Dissertations / Theses on the topic 'Islands of Langerhans'

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006.
Shuker, Suzanne, Committee Chair ; Doyle, Donald, Committee Member ; Orville, Allen, Committee Member ; Barry, Bridgette, Committee Member ; McCarty, Nael, Committee Member.

La regulación y potenciación de la proliferación de la célula β pancreática es un objetivo principal en la prevención y/o retardo de la diabetes. Su replicación se regula a través de un proceso dinámico, siendo muy elevada en el estado embrionario hasta el nacimiento y disminuyendo gradualmente durante la etapa posnatal, manteniéndose en niveles muy bajos durante la edad adulta. Las vías de señalización que regulan este descenso son poco conocidas. Este estudio pretende establecer el papel de las vías Gsα-dependientes que actúan a través del segundo mensajero AMPc en la regulación de la masa celular β durante la etapa postnatal temprana. Para esta tesis hemos generado ratones deficientes para Gsα en célula β (β-GsαKO). En primer lugar hemos realizado una caracterización fenotípica de este modelo animal tanto a nivel de la homeostasis de la glucosa como a nivel del compartimento β pancreático, mostrando una clara intolerancia la glucosa junto con una reducción en la masa celular β así como en los niveles de proliferación des de una etapa postnatal temprana. Por otro lado el estudio de los determinantes moleculares responsables de las alteraciones observadas en el establecimiento de la masa celular β, pone en manifiesto un defecto claro en la señalización Gs-AMPc/PKA así como en la señalización de la insulina/igf1. El defecto en la vía de la insulina se observa tanto a nivel de ligando como a nivel de receptor con una alteración en la distribución de las isoformas así como en la propia activación de la vía para estimular la proliferación de la célula β en respuesta a insulina. De hecho, hemos visto una relación sinérgica dependiente entre las dos vías de señalización Gs-AMPc/PKA y insulina en la potenciación de la proliferación en respuesta a diferentes mitogenos. Esta tesis revela la importancia de la señalización mediada por AMPc en el establecimiento de la masa de célula β durante la etapa posnatal temprana. Además, demuestra de forma preliminar la existencia de una conexión entre el AMPc y la señalización intracelular mediada por la insulina en la célula β.

Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of cell surface-supported thin films that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Specifically, the process of layer-by-layer (LbL) polymer self assembly was employed to generate nanothin films of diverse architecture with tunable properties directly on the extracellular surface of individual islets. Importantly, these studies are the first to report in vivo survival and function of nanoencapsulated cells, and have helped establish a conceptual framework for translating the diverse applications of LbL films to cellular interfaces. Additionally, through proper design of film constituents, coatings displaying ligands and bioorthogonally reactive handles may be generated, providing a modular strategy for incorporating exogenously derived regulators of host responses alongside native constituents of the islet surface. Towards this end, a strategy was developed to tether thrombomodulin to the islet surface in a site-specific manner, thereby facilitating local generation of the powerful anti-inflammatory agent, activated protein C. Collectively, this work offers novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond.

El trasplante de islotes ha sido reconocido como una prometedora opción de tratamiento de la diabetes tipo 1 (DT1) tras la introducción del protocolo de Edmonton, el cual enfatiza el requerimiento de un adecuado número de islotes donantes así como el uso de regímenes de inmunosupresores libres de esteroides. Además es un procedimiento quirúrgico menos invasivo y que conlleva menos complicaciones comparado con el trasplante de páncreas. A pesar de los importantes avances establecidos por el protocolo de Edmonton, el uso clínico del trasplante de islotes para el tratamiento de pacientes DT1 continúa siendo limitado, debido en gran parte a los retos post-trasplante. Tras el trasplante, los islotes son separados de su red vascular nativa, por lo que la funcionalidad y supervivencia del injerto dependerá del restablecimiento de nuevos vasos en el injerto para derivar el flujo sanguíneo al sistema vascular del huésped. Sin embargo, los estudios han demostrado que el grado de revascularización de los islotes trasplantados es considerablemente menor que el de la microvasculatura nativa de los islotes pancreáticos, incluso a los 9 meses del injerto . Ésta retrasada y insuficiente revascularización priva los nuevos islotes injertados de oxígeno y nutrientes, pudiendo provocar su muerte celular y el fallo temprano del injerto. En el estudio realizado, identificamos, por primera vez, la proteína tirosina fosfatasa 1B (PTP-1B) como una diana terapéutica, capaz de mejorar la revascularización de los injerto pancreáticos sin comprometer la masa ß-celular del injerto y la principal mediadora de la acción del tratamiento del tungstato sódico en la revascularización de islotes. Además hemos identificado el mecanismo por lo cual la PTP-1B induce la revascularización. Nuestros datos apuntan que en la ausencia de PTP-1B, los islotes pancreáticos expresan y secretan el factor de crecimiento del endotelio vascular A (VEGFA), una citoquina pro-angiogénica, mediante la activación de la PGC1 alpha y ERR-alpha de forma independiente de hipoxia. Finalmente hemos comprobado que éste mecanismo de inducción de VEGFA se conserva en islotes humanos. De ésta forma concluimos que PTP-1B es una diana prometedora en el desarrollo de nuevas terapias para la mejora de la revascularización de injertos de islotes.
Islet transplantation is considered a potentially curative treatment for type 1 diabetes, Despite the key important advances achieved by the establishment of the Edmonton protocol, islet transplantation remains clinically limited due to several challenges, which lead to massive islet loss or failure of the grafts. Therefore searching for new targets to facilitate islet revascularization may lead to improved future results in cell transplantation.Islets native architecture is characterized by a dense vessel network that, delivers oxygen, hormones, glucose, and nutrients to islet’s cells allowing them to function correctly. After transplantation, the survival and function of islet grafts must depend on the reestablishment of new vessels within the grafts to derive blood flow from the host vascular system. This vascular network is severed when islets are isolated for transplantation, and even though islets freely revascularize, they do not reach the levels of vascularization present in endogenous pancreatic islets, which results in the impairment of grafts function and survival. Altogether, the lack of a proper vascular network account as the primary responsible for early graft loss. Although the molecular mechanisms underlying islet revascularization remain elusive, a number of factors have been implicated, such as the vascular endothelial growth factor A (VEGFA), a key angiogenic molecule that acts to stimulate new vessel formation. VEGFA expression in transplanted islets is significantly impaired, which is further pronounced in prevailing hyperglycemia, and coincides with delayed and insufficient islet revascularization in diabetic mice In this thesis we identify for the first time, tyrosine phosphatase PTP-1B as a target for improving graft revascularization. We targeted PTP-1B, either by its inhibition, following a sodium tungstate treatment after transplantation, or by transplanting islets lacking PTP-1B, using a genetic model of PTP-1B knock-out, or following genetic silencing, using siRNA and shRNA Lentivirus particles. Following transplantation into the anterior chamber of the eye in diabetic mice, islet-grafts showed increased revascularization by inducing the expression of VEGF-A by ß-cells in the graft. This improved revascularization was followed by an improvement of islet-graft survival and function, as transplanted mice recovered normoglycemia and glucose tolerance. Furthermore, we demonstrated that PTP-1B induces VEGF-A expression and secretion in islets by upregulating HIF1A-independent PGC1α/ERRα signaling. Finally, we demonstrated that this regulatory mechanism is conserved in human islets. Together, these findings unravel the potential role of PTP-1B as a target for improving islet transplantation outcomes.

Orientador: Everardo Magalhães Carneiro
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Suspeita-se que a desnutrição intra-uterina e pós-natal produzam mudanças morfológicas e funcionais no pâncreas endócrino e em tecidos periféricos, que se traduzem em insulinopenia e resistência à insulina. Baseado nessas complicações avaliou-se neste trabalho a regulação da secreção de insulina em ilhotas de Langerhans de ratos e camundongos submetidos à restrição protéica e suplementados com leucina. A suplementação com leucina não modificou hábitos alimentares, ingestão hídrica e o peso corpóreo dos animais em estudo, mas alterou parâmetros bioquímicos importantes como glicose (G), ácidos graxos (AGL) em animais desnutridos. A fosforilação do receptor de insulina (IR) e de seu substrato (IRS-l) foi modificada em fígado e músculo levando a uma melhoria na homeostasia glicêmica em ratos desnutridos, a metabolismo de glicose em ilhotas de ratos controle e desnutridos teve redução após suplementação, a potencial de membrana das células B foi restaurado, o movimento de cálcio citoplasmático e a secreção de insulina estimulada por G e leucina apresentaram aumentados em ilhotas de ratos e camundongos desnutridos. Alterações ocorreram também no perfil eletroforético de proteínas citoplasmáticas após suplementação com leucina em ilhotas de ratos. Expressão gênica e protéica de proteínas chaves na cascata de sinalização de insulina como IR, IRS-l, PI3K, mTaR e S6K-l se alteraram em resposta à suplementação com leucina em ilhotas de ratos desnutridos, favorecendo vias de crescimento, em especial o aumento da PI3K a qual resultou em aumento de sua atividade em ilhotas de ratos controle e desnutridos. Por fim, podemos concluir que a suplementação com leucina promoveu modulação da sensibilidade periférica em fígado e músculo de maneira tecido específica. Isto confere uma regulação da homeostase glicêmica de maneira distinta entre animais controle e desnutridos suplementados. Além disso, direcionam para que os sinais metabólicos produzidos pela leucina devam promover seus efeitos, em ilhotas de Langerhans, via dois sensores: a GDH que controla a glutaminólise, e por outro lado está a PI3K que deve exercer seu papel, ativando vis envolvidas com a síntese protéica através da mTOR. Estes dois sensores devem atuar sinergicamente participando do rearranjo da concentração citosólica dos íons cálcio, principalmente em ilhotas de animais desnutridos que foram suplementados com leucina
Abstract: We think that intra-uterine mal nutrition and after birth produce morphological and changes in endocrine pancreas and in peripheric tissue that translate insulinopenia and insulin resistance. Based on these complications we have evaluated in this work the insulin secretion regulation in Langerhans islets from rats and mice fed a low protein diet and supplemented with leucine. The leucine supplementation hasn't changed the diet in the hybrid ingest and body weight but has changed important biochemistry standards as glucose (G) and FFA in malnutrition animals. The insulin receptor phosphorilation and its substract have been changed in liver and musc1e leading to an increase in glicemic homeostase in malnutrition rats. The glucose metabolism in control and malnutrition rats' islets had one reduction after supplementation. B cells potential membranes were restored; the citoplasmatic ca1cium movement and insulin secretion were stimulated by glucose and leucine. They have showed an increased in islets of control and malnourished rats and mice islets. Some alterations have also occurred in the citoplasmatic protein eletrophoretic profile after leucine supplementation in rats islets. The genetic and protein expression from key enzymes in the insulin cascade signalization as: IR; IRS-l; PI3K; mTOR and S6K-l have altered in leucine supplementation answer in malnutrition islets rats favoring growth pathways specially PI3K increase which resulted in an increasement of control and malnutrition rats islets activity. In the end we can conc1ude that the leucine supplementation has promoted peripheric sensibilization in liver and muscle in specific tissue manner. This confirms one glicemic homeostase regulation in distinct manner among control and malnutrition both supplemented animals. Furthermore they lead the metabolic signals produced by leucine should promote their effects in Langerhans islets throw sensor ways: GDH which controls glutaminolisis and on the otherhand is PI3K that should do its role activating growth pathways throw mTOR. These two sensors should work in synergism participating in citosolic concentration changes of ca1cium ions mainly in malnutrition animals' islets, which were supplemented.
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

Orientador: Antonio Carlos Boschero
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Em animais, a desnutrição intra-uterina exerce efeitos marcantes sobre o desenvolvimento fetal e pós-natal. Sabe-se que animais desnutridos apresentam níveis elevados de ácidos graxos plasmáticos e esses, por sua vez, são responsáveis por alterar a secreção de insulina. Neste trabalho, verificamos a expressão do fator de transcrição PDX-1, da p38/SAPK2, o metabolismo da glicose e a secreção de insulina em ilhotas de ratos mantidos durante o período fetal e da lactação com uma dieta normoprotéica (17% de proteína) ou hipoprotéica (6% de proteína). Cultivamos as ilhotas por 48 horas em meio de cultura contendo 5.6 mM/L de glicose, na ausência ou presença de0.6 mM/L de ácido palmítico. A secreção de insulina em ilhotas isoladas em resposta 16,7 mmol/L de glicose foi reduzida em ratos desnutridos, no entanto, quando na presença de ácido graxo, observou-se um aumento. Em 2.8 mmol glicose/L,houve diminuição do metabolismo da glicose em ilhotas de desnutridos .Entretanto, quando estimuladas com 16.7 mmol/L de glicose, tanto as ilhotas de desnutridos como as do controle, apresentaram acentuada redução na oxidação da glicose, na presença de ácido graxo. Os níveis de mRNA do PDX-1 e da insulina aumentaram significativamente quando na presença de ácido graxo em ambos os grupos. O efeito do ácido palmítico sobre a expressão protéica de PDX-1 e da p38/SAPK2 apresentou-se similar em ambos os grupos, mas o aumento foi muito mais evidente em ilhotas de desnutridos. Esses resultados demonstram a complexa relação entre nutrientes no controle da secreção de insulina e mostram queos ácidos graxos desempenham um papel importante na homeostasia da glicose, por afetar mecanismos moleculares e as vias de acoplamentsecreção de insulina
Abstract: A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of free fatty acids. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of B-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein diet, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/SAPK2 protein expression, in islets from young rats fed low (6%; LP) or normal (17%; C) protein diets and maintained for 48 h in culture medium containing 5.6 mmol glucose/L with or without 0.6 mmol palmitic acid/L. We also measured glucoseinduced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16.7 mmol glucose/L was reduced in LP compared to C rats. In the presence of free fatty acids, there was an increase in insulin secretion in both groups At 2.8 mmol glucose/L, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16.7 mmol glucose/L, LP and C islets showed a severe reduction in glucose oxidation in the presence of free fatty acid. The PDX-1 and insulin mRNA were significantly higher when free fatty acid was added to the culture medium in both groups of islets.The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and C islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

Orientador: Jose Roberto Bosqueiro
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Introdução e objetivos: O aumento nos níveis de glicose circulante é o principal estímulo para a secreção de insulina. A insulina se liga a receptores de membrana desencadeando diversas respostas celulares. Qualquer alteração na sensibilidade à insulina pode levar a disfunções fisiológicas como a resistência à insulina observada em pacientes diabéticos tipo 2 (T2DM). Experimentalmente, essa condição patológica pode ser mimetizada pela administração de altas doses de glicocorticóides, provendo assim um bom modelo para seu estudo. O objetivo do presente trabalho foi avaliar a plasticidade das ilhotas pancreáticas submetidas à variação na necessidade secretória de insulina por indução de resistência periférica ao hormônio por tratamento com dexametasona e posterior interrupção do tratamento. Métodos: Ratos wistar com 90 dias de vida foram tratados com dexametasona (1mg/kg, ip) por 5 dias consecutivos (DEX). Em outro grupo (DEX10), os animais foram tratados da mesma maneira e avaliados 10 dias após o último dia da administração de dexametasona. Ratos controle (CTL) receberam administração de NaCl 0,9% apenas. As ilhotas foram isoladas pelo método da colagenase. A expressão de proteínas foi feita por immunoblotting. As análises morfométricas foram realizadas microscopicamente. Resultados: O grupo DEX exibiu marcante resistência periférica à insulina, que foi revertida após o período de 10 dias no grupo DEX10. As ilhotas do grupo DEX apresentaram alterações funcionais e morfológicas como aumento da secreção de insulina estimulada por secretagogos, da área, da densidade e tendência de aumento na massa de células ß ao contrário do grupo DEX10. O conteúdo de proteínas relacionadas ao ciclo celular como a CD2 e CDK4 e a fosforilação da AKT aumentou em ilhotas do grupo DEX, mas retornou aos níveis do CTL em ilhotas DEX10. Conclusão: Estes resultados mostram a plasticidade do pâncreas endócrino haja vista a habilidade de se adaptar a situações que exigem maior ou menor demanda de insulina
Abstract: Introduction and aims: Insulin binds to plasma membrane receptors leading to a variety of cellular responses. Malfunction in any of the insulin cell signalling pathways in target tissues may lead to several conditions and diseases, like hyperglycemia, insulin resistance and type 2 diabetes mellitus (T2DM). These effects may be experimentally reproduced using high doses of glucocorticoids, providing thus a good model for the study of T2DM. The aims of this study were to evaluate the plasticity of pancreatic islets subject to variation on the need for insulin secretory induction of peripheral resistance to the hormone by treatment with dexamethasone and subsequent treatment interruption. Methods: Male wistar rats (90 days old) were treated with dexamethasone (1mg/kg, ip) for 5 consecutive days (DEX). In another group (DEX10), the animals were treated in the same way and assessed 10 days after the last day of administration. Control rats (CTL) received equivalent volume of vehicle. Protein expression was assayed trough immunoblotting. Morphometric analyses were done using a optical microscope and specific digital analysis programs. Results: DEX group showed marked peripheral insulin resistance, reverted after the recovering period in the DEX10 group. DEX islets showed functional and morphological changes, like increased insulin secretion, superficial area, population density, and a tendency for increase in the total mass content of beta cell. Cell cycle proteins CD2 and CDK4 and AKT phosphorylation were increased in the DEX group when compared to CTL group. All these effects were reverted in the group DEX10. Conclusions: These results show that the endocrine pancreas possess a plasticity regarding the capacity of pancreatic islets to adapt themselves to situations where a higher or lower demand for insulin is needed.
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular

This project investigates the role of chemokine and chemokine receptors in a model of CD4 T cell dependent cellular xenograft rejection, specifically the transplantation of fetal pig pancreas tissue to the renal subcapsular space of mice. Chemokines and chemokine receptor gene expression was assessed by cDNA arrays, and confirmed by multi-probe ribonuclease protection assay. Immunostaining for a selected chemokine, RANTES was performed to demonstrate upregulation at the protein level. These methods were applied to several different models to dissect the role Chemokines and their receptors in this process. Comparisons were made with: an allografi model, a model where indefinite xenograft survival could be achieved by short term costimulatory blockade with CTLA4-Fc and MR1, and an immunodeficient mouse recipient (RAG—1 KO, lacks B and T cells) that was reconstituted with either unfractionated leucocytes or purified CD4 T cells. The main findings were: 1. Allograft rejection and cellular xenografi rejection are THl type CD4 T cell dependent processes as shown by the common T cell chemokine genes (Ltn, IP-lO, and Mig) expressed in both models; however macrophages are the main effector cell in cellular xenografi rejection as evidenced by the selective upregulation of MCP-l and its receptor CCR2, as well as other macrophage markers 2. Of the Chemokines / receptors upregulated in this model of cellular xenograft rejection (Ltn, IP-lO, MCP-l, RANTES, MIP-lB, eotaxin) only MCP-l and IP-lO are CD4 T cell dependent, while Ltn expression is dependent upon a non-CD4 T cell leucocyte subset. 3. CTLA4-Fc and MR1 therapy resulted in indefinite fetal porcine islet survival and function in diabetic immune competent wild type C57BL/6 mice. This treatment suppresses the early upregulation of chemokines and chemokine receptors seen in untreated animals, and this corresponds with a significant reduction CD4 T cell and macrophage grafi infiltration at these time points, consistent with a role for select chemokine / receptors in the mechanism by which this therapy leads to indefinite graft survival. 4. In addition we studied the functioning of fetal porcine islet tissue in diabetic mice and found they developed and controlled glucose metabolism in a piglike manner, and different to normal mice, and thus conclude the development and function of fetal tissue in cross species transplantation is dependent upon the origins of the progenitor cells and not the xenogeneic environment i.e. nature not nurture (in this case anyway). We conclude that select chemokines and their receptors are important factors in the recruitment of effector cells mediating graft rejection in this model of cellular xenograft rejection and these chemokine pathways and networks may represent potential future therapeutic targets.

