Thesis (M.S.)--University of North Carolina Wilmington, 2008 / Includes appendixes. Title from PDF title page (viewed May 27, 2009) Includes bibliographical references (p. 112-117)

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Dr. Athanassios Sambanis; Committee Member: Dr. Barbara Boyan; Committee Member: Dr. Peter Thule.

Diabetes and congenital hyperinsulinism (CHI) are severe diseases affecting the pancreas. Current models for testing drugs to treat these diseases are in vivo in rodents or isolated rodent islets. Differences between the human and rodent pancreas, and ethical issues, mean that in vitro human models are needed. To develop a novel in vitro model for pancreatic diseases, mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) were derived from the pancreas of patients with CHI. MSCs from three forms of CHI were phenotypically normal for MSCs, and maintained the CHI-causing mutation. When compared to MSCs from bone marrow, the CHI pancreatic MSCs expressed pancreas-specific gene ISL1 and showed promoter hypomethylation of other pancreatic genes, including PDX1. The CHI pMSCs could be differentiated to cells resembling immature beta-cells, with some beta-cell gene expression (INS, PDX1), but no glucose responsive insulin secretion. CHI associated hypersecretion of insulin was not seen as the ATP-sensitive potassium KATP channels were not being expressed. Addition of the Wnt inhibitor DKK1 markedly enhanced differentiation via induction of neuronal genes. Alongside high insulin secretion, CHI also features increased proliferation. CHI MSCs were also hyperproliferative, and showed alterations to the cell cycle. These changes were related to p27Kip1 localisation, a known affected protein in CHI tissue, and CDK1, a novel regulator for CHI. iPSCs were also derived from focal CHI MSCs and were also phenotypically normal, but did not maintain the pancreatic hypomethylation present in MSCs. The CHI iPSCs were efficiently differentiated to definitive endoderm and PDX1 positive cells. Terminally differentiated iPSCs were endocrine, but were not mature beta-cells. In conclusion, authentic MSCs and iPSCs were derived for the first time from patients with CHI. These stem cells could be differentiated towards beta-cells, but mature glucose responsive beta-cells were not produced. MSC derived beta-like cells secreted insulin but did not have KATP channels, whereas iPSC derived beta-like cells had KATP channel gene expression but not INS. With further optimisation to resolve these, CHI stem cell derived beta-cells may be used for in vitro modelling. Further, the undifferentiated MSCs only show hyperproliferation associated with p27Kip1 and CDK1 and so can be a useful resource for modelling hyperproliferation seen in CHI.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic β-cells, mediated by autoreactive T cells.The resulting inflammatory response takes place in the context of a dialogue between invading immune cells and the targeted β-cells, and it is modulated by genetic susceptibility, acting on both immune and β-cells, and by inflammatory cytokines and chemokines. Stress pathways triggered within β-cells may potentiate autoimmunity, and T1D susceptibility genes shape β-cell responses to “danger signals”, innate immunity, and activation of apoptosis. However, the molecular mechanisms linking genetic variation, environmental triggers, and the signaling events promoting β-cell dysfunction and loss remain poorly clarified. Pre-mRNA splicing is a crucial mechanism for gene expression regulation, and more than 95% of the human multi-exonic primary transcripts undergo alternative splicing. Splicing dysregulation have been increasingly recognized to play a pivotal role in multiple pathologies, including autoimmune diseases. More than 15% of the mutations described in the Human Gene Mutation Database are predicted to affect splicing. Our group has shown that exposure to pro-inflammatory cytokines induces major changes on the β-cell transcriptome, affecting the splicing of genes that are key for β-cell function and survival. Importantly, our group identified that GLIS3, a susceptibility gene for both T1D and type 2 diabetes (T2D), modulates β-cell apoptosis via regulation of the splicing factor SRSF6, linking T1D genetic susceptibility and alternative splicing. The downregulation of GLIS3, either by germline mutations associated with monogenic forms of diabetes or risk single nucleotide polymorphisms, contribute to SRSF6 splicing factor downregulation. Splicing factors are the primary regulators of splicing and orchestrate functionally related transcripts into regulatory networks, therefore, oscillations of splicing factors’ expression levels have a major impact on splicing decisions. In the present study we aimed: 1. To evaluate the functional impact of SRSF6 downregulation in human pancreatic β-cells; 2. To identify the SRSF6-regulated splicing networks and to decode the SRSF6 cis-regulatory