPI. Taken all these data together, our results indicate that NADPH oxidase-dependent ROS-induced signaling pathway is involved in the differentiation of pancreatic endocrine progenitors into insulin-producing β cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Juan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 171-205). / Abstracts also in Chinese.

Ca2+ is an important mediator of stimulus-secretion coupling in beta cells of the pancreatic islets, which secrete insulin in response to elevation in plasma glucose concentration. I studied the actions of two lipids, arachidonic acid (AA) and cholesterol, on enzymatically-dissociated single beta cells of rat and mouse, using cytosolic Ca2+ ([Ca2+]i) measurement in conjunction with whole-cell patch-clamp techniques. AA, which is produced in the beta cell upon stimulation with either glucose or acetylcholine, was found to induce a large increase in [Ca2+]i that was dependent on both extracellular Ca2+ entry and intracellular Ca2+ release. Part of the AA-mediated extracellular Ca2+ entry was due to Ca2+ influx through the arachidonate-regulated Ca2+ (ARC) channels, which have not previously been reported in beta cells. The AA-mediated intracellular Ca2+ release was a result of Ca2+ mobilization from multiple inositol trisphosphate (IP3)-sensitive intracellular stores, including the endoplasmic reticulum (ER) and an acidic Ca2+ store that is probably the secretory granules. Therefore, in beta cells, the AA-mediated Ca2+ signal may amplify the [Ca2+]i rise induced by insulin secretagogues. Cholesterol is an integral component of cellular membranes and an important regulator of cellular functions. However, elevation of cholesterol level in the pancreatic islets reduces glucose-stimulated insulin secretion. I found that cholesterol overload impairs the glucose-stimulated [Ca2+]i increase in beta cells by two major mechanisms: the first is a decrease in glucose-stimulated ATP production, which is partly mediated by a decrease in glucose uptake, and the second is the reduction of voltage-gated Ca2+ current density. These effects of cholesterol may partly account for the decreased insulin secretion that develops in patients with type II diabetes, who typically exhibit hypercholesterolemia. In summary, different lipids may mediate beneficial or detrimental effects on Ca2+ regulation in rodent pancreatic beta cells.

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Orientador: Carla Beatriz Collares-Buzato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T07:00:40Z (GMT). No. of bitstreams: 1 Santos-Silva_JuniaCarolinaRebelo_M.pdf: 6726589 bytes, checksum: 09893d0d795475f8007b857273dde88e (MD5) Previous issue date: 2010 / Resumo: As junções intercelulares são especializações da membrana plasmática através das quais células dentro de um tecido podem interagir e aderirem-se umas às outras. Nas ilhotas pancreáticas, as diferentes células endócrinas se interconectam por meio das junções de oclusão, comunicante, aderente e desmossomos. Tais contatos intercelulares parecem ser cruciais para o perfeito funcionamento deste órgão, porém, pouco se sabe sobre a função fisiopatológica e composição das junções intercelulares no pâncreas endócrino. O objetivo geral desta dissertação foi estudar a importância funcional da adesão e reconhecimento celular mediados pelas junções de oclusão (JO) e de adesão (JA) nos processos de maturação e disfunção da célula beta do pâncreas endócrino, ao longo do desenvolvimento do animal e na patogênese da diabetes tipo 2, respectivamente. Nossos dados mostram que as ilhotas de fetos e recém-nascidos de ratos Wistar, cuja resposta secretora de insulina à glicose é significativamente menor, apresentam uma morfologia menos organizada, caracterizada por um formato menos definido e uma associação mais freqüente com ductos, quando comparadas às ilhotas de jovens e adultos, que são responsivas à esse secretagogo. Na ausência de contato intercelular, quando as células das ilhotas de adultos foram dispersas, a resposta secretora de insulina foi completamente inibida, porém parcialmente restabelecida quando as células foram reagregadas. Quando comparado às ilhotas de adultos, o impacto da ausência de contatos intercelulares sobre a resposta secretora de insulina à glicose foi consideravelmente menor no caso das ilhotas de ratos recém-nascidos. A imunofluorescência para NCAM e pan-caderina revelou uma distribuição diferencial destas moléculas de adesão somente nas células das ilhotas pancreáticas de jovens e adultos, o que pode estar relacionada com a citoarquitetura típica (células beta, ocupando a região central e os outros tipos celulares na periferia da ilhota) adquirida no período pré-natal. Das proteínas associadas à JA e JO estudadas, ?