Diabetes epidemic is underway. Beta cell dysfunction (BCF) and loss of beta cell mass (BCM) are known to be key events in its progression. Currently, there are no reliable techniques to estimate or follow the loss of BCM, in vivo. Non-invasive imaging and quantification of the whole BCM in the pancreas, therefore, has a great potential for understanding the progression of diabetes and the scope for early diagnosis for Type 2 diabetes. Glucagon-like peptide-1 receptor (GLP-1R) is known to be selectively expressed on the pancreatic beta cells and overexpressed on the insulinoma, a pancreatic neuroendocrine tumor (PNET). Therefore, this receptor is considered to be a selective imaging biomarker for the beta cells and the insulinoma. Exendin-4 is a naturally occurring analog of GLP-1 peptide. It binds and activates GLP-1R with same the potency and engages in the insulin synthesis, with a longer biological half-life. In this thesis, Exendin-4 precursor, DO3A-VS-Cys40-Exendin-4 labeled with [68Ga], [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 ([68Ga]Exendin-4), was evaluated in different species models, namely, immune deficient nude mice, rats, pigs, non-human primate (NHP), and clinically in one insulinoma patient by positron emission tomography (PET), for its potential in beta cell imaging and its quantification as well as for visualizing the insulinoma. From internal dosimetry, the possible number of repetitive [68Ga]Exendin-4-PET/CT scans was estimated. Pancreatic uptake and insulinoma tumor uptake of [68Ga]Exendin-4 were confirmed to be mediated by the specific binding to the GLP-1R. Pancreatic GLP-1R could be visualized and semi-quantified, for diabetic studies, except in rats. Nonetheless, we found conflicting results regarding the GLP-1R being a selective imaging biomarker for the beta cells. PET/CT scan of the patient with [68Ga]Exendin-4 has proven to be more sensitive than the clinical neuroendocrine tracer, [11C]5-HTP, as  it could reveal small metastatic tumors in liver. The kidney was the dose-limiting organ in the entire species model, from absorbed dose estimation. Before reaching a yearly kidney limiting dose of 150 mGy and a whole body effective dose of 10 mSv, 2–4 [68Ga]Exendin-4 PET/CT scans be performed in an adult human, which enables longitudinal clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma, as well as in healthy volunteers enrolled in the early phase of anti-diabetic drug development studies.

<p>The delivery of therapeutic peptides and proteins is often challenged by a short circulation half-life, necessitating frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. The covalent conjugation of therapeutic peptides and proteins, and more recently oligonucleotide-based drugs, with the “stealth” polymer poly(ethylene glycol) (PEG), termed PEGylation, is one of the most commonly used approaches to increase the in vivo half-life and reduce the immunogenicity of these therapeutic biomolecules. However, after several decades of research and clinical use, the limitations of PEGylation have begun to emerge.</p><p>Conventional methods for synthesizing peptide/protein-polymer conjugates have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of control over site and stoichiometry of conjugation, which results in heterogeneous products with significantly compromised biological activity. Additionally, anti-PEG antibodies have been induced in patients treated with PEGylated drugs and have been shown to correlate with rapid clearance of these drugs. High levels of pre-existing anti-PEG antibodies have also been found in individuals naïve to PEGylated agents, which are associated with serious first-exposure allergic reactions.</p><p>To address the synthetic limitations of PEGylation, a general approach for the high-yield synthesis of site-specific (C-terminal) and stoichiometric (1:1) peptide/protein-polymer conjugates, named sortase-catalyzed polymer conjugation, was developed. Demonstrating proof-of-concept of the approach with green fluorescent protein (GFP) as a model protein, sortase A from Staphylococcus aureus was used to site-specifically attach an initiator solely at the C-terminus of GFP, followed by in situ growth of the PEG-based brush polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) from the protein macroinitiator by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeded with high specificity and near-complete (~ 95%) product conversion. Subsequent in situ ATRP in aqueous buffer produced 1:1 stoichiometric conjugates with > 90% yield, tunable MW, low dispersity, and no denaturation of the protein. The extraordinarily high yield compares favorably to order of magnitude losses typically seen in conventional PEGylation processes.</p><p>Next, the therapeutic potential of POEGMAlation, or the conjugation of POEGMA to a peptide or protein, was demonstrated by implementing the developed sortase-catalyzed polymer conjugation strategy with exendin-4 (exendin), a therapeutic peptide for treating type 2 diabetes, to synthesize exendin-C-POEGMA conjugates with a wide and tunable range of molecular weights (MWs) and low dispersity. A single subcutaneous injection of exendin-C-POEGMA conjugates lowered blood glucose for up to 120 h in a diabetic mouse model. Most intriguingly, we showed that appending PEG as oligomeric side-chains on the conjugated POEGMA and tuning the side-chain length completely eliminated the reactivity of exendin-C-POEGMA conjugates toward patient-derived anti-PEG antibodies without compromising in vivo efficacy. Clinically, the lack of anti-PEG antigenicity of POEGMA conjugates is expected to completely eliminate serious first-exposure allergic reactions and the accelerated blood clearance of POEGMA-drug conjugates due to pre-existing anti-PEG antibodies in patients.</p><p>Collectively, these results establish POEGMAlation as a next-generation PEGylation technology that is highly useful for improving the pharmacological performance of therapeutic biomolecules while providing a timely solution to the increasing levels of pre-existing anti-PEG antibodies in patients that are seriously hindering the safety and efficacy of traditional PEGylated drugs.</p> / Dissertation

