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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Type 1 and type 2 diabetes are characterized by hyperglycemia and loss of beta-cell mass, function, and peptide hormones. Beyond hyperglycemia, diabetes is also associated with obesity and increased risk of cancer, particularly pancreatic cancer. Islet amyloid polypeptide (IAPP) is the second most abundant beta-cell hormone, co-secreted with insulin, and reduced in diabetes. Paracrine actions of endogenous IAPP have been proposed in glycemic regulation and tumour growth suppression, but remain incompletely understood. To further characterize the effects of IAPP loss, we used an IAPP-knockout mouse fed a high-fat or control diet and assessed glycemia and adiposity. We also generated a genetic mouse model of pancreatic ductal adenocarcinoma in IAPP-knockout mice to investigate IAPP loss as a potential mechanism for the association of diabetes and pancreatic cancer. Trends in IAPP-knockout mice suggest elevated adiposity and glycemia in the absence of IAPP. We observed no effect of IAPP on pancreatic ductal adenocarcinoma survival in mice, or cancer cell proliferation, death, or glycolysis.A reduction in the processing efficiency of beta-cell peptide prohormones is also observed in diabetes. Pcsk1 and Pcsk2 are the two major prohormone endoproteases within insulin granules, and are responsible for multiple steps in the proIAPP and proinsulin processing pathways. To determine whether impaired processing functions as a biomarker or driver of beta-cell dysfunction, we generated mouse models of beta-cell specific (Ins1cre-driven) Pcsk1 and Pcsk2 deficiency using Cre-lox recombination. Loss of Pcsk1 in beta cells caused severe proinsulin processing impairments and increased diabetes susceptibility in male mice, while female mice remained euglycemic. In contrast, male and female beta-cell Pcsk2-deficient mice were euglycemic with minimally impaired proinsulin processing, but severely impaired proIAPP processing. Deletion of both Pcsk1 and Pcsk2 in beta cells blocked proinsulin processing, drove hyperglycemia, increased beta-cell glucose responsiveness and mass, and reduced beta-cell maturity and proliferative capacity. In a mouse model expressing amyloidogenic human IAPP in beta cells, Pcsk1 deficiency significantly increased amyloid deposition. Collectively, these findings show that loss of beta-cell prohormone processing alters beta-cell function and drives hyperglycemia, and that endogenous IAPP does not act as a pancreatic tumour suppressor but may alter glycemia and adiposity.
Pancreatic islet inflammation and beta-cell death are features of type 2 diabetes, contributing to inadequate insulin secretion and hyperglycemia. Macrophages, innate immune cells, form a dispersed homeostatic organ found in most mammalian tissues, including insulin-producing islets. Macrophages act as local sentinel cells that can either contribute to the pro-inflammatory milieu of the islet or support beta-cell proliferation in mouse models of beta-cell regeneration. They constantly monitor their surroundings and are able to uptake particles from the blood. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in people who are newly diagnosed with type 2 diabetes and activation of TLRs facilitates proinflammatory responses in macrophages. Conversely, apoptotic cells promote a tissue repair program in macrophages, and apoptosis is thought to be the main mechanism of beta-cell death in type 2 diabetes. Thus, based on the hypothesis that the polarization of islet macrophages is adaptively shaped by changes in their microenvironment and has functional consequences on beta cells, this study was designed to evaluate the physiology of islet macrophages and beta cells in two settings: first, in response to TLR2/6- and TLR4- ligands, and secondly, following beta-cell death. Genetic and pharmacological approaches were used to thoroughly characterize islet macrophages from C57BL/6J, TLR2- and TLR4-deficient, streptozotocin (STZ)-treated, high-fat diet (HFD)-STZ-treated, and db/db mice. Macrophages were found to be major contributors to human islet IL-1β secretion in response to TLR2/6 and TLR4 ligands and reduced insulin secretion from mouse pancreatic beta cells, partly via IL-1- and IL-6-mediated decrease in insulin gene expression. Following STZ-induced beta-cell death, in diabetic db/db, and HFD+STZ mice, islet macrophages acquired a reparative state characterized by enhanced Igf1, and decreased proinflammatory cytokine expression. Depletion and adoptive transfer experiments indicated a role for macrophages in regulating insulin secretion in vivo, and IGF-1 neutralization during STZ-treatment decreased insulin secretion without affecting islet-cell turnover. Importantly, macrophages were still in a reparative state even when beta-cell death occurred during HFD feeding or severe hyperglycemia. Overall, these data may have implications for islet pathophysiology in type 2 diabetes and suggest that islet macrophages could be manipulated to either induce or resolve islet inflammation.
