Le pancréas est une glande mixte à composantes endocrine et exocrine. Le tissu endocrine, essentiellement composé de cellules bêta productrices d’insuline, joue un rôle prépondérant dans le maintient de l’homéostasie glucidique. La perte qualitative ou quantitative des cellules bêta conduit au développement de pathologies caractérisées par une hyperglycémie chronique et connues sous le nom de diabète. Le développement de stratégies thérapeutiques innovantes, thérapie cellulaire ou médecine régénérative, pour guérir le diabète repose sur une connaissance précise des mécanismes développementaux impliqués dans la formation des cellules bêta. Ainsi, au delà de l’intérêt cognitif, il est primordial de comprendre au mieux les évènements cellulaires et moléculaires qui régissent l’organogénèse pancréatique pour offrir des thérapies alternatives. Le développement embryonnaire s’effectue dans un environnement où la pression partielle en oxygène (pO2) est faible. Par ailleurs, une étude menée au sein du laboratoire a montré que la pO2 influence la différenciation des cellules bêta pancréatique in vitro. En effet, lorsque des pancréas embryonnaires sont cultivés sur filtre en hypoxie (pO2=3%), le développement des cellules bêta est drastiquement diminué comparativement à une condition de 21% d’O2. Le facteur de transcription HIF-1 (Hypoxia Inducible Factor-1), composé d’une sous-unité alpha sensible au niveau d’oxygène et d’une sous-unité bêta constitutivement présente, permet à la cellule de s’adapter à un environnement pauvre en O2, notamment en favorisant la formation de nouveaux vaisseaux sanguins au cours d’un processus appelé angiogénèse. L’objectif de ma thèse était d’étudier le rôle de HIF-1alpha au cours du développement embryonnaire du pancréas in vivo. Pour cela, nous avons utilisé des lignées murines génétiquement modifiées permettant de stabiliser constitutivement la protéine HIF-1alpha dans l’épithélium pancréatique. En utilisant ce modèle murin, nous avons montré que la différenciation endocrine et le développement des cellules bêta est altéré dans les pancréas mutants comparativement aux contrôles. Par ailleurs, en utilisant une approche pharmacologique in vitro conduisant à l’ablation des cellules endothéliales du pancréas, nous avons pu restaurer une différenciation endocrine comparable aux contrôles. Ce travail a permis d’éclaircir le rôle de HIF-1 et de la vascularisation au cours du développement embryonnaire du pancréas. Nos résultats indiquent que ces paramètres doivent être pris en compte pour améliorer les protocoles actuels permettant de générer des cellules bêta in vitro. / The pancreas is an endoderm-derived organ which is composed by both an exocrine and an endocrine compartment. Within the endocrine tissu, insulin-producing beta-cells are essential for the regulation of glucose homeostasis. The loss of beta-cells can lead to pathologies such as diabetes. Currently, people suffuring from diabetes can be treated but not permanently cured. The development of innovating therapeutical approaches, like cellular therapy or regenerative medecine, relies on the precise knowledge of the mechanisms regulating the ontogenesis of pancreatic beta-cells. Different studies have linked proper embryonic development and low-oxygen tension (pO2). Specifically, when embryonic pancreases are cultured in vitro under a hypoxic condition (pO2=3%), the beta-cells development is impaired compared to a normoxic condition (pO2=21%). Different pathways are involved in the cell adaptation to hypoxia, such as the ubiquitous Hypoxia Inducible Factor 1-alpha (HIF-1alpha). The aim of my PhD project was to elucidate the role of HIF-1alpha during pancreatic development in vivo. To do so, we used genetically modified mice allowing the constitutive stabilization of HIF-1alpha in pancreatic epithelial cells. We have shown that HIF-1alpha stabilization leads to a reduction of endocrine differentiation and beta-cells development. Moreover, using a pharmacological approach in vitro consisting in deleting endothelial cells, we rescued the endocrine differentiation in the mutant pancreases. In conclusion, my data demonstrated the negative influence of both HIF-1 and endothelial cells on endocrine differentiation processes.

