GLP-2 is a potent intestinotrophic peptide that can increase mucosal growth, intestinal blood flow, and nutrient absorption when administered exogenously. We aimed to delineate the effects of endogenous GLP-2R signalling in conditions of nutrient deprivation and excess. Using a mouse with a targeted genetic deletion of the Glp2r gene (Glp2r-/-), we addressed the hypothesis that the known GLP-2R is required for intestinal adaptation to nutrient deprivation and excess. In Chapter 2, we demonstrate that Glp2r−/− mice fasted for 24 hours and re-fed for 24 hours failed to increase intestinal growth and jejunal crypt cell proliferation compared to littermate Glp2r+/+ mice. Administration of EGF to Glp2r−/− during the re-feeding period rescued this re-feeding defect. Wildtype mice re-fed for 30, 90, and 180 minutes following a 24 hour fast displayed increased jejunal mRNA levels of the ErbB ligands amphiregulin, epiregulin and HB-EGF. Treatment with the pan ErbB inhibitor CI-1033 inhibited induction of these ErbB ligands in jejunum of mice in association with prevention of crypt cell proliferation. Re-feeding also caused an increase in jejunal p-Akt levels and treatment with CI-1033 prevented increased p-Akt levels. Moreover, re-fed Glp2r−/− mice failed to increase ErbB ligands or p-Akt levels 90 minutes following re-feeding when compared to Glp2r+/+ littermates. Therefore, the GLP-2R is essential for re-feeding induced intestinal adaptation by activating the ErbB network and p-Akt to increase crypt cell proliferation. In Chapter 3, we show that the known GLP-2R is not required for intestinal adaptation to a perceived nutrient deprivation challenge (STZ-induced diabetes) or chronic nutrient excess (high-fat diet induced glucose intolerance). Although exogenous GLP-2 administration has been previously shown to stimulate glucagon secretion, glucose homeostasis was normal in STZ-diabetic and high fat fed Glp2r−/− mice. We also developed a third model of diabetes and glucose intolerance: ob/ob: Glp2r−/−. In the absence of GLP-2R signalling, ob/ob mice display improved oral but impaired intraperitoneal glucose tolerance, elevated fed and fasted glucose levels, increased circulating glucagon, decreased beta cell and increased alpha cell mass. Taken together, these results suggest that endogenous GLP-2R signalling is essential for intestinal and islet adaptation to conditions of nutrient deprivation and excess.

Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that promotes gut growth through an insulin-like growth factor (IGF)-1 and intestinal epithelial (IE)-IGF-1 receptor (R)-dependent pathway. GLP-2 also promotes epithelial barrier function by as yet unknown mechanisms. I hypothesized that GLP-2-mediated effects on barrier function requires the IE-IGF-1R. Chronic GLP-2 treatment enhanced barrier function by decreasing gastrointestinal permeability in vivo and increasing jejunal resistance ex vivo. These responses were abolished in inducible IE-IGF-1R knockout (KO) animals. Additionally, epithelial sealing tight junctional proteins claudin-3 and -7 were upregulated by GLP-2 in control but not KO mice. Moreover, IE-IGF-1R deletion induced a shift in occludin localization from apical to intracellular domains. In contrast, in irinotecan-induced enteritis, GLP-2 normalized epithelial barrier function in control animals, but continued to be ineffective in KO mice. Collectively, the effects of GLP-2 on barrier function are dependent on the IE-IGF-1R and involve modulation of the tight junctional complex.

Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that promotes gut growth through an insulin-like growth factor (IGF)-1 and intestinal epithelial (IE)-IGF-1 receptor (R)-dependent pathway. GLP-2 also promotes epithelial barrier function by as yet unknown mechanisms. I hypothesized that GLP-2-mediated effects on barrier function requires the IE-IGF-1R. Chronic GLP-2 treatment enhanced barrier function by decreasing gastrointestinal permeability in vivo and increasing jejunal resistance ex vivo. These responses were abolished in inducible IE-IGF-1R knockout (KO) animals. Additionally, epithelial sealing tight junctional proteins claudin-3 and -7 were upregulated by GLP-2 in control but not KO mice. Moreover, IE-IGF-1R deletion induced a shift in occludin localization from apical to intracellular domains. In contrast, in irinotecan-induced enteritis, GLP-2 normalized epithelial barrier function in control animals, but continued to be ineffective in KO mice. Collectively, the effects of GLP-2 on barrier function are dependent on the IE-IGF-1R and involve modulation of the tight junctional complex.