Soaring rates of diabetes highlight the importance of controlling this global epidemic, with an estimated 415 million people thought to be living with diabetes in 2015. The defining characteristic of diabetes is elevated fasting blood glucose levels, or hyperglycemia, which if not controlled promotes long-term complications such as neuropathy, kidney failure and damage to blood vessels. Glucose homeostasis is primarily controlled by pancreatic islets, cell clusters that mediate the endocrine functions of the pancreas. To manage circulating glucose concentrations, islet beta cells synthesize proinsulin, a peptide that undergoes proteolytic cleavage to become bioactive. In this dissertation, I examine the processing of the beta-cell protein pro-islet amyloid polypeptide (proIAPP), a propeptide that is cleaved similarly to proinsulin to form the aggregation-prone mature IAPP molecule, and determine whether impairments in the processing of this protein accelerate diabetes development. First, I generated a novel immunoassay to quantify the concentration of IAPP prohormone precursors for the first time in human circulation. Following rigorous validation of this ELISA, I demonstrated that elevated levels of the NH₂-proIAPP₁-₄₈ intermediate form are characteristic of type 1 diabetic recipients of islet transplants, and children with impaired glucose tolerance. Furthermore, I elucidated that this effect was not true for patients with established type 2 diabetes, implicating the peptide intermediate as a biomarker of diabetes onset but not a marker of the diseased state. Following this, I generated a rodent transplant model in which the loss of prohormone convertase 2 (PC2), essential for proIAPP processing in rodents, led to early islet transplant failure. Using a beta cell-specific PC2 null mouse that we generated, I also demonstrated that the loss of this enzyme in beta cells promotes earlier development of diabetes. Lastly, I was successful in establishing an in vitro islet culture model in which the overexpression of a non-cleavable proIAPP substrate leads to increased islet cell death. Altogether, the work in this dissertation highlights the importance of precise prohormone processing in the pancreatic islet, and demonstrates a role for proIAPP processing intermediates as biomarkers of diabetes and contributors to beta-cell dysfunction.
Patients with type 2 diabetes experience an inevitable deterioration of glycemic control leading to long-term complications and dependence on exogenous insulin. Amyloid deposition, macrophage infiltration, and upregulation of pro-inflammatory cytokines are common pathological features of both type 2 diabetic and transplanted islets. Islet amyloid is comprised primarily of aggregates of islet amyloid polypeptide (IAPP), a peptide that is co-secreted with insulin by beta cells. IAPP fibrils share a common cross-β-sheet structure with other amyloids of mammalian and microbial origin that activate innate immune cells via interaction with pattern recognition receptors. We therefore hypothesized that IAPP aggregation acts as a local trigger for islet inflammation. We found that human IAPP, but not non-amyloidogenic rodent IAPP, induced a potent pro-inflammatory response in islets and macrophages that was amplified by autocrine/paracrine induction of interleukin-1 (IL-1). Pre-fibrillar IAPP activated the membrane-associated pattern recognition receptor Toll-like receptor 2 (TLR2) to induce expression of proIL-1β. Secretion of mature IL-1β required fibrillar IAPP and was attenuated by inhibitors of caspase-1 and the cytosolic NLRP3 inflammasome. Pancreatic islets from transgenic mice with beta cell expression of human IAPP expressed higher levels of pro-inflammatory cytokines than islets from wild-type littermates. Transgenic expression of human IAPP also altered the activation state of resident islet macrophages, the primary cell type responsible for IAPP-induced upregulation of IL-1β. Clodronate liposome-mediated macrophage depletion improved islet function in human IAPP transgenic mice. Moreover, administration of IL-1 receptor antagonist improved human IAPP-induced glucose intolerance in mouse models of islet transplantation and type 2 diabetes. Inhibition of a local IAPP-induced pro-inflammatory response mediated by islet macrophages may therefore help to explain the islet-specific effects of anti-IL-1 therapies in patients with type 2 diabetes. Collectively, these data suggest a novel – and potentially reversible – mechanism by which IAPP aggregation contributes to beta cell dysfunction and implicate the resident islet macrophage as a critical mediator of chronic islet inflammation in the setting of amyloid formation. Strategies to block TLR2 or NLRP3 activation, inhibit IL-1 signalling, or alter macrophage polarization may improve IAPP-induced islet dysfunction in type 2 diabetes and islet transplantation.