O transplante de ilhotas pancreáticas é uma terapia promissora para o tratamento da diabetes mellitus tipo 1 (DM1). No entanto, ilhotas transplantadas estão sujeitas à rejeição pelo sistema imune dos pacientes receptores, portanto faz-se necessário o desenvolvimento de mecanismos moleculares que protejam essas células. Estudos mostraram que o hormônio prolactina (PRL) é capaz de inibir a apoptose desencadeada por citocinas pró-inflamatórias sobre células beta pancreáticas e que este processo citoprotetor depende da presença da chaperona HSPB1. Foi observado que durante o desenvolvimento do DM1, as células beta pancreáticas sofrem estresse de retículo endoplasmático e que isso contribui para desencadear apoptose. O estresse de retículo endoplasmático é caracterizado pelo acúmulo de proteínas mal dobradas nessa organela resultando na ativação da resposta a proteínas mal dobradas (UPR) que tem como finalidade recuperar a homeostase celular. No presente estudo mostramos, pela primeira vez, que PRL foi capaz de proteger células beta pancreáticas contra estresse de retículo endoplasmático promovido tanto por citocinas pró-inflamatórias (TNFα, IFNγ e IL1β) quanto pelos estressores de retículo endoplasmático: tunicamicina e tapsigargina; e que HSPB1 é essencial nesse mecanismo de citoproteção. No contexto do DM1, esse hormônio parece ter um efeito modulador da UPR aumentando os níveis de BiP, antecipando a ativação de ATF6 e PERK, mantendo a via de PERK ativa por mais tempo, inibindo a via de IRE1α, e diminuindo os níveis de CHOP em tempos maiores. Coletivamente, os resultados aqui apresentados aprofundam os conhecimentos sobre a função de HSPB1, conduzindo para o desenvolvimento de estratégias que visam à atenuação da morte de células beta por meio da modulação de uma via de proteção endógena, a qual é independente da modulação do sistema imunológico. / The islet transplantation is a promising therapy for the treatment of type 1 diabetes mellitus (T1DM). However, transplanted islets are subject to rejection by the immune system of the recipient patients, therefore the development of molecular mechanisms that protect these cells is necessary. Studies have shown that the hormone prolactin (PRL) is capable of inhibiting apoptosis triggered by pro-inflammatory cytokines on pancreatic beta cells and that this cytoprotective process depends on the presence of the chaperone HSPB1. It was observed that during the development of type 1 diabetes, pancreatic beta cells undergo endoplasmic reticulum stress and that this contributes to trigger apoptosis. The endoplasmic reticulum stress is characterized by accumulation of misfolded proteins in this organelle resulting in the activation of unfolded protein response (UPR) that aims to restore cellular homeostasis. In the present study, we show for the first time that PRL was able to protect pancreatic beta cells against endoplasmic reticulum stress promoted by both pro-inflammatory cytokines (TNFα, IFNγ and IL1β) as the endoplasmic reticulum stressors: tunicamycin and thapsigargin; and HSPB1 is essential that cytoprotective mechanism. In the context of T1DM, PRL appears to have a modulating effect of the UPR by increasing the levels of BiP, anticipating the activation of ATF6 and PERK, keeping the PERK pathway active for longer, inhibiting the pathway IRE1α, and decreasing the levels of CHOP for longer times. Collectively, the results presented here deepen the knowledge of the HSPB1 function, leading to the development of strategies inducing attenuation of beta cells death through modulation of endogenous protection means, which are independent of the modulation of the immune system.

No description available.

<p>  </p> <p>Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (<em>Dhps</em>) to post-translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of <em>Dhps</em> in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein <em>Eif5a</em> is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.</p>

No description available.

O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-&#946; produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação &#946;-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação &#946;-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-&#946; através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1. / Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic &#946;-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the &#946;-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of &#946;-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of &#946;-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients.

O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-&#946; produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação &#946;-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação &#946;-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-&#946; através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1. / Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic &#946;-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the &#946;-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of &#946;-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of &#946;-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients.

