During treatment of diabetes mellitus by immunointervention or transplantation, it is necessary to monitor the markers of immune destruction or rejection of surviving insulin producing cells. An aim of this thesis is to improve the possibilities of following autoimmunity and to detect the survival of transplanted pancreatic islet in vivo. Partial aims included vitality testing of isolated islets for transplantation by measurement of respiration activity, observing the process of in vitro labeling of isolated islets with superparamagnetic iron oxide (SPIO) contrast agent for subsequent magnetic resonance imaging (MRI) of islets and observing SPIO particles transport after transplantation. We also studied a new dual paramagnetic contrast agent combined with fluorescein intended for identification of the MRI contrast agent in samples for histology. Further, we assessed autoimmune reaction by evaluation of cytokine response to specific stimulation with auto-antigens. We tried to affect beta-cells destruction by polyclonal anti- thymocyte antibodies in a mouse experimental model. A new method of the islet respiration measurement correlated with other methods of islet quality testing and it was suggested as a diagnostic test before clinical transplantation. Results obtained studying the intercellular...

Type I diabetes (T1D) is an autoimmune disorder affecting the insulin-producing beta cells of the islets of Langerhans, disrupting the glucose homeostasis regulatory system. Nowadays, islet transplantation is one of the anticipated treatments to revive the endocrinal function by injecting isolated pancreatic islets from a deceased donor into the patient’s liver’s portal vein. Regardless of the promising aspect, the main issue prior to transplantation is the inconsistent quality and low percentage of functioning islets post-transplantation. Therefore, a rapid islet functionality test with minimal complicated operation becomes necessary to tackle the pre-transplantation issue. This project revolves around the end-to-end development of an electrophysiology recording platform to monitor extracellular activities in murine pancreatic islets. A microfluidic perfusion system with hydrodynamic trapping is integrated with planar gold microelectrode arrays (MEA) as the preliminary device directly interfacing the islets. The design and fabrication of both the microfluidics and electrode devices, as well as in-house front-end electronics with analog filters and amplifiers tailored to capture the microvolt-scale signals, covered most of the project. Offline digital processing was performed in Python to analyse the recorded signals further. As a result, the complete platform and recording setup have been fully integrated, with successful islet trapping on top of electrodes and front-end electronics with 220x voltage gain and 0.1-3000 Hz bandwidth to record extracellular electrophysiology signals from intact pancreatic islets. While the current preliminary electrophysiology recordings are still quite inconclusive and require further validation, the project serves as a starting point in developing devices for extracellular electrophysiology measurement, which has not commonly been investigated specifically in pancreatic islets, and enables further exploration in the field. / Typ I-diabetes (T1D) är en autoimmun sjukdom som påverkar de insulinproducerande betacellerna på de Langerhanska öarna och stör det reglerande systemet för glukoshomeostas. Nuförtiden är ötransplantation en av de förväntade behandlingarna för att återuppliva den endokrina funktionen genom att injicera isolerade pankreasöar från en avliden donator i patientens levers portven. Oavsett den lovande aspekten är huvudfrågan före transplantation den inkonsekventa kvaliteten och låga andelen fungerande öar efter transplantationen. Därför blir ett snabbt funktionstest av öar med minimalt komplicerad operation nödvändigt för att ta itu med problemet före transplantation. Detta projekt kretsar kring end-to-end utveckling av en elektrofysiologisk inspelningsplattform för att övervaka extracellulära aktiviteter i murina pankreatiska öar. Ett mikrofluidiskt perfusionssystem med hydrodynamisk infångning är integrerat med plana guldmikroelektrodarrayer (MEA) som den preliminära enheten som direkt gränsar till öarna. Designen och tillverkningen av både mikrofluidik och elektrodenheter, såväl som intern front-end-elektronik med analoga filter och förstärkare skräddarsydda för att fånga signalerna i mikrovoltskala, täckte större delen av projektet. Offline digital bearbetning utfördes i Python för att analysera de inspelade signalerna ytterligare. Som ett resultat har den kompletta plattformen och inspelningsuppsättningen integrerats helt, med lyckad ö-infångning ovanpå elektroder och front-end-elektronik med 220x spänningsförstärkning och 0,1-3000 Hz för att registrera extracellulära elektrofysiologiska signaler från intakta pankreatiska öar. Medan de nuvarande preliminära elektrofysiologiska inspelningarna fortfarande är ganska ofullständiga och kräver ytterligare validering, fungerar projektet som en utgångspunkt för att utveckla enheter för extracellulär elektrofysiologisk mätning, som inte vanligtvis har undersökts specifikt i pankreasöar, och möjliggör ytterligare utforskning inom området.

