Academic literature on the topic 'Pancreatic β-islet cell'

Type 1 diabetes (T1D) is an autoimmune disease resulting from T cell–mediated destruction of insulin-producing β cells, and viral infections can prevent the onset of disease. Invariant natural killer T cells (iNKT cells) exert a regulatory role in T1D by inhibiting autoimmune T cell responses. As iNKT cell–plasmacytoid dendritic cell (pDC) cooperation controls viral replication in the pancreatic islets, we investigated whether this cellular cross talk could interfere with T1D development during viral infection. Using both virus-induced and spontaneous mouse models of T1D, we show that upon viral infection, iNKT cells induce TGF-β–producing pDCs in the pancreatic lymph nodes (LNs). These tolerogenic pDCs convert naive anti-islet T cells into Foxp3+ CD4+ regulatory T cells (T reg cells) in pancreatic LNs. T reg cells are then recruited into the pancreatic islets where they produce TGF-β, which dampens the activity of viral- and islet-specific CD8+ T cells, thereby preventing T1D development in both T1D models. These findings reveal a crucial cooperation between iNKT cells, pDCs, and T reg cells for prevention of T1D by viral infection.

Pancreatic islets from pregnant rats develop a transitory increase in the pancreatic β-cell proliferation rate and mass. Increased apoptosis during early lactation contributes to the rapid reversal of those morphological changes. Exposure to synthetic glucocorticoids during pregnancy has been previously reported to impair insulin secretion, but its impacts on pancreatic islet morphological changes during pregnancy and lactation have not been described. To address this issue, we assessed the morphological and molecular characteristics of pancreatic islets from rats that underwent undisturbed pregnancy (CTL) or were treated with dexamethasone between the 14th and 19th days of pregnancy (DEX). Pancreatic islets were analyzed on the 20th day of pregnancy (P20) and on the 3rd, 8th, 14th and 21st days of lactation (L3, L8, L14 and L21, respectively). Pancreatic islets from CTL rats exhibited transitory increases in cellular proliferation and pancreatic β-cell mass at P20, which were reversed at L3, when a transitory increase in apoptosis was observed. This was followed by the appearance of morphological features of pancreatic islet neogenesis at L8. Islets from DEX rats did not demonstrate an increase in apoptosis at L3, which coincided with an increase in the expression of M2 macrophage markers relative to M1 macrophage and T lymphocyte markers. Islets from DEX rats also did not exhibit the morphological characteristics of pancreatic islet neogenesis at L8. Our data demonstrate that maternal pancreatic islets undergo a renewal process during lactation that is impaired by exposure to DEX during pregnancy.

ObjectiveThe prevalence of type 2 diabetes mellitus escalates with aging although β-cell mass, a primary parameter of β-cell function, is subject to compensatory regulation. So far it is unclear whether the proliferative capacity of pancreatic islets is restricted by senescence.Materials and methodsHuman pancreatic tissue from n=20 non-diabetic organ donors with a mean age of 50.2±3.5 years (range 7–66 years) and mean body mass index of 25.7±0.9 kg/m2 (17.2–33.1 kg/m2) was morphometrically analyzed to determine β-cell volume, β-cell replication, β-cell apoptosis, islet neogenesis, and pancreatic duodenal homeobox-1 (PDX-1) expression.ResultsRelative β-cell volume in human pancreata (mean 2.3±0.2%) remains constant with aging (r=0.26, P=ns). β-cell replication (r=0.71, P=0.0004) decreases age-dependently, while β-cell apoptosis does not change significantly (r=0.42, P=0.08). Concomitantly, PDX-1 expression is downregulated with age in human pancreatic tissue (r=0.65, P=0.002). The rate of islet neogenesis is not affected by aging (r=0.13, P=ns).ConclusionsIn non-diabetic humans, aging is linked with impaired islet turnover possibly due to reduced PDX-1 expression. As β-cell replication is considered to be the main mechanism responsible for β-cell regeneration, these changes restrict the flexibility of the aging human pancreas to adapt to changing demands for insulin secretion and increase the risk for the development of diabetes mellitus in older subjects.