Diabetes mellitus is a heterogeneous group of metabolic diseases characterized by impaired blood glucose homeostasis that affects more than 415 million people worldwide and is a leading cause of mortality. The most prevalent form of diabetes is Type 2 Diabetes (T2D) that accounts for 90% of diabetes cases. An interplay of environmental and genetic risk factors contributes to etiology of T2D via a progressive loss of pancreatic beta cell function coupled with insulin resistance. Genome Wide Association Studies (GWAS) identified more than 400 independent genetic loci associated with T2D risk, although the molecular mechanisms underlying these genetic signals remain poorly understood. A comprehensive understanding of gene regulation in human pancreatic islets and identifying the role of T2D risk variants on different components of gene regulation will enlighten our insights into T2D etiology. In this work, we performed an in-depth characterization of human pancreatic islets transcriptional regulatory elements, attaining a greater granularity at transcriptional enhancers. We further identified glucose responsive enhancers which regulate glucose-dependent gene expression programs via three-dimensional chromatin interactions. This allowed us to gain insights into human islet transcriptional gene regulation and how glucose, a primary physiological stimulant of pancreatic islets, modulates human islet genome function. We also generated comprehensive transcriptome annotations in human islets using short- and long-read sequencing data along with accurate maps of transcriptional start sites. This revealed islet-specific promoters, transcript isoforms and novel coding sequences. This underscored the importance of generating transcript models in disease relevant tissue to progress in the understanding of gene regulation. Finally, these parallel efforts allowed us to create pioneer maps of genetic effects on human alternative splicing that revealed for the first time the noteworthy contribution of human islet mRNA splicing to T2D pathophysiology. These results have thus the potential to blossom in the discovery of novel T2D drug targets.

Type 1 diabetes affects one in every 400-600 children and adolescents in the US. Standard therapy with exogenous insulin is burdensome, associated with a significant risk of dangerous hypoglycemia, and only partially efficacious in preventing the long term complications of diabetes. Pancreatic islet transplantation has emerged as a promising therapy for type 1 diabetes. However, this cell-based therapy is significantly limited by inadequate islet supply (more than one donor pancreas is needed per recipient), instant blood-mediated inflammatory reaction, and loss of islet viability/function during isolation and following implantation. In particular, inadequate revascularization of transplanted islets results in reduced islet viability, function, and engraftment. Delivery of pro-vascularization factors has been shown to improve vascularization and islet function, but these strategies are hindered by insufficient and/or complex release pharmacokinetics and inadequate delivery matrices as well as technical and safety considerations. We hypothesized that controlled presentation of angiogenic cues within a bioartificial matrix could enhance the vascularization, viability, and function of transplanted islets. The primary objective of this dissertation was to enhance allogenic islet engraftment, survival and function by utilizing synthetic hydrogels as engineered delivery matrices. Polyethylene glycol (PEG)-maleimide hydrogels presenting cell adhesive motifs and vascular endothelial growth factor (VEGF) were designed to support islet activities and promote vascularization in vivo. We analyzed the material properties and cyto-compatibility of these engineered materials, islet engraftment in a transplantation model, and glycemic control in diabetic subjects. The rationale for this project is to establish novel biomaterial strategies for islet delivery that support islet viability and function via the induction of local vascularization.

Orientador: Everardo Magalhães Carneiro
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Neste estudo, investigamos os efeitos da suplementação com taurina (TAU; 2% adicionada à água de beber) sobre a tolerância à glicose e a secreção de insulina frente a diferentes secretagogos em camundongos adultos. Camundongos suplementados apresentaram aumento da tolerância à glicose e da sensibilidade à insulina. Ilhotas isoladas destes animais secretaram mais insulina em resposta à glicose e L-leucina. A oxidação da L-leucina foi maior no grupo TAU, não havendo diferenças quanto ao consumo de glicose, concentrações de ATP e expressão do transportador da glicose (GLUT) 2 e da glicoquinase (GCK). A captação de Ca2+, na presença de glicose, e a expressão protéica da subunidade ß2 do canal de Ca2+ sensível à voltagem foi maior no grupo TAU comparado ao controle (CTL). Ainda, a expressão protéica da PL (fosfolipase) C ß 2 e da PK (proteína quinase) Aa, bem como a secreção de insulina em resposta a agentes potencializadores tais como carbacol (Cch) e IBMX, foi maior nas ilhotas TAU. A mobilização intracelular de Ca2+ induzida por Cch foi também maior em ilhotas deste grupo, e observamos que a inibição da PKA reduziu a captação de Ca2+ em resposta à glicose no grupo suplementado. Além disso, ilhotas TAU secretaram mais glucagon em relação a ilhotas CTL, quando em presença de baixa concentração de glicose. Concluindo, a suplementação com TAU melhora a homeostase glicêmica e aumenta a secreção de insulina de ilhotas isoladas e incubadas na presença de nutrientes e agentes potencializadores da secreção. Os efeitos sobre a secreção estão relacionados ao melhor manejo dos íons Ca2+ pelas células insulares provenientes dos animais suplementados com TAU.
Abstract: In this study, we investigated the effects of taurine (TAU)-supplementation (2% in the drinking water) on glucose tolerance and insulin secretion stimulated by different secretagogues in adult mice. TAU-supplemented mice showed enhanced glucose tolerance and insulin sensitivity when compared to controls (CTL). In addition, their islets secreted more insulin in response to high concentrations of glucose and L-leucine. L-[U-14C]leucine oxidation was higher in TAU islets compared with CTL islets, whereas D-[U-14C]glucose oxidation, ATP levels, and the protein expression of the glucose transporter (GLUT) 2 and of glucokinase (GCK) were similar. 45Ca uptake induced by high glucose concentrations was increased in TAU islets as well as the expression of the ß2 subunit of the L-type Ca2+ channel. In addition, the insulin secretion induced by carbachol (Cch) and IBMX, but not, by forskolin and PMA was higher in TAU-supplemented compared with CTL islets. The higher insulin secretion in the presence of Cch is accompanied by an increase in the expression of PL (phospholipase) C ß 2 protein and a higher intracellular Ca2+ mobilization. Besides, TAU-supplemented islets showed increased PK (protein kinase) Aa expression. Since the increase in Ca2+ uptake induced by glucose in TAU islets was minimized by the presence of the PKA inhibitor, H89, this kinase seems to be important for the better Ca2+ handling in these islets. TAUsupplementation also turns the a-cells more sensitivity since these cells secreted more glucagons compared with CTL islets. In conclusion, TAU supplementation enhances glucose tolerance and insulin sensitivity in mice and turns the islets more sensitive to nutrients and to potentiators of secretion. The effect on insulin secretion seems to be linked to a better Ca2+ handling by ß-cells.
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

This thesis examined (1) the effect of liver transplantation on tolerance induction to islet allografts; (2) the effects of gut hormones (CCK, GLP-1) on fetal pig islets. Liver allografts in tolerant strain combinations are unique in their ability to induce tolerance to other organ and skin grafts from the same donor strain in the absence of immunosuppression. Whether liver transplantation has the same effect on islet allografts is unknown. Studies in this thesis demonstrate that the protective effect of liver transplantation on islet allografts varies with the time of liver grafting. Islet allografts in the PVG —> DA combination were rapidly rejected. Rejection was delayed, but not prevented, when islets were transplanted simultaneously with the liver. Liver transplantation protected subsequently transplanted islet allografts from rejection and reversed ongoing rejection in previously placed islet grafts, in both cases leading to the tolerance of the islet allografts. There was a progressive increase of cell infiltration from day 2 to day 7 in both rejecting and tolerant islet allografts. The intensity of infiltration did not relate to the outcome of grafts. Islet rejection was characterised by an early dominance of monocytes/macrophages and CD25+ T cells in the infiltrate, a high incidence of apoptotic B cells in grafts, and a sensitised status in the MLR. Tolerance of islet allografts was associated with increased numbers of dendritic cells in the graft infiltrates, upregulation of FasL and prominent apoptosis of alloreactive leukocytes in the spleen and islet grafts, as well as donor-specific suppression in long—term survivors. It is suggested that islet allograft tolerance induced by liver transplantation is the result of an active immune regulation process which involves the deletion of donor-specific alloreactive lymphocytes by apoptosis. Transplantation of fetal pig pancreatic islets is capable of reversing diabetes in rodents, but several months are required to achieve this. During this time fetal [3 cells proliferate, differentiate and mature in their ability to secrete insulin when challenged with glucose. The study in Chapter 6 showed that CCK, GLP-l can induce the maturation and differentiation of fetal pig [5 cells during culture of fetal pig islet cell clusters (lCCs) in vitro. Two months after the ICCs were transplanted beneath kidney capsule of SCID mice, perfusion of the graft showed that ICCs previously exposed to CCK, GLP-1 for 4 days secreted insulin in response to glucose, whereas the control grafts remained glucose unresponsive. It is suggested that exposure of fetal pig lCCs to CCK, GLP-1 is likely to be advantageous in enhancing their ability to normalise blood levels when transplanted into diabetic recipients.

La disminución de la masa de célula ß pancreática y la función en la diabetes tipo 2 (T2D) se puede atribuir a una serie de factores estresantes experimentados por el islote durante el desarrollo y la progresión de la enfermedad, incluyendo el amiloide de los islotes. El amiloide de los islotes pancreáticos se compone predominantemente del péptido humano amilina, también conocido como polipeptido amiloide del islote humano (hIAPP). Estos depósitos están implicados en el proceso de deterioro de las células ß y en la reducción de la masa celular ß e implica la agregación de monómeros solubles normales del péptido de la célula ß en oligomeros, fibrillas y, en última instancia, en depósitos de amiloide maduros. El mecanismo que subyace a la formación y agregación de hIAPP no está del todo claro. Sin embargo, varios estudios demostraron que el aumento de la síntesis de hIAPP dentro de la célula ß pancreática y su cambio conformacional a la lámina de estructura ß podría ser factores importantes para los depósitos de amiloide. El estrés del Retículo Endoplasmático (RE) ha sido implicado en la T2D relacionada con la obesidad, contribuyendo tanto a la resistencia a la insulina y como a la disfunción de las células ß del páncreas. La acumulación de proteínas mal plegadas puede perturbar el plegamiento de las proteínas en el RE y conducir a una alteración en la homeostasis del RE. Como consecuencia, la respuesta al estrés de RE se activa con el fin de restablecer el equilibrio entre la capacidad de plegamiento de proteínas y la carga de proteínas. En consecuencia, las cascadas sucesivas de transducción de señales se activan y se denominan colectivamente como la respuesta de la proteína desplegada (unfolding protein response, UPR). Este mecanismo incluye la atenuación de traslación de síntesis de proteínas, la degradación de proteínas asociadas al RE y la inducción de las chaperonas moleculares, un grupo de proteínas esenciales que ayudan al plegamiento de las proteínas recién sintetizadas, incluyendo la insulina y IAPP en la célula ß pancreática. También se sabe que muchas enfermedades neurodegenerativas están asociadas con la acumulación de proteínas mal plegadas en el RE que conducen al estrés de RE y a la pérdida progresiva de células. Dado que esta es una característica común observada también en T2D, la acumulación de agregados de la proteína IAPP puede ser responsable por la inducción o progresión del estrés de RE y en última instancia, el responsable por la inflamación de los islotes y su muerte. La célula ß pancreática se enfrenta al reto de aumentar la síntesis de proteínas durante una estimulación aguda o crónica. Esto provoca una carga en el RE, el orgánulo donde la síntesis y plegamiento de insulina y IAPP tienen lugar. Los factores plegables denominados chaperonas se unen a las proteínas secretoras desplegadas y las ayudan al plegamiento y agregación. Cada vez más pruebas sugieren que las chaperonas juegan un efecto protector importante en la disminución de la agregación de proteínas y en la fisiopatología de la deposición de amiloide. Además, la sobreexpresión de algunas chaperonas del RE puede proteger la célula contra la muerte celular causada por alteraciones de la homeostasis del RE. De interés, los ratones transgénicos que sobreexpresan la chaperona molecular BiP en células ß están protegidos contra la patogénesis de la T2D inducida por la obesidad, manteniendo la función de la célula ß y mejorando la homeostasis de la glucosa. Con la sobreexpresión de BiP también se ha demostrado que atenúa el estrés de RE inducido por ácidos grasos, asi como, la apoptosis en los hepatocitos. Además, BiP es una de las chaperonas responsables para el tráfico del IAPP a través del RE y aparato de Golgi en la célula ß pancreática humana. Del mismo modo, se ha verificado que la menor disponibilidad de la chaperona endógena, proteína disulfuro isomerasa (PDI), contribuye a los efectos tóxicos de la agregación de proteínas en la deficiencia de a1-antitripsina, lo que sugiere que ambos, BiP y PDI pueden tener potencial interés en el plegamiento y agregación de IAPP. Además, los últimos informes indican los efectos beneficiosos de determinados compuestos químicos (chaperonas farmacológicas) en el estrés de RE y en aliviar y mejorar el plegamiento de proteínas.El 4-Fenil butírico (PBA) y la taurina conjugado de ácido ursodesoxicólico (TUDCA) fueron capaces de aliviar el estrés de RE en células y animales. Aunque el papel del hIAPP en el desarrollo de la diabetes no está bien establecido, se ha sugerido que IAPP contribuye al aumento del estrés de la célula ß. Nuestro grupo ha demostrado que la agregación extracelular de hIAPP deteriora la vía ubiquitina-proteasoma y que además está implicada en el estrés de RE mediada por apoptosis de la célula ß pancreática. Las células INS1E, una línea de células ß de páncreas de rata, que expresan establemente hIAPP (hIAPP-INS1E), mostraron oligómeros intracelulares y una fuerte alteración de la insulina estimulada por glucosa así como en la secreción de IAPP. Además, la sobreexpresión de hIAPP en ratones y ratas transgénicas desencadena la apoptosis inducida por el estrés de RE en las células ß. En particular, los ratones y ratas transgénicos que expresan IAPP humano específicamente en células ß pancreáticas tienen los marcadores de estrés RE elevados (spliced X-box-binding protein-1; sXBP1, CCAAT/enhancer binding-protein homologous protein; CHOP, active caspase-12, y la acumulación de proteínas ubiquitinadas). La susceptibilidad de las células ß pancreáticas al estrés de RE y a la apoptosis inducida por IAPP nos llevó a explorar potenciales terapias para modular la respuesta de las células ß a factores de estrés de RE inducida por hIAPP, así como en aumentar sus mecanismos de protección con el fin de prevenir la apoptosis, recuperar la función y la disminución de la formación de los depósitos de amiloide en las células ß. En esta tesis, se pretende mejorar el conocimiento sobre el estrés de RE inducido por hIAPP y la posible mejoría por el tratamiento con chaperonas. Nuestra hipótesis es que hIAPP potencia estrés ER, y el tratamiento con chaperonas en modelos de células ß pancreáticas puede resultar en la mejora del estrés de RE inducido por hIAPP y en última instancia disminuir los depósitos de amiloide en los islotes pancreáticos. Estos hallazgos podrían ofrecer nuevas terapias para evitar la pérdida de la masa de células ß asociado con la formación de amiloide en la T2D. En el presente trabajo, hemos establecido inicialmente un modelo de sobreexpresión de hIAPP. Para este propósito, una línea de célula ß pancreática de rata, INS1E, fue transfectada de forma estable con hIAPP (células hIAPP-INS1E), rIAPP (células rIAPP-INS1E) o un vector vacío (células de control INS1E). El primer objetivo del trabajo fue conocer si la sobreexpresión de hIAPP se asocia con alteraciones en los niveles de marcadores de estrés de RE. Nuestro grupo observó previamente que estos clones estables en condiciones normales no mostraron cambios en la expresión de genes implicados en el estrés de RE, así como en la muerte celular. En este contexto, nuestros clones estables fueron expuestos a un inductor químico de estrés llamado tapsigargina. Dosis-respuesta y tiempo de experimentos determinaron las mejores condiciones para inducir un estrés de RE marcada sin la muerte celular excesiva. A continuación, para investigar el efecto de inductores fisiológicos de estrés de RE, las células se cultivaron en alta glucosa y palmitato (HG + PA). Los resultados obtenidos indicaron que la sobreexpresión de hIAPP activa la vía UPR, haciendo que estas células sean más sensibles al estrés que las células rIAPP-INS1E o las células control INS1E. Estos efectos están mediados a través de la regulación positiva de genes implicados en la defensa de las células ß contra el estrés de RE, incluyendo CHOP, ATF3 y sXBP1. Además, se investigó el papel de CHOP en el estrés de RE en respuesta a agentes inductores del estrés de RE, como la thapsigargin y HG + PA. La inhibición de la expresión de siRNA CHOP mostró una disminución en el marcador de apoptosis caspasa-3, como se ejemplifica en los experimentos de inmunohistoquímica. Hemos investigado posteriormente si las chaperonas endógenas y químicas eran capaces de recuperar el estrés de RE y mejorar la secreción de insulina en las células hIAPP-INS1E. Para este propósito, hemos sobreexpressado las chaperonas moleculares BiP y PDI por transducción adenoviral o tratando las células con las chaperonas químicas TUDCA y PBA. Se observó que después de la exposición a HG + PA, las células hIAPP-INS1E tratadas con las chaperonas (BiP, PDI, TUDCA o PBA) mostraron una disminución en los niveles de los marcadores de las proteínas de estrés, ATF3 y p-eIF2a. Además, TUDCA y PBA fueron capaces de reducir el estrés de RE potenciado por hIAPP y tapsigargina. Consecutivamente, hemos investigado el efecto de las chaperonas en la funcionalidad de las células hIAPP-INS1E. Para ello, las células hIAPP-INS1E se dejaron sin tratar o han sido tratadas con Ad-BiP, Ad-PDI y Ad-GFP (como control), TUDCA y PBA y se ha monitoreado su efecto sobre la secreción de insulina estimulada por glucosa. Los resultados realizados en condiciones basales de glucosa mostraron que BiP, TUDCA y PBA fueron capaces de restaurar y mejorar la secreción de insulina cuando comparadas con las células GFP o INS1E control. Curiosamente, la sobreexpresión de PDI parece tener un efecto perjudicial sobre la liberación de insulina, debido a una reducción en el contenido de insulina después de la exposición a la glucosa. A continuación y con el fin de abordar el papel de las chaperonas en condiciones de estrés fisiológicos, las células hIAPP-INS1E fueron expuestos a HG + PA en presencia de las chaperonas. Tal y como esperábamos, HG + PA causó una reducción en la secreción de insulina en las células que expresan hIAPP no transducidas en comparación con las células no tratadas. Con el tratamiento de chaperonas (BIP, PDI, TUDCA y PBA), se observó un re-establecimiento de los niveles de secreción de insulina de una manera similar que las células hIAPP-INS1E no tratados con HG+PA. Para confirmar estos resultados se midió el contenido de insulina de las células hIAPP-INS1E estimulados a 2.8 mM y 16 mM de glucosa, y se observó que los niveles de insulina fueron similares en todos los grupos. El tercer objetivo de este trabajo fue evaluar la efectividad del tratamiento en los islotes pancreáticos de ratones. Para este propósito, hemos sobreexpressado BiP y PDI en los islotes de tipo salvaje (WT) de ratón y en los islotes no-transducidos (CON) a través de una transducción adenoviral y hemos monitoreado la secreción de insulina en condiciones basales de 11 mM de glucosa. Descubrimos que la sobreexpresión de BiP aumenta la secreción de insulina en comparación con Ad-GFP y con los islotes control estimulados con 16 mM de glucosa. Además, la sobreexpresión de PDI no mostró cambios en la secreción de insulina. A continuación, se trataron islotes con las chaperonas químicas TUDCA y PBA. Hemos observado que PBA mejoró significativamente la secreción de insulina estimulada por glucosa en comparación con los islotes controles. Al contrario, el tratamiento con TUDCA no mostró diferencias en la secreción de insulina. Al mismo tiempo, hemos utilizado islotes de ratón transgénicos que sobreexpresan hIAPP (hIAPP Tg) y examinamos los efectos de las chaperonas en la secreción de insulina. La sobreexpresión de BiP aumentó los niveles de secreción de insulina a 16 mM de glucosa en comparación con islotes tratados con Ad-GFP. Se observaron efectos similares en el tratamiento con PBA. En cambio, con la sobreexpresión de PDI y el tratamiento con TUDCA no se observaron alteraciones en la secreción de insulina después de la estimulación con glucosa a pesar de los niveles en el contenido de insulina continuaran siendo similares en todos los grupos. Los mismos experimentos se realizaron en condiciones de HG+PA. Los resultados obtenidos mostraron que BiP y PDI aumentaron significativamente la secreción de insulina estimulada por glucosa cuando comparados con los islotes hIAPP Tg controles que se han cultivado a con HG+PA. Del mismo modo, con el tratamiento con TUDCA y PBA se ha restaurado los niveles de secreción de insulina, alcanzando los valores obtenidos con los islotes control Por último, se examinó si la disminución en el estrés de RE mejora la función de la célula ß y si está correlacionada con una disminución en la formación de amiloide. Después de 7 días de cultivo a 16 mM de glucosa, los islotes no transgénicos y hIAPP Tg mostraron depósitos de amiloide en comparación con islotes no transgénicos y islotes hIAPP Tg cultivados a 11 mM de glucosa (NG) como se muestra por el ensayo de tioflavina S. Después del tratamiento con las chaperonas moleculares (BiP y PDI) en alta glucosa, los resultados demostraron una disminución notable de la formación de amiloide. El mismo efecto inhibidor se observó con el tratamiento de PBA. En contraste, TUDCA mostró una ligera disminución en la formación de amiloide. En resumen, nuestros datos sugieren que el tratamiento con chaperones no sólo mejora el estrés del RE y la secreción de insulina, sino que también desempeña un importante efecto protector en el desarrollo de la formación de amiloide en un modelo de ratón con deposición de amiloide. Nuestros resultados sugieren que los tratamientos con chaperonas se pueden utilizar como una potencial terapia para el enfoque de la mejora del estrés de RE, función de las células ß y en la deposición de amiloide en T2D. En conclusión, nuestro trabajo permitió una mejor comprensión de cómo el estrés de RE afecta la función de la célula ß pancreática en un contexto de sobreexpresión de hIAPP: 1) Células hIAPP-INS1E son más sensibles a los inductores de estrés de RE que las células rIAPP y células control INS1E. 2) La inducción de estrés de RE es dependiente de la activación de CHOP, confiriendo protección a la inducción del estrés y apoptosis en nuestro modelo de células de sobreexpresión de hIAPP. 3) Las chaperonas moleculares y químicas fueron capaces de aliviar el estrés de RE inducida por las células ß que sobreexpresan hIAPP. 4) El tratamiento de con chaperonas ha llevado a una mejora en la secreción de insulina estimulada por glucosa en las células hIAPP-INS1E tanto en condiciones basales y como estresantes. 5) La sobreexpresión de BiP y PDI, así como el tratamiento con PBA potenciaron la capacidad secretora de insulina en los islotes de ratones WT en condiciones basales. 6) La sobreexpresión de BiP y el tratamiento con PBA aumentan la secreción de insulina en los islotes de ratón hIAPP Tg en condiciones normales. 7) BiP, PDI, TUDCA y PBA mejoran la secreción de insulina en el tratamiento con HG y PA en los islotes de ratón hIAPP Tg. 8) El tratamiento con chaperonas reduce la severidad del amiloide en los islotes de ratón hIAPP Tg expuestos a glucose de 16 mM durante 7 días. 9) Por lo tanto, este estudio confirma el papel de la sobreexpresión de hIAPP en el desencadenamiento de la respuesta de estrés de RE y mostró un nuevo mecanismo de protección contra el desarrollo de la formación de amiloide mediante el tratamiento con chaperonas en condiciones fisiopatológicas en un modelo de ratón con deposición de amiloide.

