Dissertations / Theses on the topic 'Pancreatic β-islet cell'

Type 1 diabetes is an autoimmune disease resulting in the selective destruction of the insulin producing β-cells in the pancreas. Pro-inflammatory cytokines and the free radical nitric oxide (NO) have been implicated in mediating the destruction of β-cells, possibly through activation of the mitogen activated protein kinases (MAPKs) JNK, ERK and p38. In addition to MAPKs, cytokine signaling also results in activation of the transcription factor nuclear factor-kappaB (NF-κB). The upstream signaling events leading to MAPK and NF-κB activation in β-cells are not well known. The work presented in this thesis therefore aims at characterizing the regulation of MAPKs and NF-κB in human islets, with emphasis on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. It was found that MEKK-1 was phosphorylated in response to the nitric oxide donor DETA/NONOate (DETA/NO), the β-cell toxin streptozotocin (STZ) and pro-inflammatory cytokines and that MEKK-1 downstream signaling in response to the same treatments involved activation of JNK but not ERK and p38. MEKK-1 was also found to be essential for cytokine-induced NF-κB activation. MEKK-1 downregulation protected human islet cells from DETA/NO-, STZ, and cytokine-induced cell death. Furthermore, overexpression of the NF-κB subunit c-Rel protected human islet cells from STZ and hydrogen peroxide-induced cell death indicating that NF-κB activity protects against cell death in human islets. In summary, these results support an essential role for MEKK-1 in the activation of JNK and NF-κB, with important consequences for human islet cell death and that strategies preventing human islets death by inhibition of the JNK pathway instead of NF-κB might be suitable.

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.

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.

Patients with type-1 diabetes lose their β-cells after autoimmune attack. Islet transplantation is a co-option for curing this disease, but survival of transplanted islets is poor. Thus, methods to enhance β-cell viability and function as well as methods to expand β-cell mass are required. The work presented in this thesis aimed to study the roles of neural crest stem cells or their derivatives in supporting β-cell proliferation, function, and survival. In co-culture when mouse boundary cap neural crest stem cells (bNCSCs) and pancreatic islets were in direct contact, differentiating bNCSCs strongly induced β-cell proliferation, and these proliferating β-cells were glucose responsive in terms of insulin secretion. Moreover, co-culture of murine bNCSCs with β-cell lines RIN5AH and β-TC6 showed partial protection of β-cells against cytokine-induced β-cell death. Direct contacts between bNCSCs and β-cells increased β-cell viability, and led to cadherin and β-catenin accumulations at the bNCSC/β-cell junctions. We proposed that cadherin junctions supported signals which promoted β-cell survival. We further revealed that murine neural crest stem cells harvested from hair follicles were unable to induce β-cell proliferation, and did not form cadherin junctions when cultured with pancreatic islets. Finally, we discovered that the presence of bNCSCs in co-culture counteracted cytokine-mediated insulin-producing human EndoC-βH1 cell death. Furthermore, these two cell types formed N-cadherin, but not E-cadherin, junctions when they were in direct contact. In conclusion, the results of these studies illustrate how neural crest stem cells influence β-cell proliferation, function, and survival which may improve islet transplantation outcome.

Dietary antioxidant curcumin derived from turmeric has been suggested to decrease the risk of many chronic diseases. Much of the existing data for curcumin stem from experiments performed at supra-physiological concentrations (μM-mM) that are impossible to attain through oral ingestion. It was therefore hypothesized that curcumin at low plasma achievable concentration, though itself not acting as a direct antioxidant might up-regulate the intracellular antioxidants and thus helping combat oxidative stress and protect β-islet cells. The results indicated that Curcumin, DMC and BDMC were able to scavenge hydroxyl radicals, but showed little scavenging ability against superoxide and nitric oxide radicals. Nanomolar concentrations of curcuminoids easily prevented the deleterious effects of H₂O₂ in pancreatic β-islet RINm5F cells. Non of the curcuminoids showed a detrimental effect on insulin secretion, but the model did not allow assessment of any potential positive effect on insulin secretion. The findings confirmed that nanomolar concentrations of curcumin offered protection in pancreatic β-islet cells against H₂O₂-indicated damage by modulating the proportion of oxidised GSH (GSSG): reduced GSH in the favour of GSH and the increasing the activity of SOD. This increase in GSH and SOD levels was, at least in part, on account of an increase in GR, SOD-1 and SOD-2 gene expression. The intracellular mechanism driving this modulation of antioxidant gene was, by virtue of blocking the H₂O₂  induced NF-κB activation.