Le diabète de type 2 (DT2) est une maladie métabolique complexe causée par des facteurs génétiques mais aussi environnementaux, tels la sédentarité et le surpoids. La dysfonction de la cellule β pancréatique est maintenant reconnue comme l’élément déterminant dans le développement du DT2. Notre laboratoire s’intéresse à la sécrétion d’insuline par la cellule β en réponse aux nutriments calorigéniques et aux mécanismes qui la contrôle. Alors que la connaissance des mécanismes responsables de l’induction de la sécrétion d’insuline en réponse aux glucose et acides gras est assez avancée, les procédés d’inhibition de la sécrétion dans des contextes normaux ou pathologiques sont moins bien compris. L’objectif de la présente thèse était d’identifier quelques-uns de ces mécanismes de régulation négative de la sécrétion d’insuline dans la cellule β pancréatique, et ce en situation normale ou pathologique en lien avec le DT2. La première hypothèse testée était que l’enzyme mitochondriale hydroxyacyl-CoA déshydrogénase spécifique pour les molécules à chaîne courte (short-chain hydroxyacyl-CoA dehydrogenase, SCHAD) régule la sécrétion d’insuline induite par le glucose (SIIG) par la modulation des concentrations d’acides gras ou leur dérivés tels les acyl-CoA ou acyl-carnitine dans la cellule β. Pour ce faire, nous avons utilisé la technologie des ARN interférants (ARNi) afin de diminuer l’expression de SCHAD dans la lignée cellulaire β pancréatique INS832/13. Nous avons par la suite vérifié chez la souris DIO (diet-induced obesity) si une exposition prolongée à une diète riche en gras activerait certaines voies métaboliques et signalétiques assurant une régulation négative de la sécrétion d’insuline et contribuerait au développement du DT2. Pour ce faire, nous avons mesuré la SIIG, le métabolisme intracellulaire des lipides, la fonction mitochondriale et l’activation de certaines voies signalétiques dans les îlots de Langerhans isolés des souris normales (ND, normal diet) ou nourries à la dière riche en gras (DIO) Nos résultats suggèrent que l’enzyme SCHAD est importante dans l’atténuation de la sécrétion d’insuline induite par le glucose et les acides aminés. En effet, l’oxydation des acides gras par la protéine SCHAD préviendrait l’accumulation d’acyl-CoA ou de leurs dérivés carnitine à chaîne courtes potentialisatrices de la sécrétion d’insuline. De plus, SCHAD régule le métabolisme du glutamate par l’inhibition allostérique de l’enzyme glutamate déshydrogénase (GDH), prévenant ainsi une hyperinsulinémie causée par une sur-activité de GDH. L’étude de la dysfonction de la cellule β dans le modèle de souris DIO a démontré qu’il existe une grande hétérogénéité dans l’obésité et l’hyperglycémie développées suite à la diète riche en gras. L’orginialité de notre étude réside dans la stratification des souris DIO en deux groupes : les faibles et forts répondants à la diète (low diet responders (LDR) et high diet responder (HDR)) sur la base de leur gain de poids corporel. Nous avons mis en lumières divers mécanismes liés au métabolisme des acides gras impliqués dans la diminution de la SIIG. Une diminution du flux à travers le cycle TG/FFA accompagnée d’une augmentation de l’oxydation des acides gras et d’une accumulation intracellulaire de cholestérol contribuent à la diminution de la SIIG chez les souris DIO-HDR. De plus, l’altération de la signalisation par les voies AMPK (AMP-activated protein kinase) et PKC epsilon (protéine kinase C epsilon) pourrait expliquer certaines de ces modifications du métabolisme des îlots DIO et causer le défaut de sécrétion d’insuline. En résumé, nous avons mis en lumière des mécanismes importants pour la régulation négative de la sécrétion d’insuline dans la cellule β pancréatique saine ou en situation pathologique. Ces mécanismes pourraient permettre d’une part de limiter l’amplitude ou la durée de la sécrétion