Diabetes incidence highly increased in the last years. According to IDF (International Diabetes Federation), 463 million people suffered this disease in 2019. The estimations of diabetic people highly increase in the upcoming years, rising approximately to 700 million diabetic patients in 2045 [1]. Type 2 diabetes (T2D) is the most common type of diabetes, representing 90% of diabetic patients. It occurs when the body becomes resistant to insulin. Body insulin resistance confirms that T2D is not only a pancreatic disease, as there are many other tissues involved, like liver, adipose tissue, or skeletal muscle. This last has a significant implication in glucose-insulin homeostasis as it is one of the main glucose-consuming organs in the body. Nowadays, to study how two tissues crosstalk between them, animal testing is the gold standard. However, the unmatching physiological behaviors compared to humans, the variability between different animals, ethical dilemmas, and the need to go for more personalized medicine activates the search for other suitable alternatives. At this point, Organs-on-a-chip appeared as a valid alternative. Organs-on-a-chip (OOC) are 3D bioengineered microfluidic cell culture platforms to simulate microphysological environments of an organ or its specific functions. Nowadays, to engineer the tissues for OOC applications, encapsulating cells inside hydrogels is the most common technique. Its beneficial properties include high water content, mechanical adjustability, and moldability to generate the desired architectures [2]. However, its small porosity limits nutrient and oxygen diffusion through it [3]. This problem is a significant limitation when pancreatic islets are encapsulated inside hydrogels due to their size (~100 μm of diameter). Pancreatic islets are cell aggregations composed of many different cells as insulin-secreting cells (Beta-cells) or glucagon-secreting cells (alpha-cells). Similarly, skeletal muscle tissue is generally encapsulated in small bundles. Skeletal muscle is a highly aligned and multinucleated tissue formed from the fusion of single cells, called myoblasts, into multinucleated cells, called myotubes. Cryogels have been proposed as a valid alternative to overcome these limitations. Cryogels are fabricated by crosslinking a prepolymer solution at sub-zero temperatures, so while the material crosslinks, water freezes, generating the desired micropore architecture. After thawing, cryogels are sponge-like scaffolds with microporous structure, high interconnected porosity, high diffusivity, fine-tuned properties, and desired internal pore architecture. This thesis developed two cryogel scaffolds made of gelatin and carboxymethylcellulose with different pore architectures to engineer pancreatic and skeletal muscle tissues. Here, we proved that the achieved pore architecture fits with the prerequisites to engineer each tissue. Moreover, the mechanical and physical properties of each scaffold highly resemble the 3D microenvironment of each tissue. In pancreatic tissue, we generate a random pore cryogel to aggregate beta-cells to form pseudoislets. We proved that these engineered pseudoislets are viable, functional responding correctly to the glucose and improving insulin response compared to monolayer results. In the skeletal muscle approach, we could develop a highly aligned pore architecture to prompt cell alignment and cell fusion. Moreover, we incorporate carbon nanotubes to enhance the electrical conductivity of the scaffold, so by applying electrical pulse stimulation, we could improve the early steps of the myogenic maturation.
La incidència de la diabetis ha augmentat considerablement en els últims anys. Segons l’IDF (International Diabetes Federation), al 2019 hi havia 463 milions de persones que patien diabetis i les estimacions estimen un augment considerable de casos, arribant als 700 milions de persones diabètiques cap al 2045 [1]. Entre els diferents tipus de diabetis, la diabetis tipus 2, és la que té major incidència en la població, corresponent al 90% dels casos de pacients amb diabetis. Aquest tipus de diabetis, succeeix quan el cos es torna resistent a la insulina. Aquesta resistència a la insulina per part dels teixits perifèrics ens prova que la diabetis no és només una malaltia del pàncreas, sinó que hi ha altres teixits relacionats, com el fetge, el teixit adipós o el múscul esquelètic. Aquest últim té un factor molt rellevant en la homeòstasi de la insulina i la glucosa, ja que és un dels principals teixits consumidors de glucosa. La interacció, però, entre aquest dos teixits encara presenta molts interrogants. Actualment, per estudiar com dos teixits interactuen entre ells, el testeig animal és el mètode més confiable. No obstant, presenta certes limitacions, com la poca similitud en quan a l’activitat dels illots, la variabilitat fisiològica entre diferents animals, dilemes ètics o la necessitat d’encarar la recerca cap a una medicina més personalitzada. Aquesta finalitat és el que ha portat als científics a buscar alternatives a l’experimentació animal. Entre moltes, una de les més prometedores són els anomenats Òrgans-en-un-xip, plataformes 3D de cultiu cel·lular combinades amb microfluídica i biomaterials que permeten simular les funcions específiques d’un òrgan. Per tal de generar el teixit dins d’aquesta plataforma, l’encapsulació de cèl·lules dins de hidrogels és la tècnica més utilitzada, degut al seu alt contingut d’aigua, la seva adaptabilitat mecànica o la possibilitat de generar una certa estructura geomètrica [2]. No obstant, la seva petita porositat, limita la difusió homogènia d’oxigen i de nutrients dins seu [3]. Aquest problema creix quan es volen encapsular illots pancreàtics en bastides d’hidrogel, degut a la seva mida (~100 μm de diàmetre). Els illots pancreàtics són agregacions de varis tipus diferent de cèl·lules, on destaquen les cèl·lules secretores de insulina (cèl·lules beta) i les secretores de glucagó (cèl·lules alfa). Per altre costat, el teixit muscular s’encapsula en petits constructes per tal d’imitar l’estructura d’aquest. El múscul esquelètic és un teixit altament alineat, amb cèl·lules multi nucleades, anomenades miotubs, que s’obtenen a partir de la fusió de cèl·lules soles, anomenades mioblasts. Per tal de solucionar aquests problemes, els criogels han aparegut com a alternativa. Els criogels, estan fabricats a temperatures sota zero, així mentres el polímer crosslinca es formen cristalls de gel. Un cop formada la matriu, la bastida es descongela i aquests cristalls es desfaran, deixant pas a espais buits, anomenats pors. Aquests, seran els que posteriorment li donaran la l’estructura porosa, altament interconnectada, amb alta permeabilitat i amb una arquitectura de pors determinada a la nostra bestida. En aquesta tesi s’han desenvolupat dos bastides de cel·lulosa carboxymetilada diferents seguint la tècnica de la criogelificació. Cada bastida ha estat dissenyada per tenir una distribució i una arquitectura de pors diferent d’acord amb la necessitat i propietat del teixit que es vulgui generar. A més, les propietats físiques i mecàniques de les dos bastides tenen alta semblança amb les propietats físiques i mecàniques de la matriu extracel·lular de cada teixit. Per el teixit pancreàtic, s’ha generat una bastida amb un diàmetre de pors similar als illots pancreàtics, per tal que, sembrant cèl·lules beta, aquestes formin pseudoillots similars als illots fisiològics. A més, s’ha demostrat que aquests illots tenen el diàmetre i la arquitectura desitjada, són viables i capaços de respondre a diferents nivells de glucosa. A més, s’ha demostrat que aquestes cèl·lules agregades en forma de pseudoillots responen millor a la glucosa que les cèl·lules configurades en distribució dispersa. En el cas del múscul esquelètic, s’ha desenvolupat una bastida amb una arquitectura de pors altament alineada per promoure l’alineament cel·lular i la fusió cel·lular. A més, s’han pogut incorporar nanotubs de carboni per millorar les propietats elèctriques de la vestida. D’aquesta manera, aplicant pulsos elèctrics per estimular el teixit, s’han pogut millorar les etapes primerenques de la maduració miogènica.

Orientador: Jose Roberto Bosqueiro
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: As células B pancreáticas possuem controle multifatorial que permite a secreção de insulina em quantidade e tempo adequados. Os glicocorticóides modulam a secreção de insulina dependendo do tempo e dose em que forem utilzados. Assim,o presente trabalho teve por objetivo analisar as alterações na expressão gênica e protéica de ilhotas de ratos tratados com dexametasona (1mg/kg, 5dias). Utilizando a técnica de cDNA Macroarray observamos que dos 1176 genes presentes na membrana, 66 tiveram sua expressão aumentada e 38 genes tiveram sua expressão diminuída. Os genes com expressão aumentada pertencem às vias de estresse celular (JNK1), inibidores do ciclo celular (p21) e vias de apoptose (Baxa e Fas). Os genes com expressão diminuída pertencem ao ciclo celular (ciclinas D1 e D2, CDK4) e vias de sinalização PI3K, AKT1 e P70. Demonstramos também aumento na expressão protéica da Bax a, redução na expressão da proteína anti-apoptótica Bcl-2, PI3K e P70. Animais tratados com dexametasona por 5 dias possuem níveis plasmáticos aumentados de insulina, triglicérides e ácidos graxos livres. Ilhotas isoladas de animais tratados com dexametasona por 5 e 10 dias apresentaram maior secreção de insulina em relação aos controles, em concentrações basais e estimulatórias de glicose e 40mM de potássio. Porém, o tratamento por 10 dias com dexametasona induziu diminuição na secreção de insulina quando comparado aos animais tratados por 5 dias. Nossos dados sugerem o tratamento com dexametasona pode modular (direta ou indiretamente) a expressão de diversos genes e proteínas envolvidas na apoptose e sobrevivência de células na ilhota pancreática. Essa modulação pode, em longo prazo, se refletir na secreção de insulina como visto nos animais tratados por 10 dias
Abstract: Insulin secretion from pancreatic B-cells is regulated by nutrients like glucose and amino acids and by neurotransmitters, and hormones. Since glucocorticoids modulate insulin secretion we investigated the effects of dexamethasone on gene and protein expression in pancreatic islets from rats treated with the glucocorticoid for 5 days (mg/kg/day). Using cDNA array analysis we found that, out of 1176 genes presented in the array, 66 were up-regulated and 38 down-regulated after dexamethasone treatment. RT-PCR confirmed the macroarray results for 4 genes whereas the expression of these transcripts was confirmed by Western blotting for the corresponding proteins. Many of the up-regulated genes are implicated in apoptosis (Bax a, Fas), cell cycle regulation (p21) and stress response (JNK1) whereas many of the down-regulated were involved in cell cycle progression (cyclins D1 and D2, and CDK4), and survival and proliferations pathways (PI3K, AKT, P70). The protein expression of Bax a was increased whereas Bcl-2, PI3K and P70 repressed. The rats treated with dexamethasone for 5 days showed higher insulin, triglicerides and free fatty acids plasma levels than controls. The insulin secretion, in response to glucose and high concentrations of K+, in islets isolated from dexamethasone-treated rats for 5 and 10 days was higher than control rats. However, after 10 days of treatment with dexamethasone the insulin secretion was lower than after 5 days, but still higher than controls. In conclusion, these data indicate that dexamethasone-treatment (directly or indirectly) modulates the expression of several genes and proteins involved in apoptosis and survival of pancreatic islet-cells, and could, thereafter, modulates the insulin secretion in rats treated for 10 days
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular

Orientador: Antonio Carlos Boschiero
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O INGAP-PP é um pentadecapeptídeo constituído pela seqüência de aminoácidos do resíduo 104 ao 118 do INGAP (Islet Neogenesis Associated Protein), o qual é expresso no pâncreas exócrino e também nas células ductais durante a neogênese de células ßpancreáticas. Nesse trabalho, analisamos o efeito do tratamento crônico (4 dias) com INGAP-PP sobre a secreção de insulina, expressão gênica e protéica das subunidades Kir6.2 e SUR1 que formam o canal de K+ATP e do fator de transcrição Foxa2 (regulador da expressão das subunidades desse canal), em ilhotas isoladas de ratos adultos. As ilhotas tratadas com INGAP-PP (10 µg/mL) secretaram significativamente mais insulina quando incubadas por 1 h em concentrações entre 2.8 e 22.2 mM de glicose em comparação às ilhotas controles. Resultados de RT-PCR mostram que ilhotas tratadas com INGAP-PP tiveram expressão gênica do Foxa2 e das subunidades SUR1 e Kir6.2 aumentada. A expressão das proteínas SUR1 e Foxa2, analisada por Western Blotting, também foi maior nas ilhotas tratadas com INGAP-PP. Quando perfundidas na presença de 22,2 mM de glicose o aumento da secreção de insulina pelas ilhotas tratadas se manifestou com um primeiro pico secretor significativamente maior do que as ilhotas controles. Em presença de 2,8 mM de glicose, ilhotas tratadas com INGAP-PP secretaram mais insulina frente à concentrações despolarizantes de KCl ou tolbutamida (100 µM). Entretanto, a secreção de insulina estimulada por tolbutamida não diferiu entre os grupos em presença de 22,2 mM de glicose. A análise do efluxo de 86Rb mostrou que as ilhotas cultivadas com INGAP-PP apresentam menor efluxo do isótopo em relação às controle. Portanto, a maior secreção de insulina frente à glicose e concentrações despolarizantes de K+ indica que o tratamento com INGAP-PP induziu alterações que tornaram as células ßmais sensíveis a agentes despolarizantes. Quando associamos estes resultados ao aumento da expressão das proteínas formadoras do canal K+ATP e à redução do efluxo de 86Rb pelas ilhotas tratadas com INGAP-PP, podemos sugerir que o aumento no número de canais KATP pode ser um dos responsáveis pelo aumento na secreção de insulina nas ilhotas tratadas com o peptídeo
Abstract: Cultured adult rat islets were used to study the effect of INGAP-PP upon: a) gene expression of Kir6.2 and SUR1 of K+ATP channels and of their transcription factor Foxa2 (RT-PCR), b) protein levels (Western blotting) of SUR1 and Foxa2, c) static and dynamic insulin secretion elicited by metabolic and non metabolic stimuli and d) 86Rb efflux from perifused islets. INGAP-PP increased significantly the expression of Kir6.2, SUR1 and Foxa2 and the protein levels of SUR1 and Foxa2. Islets cultured with INGAPPP and further incubated for 1 h with 2.8 mM glucose, significantly enhanced the release of insulin in response to 40 mM KCl, and 100 µM tolbutamide. The dose-response curve of insulin secretion to increasing glucose concentrations (2.8 to 22.2 mM) shifted to the left in INGAP-PP-cultured islets with an EC50 of 10.0 ± 0.4 vs. 13.7 ± 1.5 mM glucose of the controls (P < 0,05). In dynamic studies INGAP-PP increased significantly the first-phase of insulin secretion elicited by either 22.2 mM glucose or 100 µM tolbutamide and promotes a higher glucose-induced reduction of 86Rb efflux from perifused islets. These results confirm the enhancing effect of INGAP-PP upon insulin release induced by different secretagogues and provide new evidence that such effect is due, at least partly, to an enhanced expression of the SUR1 and Kir6.2 genes of K+ATP channels and of the Foxa2 gene that controls their expression. They would also suggest that INGAP-PP could potentially be used to maintain the capacity of cultured islets to release insulin in response to glucose and maybe for the treatment of diabetes
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular

Orientador: Antonio Carlos Boschero
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: No diabetes mellitus, radicais de oxigênio estão associados com perda de sensibilidade à glicose ou destruição das células b. Nesse trabalho, investigamos a tolerância de ilhotas de Langerhans de ratos neonatos ao estresse provocado por H2O2, seus mecanismos antioxidantes de defesa e os fatores que promovem manutenção da sensibilidade à glicose. Cultivadas com 1 mM de H2O2, as ilhotas aumentaram até 6' seu consumo de glicose e resistiram ao estresse induzido por H2O2 quando em meio contendo 20 mM de glicose. A expressão da enzima catalase em resposta à glicose se mostrou necessária a essa resistência, mas não suficiente. Em concentrações baixas de H2O2, quando a catalase tem sua atividade modulada por NADPH, observamos que mesmo proteínas sem atividade catalítica adquiriam capacidade antioxidante e eram regeneradas por NADPH. Mapeando a distribuição de atividade peroxidase na ilhota, observamos sensibilidade ao NADPH nas frações nuclear e citossólica. As ilhotas cultivadas em 20 mM de glicose e as que resistem ao H2O2 possuem em comum uma maior atividade da via das pentoses, que gera NADPH citossólico. Nessas ilhotas, verificamos que a produção citossólica de NAD(P)H limita a secreção de insulina. Tais ilhotas produzem NAD(P)H principalmente da oxidação de substratos endógenos no citossol e nas mitocôndrias, ao invés de localizarem seu uso somente no citossol, como se dá nas ilhotas mais sensíveis ao H2O2. A cultura com 20 mM de glicose produziu ilhotas com alta expressão da lançadeira de NADH glicerol-fosfato, enquanto o H2O2 selecionou ilhotas com alta expressão da lançadeira mal/asp. Como ambas as lançadeiras promovem a comunicação entre citossol e mitocôndrias, concluímos que o sistema de lançadeiras e a geração de NADPH sejam fatores críticos para a manutenção da sensibilidade à glicose nas ilhotas
Abstract: In diabetes mellitus, oxygen radicals are associated with loss of glucose-sensibility and destruction of b-cells. In this work, we investigated the tolerance of neonatal rat islets to stress induced by H2O2, the islets antioxidant defense mechanism and factors maintaining islet glucoseresponsiveness. Islets cultured with 1 mM of H2O2 increased 6 fold the glucose uptake and resisted H2O2-induced stress when cultured in media containing 20 mM of glucose. Glucose-induced catalase expression was shown to be necessary to islet cell-survival, although not sufficient. In low H2O2 concentrations, the activity of catalase is dependent on NADPH and we observed that even proteins with no catalytic activity could be antioxidants regenerated by NADPH. Mapping the peroxidase activity in islets, we observed sensibility to NADPH in nuclear and cytossolic fractions. Islets cultured with 20 mM of glucose and islets that survived after culture with H2O2 both showed increased activity of the pentose phosphate pathway, which generate cytossolic NADPH. Is theses islets, we verified that cytossolic production of NAD(P)H limits insulin secretion. Such islets generate NAD(P)H principally from oxidation of endogenous fuels in cytossol and mitochondria, in contrary of the most H2O2-sensible islets which use endogenous fuels exclusively in cytossol. Culture with 20 mM of glucose produced islets with high expression of the glycerol-phosphate NADH shuttle, where as culture with H2O2 selected islets with high expression of the mal/asp shuttle. Since both shuttles promote interchange between cytossol and mitochondria, we have concluded that the shuttle system together with NAD(P)H generation ability are critical factors in maintaining islet glucoseresponsiveness
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

Orientador: Jose Roberto Bosqueiro
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A desnutrição e a resistência periférica à insulina induzida por administração de glicocorticóides induzem compensações funcionais e morfológicas em ilhotas pan creáticas a fim de manter a homeostase glicêmica. Deste modo, investigamos as alterações desenvolvidas pelo tratamento com dexametasona (Dex) em animais submetidos à restrição protéica. Foram analisados: parâmetros metabólicos, secreção de insulina em resp osta a glicose e proteínas envolvidas na via de sinalização da insulina em ilhotas pancreáticas isoladas. Ratos submetidos à dieta hipoprotéica (LP) apresentaram características padrões que caracterizam a desnutrição como: diminuição de ganho de massa corp oral, redução dos níveis séricos de albumina, proteína total e insulina. Adicionalmente, os ratos LP exibiram aumento da sensibilidade periférica à insulina e redução da área das ilhotas pancreáticas comparadas ao grupo controle ( P < 0,05). Todos estes parâmetros apresentaram valores similares ao grupo controle nos ratos submetidos à dieta hipoprotéica e submetidos ao tratamento com Dex (LPD), exceto para o peso corpóreo ( P < 0,05). A secreção de insulina em ilhotas pancreáticas isoladas de ratos LPD aprese ntou maior responsividade à glicose, em níveis estimulatórios, comparados a secreção em ilhotas de ratos LP (P < 0,05). Paralelo aos resultados de secreção, os ratos LPD exibiram redução do conteúdo protéico de IRS-1, IRS-2 e aumento dos níveis protéicos d e p-FoxO1, p-ERK e PKC comparados ao grupo LP (P < 0,05). Concomitantemente, as ilhotas dos ratos LPD mostraram ¿se hipertrofiadas comparadas com ilhotas de ratos LP ( P < 0,05). Em conclusão, o tratamento com dexametasona reverte, ao menos parcialmente, os efeitos no metabolismo analisados e no funcionamento das ilhotas pancreáticas causados pela restrição protéica, confirmando a grande plasticidade das células ß frente a condições adversas facultativas e/ou permanentes
Abstract: Malnutrition caused by protein restriction and dexamethasone -induced insulin resistance, in vivo treatment (Dex) are conditions associated with morphological and functional alterations in pancreatic islets. Thus, the present study evaluated the dexamethasone treatment effects on the metabolic parameters, glucose-stimulated insulin secretion and proteins involved in the insulin - signalling pathway over low protein diet fed rats (LP). LP rats showed decrease in body weight, serum insulin, total serum protein, and serum albumin, patte rns that characterize the LP rats. Moreover, LP rats presented improved peripheral insulin sensibility and reduced islets area (P < 0,05). Except for the body weight (P < 0,05), all these parameters were proned to be normalized in rats exposed to a low protein diet and treated with dexamethasone (LPD), whose islets showed increased glucose stimulated insulin secretion (GSIS). In addition, LPD rats showed lower protein expression of IRS-1, IRS-2 and higher in p-FoxO1, p-ERK and PKC, while presenting pancreatic islet hypertrophy compared to LP rats islet. In conclusion, dexamethasone treatment revert the effects related to metabolism and islet function caused by diet protein restriction, confirming ß-cells wide plasticity, even in transient or lasting adverse conditions
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

In type 1 diabetes (T1D) a combination of genetic predisposition and environmental factors triggers islet inflammation (insulitis) leading to a selective and gradual destruction of the pancreatic beta cells. Beta cells mainly die through apoptosis, triggered at least in part by pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-γ. Recent findings suggest that the mitochondrial pathway of cell death is involved in this death cascade. Array analysis indicated that TNF-α+IFN-γ induces transcription factors such as NF-ĸB, STAT1, and AP-1 in beta cells. We presently aimed to examine the pathway(s) of apoptosis triggered by TNF-α+IFN-γ in beta cells.

TNF-α+IFN-γ induces beta cell apoptosis through the intrinsic pathway of cell death. This involved activation of the BH3 only proteins DP5, PUMA and Bim. Knockdown (KD) of either DP5 or PUMA or both led to a partial protection of INS-1E cells (12-20%), while silencing Bim led to about 60% protection against cytokine-induced apoptosis. Bim is transcriptionally induced by activated STAT1. TNF-α+IFN-γ also induces downregulation of Bcl-XL, an anti-apoptotic Bcl-2 gene which inhibits Bim. Knocking down Bcl-XL alone led to increase in apoptosis, but this was prevented by the parallel KD of Bim.

The ultimate goal of our research is to protect beta cells from the autoimmune assault. Previous data revealed that JunB inhibits ER stress and apoptosis in beta cells treated with IL-β+IFN-γ. Here, TNF-α+IFN-γ up-regulated the expression of JunB which was downstream of activated NF-ĸB. JunB KD exacerbated TNF-α+IFN-γ induced beta cell death in primary rat beta cells and INS-1E cells. The gene networks affected by JunB were studied by microarray analysis. JunB regulates 20-25% of the cytokine-modified beta cell genes, including the transcription factor ATF3 and Bcl-XL. ATF3 expression was increased in cytokine-treated human islets and in vitro silencing of JunB led to >60% reduction in ATF3 overexpression. We confirmed direct JunB regulation of the ATF3 promoter by its binding to an ATF/CRE site. Silencing of ATF3 aggravated TNF-α+IFN-γ induced cell death in beta cells and led to the downregulation of Bcl-XL expression in INS-1E cells. Pharmacological upregulation of JunB using forskolin led to upregulation of ATF3 and consistent protection of these cells against cytokine-induced cell death, while genetic overexpression of JunB in mice increased ATF3 expression in the pancreatic islets and reversed the pro-apoptotic effects of cytokines on beta cells (±40 % protection).

As a whole, our findings indicate that TNF-α+IFN-γ triggers beta cell apoptosis by the upregulation of the pro-apoptotic protein Bim and downregulation of the Bcl-XL protein. These deleterious effects are at least in part antagonized by JunB via activation of ATF3.

Dans le diabète de type 1 (DT1), la combinaison de facteurs génétiques de prédisposition et de l'environnement déclenche l'inflammation des îlots de Langerhans (insulite) conduisant à une destruction sélective et progressive des cellules bêta du pancréas. Les cellules bêta meurent principalement d’apoptose, déclenchée au moins en partie par les cytokines pro-inflammatoires sécrétées par les cellules immunitaires comme l’IL-β, le TNF-α l’IFN-γ. De récentes découvertes suggèrent que la voie mitochondriale de la mort cellulaire jouerait un rôle dans la mort de ces cellules. L'analyse de réseaux de gène utilisant les biopuces d’ADN indique que l’association TNF-α+IFN-γ induit l’activation de facteurs de transcription tels que NF-ĸB, STAT1 et AP-1 dans la cellule bêta. Dans ce contexte, nous avons cherché à examiner les voies de l'apoptose déclenchées par le TNF-α+IFN-γ dans la cellule bêta.

En présence de TNF-α+IFN-γ les cellules bêta meurent par apoptose via la voie intrinsèque. L’activation des protéines pro-apoptotiques « BH3-seulement » dont DP5, PUMA et Bim étaient en cause de cette apoptose. Le « knockdown »1 (KD), de DP5 ou de PUMA, ou des deux en même temps conduit à une protection partielle des cellules INS-1E (12-20%), tandis que le KD de Bim conduit à environ 60% de protection contre l’apoptose induite par cette combinaison de cytokines. La transcription de Bim est induite par STAT1 activé. Parallèlement à la régulation positive de Bim, TNF-α+IFN-γ conduit à la régulation négative de la protéine Bcl-XL. Bcl-XL est une protèine anti-apoptotique de la famille de protèines Bcl-2 qui en general inhibe Bim. Réduire l’expression de Bcl-XL seul induit une augmention de l'apoptose, alors que le KD de Bim et Bcl-XL en parallèle empêche l'apoptose.

Le but ultime de notre recherche est de protéger les cellules bêta des agressions autoimmunitaires. Les données antérieures ont révélé que JunB inhibe le stress du réticulum endoplasmique et l'apoptose dans les cellules bêta traitées avec IL-β+IFN-γ. Nous avons observé que TNF-α+IFN-γ induit l'expression de JunB qui se produit en aval de NF-ĸB activé. Il est important de noter que l’inactivation de JunB par des agents interférants de l’ARN (siRNA) exacerbe la mort des cellules primaires bêta de rat et de cellules INS-1E induite par les cytokines. Les réseaux de gènes touchés par JunB ont été étudiés grâce a l'analyse en microréseaux. JunB règule 20-25% des gènes modifiés par des cytokines dans les cellules bêta, y compris le facteur de transcription ATF3 et Bcl-XL. L’expression d’ATF3 est augmenté dans les îlots humains traités avec les cytokines et la répression in vitro de JunB conduit à une réduction de >60% de l’expression d’ATF3. Nous avons confirmé la régulation d’ATF3 par JunB en montrant que JunB est directement lié au promoteur d’ATF3 via le site ATF/CRE. La diminution d’expression d’ATF3 en presence de TNF-α+IFN-γ a aggravé la mort cellulaire induite dans les cellules bêta et a conduit à la régulation négative de l'expression de Bcl-XL dans les cellules INS-1E. L’augmentation pharmacologique de JunB dans les cellules INS-1E par l’utilisation de forskolin a conduit à la régulation positive en aval d’ATF3 et par conséquente à la protection de cellules bêta vis-a-vis de effets indésirables des cytokines. Dans cette optique, la surexpression génétique de JunB dans le modèle Ubi-JunB de souris transgénique a conduit à une surexpression d’ATF3 dans les îlots pancréatiques et a permir d’inverser les effets pro-apoptotiques de cytokines sur la cellule bêta (protection ± 40%).

Globalement, ces résultats indiquent que TNF-α+IFN-γ déclenche l'apoptose des cellules bêta par la régulation positive du gène pro-apoptotique Bim et la régulation négative du gène anti-apoptotique Bcl-XL. Ces effets indésirables sont inhibé en partie par JunB via l’activation de ATF3.

1Pas d’équivalent en français. Signifie la réduction de l’expression d’un gène via utilisation d’un siRNA (agent interférant de l’ARN).

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Orientador: Everardo Magalhães Carneiro
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A desnutrição ainda é um problema de saúde pública que afeta principalmente países em desenvolvimento e sua prevalência chega a ser crescente em algumas áreas. Vários estudos obtiveram êxito em correlacionar a má nutrição em estágios iniciais de vida com o desenvolvimento de doenças cardiovasculares e diabetes tipo 2 na vida adulta. No modelo de desnutrição pós desmame verifica-se menor secreção de insulina estimulada por glicose e outros agentes insulinotrópicos bem como menor expressão de várias proteínas envolvidas com a funcionalidade da célula b. Estudos realizados por nosso grupo e outros laboratórios mostram que a suplementação de camundongos com taurina aumenta a secreção de insulina além de regular o influxo de íons Ca2+ para as células b, etapa crucial para o processo secretório. Para avaliar os efeitos da taurina sobre animais desnutridos, utilizamos ratos wistar, machos com 21 dias de vida. Os animais receberam dieta contendo 17% de proteína (normoprotéica) (C) ou 6% de proteína (hipoprotéica) (D). Animais C e D receberam suplementação com taurina a 2,5% na água de beber por 30 dias (CT30 e DT30) ou 90 dias (CT90 e DT90). Em seguida avaliamos parâmetros biométricos e bioquímicos, tolerância à glicose, secreção de insulina estimulada por glicose e pelo agonista colinérgico carbacol, expressão de proteínas envolvidas no controle da secreção de insulina e, finalmente, registramos os movimentos citoplasmáticos de íons Ca2+ após estímulo com glicose e carbacol. Verificamos que a restrição protéica retardou o crescimento dos animais além de reduzir a concentração plasmática de proteínas totais (C = 6,81±0,04; CT30 = 7,15±0,54; CT90 = 6,87±0,19; D = 5,35±0,24; DT30 = 5,37±0,28; DT90 = 5,70±0,09 g/dl; n = 3-5) e albumina (C = 3,20±0,11; CT30 = 3,41±0,02; CT90 = 3,18±0,05; D = 2,74±0,07; DT30 = 2,49±0,09; DT90 = 2,67±0,04 g/dl; n = 5-9) sem efeito da suplementação com taurina. Os animais D se mostraram mais tolerantes à glicose e a suplementação com taurina por 90 dias restaurou parcialmente a tolerância desses animais (C = 30249±2682; CT30 = 37255±6691; CT90 = 29365±2257; D = 16916±1609; DT30 = 18791±2859; DT90 = 23425±3856 AAC; n = 5-9). Nesse trabalho mostramos que a suplementação com taurina corrige a hipoinsulinemia verificada em animais desnutridos alimentados (C = 4,97±0,34; CT90 = 3,56±0,52; D = 1,39±0,10; DT90 = 3,31±0,70 ng/ml; n = 5-8) bem como a responsividade de ilhotas isoladas a concentrações crescentes de glicose. Verificamos também que a taurina normaliza a secreção de insulina potencializada pelo carbacol (C = 9,4+0,8; CT90 = 12,4+0,7; D = 6,4+0,5; DT90 = 9+0,7 ng/ml; n = 12). As respostas secretórias foram observadas em conjunto com a regulação da expressão das proteínas SERCA3 (C = 100+21; CT90 = 174+17; D =96+90; DT90 = 149+11 % do C; n = 6), receptor muscarínico M3 (C = 100+24; CT90 = 155+80; D = 51+10; DT90 = 108+14 % do C; n = 5) e sintaxina 1 (C = 100+30; CT90 = 92+40; D = 50+12; DT90 = 77+11 % do C; n = 5) que participam do controle de diferentes etapas do processo de secreção de insulina. Por fim, verificamos que a suplementação com taurina melhorou o padrão de oscilação de íons Ca2+ após estímulo com glicose. Concluímos então que a suplementação com taurina por 90 dias restaura a sensibilidade das ilhotas à glicose e ao carbacol possivelmente pela regulação do fluxo de cálcio para as células b bem como pela modulação da expressão de proteínas que controlam o processo de secreção de insulina.
Abstract: Malnutrition still is a public health issue, especially in developing countries. Many studies correlate malnourishment during early life and the development of cardiovascular disease and type 2 Diabetes Mellitus on latter stages. Animal models of malnutrition reveal impaired insulin secretion stimulated by glucose and other insulinotropic agents as well as lower expression of key proteins for b cell function. The literature shows that taurine supplementation increases insulin secretion and regulates calcium dynamics on b cells. Male, 21 days old, wistar rats received diet containing 17% (C) or 6% (D) of protein. Both groups received taurine supplementation on the drinking water for 30 (CT 30 and DT 30) and 90 (CT90 and DT 30) days. Next we assessed biometric and biochemical parameters, glucose tolerance, glucose and carbacholstimulated insulin secretion, protein expression of muscarinic M3 receptor, Phospholipase C b2, SERCA3, Syntaxin 1 and, finally, we registered cytoplasmic Ca2+ after stimulus with glucose and carbachol. Protein restricted rats showed lower body weight, plasma proteins (C = 6,81±0,04; CT30 = 7,15±0,54; CT90 = 6,87±0,19; D = 5,35±0,24; DT30 = 5,37±0,28; DT90 = 5,70±0,09 g/dl; n = 3-5), albumin (C = 3,20±0,11; CT30 = 3,41±0,02; CT90 = 3,18±0,05; D = 2,74±0,07; DT30 = 2,49±0,09; DT90 = 2,67±0,04 g/dl; n = 5-9) and increased glucose tolerance (C = 30249±2682; CT30 = 37255±6691; CT90 = 29365±2257; D = 16916±1609; DT30 = 18791±2859; DT90 = 23425±3856 AUC; n = 5-9). Taurine supplementation had no effect upon nutritional status parameters and partially restored glucose tolerance and insulinemia to C levels. Taurine increased secretory response to glucose and carbachol (C = 9,4+0,8; CT90 = 12,4+0,7; D = 6,4+0,5; DT90 = 9+0,7 ng/ml; n = 12). It also increased protein expression of M3 receptor (C = 100+24; CT90 = 155+80; D = 51+10; DT90 = 108+14 % of C; n = 5), SERCA 3 (C = 100+21; CT90 = 174+17; D =96+90; DT90 = 149+11 % of C; n = 6) and syntaxin 1 (C = 100+30; CT90 = 92+40; D = 50+12; DT90 = 77+11 % of C; n = 5). Finally, taurine supplementation for 90 days improved Ca2+ dynamics when the islets were stimulated with glucose. In conclusion, these data show that taurine supplementation restores secretory responsiveness to glucose and carbachol possibly through Ca2+ dynamics modulation and increased expression of key proteins for insulin secretion.
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular

