Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes.  Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development.  Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse ES cell.  By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor’s activity.  mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like.  Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development.  Furthermore, the differential beta-like cells secreted C-peptide (insulin) in response to KC1 and IBMX, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells.  Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta cells in the developing endocrine pancreas.  Collectively, we show that this method of <i>in vitro</i> differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.

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.

Transplantation of insulin producing cells is currently the only cure for type 1 diabetes. However, even though the Edmonton protocol markedly increased the success rate of pancreatic islet transplantation, the long term insulin independence is still very poor. An adequate engraftment is critical for islet graft survival and function. In the present thesis, isolated islet endothelial cells were found to have a low proliferatory and migratory capacity towards vascular endothelial growth factor (VEGF), but this could be reversed by using neutralizing antibodies to the angiostatic factors thrombospondin-1, endostatin or alpha1-antitrypsin. In the adult islet endothelial cell, VEGF may act as a permeability inducer more than an inducer of angiogenesis. p38 MAP kinase activity has been shown to serve as a switch between these properties of VEGF. Inhibition of p38 MAP kinase by daily injections of SB203580 in the early posttransplantation phase lead to a redistribution of the islet graft blood vessels from the stroma into the endocrine tissue and this was accompanied by a higher oxygen tension. Besides transports of oxygen and nutrients, beta-cells may require signals from the endothelial cells for their growth and differentiation. It was demonstrated that islet endothelial cells secrete factors, including laminin, that have positive effects on beta-cell insulin release and insulin content. Our results suggest that improved revascularization of transplanted islets may be achieved by either inhibition of angiostatic factors, or by blocking p38 MAPkinase activity, in the implanted tissue. Islet endothelial cells have a supportive paracrine role for beta-cells that might be hampered by the normally poor revascularization.

This thesis is focused on the role of mitochondria in pancreatic β-cells and brown adipose tissue (BAT). Two main aspects of mitochondria were explored; mitochondrial functional efficiency and the interrelationship between mitochondrial shape and function. Mitochondria in β-cells were found to exhibit heterogeneity in mitochondrial membrane potential. This functional diversity decreased when cells were challenged with glucose stimuli, suggesting that at higher fuel levels low-activity mitochondria are recruited into a pool of high-activity mitochondria. Glucolipotoxic conditions increased the functional diversity suggesting that this may be of importance for diabetes pathophysiology. To examine mitochondrial efficiency in intact islets a high throughput islet respirometry method was developed. Due to increased uncoupling, islets from a diabetic animal model exhibit lower respiratory efficiency. Glucose, free fatty acids and amino acids all decreased respiratory efficiency. A large portion of the respiratory efficiency was mediated by reactive oxygen species and the adenine nucleotide translocase. In β-cells mitochondria were found to undergo cycles of fusion and fission. During glucolipotoxicity mitochondria fragmented and lost their fusion ability. Knock down of the fission protein Fis1 rescued the β-cells from glucolipotoxic induced cell death. BAT mitochondria also showed fusion and fission. The mitochondrial dynamics proteins Mfn2 and Drp1 were shown to strongly affect BAT mitochondrial morphology. In response to a combination of adrenergic and free fatty acid stimuli mitochondria drastically changed from long filamentous structures to fragmented spheres. Inhibiting fission by the negative form of Drp1 decreased BAT response to adrenergic stimuli by half. In conclusion, mitochondrial efficiency may be of importance for normal as well as compromised β-cell and islet function. Mitochondrial morphology appears critical for mitochondrial function in β-cells and BAT. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

published_or_final_version / Biochemistry / Master / Master of Medical Sciences

No description available.

We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

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