<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>

Transplantation of pancreatic islets is a potential treatment of type 1 diabetes that aims to restore normal blood glucose control. By encapsulating the islets in alginate, they can be protected from rejection. The aim of this thesis was to study the biology of encapsulated islets and to use the technique of microencapsulation to study the effect of transplantation in a system that is separated from direct contact with the vascular system and the host tissue at the transplantation site. Encapsulated islets can effectively reverse hyperglycaemia after transplantation into the peritoneal cavity of diabetic mice. A period of culture before encapsulation and transplantation did not affect their insulin release or curative capability. Pre-culture with exendin-4 improved insulin secretion, but not to the extent that the long term outcome in our transplantation model was improved. Despite being able to reach and retain normoglycaemia, microencapsulated islets transplanted intraperitoneally decreased in size. More specifically the number of beta cells in each individual islet was decreased. However, in contrast to previous studies using non-encapsulated islets, the alpha cell number was maintained, and thus the capsule seems to protect these peripherally located and otherwise exposed cells. As the capsule also prevents revascularisation of the islets, the model was used to study the importance of vascular supply for islet amyloid formation. Islet amyloid is a possible reason for the long-term failure of transplanted islets. It is likely that their low vascular density causes a disturbed local clearance of IAPP and insulin that starts the aggregation of IAPP. Indeed, encapsulated islets had an accelerated amyloid formation compared to normal islets, and might serve as a model for further studies of this process. In conclusion, although revascularisation is not a prerequisite for islet graft function, it plays an important role for islet transplantation outcome.

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: Stroke is caused from the occlusion of any cerebral artery leading to cerebral ischemia, brain damage and consequent neurological impairments and disability. The primary causes of mortality in western populations is stroke. Diabetes type 2 is a high risk factor for stroke. Stroke leads to an observable increase of neural stem cell proliferation in the subventricular zone and enhances neurogenesis in the adult rodent and human brain which suggest a mechanism contributing to stroke recovery. Neurogenesis in type 2 diabetes patients is impaired. However, whether stroke-induced neurogenesis is impaired in diabetes has not been studied. Exendin-4 is a drug for clinical treatment of type 2 diabetes which has been shown to have neuroprotective properties in animal studies. However whether Exendine-4 leads to increased neurogenesis after  stroke in the diabetic brain has not been previously studied.  Aims: The specific aims of this project were to determine whether stroke-induced stem cell proliferation is impacted by diabetes in the mouse, and if Exendine-4 regulates stroke-induced stem cell proliferation in normal and diabetic mice. Material and Methods: Aged obese/type 2 diabetic mice were subjected to stroke. The Exendin-4 treatment was started 1.5 hours thereafter. Treatment was continued for one week before animals were sacrificed. Brains were isolated and the neurons were immunostained using the specific proliferation marker Ki67. Neural stem cell proliferation was quantified by counting Ki67+ cells in the ipsilateral (subventricular zone in stroke hemisphere).The estimation was assessed by stereological counts of proliferating stem cell in the subventricular zone.  Results: The number of proliferating stem cell after stroke was statistically significantly higher in the normal mice versus diabetic mice. The effect was present in both sides (control and stroke) of the subventricular zone. Exendine-4 treatment induced statistically significant increased of  stem cell proliferation in normal mice but not in diabetic mice.   Conclusions: The result of this study shows that type 2 diabetes decreased the proliferation of neural stem cell in the subventricular zone and that Exendin-4 enhanced the subventricular proliferation in a preclinical model of clinical relevance. The data suggest that the Exendin-4 treatment could be administered to normal patients suffering from stroke in the ambulance or in the emergency room although more studies are needed.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22363号 / 医博第4604号 / 新制||医||1043(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 川口 義弥, 教授 富樫 かおり, 教授 上本 伸二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Islet transplantation is a possible curative treatment for type 1 diabetes (T1D). Currently the liver dominates as implantation site, despite the many challenges encountered at this site. Acute hypoxia in islets transplanted to muscle and omentum, two possible alternative sites, was prevailing. However, it was rapidly reversed at both implantation sites, in contrast to when islets were transplanted intraportally. At the intramuscular site hypoxia was further relieved by co-transplantation of an oxygen carrier, polymerized hemoglobin, which also improved the functional outcome. The complement system was activated after islet transplantation to muscle, but did not hamper graft function. Both mouse and human islets transplanted to omentum become well re-vascularized and have a functional blood flow and oxygenation comparable with that of endogenous islets. Animals transplanted with islets to the omentum had a superior graft function compared with animals receiving intraportal islet grafts. Alloxan-diabetic animals were cured with a low number of islets both when the islets were implanted in the omentum and muscle. The islet grafts responded adequately to both glucose and insulin and displayed a favorable mRNA gene expression profile. A challenge in diabetes research and in islet transplantation is that there are no established techniques for quantifying beta-cell mass in vivo. By using radiolabeled Exendin-4, a GLP-1 receptor agonist, beta-cell mass after transplantation to muscle of mice was quantified. The results may well be translated to the clinical setting. By comparing the pancreatic accumulation of [11C]5-hydroxy tryptophan ([11C]5-HTP) as detected by positron emission tomography (PET) in T1D patients with that of healthy controls, a 66% decrease was observed. This may in fact represent the loss of beta-cells, taking into account that other cells within the islets of Langerhans are largely unaffected in T1D.  In conclusion, the data presented support the use of alternative implantation sites for islet transplantation. In addition to improving the functional outcome this may enable more transplantations since the number of transplanted islets may be reduced. The techniques investigated for quantifying transplanted and endogenous beta-cell mass may greatly improve our knowledge of the pathophysiology of T1D and become a valuable tool for evaluation of beta-cell mass.