Orientador: Antonio Carlos Boschero
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A obesidade atingiu proporções alarmantes constituindo-se num fator de risco para o desenvolvimento de várias doenças. O aumento da resistência periférica à insulina acompanha esta patologia e a incapacidade da célula beta pancreática em suprir a maior necessidade por insulina leva ao desenvolvimento de intolerância à glicose, hiperglicemia e diabetes. Por esta razão, é importante investigar mecanismos que tornem a célula beta capaz de aumentar sua capacidade secretória. A exemplo da obesidade, resistência periférica à insulina é também observada durante a prenhez. No entanto, neste caso, a célula beta é capaz de aumentar a produção e secreção do hormônio, mantendo a tolerância à glicose em condições adequadas. Diante disso,decidimos investigar a sensibilidade à insulina e a consequente resposta das células beta pancreáticas durante a prenhez em ratas obesas. Observamos que a alimentação com a dieta de cafeteria aumentou o ganho de peso, bem como os depósitos de gordura das ratas. Ratas obesas não-prenhes (Caf) e prenhes (CafP) apresentaram tolerância à glicose diminuída, associada a um aumento da insulina plasmática em resposta à sobrecarga de glicose no grupo CafP. Apesar disso, as glicemias de jejum e pós-prandial foram normais nos dois grupos. No entanto, as ratas Caf e CafP apresentaram hiperinsulinemia (jejum e alimentado), aumento do índice insulina/glicose e do AGL plasmático (alimentado). Ainda, houve redução na sinalização da insulina no fígado e músculo esquelético das ratas Caf e CafP, aos 15 e aos 19 dias de prenhez, de forma mais exacerbada do que a redução observada nas ratas controle prenhes. Em paralelo, as ilhotas isoladas das ratas Caf secretaram menos insulina em resposta a diferentes estímulos. Contudo, o conteúdo total de insulina, a secreção estimulada por PMA (ativador da PKC), a produção decompostos redutores e a oxidação de glicose, na presença de 11,1 mmol/L do açúcar, foram similares entre as ratas Caf e as controle não-prenhes. Entretanto, as ilhotas isoladas das ratas Caf apresentaram redução na mobilização do Ca2+ citoplasmático livre frente à glicose ou tolbutamida, acompanhada pela redução da expressão gênica da subunidade ?1.2 do canal de cálcio voltagem-dependente (CaVa1.2), e da Ca2+- ATPase do retículo endoplasmático tipo 2a. Independente da dieta, a prenhez aumentou a secreção de insulina em resposta à glicose, a produção de compostos redutores, a oxidação de glicose, a amplitude e a frequência das oscilações do Ca2+ citoplasmático e, a expressão gênica do CaVa1.2. Concluindo, a prenhez nas ratas obesas melhorou o manejo do Ca2+ e restaurou a secreção de insulina por ilhotas isoladas. Contudo, esta restauração não foi suficiente para vencer o aumento da resistência periférica à insulina e normalizar a tolerância à glicose nas ratas obesas
Abstract: The incidence of obesity reached alarming levels worldwide. This illness constitutes a risk factor for the development of several other diseases. The augmented peripheral insulin resistance accompanies this pathology, and the failure of the pancreatic beta cell to overcome the higher demand for insulin causes glucose intolerance, hyperglycemia and diabetes. For this reason, it became interesting to investigate mechanisms that make the beta cell capable to increases its secretory capacity. As obesity, peripheral insulin resistance is also observed during pregnancy. Nevertheless, in this situation, the beta cell is capable to enhance insulin production and release, maintaining glucose tolerance at adequate levels. Therefore, we decided to investigate insulin sensibility and the consequent beta cell response during pregnancy in obese rats. We observed that cafeteria diet enhanced weight gain and fat pads in rats. Despite no differences were noticed in obese non-pregnant (Caf) and pregnant (CafP) rats, during fast and fed states, the glucose tolerance was diminished in these rats, associated with an augmented plasma insulin levels in response to a glucose load inCafP rats. However, Caf and CafP rats had hyperinsulemia (fast and fed), higher insulin/glucose index, and enhanced plasma FFA (fed state). In addition, we observed a reduction in insulin signaling in liver and skeletal muscle from Caf and CafP rats, at15th and 19th days of pregnancy, higher than that registered in control pregnant rats. Also, there was a reduction in insulin secretion induced by different stimuli in is lets from Caf rats. However, total islet insulin content, PMA-stimulated insulin secretion, production of reducing equivalents, and glucose oxidation in the presence of 11.1mmol/L glucose, were similar between islets from Caf and non-pregnant control rats .Nevertheless, glucose- and tolbutamide-induced Ca2+ mobilization, a1.2 subunit of thevoltage sensitive Ca2+ channel (CaVa1.2), and sarcoendoplasmic reticulum Ca2+ATPase 2a gene expression were reduced in islets from Caf rats. Independently of the diet, pregnancy enhanced glucose stimulated insulin secretion, reducing equivalents production, glucose oxidation, amplitude and frequency of cytoplasm Ca2+ oscillations, and CaVa1.2 gene expression. In conclusion, although pregnancy improved Ca2+ handling and restored insulin secretion in cafeteria diet-induced obese rats, this restoration was not enough to overcome the increase in peripheral resistance and normalize glucose tolerance in these obese rats
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

Orientadores: Antonio Carlos Boschiero, Alex Rafacho
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Administração de glicocorticóides em roedores e humanos aumenta a resistência à insulina (RI). A RI, provocada por dexametasona, leva a hiperinsulinemia por aumento da secreção do hormônio pelas ilhotas pancreáticas. Recentemente, demonstrou-se que a AS160, uma GAP (proteína ativadora de GTPase), participa no tráfego de vesículas em diferentes tipos celulares que, por sua vez, pode ser alterado por dexametasona. Neste trabalho, avaliamos possível participação da AS160 na secreção de insulina em ilhotas de ratos RI por dexametasona, para isto foram avaliadas proteínas envolvidas no processo de secreção; pAS160, Akt e AMPK. Ratos Wistar adultos foram tratados com o glicocorticóide (DEX) com 1mg/kg (ip) de peso corporal, ou salina (CTL), durante 5 dias. Ao final do período de tratamento, os ratos foram submetidos a um Teste de Tolerância à Glicose intraperitoneal (ipGTT) e, após sacrifício, amostras de sangue foram coletadas para dosagem de insulina. As ilhotas pancreáticas foram isoladas por digestão do pâncreas com colagenase. As proteínas insulares foram avaliadas por Western Blot e os genes por RCP-TR. A insulina, contida nas amostras de sangue e nas incubações de ilhotas, foi medida por radioimunoensaio (RIA). A razão pAS160/AS160 foi aumentada nas ilhotas DEX (P<0,05). Nestas ilhotas, resultados semelhantes foram observados para a razão pAkt/Akt (P<0,05). O tratamento com DEX também aumentou a expressão gênica e protéica da Rab27A (P<0,05), contudo, reduziu significativamente sua associação com a AS160 (P<0,05). A associação entre essas duas proteínas foi observada pela primeira vez nas ilhotas neste trabalho. O tratamento com DEX também reduziu as expressões gênica e protéica bem como a fosforilação da AMPK. A secreção de insulina foi maior nas ilhotas DEX comparado à CTL e, em ambas, a secreção foi diminuída pela wortmanina (inibidor da PI3K). Ilhotas de ratos CTL e DEX, tratados com anti-sense anti-AS160, tiveram o conteúdo protéico da AS160 reduzido em ± 80%, comparado ao CTL (P<0,05). Nas ilhotas de ratos CTL knockdown, a secreção de insulina foi maior que nos CTL e, nas ilhotas dos DEX knockdown a secreção foi semelhante às DEX. Concluindo, o aumento da secreção de insulina em ilhotas de ratos RI por dexametasona envolve a participação da AS160 e, essa potencialização parece ser mediada pela via PI3K/Akt. Esse aumento de secreção parece também ser diretamente proporcional ao aumento da dissociação entre a Rab27A e a AS160
Abstract: It is well known that glucocorticoids induce insulin resistance (IR). It is also known that dexamethasone-induced IR is linked to increased levels of plasma insulin due to higher insulin secretion by pancreatic islets. Recent findings show that the Rab-GTPase AS160 plays a role in the traffic of vesicles in different cells type that, in turn, may be affected by dexamethasone. Here, we evaluated the participation of AS160 in the insulin secretion in islets from dexamethasone treated rats. Adult rats were treated with dexamethasone (DEX) with 1.0 mg/kg, body weight (ip) or saline (CTL) for 5 consecutive days. Insulin resistance was evaluated by intraperitoneal Glucose Tolerance Test (ipGTT). After, the rats were sacrificed and the islets isolated by the digestion of their pancreases with collagenase. Protein was measured by Western- Blot, and insulin by RIA. AS160 expression, phosphorylation, and the pAS160/AS160 ratio were increased in DEX islets (P<0.05). Similar results were observed for Akt (P<0.05). Dexamethasone also increased Rab27a protein and gene expression but significantly reduced its association with AS160. The association between these two proteins was observed in pancreatic islets for the first time in this work. AMPK gene and protein expression as well as phosphorylation were reduced by Dexamethasone (P<0.05). The insulin secretion was higher in DEX compared with CTL islets (P<0.05). Both secretions were reduced by wortmanin. Islets from CTL and DEX rats, treated with anti-sense anti-AS160, showed ± 80% reduction on its expression. The CTL knockdown islets secreted more insulin than CTL and the DEX knockdown secreted similar amount of insulin than DEX islets. In conclusion, these results indicated that AS160 participates in the increased insulin secretion in islets from DEX rats, and this effect seems to be dependent on the activation of the PI3K/Akt pathway. The increase in insulin secretion also depends on the dissociation between Rab27a and AS160
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular

Un aspecte central, tant en el desenvolupament de la diabetis tipus 1 com en el de la tipus 2, és la reducció del nombre de cèl·lules beta productores d’insulina, i en ambdues condicions la pèrdua de massa beta s’ha atribuït en gran mesura a l’augment de la mort de les cèl·lules beta. A més, diversos treballs han posat de relleu la contribució fonamental de la replicació de les cèl·lules beta pel manteniment fisiològic i la regeneració de la massa de cèl·lules beta en models amb massa beta reduïda, la qual cosa pot suggerir que l’alteració de la replicació de les cèl·lules beta també pot contribuir de manera important a la reducció de la massa beta en la diabetis. El trasplantament d’illots és una teràpia prometedora per la curació de la diabetis mellitus, amb la que s’ha aconseguit un control acurat de la glucèmia de l’individu. Ara bé, el trasplantament d’illots presenta algunes limitacions importants, una de les principals és la baixa disponibilitat d’òrgans i la seva elevada demanda, agreujada per l’alt nombre d’illots requerits per restablir la normoglucèmia, probablement a causa de la important pèrdua de massa beta que té lloc els primers dies post-trasplantament. Aquesta pèrdua es deu als alts nivells d’apoptosi i de necrosi de les cèl·lules beta i a la seva limitada proliferació incapaç de compensar la mort. La interleucina 1 (IL-1) té un paper clau en la destrucció de les cèl·lules beta pancreàtiques que es dóna en la diabetis mellitus tipus 1 i tipus 2. A més, la citocina juga un paper important en l’increment de la mort beta que té lloc durant els primers dies després del trasplantament d’illots pancreàtics. El factor de creixement similar a la insulina 2 (IGF2) és un potent factor de creixement que té un rol important promovent la diferenciació i la proliferació cel·lular i actuant com a factor de supervivència en limitar l’apoptosi en diferents tipus cel·lulars. Per tant, IGF2 pot tenir un doble rol beneficiós en la massa de cèl·lules beta, actuant com a mitogen i com a factor de supervivència per les cèl·lules beta. La hipòtesi general del projecte és que el pronòstic del trasplantament d’illots pot millorar amb la utilització d’estratègies orientades a incrementar la supervivència i la replicació de les cèl·lules beta. L’objectiu general és determinar si la sobreexpressió d’IGF2 en els illots pancreàtics millora la supervivència i la regeneració de les cèl·lules beta pancreàtiques. L’objectiu de l’estudi in vitro fou caracteritzar l’efecte de la IL-1β sobre la replicació i supervivència de les cèl·lules beta i la potencial modulació per IGF2. L’objectiu de l’estudi in vivo fou determinar l’efecte de la sobreexpressió d’IGF2 sobre la massa beta trasplantada. En l'estudi in vitro, illots de rata control no infectats i illots infectats amb l’adenovirus que codifica per l’IGF2 (Ad-IGF2) es van cultivar durant 48h a 5,5 o 22,2 mM de glucosa en presència o absència de 1, 10, 30, i 50 U/ml d'IL-1β. La proliferació de les cèl·lules beta es va veure dràsticament reduïda quan els illots es van exposar a 10 U/ml d’IL-1β i es va suprimir gairebé completament en els illots exposats a 30 i 50 U/ml d’IL-1β. Concentracions més elevades de la citocina van ser necessàries per augmentar l’apoptosi de les cèl·lules beta de manera significativa. Malgrat la sobreexpressió d’IGF2 té un fort efecte mitogènic sobre les cèl·lules beta, IGF2 va preservar la replicació de les cèl·lules beta només en els illots exposats a 10 U/ml d’IL-1β, i no va tenir efecte en els illots exposats a altes concentracions de la citocina (30 i 50 U/ml). En contrast, la sobreexpressió d’IGF2 va presentar una clara protecció contra l’apoptosi induïda per IL-1β, pel que van ser necessàries concentracions superiors de la citocina per induir un augment significatiu de l’apoptosi respecte els illots control. Aquests resultats indiquen que la replicació de les cèl·lules beta és altament sensible als efectes deleteris de la IL-1β, com s’observa per la inhibició de la replicació per concentracions d’IL-1β relativament baixes, i la supressió quasi completa de la replicació de les cèl·lules beta per altes concentracions de la citocina. A més, els efectes inhibitoris de la IL-1β sobre la replicació de les cèl·lules beta no es van modificar per la glucosa, i van ser només modestament evitats per la sobreexpressió d’IGF2, en contrast amb la protecció proporcionada per la glucosa i per la sobreexpressió d’IGF2 contra l'apoptosi induïda per la IL-1β. En l'estudi in vivo, illots infectats amb l’adenovirus que codifica per l’IGF2 (grup Ad-IGF2), la luciferasa (grup control Ad-Luc), o amb illots no infectats (grup control) van ser trasplantats a rates Lewis diabètiques per estreptozotocina. Es van trasplantar 800 illots, que es una massa beta subcrítica, o 500 illots, que és una massa clarament insuficient per restaurar la normoglicèmia. Les rates trasplantades amb 800 illots que sobreexpressaven IGF2 van mostrar una millor evolució metabòlica respecte els grups control. Com era d'esperar, les rates trasplantades amb 500 illots que sobreexpressaven IGF2 o amb illots control van presentar una hiperglucèmia similar al llarg de tot l'estudi, assegurant condicions metabòliques comparables entre ambdós grups. La replicació de les cèl·lules beta va ser superior en el grup Ad-IGF2 vs el grup control els dies 3, 10, i 28 després del trasplantament. La massa beta es va reduir de manera similar el dia 3 després del trasplantament en el grup Ad-IGF2 i el grup control, es va incrementar el dia 10 i a dia 28 va ser superior en els empelts que sobreexpressaven IGF2. L'apoptosi es va incrementar de manera similar en ambdós grups, Ad-IGF2 i control, després del trasplantament. No es van trobar diferències en la secreció d'insulina entre els illots Ad-IGF2 i els illots control no infectats. En resum, la sobreexpressió de IGF2 en illots trasplantats va augmentar la replicació de cèl·lules beta, aquest increment de la replicació en els empelts d'illots que sobreexpressen IGF2 es va traduir en la regeneració de la massa beta trasplantada, fet que es reflectí en la millora del pronòstic del trasplantament. En conjunt, aquests resultats suggereixen que estratègies dirigides a augmentar la proliferació beta poden ser útils per induir la regeneració de la massa beta pel tractament de la diabetis i en el trasplantament d’illots.
β-Cell mass reduction has a central role in the development of type 1 and type 2 diabetes, and in both conditions the loss of β-cells has been largely attributed to increased β-cell death. Furthermore several reports have highlighted the fundamental contribution of β-cell replication to the physiological maintenance of β-cell mass, and to β-cell mass regeneration in models with reduced β-cell mass. This may suggest that impaired β-cell replication could contribute to the reduction of β-cell mass in diabetes. Islet transplantation restores normoglycemia in type 1 diabetic patients. However, islet transplantation presents some important limitations. A basic restriction is the low organ availability and high requirement, which is exacerbated by the high islet mass that must be transplanted to achieve normoglycemia, probably due to the massive destruction of islets taking place in the initial days after transplantation due to increased beta-cell apoptosis, necrosi and impaired beta-cell replication. IL-1β is an important contributor to β-cell damage in type 1 diabetes, and recently it has also been related to the development of type 2 diabetes. IL-1beta also could contribute to the dramatic beta cell loss that takes place after islet transplantation IGF2 is a growth promoting peptide which is able to stimulate cell differentiation, proliferation and survival. Thus, IGF2 may play a dual beneficial role on β-cell mass, acting both as a mitogenic and as a survival factor for β-cells. We investigated in vitro and in vivo effects of the IGF2 overexpression on β-cell survival and regeneration. The aim of the in vitro study was to characterise the effect of IL-1β on β-cell replication, and the potential modulation by IGF2. Since the induction of β-cell proliferation by IGFs is dependent on ambient glucose concentration, low and high glucose concentrations were used to better define the effects of IL-1β and IGF2 on β-cell replication. The aim of the in vivo study was to determine the effect of IGF2 overexpression on β-cell mass in transplanted islets. In the in vitro study, control-uninfected and adenovirus encoding for IGF2 (Ad-IGF2)-infected rat islets were cultured at 5.5 or 22.2  mmol/l glucose with or without 1, 10, 30, and 50 U/ml of IL-1β. β-Cell replication was markedly reduced by 10 U/ml of IL-1β and was almost nullified with 30 or 50 U/ml of IL-1β. Higher concentrations of IL-1β were required to increase β-cell apoptosis. Although IGF2 overexpression had a strong mitogenic effect on β-cells, IGF2 could preserve β-cell proliferation only in islets cultured with 10 U/ml IL-1β, and had no effect with 30 and 50 U/ml of IL-1β. In contrast, IGF2 overexpression induced a clear protection against IL-1β-induced apoptosis, and higher concentrations of the cytokine were needed to increase β-cell apoptosis in Ad-IGF2-infected islets. These results indicate that β-cell replication is highly sensitive to the deleterious effects of the IL-1β as shown by the inhibition of replication by relatively low IL-1β concentrations, and the almost complete suppression of β-cell replication with high IL-1β concentrations. Likewise, the inhibitory effects of IL-β on β-cell replication were not modified by glucose, and were only modestly prevented by IGF2 overexpression, in contrast with the higher protection against IL-1β-induced apoptosis afforded by glucose and by IGF2 overexpression. In the in vivo study, islets infected with adenovirus encoding for IGF2 (Ad-IGF2 group), for luciferase (Ad-Luc control group), or with uninfected islets (control group) were syngeneically transplanted to streptozotocin-diabetic Lewis rats. Eight hundred islets, a minimal mass model to restore normoglycemia, or 500 islets, a clearly insufficient mass, were transplanted. Rats transplanted with 800 Ad-IGF2 islets showed a better metabolic evolution than control groups. As expected, rats transplanted with 500 Ad-IGF2 or control islets maintained similar hyperglycemia throughout the study, ensuring comparable metabolic conditions among both groups. β-Cell replication was higher in Ad-IGF2 group than in control group on days 3, 10, and 28 after transplantation. β-Cell mass was similarly reduced on day 3 after transplantation in Ad-IGF2 and control group, it increased on day 10, and on day 28 it was higher in Ad-IGF2 than in control group. Apoptosis was similarly increased in Ad-IGF2 and control islets after transplantation. No differences in insulin secretion were found between Ad-IGF2 and uninfected control islets. In summary, IGF2 overexpression in transplanted islets increased β-cell replication, induced the regeneration of the transplanted β-cell mass, and had a beneficial effect on the metabolic outcome reducing the β-cell mass needed to achieve normoglycemia. Taken together, these results suggest that strategies aimed to preserve or increase the engrafted β-cell mass may be useful for inducing the regeneration of beta mass for the treatment of diabetes and in islet transplantation.

