O transplante de ilhotas microencapsuladas constitui uma alternativa terapêutica interessante para o Diabetes Mellitus tipo 1, permitindo um melhor controle glicêmico e eliminando a necessidade de imunossupressão. Entretanto, a manutenção a longo prazo da viabilidade das células-&#946; ainda é um desafio. No isolamento, a perda da matriz extracelular e as condições hipóxicas subsequentes afetam decisivamente a sobrevivência e funcionalidade das ilhotas. Objetivo Para diminuir o estresse sobre o enxerto, levando a um sucesso prolongado do transplante, propôs-se a adição de perfluorocarbono (PFC) ou laminina (LN), moléculas associadas respectivamente à oxigenação e interações célula-célula, ao biomaterial baseado em alginato, Biodritina, adequado ao encapsulamento celular. Metodologia Para testar a estabilidade das formulações PFC-Biodritina e LN-Biodritina, microcápsulas foram submetidas a diferentes estresses (rotacional, osmótico, temperatura e cultura) por 7 e 30 dias. A pureza do biomaterial foi avaliada pela coincubação com macrófagos murinos RAW264.7, por 3, 9 e 24h, quando a ativação dos macrófagos foi observada pela expressão gênica de IL- 1&#946; e TNF&#945;. Microcápsulas implantadas i.p. em camundongos foram recuperadas após 7 ou 30 dias, para análises de biocompatibilidade. A expressão de níveis de mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulina 1 e 2), proteínas (Bax, Bcl-XL e Xiap) e a atividade de Caspase3 foram avaliadas em ilhotas microencapsuladas com PFC- e LN-Biodritina, após cultura de 48h em condições de normóxia e hipóxia (<2% O2). Camundongos diabéticos foram transplantados com ilhotas encapsuladas nas diferentes formulações e os animais foram monitorados pelas variações de massa corporal, glicêmicas e pela funcionalidade do enxerto (TOTGs). As ilhotas foram recuperadas de animais normo ou hiperglicêmicos e uma análise de biocompatibilidade das cápsulas foi realizada, assim como a avaliação funcional das células-&#946;. Após o explante, a glicemia dos animais normoglicêmicos foi monitorada para se atestar a eficiência das ilhotas transplantadas. Resultados Microcápsulas de PFC- e LN-Biodritina são tão estáveis e biocompatíveis quanto as de Biodritina. Para ilhotas encapsuladas em ambos os materiais, em normóxia ou hipóxia, observou-se uma modulação gênica que sugere proteção contra apoptose. Adicionalmente, encontrou-se uma diminuição na expressão de genes indicadores de estresse (mcp1, hsp70). Uma diminuição nos níveis de mRNA de ldh foi vista para PFC-Biodritina, mas o oposto foi encontrado para LN-Biodritina. As diferenças encontradas na expressão proteica sugerem o mesmo padrão anti-apoptótico. Caspase3 não foi modulada por nenhum biomaterial. Nos experimentos de transplante, apenas LN-Biodritina levou reversão prolongada do diabetes, com 60% dos animais normoglicêmicos, 198 dias pós-cirurgia, comparado a 9% do grupo Biodritina. O TOTG demonstrou que camundongos transplantados com ilhotas encapsuladas secretaram mais insulina do que controles, 60 (LN-Biodritina) ou 100 (PFC- e LN-Biodritina) dias pós-cirurgia. O explante restabeleceu a hiperglicemia nos camundongos. Microcápsulas recuperadas de animais hiperglicêmicos apresentavam uma extensa adesão celular. Testes de secreção de insulina in vitro demonstraram que somente ilhotas do grupo normoglicêmico responderam às variações da concentração de glicose. Conclusão A adição de moléculas bioativas à Biodritina é capaz de diminuir o estresse em ilhotas isoladas e tem o potencial de melhorar a terapia pelo transplante de ilhotas. / Transplantation of microencapsulated