Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by the inflammation of the insulin-producing pancreatic beta cells, eventually leading to beta cell loss and the inability to maintain glucose homeostasis. Understanding the mechanisms of beta cell-intrinsic factors that influence the maintenance of cellular defenses and contribute to cell death when deregulated will be crucial in efforts to treat or prevent beta cell loss in individuals who are prone to autoimmunity. Through my thesis work, I have investigated beta cell-specific etiologies of T1D through both a candidate-based approach using beta cell specific deletion of a susceptibility gene and an unbiased global exploration of beta cell factors that regulate the predisposition to insulitic injury. Protein tyrosine phosphatase N2 (PTPN2) is a T1D candidate gene that has been shown to be critical for modulating inflammation by regulating T cell activation. PTPN2 is also highly expressed in human and murine beta cells and it has been shown to be critical for beta cell function in vivo and inhibit inflammatory stimuli-mediated beta cell apoptosis in vitro, suggesting that PTPN2 mediated defense against inflammation is two pronged negative regulation of inflammatory immune cells and elevation of a beta cell intrinsic defense. To examine whether PTPN2 regulates beta cell loss upon cytotoxic stimuli by bolstering beta cell defense mechanisms in vivo, I deleted PTPN2 in the beta cells (Ptpn2 beta-KO) and subjected the mice to the diabetogenic agent streptozotocin (STZ). Animals deficient in beta cell PTPN2 are more susceptible to STZ induced diabetes and have poor survival due to hyperglycemia. While investigating the mechanism of PTPN2-mediated beta cell defense, I have discovered that PTPN2 interacts with pyruvate kinase M2 (PKM2), a key metabolic enzyme that normally resides in the cytosol. In response to STZ, PKM2 translocates to the nuclei of diabetic beta cells, and the lack of PTPN2 results in the hyper-accumulation of nuclear PKM2, suggesting that PTPN2 mediates nuclear export of PKM2 in stressed beta cells. In the nucleus, PKM2 mediates the transcriptional activation of key proapototic genes, which is attenuated when I modulate nuclear PKM2 ex vivo, in effect reconstituting the function of PTPN2. Together, deregulation of PTPN2 mediated nuclear export of PKM2 leading to excessive transcriptional activation of proapoptotic genes may be the mechanism for exacerbated diabetes in the Ptpn2 beta KO mice. To identify novel candidates that function in the beta cells to influence beta cell susceptibility to insulitic injury, I established RNA transcriptome and CpG dinucleotide methylome profiles of islets isolated from insulitis susceptible NOD and insulitis resistant NOR mice, prior to the onset of insulitis. Integrating these profiles with the genes nested in the human diabetic loci from the genome wide association studies, I identified several novel candidate genes that may be involved in T1D pathogenesis in a beta cell specific manner. Moreover, I also examined non CpG methylation, which appears to influence gene expression independently of CpG methylation. Collectively, my studies have expanded the understanding of beta cell-specific factors that regulate cellular defense to insulitis and may have expanded the therapeutic possibilities by implicating PKM2, inhibition of which is the focus of many cancer therapy research.

Pancreatic beta cell specification is a complex process, requiring proper function of numerous transcription factors. Nkx2.2 is a transcription factor that is crucial for beta cell formation, and is expressed early and throughout pancreatic development. Nkx2.2-/- mice display complete loss of the beta cell lineage and defects in the specification of other endocrine cell types, demonstrating the importance of Nkx2.2 in establishing proper endocrine cell ratios. Recent studies have also demonstrated a role for Nkx2.2 within the mature beta cell to maintain identity and function. This thesis work investigated the timing of pancreatic beta cell specification and the mechanism of this process. In these studies, Nkx2.2 was ablated specifically within the Ngn3-expressing endocrine progenitor population in vivo. These mice displayed defects similar to Nkx2.2-/- mice. Surprisingly, the disruption of endocrine cell specification did not require loss of expression of multiple essential transcription factors known to function downstream of Nkx2.2, including Ngn3, Rfx6, and NeuroD1. While these factors are all necessary for beta cell specification, their preserved expression did not rescue beta cell formation. ChIP-Seq analyses also revealed co-occupancy of Nkx2.2, Rfx6, and NeuroD1 near endocrine-related genes, suggesting Nkx2.2 may cooperate with its downstream targets to regulate beta cell fate. These results have revealed a unique requirement for Nkx2.2 during a critical window of beta cell development. In addition, the role of a conserved domain of Nkx2.2, the specific domain (SD), was assessed using Nkx2.2SDmutant mice. Transcriptional profiling of Nkx2.2SDmutant endocrine progenitors revealed a critical role for the SD domain in regulating the transcription of endocrine fate genes early in the process of endocrine differentiation. In addition, beta cell-specific deletion of the Nkx2.2 SD domain resulted in hyperglycemia, glucose intolerance and dysregulation of beta cell functional genes. This suggests the SD domain is important for mediating Nkx2.2 function within the beta cell to maintain glucose homeostasis. Together, these results have elucidated a critical developmental window for beta cell specification and demonstrated an essential role for Nkx2.2 and specifically its SD domain in this process. Furthermore, these studies suggest that beta cell transcription factors may also regulate endocrine fate in a combinatorial manner, and exert changes within the endocrine progenitor lineage. These findings have provided us with a better understanding of in vivo pancreatic development, and will improve current research efforts to differentiate beta cells in vitro from hPSCs.

The acquisition of beta cell identity and function is a multistage process that involves the sequential regulation of specific factors and signals. The maintenance of beta cell identity and function is a process of comparable importance that requires persistent and continuous regulation. Loss of beta cell identity and/or reprogramming represents an important feature of beta cell dysfunction in genetic models of diabetes, as well as in patients with type 1 and type 2 diabetes. The factors and mechanisms involved in the acquisition and maintenance of beta cell identity are still not well understood. Nevertheless, several beta cell developmental transcription factors have been found to be important in the maintenance of its functional identity during the postnatal stage. Nkx2.2 is a transcription factor that is critical for the development and differentiation of beta cells both in mice and humans. In adults, Nkx2.2 is expressed in the entire beta cell population. However, due to the perinatal lethality of the Nkx2.2 null mice, the study of its function in adult beta cells has remained elusive. For my dissertation work, I explored the function and mechanism of action of Nkx2.2 in the adult beta cell. I deleted Nkx2.2 specifically in beta cells during their maturation and in adults. Deletion of Nkx2.2 in beta cells caused rapid onset of diabetes due to the loss of insulin and the down-regulation of many beta cell functional genes. Concomitantly, Nkx2.2-deficient beta cells acquired non-beta cell endocrine features, resulting in populations of completely reprogrammed cells and bi-hormonal cells that have hybrid endocrine cell morphological characteristics. Molecular analysis in mouse and human islets revealed that Nkx2.2 is a conserved master regulatory protein that controls the acquisition and maintenance of a functional monohormonal beta cell identity by directly activating critical beta cell genes, and actively repressing genes that specify the alternative islet endocrine cell lineages. This study demonstrates the highly volatile nature of the beta cell; it is necessary to actively maintain expression of genes involved in beta cell function, but to also maintain repression of closely related endocrine gene programs. These findings have potential applications that include the optimization of iPS cell differentiation protocols that aim to differentiate functional beta cells that remain safely locked into that identity state; as well as in future therapies that attempt to restore beta cells into a functional state.

published_or_final_version / Biochemistry / Master / Master of Medical Sciences

No description available.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

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.

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.

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.