d’insuline suite à un repas chez la cellule saine, et d’autre part de préserver la fonction de la cellule β en retardant l’épuisement de celle-ci en situation pathologique. Certaines de ces voies peuvent expliquer l’altération de la sécrétion d’insuline dans le cadre du DT2 lié à l’obésité. À la lumière de nos recherches, le développement de thérapies ayant pour cible les mécanismes de régulation négative de la sécrétion d’insuline pourrait être bénéfique pour le traitement de patients diabétiques. / Type 2 diabetes (T2D) is a complex metabolic disease caused by genetic as well as environmental factors, such as sedentarity and obesity. Pancreatic β cell dysfunction is now recognized as the key factor in T2D development. Our laboratory is studying the mechanisms of regulation of insulin secretion by the pancreatic β cell in response to nutrients. While the knowledge of the mechanisms responsible for initiation of insulin secretion in response to glucose and fatty acids is quite advanced, the inhibitory processes of insulin secretion in normal or pathological situations are still poorly understood. This doctoral thesis has focused on the identification of some of the mechanisms responsible for negative regulation of insulin secretion in pancreatic β cell. We have addressed this issue under normal situation or pathological conditions related to T2D. We first tested the hypothesis by which a mitochondrial enzyme, short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), negatively regulates glucose-induced insulin secretion (GIIS) by limiting the concentrations of some fatty acids and their derivatives such as acyl-CoA or acyl-carnitine molecules in the β cell. For this purpose, the downregulation of SCHAD by RNA interference (RNAi) was used in the pancreatic β cell line INS832/13. Then, we tested wether a prolonged administration of high-fat diet to mice (diet-induced obesity mouse model, DIO) would modulate intracellular metabolic and molecular pathways responsible for inhibition of insulin secretion. C57BL/6 mice were therefore fed a high-fat diet for 8 weeks followed by insulin secretion, intracellular lipid metabolism, mitochondrial function and intracellular signaling measurements on isolated pancreatic islets of Langerhans of those mice. Our results suggest that SCHAD negatively regulates GIIS and amino acid-induced insulin secretion. We propose that fatty acid oxidation by SCHAD would prevent the accumulation of short-chain acyl-CoAs or acyl-carnitines capable of potentiating insulin secretion. In addition, SCHAD regulates glutamate metabolism by the allosteric inhibition of glutamate dehydrogenase (GDH) preventing the hyperinsulinemia caused by excessive GDH activity. The study of β cell dysfunction in the DIO mouse model stratified LDR and HDR highlighted various fatty acid metabolism pathways involved in the reduction of GIIS. A decrease in the triglycerides/free fatty acid (TG/FFA) cycling associated with an increase in fatty acid oxidation and intracellular accumulation of cholesterol was shown to contribute to the decreased GIIS in DIO-HDR mice. Furthermore, alteration of AMP-activated kinase (AMPK) and protein kinase C epsilon (PKC epsilon) signaling pathways would be responsible for those alterations in metabolic pathways observed in DIO islets and cause decreased insulin secretion. In summary, we have shed light on important pathways negatively regulating insulin secretion in pancreatic β cell. These pathways could either limit the amplitude or duration of insulin secretion after a meal, or help to preserve β-cell function by delaying exhaustion. Some of those signaling pathways could explain the altered insulin secretion observed in T2D obese patients. In light of our research, the development of therapies targeting pathways that negatively regulate insulin secretion may be beneficial for treating diabetic patients.