It is thought that differentiation of β-cell precursors into mature cells is largely autonomous, but under certain conditions differentiation can be modified by external factors. The factors that modify β-cell differentiation have not been identified. In this study, we tested whether adult islet cells can affect the differentiation process in mouse and human pancreatic anlage cells. We assessed β-cell proliferation and differentiation in mouse and human pancreatic anlage cells cocultured with adult islet cells or βTC3 cells using cellular, molecular, and immunohistochemical methods. Differentiation of murine anlage cells into β-cells was induced by mature islet cells. It was specific for β-cells and not a general feature of endodermal derived cells. β-Cell differentiation required cell-cell contact. The induced cells acquired features of mature β-cells including increased expression of β-cell transcription factors and surface expression of receptor for stromal cell-derived factor 1 and glucose transporter-2 (GLUT-2). They secreted insulin in response to glucose and could correct hyperglycemia in vivo when cotransplanted with vascular cells. Human pancreatic anlage cells responded in a similar manner and showed increased expression of pancreatic duodenal homeobox 1 and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and increased production of proinsulin when cocultured with adult islets. We conclude that mature β-cells can modify the differentiation of precursor cells and suggest a mechanism whereby changes in differentiation of β-cells can be affected by other β-cells. Mature β cells affect differentiation of pancreatic anlage cells into functional β cells. The differentiated cells respond to glucose and ameliorate diabetes.

Islets of Langerhans are microorgans scattered throughout the pancreas, and are responsible for synthesizing and secreting pancreatic hormones. While progress has recently been made concerning cell differentiation of the islets of Langerhans, the mechanism controlling islet morphogenesis is not known. It is thought that these islets are formed by mature cell association, first differentiating in the primitive pancreatic epithelium, then migrating in the extracellular matrix, and finally associating into islets of Langerhans. This mechanism suggests that the extracellular matrix has to be degraded for proper islet morphogenesis. We demonstrated in the present study that during rat pancreatic development, matrix metalloproteinase 2 (MMP-2) is activated in vivo between E17 and E19 when islet morphogenesis occurs. We next demonstrated that when E12.5 pancreatic epithelia develop in vitro, MMP-2 is activated in an in vitro model that recapitulates endocrine pancreas development (Miralles, F., P. Czernichow, and R. Scharfmann. 1998. Development. 125: 1017–1024). On the other hand, islet morphogenesis was impaired when MMP-2 activity was inhibited. We next demonstrated that exogenous TGF-β1 positively controls both islet morphogenesis and MMP-2 activity. Finally, we demonstrated that both islet morphogenesis and MMP-2 activation were abolished in the presence of a pan-specific TGF-β neutralizing antibody. Taken together, these observations demonstrate that in vitro, TGF-β is a key activator of pancreatic MMP-2, and that MMP-2 activity is necessary for islet morphogenesis.

Type 2 diabetes is caused by impaired insulin secretion and/or insulin resistance. Loss of pancreatic β-cell mass detected in human diabetic patients has been considered to be a major cause of impaired insulin secretion. Additionally, apoptosis is found in pancreatic β-cells; β-cell mass loss is induced when cell death exceeds proliferation. Recently, however, β-cell dedifferentiation to pancreatic endocrine progenitor cells and β-cell transdifferentiation to α-cell was reported in human islets, which led to a new underlying molecular mechanism. Hyperglycemia inhibits nuclear translocation and expression of forkhead box-O1 (FoxO1) and induces the expression of neurogenin-3 (Ngn3), which is required for the development and maintenance of pancreatic endocrine progenitor cells. This new hypothesis (Foxology) is attracting attention because it explains molecular mechanism(s) underlying β-cell plasticity. The lineage tracing technique revealed that the contribution of dedifferentiation is higher than that of β-cell apoptosis retaining to β-cell mass loss. In addition, islet cells transdifferentiate each other, such as transdifferentiation of pancreatic β-cell to α-cell and vice versa. Islet cells can exhibit plasticity, and they may have the ability to redifferentiate into any cell type. This review describes recent findings in the dedifferentiation and transdifferentiation of β-cells. We outline novel treatment(s) for diabetes targeting islet cell plasticity.

AbstractDiabetes mellitus is a significant public health problem worldwide. It encompasses a group of chronic disorders characterized by hyperglycemia, resulting from pancreatic islet dysfunction or as a consequence of insulin-producing β-cell death. Organ-on-a-chip platforms have emerged as technological systems combining cell biology, engineering, and biomaterial technological advances with microfluidics to recapitulate a specific organ’s physiological or pathophysiological environment. These devices offer a novel model for the screening of pharmaceutical agents and to study a particular disease. In the field of diabetes, a variety of microfluidic devices have been introduced to recreate native islet microenvironments and to understand pancreatic β-cell kinetics in vitro. This kind of platforms has been shown fundamental for the study of the islet function and to assess the quality of these islets for subsequent in vivo transplantation. However, islet physiological systems are still limited compared to other organs and tissues, evidencing the difficulty to study this “organ” and the need for further technological advances. In this review, we summarize the current state of islet-on-a-chip platforms that have been developed so far. We recapitulate the most relevant studies involving pancreatic islets and microfluidics, focusing on the molecular and cellular-scale activities that underlie pancreatic β-cell function.

