Marked by a defect in the production of the Beta (β)-globin chain that make-up hemoglobin, Beta (β)-thalassemia is the most prevalent form of inherited single-gene disorders in the world. To understand the molecular mechanisms that govern the expression of the β-globin polypeptide encoded by the β-globin locus, we examined closely the enzymes involved in the epigenetic regulation of gene expression through histone 3 lysine 9 mono and di-methylation (H3K9 me1/2). G9a-like protein (GLP), a mammalian methyltransferase involved in the establishment and maintenance of H3K9 me1/2 mark at euchromatin, regions was found to be critical for the full activation of the adult β-globin genes in vivo during Murine erythroleukemia cell line (MEL) differentiation. Though it was initially hypothesized that GLP binding to H3K9 me1/2 mark through its Ankyrin domain was key to its activating function, we found that Flag- GLP ankyrin mutants E817R and W791A unable to bind to the methyl mark, are able to activate β-globin genes as well as their wild-type counterpart. Additionally, this study found that the embryonic gene εγ, known to be re-activated after G9a KD at the mRNA level, was effectively transcribed at the protein level using Triton Urea Acetic acid (TAU) western blots, thereby identifying potential therapeutic applications for treatment for β-thalassemia patients.

Enteroendocrine hormones released from the intestine following food intake have several roles in the control of metabolism, some of which are exploited therapeutically for the treatment of type 2 diabetes. Within this thesis, focus has been on the receptors of the gut hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2). In recent years there has been a surge of interest in the enteroendocrine hormones particularly due to the success of GLP-1 mimetics in the treatment of type 2 diabetes. GLP-1 is an incretin hormone, which enhances glucose induced insulin secretion by binding GLP-1 receptors (GLP1R) on pancreatic β-cells. Despite the therapeutic success, several extra-pancreatic clinical effects of GLP-1 remain unexplained. Here, a GLP1R monoclonal antagonistic antibody that can block GLP1R signalling in vivo has been developed and characterised, providing a new tool for the study of GLP1R physiology. GIP is the second incretin hormone, initially referred to as the 'ugly duckling' incretin hormone due to it's ineffectiveness in inducing insulin secretion in type 2 diabetic patients. Aside from the incretin actions, GIP is thought to be involved in the regulation of high-fat diet (HFD) induced obesity. A new transgenic mouse model expressing a fluorescent reporter under the control of the Gipr promoter has been used here to identify GIPR expressing cells. This model showed GIPR expression in the pancreas, adipose tissue, duodenum and nodose ganglia. Surprisingly GIPR expressing cells were found centrally, in areas of the hypothalamus involved in the regulation of food intake and energy expenditure. We consequently sought to investigate the function of GIPR expressing hypothalamic cells. GLP-2, unlike GLP-1 and GIP, is not an incretin hormone. Rather, GLP-2 has been implicated in the regulation of epithelial cell proliferation and apoptosis within the intestine. Therapeutically, an analogue of GLP-2 is used for the treatment of short bowel syndrome. A common missense mutation in the GLP-2 receptor (GLP2R), D470N, has been found to be associated with type 2 diabetes, and here we sought to understand the mechanism underlying this association. The D470N mutant has decreased β-arrestin recruitment, though the significance of this finding will need further research. Overall; the new monoclonal antagonistic GLP1R antibody will help to further understand GLP1R physiology, the new transgenic GIPR mouse model has contributed to the understanding of GIPR localisation, and cell based assays have identified functional implications of a polymorphism in the GLP2R associated with an increased risk of diabetes. It is hoped that further understanding of the physiology of these gut hormone receptors will be critical in the development of new therapeutics for diabetes and obesity.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第18366号 / 人博第679号 / 新制||人||163(附属図書館) / 25||人博||679(吉田南総合図書館) / 31224 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦, 教授 津田 謹輔 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

