Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut non- endocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and co-localized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.

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.

G-protein coupled receptors (GPCRs) have been shown to interact with an array of accessory proteins that modulate their function. I hypothesize that the GLP-1R, a B-class GPCR, similarly has interacting proteins that regulate its signaling. An unliganded human GLP-1R was screened using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay and a human fetal brain cDNA prey library to reveal 38 novel interactor proteins. These interactions were confirmed by co-immunoprecipitation and immunofluorescence. When co-expressed with the GLP-1R in cell lines, 15 interactors significantly attenuated GLP-1-induced cAMP accumulation. Interestingly, SiRNA-mediated knock down of three selected novel interactors, SLC15A4, APLP1 and AP2M1, significantly enhanced GLP-1-stimulated insulin secretion from the MIN6 beta cells. In conclusion, this present work generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling; and the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.

G-protein coupled receptors (GPCRs) have been shown to interact with an array of accessory proteins that modulate their function. I hypothesize that the GLP-1R, a B-class GPCR, similarly has interacting proteins that regulate its signaling. An unliganded human GLP-1R was screened using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay and a human fetal brain cDNA prey library to reveal 38 novel interactor proteins. These interactions were confirmed by co-immunoprecipitation and immunofluorescence. When co-expressed with the GLP-1R in cell lines, 15 interactors significantly attenuated GLP-1-induced cAMP accumulation. Interestingly, SiRNA-mediated knock down of three selected novel interactors, SLC15A4, APLP1 and AP2M1, significantly enhanced GLP-1-stimulated insulin secretion from the MIN6 beta cells. In conclusion, this present work generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling; and the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.

Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut non- endocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and co-localized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver dysfunction in the western world and one of the main contributors to cirrhosis and potentially liver cancer and liver failure. A variety of behaviors and other factors can predispose certain individuals to an increased risk of developing NAFLD and non-alcoholic steatohepatitis (NASH). These factors include, but are not limited to, obesity, insulin resistance, hyperglycemia, and high levels of fat in the blood. The result is the accumulation of fat in hepatocytes that over time leads to inflammation and scarring of the liver and ultimately liver damage. At present, the mainstay of therapy remains weight loss through dietary modification and lifestyle change. Due to the lack of specific targeted pharmacotherapies, there is great interest from the scientific community to find a potential therapy that can provide benefit to the many patients in need. Some existing medications, including pioliglitazones and angiotensin receptor antagonists, may be repurposed to help treat this condition. Vitamin E may improve the histology in NASH, but safety issues limit its use. Other drugs, such as farnesoid X receptor agonist, obeticholic acid, are also in clinical trials with great hope for the future. However, as the cause of NAFLD and thereby NASH is poorly understood, there is a need for further research in the field to better understand the pathophysiology of the disease and the potential for pharmacotherapy for treatment of the disease. There has, however, been evidence that the antidiabetic drug class known as glucagon-like receptor agonists (GLP-1 RAs) has been shown to reduce liver damage in patients with non-alcoholic steatohepatitis. Liraglutide, currently a drug for type 2 diabetes and obesity, has been shown to provide great benefit in type 2 diabetes and obesity and after reviewing multiple studies, seems to provide a potential treatment also for patients with NAFLD and NASH. However, more research needs to be done to confirm this hypothesis. Its more potent version, called semaglutide, is currently in phase 2 clinical trials and provides great hope in potentially further reducing liver damage. This class of drugs provides a huge opportunity to address an unmet clinical need that could benefit millions of patients worldwide. .

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.