RNA binding elements.To fulfil these aims, human insulin-producing EndoC-βH1 cells were subjected to RNA sequencing (under control conditions or following SRSF6 knock down for 48h) to identify transcriptome-wide alternative splicing events regulated by SRSF6, and to individual-nucleotide resolution UV crosslinking and immunoprecipitation followed by high-throughput sequencing (iCLIP) to determine the SRSF6 mechanistic model of splicing regulation, its associated cis-regulatory elements and directly bound transcripts in human β-cells. We observed that SRSF6 depletion has a major impact on human pancreatic β-cell function and survival, leading to β-cell apoptosis and impaired insulin secretion. SRSF6 downregulation affects the splicing of transcripts involved in central pathways for β-cell function and survival, such as insulin secretion (e.g. INSR, SNAP25), apoptotic regulators (e.g. BCL2L11 (or BIM), BAX), and the mitogen-activated protein kinases (MAPKs) signaling pathway (e.g. MAPK8, MAPK9, MAP3K7). SRSF6 silencing potentiates the generation of constitutively active isoforms of pro-apoptotic inducers – BAX-β, and BIM-Small - leading to apoptosis activation, and also of different members of the MAPK signaling pathway contributing to the hyper-phosphorylation of the pathway, leading to activation of down-stream transcription factors and consequent β-cell apoptosis. These data indicate that specific splicing networks, regulated through diabetes susceptibility genes, control key pathways and processes involved in the function and survival of β-cells. The iCLIP analysis has shown that SRSF6 recognizes more than 100,000 of RNA binding sites in protein coding sequences, and it regulates splicing by preferentially binding into exons through a purine-rich consensus motif consisting of GAA triplets. The number of triplets in direct sequence correlates with increasing binding site strength. The SRSF6 binding position affects the splicing outcome, possibly resulting from the competition between alternative exons and their flanking constitutive exons for SRSF6 tethering. We identified SRSF6 binding sites on SRSF6-regulated cassette exons of several susceptibility genes for both T1D and T2D, and as a proof-of-concept, modulated the splicing of the LMO7 susceptibility gene using antisense oligonucleotides.In conclusion, our data suggest that SRSF6 is a master splicing regulator in pancreatic β-cells, downstream of the diabetes susceptibility gene GLIS3. SRSF6 silencing potentiates the splicing of constitutively active pro-apoptotic variants (BAX-β and BIM-Small), and exacerbates the MAPK signalling pathway. SRSF6 recognizes specific purine-rich RNA binding motifs, with important implications for the interpretation of sequence variants. This work unveiled a novel regulatory layer for β-cell demise and diabetes genetic susceptibility, namely through splicing mis-regulation. These observations raise the possibility that splicing networks regulated by candidate genes for diabetes contribute to β-cell dysfunction and death in diabetes. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

In 2011, Diabetes and prediabetes affected 9 million Canadians and 366 million people worldwide (Canadian Diabetes Website). The underlying pathophysiology of diabetes is beta cell dysfunction leading to loss of appropriate insulin secretion and resulting in hyperglycemia. I have focused on identifying critical molecular regulators of beta cell function and insulin secretion. The CRTC2-CREB pathway is required for maintaining beta cell mass and insulin secretion. I propose that identifying kinases that regulate CRTC-CREB activity will identify other important regulators of pancreatic beta cell survival and function. First, I have identified several AMP kinases as inhibitors of CRTC2-CREB that are activated by an upstream kinase, LKB1. I then went on to generate mice with a beta cell-specific deletion of LKB1 during adulthood. Loss of LKB1 increased insulin secretion and glucose clearance through enhanced beta cell mass and proliferation. The increased insulin secretion was largely the result of loss of AMPK activity and consequent constitutive mTor activity. AMPK is activated under starvation conditions and as such is thought to be a critical regulator of beta cell function. However, the decrease of AMPK activity in high glucose has been a strong argument against it being a critical effector of insulin secretion. I provide genetic evidence supporting the idea that AMPK activity attenuates insulin secretion. During periods of starvation where AMPK activity is high there is a chronic dampening effect on events that prepare beta cells for the next round of insulin secretion. Surprisingly, another downstream kinase of LKB1, SIK2, has opposing functions in the beta cell. I present evidence that the LKB1-AMPK axis attenuates beta cell functions and that targeting this pathway in beta cells may be of therapeutic benefit for T2D.