The development of a bioartificial pancreas (BAP) has the potential to substantially improve the treatment of insulin-dependent diabetes. Composed of insulin-secreting cells encapsulated in a hydrogel material, a BAP may provide superior glycemic regulation compared with conventional exogenous insulin-delivery therapies. Towards this goal, β- cells or islets encapsulated in alginate microcapsules remain a promising approach. Due to the limited supply of human islets, alternative cell sources are under investigation for incorporation into a BAP, including porcine islets and β- cell lines. Several challenges remain to clinical implementation, including loss of islet or β- cell function and viability following transplantation and host response to the transplanted microcapsules. The objective of this work was to evaluate strategies to improve a BAP by supporting the function and survival of encapsulated islets and β -cells. Towards this goal, two areas were explored: 1) the provision of pro-survival and insulinotropic factors, namely, CXCL12 and GLP-1 (or a GLP-1 analog, Exendin-4), to encapsulated islets and β-cells and 2) modification of the alginate microcapsule to confer long-term resistance to host cell adhesion. To achieve the first objective, methods to deliver both pro-survival and insulinotropic factors to a BAP were developed and their effects on encapsulated β-cells and porcine islets were studied, both in vitro and in vivo. Results demonstrate that delivery of pro-survival and insulinotropic factors is a promising strategy to prolong the survival and function of a BAP. To reduce host cell adhesion to the microcapsule, we employed covalent conjugation of PEG to the surface of alginate-PLL capsules to replace the un-crosslinked layer of alginate used in traditional alginate-PLL-alginate (APA) microcapsules. Results demonstrate that while PEGylation of alginate-PLL microcapsules initially reduced host cell adhesion over 2 weeks in vivo compared with APA capsules, the PEG coating did not provide long-term protection over 3 months. Taken together, these studies represent a multipronged approach towards improving the duration of BAP function, with the ultimate goal of advancing this technology to the clinic.

Le projet de cette thèse porte sur le développement de dépôts lipidiques ectopique et leur modulation suite à des intervenions thérapeutiques par imagerie résonance magnétique.Dans notre première étude, nous avons établi l’ordre chronologique d’apparition de graisses ectopiques et d’anomalies cardiaques dans un modèle de souris soumises à un régime riche en graisse et en sucre. Un traitement de courte durée à l’exendine-4 permet une amélioration de tous les paramètres altérés. Dans la deuxième étude, nous avons évalué l’impact d’un traitement de l’obésité sur les dépôts ectopique de graisse cardiaque (TAE et stéatose), hépatique et pancréatique à deux temps (6 mois et 32 mois) après chirurgie bariatrique. Nous avons montré que ce traitement chirurgical permet une réduction de tous ces dépôts, avec une cinétique différente. Enfin, dans la troisième étude, nous nous sommes intéressés à l’effet du poids de naissance sur le développement de tissu adipeux épicardique. Cette étude nous a permis de mettre en évidence qu’il existe une accumulation plus importante de TAE à l’âge adulte lorsque le poids de naissance est augmenté ; et que les paramètres poids de naissance et croissance entre 2 et 12 ans, jouent un rôle important dans la mise en place de ce dépôts de graisse ectopique. En somme, ces résultats permettent une avancée dans la compréhension du développement des dépôts de graisses et de leur modulation. / The project of this thesis mainly focuses on ectopic lipid deposition development and their flexibility following therapeutic intervention. In our first study, we set out chronological order of ectopic fat onset and cardiac abnormalities in a high fat high sucrose mice model. Short duration exendin-4 treatment reverses every altered parameter. In the second study, we assessed treatment of obesity effect on cardiac ectopic fat deposition (EAT and steatosis), as well as hepatic and pancreatic fat at two different time points (6 months and 32 months) after bariatric surgery. We show significant reduction of every ectopic fat deposition, however in different kinetic. Finally, in a third study, we investigate birth weight effect on epicardial adipose tissue development. This study demonstrate important EAT accumulation in adulthood when birth weight is increased. Furthermore, birth weight and catch up growth in childhood between 2 and 12 years parameters impact significantly the development of epicardial fat.In summary, these results provide better understanding of ectopic fat deposition development and modulation.