islets represents an attractive therapeutical approach to treat type 1 Diabetes Mellitus, accounting for an improved glycemic control and the abolishment of immunosuppressive therapies. However, maintenance of long-term &#946;-cell viability remains a major problem. During islet isolation, the loss of extracellular matrix interactions and the hypoxic conditions thereafter dramatically affect &#946;-cell survival and function. Objective To lessen the burden of islet stress and achieve a better outcome in islet transplantation we tested the addition of perfluorocarbon (PFC) or laminin (LN), molecules associated respectively with oxygenation and cell-cell interaction, to Biodritin, an alginate-based material suitable for cell microencapsulation. Methodology To test the stability of PFC-Biodritin and LN-Biodritin composites, microcapsules were subjected to different stresses (rotational, osmotic, temperature and culture) for 7 and 30 days. To assess biomaterial purity microcapsules were co-incubated with RAW264.7 murine macrophage cell line for 3, 9 and 24h and macrophage activation was detected through mRNA levels of IL-1&#946; and TNF&#945;. Microcapsules were implanted i.p. in mice and retrieved after 7 or 30 days, for biocompatibility analyses. Gene expression at mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulin 1 and 2) and protein (Bax, Bcl-XL and Xiap) levels, together with Caspase3 activity, were evaluated in islets microencapsulated in PFC- or LN-Biodritin, upon culturing for 48h in normoxic or hypoxic (<2% O2) conditions. Diabetic mice were transplanted with PFC- or LN-Biodritin microencapsulated islets, followed by assessments of body weight, glycemia and graft function by oral glucose tolerance tests (OGTTs). Microencapsulated islets were retrieved from normoglycemic or hyperglycemic mice and biocompatibility analyses of the beads together with a functional assessment of the graft followed. After graft removal, normoglycemic animals had their glycemias monitored to attest the efficacy of the transplanted islets. Results PFC- and LN-Biodritin microcapsules were as stable and biocompatible as Biodritin. For both biomaterials in normoxia and hypoxia a modulation in gene expression was observed in islets associated with a protection against apoptosis. Also, a decreased expression of stress-related genes (mcp1, hsp70) was evidenced. ldh mRNA levels were down-regulated in PFC-Biodritin microencapsulated islets but upregulated in the presence of LN. Increased levels of insulin mRNA were observed. The differences seen in protein expression indicated the same anti-apoptotic pattern. Caspase3 activity was not different between groups. Concerning diabetes reversal experiments, only mice transplanted with LN-Biodritin microencapsulated islets presented a better outcome, with 60% remaining euglycemic at 198 days post-surgery, compared with 9% for the Biodritin group. OGTT showed that mice transplanted with encapsulated islets secreted more insulin than normal mice, 60 (LN-Biodritin) or 100 days (PFC- and LN-Biodritina) posttransplant. Hyperglycemia was achieved after the retrieval of microcapsules showing graft efficacy. Retrieved microcapsules revealed an extensive overgrowth in most beads from hyperglycemic mice. A static glucose stimulated insulin secretion test revealed that only islets from normoglycemic subjects were able to secrete insulin according to glucose concentration. Conclusion- The addition of bioactive molecules to Biodritin may lessen the stress of isolated islets and have the potential to improve islet transplantation therapy.