Type I diabetes mellitus (T1DM) is characterized by insulin deficiency due to β cells death caused by inflammation and immune reaction. Pro-inflammatory cytokines play a key role in T1DM pathogenesis by activating the pro-apoptotic pathway. Cytokine-activated NF-κB and STAT1 signalling also leads to oxidative stress and triggers the antigen presentation pathway. Stressful stimuli and self-defence responses combined cause mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which progress to β cell functional impairments and death. Some molecular mechanisms involved in the progressive loss of functions are still unknown. In this contest, a study, which analyses proteomic changes of β cells upon cytokine prolonged exposure, can be useful. The global effects of pro-inflammatory cytokines, IL-1β and INF-γ, on protein expression, Nε-acetylation, and thermal stability was studied using different proteomic approaches and β cell models (rat INS-1E cells and human pancreatic islets). At first, the impact of cytokines on β cell insulin secretion, survival and apoptosis activation was examined to confirm functional impairment. Differential expression proteomics showed that cytokines dysregulated the expression of many proteins, which are components of pathways involved in T1DM pathogenesis. The study of protein lysine acetylome highlighted 83 and 242 differentially modified proteins in human islets and INS-1E cells, respectively. Most proteins are related to metabolic, mitochondrial dysfunction, inflammation, and insulin secretion pathways. In INS-1E cells, an analysis of Proteome Integral Solubility Alteration (PISA) was also performed. This technique allowed to identify 504 proteins, which thermal stability was modified by cell exposure to cytokines. Many of these proteins participate to protein synthesis, protein trafficking, and antigen presentation or are directly involved in mediating cytokine effects. Overall, this multi-level proteomic analysis unveiled new potential players of cytokine-induced β cells dysfunction.

The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied in vitro in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound.

It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which in vivo may increase a virus’s ability to persist in islets.

Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect.

All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis in vivo.

Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study.

To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells in vivo, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction in vivo.

Type 2 diabetes mellitus is a heterogeneous group of metabolic diseases characterized by increased levels of blood glucose due to insulin resistance and alteration of insulin secretion by pancreatic β cells. Recent studies suggest that β cell loss in T2D results from endoplasmic reticulum stress which can cause an alteration in the processing of PI to INS. In particular, it has been reported that the increased circulating levels of PI and elevated PI/INS ratio are well-known abnormalities in type 2 diabetes. Several studies have hypothesized that an elevated PI/INS ratio was caused by increased secretory demand on β cells due to insulin resistance and hyperglycemia. However, an in-depth analysis of PI/INS expression pattern inside the pancreatic islets during metabolic alteration is not entirely clear. For this reason, the first aim of this work was: (i) to analyze PI and INS expression pattern in pancreatic islets of tissue biopsies of patients undergoing partial pancreatectomy (PP) with normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and type 2 diabetes, in order to explore the in-situ alterations that occur in islets during metabolic stress. Given the enormous heterogeneity between pancreatic islets of different donors but also between islets belonging to the same donors, the second aim of project thesis was: (ii) to perform single islet phenotyping by characterizing histological and molecular aspects, in order to investigate the underlying molecular mechanisms driving β cell failure at single islet level with the aim to specifically determine the cues leading to intracellular alteration of PI and insulin during metabolic stress. In order to explore the alterations that occur in PI and INS staining pattern in pancreatic islets, OGTT (Oral Glucose Tolerant Test) was performed in n=19 patients scheduled for PP, classified into n=5 NGT, n=9 IGT and n=5 T2D. Frozen sections of pancreatic tissue biopsy were stained for INS and PI through double immunofluorescence staining. Image analysis was performed through Volocity software. In-situ staining measurements were correlated with patients’ clinical parameters. Subsequently, we evaluated the expression of ER stress genes and β cell mature phenotype-related genes in microdissected pancreatic islets collected from n=4 NGT, n=7 IGT and n=4 T2D patients. Given the high heterogeneity among pancreatic islets we also performed a single islets phenotyping analysis in n=88 islets from n=3 NGT, n=3 IGT and n=3 T2D patients. Results showed that in pancreatic islets of IGT and T2D patients, PI intracellular localization was altered. The colocalization coefficient INS-PI as well as PI levels and PI/INS ratio gradually increased from NGT to IGT and T2D pancreatic islets and were related to the loss of glucose tolerance and impaired β-cell function. PDIA1, GRP78 and XBP1, genes involved in unfolded protein response (UPR) were significantly upregulated in pancreatic islets of IGT and T2D patients compared to NGT and were positively correlated with in-situ PI/INS ratio and colocalization, with in-vivo measurements of glucose intolerance and β cell functional reduction. Single islets phenotyping approach revealed a progressively increased heterogeneity from NGT to IGT and T2D patients. Of note, in-situ PI/INS ratio and colocalization were positively correlated with the expression of UPR genes and negatively with those associated to β cell identity. In conclusion, we demonstrated that: (i) the in-situ expression patterns of proinsulin and insulin are altered in prediabetic and diabetic patients reflecting their metabolic profiles; (ii) the molecular mechanism behind this in-situ alteration involves the ER stress and the establishment of UPR within the β cell; (iii) single islets phenotyping analysis in T2D pancreas reveals a high heterogeneity among pancreatic islets in terms of ER stress and β cell differentiation profile.

Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

Les îlots pancréatiques sont le principal capteur de glycémie et intègrent toutes les informations métaboliques et hormonales pour adapter en temps réel la sécrétion des hormones, telles que l'insuline par les cellules β majoritaires et le glucagon par les cellules α. Dans le diabète de type 1 (DT1) les cellules β sont détruites par réaction auto-immune et dans le DT2 la masse de cellules β, la fonction et le réseau intra-îlot sont altérés. La réactivité de ces micro-organes est due à leurs propriétés électriques, encodant rapidement l’information, et aux communications entre cellules β/β et β/non-β. Cependant des outils non-invasifs à haute résolution et long terme pour les analyser manquent. L’électrophysiologie extracellulaire par multi-electrode arrays (MEAs) le permet en mesurant des signaux cellulaires mais aussi multicellulaires (SPs) dus aux couplages entre cellules β. J’ai donc utilisé les MEAs (i) pour explorer la physiologie/physiopathologie des îlots et (ii) pour des applications en diabétologie. J’ai montré que la cinétique biphasique de la sécrétion d’insuline était encodée par les SPs avec des changements dynamiques de couplages entre cellules β. Une hormone intestinale importante (GLP-1) augmente la 2de phase alors que des conditions diabétiques (glucotoxicité) réduisent la 1re. La réponse aux nutriments requiert de plus la coopération avec les cellules α, car leur suppression (modèle inductible GluDTR) réduit l’activité basale et la 2de phase des cellules β en présence d’un mélange physiologique d’acides aminés. J’ai également caractérisé électrophysiologiquement les cellules β humaines dérivées de cellules souches pluripotentes induites (iPSC), déterminé leurs couplages, établi leur contrôle qualité et démontré l’impact fonctionnel d’une mutation d’intérêt (ZnT8) éditée par CRISPR/Cas9. Un contrôle qualité fonctionnel des îlots humains avant greffe chez des patients DT1 a également été réalisé et comparé aux résultats cliniques. Enfin, mes enregistrements de SPs analysées par microélectronique en temps réel ont permis de valider un modèle in silico de biocapteur dans un simulateur de référence de patients DT1. En conclusion, mes travaux montrent (i) l’importance des communications intra-îlots dans leur adaptation physiologique dynamique, (ii) et que l’exploitation des SPs ouvre des applications allant du pancréas artificiel à la thérapie cellulaire personnalisée
Pancreatic islets are the main sensor of glycaemia and they integrate all the metabolic and hormonal inputs to adapt in real time the secretion of hormones such as insulin by β cells and glucagon by α cells. In type 1 diabetes (T1D) β cells are destroyed by immune attack, and in T2D, β cell mass, function and the intra-islet network are altered. The islet micro-organs are highly reactive due to their electrical properties encoding rapid information and due to intercellular communications between β cells and β/non-β cells. Nevertheless, non-invasive, high resolution and long-term approaches for analysis are still lacking. Extracellular electrophysiology with multi-electrode arrays (MEAs) allows this analysis of islets by measuring both cellular as well as multicellular signals (SPs) due to β cell coupling. During my PhD, I used MEAs (i) to explore islet physiology/pathophysiology and (ii) for biotechnological applications in diabetology. I have shown that biphasic kinetics of insulin secretion are encoded by SPs through dynamic changes in β cell coupling. An important intestinal hormone (GLP-1) increases the 2nd phase of β-cell activity while diabetic conditions (glucotoxicity) reduce the 1st phase. Islet responses to nutrients also require α/β cell cooperation since α cell ablation in the inducible GluDTR mice model reduced both the basal and 2nd phase of β cell activity generated by glucose and a physiological mix of amino acids. I have also performed the electrophysiological characterization of human β cells derived from induced pluripotent stem cells (iPSC), determined their coupling, established their quality control and shown the functional impact of a mutation of interest (ZnT8) edited by CRISPR/Cas9. A functional quality control of human islets prior to transplantation in T1D patients was also performed for correlations with clinical data. Finally, my SP recordings analyzed in real time by microelectronics has contributed to validate an in silico model of biosensor in a FDA-approved simulator of T1D patients. In conclusion, my work demonstrates (i) the role of intra-islet communications in the dynamic physiological adaptation of these micro-organs, (ii) and that detailed characterization of SPs opens new applications from artificial pancreas to personalized cell therapy