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A manutenção da homeostase energética é, sem duvida, o aspecto fisiológico mais importante para a garantia de sobrevivência animal. Doenças crônicas como câncer são capazes de alterar esta homeostase, agravada principalmente pela presença do tumor. O conjunto de sintomas envolvidos neste quadro de desequilíbrio metabólico, comum a diversas doenças, foi agrupado e caracterizado como uma síndrome metabólica denominada caquexia que, entre outros fatores, leva à progressiva perda de peso, perda de tecido muscular e adiposo, e está frequentemente associado a sintomas de anorexia e inflamação sistêmica. Na caquexia induzida por câncer, o desequilíbrio no metabolismo de carboidratos é um sintoma frequente, especialmente alterações no perfil secretório de insulina. Da mesma forma, a presença tumoral gera um estado de inflamação sistêmica que há algum tempo tem sido apontada como principal responsável pela alteração funcional de diversos órgãos, acarretando na progressão da síndrome. No entanto, não há trabalhos na literatura que correlacionem o papel da inflamação sistêmica gerada pelo desenvolvimento tumoral com a alteração no metabolismo de carboidratos, principalmente aquelas relacionadas à secreção de insulina. Em resultados anteriores evidenciamos que camundongos portadores do tumor sólido de Ehrlich (TSE) exibiram drástica diminuição na capacidade secretória de insulina, hipoinsulinemia, maiores sensibilidade a este hormônio e tolerância à glicose. Deste trabalho, inúmeras possibilidades de estudo surgiram, dentre elas a investigação do quadro inflamatório na ilhota como possível causador das alterações na secreção de insulina. Diante da escassez de trabalhos nesta área e diante da importância da adequada secreção de insulina e da homeostase glicêmica na melhora da sobrevida de portadores de doenças malignas, foi proposta do presente estudo avaliar...
The maintenance of energy homeostasis is one of the most crucial physiological tasks to ensure long-term survival in animals. Chronic diseases, as cancer, are capable to lead to homeostasis imbalance mainly by tumor presence and the cluster of symptoms involved in this process was named cachexia. Cancer cachexia is a metabolic syndrome characterized by a marked weight loss, loss of muscle and adipose tissues and is frequently associated with anorexia and systemic inflammation. Alterations in carbohydrate metabolism are frequent in cancer cachexia, especially insulinemic alterations. In the same way, tumor presence promotes severe systemic inflammation in the host, and it was demonstrated that this is responsible for functional alterations in many organs leading to cachexia progression. However, there are no studies that correlate the role of systemic inflammation with the alterations in carbohydrate metabolism, mainly related with alterations in insulin secretion. In previous studies we demonstrated that solid Ehrlich carcinoma-bearing mice (SET) showed drastic decrease in insulin secretion, hipoinsulinemia, increase in insulin sensitivity and glucose tolerance. With these results, numerous opportunities for study appeared, among then the establishment of inflammatory response in islets. Considering the lack of studies in this area and considering the importance of proper insulin secretion and glicemic homeostasis to the survival of cancer patients, we evaluated inflammatory response components in pancreatic islets of solid Ehrlich tumor-bearing mice in order to seek consistent information about possible mechanisms involved in alterations in insulin secretion in SET group. For this, pancreatic islets of control (CTL) and SET bearing mice were analyzed 14 days after tumor inoculation for determination of the expression of the proinflammatory citokynes TNF-α, IL-1β, IFNγ, IFN-α, IL-6 e IL-8, and ...

Orientador: José Roberto Bosqueiro
Banca: José Maurício Sforcin
Banca: Silvana Bordin
Resumo: A manutenção da homeostase energética é, sem duvida, o aspecto fisiológico mais importante para a garantia de sobrevivência animal. Doenças crônicas como câncer são capazes de alterar esta homeostase, agravada principalmente pela presença do tumor. O conjunto de sintomas envolvidos neste quadro de desequilíbrio metabólico, comum a diversas doenças, foi agrupado e caracterizado como uma síndrome metabólica denominada caquexia que, entre outros fatores, leva à progressiva perda de peso, perda de tecido muscular e adiposo, e está frequentemente associado a sintomas de anorexia e inflamação sistêmica. Na caquexia induzida por câncer, o desequilíbrio no metabolismo de carboidratos é um sintoma frequente, especialmente alterações no perfil secretório de insulina. Da mesma forma, a presença tumoral gera um estado de inflamação sistêmica que há algum tempo tem sido apontada como principal responsável pela alteração funcional de diversos órgãos, acarretando na progressão da síndrome. No entanto, não há trabalhos na literatura que correlacionem o papel da inflamação sistêmica gerada pelo desenvolvimento tumoral com a alteração no metabolismo de carboidratos, principalmente aquelas relacionadas à secreção de insulina. Em resultados anteriores evidenciamos que camundongos portadores do tumor sólido de Ehrlich (TSE) exibiram drástica diminuição na capacidade secretória de insulina, hipoinsulinemia, maiores sensibilidade a este hormônio e tolerância à glicose. Deste trabalho, inúmeras possibilidades de estudo surgiram, dentre elas a investigação do quadro inflamatório na ilhota como possível causador das alterações na secreção de insulina. Diante da escassez de trabalhos nesta área e diante da importância da adequada secreção de insulina e da homeostase glicêmica na melhora da sobrevida de portadores de doenças malignas, foi proposta do presente estudo avaliar ...
Abstract: The maintenance of energy homeostasis is one of the most crucial physiological tasks to ensure long-term survival in animals. Chronic diseases, as cancer, are capable to lead to homeostasis imbalance mainly by tumor presence and the cluster of symptoms involved in this process was named cachexia. Cancer cachexia is a metabolic syndrome characterized by a marked weight loss, loss of muscle and adipose tissues and is frequently associated with anorexia and systemic inflammation. Alterations in carbohydrate metabolism are frequent in cancer cachexia, especially insulinemic alterations. In the same way, tumor presence promotes severe systemic inflammation in the host, and it was demonstrated that this is responsible for functional alterations in many organs leading to cachexia progression. However, there are no studies that correlate the role of systemic inflammation with the alterations in carbohydrate metabolism, mainly related with alterations in insulin secretion. In previous studies we demonstrated that solid Ehrlich carcinoma-bearing mice (SET) showed drastic decrease in insulin secretion, hipoinsulinemia, increase in insulin sensitivity and glucose tolerance. With these results, numerous opportunities for study appeared, among then the establishment of inflammatory response in islets. Considering the lack of studies in this area and considering the importance of proper insulin secretion and glicemic homeostasis to the survival of cancer patients, we evaluated inflammatory response components in pancreatic islets of solid Ehrlich tumor-bearing mice in order to seek consistent information about possible mechanisms involved in alterations in insulin secretion in SET group. For this, pancreatic islets of control (CTL) and SET bearing mice were analyzed 14 days after tumor inoculation for determination of the expression of the proinflammatory citokynes TNF-α, IL-1β, IFNγ, IFN-α, IL-6 e IL-8, and ...
Mestre

Lau Tung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 142-157).
Abstracts in English and Chinese.
Abstract --- p.i
摘要 --- p.iii
Acknowledgements --- p.v
Table of Contents --- p.vi
List of Abreviations --- p.x
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- Pancreas and its functions --- p.1
Chapter 1.1.1 --- Structure of pancreas --- p.1
Chapter 1.1.2 --- Exocrine function --- p.4
Chapter 1.1.3 --- Endocrine function --- p.7
Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.7
Chapter 1.1.3.2 --- Regulation of insulin secretion --- p.10
Chapter 1.1.3.3 --- Mechanism for glucose-stimulated insulin release --- p.14
Chapter 1.1.3.4 --- Bi-phase response of insulin secretion --- p.16
Chapter 1.2 --- Pancreatic Renin-Angiotensin System --- p.19
Chapter 1.2.1 --- Circulating RAS and local RAS --- p.19
Chapter 1.2.2 --- RAS inhibitors --- p.25
Chapter 1.2.2.1 --- Angiotensin converting enzyme inhibitor --- p.25
Chapter 1.2.2.2 --- Non-specific Ang II receptor blocker --- p.28
Chapter 1.2.2.3 --- Specific AT1 receptor antagonist --- p.29
Chapter 1.2.2.4 --- Specific AT2 receptor antagonist --- p.30
Chapter 1.2.3 --- RAS and Pancreas --- p.30
Chapter 1.2.3.1 --- Expression and localization of pancreatic RAS --- p.30
Chapter 1.2.3.2 --- Regulation of pancreatic RAS and its clinical relevance --- p.32
Chapter 1.3 --- Islet Transplantation and RAS --- p.34
Chapter 1.3.1 --- Whole pancreas and islet transplantation --- p.34
Chapter 1.3.2 --- Problems encountered in islet transplantation --- p.36
Chapter 1.3.3 --- Potential role of RAS in islet transplantation --- p.38
Chapter 1.4 --- Diabetes Mellitus and RAS --- p.40
Chapter 1.4.1 --- Diabetes Mellitus --- p.40
Chapter 1.4.2 --- Type 1 diabetes and its animal model --- p.42
Chapter 1.4.3 --- Type 2 diabetes and its animal model --- p.44
Chapter 1.4.4 --- RAS blockade in diabetes patients --- p.46
Chapter 1.4.5 --- Potential role of RAS in Diabetes Mellitus --- p.47
Chapter 1.5 --- Aims of Study --- p.49
Chapter Chapter 2 --- Materials and Methods
Chapter 2.1 --- Experimental animals and mouse models --- p.50
Chapter 2.1.1 --- Experimental animals for islet isolation and transplantation --- p.50
Chapter 2.1.2 --- Mouse model for type 2 diabetes --- p.51
Chapter 2.2 --- Islet isolation and transplantation --- p.52
Chapter 2.2.1 --- Enzymatic islet isolation --- p.52
Chapter 2.2.2 --- Islet transplantation --- p.53
Chapter 2.3 --- Biological assay on islet functions --- p.53
Chapter 2.3.1 --- Measurement of islet insulin release --- p.53
Chapter 2.3.2 --- Measurement of islet glucose oxidation rate --- p.56
Chapter 2.3.3 --- Measurement of islet (pro)insulin biosynthesis --- p.59
Chapter 2.3.4 --- Measurement of islet total protein synthesis --- p.60
Chapter 2.4 --- Chronic losartan treatment --- p.62
Chapter 2.5 --- Perfusion experiment of transplanted islet graft --- p.62
Chapter 2.6 --- Insulin content of the islet graft --- p.63
Chapter 2.7 --- Islet graft (pro)insulin and total protein biosynthesis --- p.64
Chapter 2.8 --- Real-time RT-PCR Analysis --- p.64
Chapter 2.8.1 --- Design of primers and probes --- p.67
Chapter 2.8.2 --- Use of internal control --- p.69
Chapter 2.8.3 --- RT-PCR reaction --- p.69
Chapter 2.8.4 --- Calculation using the comparative CT method --- p.70
Chapter 2.9 --- Western Blot Analysis --- p.71
Chapter 2.10 --- Immunocytochemistry --- p.72
Chapter 2.11 --- Statistical data analysis --- p.73
Chapter Chapter 3 --- Results
Chapter 3 .1 --- Effect of Angiotensin II and Losartan on islet insulin release --- p.74
Chapter 3.1.1 --- Insulin release from normal islets --- p.74
Chapter 3.2 --- "Effect of Angiotensin II and Losartan on islet glucose oxidation rate, (pro)insulin and total protein biosynthesis" --- p.77
Chapter 3.2.1 --- Glucose oxidation rate of isolated normal islets --- p.77
Chapter 3.2.2 --- (pro)insulin and total protein biosynthesis of isolated normal islets --- p.77
Chapter 3.3 --- Regulation of RAS components in islet transplantation --- p.81
Chapter 3.3.1 --- Expression of RAS components in endogenous islets and transplanted islets --- p.81
Chapter 3.3.2 --- Localization of AT1-receptor in endogenous islets --- p.87
Chapter 3.3.3 --- Expression of AT1-receptor protein in endogenous and transplanted islets --- p.89
Chapter 3.3.4 --- Relative abundance of RAS components in kidney and liver --- p.91
Chapter 3.3.5 --- Insulin release from perfused transplanted islet graft --- p.93
Chapter 3.3.5 --- (pro)insulin and total protein biosynthesis of transplanted islet graft --- p.96
Chapter 3.4 --- Effect of Angiotensin II and losartan on diabetic islets --- p.99
Chapter 3.4.1 --- Expression of RAS components in diabetic pancreas --- p.99
Chapter 3.4.2 --- Localization of AT1 receptors in diabetic pancreas --- p.105
Chapter 3.4.3 --- Insulin release from islets of type 2 diabetic mice --- p.107
Chapter 3.4.4 --- (pro)insulin and total protein biosynthesis of islets from type 2 diabetic mice --- p.112
Chapter Chapter 4 --- Discussion
Chapter 4.1 --- Effect of angiotensin II and losartan on islet insulin release --- p.116
Chapter 4.2 --- Existence of local RAS in pancreatic islets --- p.119
Chapter 4.3 --- Regulation of islet RAS components in transplanted islets --- p.122
Chapter 4.4 --- Clinical relevance of islet RAS in transplantation --- p.125
Chapter 4.5 --- Regulation of islet RAS by type 2 diabetes --- p.126
Chapter 4.6 --- Clinical relevance of islet RAS in type 2 diabetes --- p.134
Chapter 4.7 --- Conclusion --- p.140
Chapter 4.8 --- Further studies --- p.141
Chapter Chapter 5 --- Bibliography --- p.142

Thesis (M.Sc.)--University of Alberta, 2009.
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Surgery, Department of Surgery, University of Alberta. Title from pdf file main screen (viewed on July 28, 2009). Includes bibliographical references.

by Hinny Shuk-Yee Lam.
Year shown on spine: 1997.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.
Includes bibliographical references (leaves 92-117).
Declaration --- p.i
Acknowledgements --- p.ii
Abstract --- p.iii
Table of Contents --- p.v
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Pancreas and Islets of Langerhans --- p.1
Chapter 1.1.1 --- Islet Hormones and Glucose Balance --- p.3
Chapter 1.1.2 --- Glucagon and Its Derived Peptides --- p.4
Chapter A. --- Tissue-specific Post-translational Processing --- p.4
Chapter B. --- Features of Proglucagon Gene --- p.6
Chapter 1.1.3 --- Insulin and Features of Its Gene --- p.9
Chapter 1.2 --- Regulation of Islet Hormone Secretion --- p.12
Chapter 1.2.1 --- Endocrine Control --- p.12
Chapter A --- GIP --- p.13
Chapter B. --- Truncated GLP-1 --- p.13
Chapter 1.2.2 --- Paracrine Control --- p.14
Chapter 1.2.3 --- Neuroendocrine Control --- p.15
Chapter 1.3 --- Neuropeptide Y --- p.16
Chapter 1.3.1 --- NPY in Central Nervous System --- p.17
Chapter 1.3.2 --- NPY in Pancreas --- p.17
Chapter 1.3.3 --- NPY and Islet Hormones --- p.18
Chapter 1.4 --- Synthesis and Secretion --- p.19
Chapter 1.5 --- Objectives of Study --- p.23
Chapter Chapter 2 --- Materials and Methods --- p.26
Chapter 2.1 --- Effects of NPY on Islet Gene Expression --- p.26
Chapter 2.1.1 --- Tissue Culture --- p.26
Chapter A. --- Materials --- p.26
Chapter B. --- Maintenance and Passage --- p.26
Chapter C. --- Experimental Protocol --- p.28
Chapter 2.1.2 --- Total RNA Isolation --- p.28
Chapter A. --- Materials --- p.28
Chapter B. --- Extraction Using FastPrep System --- p.29
Chapter C. --- Quantification of RNA --- p.30
Chapter D. --- Preparation of Reagents --- p.30
Chapter 2.1.3 --- Northern Blot Analysis --- p.31
Chapter A. --- Materials --- p.31
Chapter B. --- Formaldehyde Gel Electrophoresis --- p.32
Chapter C. --- Transfer onto Nylon Membrane --- p.33
Chapter D. --- Labeling of cDNA Probes --- p.34
Chapter E. --- Hybridization and Autoradiography --- p.35
Chapter F. --- Preparation of Reagents --- p.36
Chapter 2.1.4 --- Preparation of cDNA Probe --- p.37
Chapter A. --- Materials --- p.37
Chapter B. --- Preparation of Competent Cells --- p.37
Chapter C. --- Transformation --- p.38
Chapter D. --- Plasmid DNA Isolation --- p.39
Chapter E. --- Restriction Enzyme Digestion --- p.41
Chapter F. --- Agarose Gel Electrophoresis --- p.42
Chapter G. --- Isolation of DNA Fragments --- p.42
Chapter H. --- Preparation of Reagents --- p.43
Chapter 2.1.5 --- Data Analysis --- p.46
Chapter 2.2 --- Effects of NPY on Cytosolic Calcium --- p.46
Chapter 2.2.1 --- Tissue Culture --- p.47
Chapter 2.2.2 --- Confocal Laser Scanning Microscopy --- p.47
Chapter A. --- Materials --- p.47
Chapter B. --- Loading of Dye --- p.48
Chapter C. --- Cytosolic Calcium Measurement --- p.49
Chapter D. --- Preparation of Reagents --- p.49
Chapter Chapter 3 --- Results --- p.51
Chapter 3.1 --- Studies on Islet Gene Expression --- p.51
Chapter 3.1.1 --- Effect of NPY on Proglucagon Expression --- p.51
Chapter A. --- Effect at 11 mM Glucose --- p.51
Chapter B. --- Effect at 5 mM Glucose --- p.52
Chapter 3.1.2 --- Effect of NPY on Proinsulin Expression --- p.52
Chapter 3.1.3 --- "Effect of PYY, PP and FSK on Proglucagon Expression" --- p.53
Chapter 3.2 --- Studies on Cytosolic Calcium --- p.65
Chapter 3.2.1 --- Features of InRlG9 Cells --- p.65
Chapter 3.2.2 --- Effect of NPY on Cellular Calcium Level --- p.66
Chapter Chapter 4 --- Discussion --- p.77
Chapter Chapter 5 --- References --- p.92