El presente informe expositivo se basó en la experiencia del autor, en la elaboración de proyectos, asesoramiento, construcción y mantenimiento (mecánico, eléctrico y electrónico) de las estaciones de servicio de gas natural vehicular (GNV), gas licuado de petróleo (GLP) y combustibles líquidos, así como también de la Norma Técnica Peruana NTP 111.019 y de la Norma Técnica de Asociación NFPA70:2002 (National Electric Code) quienes establecieron los requisitos mínimos de instalación y seguridad que deben cumplir las estaciones de servicio.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by the enteroendocrine L-cell that potently stimulates insulin secretion. Although signaling pathways promoting GLP-1 secretion are well characterized, the mechanism by which GLP-1 containing granules fuse to the L-cell membrane remain elusive. RT-PCR and protein analysis indicate that vesicle-associated membrane protein 2 (VAMP2) is expressed and localized to secretory granules in the murine GLUTag L-cell model. VAMP2, but not VAMP1, interacted with the core SNARE complex protein, Syntaxin 1a, in GLUTag cells. Tetanus toxin (TetX) cleavage of VAMP2 in GLUTag cells prevented glucose-dependent insulinotropic peptide (GIP)- and oleic acid (OA)-stimulated GLP-1 secretion, as well as K+-stimulated exocytosis from GLUTag cells. Although components of membrane rafts were detected in GLUTag cells, their role in GLP-1 secretion remains to be determined. Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag cell.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by the enteroendocrine L-cell that potently stimulates insulin secretion. Although signaling pathways promoting GLP-1 secretion are well characterized, the mechanism by which GLP-1 containing granules fuse to the L-cell membrane remain elusive. RT-PCR and protein analysis indicate that vesicle-associated membrane protein 2 (VAMP2) is expressed and localized to secretory granules in the murine GLUTag L-cell model. VAMP2, but not VAMP1, interacted with the core SNARE complex protein, Syntaxin 1a, in GLUTag cells. Tetanus toxin (TetX) cleavage of VAMP2 in GLUTag cells prevented glucose-dependent insulinotropic peptide (GIP)- and oleic acid (OA)-stimulated GLP-1 secretion, as well as K+-stimulated exocytosis from GLUTag cells. Although components of membrane rafts were detected in GLUTag cells, their role in GLP-1 secretion remains to be determined. Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag cell.

Type 1 diabetes mellitus (T1D) is an autoimmune disorder that targets the insulin-producing β-cells. The gut may play a role in the pathogenesis of T1D, as genetically-susceptible individuals and animal models of T1D exhibit increased intestinal permeability and improving gut barrier function can interfere with the onset of diabetes. Moreover gut-derived peptides are capable of modifying barrier function and regulate β-cell mass via effects on proliferation and apoptosis. I tested whether chronic administration of glucagon-like peptide-2 (GLP-2), a peptide which potently improves gut barrier function, modifies diabetes onset in a mouse model of T1D, the non obese diabetic (NOD) mouse. Although chronic treatment with a long-acting GLP-2 analogue was associated with improved intestinal barrier function, it failed to delay the onset of T1D. Once the autoimmune attack is initiated, pathogenic T-cells infiltrate the islets and trigger the death of β-cells. Studies in animal models have revealed that β-cells exhibit a compensatory response in the initial stages of the immune attack, which eventually fails, resulting in β-cell mass deficiency and onset of T1D. Glucagon-like peptide-1 (GLP-1) exerts both proliferative and anti-apoptotic actions on β-cells. I hypothesized that chronic activation of the GLP-1 receptor (GLP-1R) would delay or prevent the loss of functional β-cell mass in the NOD mouse. I have shown that chronic administration of the GLP-1R agonist exendin-4 significantly delayed the onset of diabetes and enhanced β-cell mass. Furthermore, GLP-1R activation was associated with a reduction of islet-infiltrating immune cells, as well as changes in lymphocyte subpopulations. Consequently, I addressed whether the GLP-1R has a role in the immune system of NOD and C57Bl/6 mice. GLP-1R mRNA transcripts were detectable in several immune subpopulations, and GLP-1R activation was associated with cAMP production in primary splenocytes and thymocytes. Furthermore I demonstrated that GLP-1R signaling controls proliferation of thymocytes and lymphocytes, and is required for maintaining peripheral regulatory T-cells. In summary, these studies establish that while GLP-2R activation is not sufficient to modify disease onset in a murine model of T1D, GLP-1R activation reduces the extent of diabetes development by exerting actions on β-cells and the immune system.