Fan, Rongrong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 130-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Poon, Chui Wa Christina. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / PUBLICATIONS --- p.x / Abstracts --- p.x / ABBREVIATIONS --- p.xii / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1. --- Diabetes --- p.1 / Chapter 1.1.1. --- Overview --- p.1 / Chapter 1.1.2. --- Diagnostic Criteria of Type-2 Diabetes --- p.2 / Chapter 1.1.3. --- Type-2 Diabetes (T2DM) --- p.3 / Chapter 1.1.3.1. --- Impaired Insulin Synthesis and Insulin Secretory Defects in Type-2 Diabetes --- p.3 / Chapter 1.1.3.2. --- β-Cell Dysfunction --- p.5 / Chapter 1.1.3.3. --- Insulin Resistance --- p.5 / Chapter 1.1.4. --- Glucose Toxicity --- p.6 / Chapter 1.1.4.1. --- Fasting Hyperglycemia --- p.8 / Chapter 1.1.4.2. --- Postprandial Hyperglycemia --- p.8 / Chapter 1.2. --- Oxidative Stress --- p.8 / Chapter 1.2.1. --- ROS and Mitochondria --- p.8 / Chapter 1.2.2. --- ROS Production by Mitochondria --- p.9 / Chapter 1.2.3. --- The Relationship of Glucose Recognition by β-cells and Oxidative Stress --- p.11 / Chapter 1.2.4. --- Important Roles of Glutathione in Pancreatic β-cells and Glutathione Synthesis --- p.14 / Chapter 1.2.5. --- N-acetyl-L-cysteine - A Potential Drug Treatment for Type-2 Diabetes? --- p.17 / Chapter 1.3. --- Role of F-actin Cytoskeleton on Glucose-induced Insulin Secretion --- p.18 / Chapter 1.4. --- Current Clinical Treatments for Type-2 Diabetes Mellitus --- p.21 / Chapter 1.4.1. --- Metformin --- p.22 / Chapter 1.4.2. --- Sulfonylureas --- p.22 / Chapter 1.4.3. --- Thiazolidinediones --- p.23 / Chapter 1.4.4. --- Glinides (Meglitinide Analogues) --- p.23 / Chapter 1.4.5. --- α-Glucosidase (AG) Inhibitors --- p.24 / Chapter 1.4.6. --- Dipeptidyl Peptidase-4 (DPP-4) Inhibitors --- p.24 / Chapter 1.4.7. --- (Clinical) Antioxidant Treatment --- p.24 / Chapter 1.5. --- Animal Models Used in Type-2 Diabetes Research --- p.25 / Chapter 1.6. --- Aims of Study --- p.27 / Chapter 2. --- RESEARCH DESIGN & METHODS --- p.28 / Chapter 2.1. --- Materials --- p.28 / Table 1. Sources and concentrations of drugs tested in this study: --- p.28 / Culture Medium - --- p.29 / General Reagents --- p.29 / Chapter 2.2. --- Isolation of Islets of Langerhans and Single Pancreatic β-Cells --- p.31 / Chapter 2.3. --- Measurement of Mitochondrial ROS Levels --- p.32 / Chapter 2.4. --- Measurement of Islets Insulin Release and Insulin Content --- p.34 / Chapter 2.4.1. --- Preparation of Samples --- p.34 / Chapter 2.4.2. --- Enzyme-Link Immunosorbent Assay (ELISA) --- p.35 / Chapter 2.5. --- Immunocytochemistry --- p.35 / Chapter 2.6. --- Data and Statistical Analysis --- p.37 / Chapter 3. --- RESULTS --- p.38 / Chapter 3.1. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Releasable Insulin Levels and Insulin Contents in Response to Low Glucose (5 mM) and High Glucose (15 mM) of Isolated Pancreatic Islets of (db+/m+) and (db+/db+) Mice" --- p.38 / Chapter 3.1.1. --- Effect of L-NAC on Insulin Secretion and Insulin Contents --- p.38 / Chapter 3.1.2. --- Effect of Cytochalasin B on Insulin Secretion and Insulin Contents --- p.39 / Chapter 3.1.3. --- Effect of 4-Phenyl Butyric Acid on Insulin Secretion and Insulin Contents --- p.43 / Chapter 3.1.4. --- Effect of Ursodeoxycholic Acid on Insulin Secretion and Insulin Contents --- p.46 / Chapter 3.1.5. --- Effect of Hydrogen Peroxide on Insulin Secretion and Insulin Contents --- p.49 / Chapter 3.1.6. --- Effect of Jasplakinolide on Insulin Secretion and Insulin Contents --- p.53 / Chapter 3.1.7. --- Effect of Thapsigargin on Insulin Secretion and Insulin Contents --- p.57 / Chapter 3.1.8. --- Effect of BSO on Insulin Secretion and Insulin Contents --- p.61 / Chapter 3.2. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Mitochondrial ROS Levels in Response to High Glucose (15 mM) Challenge in Isolated Single Pancreatic β-Cells of (db +/m+) and (db +/db +) Mice" --- p.65 / Chapter 3.2.1. --- "Effects of L-NAC (20 mM), 4-Phenyl Butyric Acid (4-PBA) (1 mM), Ursodeoxycholic Acid (UA) (500 μg/ml), H202 (200 μM), Thapsigargin (0.5 μM) and DL-Buthionine-[S,R]-Sulfoximine (BSO) (0.1 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge" --- p.65 / Chapter 3.2.2. --- "Effects of L-NAC (20 mM), Cytochalasin B (10 μM) and Jasplakinolide (5 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge_" --- p.76 / Chapter 3.3. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on F-actin Cytoskeleton Levels Incubated in Low Glucose (5 mM) and High Glucose (15 mM) Medium in Single Pancreatic β-Cells of Non-Diabetic (db +/m+) and Diabetic (db +/db +) Mice" --- p.81 / Chapter 4. --- DISCUSSION --- p.100 / Chapter 4.1. --- General Discussion --- p.100 / Chapter 5. --- SUMMARY --- p.120 / Chapter 6. --- FUTURE PERSPECTIVES --- p.121 / Chapter 7. --- REFERENCES --- p.123

囊性纖維化（CF）是由囊性纖維化跨膜電導調節器（CFTR）的突變引起的一種隱性遺傳病。CF病人的肺、肝、胰腺、腸道與生殖道受到嚴重影響，其中有50%的成年病人患有糖尿病。由CF引起的糖尿病被稱為CF相關糖尿病（CFRD）, 关于它的病因至今仍然存有爭議。2007年，人們發現CFTR在分泌胰島素的胰島β細胞上有表達。儘管如此，β細胞上的CFTR与糖尿病发病的关系却一直被忽略。我們的研究目標是闡述β細胞上的CFTR在胰島素分泌中的作用。 / 在β細胞上，葡萄糖刺激的胰島素分泌伴隨著複雜的電活動，這種電活動被描述為細胞膜電位去极化疊加的動作電位的爆發。葡萄糖引起的ATP敏感的鉀離子通道（K[subscript Asubscript Tsubscript P]）的關閉被普遍認為是β細胞去極化的初始事件，初始的去極化啟動了電壓依賴的鈣離子通道，由此產生的鈣離子內流成為構成動作電位的去極化電流，引起了細胞內鈣離子的震盪，從而引起胰島素的釋放。雖然氯離子電流被認為參與了β細胞去極化電流，但是，人們仍然不能確定是哪一種氯離子通道介導了這個去極化電流。在我們研究的第一部分，CFTR被證明功能性的表達在β細胞上，並且可以被葡萄糖激活。CFTR可以被葡萄糖激活这一性质，在CFTR超表達的CHO 细胞上被進一步驗證。在原代培養的β細胞與β細胞株RIN-5F细胞中的葡萄糖引起的全細胞電流、膜電位的去極化、動作電位的幅度與頻率、鈣震盪和胰島素的分泌可以被CFTR的抑制劑或缺陷所降低。與野生型小鼠相比，CFTR基因敲除的小鼠，禁食之後，具有更高的血糖濃度，然而其胰島素的濃度低。 / 我們研究中的第二部分，利用了數學模型去闡明CFTR 在胰島素分泌的電活動中的角色。結果顯示， CFTR電導的減低可以使細胞的細胞膜去極化，從而導致需要更高的電刺激去引發動作電位，这些結果證明了CFTR對於维持細胞膜電位的貢獻。同時增加細胞內氯離子濃度和CFTR的電導可以引起更大頻率的膜電位的震盪，這一點證明了氯離子對於細胞膜電位震盪有著重要的作用。在数学模型中，CFTR電導的降低可以消除通過改變ATP/ADP值所引起的電火花， 這與我們在試驗中發現的CFTR參與了葡萄糖引起的動作電位是一致的。總而言之，我們的数学模型證明了CFTR對於胰島素的分泌是非常重要的，它通過介導氯離子外流對細胞膜電位的產生貢獻並且參與了電火花的產生，所有這些都進一步驗證了我們在實驗部分的發現。 / 综上所述，現有的研究揭示了CFTR，通過對β細胞膜電位作用與参与了動作電位的產生，在葡萄糖刺激胰島素分泌过程中的鮮為人知的重要角色。這個發現為揭示CFRD的病理機制提供了全新的視角，並且可能為開發治療CFRD的方法帶来了曙光。 / Cystic fibrosis (CF) is a recessive autosomal genetic disease resulted from mutations of cystic fibrosis transmembrane conductance regulator (CFTR). CF affects critically the lung, liver, pancreas, intestine and reproductive tract. CF patients also exhibit a high percentage of diabetes, which almost reach 50% in adult. The pathological cause of diabetes in CF patients, also called CF related diabetes (CFRD), is still controversial. It has been reported that CFTR expressed in the islet β cells, which is responsible for insulin secretion. However, the exact role of CFTR in islet β-cell and its relation to diabetes have been ignored. The present study aims to elucidate the role of CFTR in the process of insulin secretion by pancreatic islet β cells. / Glucose-stimulated insulin secretion is associated with a complex electrical activity in the pancreatic islet β-cell, which is characterized by a slow membrane depolarization superimposed with bursts of action potentials. Closing ATP-sensitive K⁺ channels (K[subscript Asubscript Tsubscript P]) in response to glucose increase is generally considered the initial event that depolarizes the β-cell membrane and activates the voltage-dependent Ca²⁺ channels, which