- e ?-cateninas e ZO-1 (mas não a ocludina) foram expressas nas células endócrinas das ilhotas de todos os grupos experimentais. De forma geral, a imunofluorescência para tais proteínas revelou uma marcação intercelular menos definida nas células beta de ilhotas de recém-nascidos e fetos em comparação com os animais jovens e adultos. Isto pode indicar uma menor interação/adesão celular, que por sua vez, pode estar relacionada com a resposta secretora deficitária de insulina das ilhotas pancreáticas verificada na fase perinatal. Como modelo de diabetes do tipo 2, utilizamos camundongos C57, machos, alimentados com dieta hiperlipídica por 8 meses desde os 21 dias de idade. Após este período em dieta, os animais tornaram-se obesos e pré-diabéticos, apresentando moderada hiperglicemia e significativa hiperinsulinemia ao final desta dieta. Não observamos diferenças morfológicas marcantes entre os grupos e nem alterações significativas na distribuição intercelular da ZO-1, ?- e ?-cateninas nas ilhotas do grupo tratado em comparação ao grupo controle. Entretanto, verificou-se uma maior marcação intercelular para Ecaderina e uma maior associação de F-actina à membrana na região de contato intercelular nas células endócrinas das ilhotas do grupo pré-diabético em relação ao controle. Em conclusão, os contatos intercelulares mediados pelas proteínas associadas à JA e à JO parecem desempenhar um papel no processo de maturação do pâncreas do endócrino durante o desenvolvimento animal e na patogênese da diabetes do tipo 2 / Abstract: Intercellular junctions are specializations of the plasma membrane that allow cells within a tissue to interact and adhere to each other. In endocrine pancreas, the different endocrine cells are interconnected by tight, gap and adherens-type junctions. Such intercellular contacts seem to be crucial for the function of this organ, however, little is known about the pathophysiological role and biochemistry of intercellular junctions in the endocrine pancreas. The aim of this thesis was to study the importance of cell-cell recognition and adhesion mediated by proteins associated to tight and adherens junctions in pancreatic islets, with emphasis on the process of insulin secretion, along the animal development and in pathogenesis of type 2 diabetes. Pancreatic islets of foetuses (F) and newborn (N) Wistar rats, that display a relatively poor insulin secretory response to glucose, present an immature morphology and a less defined cytoarchitecture when compared to islets from young (Y) and adult (A) rats, that are responsive to glucose. The immunofluorescence for N-CAM and pan-cadherin, adhesion molecules that are important in cell segregation, revealed a differential distribution of these proteins only in cells of the islets from Y and A. A lower junctional content of ?-and ?-catenins and ZO-1 in islet cells was seen in F and N in comparison with Y and A. In addition, we found that in the absence of intercellular contact, the glucose-stimulated insulin secretion was completely blocked in A islets. In contrast, the impact of the disruption of cell-cell adhesion on insulin secretory response of N islets was relatively small. As a model of type 2 diabetes, we employed male C57 mice, fed on high-fat (HF) diet for 8 months since they were 21 days old. After HF diet, these mice became obese and pre-diabetic (displaying moderate hyperglycemia and marked hyperinsulinaemia). We did not observe marked differences either in morphology either in the intercellular distribution of ZO-1, ?- and ?