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

O transplante de ilhotas pancreáticas humanas é considerado uma estratégia promissora para curar pacientes portadores de Diabetes Mellitus tipo 1. Entretanto, sua eficiência é dramaticamente afetada pelo rendimento das ilhotas no processo de isolamento/purificação e pela viabilidade das células após o transplante. As ilhotas pancreáticas isoladas são obtidas através da perfusão do pâncreas com colagenase e purificação em gradiente de densidade. As espécies reativas de oxigênio (ERO) exercem um papel importante durante a obtenção e o transplante de ilhotas pancreáticas humanas, contribuindo significativamente para diminuir a viabilidade dessas células. Nesse trabalho foram avaliadas as respostas oxidativas de ilhotas pancreáticas humanas durante os processos de isolamento/purificação e cultivo. As atividades da superóxido dismutase (SOD), da catalase, bem como os níveis de oxidação em proteínas mostraram-se, na maioria dos casos, aumentados, principalmente durante a etapa de purificação das ilhotas em gradiente de Ficoll e no período de cultura das ilhotas. Esses resultados indicam que a purificação em gradiente de Ficoll parece ser uma etapa crítica de geração das ERO, assim como longos períodos de cultivo. Porém, verificou-se que influências advindas dos diferentes doadores (idade, causa- mortis, estilo de vida, etc.) e condições de preservação do órgão (tempo de isquemia, solução de conservação, etc.) poderiam estar relacionadas à discrepância de alguns resultados encontrados. Com o intuito de minimizar tais variáveis, optou-se por estudar os efeitos relacionados ao Ficoll em células de insulinoma INS-1E, um modelo celular fisiologicamente semelhante. Para tanto, as atividades das enzimas antioxidantes SOD, catalase, glutationa peroxidase (GPx) e glutationa redutase (GR), assim como os danos oxidativos em proteínas e lipídeos, os níveis de glutationa reduzida (GSH) e de glutationa oxidada (GSSG), a viabilidade celular e os níveis de algumas enzimas envolvidas no processo apoptótico como p38, JNK-1, ERK 1-2 e PI3-K expostas a polissacarose (1100 mg/mL), um genérico do Ficoll, foram determinadas. De acordo com os resultados, as atividades da SOD, catalase e GPx presentes em amostras expostas a polissacarose mostraram-se aumentadas. Em cultura, a atividade de isoforma mitocondrial da SOD (Mn-SOD) de células INS-1E correspondeu a 50% da atividade total da SOD. Na presença da polissacarose, a atividade da Mn-SOD aumentou para 80% do total. Além disso, a oxidação de lipídios e de proteínas aumentou e os níveis de GSH e GR diminuíram discretamente. Estes resultados mostraram que a exposição dessas células a polissacarose está associada com o estresse oxidativo. Entretanto, tal exposição não foi responsável pela diminuição da viabilidade celular embora os níveis protéicos de JNK-1, ERK1-2 e PI3-K tenham se mostrado consideravelmente aumentados e os níveis de p38, diminuídos. Os níveis de expressão e a atividade de enzimas antioxidantes são conhecidamente baixos em ilhotas pancreáticas. A N-acetilcisteína (NAC) foi adicionada em cultura de células para prevenir o estresse oxidativo. Nessas condições, a NAC foi capaz de proteger as células INS-1E do estresse oxidativo induzido. Esses resultados sugerem que a exposição à polissacarose está associdada ao estresse oxidativo em células INS-1E e que a NAC foi capaz de prevenir a morte celular de células INS-1E expostas a ERO através do aumento intracelular de GSH. / Human pancreatic islet transplantation is considered a promising strategy to cure the cure Diabetes Mellitus type I. However, transplantation efficiency is dramatically affected by sub-optimum islet recovery in the isolation/purification procedure and islet viability after transplantation. Isolated pancreatic islets are obtained through collagenase perfusion and cell purification in a Ficoll gradient. Reactive oxygen species (ROS) play an important role during human pancreatic islet isolation and may contribute to the decrease in cell viability. The aim of this study was evaluated the response of human pancreatic islets during its isolation/purification and culture time. Activities of superoxide dismutase (SOD) and catalase as well as protein oxidation levels increased in most of analyzed samples, mainly during the Ficoll gradient islet purification step and further culture. Ficoll seems to be the critical step for ROS generation. Nevertheless, it was observed that donors characteristics (aging, cause of death, habits, etc.) and organ preservation conditions (ischemic time, preservation solution, etc.) may be related to our results. To minimize these variations, a physiological cellular model based on INS-1E cells was chosen. The antioxidant enzymes SOD, catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) activities as well the oxidative damage to proteins and lipids, reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, cellular viability and the protein levels of some enzymes responsible for apoptotic signaling like p38, JNK-1, ERK 1-2 and PI3-K upon exposure to polysucrose (1100 mg/mL), a similar of Ficoll, were determined. The SOD, catalase and GPx in samples exposed to polysucrose displayed hight activities. In all cultures, the activity of mitochondrial isoform of SOD (Mn-SOD) corresponds to 50% of total SOD activity. In the presence of polysucrose, the activity of Mn-SOD increased up to 80%. Lipids and protein oxidation levels were also increased and the GSH levels with the GR activity decreased. These results indicated that the exposure of INS-1E cells to polysucrose is associated with oxidative stress. However, the polysucrose exposure was not responsible for cell death although JNK-1, ERK1-2 and PI3-K levels showed hight levels but not p38, upon polysucrose exposure. The expression and activities of antioxidants enzymes are known to be very low in pancreatic islets. N-acetylcysteine (NAC) was added to the INS-1E cultures to prevent oxidative stress. Under these conditions, NAC was able to protect INS- 1E cells from induced oxidative damage by increasing intracellular GSH levels. Taken together, these results suggest that the exposure to polysucrose is related to the oxidative stress in INS-1E cells and NAC seems to be able to maintain cell viability.