constitutes the major depolarizing component of the bursting action potentials giving rise to the cytosolic calcium oscillations that trigger insulin release. While Cl⁻ has been implicated in an unknown depolarization current of the β-cell, the responsible Cl⁻ channel remains unidentified. In the first part of our study, we show functional expression of CFTR and its activation by glucose in the β-cell. Activation of CFTR by glucose was also demonstrated in CHO cell over-expression system. The glucose-elicited whole-cell currents, membrane depolarization, electrical bursts (both magnitude and frequency), Ca²⁺ oscillations and insulin secretion could be abolished or reduced by inhibitors/knockdown of CFTR in primary mouse β-cells or RIN-5F β-cell line, or significantly attenuated in isolated mouse islet β-cells from CFTR mutant mice compared to that of wildtype. Significantly increased blood glucose level accompanied with reduced level of insulin is found in CFTR mutant mice compared to the wildtype. The results strongly indicate a role of CFTR in the process of insulin secretion. / In the second part of our study, mathematical model is built up to clarify the role of CFTR in the electrical activity during insulin secretion. It is shown that reduction of CFTR conductance hyperpolarizes the membrane of the β-cell, for which it requires a larger electrical stimulus to evoke an action potential, indicating the contribution of CFTR to the membrane potential as demonstrated by our experimental results. Increase in intracellular Cl⁻ concentration and the conductance of CFTR result in higher frequency of membrane potential oscillations, demonstrating that Cl⁻ is crucial for the membrane potential oscillations. The electrical spikes induced by increase of ATP/ADP in the model are abolished by decreasing CFTR conductance, which is consistent with our findings that CFTR is involved in the generation of action potentials induced by glucose. In other word, our model demonstrates that CFTR is crucial for insulin secretion by its contribution to membrane potential and participating in the generation of electrical spikes via conducting Cl⁻ efflux, which confirms our findings in the experimental study. / Taken together, the present study reveals a previously unrecognized important role of CFTR in glucose-stimulated insulin secretion via contributing to the membrane potential and the participating in the generation of action potential in islet β cells. This finding sheds new light into the understanding of the pathogenesis of CFRD and may provide grounds for the development of new therapeutic approaches for CFRD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guo, Jinghui. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 156-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要： --- p.iii / Acknowledgement: --- p.v / LIST OF PUBLICATIONS --- p.vi / Declaration --- p.viii / ABBREVIATIONS --- p.xi / LIST OF FIGURES --- p.xiii / Chapter Chapter 1: --- General introduction --- p.1 / Chapter 1.1 --- The function of islet β cells and diabetes --- p.1 / Chapter 1.1.1 --- The introduction of the pancreas --- p.1 / Chapter 1.1.2. --- Glucose metabolism and blood glucose regulation --- p.6 / Chapter 1.1.2.2 --- Blood glucose regulation --- p.7 / Chapter 1.1.3 --- Insulin secretion by the islet β cell --- p.10 / Chapter 1.1.4 --- Diabetes --- p.14 / Chapter 1.2 --- Cystic fibrosis-related diabetes --- p.17 / Chapter 1.2.1 --- Cystic fibrosis --- p.17 / Chapter 1.2.2 --- CFTR --- p.19 / Chapter 1.3 --- Mathematical model for insulin secretion --- p.25 / Chapter 1.4 --- Aim and hypothesis --- p.27 / Chapter 1.4.1 --- CFTR may be activated by glucose --- p.27 / Chapter 1.4.2 --- Activation of CFTR may depolarize the membrane potential --- p.28 / Chapter 1.4.3 --- CFTR-mediating Cl-efflux may be involved in the generation of electrical spikes --- p.28 / Chapter 1.4.4 --- Calcium oscillation depends on CFTR --- p.28 / Chapter 1.4.5 --- Insulin secretion --- p.29 / Chapter 1.5 --- Approaches to test the hypothesis --- p.29 / Chapter Chapter 2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Cell culture --- p.31 / Chapter 2.1.1 --- RIN-5F cell --- p.31 / Chapter 2.1.2 --- CHO cell --- p.31 / Chapter 2.2 --- Islet isolation and culture --- p.32 / Chapter 2.3 --- CFTR knockdown --- p.33 / Chapter 2.4 --- Western blot --- p.35 / Chapter 2.5 --- Immunofluorescence --- p.37 / Chapter 2.6 --- Membrane potential (Vm) measurement --- p.38 / Chapter 2.7 --- Intracellular chloride imaging --- p.39 / Chapter 2.8 --- Intracellular calcium imaging --- p.40 / Chapter 2.9 --- Patch-clamp --- p.40 / Chapter 2.10 --- Blood glucose measurement --- p.42 / Chapter 2.11 --- Insulin ELISA --- p.42 / Chapter 2.12 --- Statistics --- p.42 / Chapter Chapter 3: --- Contribution of CFTR on the eletrophysiological properties in insulin secretion --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Results --- p.45 / Chapter 3.2.1 --- Functional expression of CFTR in mouse islet β cells --- p.45 / Chapter 3.2.2 --- CFTR activation by glucose --- p.46 / Chapter 3.2.3 --- Involvement of CFTR in the maintenance of membrane potential of islet β cells --- p.47 / Chapter 3.2.4 --- CFTR is involved in the generation of spikes induced by glucose --- p.50 / Chapter 3.2.5 --- Generation of spikes burst in the β cell depends on intracellular chloride. --- p.52 / Chapter 3.2.6 --- Inhibition/mutation of CFTR attenuates calcium oscillation induced by glucose --- p.53 / Chapter 3.2.7 --- Inhibition/mutation of CFTR impairs insulin secretion --- p.53 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4: --- Mathematical model for the role of CFTR in the process of insulin secretion in islet β cell --- p.74 / Chapter 4.1 --- Introduction to the mathematical modeling in the process of insulin secretion --- p.74 / Chapter 4.2 --- Methods --- p.77 / Chapter 4.2.1 --- Components in the model --- p.77 / Chapter 4.2.2 --- Assumptions and approaches in modeling --- p.78 / Chapter 4.2.3 --- Modeling ion channels and transporters --- p.79 / Chapter 4.2.3.1 --- KATP channel --- p.79 / Chapter 4.2.3.2 --- Sodium channel --- p.82 / Chapter 4.2.3.3 --- Voltage Dependent calcium channel --- p.83 / Chapter 4.2.3.4 --- NCX --- p.84 / Chapter 4.2.3.5 --- Na-K pump --- p.85 / Chapter 4.2.3.6 --- Kv channel --- p.87 / Chapter 4.2.3.7 --- Ca pump --- p.88 / Chapter 4.2.3.9 --- CFTR --- p.90 / Chapter 4.2.3.10 --- NKCC --- p.91 / Chapter 4.3 --- Results --- p.93 / Chapter 4.3.1 --- Role CFTR in regulation of the basal membrane potential in β cells --- p.93 / Chapter 4.3.2 --- Role of intracellular chloride concentration in the burst spikes induced by glucose --- p.95 / Chapter 4.3.3 --- Role of CFTR in the burst spikes induced by glucose --- p.96 / Chapter 4.4 --- Discussion --- p.105 / Chapter Chapter 5: --- General discussion and conclusion --- p.109 / Chapter 5.1 --- General discussion --- p.109 / Chapter 5.1.1 --- Role of CFTR in endocrine pancreas and diabetes --- p.109 / Chapter 5.1.2 --- Role of CFTR as a cell metabolic sensor --- p.111 / Chapter 5.1.3 --- Role of CFTR in excitable cells --- p.113 / Chapter 5.2 --- Conclusion --- p.114 / Appendix A --- p.115 / Appendix B --- p.118 / Reference: --- p.156