The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation may impair islet function.

In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. In vitro, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. In vivo, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.

Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The in vitro data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE₂ production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.

Oscillations in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca²⁺] _i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca²⁺] _i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca²⁺] _i in isolated mouse β-cells into sustained elevation. Increased Ca²⁺ entry promoted the reappearance of the slow [Ca²⁺] _i oscillations. The [Ca²⁺] _i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca²⁺] _i oscillations due to periodic entry of Ca²⁺ as well as with transients evoked by mobilization of intracellular stores. The [Ca²⁺] _i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca²⁺] _i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca²⁺] _i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca²⁺ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca²⁺] _i oscillations. Nevertheless, there was an excessive firing of [Ca²⁺] _i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca²⁺] _i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

Embryonic stem cells (ES-cells) are an excellent source for generating insulin-producing β-islet cells for potential use in the management of diabetes mellitus. Although many protocols have been developed to promote the differentiation of ESCs into β-islet cells, they are limited in terms of (i) low efficiency of differentiation and (ii) generation of functionally immature β-islet cells with inefficient glucose stimulated insulin secretion (GSIS). The present study is aimed at understanding the differentiation and functional maturity of pancreatic β-islet cell. Earlier in our lab EGFP-expressing transgenic mouse ‘GS-2’ ES-cell line was derived (Singh et al., 2012). Through the embryoid body (EB) formation and differentiation method (day 2 through 21), we obtained spontaneous differentiation of GS-2 ES-cells to three germ-lineage cell types, with the expression of molecular markers of definitive endoderm (DE; sox17) and hepatic lineage (afp). However, we were unable to detect the expression of markers of pancreatic progenitors (pdx1) and β-islet cells (ins2). By improvising the spontaneous differentiation protocol i.e. increasing cell seeding density for EB, we were able to detect expression of pancreatic progenitor marker, pdx-1. However, we were still unable to detect expression of β-islet marker ins1 and ins2. In view of this, we employed an induction protocol by inclusion of laminin, nicotinamide and insulin (Wobus et al., 2006). This resulted in the differentiation of GS-2 ES-cells to DE-like cells by day 14, followed by the appearance of pancreatic progenitor-like clusters by day 21 and significantly, β-islet-like clusters by day 33. This sequential appearance of progenitors and β-islet cells was accompanied by the expression of pdx1, ins1and ins2. However, expression of more important β-islet marker ins1 appeared to be low. Also, we were unable to detect expression of glucose transporter, glut-2 indicating, this model system may not be suitable for testing molecules to improve GSIS or functional maturity. Therefore, we found an alternative model system, Islet Cell Aggregates (ICAs) from day1-2 old neonatal mice that are considered functionally immature or have inefficient GSIS. Sirtuin-1 activator SRT1720 was found as the molecule to improve functional maturity of immature β-islet cells. When functionally immature β-islet cells from day1-2 old neonatal mice were treated with SRT1720 (5μM) for 24 hours, their GSIS improved by 1.5 fold. We envisage that this molecule may be used to improve functional immaturity of PSC-derived functionally-immature β-islet cells