Background. Islet amyloid polypeptide (IAPP, also known as amylin) is a 37-amino acid peptide principally co-secreted with insulin from the beta-cells of the pancreatic islets. Some of the physiological actions of human amylin (hIAPP) include glucose regulation, suppression of appetite and stimulation of renal sodium and water reabsorption. Amylin deficiency and diminished post-prandial amylin response have been reported in advanced stages of type 1 and type 2 diabetes. In autopsy specimens of type 2 diabetes, amyloid is found in 40--90% of cases. During the characterization of islet morphology of aged hIAPP transgenic mice, I observed pathological features suggestive of immune dysregulation. Review of literature also suggested possible immuno-modulating functions of human amylin in in vitro experiments. Since autoimmunity and innate immunity are implicated in aging and diabetes, I explored the immunological role of amylin with particular focus on CD4+CD25+ T regulatory cells and toll-like receptors (TLR) which are known mediators of autoimmunity and innate immunity respectively. / Conclusions. Human amylin may play an important role in modulating immunity mainly through stimulating CD4+CD25+ Treg cells, decreasing PLN and altering expression of TLR-4 and cytokines. If these findings are confirmed in in vivo model, human amylin has the potential to become a novel and promising therapy to prevent and reverse autoimmune disease such as autoimmune type 1 diabetes. / Hypothesis. Human amylin may have immunomodulating effects which may have implications on pathogenesis of autoimmune type 1 diabetes. / Materials and methods. Male hemizygous hIAPP transgenic mice (n=32) and their nontransgenic littermates (n=20) were fed with normal chow and studied longitudinally up to 18 months of age with measurement of plasma insulin, glucose and amylin at regular intervals. Detailed oral glucose tolerance test, intra-peritoneal insulin tolerance test, insulin and amylin protein expression were examined at 3, 7, 12 and 18 months of age. Histological changes of pancreas and spleen including changes in CD4+CD25+ T regulatory cells and cytokines were examined at 12 and 18 months. / Objectives. (1) I systemically characterized the morphological, functional and immune regulatory role of human amylin in aged hIAPP transgenic mice which include metabolic profiles, plasma levels of amylin and insulin as well as morphological changes of pancreatic lymph nodes (PLN). (2) I then examined splenic expression of TLR-4 associated changes in cytokines (TNF-alpha, TGF-beta, and IL-6). (3) I also examined the expression level of receptor activity modifying proteins (RAMPs) in pancreas and spleen. (4) I finished by investigating the role of human amylin on stimulating CD4+CD25+ T regulatory (Treg) cells in hIAPP transgenic mice and peripheral blood monocytes (PBMC) from healthy subjects. / Results. (1) With aging, the hIAPP transgenic mice demonstrated increased plasma amylin, decreased plasma insulin, reduced insulin to amylin ratio and improved insulin sensitivity (p&lt;0.05). (2) The aged hIAPP transgenic mice showed changes in immune function as indicated by: (a) Reduced number and size of PLN (p&lt;0.05). (b) Decreased expression level of TLR-4 in splenocytes (p&lt;0.05). (c) Increased expression of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) protein but decreased level of IL-6 in splenocytes (p&lt;0.05). (3) The changes in the levels of immune cytokines such as IL-1, IL-2, IL-4, IL-10, IL-17, interferon-gamma and GM-CSF were similar between hIAPP transgenic and nontransgenic mice (p>0.05). (4) The levels of RAMP1, RAMP2, and RAMP3 were higher in the spleen of hIAPP transgenic mice than nontransgenic mice (p&lt;0.05). (5) The hIAPP transgenic mice showed higher percentage of CD4+CD25+ Treg cells compared with nontransgenic littermates. Treatment with human amylin, but not rat amylin, increased the percentage of FoxP3+CD4+CD25+ Treg cells in both splenic T lymphocytes of hIAPP transgenic mice and PBMCs of healthy subjects ex vivo (p&lt;0.05). / He, Lan / Adviser: Juliana C.N. Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 176-199). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Os ácidos graxos (AG) podem influenciar o processo secretório de insulina induzido pela glicose. Os AG &#969;3 interferem em diversos processos fisiológicos, sendo que nas ilhotas pancreáticas, os AG &#969;3 colaboram para a diminuição da lipotoxicidade induzida pelo ácido palmítico. Ao ácido palmítico são atribuídos efeitos deletérios em diversos tecidos, assim como nas células &#946;, onde ele promove a alteração da composição dos fosfolípides de membrana, do potencial elétrico da mesma e consequentemente do processo de extrusão dos grânulos de insulina. A exposição crônica das células &#946; ao excesso de ácido palmítico é tóxica, provocando diminuição da resposta secretória de insulina, redução da oxidação e captação de glicose e aumento de espécies reativas de oxigênio (EROs) que, em quantidades suprafisiológicas, irão contribuir para a falência e morte da célula &#946;. As EROs podem ser de origem mitocondrial, através do metabolismo dos nutrientes ou ainda proveniente da ativação do complexo enzimático NADPH oxidase, o qual é modulado pela glicose e pelos AG, incluindo o ácido palmítico. Em contrapartida, os AG &#969;3 exercem efeitos anti-inflamatórios e antioxidantes em diversos sistemas, contribuindo para melhora de perfil lipídico e resistência periférica à insulina. O objetivo deste trabalho foi verificar o possível efeito protetor dos AG &#969;3 contra os efeitos deletérios do ácido palmítico em células &#946; pancreáticas. Nas células &#946;, a partir dos resultados obtidos, a presença de AG &#969;3 mostrou-se eficaz para prevenir o dano secretório e o aumento de EROs, além de contribuir para manutenção da viabilidade celular e da captação de glicose nas ilhotas tratadas com ácido palmítico, desempenhando um importante papel protetor na célula &#946;. / Fatty acids (FA) may influence the process of glucose-induced insulin. The &#969;3 FA interferes in several physiological processes, and in the pancreatic islets collaborate to decrease the lipotoxicity induced by palmitic acid. Palmitic acid induces deleterious effects in several tissues, as well as in &#946; cells, where it promotes the alteration of the membrane phospholipid composition, the plasma membrane electric potential, and consequently, the process of the insulin granules extrusion. Chronic exposure of &#946; cells to high concentration of palmitic acid is toxic, leading to decreased insulin secretory response, reduced oxidation and uptake of glucose, and an increase in reactive oxygen species (ROS) which, in supraphysiological amounts, will contribute to &#946;-cell failure and death. ROS may be of mitochondrial origin, through the metabolism of nutrients or even from the activation of the enzymatic complex NADPH oxidase, which is modulated by glucose and FA, including palmitic acid. In contrast, &#969;3 FA exerts anti-inflammatory and antioxidant effects in several systems, contributing to the improvement of lipid profile and peripheral resistance to insulin. The aim of this study was verify the protective possible effect of AG &#969;3 against the deleterious effects of palmitic acid on pancreatic &#946; cells. Our results shown that the presence of &#969;3 FA was effective in preventing secretory damage and increase of EROs, also contributing to the maintenance of cell viability and glucose uptake in the islets treated with palmitic acid, playing an important &#946;-cell protective role.