Islet transplantation has great promise as a treatment for patients with insulin-dependent diabetes but its long-term success is limited by progressive graft dysfunction. Many measures of cell dysfunction in transplantation are similar to those observed in the type 2 diabetic cell. We focused upon two particular genes underlying pathology of the latter, namely islet amyloid polypeptide (IAPP) and C/EBP homologous protein (CHOP). We hypothesized that CHOP and islet amyloid composed of IAPP played distinct roles in progressive dysfunction and loss of the transplanted cell. Human islets and murine islets expressing the IAPP transgene developed islet amyloid following transplantation. Amyloid deposition correlated with loss of glycemic control and was exacerbated by transplantation of a marginal islet mass. Further, pre-existing amyloid in human islets prior to transplantation correlated with graft dysfunction at one year following islet transplantation into human allograft recipients. We tested several strategies to protect against islet amyloid toxicity in a pre-clinical model of human islet culture. Human IAPP deposition and toxicity was abrogated by siRNA against IAPP and by peptide inhibitors of IAPP aggregation. As an alternate strategy, transplantation of porcine islets expressing minimally amyloidogenic forms of IAPP yields excellent long-term outcomes. Islet amyloid deposition and toxicity is particularly rapid in the transplanted islet. This phenomenon may relate to factors in the transplant environment. We demonstrated that heparin, used in clinical islet transplantation, potentiates amyloid deposition in human islets. In addition, it exacerbates IAPP toxicity to cultured cells and accelerates graft failure in marginal mass human islet grafts. CHOP is activated by prolonged endoplasmic reticulum or oxidative stress. We demonstrated that CHOP immunostaining is increased in marginal mass islet grafts. Transplantation of islets in which CHOP has been deleted or silenced by RNA significantly improves glycemic normalization in marginal mass grafts and reduces apoptosis. These data suggest that CHOP plays a detrimental role in islet graft dysfunction. These studies demonstrate a role for two independent non-immune factors in mediating islet graft dysfunction and for which therapeutic modulation may improve cell function and survival in both islet transplantation and type 2 diabetes.
Type 1 diabetes is an autoimmune disorder characterized by destruction of insulin-producing beta cells by autoreactive T cells. Despite management of type 1 diabetes with insulin therapy, affected individuals face devastating complications, such as blindness and kidney failure. Islet transplantation has emerged as a promising therapy to replace beta cells; unfortunately, it is still challenged by a high rate of graft failure due to allo- and recurrent auto-immune responses. The pathogenesis of type 1 diabetes results from a combination of genetic and environmental factors that cause an imbalance in regulatory mechanisms, including defects in T regulatory (Treg) and natural killer T (NKT) cells. We believe that enhancing the activity of these cells locally may have the desirable effect of modulating the immune response against beta cells. Interestingly, the chemokines CCL22 and CCL17 preferentially recruit Tregs. We hypothesized that expression of these chemokines in islets would prevent beta cell destruction by enhancing influx of immunoregulatory cells. We used viral vectors to induce expression of CCL22 or CCL17 in islets and tested this hypothesis in models of spontaneous and recurrent autoimmune diabetes in the non-obese diabetic mouse. We found that CCL22 expression in pancreatic islets prevented diabetes development and protected islet transplants from recurrent autoimmunity, although not indefinitely. CCL22-expressing islets recruited Tregs, invariant NKT and plasmacytoid dendritic cells, resulting in a tolerogenic milieu characterized by lower IFNγ levels and increased expression of indoleamine 2,3-dioxygenase. CCL22 induced expression of CTLA-4, ICOS and CD62L on Tregs and enhanced their ability to modulate dendritic cell function, indicatory of a superior suppressive function. Notably, depletion of Tregs abrogated CCL22’s protective effect, suggesting that CCL22 modulatory properties are dependent on Tregs. When comparing CCL22 and CCL17, we found similar recruitment of Tregs and invariant NKT cells, but unlike CCL22, CCL17 did not impact Treg function. Nevertheless, CCL17 expression in islet grafts was equally protective from recurrent autoimmunity. Our findings suggest a novel strategy for protecting beta cells from immune attack, by using chemokines CCL22 or CCL17 to harness the natural regulatory properties of immune cells such as Tregs and invariant NKT cells.