Indiana University-Purdue University Indianapolis (IUPUI)
Diabetes mellitus affects an estimated 285 million people worldwide, and a central component of diabetes pathophysiology is diminished pancreatic islet beta cell function resulting in the inability to manage blood glucose effectively. The beta cell is a highly specialized metabolic factory that possesses a number of specialized characteristics, chief among these a highly developed endoplasmic reticulum (ER). The sarco endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen, and while the Pancreatic and duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in beta cell development and function, recent data also implicate Pdx-1 in the maintenance of ER health. Our data demonstrates that a decrease of beta cell Pdx-1 occurs in parallel with decreased SERCA2b expression in models of diabetes, while in silico analysis of the SERCA2b promoter reveals multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. Results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using an ER-targeted D4ER adenovirus and fluorescence lifetime imaging microscopy. Co-transfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity three-fold relative to the empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/- mice were fed high fat diet for 8 weeks. Isolated islets from these mice demonstrated increased expression of spliced Xbp1, signifying ER stress, while subsequent SERCA2b overexpression in isolated islets reduced spliced Xbp1 levels to that of wild-type controls. These results identify SERCA2b as a direct transcriptional target of Pdx-1 and define a novel role for altered ER Ca2+ regulation in Pdx-1 deficient states.

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

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.

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

Indiana University-Purdue University Indianapolis (IUPUI)
The islet beta cell is unique in its ability to synthesize and secrete insulin for use in the body. A number of factors including proinflammatory cytokines, free fatty acids, and islet amyloid are known to cause beta cell stress. These factors lead to lipotoxic, inflammatory, and ER stress in the beta cell, contributing to beta cell dysfunction and death, and diabetes. While transcriptional responses to beta cell stress are well appreciated, relatively little is known regarding translational responses in the stressed beta cell. To study translation, I established conditions in vitro with MIN6 cells and mouse islets that mimicked UPR conditions seen in diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Chronic exposure of beta cells to proinflammatory cytokines (IL-1 beta, TNF-alpha, IFN-gamma), or to the saturated free fatty acid palmitate, led to changes in global beta cell translation consistent with attenuation of translation initiation, which is a hallmark of ER stress. In addition to changes in global translation, I observed transcript specific regulation of ribosomal occupancy in beta cells. Similar to other privileged mRNAs (Atf4, Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes during the UPR, whereas the mRNA encoding a proinsulin processing enzyme (Cpe) partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5’ untranslated region of mouse Pdx1 (between bp –105 to –280) contained elements that promoted translation under both normal and UPR conditions. In contrast to regulation of translation initiation, deoxyhypusine synthase (DHS) and eukaryotic translation initiation factor 5A (eIF5A) are required for efficient translation elongation of specific stress relevant messages in the beta cell including Nos2. Further, p38 signaling appears to promote translational elongation via DHS in the islet beta cell. Together, these data represent new insights into stress induced translational regulation in the beta cell. Mechanisms of differential mRNA translation in response to beta cell stress may play a key role in maintenance of islet beta cell function in the setting of diabetes.