-catenins in islets between the HF-treated and control groups. However, there was a stronger immunoreaction for Ecadherin at the cell-cell contact site and an increased association of F-actin to the plasma membrane of islet endocrine cells from pre-diabetic mice as compared to control ones. In conclusion, the intercellular contacts mediated by adherens and tight junction and their constitutive proteins seem to play a role in the developmental maturation of the endocrine pancreas and in the pathogenesis of type 2 diabetes / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Orientador: Carla Beatriz Collares Buzato / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T09:31:49Z (GMT). No. of bitstreams: 1 Carvalho_CarolinaPradodeFranca_D.pdf: 11012408 bytes, checksum: 13da099b8bd43a93f7783c961a08d48f (MD5) Previous issue date: 2009 / Resumo: O objetivo central dessa tese foi estudar o papel das junções comunicantes (GJs) no processo de maturação da célula B e na patogênese da diabetes tipo 2. Para isso, foram utilizados dois modelos animais. No modelo in vivo de maturação da célula B e do processo secretório de insulina foram empregados ratos em vários estágios do desenvolvimento (fetal, neonatal, jovem e adulto). No modelo de diabetes tipo 2 foram utilizados camundongos C57BL/6, wild-type e knockout para o gene do receptor de LDL (LDLR -/-), alimentados com dieta hiperlipídica. No primeiro modelo de estudo, observamos, por técnicas morfológicas (imunocitoquímica) e de biologia molecular (Western Blot, RT-PCR semi-quantitativo e qPCR) que a expressão gênica e protéica de Cx36 e Cx43, se altera durante o desenvolvimento. Os dados de microinjeção intracelular de traçadores confirmam tais resultados que sugerem que ilhotas de ratos recém-nascidos apresentam menor número de canais intercelulares associados à membrana e, conseqüentemente, um menor acoplamento intercelular mediado pelas junções comunicantes em comparação às ilhotas de adultos. Esses resultados estão de acordo com a observação de que a célula B durante o período perinatal mostra uma menor resposta secretória à glicose e sugerem a participação da comunicação intercelular mediada pelas junções comunicantes no processo de maturação da célula B. No caso do modelo in vivo de diabetes tipo 2, o estudo iniciou com a caracterização do quadro metabólico e de aspectos da morfologia e morfometria do pâncreas endócrino de animais submetidos ao tratamento com a dieta hiperlipídica. Os dados obtidos demonstram que os animais submetidos a essa dieta por um curto período de tempo já apresentam alterações metabólicas típicas da fase inicial da diabetes (hiperglicemia moderada, hiperinsulinemia, intolerância á glicose e resistência à insulina), bem como modificações de parâmetros da morfometria do pâncreas endócrino. Posteriormente, foi analisada a expressão e localização celular das proteínas juncionais (Cx36, Cx43 e ZO-1) e o aspecto funcional dos canais formados pela GJ, através da microinjeção de marcadores intracelulares. Observamos que os animais tratados com dieta hiperlipídica apresentam tendência de diminuição de expressão de Cx36 acompanhada por alteração no padrão de marcação para essa proteína. Esses dados estão de acordo os de microinjeção que revelam uma pequena diminuição do grau de acoplamento celular após o tratamento com a dieta hiperlipídica. As alterações observadas para Cx36 foram acompanhadas por modificações no grau de expressão e de localização celular de ZO-1, proteína envolvida em processos como a organização dos canais intercelulares da GJ na membrana e no turnover de vários subtipos de conexina. Esses resultados indicam que a comunicação intercelular, particularmente a mediada por canais formados pela Cx36 parecem desempenhar importante papel na patogênese da diabetes tipo 2, desde sua fase inicial. / Abstract: Intercellular communication and adhesion among pancreatic B-cells are crucial for a proper insulin biosynthesis and secretion. The aim of this work was to investigate the role of gap junction (GJ) mediated-intercellular communication in the processes of maturation and dysfunction of B-cells. It is well known that fetal and neonatal rat pancreatic B-cells exhibit a reduced insulin secretory response to glucose and to other secretagogues as compared to adult ones. Based on this finding, we have used Wistar rats at different stages of development (fetal, neonatal, young and adult) as a model of B-cell maturation. As a model of type 2 diabetes, C57BL/6 mice (wild-type) and LDL receptor-deficient mice were fed a high fat diet up to 60 days. The cellular expression of GJ connexin (Cx) subtypes found in the endocrine pancreas (Cx36, Cx43 and Cx45) was assessed by immunohistochemistry, Western Blot, quantitative and semi-quantitative RTPCR. Our results indicate that young and adult endocrine pancreas express relatively high levels of Cx36 on B-cells in comparison with the other groups. Meanwhile, fetal and neonatal islets express predominantly Cx43 on non