Type 1 diabetes mellitus (T1D) is an autoimmune disorder that targets the insulin-producing β-cells. The gut may play a role in the pathogenesis of T1D, as genetically-susceptible individuals and animal models of T1D exhibit increased intestinal permeability and improving gut barrier function can interfere with the onset of diabetes. Moreover gut-derived peptides are capable of modifying barrier function and regulate β-cell mass via effects on proliferation and apoptosis. I tested whether chronic administration of glucagon-like peptide-2 (GLP-2), a peptide which potently improves gut barrier function, modifies diabetes onset in a mouse model of T1D, the non obese diabetic (NOD) mouse. Although chronic treatment with a long-acting GLP-2 analogue was associated with improved intestinal barrier function, it failed to delay the onset of T1D. Once the autoimmune attack is initiated, pathogenic T-cells infiltrate the islets and trigger the death of β-cells. Studies in animal models have revealed that β-cells exhibit a compensatory response in the initial stages of the immune attack, which eventually fails, resulting in β-cell mass deficiency and onset of T1D. Glucagon-like peptide-1 (GLP-1) exerts both proliferative and anti-apoptotic actions on β-cells. I hypothesized that chronic activation of the GLP-1 receptor (GLP-1R) would delay or prevent the loss of functional β-cell mass in the NOD mouse. I have shown that chronic administration of the GLP-1R agonist exendin-4 significantly delayed the onset of diabetes and enhanced β-cell mass. Furthermore, GLP-1R activation was associated with a reduction of islet-infiltrating immune cells, as well as changes in lymphocyte subpopulations. Consequently, I addressed whether the GLP-1R has a role in the immune system of NOD and C57Bl/6 mice. GLP-1R mRNA transcripts were detectable in several immune subpopulations, and GLP-1R activation was associated with cAMP production in primary splenocytes and thymocytes. Furthermore I demonstrated that GLP-1R signaling controls proliferation of thymocytes and lymphocytes, and is required for maintaining peripheral regulatory T-cells. In summary, these studies establish that while GLP-2R activation is not sufficient to modify disease onset in a murine model of T1D, GLP-1R activation reduces the extent of diabetes development by exerting actions on β-cells and the immune system.

The N-terminal tails of histones are subject to many chemical modifications that are involved in a variety of biological functions. Histone methylation is a major epigenetic modification found in both single and multicellular organisms and is directly involved in the regulation of gene expression. Methylation of lysine 9 of histone 3 (H3K9) has been shown to have diverse functions depending on the number of methyl groups added; H3K9me1 marks the active promoters, while H3K9me2 and H3K9me3 are present within inactive gene promoters and pericentric heterochromatin. G9a, also known as euchromatic histone-lysine N-methyltransferase 2 (Ehmt2), is a histone methylase that catalyses addition of mono- and dimethyl groups to H3K9 in euchromatic regions of the genome to silence genes. Therefore, it is a vital component of the gene expression regulation machinery. In mouse embryonic stem (ES) cells, G9a forms a stable heterodimer with the G9a-like protein (GLP or Ehmt1), which is further stabilised by the C2H2-type zinc finger protein, widely interspaced zinc finger protein (WIZ). These three proteins form the core G9a complex, which is essential for mouse development. Lack of any G9a complex member leads to embryonic lethality at E9.5 with severe growth defects. The ankyrin repeat domain of G9a/GLP can bind to H3K9me1/2 with high affinity in vitro (Collins et al. 2008). This enables the self-recruitment of the G9a complex to sites with H3K9me1/2 and maintenance of the mark. However, the initial recruitment of the G9a complex to sites lacking H3K9me1/2 mark during differentiation is poorly understood. Neither G9a nor GLP has a DNA/RNA binding domain, so recruitment of the G9a complex to specific sites must be mediated by other binding partners of the G9a complex. Using mass spectrometry, I was able to identify