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. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

La sécrétion d'insuline stimulée par les incrétines tels le GLP-1 (glucagon-like peptide-1) et le GIP (glucose-dependent insulinotropic polypeptide), a un rôle majeur dans la régulation du métabolisme des glucides, puisque les incrétines sont responsables de 50 à 70 % de la réponse postprandiale de la cellule beta du pancréas endocrine. Les mécanismes impliquant l'activation de l'adénylate cyclase, l'augmentation intracellulaire de l'AMP cyclique (AMPc) et, en conséquence, l'activation de certaines protéines AMPc-dépendantes, sont responsables de l'effet sécrétagogue des incrétines. Cependant, l'effet du GLP-1 sur la sécrétion d'insuline est préservé même en absence de l'activation de ces protéines, ce qui suggère que des mécanismes indépendants de l'AMPc sont également impliqués. Dans la présente étude, nous nous sommes intéressés à élucider ces mécanismes. Nous avons observé que la voie de la kinase AMP-dépendante (AMPK), qui semble avoir un rôle dans la sécrétion d'insuline stimulée par des nutriments, était également modulée par le GLP-1 dans des cellules beta du pancréas. Nous avons ensuite démontré que l'activation de cette voie réduisait l'effet du GLP-1 sur la sécrétion d'insuline. Ainsi, notre étude a permis l'identification d'une nouvelle voie contribuant à l'effet du GLP-1 sur la sécrétion d'insuline.

Titre de l'écran-titre (visionné le 30 janvier 2024) / La grossesse est un moment durant lequel de nombreux changements physiologiques se produisent. Des apports énergétiques adéquats sont donc importants durant cette période cruciale pour assurer une santé optimale de la mère et du bébé à venir. Alors que les recommandations spécifient une augmentation des besoins énergétiques aux deuxième et troisième trimestres, la littérature suggère que les apports des personnes enceintes demeurent relativement stables tout au long de la grossesse. Le présent mémoire se concentre sur certains facteurs biologiques qui influencent les apports énergétiques, soit les sensations d'appétit et les hormones reliées à la prise alimentaire, spécifiquement le glucagon-like peptide-1 (GLP-1), pour tenter d'élucider la stabilité d'apports observée. La littérature contient des articles qui explorent les liens entre les hormones régulant la prise alimentaire, les sensations d'appétit et les apports énergétiques au sein de la population générale, mais les articles adressant ces sujets en contexte gestationnel sont limités. Par conséquent, l'objectif du présent mémoire est d'examiner la réponse du GLP-1 durant la grossesse en association avec les sensations d'appétit et les apports énergétiques usuels. Les résultats de notre étude indiquent que la réponse postprandiale du GLP-1 est plus faible au troisième trimestre comparativement au premier. D'un point de vue physiologique, ceci pourrait suggérer une diminution de la satiété, donc une augmentation de la faim en fin de grossesse. Dans le même ordre d'idée, des sensations postprandiales de faim plus élevées en fin de grossesse, comparativement au début, ont été observées. Cependant, peu d'associations entre le GLP-1 et les sensations d'appétit ont été notées. Puis, aucune association entre le GLP-1 et les apports énergétiques n'a été observée. Ces résultats fournissent de nouvelles données sur le GLP-1 en contexte gestationnel, tout en mettant en lumière le besoin d'approfondir la recherche concernant les facteurs pouvant influencer les apports énergétiques durant la grossesse. / Pregnancy is characterized by many physiological changes. Adequate energy intake is therefore important during this crucial period to ensure optimal health for both mother and baby. While recommendations specify an increase in energy needs during the second and third trimesters, the literature suggests that the intakes of pregnant people remain relatively stable throughout pregnancy. This dissertation focuses on certain biological factors that influence energy intake, notably appetite sensations and hormones related to food intake, specifically glucagon-like peptide-1 (GLP-1), to explain the observed stability of intakes. The literature contains articles exploring the links between hormones regulating food intake, appetite sensations and energy intake in the general population, but articles addressing these topics during pregnancy are limited. Therefore, the aim of the present dissertation is to examine GLP-1 responses during pregnancy in association with appetite sensations and usual energy intake. Data from our study indicates that the postprandial GLP-1 response is lower in the third trimester compared to the first. From a physiological point of view, this could suggest a decrease in satiety and an increase in hunger towards the end of gestation. Along the same lines, higher postprandial feelings of hunger at the end of pregnancy, compared with the beginning, were observed. However, few associations between GLP-1 and appetite sensations were noted. Furthermore, there was no association between GLP-1 and energy intake. These findings provide new data on GLP-1 in the gestational context, while highlighting the need for further research into factors that may influence energy intake during pregnancy.