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. .

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

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.

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.

Analisamos a ação da liraglutida na flora intestinal e perda de peso de dois modelos de obesidade: por dieta hiperlipidica (HFD) e obesidade genética (ob/ob). Os modelos foram tratados com o fármaco durante duas semanas. Perfis metabólicos foram feitos por meio de testes glicêmicos e insulínicos, histologia do fígado, região cecal e coxins gordurosos, ingestão alimentar, peso corporal e metagenômica do conteúdo cecal. O tratamento induziu perda de peso com melhora dos níveis glicêmicos e redução da inflamação na região cecal e do fígado e foi capaz de reduzir o acúmulo de gordura hepática promovendo a redução da EHNA. A metagenômica mostrou mudança taxonômica geral, bem como a abundância relativa de bactérias envolvidas com peso e controle glicêmico:redução de Proteobacterias e aumento de Akkermansia muciniphila. Apresentamos evidências do fármaco revertendo DGHNA/EHNA e a perda de peso associados às mudanças da microbiota. Sugerimos uma lista de alvos bacterianos que podem interferir no metabolismo energético para o controle clinico de doenças metabólicas. / The study analyzed the effects of liraglutide on gut microbiota and weight-loss in two obesity model: induced by high fat diet (HFD) and genetic obese mice (ob/ob). Models were treated with liraglutide for two weeks. Metabolic profiles were measured by glycemic and insulin test, histological liver, cecal region and fat pad morphologies, food intake, body weight and metagenomic of cecal contents. The treatment induced weight-loss, improvement of glycemic parameters and reduction of inflammatory cells in the cecum and the liver and reduced fat accumulation in liver reverting NASH. The metagenomic showed a general changed in taxonomic structure as well the relative abundance of weight-relevant:reduction of Proteobacteria and increases of Akkermansia muciniphila. We showed evidences that liraglutide leads to improvement of NASH and weight loss associated with changes in microbiota. Moreover, by the profile of the gut microbiota, we present a bacterial target list that may affect energetic metabolism inducing a metabolic clinical controlled profile.