Wong, Mei Fung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 207-251).
Abstracts in English and Chinese.
ABSTRACT --- p.i
摘要 --- p.iv
ACKNOWLEDGEMENTS --- p.vi
PUBLICATIONS BASED ON WORK IN THIS THESIS --- p.vii
ABBREVIATIONS --- p.viii
TABLE OF CONTENTS --- p.x
Chapter CHAPTER 1 --- INTRODUCTION --- p.1
Chapter 1.1 --- Diabetes Mellitus --- p.1
Chapter 1.2 --- Structure and Functions of the Pancreas --- p.2
Chapter 1.2.1 --- Size of Pancreatic β-Cells --- p.4
Chapter 1.2.2 --- Signaling Pathways of Insulin Secretion from Pancreatic β-Cells --- p.4
Chapter 1.3 --- Classification of Diabetes --- p.6
Chapter 1.3.1 --- Type 1 Diabetes --- p.6
Chapter 1.3.2 --- Type 2 Diabetes --- p.8
Chapter 1.4 --- Pathologies of Type 2 Diabetes --- p.9
Chapter 1.4.1 --- Hyperglycemia --- p.9
Chapter 1.4.1.1 --- A dvanced Glycosylation End Products --- p.11
Chapter 1.4.1.2 --- Protein Kinase C Activation --- p.13
Chapter 1.4.1.3 --- The Glucosamine Pathway --- p.14
Chapter 1.4.1.4 --- Oxidative Stress --- p.15
Chapter 1.4.2 --- Insulin Resistance --- p.15
Chapter 1.4.3 --- Loss of β-Cell Mass and β-Cell Dysfunction --- p.18
Chapter 1.5 --- Complications of Diabetes Mellitus --- p.21
Chapter 1.5.1 --- Cardiovascular Diseases --- p.21
Chapter 1.5.2 --- Diabetic Retinopathy --- p.22
Chapter 1.5.3 --- Diabetic Nephropathy --- p.23
Chapter 1.5.4 --- Neuropathy --- p.24
Chapter 1.6 --- Anti-Diabetic Drugs for Type 2 Diabetes Mellitus --- p.25
Chapter 1.6.1 --- Secretagogues --- p.25
Chapter 1.6.2 --- Sensitizers --- p.26
Chapter 1.6.3 --- Alpha-Glucosidase Inhibitors --- p.27
Chapter 1.6.4 --- Peptide Analogs --- p.27
Chapter 1.6.4.1 --- Incretin Mimetics --- p.27
Chapter 1.6.4.2 --- Dipeptidyl Peptidase-4 Inhibitors --- p.28
Chapter 1.7 --- Insights of Porcine Islets in Treatment of Diabetics --- p.28
Chapter 1.8 3 --- -Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMG CoA Reductase) --- p.31
Chapter 1.8.1 --- Statins --- p.32
Chapter 1.8.2 --- Pleiotropic Effects of Statins --- p.36
Chapter 1.8.2.1 --- Statins and Isoprenylated Proteins --- p.36
Chapter 1.8.2.2 --- Statins and Endothelial Functions --- p.38
Chapter 1.8.2.3 --- Statins and Platelet Functions --- p.39
Chapter 1.8.2.4 --- Statins and Plaque Stability --- p.39
Chapter 1.8.2.5 --- Statins and Vascular Inflammation --- p.40
Chapter 1.9 --- Clinical Studies of Statins on Diabetics --- p.41
Chapter 1.10 --- Possible Factors Involved in Simvastatin-Regulated Insulin Secretion --- p.44
Chapter 1.10.1 --- AMP-Activated Protein Kinase --- p.44
Chapter 1.10.2 --- Caveolin-1 --- p.46
Chapter 1.10.3 --- Sterol-Regulatory Elementary Binding Protein --- p.50
Chapter 1.10.4 --- Protein Phosphatase 2A --- p.52
Chapter 1.10.5 --- Calcium Sensing Receptor --- p.55
Chapter 1.11 --- Objectives of Study --- p.59
Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.60
Chapter 2.1 --- Materials --- p.60
Chapter 2.1.1 --- Solutions --- p.60
Chapter 2.1.2 --- Antibodies --- p.63
Chapter 2.2 --- Methods --- p.64
Chapter 2.2.1 --- Maintenance of Pancreas Function --- p.64
Chapter 2.2.2 --- Islet Isolation --- p.65
Chapter 2.2.3 --- Hematoxylin and Eosin (H&E) Staining --- p.65
Chapter 2.2.4 --- Simvastatin and Simvastatin-Na+ --- p.66
Chapter 2.2.5 --- AICAR --- p.67
Chapter 2.2.6 --- Compound C --- p.67
Chapter 2.2.7 --- Incubation of Islets --- p.67
Chapter 2.2.8 --- Western Blot --- p.68
Chapter 2.2.9 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.69
Chapter 2.2.10 --- Statistical Analysis --- p.71
Chapter CHAPTER 3 --- HISTOLOGY OF PORCINE PANCREATIC ISLETS OF LANGERHANS --- p.72
Chapter 3.1 --- Comparison of Sizes of Porcine Pancreatic Islets in Histological Sections of Pancreas --- p.72
Chapter CHAPTER 4 --- PROTEIN EXPRESSION OF HMG COA REDUCTASE --- p.75
Chapter 4.1 --- Effect of Incubation Time on HMG CoA Reductase Expression --- p.75
Chapter 4.2 --- Short-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.78
Chapter 4.3 --- Long-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.81
Chapter 4.4 --- Effect of Osmolality on HMG CoA Reductase Expression --- p.83
Chapter 4.5 --- Effect of Simvastatin on Ser871 p-HMG CoA Reductase Expression --- p.87
Chapter CHAPTER 5 --- EVALUATION OF THE ROLE OF SIMVASTATIN IN INSULIN SECRETION VIA HMG CO A REDUCTASE REGULATION --- p.90
Chapter 5.1 --- Effect of Simvastatin on Insulin Secretion --- p.90
Chapter 5.2 --- Effect of Different Concentrations of Simvastatin on Insulin Secretion --- p.94
Chapter 5.3 --- Effect of Simvastatin on Insulin Content --- p.96
Chapter CHAPTER 6 --- ROLE OF AMPK EXPRESSION IN INSULIN SECRETION PATHWAY --- p.100
Chapter 6.1 --- Effect of Simvastatin on Thr172 p-AMPK α and AMPK α1 Expressions --- p.100
Chapter 6.2 --- Evaluation of the Role of Simvastatin in AMPK Regulation --- p.104
Chapter 6.3 --- Evaluation of the Role of PP2A in AMPK Regulation --- p.108
Chapter 6.4 --- Evaluation of the Role of Simvastatin on Insulin Secretion via AMPK Regulation --- p.111
Chapter 6.4.1 --- AMPK Regulation on Releasable Insulin Secretion --- p.111
Chapter 6.4.2 --- AMPK Regulation on Non-Releasable Insulin Content and Total Insulin Content --- p.112
Chapter CHAPTER 7 --- EFFECT OF SIMVASTATIN ON THE EXPRESSION OF REGULATORY PROTEINS INVOLVED IN INSULIN SECRETION --- p.119
Chapter 7.1 --- Effect of Simvastatin on SREBP-2 Expression --- p.119
Chapter 7.2 --- Effect of Simvastatin on Caveolin-1 Expression --- p.121
Chapter 7.3 --- Effect of Simvastatin on Calcium Sensing Receptor Expression --- p.123
Chapter CHAPTER 8 --- EFFECT OF SIMVASTATIN-NA+ ON INSULIN SECRETION --- p.126
Chapter 8.1 --- Effect of Simvastatin-Na+ on HMG CoA Reductase Expression --- p.126
Chapter 8.2 --- Effect of Simvastatin-Na+ on Insulin Secretion --- p.128
Chapter 8.3 --- Effect of Different Concentrations of Simvastatin-Na+ on Insulin Secretion --- p.130
Chapter 8.4 --- Effect of Simvastatin-Na+ on Insulin Content --- p.132
Chapter CHAPTER 9 --- EFFECT OF PRAVASTATIN ON INSULIN SECRETION --- p.136
Chapter 9.1 --- Effect of Pravastatin on Insulin Secretion --- p.136
Chapter 9.2 --- Effect of Pravastatin on Insulin Content --- p.138
Chapter CHAPTER 10 --- EFFECT OF METHYL-B-CYCLODEXTRIN ON INSULIN SECRETION --- p.142
Chapter 10.1 --- Effect of Methyl-β-cyclodextrin on Insulin Secretion --- p.142
Chapter 10.2 --- Effect of Methyl-β-cyclodextrin on Insulin Content --- p.144
Chapter CHAPTER 11 --- DISCUSSION --- p.149
Chapter 11.1 --- Importance of Studying Porcine Pancreatic Islets and Islet Distribution --- p.150
Chapter 11.2 --- Screening of Concentration and Incubation Time of Simvastatin on Porcine Pancreatic Islets --- p.152
Chapter 11.3 --- Glucose-Independent Effect of Simvastatin on Protein Expression of HMG CoA Reductase --- p.154
Chapter 11.4 --- Role of AMPK in HMG CoA Reductase-Modulated Insulin Secretion --- p.159
Chapter 11.5 --- Role of SREBP-2 in Simvastatin-Modulated Regulation --- p.174
Chapter 11.6 --- Role of Calcium Sensing Receptor in Simvastatin-Modulated Regulation --- p.175
Chapter 11.7 --- Role of Caveolin-1 in Simvastatin-Modulated Regulation --- p.179
Chapter 11.8 --- "Effects of Simvastatin-Na+， Pravastatin and Methyl-β-cyclodextrin, and Importance of Endoplasmic Reticulum in Insulin Secretion" --- p.183
Chapter CHAPTER 12 --- CONCLUSIONS AND FURTHER STUDIES --- p.197
Chapter 12.1 --- Conclusions --- p.197
Chapter 12.2 --- Further Studies --- p.203
REFERENCES --- p.207

Metabolic syndrome defines a cluster of related comorbidities including obesity, Type 2 diabetes, fatty liver disease, and cardiovascular diseases. Increasingly prevalent in Western countries, metabolic syndrome diseases are a major focus of efforts to understand the complex genetics that underlie disease risk and severity. Immunoglobulin domain-containing receptor 2 (ILDR2) is an ER transmembrane protein first identified as a candidate genetic modifier of diabetes susceptibility in the context of obesity. Obese, leptin-deficient mice with hypomorphic Ildr2 expression had hypoinsulinemic hyperglycemia with reduced beta cell mass, suggesting that ILDR2 plays a role in maintain beta cell mass and function. Further studies proposed a role for ILDR2 in hepatic lipid metabolism as Ildr2 shRNA-mediated knockdown (KD) caused hepatic steatosis in mice. The goal of this thesis work is to clarify the role of ILDR2 in diabetes and hepatic steatosis in an effort to elucidate the specific mechanism of ILDR2. We developed a conditional Ildr2 knockout (KO) allele, enabling tissue-specific ablation in mice. Liver-specific and hepatocyte-specific KO mice did not develop hepatic steatosis. However, liver-specific KO mice treated with adenoviral Ildr2 shRNA accumulated hepatic triglycerides, suggesting off-target effects of the shRNA. Using RNA sequencing and sequence alignment, several gene candidates for shRNA off-targeting effect were identified. Future studies are proposed to elucidate role(s) of these genes in the previously described phenotype of Ildr2 KD mice. I conclude that Ildr2 ablation may contribute to the development of hepatic steatosis, but does not play a major role in hepatic lipid metabolism. We also developed beta cell-specific (RIP2-cre) and pancreas-specific (Pdx-cre) Ildr2 KO mice and characterized them for diabetic phenotypes. Pancreas-specific KO mice displayed impaired glucose tolerance, reduced insulin secretion and decreased calcium signaling in islets. These results confirm a role for ILDR2 in islet cell function. Experiments performed in RIP2-cre beta cell-specific KO mice were confounded by effects of the Cre construct, prohibiting definitive conclusions about the role of ILDR2 in the beta cell. Additionally, because Ildr2 is expressed at low levels in beta cells, we propose that ILDR2 may function in islet macrophages. Overall, this work defines the metabolic functions of ILDR2, clarifying its role in hepatic lipid metabolism, and confirming its role in islet cell function. In addition, I discuss preliminary evidence suggesting that ILDR2 may function in the brain to regulate body weight and metabolism.

Diabetes is a complex group of metabolic disorders with genetic, immunological, and environmental etiologies. Decades of diabetes research have elucidated many genetic drivers of normal islet function and dysfunction. Furthermore, genome wide associated studies (GWAS) have discovered that most diabetes susceptibility loci fall outside of coding regions, which suggests a role for noncoding elements in the development of disease. This highlights our incomplete understanding of the islet regulome and suggests the need for detailed functional analyses of noncoding genes to precisely determine their contribution to diabetes susceptibility and disease progression. Transcriptome analyses have revealed that the eukaryotic genome is pervasively transcribed. Strikingly, only a small proportion of the transcriptome is subsequently translated into protein; the majority is made up non-protein coding RNAs (ncRNAs). The most abundant class of these ncRNAs are called long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides that lack protein-coding potential. The establishment of lncRNAs, once dismissed as genomic dark matter, as essential gene regulators in many biological processes has redefined the central role for RNA in cells. While evidence suggests a role for lncRNAs in islets and diabetes, in vivo functional characterization of islet lncRNAs is lacking. For my thesis project, I sought to understand the lncRNA regulatory mechanisms that promote pancreas development and function. We conducted comparative transcriptome analyses between embryonic mouse pancreas and adult mouse islets and identified several pancreatic lncRNAs that lie in close proximity to essential pancreatic transcription factors. One of the candidate lncRNAs, Pax6 Upstream Antisense RNA (Paupar), mapped near Pax6, a gene encoding an essential pancreatic regulatory protein. We demonstrate Paupar is enriched in glucagon-producing alpha cells where it promotes the alternative splicing of Pax6 to an isoform required for activation of essential alpha cell genes. Consistently, deletion of Paupar in mice resulted in dysregulation of Pax6 alpha cell target genes and corresponding alpha cell dysfunction. These findings illustrate a distinct mechanism by which lncRNAs can contribute to cell-specific regulation of broadly expressed transcription factors to coordinate critical functions within a cell.

Ng, Ka Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 179-204).
Abstracts in English and Chinese.
Abstract --- p.I
摘要 --- p.IV
Publications --- p.VII
Acknowledgements --- p.VIII
Table of contents --- p.IX
List of figures --- p.XV
List of tables --- p.XVII
List of abbreviations --- p.XVIII
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- The Pancreas --- p.2
Chapter 1.1.1 --- Anatomy of Pancreas --- p.2
Chapter 1.1.2 --- The Exocrine Pancreas --- p.4
Chapter 1.1.3 --- The Endocrine Pancreas --- p.5
Chapter 1.1.3.1 --- Structure of Islets --- p.5
Chapter 1.1.3.2 --- "Functions of α-, β-, y-, ð-, Σ-and PP-cells in Islets" --- p.7
Chapter 1.1.4 --- Overview of Pancreas Development --- p.9
Chapter 1.1.4.1 --- Organ Morphology --- p.10
Chapter 1.1.4.2 --- Cyto-differentiation --- p.12
Chapter 1.1.4.3 --- Control by Transcriptional Factors --- p.14
Chapter 1.1.5 --- Postnatal Pancreas Development and Regeneration --- p.18
Chapter 1.1.5.1 --- Proliferation of Pre-existing β-cells --- p.19
Chapter 1.1.5.2 --- Neogenesis from Precursor Cells --- p.20
Chapter 1.1.5.3 --- Transdifferentiation of other Cells --- p.20
Chapter 1.2 --- Diabetes Mellitus --- p.22
Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus and Current Treatments --- p.24
Chapter 1.2.1.1 --- Type I Diabetes Mellitus --- p.24
Chapter 1.2.1.2 --- Type II Diabetes Mellitus --- p.25
Chapter 1.2.1.3 --- Gestational Diabetes --- p.27
Chapter 1.2.1.4 --- Secondary Diabetes --- p.28
Chapter 1.3 --- Stem Cell therapy --- p.29
Chapter 1.3.1 --- Stem Cell --- p.29
Chapter 1.3.1.1 --- Mesenchymal Stem Sell --- p.31
Chapter 1.3.1.2 --- Embryonic Stem Cell --- p.35
Chapter 1.3.1.3 --- Induced Pluripotent Stem Cell --- p.36
Chapter 1.3.2 --- Islets Engineering --- p.37
Chapter 1.3.2.1 --- Genetic Modification --- p.37
Chapter 1.3.2.2 --- Directed Differentiation --- p.38
Chapter 1.3.2.3 --- Microenvironment --- p.38
Chapter 1.3.2.4 --- In vivo Regeneration --- p.39
Chapter 1.3.2.5 --- Cell Fusions --- p.40
Chapter 1.3.2.6 --- Combinatory Treatments --- p.40
Chapter 1.4 --- The Vitamin A & Vitamin D System --- p.42
Chapter 1.4.1 --- The Vitamin A --- p.42
Chapter 1.4.2 --- Vitamin A Metabolism --- p.44
Chapter 1.4.3 --- Roles of vitamin A in Pancreatic Development --- p.46
Chapter 1.4.4 --- The Vitamin D --- p.48
Chapter 1.4.5 --- Vitamin D Metabolism --- p.49
Chapter 1.4.6 --- Metabolic Functions of Vitamin D in Islets --- p.51
Chapter 1.4.7 --- Cod Liver Oil --- p.53
Chapter 1.4.8 --- Interactions between Vitamin A and Vitamin D --- p.53
Chapter 1.5 --- The Relations of Liver and Pancreas Development --- p.55
Chapter 1.5.1 --- Endoderm Induction for Hepatic and Pancreatic Differentiation of ESCs --- p.55
Chapter 1.5.2 --- Bipotential Precursor Population within Embryonic Endoderm --- p.56
Chapter 1.5.3 --- Pancreatic Islets Promote Mature Liver Hepatocytes Proliferation --- p.57
Chapter 1.5.4 --- Transdifferentiation --- p.57
Chapter 1.5.5 --- Transplantation in Liver Niche Promotes Maturation of Insulin-Producing Cells --- p.60
Chapter 1.5.6 --- Neuronal Relay from the Liver to Pancreatic --- p.61
Chapter 1.5.7 --- Development of Islets in the Nile Tilapia --- p.62
Chapter 1.6 --- The Insulin-like Growth Factor-I (IGF1) --- p.64
Chapter 1.6.1 --- IGF1 System --- p.64
Chapter 1.6.2 --- IGF 1 Regulation --- p.65
Chapter 1.6.3 --- Roles of IGF 1 in Pancreatic Development and Regeneration --- p.68
Chapter 1.7 --- Aims and Objectives of Study --- p.70
Chapter Chapter 2 --- General Materials and Methods
Chapter 2.1 --- Pancreatic progenitor cells (PPCs) and liver stromal cells (LSCs) isolation and cell culture --- p.72
Chapter 2.1.1 --- Tissue procurement --- p.72
Chapter 2.1.2 --- PPC and LSC culture --- p.72
Chapter 2.1.3 --- "Treatments of vitamin A, vitamin D and IGF 1" --- p.76
Chapter 2.1.4 --- "Cell culture of Caco-2, HepG2 and DU-145" --- p.76
Chapter 2.2 --- Induction of Islet-like Cell Clusters (ICCs) Differentiation --- p.77
Chapter 2.2.1 --- In vitro Directed Differentiation --- p.77
Chapter 2.2.2 --- In vitro LSC Microenvironment --- p.77
Chapter 2.3 --- RNA Expression Detection --- p.79
Chapter 2.3.1 --- RNA isolation --- p.79
Chapter 2.3.2 --- Reverse Transcription --- p.79
Chapter 2.3.3 --- Polymerase Chain Reaction (PCR) --- p.80
Chapter 2.3.4 --- Realtime PCR --- p.81
Chapter 2.4 --- Immunocytochemistry --- p.83
Chapter 2.5 --- Western Blotting --- p.85
Chapter 2.5.1 --- Protein extraction and quantification --- p.85
Chapter 2.5.2 --- Western Blotting --- p.85
Chapter 2.6 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.87
Chapter 2.6.1 --- Detection of cell viability --- p.87
Chapter 2.6.2 --- Detection of cell proliferation --- p.87
Chapter 2.6.3 --- Measurement of Cell death --- p.88
Chapter 2.6.4 --- Measurement of IGF 1 level in condition medium --- p.89
Chapter 2.6.5 --- Measurement of glucose induced insulin secretion --- p.90
Chapter 2.7 --- Regeneration model --- p.92
Chapter 2.7.1 --- Regeneration model in neonatal-STZ rat --- p.92
Chapter 2.7.2 --- Change in IGF1 expression in pancreas and liver --- p.92
Chapter 2.8 --- Statistical Data Analysis --- p.93
Chapter Chapter 3 --- Vitamin D and vitamin A receptor expression and the proliferative effects of ligand activation of these receptors on the development of pancreatic progenitor cells derived from human fetal pancreas. (Stem Cell Rev. 2011;7:53-63)
Chapter 3.1 --- Abstract --- p.95
Chapter 3.2 --- Introduction --- p.97
Chapter 3.3 --- Materials and Methods --- p.101
Chapter 3.3.1 --- Fetal Tissue Procurement --- p.101
Chapter 3.3.2 --- Culture of Pancreatic Progenitor Cells --- p.101
Chapter 3.3.3 --- RNA Expression Analysis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.102
Chapter 3.3.4 --- Western Blot Analysis --- p.103
Chapter 3.3.5 --- Immunocytochemstry --- p.105
Chapter 3.3.6 --- PPC Proliferation Assays --- p.106
Chapter 3.3.7 --- PPC Cell Death Assays --- p.107
Chapter 3.3.8 --- Statistical Data Analysis --- p.108
Chapter 3.4 --- Results --- p.110
Chapter 3.4.1 --- "Expression and Localization of RAR, VDR and RXR, CYP26 and CYP24 in PPCs" --- p.110
Chapter 3.4.2 --- Incubation of PPC with atRA Enhances PPC Viability due to Increased Proliferation and Anti-apoptosis --- p.111
Chapter 3.4.3 --- Incubation of PPCs with Calcitriol Enhances Viability due to Increased Proliferation --- p.111
Chapter 3.4.4 --- Both atRA and Calcitriol Induce Up-regulation of both the RAR and the VDR but not the RXR --- p.112
Chapter 3.4.5 --- Combination Treatment with atRA and Calcitriol on Cell Viability and NGN3 Expression --- p.112
Chapter 3.5 --- Discussion --- p.114
Chapter Chapter 4 --- Human fetal liver stromal cell co-culture enhances the growth and differentiation of pancreatic progenitor cells into islet-like cell clusters (In submission to Gastroenterology)
Chapter 4.1 --- Abstract --- p.128
Chapter 4.2 --- Introduction --- p.129
Chapter 4.3 --- Materials and Methods --- p.133
Chapter 4.3.1 --- Use of human and animal tissues --- p.133
Chapter 4.3.2 --- "Cell preparation, characterizations and Differentiation" --- p.133
Chapter 4.3.3 --- Examination of PPC growth and ICC differentiation and functions with LSC co-culture --- p.133
Chapter 4.3.3 --- Identification of growth factors and investigation of their effects --- p.134
Chapter 4.3.4 --- Statistical Analysis --- p.135
Chapter 4.4 --- Results --- p.136
Chapter 4.4.1 --- "Isolation, Culture and Characterizations of LSCs" --- p.136
Chapter 4.4.2 --- Establishment of LSC co-culture system --- p.136
Chapter 4.4.3 --- LSC co-culture enhances PPC-derived ICC differentiation --- p.137
Chapter 4.4.4 --- Differential expression of mRNA for cytokines and growth factors between 1st and 2nd trimester LSCs --- p.138
Chapter 4.4.5 --- Characterization of IGF 1 receptors in PPCs and the effects of exogenous IGF1 on PPC growth and ICC differentiation --- p.139
Chapter 4.4.6 --- Neutralizing antibodies against IGF1R inhibit ICC differentiation --- p.140
Chapter 4.5 --- Discussion --- p.142
Chapter 4.6 --- Supplementary Materials and Methods --- p.147
Chapter 4.6.1 --- Cell Preparation and culture --- p.147
Chapter 4.6.2 --- In Vitro ICC differentiation --- p.148
Chapter 4.6.3 --- RNA expression analysis --- p.149
Chapter 4.6.4 --- Immunocytochemistry --- p.149
Chapter 4.6.5 --- PPC viability and cell count assays --- p.150
Chapter 4.6.6 --- IGF1 and insulin ELISA --- p.151
Chapter 4.6.7 --- Western blotting analysis --- p.152
Chapter 4.6.8 --- Neonatal streptozotocin regeneration model --- p.153
Chapter Chapter 5 --- General Discussion and Future Studies
Chapter 5.1 --- General Discussion --- p.165
Chapter 5.1.1 --- Proliferative effects and enhance expression of NGN3 by vitamin A and vitamin D on PPC --- p.166
Chapter 5.1.2 --- Induction of PPC derived ICCs by LSCs --- p.169
Chapter 5.1.3 --- Potential effects of liver stroma derived IGF1 on PPC derived ICCs differentiation --- p.172
Chapter 5.1.4 --- Significance of islet engineering in the management of diabetes --- p.174
Chapter 5.1.5 --- Conclusions --- p.176
Chapter 5.2 --- Future Studies --- p.177
Chapter Chapter 6 --- Reference
Reference --- p.180