Indiana University-Purdue University Indianapolis (IUPUI) Indiana University School of Medicine
The islet β cell is central to the maintenance of glucose homeostasis as the β cell is solely responsible for the synthesis of Insulin. Therefore, better understanding of the molecular mechanisms governing β cell function is crucial to designing therapies for diabetes. Pdx1, the master transcription factor of the β cell, is required for the synthesis of proteins that maintain optimal β cell function such as Insulin and glucose transporter type 2. Previous studies showed that Pdx1 interacts with the lysine methyltransferase Set7/9, relaxing chromatin and increasing transcription. Because Set7/9 also methylates non-histone proteins, I hypothesized that Set7/9-mediated methylation of Pdx1 increases its transcriptional activity. I showed that recombinant and cellular Pdx1 protein is methylated at two lysine residues, Lys123 and Lys131. Lys131 is involved in Set7/9 mediated augmented transactivation of Pdx1 target genes. Furthermore, β cell-specific Set7/9 knockout mice displayed glucose intolerance and impaired insulin secretion, accompanied by a reduction in the expression of Pdx1 target genes. Our results indicate a previously unappreciated role for Set7/9 in the maintenance of Pdx1 activity and β cell function. β cell function is regulated on both the transcriptional and translational levels. β cell function is central to the development of type 1 diabetes, a disease wherein the β cell is destroyed by immune cells. Although the immune system is considered the primary instigator of the disease, recent studies suggest that defective β cells may initiate the autoimmune response. I tested the hypothesis that improving β cell function would reduce immune infiltration of the islet in the NOD mouse, a mouse model of spontaneous type 1 diabetes. Prediabetic NOD mice treated with pioglitazone, a drug that improves β cell function, displayed an improvement in β cell function, a reduction in β cell death, accompanied by reductions in β cell autoimmunity, indicating that β cell dysfunction assists in the development of type 1 diabetes. Therefore, understanding the molecular mechanisms involved in β cell function is essential for the development of therapies for diabetes.

Le diabète de type 2 (DT2) résulte d’une résistance à l’insuline par les tissus périphériques et par un défaut de sécrétion de l’insuline par les cellules β-pancréatiques. Au fil du temps, la compensation des îlots de cellules β pour la résistance à l’insuline échoue et entraine par conséquent une baisse progressive de la fonction des cellules β. Plusieurs facteurs peuvent contribuer à la compensation de la cellule β. Toutefois, la compréhension des mécanismes cellulaires et moléculaires sous-jacents à la compensation de la masse de la cellule β reste à ce jour inconnue. Le but de ce mémoire était d’identifier précisément quel mécanisme pouvait amener à la compensation de la cellule β en réponse à un excès de nutriments et plus précisément à l’augmentation de sa prolifération et de sa masse. Ainsi, avec l’augmentation de la résistance à l’insuline et des facteurs circulants chez les rats de six mois perfusés avec du glucose et de l’intralipide, l’hypothèse a été émise et confirmée lors de notre étude que le facteur de croissance HB-EGF active le récepteur de l’EGF et des voies de signalisations subséquentes telles que mTOR et FoxM1 impliquées dans la prolifération de la cellule β-pancréatique. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la compensation de la masse de la cellule β dans un état de résistance à l’insuline et peuvent servir de nouvelles approches thérapeutiques pour prévenir ou ralentir le développement du DT2.
Type 2 diabetes (T2D) results from insulin resistance in peripheral tissues and impaired insulin secretion from the pancreatic β-cell. Over the time, compensation of the β cell islets for insulin resistance fails, and therefore leads to a gradual decline in β-cell function. Several factors may contribute to β-cell compensation. However, the cellular and molecular mechanisms underlying β-cell compensation remain unknown. The purpose of this thesis was to identify what mechanism could lead to β cell compensation in response to nutrients excess and specifically the increase in proliferation and β-cell mass. Thus, with increasing insulin resistance and circulating factors in the 6 month rats infused with glucose + intralipid, the hypothesis was made and confirmed in our study that the growth factor HB-EGF would activate the EGF receptor, and subsequent signaling pathways such as mTOR and FoxM1, both involved in the proliferation of the pancreatic beta-cell. Collectively, these results allow us to understand better the molecular mechanisms involved in the β cell compensation in the insulin resistance state and may serve as a potential new therapeutic approach to prevent or delay T2D development.