Le diabète de type 2 (DT2) est une maladie métabolique complexe causée par des facteurs génétiques mais aussi environnementaux, tels la sédentarité et le surpoids. La dysfonction de la cellule β pancréatique est maintenant reconnue comme l’élément déterminant dans le développement du DT2. Notre laboratoire s’intéresse à la sécrétion d’insuline par la cellule β en réponse aux nutriments calorigéniques et aux mécanismes qui la contrôle. Alors que la connaissance des mécanismes responsables de l’induction de la sécrétion d’insuline en réponse aux glucose et acides gras est assez avancée, les procédés d’inhibition de la sécrétion dans des contextes normaux ou pathologiques sont moins bien compris. L’objectif de la présente thèse était d’identifier quelques-uns de ces mécanismes de régulation négative de la sécrétion d’insuline dans la cellule β pancréatique, et ce en situation normale ou pathologique en lien avec le DT2. La première hypothèse testée était que l’enzyme mitochondriale hydroxyacyl-CoA déshydrogénase spécifique pour les molécules à chaîne courte (short-chain hydroxyacyl-CoA dehydrogenase, SCHAD) régule la sécrétion d’insuline induite par le glucose (SIIG) par la modulation des concentrations d’acides gras ou leur dérivés tels les acyl-CoA ou acyl-carnitine dans la cellule β. Pour ce faire, nous avons utilisé la technologie des ARN interférants (ARNi) afin de diminuer l’expression de SCHAD dans la lignée cellulaire β pancréatique INS832/13. Nous avons par la suite vérifié chez la souris DIO (diet-induced obesity) si une exposition prolongée à une diète riche en gras activerait certaines voies métaboliques et signalétiques assurant une régulation négative de la sécrétion d’insuline et contribuerait au développement du DT2. Pour ce faire, nous avons mesuré la SIIG, le métabolisme intracellulaire des lipides, la fonction mitochondriale et l’activation de certaines voies signalétiques dans les îlots de Langerhans isolés des souris normales (ND, normal diet) ou nourries à la dière riche en gras (DIO) Nos résultats suggèrent que l’enzyme SCHAD est importante dans l’atténuation de la sécrétion d’insuline induite par le glucose et les acides aminés. En effet, l’oxydation des acides gras par la protéine SCHAD préviendrait l’accumulation d’acyl-CoA ou de leurs dérivés carnitine à chaîne courtes potentialisatrices de la sécrétion d’insuline. De plus, SCHAD régule le métabolisme du glutamate par l’inhibition allostérique de l’enzyme glutamate déshydrogénase (GDH), prévenant ainsi une hyperinsulinémie causée par une sur-activité de GDH. L’étude de la dysfonction de la cellule β dans le modèle de souris DIO a démontré qu’il existe une grande hétérogénéité dans l’obésité et l’hyperglycémie développées suite à la diète riche en gras. L’orginialité de notre étude réside dans la stratification des souris DIO en deux groupes : les faibles et forts répondants à la diète (low diet responders (LDR) et high diet responder (HDR)) sur la base de leur gain de poids corporel. Nous avons mis en lumières divers mécanismes liés au métabolisme des acides gras impliqués dans la diminution de la SIIG. Une diminution du flux à travers le cycle TG/FFA accompagnée d’une augmentation de l’oxydation des acides gras et d’une accumulation intracellulaire de cholestérol contribuent à la diminution de la SIIG chez les souris DIO-HDR. De plus, l’altération de la signalisation par les voies AMPK (AMP-activated protein kinase) et PKC epsilon (protéine kinase C epsilon) pourrait expliquer certaines de ces modifications du métabolisme des îlots DIO et causer le défaut de sécrétion d’insuline. En résumé, nous avons mis en lumière des mécanismes importants pour la régulation négative de la sécrétion d’insuline dans la cellule β pancréatique saine ou en situation pathologique. Ces mécanismes pourraient permettre d’une part de limiter l’amplitude ou la durée de la sécrétion d’insuline suite à un repas chez la cellule saine, et d’autre part de préserver la fonction de la cellule β en retardant l’épuisement de celle-ci en situation pathologique. Certaines de ces voies peuvent expliquer l’altération de la sécrétion d’insuline dans le cadre du DT2 lié à l’obésité. À la lumière de nos recherches, le développement de thérapies ayant pour cible les mécanismes de régulation négative de la sécrétion d’insuline pourrait être bénéfique pour le traitement de patients diabétiques. / Type 2 diabetes (T2D) is a complex metabolic disease caused by genetic as well as environmental factors, such as sedentarity and obesity. Pancreatic β cell dysfunction is now recognized as the key factor in T2D development. Our laboratory is studying the mechanisms of regulation of insulin secretion