Another growth factor candidate is a recently recognized bioactive peptide, islet-neogenesis associated protein (INGAP). A master pancreatic transcription factor, pancreatic duodenal homeobox-1 (Pdx-1), was overexpressed in PSCs by the adenovirus-mediated transfer method in the present study. With the infection of adenovirus expressing Pdx-1, several beta-cell developmental genes, including Isl-1, Beta2, Nkx2.2, Nkx6.1 and the endogenous Pdx-1 were found to be upregulated temporally in our PSCs-derived ICCs. Meanwhile, previous study has shown that Pdx-1/INGAP-positive cells represent a new stem cell subpopulation during early stage of pancreatic development. We thus explore whether any functional integration of Pdx-1 and INGAP in the growth and functional maturation of PSCs. In order to achieve this proposition, the effects of over-expressing PSCs with the Pdx-1 adenovirus in conjunction with the treatment of INGAP were then investigated. Interestingly, differentiation of the PSC-derived ICCs was not further enhanced by the synergistic treatment of Pdx-1 and INGAP when compared to those ICCs infected with adenovirus expressing Pdx-1 alone, as revealed by the endogenous Pdx-1 and insulin gene expression and their C-peptide content. These data might provide some clues to the intricate interaction between Pdx-1 and INGAP in regulating the ICC and/or the pancreatic endocrine differentiation. (Abstract shortened by UMI.)
Due to the scarcity of fetal pancreas for generating functional insulin-secreting cell clusters for sufficient islet transplantation, we targeted for searching pancreatic stem/progenitor cells. Putative PSCs can be aggregated and differentiated into islet-like cell clusters (ICCs) when exposed to serum-free medium containing various conventional growth factors, including HGF, GLP-1, betacellulin and nicotinamide.
Fetal pancreatic tissue consisting of immature progenitor cells serves as a potential source of stem cells as they possess a higher replicative capacity and longevity than their adult counterparts.
Two novel candidates and a key pancreatic transcription factor on the PSC/ICC proliferation and differentiation were investigated in the present study. One of them is a ubiquitously expressed multi-PDZ-domain protein, PDZ-domain-containing 2 (PDZD2), which was previously found to express in the mouse beta cells and exhibit mitogenic effects in beta cell line. Results showed that PDZD2 was detected in high levels in both human fetal pancreas and in PSCs. Results indicate the potential involvement of PDZD2 in regulating PSCs proliferation and differentiation and pancreatic development.
Suen Po Man, Ada.
"July 2007."
Adviser: P.S. Leung.
Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0051.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 194-214).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Ma, Man Ting.
"July 2010."
Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 186-207).
Abstracts in English and Chinese.
Abstract --- p.I
List of Publications --- p.VI
Acknowledgements --- p.VIII
Table of Contents --- p.X
List of Figures --- p.XV
List of Tables --- p.XVIII
List of Abbreviations --- p.XIX
Chapter CHAPTER1 --- INTRODUCTION
Chapter 1.1 --- The Pancreas --- p.2
Chapter 1.1.1 --- Structure of pancreas --- p.2
Chapter 1.1.2 --- Structure and function of exocrine pancreas --- p.6
Chapter 1.1.3 --- Structure and function of endocrine pancreas --- p.9
Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.9
Chapter 1.1.3.2 --- Glucose-stimulated insulin secretion from islets --- p.12
Chapter 1.2 --- Type 1 Diabetes Mellitus (T1DM) --- p.14
Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus --- p.14
Chapter 1.2.2 --- Autoimmunity in T1DM --- p.17
Chapter 1.2.3 --- Management ofTlDM --- p.20
Chapter 1.2.3.1 --- Insulin replacement --- p.20
Chapter 1.2.3.2 --- Pancreas and islet transplantation --- p.21
Chapter 1.2.3.3 --- Stem-cell-based transplantation --- p.22
Chapter 1.3 --- The Adaptive Immune System --- p.26
Chapter 1.3.1 --- T-lymphocytes --- p.26
Chapter 1.3.2 --- B-lymphocytes --- p.29
Chapter 1.3.3 --- Major histocompatibility complex (MHC) --- p.30
Chapter 1.3.3.1 --- Classification of MHC molecules --- p.30
Chapter 1.3.3.2 --- Structure of MHC class I and II molecules --- p.32
Chapter 1.3.3.3 --- Function and regulation of MHC molecules --- p.34
Chapter 1.3.4 --- HLA-G and its immuno-modulatory properties --- p.36
Chapter 1.4 --- Transplantation Rejection --- p.40
Chapter 1.4.1 --- Mechanisms involved in transplantation rejection --- p.40
Chapter 1.4.2 --- Immunobiology of rejection --- p.41
Chapter 1.4.2.1 --- Direct allorecognition pathway --- p.42
Chapter 1.4.2.2 --- Indirect allorecognition pathway --- p.43
Chapter 1.4.2.3 --- Semi-direct allorecognition pathway --- p.43
Chapter 1.4.3 --- Xenotransplantation --- p.46
Chapter 1.5 --- Cytokines and Immunity --- p.48
Chapter 1.5.1 --- Interferons --- p.48
Chapter 1.5.1.1 --- Interferon-γ and its immune regulation --- p.49
Chapter 1.5.1.2 --- Effect and kinetics of interferon-γ on MHC molecules expression --- p.53
Chapter 1.5.1.3 --- Regulation of interferon-γ production --- p.56
Chapter 1.5.2 --- Interlukins --- p.58
Chapter 1.5.2.1 --- IL-10 and its immune regulation --- p.58
Chapter 1.5.2.2 --- IL-10 and HLA-G --- p.59
Chapter 1.6 --- Stem Cells and their Immunogenicity --- p.62
Chapter 1.6.1 --- Embroynic stem cells --- p.62
Chapter 1.6.2 --- Mesenchymal stem cells --- p.64
Chapter 1.6.3 --- Neural stem cells --- p.68
Chapter 1.6.4 --- Fetal stem cells --- p.69
Chapter 1.6.5 --- Potential immuno-study in human fetal pancreatic stem cells --- p.70
Chapter 1.7 --- Aims and Objectives of study --- p.72
Chapter CHAPTER2 --- MATERIALS AND METHODS
Chapter 2.1 --- Isolation of Pancreatic Progenitors (PPCs) from Human Fetal Pancreas and Induction of Islet-like Cell Cluster (ICCs) Differentiation --- p.75
Chapter 2.1.1 --- Tissue procurement --- p.75
Chapter 2.1.2 --- Tissue processing and PPCs culture --- p.75
Chapter 2.1.3 --- In vitro differentiation of PPCs into ICCs --- p.78
Chapter 2.1.4 --- Interferon-γ and IL-10 treatment --- p.80
Chapter 2.2 --- Cell culture of human placental Choriocarcinoma JEG-3 Cell Line --- p.81
Chapter 2.3 --- RNA Expression Detection --- p.82
Chapter 2.3.1 --- RNA isolation --- p.82
Chapter 2.3.2 --- Reverse transcriptase (RT) --- p.83
Chapter 2.3.3 --- Design of primers for Polymerase Chain Reaction (PCR) and Real-time PCR --- p.84
Chapter 2.3.4 --- PCR --- p.86
Chapter 2.3.5 --- Real-time PCR analysis --- p.88
Chapter 2.3.6 --- Calculation using the comparative CT method --- p.90
Chapter 2.4 --- Flow Cytometry --- p.91
Chapter 2.5 --- Western Blotting Analysis --- p.93
Chapter 2.5.1 --- Protein extraction and quantification --- p.93
Chapter 2.5.2 --- Western blotting --- p.93
Chapter 2.6 --- Mixed Lymphocyte Reaction (MLR) --- p.95
Chapter 2.6.1 --- Isolation of peripheral blood mononuclear cells (PBMCs) --- p.95
Chapter 2.6.2 --- PPC-PBMCs MLR --- p.98
Chapter 2.6.3 --- ICC-PBMCs MLR --- p.98
Chapter 2.6.4 --- Proliferation assay --- p.99
Chapter 2.7 --- ICC Transplantation --- p.101
Chapter 2.7.1 --- Streptozotocin-induced diabetic animals for transplantation --- p.101
Chapter 2.7.2 --- Procedures of ICCs transplantation --- p.102
Chapter 2.8 --- Histological Analysis of ICC Graft --- p.105
Chapter 2.8.1 --- H&E staining --- p.105
Chapter 2.8.2 --- DAB staining --- p.106
Chapter 2.8.3 --- Immunofluorescence staining --- p.107
Chapter 2.9 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.109
Chapter 2.10 --- Statistical Data Analysis --- p.110
Chapter CHAPTER3 --- RESULTS
Chapter 3.1 --- Immuno-characterization of PPCs and ICCs --- p.112
Chapter 3.2 --- Effect of cytokines on immune-properties of PPCs and ICCs --- p.115
Chapter 3.2.1 --- Effect of lFN-γ on MHC-I expression in PPCs --- p.115
Chapter 3.2.2 --- Effect of lFN-γ and IL-10 on HLA-G expression in PPCs and ICCs --- p.119
Chapter 3.2.3 --- Effect of IFN-γ on B7H4 expression in PPCs --- p.123
Chapter 3.3 --- Comparison of immune-properties of PPCs and ICCs from 1st and 2nd trimester --- p.125
Chapter 3.3.1 --- Differential expression of MHC molecules in PPCs --- p.125
Chapter 3.3.2 --- Different immune-related gene expression in PPCs and ICCs --- p.128
Chapter 3.3.3 --- Comparison of IFN-γ activated MHC molecules expression in PPCs/ICCs --- p.134
Chapter 3.3.4 --- Comparison of other IFN-γ activated genes expression in PPCs --- p.139
Chapter 3.4 --- Mixed lymphocyte reaction of PPCs from 1st and 2nd trimester --- p.143
Chapter 3.4.1 --- Effect of PPCs on proliferation of PBMC --- p.143
Chapter 3.4.2 --- Effect of ICCs on proliferation of PBMC --- p.145
Chapter 3.4.3 --- Effect of PPCs on cytokine production in PBMC --- p.149
Chapter 3.5 --- Xenotransplantation of ICCs into diabetic mouse model --- p.152
Chapter 3.5.1 --- Blood glucose level of diabetic mice after transplantation --- p.152
Chapter 3.5.2 --- Histological evaluation of transplanted ICCs grafts --- p.154
Chapter 3.5.3 --- Infiltration of CD45 into transplanted grafts of 1st and 2nd trimester --- p.158
Chapter CHAPTER4 --- DISCUSSION
Chapter 4.1 --- Expression of selected immuno-regulated genes in PPCs and ICCs --- p.163
Chapter 4.2 --- Effect of IFN-g and IL-10 on expression of immuno-regulated genes in PPCs and ICCs --- p.166
Chapter 4.3 --- In vitro studies on immunogenicity of PPCs and ICCs from first and second trimester --- p.171
Chapter 4.3.1 --- Immune-related genes expression --- p.171
Chapter 4.3.2 --- IFN-γ activated gene expression --- p.173
Chapter 4.3.3 --- Mixed lymphocyte reaction --- p.175
Chapter 4.3.4 --- Cytokine production of PBMC in MLR --- p.179
Chapter 4.4 --- In vivo Xenotransplantation of ICCs into diabetic mouse model --- p.181
Chapter 4.5 --- Conclusion --- p.187
Chapter 4.6 --- Further studies --- p.188
Chapter CHAPTER5 --- BIBLIOGRAPHY
Bibliography by Alphabetical Order --- p.189

Fetal pig islet-like cell clusters (ICCs) were transplanted into the thymus or omentum of STZ-induced diabetic pigs immunosuppressed with cyclosporine (CsA) and deoxyspergualin (DSG), as a potential treatment for type 1 diabetes. C-peptide levels in response to glucagon and arginine significantly increased over time using 1 litter of ICCs with highest levels obtained at 100 days post-transplantation. Increasing the number of ICCs to 2 litters was not advantageous. Histology of the graft showed all 4 pancreatic endocrine cells. Normoglycaemia was achieved for transient periods without insulin administration in 4 out of 12 pigs. These results suggest sub-optimal insulin production, possibly due to the adverse effects of CsA on the grafted β cells. The effect of CsA on adult porcine β cells was investigated and adverse effects were shown. Renal toxicity and adverse changes to the haematological parameters did not occur despite high CsA levels although minimal toxicity to the liver was observed. The results indicate that the use of CsA monotherapy in the maintenance phase to prevent rejection of allografted pancreatic β cells may become a subsequent problem over time. As an alternative to chronic immunossuppression, anti-CD3 monoclonal antibody was administered for 10 days in pigs. Using anti-CD3 alone, only 1 out 4 pigs showed cells positive for insulin. The addition of a 5-day CsA course administered the day before transplantation did not promote allograft survival. The use of DSG for 10 days with anti-CD3 promoted graft survival with the histology showing the 4 endocrine cells 3 weeks post-transplantation. An attempt was made to replace any form of immunossuppression by encapsulating fetal pig ICCs in barium alginate, which were able to remain viable when transplanted in NOD/SCID mice. Fibrosis was detected in BALB/c mice transplanted with encapsulated fetal ICCs suggesting that fetal pig ICCs shed antigens that elicit an immune response. Results from this study show that although fetal pig ICCs may be a viable source of insulin-producing cells, the use of CsA to prevent rejection has adverse effects on graft function. Encapsulation as well as transient immunosuppression is worthy of further investigation to prevent rejection of fetal pig ICCs.

Pancreatic beta cell specification is a complex process, requiring proper function of numerous transcription factors. Nkx2.2 is a transcription factor that is crucial for beta cell formation, and is expressed early and throughout pancreatic development. Nkx2.2-/- mice display complete loss of the beta cell lineage and defects in the specification of other endocrine cell types, demonstrating the importance of Nkx2.2 in establishing proper endocrine cell ratios. Recent studies have also demonstrated a role for Nkx2.2 within the mature beta cell to maintain identity and function. This thesis work investigated the timing of pancreatic beta cell specification and the mechanism of this process. In these studies, Nkx2.2 was ablated specifically within the Ngn3-expressing endocrine progenitor population in vivo. These mice displayed defects similar to Nkx2.2-/- mice. Surprisingly, the disruption of endocrine cell specification did not require loss of expression of multiple essential transcription factors known to function downstream of Nkx2.2, including Ngn3, Rfx6, and NeuroD1. While these factors are all necessary for beta cell specification, their preserved expression did not rescue beta cell formation. ChIP-Seq analyses also revealed co-occupancy of Nkx2.2, Rfx6, and NeuroD1 near endocrine-related genes, suggesting Nkx2.2 may cooperate with its downstream targets to regulate beta cell fate. These results have revealed a unique requirement for Nkx2.2 during a critical window of beta cell development. In addition, the role of a conserved domain of Nkx2.2, the specific domain (SD), was assessed using Nkx2.2SDmutant mice. Transcriptional profiling of Nkx2.2SDmutant endocrine progenitors revealed a critical role for the SD domain in regulating the transcription of endocrine fate genes early in the process of endocrine differentiation. In addition, beta cell-specific deletion of the Nkx2.2 SD domain resulted in hyperglycemia, glucose intolerance and dysregulation of beta cell functional genes. This suggests the SD domain is important for mediating Nkx2.2 function within the beta cell to maintain glucose homeostasis. Together, these results have elucidated a critical developmental window for beta cell specification and demonstrated an essential role for Nkx2.2 and specifically its SD domain in this process. Furthermore, these studies suggest that beta cell transcription factors may also regulate endocrine fate in a combinatorial manner, and exert changes within the endocrine progenitor lineage. These findings have provided us with a better understanding of in vivo pancreatic development, and will improve current research efforts to differentiate beta cells in vitro from hPSCs.