Les diabètes de type 1 et de type 2 sont caractérisés par une sécrétion insuffisante d’insuline et une diminution de la masse des cellules bêta. Pouvoir régénérer une masse de cellules bêta fonctionnelle est donc un enjeu thérapeutique dans le traitement du diabète. Dans cet optique, nous cherchons à identifier des facteurs et mécanismes permettant d’augmenter la masse de cellules bêta. Nous nous sommes intéressés aux mécanismes de plasticité des cellules bêta dans un contexte d’insulino résistance.Dans un modèle murin d’insulino-résistance provoquée par administration chronique de glucocorticoïdes, nous avons mis en évidence une adaptation de fonction des cellules bêta par hypersécrétion d’insuline. De manière intéressante une augmentation continue et progressive de la masse des cellules bêta par prolifération mais surtout par néogénèse de cellules bêta a pu être observée. Bien que la néogénèse de cellules bêta ait été décrite dans d’autres modèles murins comme un processus récapitulant le programme de différenciation fœtale c’est à dire dérivant de cellules canalaires marquées par l’expression de Sox9 et re-exprimant Ngn3, nos expériences de lignage endocrine ont révélé que les cellules bêta néoformées ne dérivent pas des cellules Sox9 ou Ngn3. L’invalidation du récepteur aux glucocorticoïdes (GR) dans le pancréas n’altère pas l’adaptation pancréatique par néogénèse dans notre modèle d’hypercorticisme, suggérant un effet indirect des GC sur la néogénèse de cellules bêta. Cette hypothèse a pu être confirmée par la mise en évidence de la présence dans le sérum des souris CORT d’un facteur capable de stimuler la néogénèse des cellules bêta in vitro. Enfin après déplétion totale des cellules bêta, l’administration de GC permet une restauration partielle de la masse de cellules béta par néogénèse.Nos résultats apportent la preuve d’une néogénèse active et induite de cellules bêta dans le pancréas adulte de souris insulino-résistantes. Cette adaptation pancréatique résulte d’une communication inter organe adaptative et l’identification du facteur pro-néogénique représente une piste thérapeutique pour les pathologies liées aux déficiences du pancréas endocrine. / Type 1 and type 2 diabetes are characterized by an insufficient insulin secretion and a decrease of beta cell mass. Regenerate a functional beta cell mass is a therapeutic issue in the treatment of diabetes. In this context we search to identify factors and mechanisms for increasing beta cell mass. We investigated mechanisms of beta cell plasticity in a context of insulin resistance.In a mouse model of insulin resistance caused by chronic administration of glucocorticoids, we demonstrated an adaptation of beta cell function by an important increase of insulin secretion. Interestingly, a continuous and progressive increase in the mass of beta cells by proliferation but especially by neogenesis of beta cells was observed.Although beta cell neogenesis has been described in other mouse models as a process recapitulating the fetal differentiation program deriving from ductal cells labeled with Sox9 expression and re-expressing Ngn3, our endocrine lineage model revealed that neoformed beta cells do not derive from Sox9 or Ngn3 cells. Inactivation of the glucocorticoid (GR) receptor in the pancreas does not alter pancreatic adaptation by neogenesis in our model of hypercorticism, suggesting an indirect effect of GCs on beta cell neogenesis. This hypothesis could be confirmed by demonstrating the presence in the serum of CORT mice of a factor able to stimulate neogenesis of beta cells in vitro. Finally, after complete depletion of beta cells, GC administration allows a partial restoration of the beta cells mass by neogenesis.Our results provide evidence of an active and induced beta-cell neogenesis in the adult pancreas of insulin-resistant mice. This pancreatic adaptation results from an inter-organ adaptive communication and the identification of the pro-neogenic factor represents a therapeutic track for pathologies related to endocrine pancreas deficiencies.