by the pancreatic β cell in response to nutrients. While the knowledge of the mechanisms responsible for initiation of insulin secretion in response to glucose and fatty acids is quite advanced, the inhibitory processes of insulin secretion in normal or pathological situations are still poorly understood. This doctoral thesis has focused on the identification of some of the mechanisms responsible for negative regulation of insulin secretion in pancreatic β cell. We have addressed this issue under normal situation or pathological conditions related to T2D. We first tested the hypothesis by which a mitochondrial enzyme, short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), negatively regulates glucose-induced insulin secretion (GIIS) by limiting the concentrations of some fatty acids and their derivatives such as acyl-CoA or acyl-carnitine molecules in the β cell. For this purpose, the downregulation of SCHAD by RNA interference (RNAi) was used in the pancreatic β cell line INS832/13. Then, we tested wether a prolonged administration of high-fat diet to mice (diet-induced obesity mouse model, DIO) would modulate intracellular metabolic and molecular pathways responsible for inhibition of insulin secretion. C57BL/6 mice were therefore fed a high-fat diet for 8 weeks followed by insulin secretion, intracellular lipid metabolism, mitochondrial function and intracellular signaling measurements on isolated pancreatic islets of Langerhans of those mice. Our results suggest that SCHAD negatively regulates GIIS and amino acid-induced insulin secretion. We propose that fatty acid oxidation by SCHAD would prevent the accumulation of short-chain acyl-CoAs or acyl-carnitines capable of potentiating insulin secretion. In addition, SCHAD regulates glutamate metabolism by the allosteric inhibition of glutamate dehydrogenase (GDH) preventing the hyperinsulinemia caused by excessive GDH activity. The study of β cell dysfunction in the DIO mouse model stratified LDR and HDR highlighted various fatty acid metabolism pathways involved in the reduction of GIIS. A decrease in the triglycerides/free fatty acid (TG/FFA) cycling associated with an increase in fatty acid oxidation and intracellular accumulation of cholesterol was shown to contribute to the decreased GIIS in DIO-HDR mice. Furthermore, alteration of AMP-activated kinase (AMPK) and protein kinase C epsilon (PKC epsilon) signaling pathways would be responsible for those alterations in metabolic pathways observed in DIO islets and cause decreased insulin secretion. In summary, we have shed light on important pathways negatively regulating insulin secretion in pancreatic β cell. These pathways could either limit the amplitude or duration of insulin secretion after a meal, or help to preserve β-cell function by delaying exhaustion. Some of those signaling pathways could explain the altered insulin secretion observed in T2D obese patients. In light of our research, the development of therapies targeting pathways that negatively regulate insulin secretion may be beneficial for treating diabetic patients.

The prevalence of diabetes is approaching epidemic proportions worldwide. There is currently no cure for type 1 diabetes, and successful treatment requires constant monitoring of blood sugars and use of exogenous insulin to prevent hyperglycemia. Diabetes will be curable when pancreatic β-islet cells can be transplanted into diabetes patients without requiring long-term immunosuppression. This will require learning more about the induction of functional tolerance, a state that maintains the competence of the immune system to most antigens but protects graft-specific antigens from immune rejection, permitting transplantation. One known mechanism of peripheral tolerance is T cell anergy, a phenotype of hypo-reponsiveness in CD4+ T cells. The focus of this thesis is a description of factors shown to be specific to the induction and maintenance of T cell anergy, whose loss reverses the anergic phenotype, restoring the ability of the cells to proliferate in response to antigen. The first of these is Egr-2, a zinc-finger transcription factor, whose presence is required for the induction of anergy induced in T cell clones by TCR stimulation in the absence of costimulation. Egr-2 is shown to be important to anergy induction but not anergy maintenance. In contrast, a negative costimulation receptor, PD-1, is shown to be necessary for the maintenance of anergy. It is possible that learning more about the genetic factors that orchestrate T cell anergy will prove useful in the development of tolerance-based protocols for organ and tissue transplantation without the use of long-term immunosuppression.