<p><strong>Aim: </strong>To investigate the quality of life and the social life situation, with special focus on the consequenses of fear of hypoglycemia (FoH), in Islet transplanted patients.</p><p><strong>Method: </strong>11 patients were included, four women and seven men, who have been Islet tranplanted at Uppsala University Hospital during the years 2001-2009. Two questionaires, Short Form 36 (SF-36) and the Swedish version Hypoglycemia Fear Survey (Swe-HFS) were used to investigate the quality of life, in relation to fear of hypoglycemia. Also, telephone interviews were conducted to investigate the patients social life situation in relation to FoH, after Islet transplantation and were analysed using content analysis method.</p><p><strong>Results:</strong> The mean value for quality of life was lower than that in the normal population. Three out of ten patients experienced FoH. Three predominant themes were revealed, one theme associated with pre- transplant, was “Struggle for control of Social Life Situation” and two themes associated with post-transplant, were “Regain power and controll of  Social Life Situation” and “At Peace with the balance between the Present and the Future”.</p><p><strong>Conclusion:</strong> The patients experienced improved control over social life situation and quality of life in relation to FoH may been improved following islet tranplantation.</p> / <p><strong>Syfte: </strong>Att undersöka ö-cellstransplanterade patienters livskvalitet och sociala livssituation med speciellt fokus på oro/rädsla för hypoglykemi.</p><p><strong>Metod</strong>: I studien inkluderades 11 patienter, fyra kvinnor och sju män, vilka genomgått ö-cellstransplantation vid Akademiska Sjukhuset i Uppsala under perioden 2001-2009. Två frågeformulär, Short Form 36 (SF-36) och den svenska versionen av Hypoglycemia Fear Survey (Swe-HFS) användes för att undersöka patienternas livskvalitet relaterat till oro/rädsla för hypoglykemi. Telefonintervjuer genomfördes för att undersöka patienternas sociala livssituation efter genomgången ö-cellstransplantation relaterat till oro/rädsla för hypoglykemi och analyserats med innehållsanalys.</p><p><strong>Resultat: </strong>Medelvärdet för hälsorelaterad livskvalitet var lägre jämfört med normalbefolkningen och tre av tio deltagare upplevde oro/rädsla för hypoglykemi. Tre övergripande teman med koppling till social livssituation identifierades, ett tema före genomgången ö-cellstransplantation, var ”Kampen om kontroll över social livssituation”, och två teman efter genomförd transplantation, var ”Återtagande av makt och kontroll över social livssituation” samt ”Tillfreds med balans mellan nuet och framtiden”.<strong></strong></p><p><strong>Konklusion: </strong>Patienterna upplevde att kontrollen över den sociala livssituationen och livskvalitet i relation till oro/rädsla för hypoglykemi förbättrades efter genomgången ö-cellstransplantation.</p>

<p>The blood vessels of the pancreatic islets are of crucial importance for oxygen and metabolite supply as well as dispersal of secreted hormones. In addition to this, endothelial cells have an important role in the revascularization process after islet transplantation. Previous studies have reported signs of poor engraftment of transplanted islets, presumably due to impaired revascularization. The aims of this thesis were to investigate the revascularization process of transplanted islets and to examine the role of islet endothelial cells. In this context, the lectin Bandeiraea simplicifolia was found to stain endothelium of both endogenous and transplanted pancreatic islets. By using this lectin we investigated the vascular density of both endogenous and islets transplanted syngeneically beneath the renal capsule, into the spleen or intraportally into the liver of normoglycemic C57BL/6 mice. One month post-transplantation, a time point when the grafts are assumed to be completely revascularized, the vascular density was decreased at all three implantation sites when compared to endogenous islets. Furthermore, most of the blood vessels were located in the graft connective tissue stroma. Similar results were obtained when islet transplant vascular density was determined six months post-transplantation and in cured diabetic animals after one month. In order to evaluate the function of intraportally transplanted islets, we developed a method to retrieve such islets. We treated the implantation organ (liver) first enzymatically (collagenase) and then mechanically, thereafter we could re-isolate the transplanted islets for further in vitro studies. The retrieved islets had a decreased insulin relase, insulin content and glucose oxidation rate when compared to non-transplanted control islets. To understand the role of islet endothelium in the revascularization of transplanted islets we performed angiogenesis GEArray studies on islet endothelial cells, from non-cultured, cultured and transplanted islets. We found that the islet endothelium expressed mRNA for both inhibitors and inducers of angiogenesis, and that this expression differed with time. The functional consequences of this remain to be determined. In summary, the results presented above provide a useful platform for future studies of the morphology and function of islet endothelial cells, especially with a view for elucidating changes induced by islet transplantation.</p>