B-cells located at the islet periphery. Islet microinjection of GJ tracers revealed that neonatal B-cells display lower intercellular exchange of the cationic molecule Ethidium Bromide (EB) in comparison with the adult ones, which is in accordance with their differences in Cx36 expression. No significant differences were found in expression and localization of Cx45 among all animal groups. Regarding the animal type 2 diabetic model, a metabolic characterization showed that the high-fat diet for 60 days induces glucose intolerance, insulin resistance, hyperinsulinemia and moderate hyperglycemia in both mice groups (LDLr knockout and wild-type groups). High-fat fed mice showed a subtle decrease in Cx36 islet expression and in the B-cell intercellular exchange of EB in comparison with the chow-fed group (control). We also observed an alteration in the pattern of Cx36 labeling in immunohistochemistry assays. The high-fat diet induced a redistribution of Cx36 within the islets from a disorganized pattern (more commonly seen in control mice) to a sub domain-pattern where Cx36 plaques demarcate groups of B-cells. These changes in Cx36 islet distribution observed in high-fat fed mice were accompanied by a similar pattern of immunolabeling for ZO-1, but an increase in its islet expression, a tight junctional protein that seem to be involved in Cx turnover and membrane organization of Cx-made channels. Taken all together, these findings suggest that cell-cell coupling mediated by GJ may play an important role in the pancreatic B-cell maturation observed during endocrine pancreas development as well as in the early stages of type 2 diabetes pathogenesis. / Doutorado / Histologia / Mestre em Biologia Celular e Estrutural

Orientador: Carla Beatriz Collares Buzato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:09:15Z (GMT). No. of bitstreams: 1 Maschio_DanielaAparecida_M.pdf: 3472469 bytes, checksum: c4faacd3683560adfb7bc1ddb9464f73 (MD5) Previous issue date: 2014 / Resumo: Tem havido um grande interesse na determinação das vias envolvidas na proliferação das células beta pancreáticas e a aplicação deste conhecimento em terapias moleculares e celulares da diabetes. Em especial, a via de sinalização da Wnt/beta-catenina (ou via Wnt canônica) tem sido pouco investigada no pâncreas endócrino. Em determinados tecidos/órgãos, é sabido que a proteína beta-catenina constitui não somente um componente estrutural das junções de adesão, mas também é uma molécula sinalizadora juntamente com a Wnt, participando de vários processos celulares, tais como diferenciação e proliferação. Hiperplasia da célula beta parece ocorrer em certas condições experimentais e in vivo, como no estado de resistência periférica à insulina. Entretanto, as vias intracelulares envolvidas nesse processo ainda permanecem desconhecidas. Os objetivos desta Dissertação de Mestrado foram: 1) verificar se as alterações metabólicas induzidas pela exposição à dieta hiperlipídica (DHL) por um curto período de tempo (60 dias) são acompanhadas por alterações morfométricas compensatórias do pâncreas endócrino de camundongos C57BL/6; 2) investigar o possível envolvimento da via de sinalização da Wnt/beta-catenina no processo de proliferação da célula beta neste modelo, analisando-se a localização celular (por imunofluorescência indireta), o conteúdo proteico (por immunoblotting) e a expressão gênica (por PCR de Tempo Real ou qPCR) de proteínas associadas à via Wnt/beta-catenina (a saber, beta-catenina, Ciclina D1/2, c-Myc, GSK-3? e Axina2 e, 3) analisar a expressão da proteína beta-catenina não fosforilada (forma ativa da via) em ilhotas não hiperplásicas de animais tratados com a DHL por apenas 30 dias. Nossos resultados demonstraram que, após 60 dias de tratamento com DHL, os animais se tornaram obesos e apresentaram acentuadas alterações metabólicas, tais como hiperglicemia em jejum e pós prandial, hiperinsulinemia em jejum e pós-prandial e, ainda, uma significativa resistência periférica à insulina (administrada intraperitonealmente), sendo esses animais, portanto, caracterizados como pré-diabéticos. Este quadro foi acompanhado por um aumento significativo da massa relativa de células beta e do seu número por ilhota, o que indica hiperplasia deste tipo celular no pâncreas endócrino, provavelmente compensatória ao quadro de resistência periférica à insulina apresentado pelos animais do grupo tratado. Como mostrado por