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets α and δ cells resided at the periphery of the β-cell core. However, human islets were markedly different in that α, β, and δ cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of α, β, and δ cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. Precisely monitoring pancreatic β-cell proliferation and islet growth is a challenging area of research. We have developed a method to capture the distribution of β-cells in the intact pancreas of transgenic mice with fluorescence-tagged β-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Total β-cell area and islet number and size distribution are quantified with reference to specific parameters and location for each islet and for small clusters of β-cells. The entire distribution of islets can now be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and a qualitative comparison of changes in overall β-cell area at a glance.

The transcription factor Pax4 plays an essential role in the development of insulin-producing β cells in pancreatic islets. Ectopic Pax4 expression not only promotes β cell survival but also induces α-to-β cell transdifferentiation. This dual functionality of Pax4 makes it an appealing therapeutic target for the treatment of insulin-deficient type 1 diabetes (T1D). In this study, we demonstrated that Pax4 gene delivery by an adenoviral vector, Ad5.Pax4, improved β cell function of mouse and human islets by promoting islet cell survival and α-to-β cell transdifferentiation, as assessed by multiple viability assays and lineage-tracing analysis. We then explored the therapeutic benefits of Pax4 gene delivery in the context of islet transplantation using T1D mouse models. Both mouse-to-mouse and human-to-mouse islet transplantation, via either kidney capsule or portal vein, were examined. In all settings, Ad5.Pax4-treated donor islets (mouse or human) showed substantially better therapeutic outcomes. These results suggest that Pax4 gene delivery into donor islets may be considered as an adjunct therapy for islet transplantation, which can either improve the therapeutic outcome of islet transplantation using the same amount of donor islets or allow the use of fewer donor islets to achieve normoglycemia.

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.

Since the introduction of the Edmonton Protocol in 2000, islet transplantation has been emerging as promising therapy for Type I diabetes mellitus (T1DM) and currently is the only therapy that can achieve glycemic control without the need for exogenous insulin. Transplanting islet cells has several advantages over transplanting a whole pancreas in that it involves only a minor surgical procedure with low morbidity and mortality, and at a significantly lower cost. However, an obstacle to realizing this goal is a lack of an islet potency index as required by the U.S. Food and Drug Administration (FDA) biologics licensing, as well as a more complete understanding of the physiological mechanisms governing islet and β-cell physiology. Recently, the University of Illinois at Chicago (UIC) has developed a microfluidic platform that can mimic in vivo islet microenvironments through precise and dynamic control of perifusing culture media and oxygen culture levels; all while measuring functionally relevant factors including intracellular calcium levels, mitochondrial potentials, and insulin secretion. By developing an understanding of the physiology and pathophysiology of islets we can more effectively develop strategies that reduce metabolic stress and promote optimization in order to achieve improved success of islet transplantation and open new clinical avenues. The presentation begins by introducing key issues in the field of pancreatic islet transplantation as a clinical therapy for T1DM. This is followed by brief review various technologies that have been developed to study islet cells. The presentation then describes the design, application, and evolution of UIC’s microfluidic-based multimodal islet perifusion and live-cell imaging system for the study of pancreatic islet and β-cell physiology. The article then concludes presenting initial findings from studies seeking to develop an islet potency test.

Islet cell transplantation has already shown improved control of glucose levels and the potential to achieve insulin independence in type 1 diabetes mellitus, however there is a shortage of organ donors needed to match patient needs [1–2]. In the search for alternative sources of islets, many cell types have shown signs of β-cell differentiation by secreting c-peptide, insulin, and glucagon [3–5]. When maintained in serum-free medium, human epithelial-like pancreatic adenocarcinoma (PANC-1) cells and human-islet derived precursor cells (hIPCs) can go through a morphological transition and cluster [6]. These islet-like cell aggregates subsequently express glucagon, somatostatin, and insulin, indicating that clustering may play an important role in differentiation towards β-cells [7].