Butylparaben (Butyl p-hydroxybenzoic acid) is a widely used cosmetic and pharmaceutical preservative that has been recently shown to induce oxidative stress and have endocrine disrupting effects in rodents, and promote adipocyte conversion of human adipose cells. Embryonic development is extremely sensitive to oxidative stress due to changes in cell growth, development and differentiation that occur during this life stage. Fluctuations in redox potentials play critical roles in normal embryonic development by guiding these cell signaling, cell-fate decisions and apoptosis. The most prevalent endogenous antioxidant that defends against oxidative stress is glutathione (GSH), which scavenges reactive oxygen species. The low antioxidant capacity of pancreatic beta cells suggests that they are sensitive target tissues of oxidative stress; this has yet to be investigated during embryonic development. Here, we aim to 1) determine whether embryonic exposure to butylparaben prompts structural and functional changes in the developing endocrine pancreas and 2) determine whether oxidative stress may be involved. Transgenic insulin-GFP zebrafish embryos were treated daily with 250, 500, 1,000 and 3,000 nM butylparaben starting at 3 hours post fertilization (hpf). Pancreatic islet and whole embryo morphological development were examined daily until 7 days post fertilization (dpf). Redox potentials were measured at 24 and 28 hpf using HPLC. Area of the pancreatic islet increased over time with increasing butylparaben exposure in a dose-dependent manner by as much as a 55% increase in islet area at 3 dpf when compared to controls. Butylparaben concentrations of 500 and 1,000 nM increased GSH by 10 and 40%, respectively, and decreased oxidized glutathione disulfide by 37 and 59%. GSH redox potentials were only significant in embryos collected at 28 hpf and became more reduced with 500 and 1,000 nM butylparaben exposure, decreasing redox potentials by 7 and 18 mV, respectively. Cysteine redox potentials also became more reduced, decreasing by 17 and 28 mV. Our data show that butylparaben-induced redox potential disruptions that may be responsible for the effects on pancreatic islet structure and function, but further studies are needed to determine how and if that directly affects pancreas development.

Sex-dependent differences in the prevalence of diabetes and cardiovascular diseases are well established. The objective of this project is to investigate the molecular mechanisms by which estrogen modulates chronic disease progression. Our lab, and others, have previously implicated endoplasmic reticulum (ER) stress in the development and progression of diabetes and cardiometabolic disease. We hypothesize that estrogens protect pancreatic beta cell health, and slow the progression of cardiometabolic disease, by modulating the unfolded protein response (UPR) in response to ER stress. Two distinct mouse models were used in these studies. The ApoE-/-Ins2+/Akita mouse model of hyperglycemia-induced atherosclerosis, in which females are significantly protected from hyperglycemia and atherosclerosis relative to males, and the TALLYHO/JngJ mouse model, in which females are protected from chronic hyperglycemia relative to males. We found that ovariectomy of female ApoE-/-Ins2+/Akita or TALLYHO/JngJ mice promoted chronic hyperglycemia. Supplementation with exogenous 17-beta estradiol significantly lowered blood glucose levels in ovariectomized ApoE-/-Ins2+/Akita mice and reduced atherosclerotic lesion development in both male and ovariectomized female mice. Pancreatic islets from sham operated ApoE-/-Ins2+/Akita female mice showed a significant increase in the expression of protective UPR factors and a decrease in pro-apoptotic factors, compared to males or ovariectomized females. To determine if alleviating ER stress could moderate hyperglycemia, male and ovariectomized female TALLYHO/JngJ mice were treated with the chemical chaperone 4-phenylbutryic acid (4-PBA). We showed that 4-PBA treatment significantly lowered fasting blood glucose levels and improved glucose tolerance. The results of this thesis suggest that estrogens play a protective role in the maintenance of beta cell health and blood glucose regulation by activating the adaptive UPR. This mechanism may explain the protection observed in premenopausal women and may lead to the development of targeted therapies to treat diabetes and cardiometabolic diseases. / Thesis / Doctor of Philosophy (PhD) / People who suffer from diabetes mellitus have a higher risk of developing heart attack and stroke compared to those who do not have diabetes. Moreover, the risk of heart attack and stroke is higher in men than in women. We still do not understand the underlying reasons for these differences. This thesis project has used unique mouse models that display many of the same sex differences in disease progression that we see in humans to study the pathways and mechanisms that promote diabetes development. Specifically, we examined the protective effects of estrogen towards the development of diabetes and cardiovascular disease and how this hormone affected specific cells and tissues. The results of these studies are important because they will provide more information regarding the effects of menopause and aging on chronic disease progression in women.