Les acides biliaires sont depuis longtemps connus pour leur propriété d’agents solubilisant des graisses et des vitamines liposolubles, permettant ainsi une absorption efficace de ces nutriments lors de la digestion. Depuis les années 2000, plusieurs équipes ont montré que ces molécules étaient également dotées de propriétés de signalisation, en particulier via l’activation de deux récepteurs : le récepteur nucléaire FXR, et le récepteur membranaire TGR5. Le récepteur TGR5 est exprimé dans de très nombreux tissus, dont les muscles lisses et squelettiques, le tissu adipeux brun, la vésicule biliaire, mais aussi certaines cellules immunitaires et certaines populations cellulaires intestinales telles que les cellules entéroendocrine L. Selon le tissu étudié, l’activation de TGR5 peut être suivie de nombreux effets biologiques. En particulier, au niveau intestinal, l’activation de ce récepteur stimule la sécrétion de l’hormone incrétine GLP-1.Les hormones incrétines sont impliquées dans la régulation de la glycémie, en particulier dans la phase postprandiale, où elles concourent à potentialiser l’action de l’insuline, hormone hypoglycémiante majeure. Or, dans un contexte de diabète, et en particulier de diabète de type 2, l’organisme est devenu moins sensible à l’insuline, ce qui se traduit par un défaut de gestion de la glycémie, pouvant entrainer à terme des complications graves, telles que des amputations, une cécité, ou encore des problèmes cardiovasculaires. La prévalence et l’incidence de cette pathologie ont conduit l’OMS à la considérer comme la première épidémie d’origine non-infectieuse, ce qui illustre son impact sur la santé publique et le besoin médical constant qu’elle génère.Dans ce contexte, TGR5 apparait comme cible thérapeutique potentielle attrayante, de par l’effet GLP-1 sécrétagogue consécutif à son activation. En effet, parmi les thérapies antidiabétiques, deux classes de molécules basent leur efficacité sur une augmentation de la signalisation de la voie incrétine : les incrétinopotentiateurs (inhibiteurs de la DDP4, enzyme responsable de la faible demi-vie du GLP-1) et les incrétinomimétiques (agonistes synthétiques du récepteur au GLP-1). Récemment, cette dernière classe a également fait son apparition dans l’arsenal thérapeutique de l’obésité, confirmant l’intérêt de cette voie de signalisation dans les pathologies issues d’un désordre métabolique. L’obtention de composés GLP-1 sécrétagogues s’avère ainsi prometteuse et représente une approche complémentaire aux deux autres classes.L’objectif de ce travail était donc d’obtenir des agonistes puissants, sélectifs et originaux du récepteur TGR5. Afin de diminuer les risques d’effets indésirables, on-target ou off-target, nous avons choisi de profiter de la localisation intestinale de notre cible d’intérêt en concevant nos composés de manière à limiter l’exposition au seul tractus gastro-intestinal, en limitant leur absorption. Ainsi, nous avons cherché à obtenir des composés non systémiques GLP-1 sécrétagogues.La stratégie employée pour aboutir à ces composés a été le développement de molécules chimériques constituées d’une partie agoniste de TGR5, le pharmacophore, liée à un groupement permettant de limiter la perméabilité membranaire et donc l’absorption intestinale, le kinétophore. Après avoir optimisé la partie pharmacophore et identifié une position permettant l’ajout de différents types de kinétophores sans impact majeur sur l’activité du composé, nous avons pu obtenir plusieurs agonistes de TGR5 puissants, originaux, et dotés d’une très faible perméabilité membranaire. L’étude in vivo de ces molécules a ensuite permis de valider d’une part leur activité GLP-1 sécrétagogue, et d’autre part leur faible exposition systémique. Enfin, l’évaluation du potentiel thérapeutique d’un des meilleurs composés dans des modèles murins de diabète a récemment pu être initiée. / Bile acids have long been known as lipid solubilizing agents, enabling efficient absorption of nutrients and vitamins during digestion. Since 2000, several teams have demonstrated the signaling properties of these molecules, especially through the activation of two receptors : the nuclear receptor FXR and the membrane receptor TGR5.The TGR5 receptor is expressed in various tissues, such as smooth and skeletal muscles, brown adipose tissue, gallbladder, but also on some immune or intestinal cell lines such as the enteroendocrine L cells. Depending on the studied tissue, TGR5 activation can trigger various biological effects. In the intestine, its activation can stimulate the secretion of an incretin hormone, the GLP-1.Incretin hormones play a role in glycaemia regulation, especially during the postprandial phase during which they potentiate the action of the insulin, the main hypoglycemic hormone. Diabetes mellitus correspond to a decreased response of the organism to insulin signaling. This leads to a default in the glycaemia handling that can lead to serious complications, such as amputation, blindness, or cardiovascular problems. Prevalence and incidence of this disease have lead the WHO to define diabetes as the first non-infectious epidemic, illustrating its impact on public health and the constant need for new therapeutic opportunities.In this context, TGR5 appears as an appealing potential therapeutic target, especially because of the GLP-1 secretagogue effect triggered by its activation. Indeed, among the antidiabetic therapeutic options, two classes of drugs work by increasing the incretin signaling: the incretinopotentiators (inhibitors of the DPP4, which is the enzyme responsible for the very short half-life of GLP-1), and the incretinomimetics (synthetic agonists of the GLP-1 receptor). Recently, this last class has also been approved to treat obesity. This demonstrates the interest of this signaling pathway in the treatment of metabolic disorders. Hence, GLP-1 secretagogue compounds may prove to be an interesting approach, and could complement the two other classes.The aim of this work was then to obtain potent, selective and original agonists of the TGR5 receptor. In order to decrease the risk of on-target and off-target effects, we decided to take advantage of the intestinal localization of our target by designing compounds that would only expose the gastro-intestinal tract, by limiting their absorption. Thus, we wanted to obtain non systemic GLP-1 secretagogue compounds.Our strategy was to develop chimeric compounds consisting of a pharmacophore part, which would be a potent and selective agonist of TGR5, linked to a kinetophore part, which would decrease membrane permeability. After having optimized the pharmacophore part and having identified a position where we could link various kinetophore moieties with only weak impact on the activity, we obtained several potent TGR5 agonists with very low membrane permeability. In vivo evaluations of these compounds have validated both their GLP-1 secretagogue activity and their low systemic exposure. In the end, evaluation of our lead compound on mouse model of diabetes was recently started.

