京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18501号 / 医博第3921号 / 新制||医||1005(附属図書館) / 31387 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 横出 正之, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Type 2 diabetes has reached epidemic proportions worldwide. The resulting increase in chronic and costly diabetes related complications has potentially catastrophic implications for healthcare systems, and economics and societies as a whole. One of the key pathological factors leading to type 2 diabetes is insulin resistance (IR), which is the reduced or impaired ability of the body to make use of available insulin to maintain safe glucose concentrations in the bloodstream. It is essential to understand the physiology of glucose and insulin when investigating the underlying factors contributing to chronic diseases such as diabetes and cardiovascular disease. For many years, clinicians and researchers have been working to develop and use model-based methods to increase understanding and aid therapeutic decision support. However, the majority of practicable tests cannot yield more than basic metrics that allow only a threshold-based assessment of the underlying disorder. This thesis gives an overview on several dynamic model-based methodologies with different clinical applications in assessing glycaemia via measuring effects of treatment or medication on insulin sensitivity. Other tests are clinically focused, designed to screen populations and diagnose or detect the risk of developing diabetes. Thus, it is very important to observe sensitivity metrics in various clinical and research settings. Interstitial insulin kinetics and their influence on model-based insulin sensitivity observation was analysed using data from the clinical pilot study of the dynamic insulin sensitivity and secretion (DISST) test and the glucose-insulin PK-PD models. From these inputs, a model of interstitial insulin dose-response that best links insulin action in plasma to response in blood glucose levels was developed. The critical parameters influencing interstitial insulin pharmacokinetics (PKs) are saturation in insulin receptor binding (αG) and the plasma-interstitium diffusion rate (nI). Population values for these parameters are found to be [αG, nI]=[0.05,0.055]. Critically ill patients are regularly fed via constant enteral (EN) nutrition infusions. The impact of incretin effects on endogenous insulin secretion in this cohort remains unclear. It is hypothesised that the identified SI would decrease during interruptions of EN and would increase when EN is resumed, where, for short periods around transition, the true patient SI would be assumed constant. The model-based analysis was able to elucidate incretin effects by tracking the identified model-based insulin sensitivity (SI) in a cohort of critically ill patients. Thus, changes in model-based SI given the fixed assumed endogenous secretion by the model would support the presence of an EN-related incretin effect in the population of non-diabetic, critically ill patients studied. The PD feedback-control model of Uen was designed to investigate endogenous insulin secretion amongst subjects with different metabolic states and levels of insulin resistance. The underlying effects that influence insulin secretion i.e. incretin effects were also defined by tracking the control model gain/response and the identified insulin sensitivity (SI) using intravenous (IV) bolus and oral glucose responses of insulin sensitivity tests. This new PD control model allowed the characterisation of both static (basal) and dynamic insulin responses, which defined the pancreatic β-cell glucose sensitivity parameters. However, incretin effects were unobserved during oral glucose responses as the PD control gains failed to simulate the true endogenous insulin secretion due to potentially inaccurate glucose appearance rates and low data resolution of glucose concentrations. The net effect of haemodialysis (HD) treatment on glycaemic regulation and insulin sensitivity in a critically ill cohort was investigated. It was hypothesized that the observed SI would decrease during HD due to enhanced insulin clearance compared to the model, and would be recaptured again when HD is stopped. The changes in model-based SI metric at HD transitions in a cohort of critically ill patients were evaluated. Significant changes of -29% in model-based SI was observed during HD therapy. However, there were insignificant changes when HD treatment was ended. Thus, the changes in model-based SI would thus offer a unique observation on insulin kinetics and action in this population of critically ill patients with ARF that would better inform metabolic care.

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

Introduction – Le but de ce travail était de développer un modèle expérimental endoscopique de chirurgie bariatrique par technique NOTES, simple, reproductible et de le valider à la fois sur le plan clinique (faisabilité, tolérance) et sur le plan métabolique (physiopathologie et mode d’action). Matériels et méthodes - Les études furent menées au sein du CERC et de l’unité INSERM UMRS 1260. L’animal étudié fut le cochon domestique, âgé de 3 à 4 mois, sains et non obèse. Les endoscopies étaient réalisées à l’aide d’un gastroscope double canal opérateur et de matériel d’endoscopie couramment disponible. Plusieurs étapes de développement furent mises en place de la confection d’un modèle animal d’anastomose gastro-jéjunale par technique endoscopique NOTES exclusive à l’évaluation de l’impact métabolique d’un modèle de by-pass gastrique à visée bariatrique utilisant l’anastomose gastro-jéjunale par apposition prothétique luminale. Résultats – Le modèle d’anastomose gastro-jéjunale choisi pour sa faisabilité et son innocuité fut développé par apposition des lumières digestives gastriques et grêliques avec prothèse en technique NOTES trans digestive. L’évaluation métabolique expérimentale du by-pass gastrique utilisant ce mode opératoire permit de constater une amélioration significative de l’insulino-résistance non véhiculées par le GLP-1. Conclusion – Le concept d’anastomose digestive par NOTES exclusive et apposition luminale prothétique est établi et le modèle de by-pass endoscopique utilisant ce mode opératoire est faisable semblant présenter un impact métabolique significatif. / Introduction - The aim of this study was to develop an endoscopic experimental model of NOTES bariatric surgery using a simple, reproducible technique and to validate it both clinically (feasibility, tolerance) and metabolically (pathophysiology and action plan). Materials and Methods - The studies were conducted within the CERC and INSERM UMRS 1260 unit. The animal was the pig, aged 3 to 4 months, healthy and not obese. Endoscopies were performed using a dual channel gastroscope operator and the endoscopy equipment currently available. Several development stages were set up from the development of an animal model of gastrointestinal anastomosis using an exclusive endoscopic NOTES procedure to the evaluation of the metabolic impact of gastric bypass bariatric model described using the gastrointestinal anastomosis by luminal apposing stent technique. Results - The gastro-jejunal anastomosis model chosen for its feasibility and safety was developed by luminal apposing gastric and small bowel digestive lumens with a specific metallic covered stent and NOTES technique. The experimental evaluation of the metabolic gastric bypass using this procedure allowed to observe a significant improvement in insulin resistance none mediated by GLP-1 and incretin effect. Conclusion - The concept of anastomosis with luminal apposing stent and exclusive NOTES technique is established and endoscopic bypass model using this procedure is feasible pretend to present a significant metabolic impact.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22321号 / 医博第4562号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川上 浩司, 教授 浅野 雅秀, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13450号 / 論医博第2243号 / 新制||医||1054(附属図書館) / (主査)教授 稲垣 暢也, 教授 妹尾 浩, 教授 小濱 和貴 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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