Indiana University-Purdue University Indianapolis (IUPUI) / Type I diabetes is an autoimmune disorder characterized by the loss of insulin producing islet cell mass. While daily insulin injection provides an easy means of glycemic control, it does not prevent long-term complications associated with diabetes. Islet transplantation has been suggested as a permanent cure for type 1 diabetes. However, the recurrence of host immunity and shortage of donor islets hinder the prevalence of islet transplantation. Biomaterial strategies provide an alternative route to solving the problems associated with host immune response and shortage of donor islets. One highly recognized platform for achieving these goals are hydrogels, which are hydrophilic crosslinked polymers with tissue-like elasticity and high permeability. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, including encapsulation of pancreatic islets and serving as a material platform for pseudo-islet differentiation. PEG hydrogels formed by mild and rapid thiol-ene photo-click reactions are particularly useful for studying cell behaviors in three-dimension (3D). Thiol-ene PEG-based hydrogels can be rendered biodegradable if appropriate macromer and cross-linker chemistry is employed. However, the influence of hydrogel matrix properties on the survival, growth, and morphogenesis of cells in 3D has not been fully evaluated. This thesis aims at using norbornene-functionalized PEG macromers to prepare thiol-ene hydrogels with various stiffness and degradability, from which to study the influence of hydrogel properties on pancreatic cell fate processes in 3D. Toward establishing an adaptable hydrogel platform for pancreatic tissue engineering, this thesis systematically studies the influence of hydrogel properties on encapsulated endocrine cells (e.g., MIN6 beta-cells) and exocrine cells (PANC-1 cells), as well as human mesenchymal stem cells (hMSC). It was found that thiol-ene photo-click hydrogels provide a cytocompatible environment for 3D culture of these cells. However, cell viability was negatively affected in hydrogels with higher cross-linking density. In contrast to a monolayer when cultured on a 2D surface, cells with epithelial characteristic formed clusters and cells with mesenchymal features retained single cell morphology in 3D. Although cells survived in all hydrogel formulations studied, the degree of proliferation, and the size and morphology of cell clusters formed in 3D were significantly influenced by hydrogel matrix compositions. For example: encapsulating cells in hydrogels formed by hydrolytically degradable macromer positively influenced cell survival indicated by increased proliferation. In addition, when cells were encapsulated in thiol-ene gels lacking cell-adhesive motifs, hydrolytic gel degradation promoted their survival and proliferation. Further, adjusting peptide crosslinker type and immobilized ECM-mimetic bioactive cues provide control over cell fate by determining whether observed cellular morphogenesis is cell-mediated or matrix-controlled. These fundamental studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for pancreatic tissue engineering

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) / Type 2 diabetes manifests from peripheral insulin resistance and a loss of functional beta cell mass due to decreased beta cell function, survival, and/or proliferation. Beta cell stressors impair each of these factors by activating stress response mechanisms, including endoplasmic reticulum (ER) stress. The glucolipotoxic environment of the diabetic milieu also activates a stress response in beta cells, resulting in death and decreased survival. Whereas the cell cycle machinery (comprised of cyclins, kinases, and inhibitors) regulates proliferation, its involvement during beta cell stress in the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two independent pharmacologic inducers of beta cell stress - dexamethasone and thapsigargin. In addition, glucolipotoxic stress mimicking the diabetic milieu also induced p21. To further investigate p21’s role in the beta cell, p21 was adenovirally overexpressed in 832/13 cells and rat islets. As expected given p21’s role as a cell cycle inhibitor, p21 overexpression decreased [3H]-thymidine incorporation and blocked the G1/S and G2/M transitions as quantified by flow cytometry. Interestingly, p21 overexpression activated apoptosis, demonstrated by increased annexin- and propidium iodide-double-positive cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the anti-apoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the pro-apoptotic proteins Bax and Bak. Therefore, the intrinsic apoptotic pathway is central for p21-mediated cell death. Like glucolipotoxicity, p21 overexpression inhibited the insulin cell survival signaling pathway while also impairing glucose-stimulated insulin secretion, an index of beta cell function. Under both conditions, phosphorylation of insulin receptor substrate-1, Akt, and Forkhead box protein-O1 was reduced. p21 overexpression increased Bim and c-Jun N-terminal Kinase, however, siRNA-mediated reduction or inhibition of either protein, respectively, did not alter p21-mediated cell death. Importantly, islets of p21-knockout mice treated with the ER stress inducer thapsigargin displayed a blunted apoptotic response. In summary, our findings indicate that p21 decreases proliferation, activates apoptosis, and impairs beta cell function, thus being a potential target to inhibit for the protection of functional beta cell mass.

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.

No description available.