<p><strong><p>Abstract</p><em><p>Background and aim:</p>Diabetes mellitus (DM) is a chronic and progressive illness that affects all type of populations and ages. According to World health organization (WHO) by 2030 it will be 366 million people effected world wild. Many new drugs are Glucagon-like peptide-1 (GLP-1) based therapy for treatment of type 2diabetes. GLP-1 is released from the intestinal L-cells, and is a potent stimulator of glucose-dependent insulin secretion. The aim of this study was to investigate the effect of GLP-1 and its stable analogs on cell proliferation of GLP-1 secreting GLUTag cells. <em><p>Material and methods:</p>GluTag cells were incubated for 48h in DMEM medium containing (0.5% fetal bovine serum and 100 IU/ml penicillin and 100 μg/ml streptomycin and 3mM glucose concentration) in the present or absence of the agents. DNA synthesis was measured using 3H- thymidine incorporation and Ki67 antigen staining. Western blot were performed to investigate the present of GLP-1 receptor in GLUTag cells. <em><p>Results/conclusions:</p><p>These results suggest that GLP-1 regulates proliferation of the GLP-1-secreting cell through a feedback mechanism via its receptor. Since serum GLP-1 levels are decreased in type 2 diabetic patients, the effect of GLP-1 on the GLP-1-secreting cell proliferation suggested here provides a novel beneficial long-term effect of the incretin-based drugs in clinical practice i.e. through increase of the GLP-1-secreting cell mass, augmenting the incretin effect. In addition, the feedback mechanism action of GLP-1 reveals a new insight in regulation manner of the L-cell proliferation.</p>GLP-1(7-36) increased cell proliferation in GLUTag cells, an effect which was blocked by the GLP-1 receptor antagonist exendin(9-39). The GLP-1 receptor was expressed in GluTag cells. <em><p>Keywords:</p>Incretin hormone<em>, GLP-1, GLP-1 receptor, Exendin-4, Diabetes </em></em></em></em></em></strong></p>

Abstract Background and aim: Diabetes mellitus (DM) is a chronic and progressive illness that affects all type of populations and ages. According to World health organization (WHO) by 2030 it will be 366 million people effected world wild. Many new drugs are Glucagon-like peptide-1 (GLP-1) based therapy for treatment of type 2diabetes. GLP-1 is released from the intestinal L-cells, and is a potent stimulator of glucose-dependent insulin secretion. The aim of this study was to investigate the effect of GLP-1 and its stable analogs on cell proliferation of GLP-1 secreting GLUTag cells. Material and methods: GluTag cells were incubated for 48h in DMEM medium containing (0.5% fetal bovine serum and 100 IU/ml penicillin and 100 μg/ml streptomycin and 3mM glucose concentration) in the present or absence of the agents. DNA synthesis was measured using 3H- thymidine incorporation and Ki67 antigen staining. Western blot were performed to investigate the present of GLP-1 receptor in GLUTag cells. Results/conclusions: These results suggest that GLP-1 regulates proliferation of the GLP-1-secreting cell through a feedback mechanism via its receptor. Since serum GLP-1 levels are decreased in type 2 diabetic patients, the effect of GLP-1 on the GLP-1-secreting cell proliferation suggested here provides a novel beneficial long-term effect of the incretin-based drugs in clinical practice i.e. through increase of the GLP-1-secreting cell mass, augmenting the incretin effect. In addition, the feedback mechanism action of GLP-1 reveals a new insight in regulation manner of the L-cell proliferation. GLP-1(7-36) increased cell proliferation in GLUTag cells, an effect which was blocked by the GLP-1 receptor antagonist exendin(9-39). The GLP-1 receptor was expressed in GluTag cells. Keywords: Incretin hormone, GLP-1, GLP-1 receptor, Exendin-4, Diabetes