Le diabète de type 2 (DT2) est caractérisé par une résistance des tissus périphériques à l’action de l’insuline et par une insuffisance de la sécrétion d’insuline par les cellules β du pancréas. Différents facteurs tels que le stress du réticulum endoplasmique (RE) et l’immunité innée affectent la fonction de la cellule β-pancréatique. Toutefois, leur implication dans la régulation de la transcription du gène de l’insuline demeure imprécise. Le but de cette thèse était d’identifier et de caractériser le rôle du stress du RE et de l’immunité innée dans la régulation de la transcription du gène de l’insuline. Les cellules β-pancréatiques ont un RE très développé, conséquence de leur fonction spécialisée de biosynthèse et de sécrétion d’insuline. Cette particularité les rend très susceptible au stress du RE qui se met en place lors de l’accumulation de protéines mal repliées dans la lumière du RE. Nous avons montré qu’ATF6 (de l’anglais, activating transcription factor 6), un facteur de transcription impliqué dans la réponse au stress du RE, lie directement la boîte A5 de la région promotrice du gène de l’insuline dans les îlots de Langerhans isolés de rat. Nous avons également montré que la surexpression de la forme active d’ATF6α, mais pas ATF6β, réprime l’activité du promoteur de l’insuline. Toutefois, la mutation ou l’absence de la boîte A5 ne préviennent pas l’inhibition de l’activité promotrice du gène de l’insuline par ATF6. Ces résultats montrent qu’ATF6 se lie directement au promoteur du gène de l’insuline, mais que cette liaison ne semble pas contribuer à son activité répressive. Il a été suggéré que le microbiome intestinal joue un rôle dans le développement du DT2. Les patients diabétiques présentent des concentrations plasmatiques élevées de lipopolysaccharides (LPS) qui affectent la fonction de la cellule β-pancréatique. Nous avons montré que l’exposition aux LPS entraîne une réduction de la transcription du gène de l’insuline dans les îlots de Langerhans de rats, de souris et humains. Cette répression du gène de l’insuline par les LPS est associée à une diminution des niveaux d’ARNms de gènes clés de la cellule β-pancréatique, soit PDX-1 (de l’anglais, pancreatic duodenal homeobox 1) et MafA (de l’anglais, mammalian homologue of avian MafA/L-Maf). En utilisant un modèle de souris déficientes pour le récepteur TLR4 (de l’anglais, Toll-like receptor), nous avons montré que les effets délétères des LPS sur l’expression du gène de l’insuline sollicitent le récepteur de TLR4. Nous avons également montré que l’inhibition de la voie NF-kB entraîne une restauration des niveaux messagers de l’insuline en réponse à une exposition aux LPS dans les îlots de Langerhans de rat. Ainsi, nos résultats montrent que les LPS inhibent le gène de l’insuline dans les cellules β-pancréatiques via un mécanisme moléculaire dépendant du récepteur TLR4 et de la voie NF-kB. Ces observations suggèrent ainsi un rôle pour le microbiome intestinal dans la fonction de la cellule β du pancréas. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la répression du gène de l'insuline en réponse aux divers changements survenant de façon précoce dans l’évolution du diabète de type 2 et d'identifier des cibles thérapeutiques potentielles qui permettraient de prévenir ou ralentir la détérioration de l'homéostasie glycémique au cours de cette maladie, qui affecte plus de deux millions de Canadiens.
Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion from the pancreatic β-cell. Endoplasmic reticulum (ER) stress and innate immunity have both been reported to alter pancreatic β-cell function. However, it is not clear whether these factors can affect the transcription of the insulin gene. The aim of this thesis was to assess the role of ER stress and innate immunity in the regulation of the insulin gene. Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. In a first study, using several approaches we showed that ATF6 (activating transcription factor 6), a protein implicated in the ER stress response, directly binds to the A5/Core of the insulin gene promoter in isolated rat islets. We also showed that overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibits the activity of an insulin promoter-reporter construct. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity. In recent years, the gut microbiota was proposed has an environmental factor increasing the risk of type 2 diabetes. Subjects with diabetes have higher circulating levels of lipopolysaccharides (LPS) than non-diabetic patients. Recent observations suggest that the signalling cascade activated by LPS binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. We showed that exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of two key transcription factors of the insulin gene, PDX-1 (pancreatic duodenal homeobox 1) and MafA (mammalian homologue of avian MafA/L-Maf). LPS repression of insulin, PDX-1 and MafA expression was not observed in islets from TLR4-deficient mice and was completely prevented in rat islets by inhibition of the NF-kB signalling pathway. These results demonstrate that LPS inhibits β-cell gene expression in a TLR4-dependent manner and via NF-kB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function. Our findings provide a better understanding of the molecular mechanisms underlying insulin gene repression in type 2 diabetes, and suggest potential therapeutic targets that might prevent or delay the decline of β-cell function in the course of type 2 diabetes, which affects more than two million Canadians.