a number of zinc finger proteins as binding partners of G9a. Among these, WIZ was identified in stoichiometric amounts to G9a and GLP, and is a potential DNA binding protein similar to other C2H2-type zinc fingers. The aim of this study was to determine the role of WIZ in the recruitment of the G9a complex to specific sites. I showed that knockdown of WIZ had no significant effect on the chromatin binding of G9a in undifferentiated mouse ES cells, which indicates WIZ is dispensable in the maintenance of H3K9me2. However, I observed a 30% decrease in the G9a levels upon WIZ knockdown, which shows that WIZ might have a role in stabilising G9a. Using recombinant WIZ zinc finger pairs, I was able to show that the 3rd and 4th zinc finger of WIZ bind DNA in vitro. Furthermore, using the systematic evolution of ligands exponential enrichment (SELEX) approach I demonstrated that the zinc fingers of WIZ preferentially bind to G-rich double-stranded DNA sequences. Binding site analysis with synthetic DNA indicated that WIZ ZF3-4 require two binding sites that are a certain distance apart from each other for efficient binding. In addition, ZF3-4 binds ssDNA with higher affinity than dsDNA, and binding to ssDNA is sequence-independent. This study shows for the first time that mouse WIZ zinc finger pairs can bind DNA and RNA in vitro. Therefore, sequence-specific recruitment of G9a might be mediated by WIZ during differentiation. Furthermore, DNA binding preference of WIZ might suggest that WIZ-mediated recruitment of G9a to establish H3K9me2 could occur at the R-loops where G-rich DNA forms a hybrid with newly transcribed RNA or at the G-rich repetitive sequences.

Glucagon-like peptide-1 (GLP-1) has received much attention as a novel diabetes therapeutic due to its pleotropic blood glucose-lowering effects, including enhancement of glucose-stimulated insulin secretion, inhibition of gastric emptying and glucagon secretion, and promotion of beta-cell survival and proliferation. GLP-1 is produced in the intestinal L-cell via processing of the proglucagon precursor by prohormone convertase (PC) 1/3. Proglucagon is also expressed in the pancreatic alpha-cell; however, in this tissue PC2 is typically expressed instead of PC1/3, resulting in differential cleavage of proglucagon to yield glucagon as the major product. We hypothesized that expression of PC1/3 in the alpha-cell would induce GLP-1 production in this tissue, and that this intervention would improve islet function and survival. Initial studies in alpha-cell lines demonstrate that adenoviral delivery of PC1/3 to alpha-cells increases GLP-1 production. By encapsulating and transplanting either PC1/3- or PC2-expressing alpha-cells, the following studies show that while PC2-expressing alpha-cells increase fasting blood glucose and impair glucose tolerance, PC1/3-expressing alpha-cells decrease fasting blood glucose and dramatically improve glucose tolerance in normal mice and in mouse models of diabetes. We further show that transplantation of PC1/3-expressing alpha-cells prevents streptozotocin (STZ)- induced hyperglycemia. We also found that PC1/3-expressing alpha-cells also improve cold-induced thermogenesis in db/db mice, demonstrating a previously unappreciated effect of one or more of the PC1/3-derived proglucagon products. Studies in isolated mouse islets demonstrate that adenoviral delivery of PC1/3 to isolated islets increases islet GLP-1 secretion and improves glucose-stimulated insulin secretion and islet survival. Experiments with diabetic mice show that these GLP-1-producing islets are better able to restore normoglycemia in recipient mice following islet transplantation. Taken together, these studies demonstrate that the alpha-cell can be induced to process proglucagon into PC1/3-derived products, and that this shift redirects the alpha-cell from a hyperglycemia-promoting fate to a blood glucose-lowering one. This research opens up avenues for further investigating the therapeutic potential of inducing islet GLP-1 production in isolated human islets and in vivo in diabetes patients, and may represent a novel way to intervene in the progressive loss of beta-cells that characterizes diabetes.