Islet transplantation is a promising treatment for diabetes; however, most transplant recipients exhibit progressive loss of graft function. Islet function in transplant recipients shares similarities with subjects with type 2 diabetes including impaired glucose-stimulated insulin secretion, decreased beta-cell mass associated with amyloid formation, and defective proinsulin processing resulting in disproportionate secretion of intact proinsulin and proinsulin intermediates. We hypothesized that processing of the beta-cell prohormones, proinsulin and pro-islet amyloid polypeptide (proIAPP), will be impaired in islet transplant recipients as in patients with type 2 diabetes. Human islet transplant recipients were found to have impaired proinsulin processing manifest as elevated proinsulin/C-peptide ratios (TP/CP). The TP/CP ratio was significantly elevated in both islet allo- and auto-transplant recipients relative to controls. Furthermore, the TP/CP was greater in those recipients that received sub-optimal numbers of islets transplanted, suggesting that beta-cell dysfunction is exacerbated in the face of increased secretory demand due to insufficient islet mass. In a mouse model of islet transplantation, proinsulin processing was found to decline over time following transplantation, resulting in elevated proinsulin/insulin ratios. Amyloid deposits, a common pancreatic lesion in type 2 diabetes, were also found in human islet transplants and were associated with reduced beta-cell mass. Since IAPP, like insulin, is also processed within the beta cell from its precursor proIAPP, and since proinsulin processing is impaired in islet transplants and type 2 diabetes, we hypothesized that proIAPP processing will also be impaired in these conditions. To quantify proIAPP levels in humans, an immunoassay was developed. Circulating proIAPP levels in normal subjects were found to be in the low picomolar range and the ratio of proIAPP/IAPP was approximately 30%. In a small cohort of type 2 diabetic subjects, the proIAPP/IAPP ratio tended to be lower. These studies demonstrate that impaired proinsulin processing is a characteristic of transplanted islets and that the proinsulin/insulin or proinsulin/C-peptide ratios may serve as markers of graft dysfunction in islet transplantation. In addition, the proIAPP/IAPP ratio can now also be evaluated as a marker of beta-cell dysfunction in islet transplants and type 2 diabetes.
Diabetes mellitus is both a metabolic and inflammatory syndrome, characterized by hyperglycemia in the presence of insufficient beta-cell mass and high levels of circulating proinflammatory cytokines. Determining the mechanism behind this chronic inflammation is important for developing therapeutic targets, as controlling inflammation may prevent the progression of diabetes, development of secondary disease, and failure of islet allografts. Toll-like receptors (TLRs), a family of pattern recognition receptors of the innate immune system, are important for regulating adaptive immunity. The aim of this project was to determine the role of Toll-like receptor signalling in islet transplantation and normal beta-cell function. The role of TLR signalling in islet transplantation was assessed using a full major histocompatability complex mismatch murine islet allograft model. TLRs are capable of sensing both exogenous and endogenous ligands. Excessive TLR activation during transplantation may promote allograft rejection. To test this hypothesis, islets from TLR-deficient mice were transplanted into chemically-induced diabetic recipients, and graft survival was assessed by monitoring blood glucose levels. Islet grafts deficient for TLR4, or for TLR signalling molecules TRIF and MyD88, had similar graft failure rates to islet grafts from wild-type controls, indicating that islet allograft rejection occurs independently of donor TLR signalling in mice. To determine whether lack of TLR signalling via TRIF and MyD88 affects normal beta-cell function, mice deficient for TRIF or MyD88 were assessed for glucose tolerance, insulin sensitivity and in vitro glucose-stimulated insulin secretion. As TLR-induced inflammation has been demonstrated to enhance insulin resistance and diabetes-associated inflammation, lack of TLR signalling under non-pathological conditions may enhance beta-cell function. Interestingly, mice lacking TRIF demonstrated impaired glucose tolerance, hyperglycemia, hyperinsulinemia and impaired glucose-stimulated insulin secretion. Mice lacking MyD88 had similar glucose tolerance and insulin sensitivity to littermate controls, but displayed a mild impairment in glucose-stimulated insulin secretion. The results of this thesis demonstrate that while TLR signalling is not likely to be essential for islet allograft rejection, TLR signalling may contribute to normal beta-cell function. These findings point to a previously unrecognized role for TLR signalling in glucose homeostasis.
Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Background. Epstein-Barr virus (EBV) infection is a major cause of malignancy worldwide. Maternal antibodies are thought to prevent EBV infection because infection is uncommon in early infancy. Additionally, maternal HIV infection is associated with an increased incidence of EBV infection in exposed infants possibly resulting from impaired transfer of EBV maternal antibodies. EBV, possibly in its acute form manifesting as infectious mononucleosis (IM), is also the highest environmental risk factor for the development of multiple sclerosis (MS), a devastating neurologic disease caused by autoimmune destruction of myelin. IM can be diagnosed by the presence of heterophile antibodies which target an antigen that is akin to certain myelin glycans in the brain. We hypothesized that maternal antibodies can protect against EBV acquisition by studying infants with and without maternal HIV exposure. Furthermore, , since the degeneration of myelin is a hallmark of MS, we hypothesized that EBV infection, specifically IM, primes the immune system to cross-react and attack myelin.Methods. Ugandan infants were followed for EBV acquisition from birth and measured antibody binding to EBV glycoproteins involved in B cell and epithelial cell entry, as well as viral neutralization and antibody-dependent cellular cytotoxicity (ADCC). Serum antibodies against major brain glycans were measured using a Luminex based glycan array for 6 infant samples (pre-post EBV infection) and 9 adults with IM. Results. HIV-exposed uninfected infants had significantly higher titers than HIV-unexposed uninfected infants for all EBV-binding and neutralizing antibodies measured (p
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