immunoblotting, houve um aumento significativo na expressão de proteínas ativadoras ou alvo da via, como beta-catenina ativada (não fosforilada) e Ciclina D1/2 em ilhotas dos animais pré-diabéticos. Quanto às proteínas Axina2 e GSK-3? (inibidoras da via), não foi observada alteração significativa na expressão de Axina2, mas supreendemente houve aumento do conteúdo celular de GSK-3? nas ilhotas do grupo pré-diabético. A imunofluorescência para beta-catenina ativada mostrou a presença desta proteína tanto na região de contato intercelular como no citoplasma e núcleo das células beta para ambos os grupos. As outras três proteínas relacionadas à via, Ciclina D1/2, GSK-3? e Axina2, por sua vez, apresentaram uma distribuição exclusivamente citoplasmática nas células endócrinas das ilhotas pancreáticas, o que está de acordo com as suas respectivas funções. A análise por qPCR não revelou alteração significativa no conteúdo celular de RNAm de ?-catenina, mas uma tendência de aumento na expressão gênica de Ciclina D1 e c-Myc, genes alvo da via, em ilhotas hiperplásicas dos animais pré-diabéticos. Ainda, por immunoblotting para beta-catenina ativada, não observamos aumento significativo da expressão proteica dessa proteína em ilhotas do grupo tratado com DHL por apenas 30 dias, os quais não desenvolveram hiperplasia do pâncreas endócrino. Em conclusão, nossos dados sugerem que a via Wnt/beta-catenina parece estar ativada na pré-diabetes experimental, e provavelmente participa do processo de hiperplasia compensatória das células beta pancreática neste estado metabólico / Abstract: The role of the Wnt/beta-catenin signaling pathway (as known as the canonical Wnt pathway) in the endocrine pancreas physiology has not been thoroughly explored. In certain tissues/organs, it is known that beta-catenin, besides being a structural component of adhesion junctions, participates as a key protein in the Wnt signaling pathway, therefore being involved in crucial cellular processes such as differentiation and proliferation. Beta cell hyperplasia appears to occur under certain experimental conditions, and in vivo during the peripheral insulin resistance state. However, the intracellular pathways involved in this process are still unknown. The objectives of this Master's Dissertation were as follows: 1) to investigate whether the metabolic changes induced by exposure of adult male C57BL/6 mice to a high-fat diet (HFD) for a relatively short period of time (30 or 60 days) are accompanied by compensatory morphometric changes of the endocrine pancreas indicative of beta cell hyperplasia; and 2) to study the possible involvement of the pathway Wnt/?-catenin signaling in the process of beta cell proliferation in this animal model. For this, we carried out the analysis of the cellular localization (by indirect immunofluorescence), the protein cell content (by immunoblotting) and the gene expression (by PCR or Real-Time qPCR) of proteins associated to the Wnt/beta-catenin pathway (i.e., ?-catenin, Cyclin D1/2, c-Myc, Axin2 and GSK-3?). Our results showed that, after 60 days of treatment with HFD, the animals became obese, as well as, hyperglycemic, hyperinsulinemic (both at fast and fed states) and resistant to intraperitoneally injected insulin, being therefore characterized as prediabetic. This metabolic condition was accompanied by a significant increase in the relative mass of beta cells and the number of beta cells per islet, which indicates hyperplasia of this pancreatic endocrine cell, probably compensatory to the relatively higher insulin demand presented by the HFD-treated animals. As shown by immunoblotting, there was a significant increase in islet expression of the activator and target proteins, such as the active (unphosphorylated) beta-catenin and Cyclin D1/2 in prediabetic animals. Regarding Axina2 and GSK- 3? proteins (antagonists of the pathway), no changes were observed concerning Axin2 islet content between the experimental groups, but surprisingly there was a significant increase in cellular content of GSK-3? in islet homogenates from the prediabetic group. The immunofluorescence for active beta-catenin showed the presence of this protein at the intercellular contact region as well as within the cytoplasm and nucleus of beta cells in both groups. Meanwhile, Cyclin D1/2, GSK-3? and Axin2 displayed an exclusively cytoplasmic distribution in pancreatic endocrine cells, which is in accordance with their respective functions. The qPCR analysis revealed no significant change in mRNA expression of ?