GLP-2 is a potent intestinotrophic peptide that can increase mucosal growth, intestinal blood flow, and nutrient absorption when administered exogenously. We aimed to delineate the effects of endogenous GLP-2R signalling in conditions of nutrient deprivation and excess. Using a mouse with a targeted genetic deletion of the Glp2r gene (Glp2r-/-), we addressed the hypothesis that the known GLP-2R is required for intestinal adaptation to nutrient deprivation and excess. In Chapter 2, we demonstrate that Glp2r−/− mice fasted for 24 hours and re-fed for 24 hours failed to increase intestinal growth and jejunal crypt cell proliferation compared to littermate Glp2r+/+ mice. Administration of EGF to Glp2r−/− during the re-feeding period rescued this re-feeding defect. Wildtype mice re-fed for 30, 90, and 180 minutes following a 24 hour fast displayed increased jejunal mRNA levels of the ErbB ligands amphiregulin, epiregulin and HB-EGF. Treatment with the pan ErbB inhibitor CI-1033 inhibited induction of these ErbB ligands in jejunum of mice in association with prevention of crypt cell proliferation. Re-feeding also caused an increase in jejunal p-Akt levels and treatment with CI-1033 prevented increased p-Akt levels. Moreover, re-fed Glp2r−/− mice failed to increase ErbB ligands or p-Akt levels 90 minutes following re-feeding when compared to Glp2r+/+ littermates. Therefore, the GLP-2R is essential for re-feeding induced intestinal adaptation by activating the ErbB network and p-Akt to increase crypt cell proliferation. In Chapter 3, we show that the known GLP-2R is not required for intestinal adaptation to a perceived nutrient deprivation challenge (STZ-induced diabetes) or chronic nutrient excess (high-fat diet induced glucose intolerance). Although exogenous GLP-2 administration has been previously shown to stimulate glucagon secretion, glucose homeostasis was normal in STZ-diabetic and high fat fed Glp2r−/− mice. We also developed a third model of diabetes and glucose intolerance: ob/ob: Glp2r−/−. In the absence of GLP-2R signalling, ob/ob mice display improved oral but impaired intraperitoneal glucose tolerance, elevated fed and fasted glucose levels, increased circulating glucagon, decreased beta cell and increased alpha cell mass. Taken together, these results suggest that endogenous GLP-2R signalling is essential for intestinal and islet adaptation to conditions of nutrient deprivation and excess.

Introduction: Appetite is often impaired in patients with gastrointestinal inflammation. Up to 75% of hospitalised Crohn's disease (CD) patients are malnourished. Recent animal research has suggested that immune mediated upregulation of enteroendocrine cell (EEC) activity plays a mechanistic role in the appetite and feeding disturbance observed during gut inflammation. The role of EEC in producing factors regulating satiety and intestinal growth is well recognised but work on their use as therapeutic targets or agents in inflammatory bowel disease (IBD) is still in its infancy. EEC peptide dynamics are further controlled through dipeptidyl peptidase (DP4) protease metabolism but no data are yet available on its expression in IBD. My aim is to understand the roles of EEC in appetite control and the maintenance of gut mucosal integrity in intestinal inflammation. Methodology: Patients with CD and healthy controls were studied. Symptoms were assessed using visual analogue scores (VAS). Gut hormone responses to a test meal were studied using a multiplex-ELISA technique, and correlated to symptoms. At the tissue level, EEC markers and transcription factors were quantified using immunohistochemistry, quantitative polymerase chain reaction (qPCR) and western blotting techniques. The same techniques were used to study DP4 expression. The effects of glucagon-like peptide-2 (GLP-2) on a gut model of the epithelial barrier were studied by measuring the transepithelial electrical resistance (TEER) across GLP-2 exposed Caco-2 cell monolayers after cytokine exposure. Tight junction protein expression in naïve and GLP-2 exposed cells was quantified by western blotting. Main Results: CD patients with active inflammation displayed a significant reduction in appetite. At the tissue level, GLP-1 and chromogranin A (CgA) were significantly upregulated. At the mRNA level significant increased expression was noted for CgA, glucagon-like peptide-1 (GLP-1), ubiquitination factor 4a and neurogenin 3. At the plasma level, total polypeptide YY (PYY) was increased. A significant correlation was seen between postprandial PYY responses and symptoms of nausea and bloating. Ghrelin, was 3-fold higher in the CD group compared to controls, and showed a reversed postprandial response with a significant correlation with the CD activity index (CDAI). Protein DP4 expression was significantly decreased at the tissue and plasma level in CD. GLP-2 increased tight junction protein expression in Caco-2 cells and maintained stable TEER and tight junction protein expression after cytokine exposure. Conclusions: The data presented are compatible with a potential role of EEC in appetite dysregulation in intestinal inflammation. An enhanced EEC response to food intake may directly affect appetite in such patients through increased gut-brain signalling. These may present tractable therapeutic targets. The decrease in mucosal DP4 expression in CD may make bioactive GLP-2 more available in the affected gut, hence improving gut mucosal integrity in intestinal inflammation. This pilot work has shown that GLP-2 has a role in maintaining gut mucosal integrity in intestinal inflammation through a positive effect on tight junction protein expression.

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

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.