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes mellitus is a group of metabolic diseases characterized by disordered insulin secretion from the pancreatic β cell and chronic hyperglycemia. In order to maintain adequate levels of insulin secretion, the β cell relies on a highly developed and active endoplasmic reticulum (ER). Calcium localized in this compartment serves as a cofactor for key proteins and enzymes involved in insulin production and maturation and is critical for ER health and function. The ER Ca2+ pool is maintained largely through activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) pump, which pumps two Ca2+ ions into the ER during each catalytic cycle. The goal of our research is to understand the molecular mechanisms through which SERCA2 maintains β cell function and whole body glucose metabolism. Our previous work has revealed marked dysregulation of β cell SERCA2 expression and activity under diabetic conditions. Using a mixture of pro-inflammatory cytokines to model the diabetic milieu, we found that SERCA2 activity and protein stability were decreased through nitric oxide and AMP-activated protein kinase (AMPK)mediated signaling pathways. Moreover, SERCA2 expression, intracellular Ca2+ storage, and β cell death under diabetic conditions were rescued by pharmacologic or genetic inhibition of AMPK. These findings provided novel insight into pathways leading to altered β cell Ca2+ homeostasis and reduced β cell survival in diabetes. To next define the role of SERCA2 in the regulation of whole body glucose homeostasis, SERCA2 heterozygous mice (S2HET) were challenged with high fat diet (HFD). Compare to wild-type controls, S2HET mice had lower serum insulin and significantly reduced glucose tolerance with similar adiposity and systemic and tissue specific insulin sensitivity, suggesting an impairment in insulin secretion rather than insulin action. Consistent with this, S2HET mice exhibited reduced β cell mass, decreased β cell proliferation, increased ER stress, and impaired insulin production and processing. Furthermore, S2HET islets displayed impaired cytosolic Ca2+ oscillations and reduced glucose-stimulated insulin secretion, while a small molecule SERCA2 activator was able to rescue these defects. In aggregate, these data suggest a critical role for SERCA2 and the maintenance of ER Ca2+ stores in the β cell compensatory response to diet induced obesity.

Indiana University-Purdue University Indianapolis (IUPUI) / Heat shock protein (HSP) 90 is a molecular chaperone that regulates diverse cellular processes by facilitating activities of various protein clients. Recent studies have shown serum levels of the alpha cytoplasmic HSP90 isoform are elevated in newly diagnosed type I diabetic patients, thus distinguishing this protein as a potential biomarker for pre-clinical type I diabetes mellitus (TIDM). This phase of disease is known to be associated with various forms of beta cell stress, including endoplasmic reticulum stress, insulitis, and hyperglycemia. Therefore, to test the hypothesis that HSP90 is released by these cells in response to stress, human pancreatic beta cells were subjected to various forms of stress in vitro. Beta cells released HSP90 in response to stimulation with a combination of cytokines that included IL-1β, TNF-α, and IFN-γ, as well as an agonist of toll-like receptor 3. HSP90 release was not found to result from cellular increases in HSP90AA1 gene or HSP90 protein expression levels. Rather, cell stress and ensuing cytotoxicity mediated by c-Jun N-terminal kinase (JNK) appeared to play a role in HSP90 release. Beta cell HSP90 release was attenuated by pre-treatment with tauroursodeoxycholic acid (TUDCA), which has been shown previously to protect beta cells against JNK-mediated, cytokine-induced apoptosis. Experiments here confirmed TUDCA reduced beta cell JNK phosphorylation in response to cytokine stress. Furthermore pharmacological inhibition and siRNA-mediated knockdown of JNK in beta cells also attenuated HSP90 release in response to cytokine stress. Pharmacological inhibition of HSP90 chaperone function exacerbated islet cell stress during the development of TIDM in vivo; however, it did not affect the overall incidence of disease. Together, these data suggest extracellular HSP90 could serve as a biomarker for preclinical TIDM. This knowledge may be clinically relevant in optimizing treatments aimed at restoring beta cell mass. The goal of such treatments would be to halt the progression of at-risk patients to insulin dependence and lifelong TIDM.