-catenin, but a tendency of increase in gene expression of Cyclin D1 and c-Myc, target genes of the pathway, in hyperplastic islets of the prediabetic animals. Additionally, the immunoblotting of active beta-catenin in homogenates of non-hyperplastic islets, isolated from mice fed a HFD for only 30 days, showed no significant change in expression this protein as compared to the control group. In conclusion, our data suggest that the Wnt/beta-catenin pathway may be activated during the process of compensatory hyperplasia of the beta cells seen in our animal model of obesity-associated prediabetes / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Orientadores: Henrique Marques Barbosa de Souza, Carla Beatriz Collares Buzato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T12:54:45Z (GMT). No. of bitstreams: 1 Hernandes_LeticiaHelenaPinto_M.pdf: 22200442 bytes, checksum: 95e81ba6b7934db5b7e01d5eea75202c (MD5) Previous issue date: 2014 / Resumo: O Diabetes Mellitus tipo 2 (T2DM) representa uma das principais doenças do mundo moderno, e é caracterizado pelo comprometimento da homeostase glicêmica do organismo, resultante de uma combinação da redução da sensibilidade periférica à insulina e o comprometimento da função das células-'beta'. Estudos recentes sugerem que Coup-TFII possui um papel importante na homeostase glicêmica e no metabolismo energético. Apesar destes trabalhos representarem fortes indícios, ainda é desconhecido se Coup-TFII desempenharia este papel modulador da homeostase glicêmica e do metabolismo energético em indivíduos com T2DM. O objetivo deste trabalho foi investigar o padrão de expressão de Coup-TFII em camundongos apresentando um quadro pré-diabético. Para isto, foram utilizados camundongos C57BL/6 tipo selvagem tratados com dieta hiperlipídica (dHL) por 60 dias (animais dHL). A caracterização metabólica revelou que estes animais apresentaram características de quadro pré-diabético, i.e., sobrepeso, resistência periférica à insulina associada à moderada hiperglicemia, significativa hiperinsulinemia (resultante da hiperplasia compensatória de células-'beta') e distúrbios na secreção de insulina, tanto no estado basal (2,8 mM de glicose), quanto em condição estimulatória (16,8 mM de glicose). A análise da expressão de Coup-TFII por qPCR revelou uma redução dos níveis transcricionais deste gene em ilhotas dos animais dHL. Estes resultados indicam que a relação negativa previamente demonstrada entre hiperglicemia, hiperinsulinemia e reduzida secreção de insulina e a expressão de Coup-TFII também ocorre no quadro pré-diabético desenvolvido pelos animais dHL. Interessantemente, análise do conteúdo proteico de Coup-TFII revelou um aumento significativo de 33,6% nos animais dHL, resultado que contrasta com a redução da transcrição deste gene nas células-'beta' destes animais. Uma possível explicação seria pelo papel fundamental que Coup-TFII exerce no processo de proliferação das células-'beta' dependente da via Wnt/'beta'-catenina, observada durante o desenvolvimento e potencialmente atuando na hiperplasia compensatória nos animais dHL. Além disso, trabalhos recentes demonstraram que Coup-TFII atua como um regulador negativo de sua própria transcrição em células-'beta'. Portanto, com 60 dias de tratamento o quadro de hiperplasia nos animais dHL demandaria maiores níveis da proteína de Coup-TFII em comparação com os animais Ctr. Uma vez elevado, este nível proteico de Coup-TFII, juntamente com os efeitos das alterações metabólicas, teriam um efeito negativo sobre a transcrição de Coup-TFII nos animais dHL / Abstract: Type 2 diabetes mellitus (T2DM) is one of the most important diseases in the modern world, and is characterized by the compromise of the glycemic homeostasis and a result of a combination of insulin resistance and impaired 'beta'-cell function. Recent studies show that nuclear receptor COUP- TFII (Chicken ovalbumim upstream promoter transcription factor II) plays an important role in regulating glycemic homeostasis and energy metabolism. Although these results are a strong hint, it is still unknown whether Coup-TFII would have a role modulating glycemic homeostasis and energy metabolism in individuals suffering from T2DM. The aim of this work was to investigate the expression pattern of the transcription factor Coup-TFII in mice displaying