<p>Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.</p>

<p>In this study, metastasizing serotonin-producing ileocaecal carcinoid tumours (MSPCs) were examined for biological characteristics that could be used to define clinically relevant subgroups within this patient population. Possible targets for new treatment options were also explored.</p><p>It was found that MSPCs share several biological characteristics such as expression of serotonin, tachykinins (TKs), chromogranin A, islet autoantigen-2 and connective tissue growth factor (CTGF). TKs and serotonin were demonstrated in the same endocrine tumours in the gut and lung. IA-2 expression was shown to be up-regulated in MSPCs, possibly in connection with active hormone secretion. CTGF expression was high in tumour areas adjacent to extensive stroma expressing alpha-smooth muscle actin. This indicated myofibroblast differentiation, which may be associated with fibrosis-related complications prevalent in patients with MSPCs. When compared with other endocrine tumours, MSPCs behaved as a relatively homogeneous group, though within the MSPC population several subgroups could be defined. Patients with tumours displaying either a solid growth pattern and/or a Ki67 index ≥1% had a less favourable prognosis than those who did not. Another group of patients, who had increased plasma TK concentrations, were more likely to suffer from severe diarrhea. This information should be considered when discussing clinical treatment and when undertaking tumour biological studies. New treatment possibilities, such as drugs that specifically target TK receptors and antibodies to CTGF, are also discussed.</p><p>In conclusion, MSPCs comprise a clinically relevant tumour group with similar biological features that are distinct from other endocrine tumours. Subgroups of patients within this patient category can be defined which may be relevant when establishing prognosis and when selecting future treatment modalities.</p>

<p>Cytokines are soluble signalling mediators within the immune system, and have been shown to be of importance in the development of type 1 diabetes (T1D). This thesis studied the production of cytokines in experimental models of T1D and during transplantation of insulin-producing islets of Langerhans. </p><p>We have demonstrated that the transcriptional TNFα-inhibitor MDL 201,449A, previously shown to reduce immune-mediated diabetes induced in mice by multiple low doses of streptozotocin, was not TNFα-specific, but also inhibited IFNγ and IL-10 in spleen cells. Furthermore, when the inhibitor was removed from in vitro cultures, a rebound phenomenon of increased cytokine secretion occurred.</p><p>The thesis also investigated whether plastic adhesion, a method generally employed to deplete macrophages, influenced cytokine production in spleen cells. We observed that plastic adhesion increased TNFα, IFNγ and IL-10 release, and decreased IL-4 secretion. Plastic adhesion depleted only ~30% of the macrophages, but as much as ~60% of the regulatory T cells. </p><p>Thirdly, we found that “control” treatments for islet transplantations, i.e. syngeneic and sham transplantations, exerted a clear effect on cytokine production from spleen cells, possibly due to a decrease in regulatory T cells that may be caused by the surgery and/or anaesthesia. Moreover, spleen cells from mice exposed to surgery exhibited a decreased proliferative capacity to concanavalin A stimulation. We also perceived a marked difference in cytokine response depending on the mouse strain used in the experiments.</p><p>Finally, we aimed to elucidate if, besides autoimmune activities, also high glucose- and free fatty acid concentrations as seen in diabetes could cause changes in cytokine production. We observed that spleen cells cultured in varying glucose concentrations had different cytokine production profiles. The free fatty acid palmitate might also influence cytokine release, but this effect was obscured by the cytokine-suppressive action of the ethanol used to dissolve the palmitate.</p>