pre-diabetic condition. To this aim, C57BL/6, wildtype mice were treated with high fat diet (dHL, from the Portuguese dieta hiper lipídida) for 60 days (dHL mice). Metabolic characterization confirmed that dHL mice display characteristics of pre-diabetic condition, i.e., overweigh, insulin resistance associated with moderate hyperglycemia, hyperinsulinemia (as a result of hyperplasia of 'beta'-cells) and impaired insulin secretion both, in basal and high blood glucose concentrations. Gene expression analysis for Coup-TFII by qPCR revealed a significant reduction of its transcripts in pancreatic islet of dHL animals. These results indicate that the previously described negative relationship between hyperglycemia, hyperinsulinemia and imparted insulin secretion and Coup-TFII expression is also happening in the pre-diabetic condition developed by dHL animals. Interestingly, analysis of the protein expression of Coup-TFII reveled a significant increase of 33,6% in dHL animals, a result that the contrasts with the reduction of Coup-TFII transcripts observed in these animals. One possible explanation is the essential role that Coup-TFII plays in the Wnt/'beta'-catenin-dependent 'beta'-cell proliferation, in addition to the negative feedback loop observed in 'beta'-cells for Coup-TFII on its own expression. By 60 days of treatment, 'beta'-cell proliferation during hyperplasia in dHL animals would require levels of Coup-TFII protein higher then in control animals. Once increased, this higher protein level, in addition to the metabolic alterations, would have a negative effect on Coup-TFII transcription in dHL animals / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

La prévalence de l’obésité et du diabète de type 1 (DT1) s’accroit dans le monde à une vitesse alarmante. L’augmentation du poids corporel, de la quantité d’acides gras libres (AGL) circulant et de la résistance à l’insuline peut induire un stress du réticulum endoplasmique (RE) dans les cellules beta du pancréas, ce qui pourrait favoriser l’inflammation. Afin de tester cette hypothèse, nous avons exposé des cellules beta à un léger stress chronique du RE induit par de l’acide ciclopiazonique (ACP) ou l’AGL palmitate et les avons ensuite traitées avec une faible dose d’interleukine 1β (IL-1β) ou de facteur de nécrose tumorale (TNF-α). L’addition d’IL- 1β, mais pas de TNF-α, a conduit à l’augmentation de la production de marqueurs pro-inflammatoires et de chimiokines. Cette différence de résultat en fonction de l’exposition à l’IL-1β ou au TNF-α peut-être au moins partiellement expliquée par un usage différentiel du complexe de la kinase IκB (IKK) et de la voie du facteur nucléaire κ-B (NF-κB), menant à terme à une activation plus élevée par l’IL-1β en comparaison à TNF-α. L’analyse des branches de la réponse de la protéine dépliée impliquées a révélé que la voie de l’Inositol-requiring enzyme 1 (IRE1) / X-box binding protein 1 spliced (XBP1s) est le médiateur clé dans le couplage avec la réponse inflammatoire déclenchée par l’IL-1β. En effet, le knockdown d’IRE1 ou de XBP1 a permis d’éviter l’exacerbation de l’activité du promoteur NF-κB et l’expression des gènes cibles de NF-κB. Les mécanismes impliqués dans la régulation XBP1-dépendante de la réponse pro-inflammatoire sont partiellement dépendant de la modulation de la Forkhead box O1 (FoxO1). Le stress du RE et l’IL-1β participent aussi à un autre évènement crucial dans le développement du DT1, à savoir la mort progressive des cellules beta, qui est également exacerbée par la combinaison ACP + IL-1β. Contrairement à l’inflammation, la voie IRE1/XBP1 n’est pas impliquée dans l’apoptose cellulaire induite par la combinaison d’ACP et d’IL-1β. Dans ce contexte, nous suggérons que le devenir de la cellule est décidé par la balance entre les protéines Bcl-2 anti-apoptotiques et pro-apoptotiques. Ainsi, nous avons montré que le gène A1 associé à Bcl-2 (A1) est négativement régulé par le stress du RE tandis que l’IL-1β active la protéine BH3-only Bim, aboutissant à une apoptose accrue des cellules beta. En conclusion, nos données suggèrent que le stress du RE est un facteur sensibilisation pour l’induction de l’inflammation des ilots pancréatiques et de l’apoptose des cellules beta. Ceci pourrait fournir une explication mécanistique à l’augmentation parallèle observée de l’obésité infantile et de la fréquence du DT1.\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished