京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12944号 / 論医博第2096号 / 新制||医||1010(附属図書館) / 32203 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 川口 義弥, 教授 横出 正之, 教授 稲垣 暢也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24965号 / 医博第5019号 / 新制||医||1069(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 波多野 悦朗, 教授 中本 裕士 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Aggregation of proteins into amyloid deposits is a common feature among dozens of diseases. Two such diseases that feature amyloid deposits are Alzheimer's disease (AD) and type 2 diabetes (T2D). AD toxicity has been associated with the aggregation and accumulation of the amyloid β-peptide (Aβ); Aβ exerts its toxic effects through interactions with neuronal cell membranes. A characteristic feature of T2D is the deposition of the islet amyloid polypeptide (IAPP) in the pancreatic islets of Langerhans. It is currently unknown if IAPP aggregation is a cause or consequence of T2D, but it does lead to β-cell dysfunction and death, exacerbating the effects of diabetes. Characterizing the fundamental interactions between both Aβ and IAPP with lipid membranes and in solution will give greater insight into mechanisms of toxicity exhibited by amyloid proteins. In this work, molecular dynamics (MD) simulations were used to study the secondary, tertiary, and quatnary structure of Aβ and IAPP, in addition to peptide-membrane interactions and membrane perturbation as independently caused by both peptides. Studies were conducted to address the following questions: (1) what influence do solution conditions and oxidation state have on monomeric Aβ] (2) how and in what way does monomeric Aβ interact with model lipid membranes and what role does sequence play on these peptide-membrane interactions; (3) can MD simulations be utilized to understand Aβ tetramer formation, rearrangement, and tetramer-membrane interactions; (4) how does IAP interact with model membranes and how does that vary from non-toxic (rat) IAPP peptide-membrane interactions. These studies led to conclusions that showed variance in lipid affinity and degree of perturbation as based on peptide sequence, in addition to insight into the type of perturbation caused to membranes by these amyloid peptides. Understanding the differences in peptide-membrane interactions of amyloidogenic and non-amyloidogenic (rat) peptides gave insight into the overall mechanism of amyloidogenicity, leading to the detection of specific amino acids essential in peptide-membrane perturbation. These residues can then be targeted for novel therapeutic design to attenuate the perturbation and potential cell death as caused by these peptides. / Ph. D.

To the Editor: The recent fndings of Hummel et al demonstrate the potential clinical benefts of identifying children with presymptomatic type 1 diabetes [1]. The authors compared children who developed type 1 diabetes after a previous diagnosis of presymptomatic type 1 diabetes in the Fr1da study with those in a registry cohort of children diagnosed without a prediagnosis [aus dem Brief]

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.

La transplantation d’îlots de Langerhans microencapsulés est un traitement prometteur du diabète de type 1. La microcapsule protège l’îlot du système immunitaire, tout en permettant la diffusion de petites molécules. Comme la microcapsule empêche la revascularisation des îlots, leur oxygénation se fait par diffusion d’oxygène et ils sont exposés à l’hypoxie. Le manque d’oxygène est un facteur limitant dans la survie des îlots microencapsulés. Il est connu que les plus petits îlots sont plus résistant à l’hypoxie à cause d’une meilleure diffusion de l’oxygène. À cette fin, les agrégats de cellules dispersées d’îlots seront étudiés. Lorsque les cellules des îlots sont dispersées, elles ont la propriété de se ré-assembler dans une structure semblable à celle des îlots. La présente étude a permis de mettre au point une technique de formation des agrégats, de les caractériser et de comparer la résistance à l’hypoxie des îlots et des agrégats. Ceux-ci ont une structure semblable aux îlots et ils sont de plus petite taille. Pour cette raison, ils sont plus viables après un choc hypoxique tout en renversant efficacement l’hyperglycémie de souris diabétiques. Les agrégats sont une alternative intéressante pour la transplantation d’îlots microencapsulés puisque leur oxygénation est plus efficace. / Transplantation of microencapsulated islets of Langerhans is a promising treatment for type 1 diabetes mellitus. The microcapsule allows the diffusion of small molecules, while protecting the islet from the antibodies and immune cells. However, microcapsule prevents islet revascularization, thus oxygenation depends on diffusion and islets are exposed to hypoxia. Poor oxygenation is a major limitation in microencapsulated islet survival. It was shown that smaller islets are more resistant to hypoxia because of a better oxygen diffusion. In this study, dispersed islet cell aggregates will be used to improve the oxygenation. When islet cells are dispersed into single cells, they have the ability to re-associate into an islet-like structure. This study allowed to set up a technique to form aggregates, to characterized them and to compare the resistance to hypoxia of islets and aggregates. Aggregates have a similar structure than islets and they are smaller. For this reason, they survive better to a hypoxic treatment, while restoring efficiently normoglycemia in diabetic mices. Aggregates are an interesting solution for microencapsulated islet transplantation because they have a better oxygenation.

Lam, Kwok Lim. / "October 2010." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 142-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Made available in DSpace on 2017-07-21T19:59:54Z (GMT). No. of bitstreams: 1 PALLOMA HOCAYEN.pdf: 1571020 bytes, checksum: 2b66eed389dee29aecb38d9310ca585e (MD5) Previous issue date: 2012-05-09 / In recent years diabetes has been gaining huge proportions, due to changes in the habits of life, poor diet and physical inactivity are key factors in disease development. To this end, various species of plants have been used etnofarmacologicamente or in clinical trials or to treat and combat the symptoms of Diabetes Mellitus. The present study used the methanol extract of the plant Bracharis dracunculifolia (rosemary field) belonging to the Asteraceae family, with the objective of evaluating the effect of his administration on hypothalamic obesity in animal model of MSG. The methodology was based on the preparation of the extract metabolic B. dracunculifolia by means of drying processes, spraying, orbital agitation of the extract in methanol, filtration and addition of rotaevaporação toxicity test of the extract in the brine shrimp. To obtain MSG animals, were used in newborn animals still injection of monosodium glutamate (MSG) for five consecutive days at a dose of 4 mg / g body weight to induce obesity. Treatment with methanol extract was carried out on animals at 180 days of life, for 30 days. Weight was evaluated, consumption of water and feed throughout the experiment. And after the animals have been sacrificed held in the isolation of pancreatic islets which were incubated with increasing concentrations of glucose (5.6 mM, 8.3 mM, 16.7 mM). The results obtained in the study demonstrated low toxicity of the extract of B. dracunculifolia. The MSG animals had proven by induction of obesity increased Lee index, and higher levels of visceral and subcutaneous fat compared to controls. The MSG animals weighed less, consumed less water and less food and had impaired glucose tolerance. The biochemical parameters did not obtain significant differences, except for triglycerides which showed to be higher in MSG animals, not having succeeded in restoring the extract this parameter. In the isolation of pancreatic islets, MSG animals that received the extract of B. dracunculifolia showed a significant secretion of insulin by the islets isolated, noting that the concentration of 8.3 mM and 16.7 mM was obtained increased secretion of insulin. It is concluded that the methanol extract of B. dracunculifolia obtained significant effect on insulin secretion in pancreatic islet cell isolation, noting the results obtained in the group MSG (MP), which were hyperinsulinemia because of a possible enhancement of oxidative stress, especially the high antioxidant content present in the extract B. dracunculifolia used in the study. / Nos últimos anos o Diabetes vem ganhando grandes proporções, devido a mudanças nos hábitos de vida das pessoas, má alimentação e sedentarismo, que são fatores centrais no desenvolvimento da doença. Para tanto, várias espécies de plantas vêm sendo utilizadas etnofarmacologicamente ou em ensaios clínicos para tratar e ou combater os sintomas do Diabetes Mellitus. O presente estudo utilizou o extrato metanólico da planta Bracharis dracunculifolia (alecrim do campo), pertencente a família Asteraceae, com o objetivo de avaliar o efeito de sua administração na obesidade hipotalâmica em modelo de animais MSG. A metodologia se baseou na preparação do extrato metabólico de B. dracunculifolia, por meio de processos de secagem, pulverização, agitação orbital do extrato em metanol, filtragem e rotaevaporação além do teste de toxicidade do extrato frente a Artemia salina. Para a obtenção de animais MSG, foram aplicados nos animais ainda neonatos injeção de glutamato monossódico (MSG), durante cinco dias consecutivos, na dose de 4mg/g de peso corporal, para indução da obesidade. O tratamento com o extrato metanólico foi realizado nos animais aos 180 dias de vida, durante 30 dias. Foi avaliado o peso, consumo de água e ração durante todo o experimento. E após os animais terem sido sacrificados realizou-se o isolamento de ilhotas pancreáticas, que foram incubadas em concentrações crescentes de glicose (5,6mM; 8,3mM; 16,7mM). Os resultados obtidos no estudo demonstraram baixa toxicidade do extrato de B. dracunculifolia. Os animais MSG obtiveram indução da obesidade comprovada pelo aumento do Índice de Lee, e maior teor de gorduras viscerais e subcutânea em relação aos controles. Os animais MSG apresentaram menor peso, consumiram menos água e menos ração e apresentaram intolerância a glicose. Os parâmetros bioquímicos não obtiveram diferenças significativas, com exceção dos triglicerídeos que apresentou-se maior nos animais MSG, não tendo o extrato conseguido restaurar este parâmetro. No isolamento de ilhotas pancreáticas, os animais MSG que receberam o extrato de B. dracunculifolia apresentaram uma secreção significativa de insulina pelas ilhotas isoladas, ressaltando que na concentração de 8,3mM e 16,7mM foi obtida maior secreção de insulina. Conclui-se que o extrato metanólico de B. dracunculifolia obteve efeito significativo da secreção de insulina, no isolamento de ilhotas pancreáticas, ressaltando os resultados obtidos no grupo MSG (MP), que se apresentaram hiperinsulinêmicos, devido a uma possível melhora do stress oxidativo, destacando-se o alto teor antioxidante presente no extrato de B. dracunculifolia utilizado no estudo.

Ce travail scientifique a abordé la problématique du vieillissement des îlots pancréatiques et l’effet de la senescence endothéliale et des microparticules (MPs) sur la fonction des îlots. Nous avons exploré l’impact du vieillissement du pancréas sur la morphologie, le devenir et la fonction de l’îlot pancréatique par analyse comparative entre pancréas de rats jeunes et d’âge moyen et le rôle des MPs endothéliales pro-sénescentes sur la fonction des îlots et leur sénescence prématurée. Nos résultats in vivo montrent que le pancréas est un organe précocement sensible au stress oxydant s’accumulant avec l’âge. Il conduit à la surexpression des marqueurs procoagulants et de senescence sans apparition d’apoptose. In vitro, les MPs de cellules endothéliales sénescentes ont un effet pro-sénescent sur les îlots pancréatiques isolés de rats jeunes avec une activité SA-β-galactosidase caractéristique, la surexpression des marqueurs p53, p21 et p16 et la réduction de la capacité de la sécrétion d’insuline en réponse au glucose. L’ensemble de nos résultats in vivo et in vitro désigne la contribution de la sénescence endothéliale comme une cause probable à la dysfonction de greffon. / This scientific work has tackled the question of the pancreatic islets aging and the effect of endothelial senescence and microparticles (MPs) on islet function. We investigated the impact of aging on pancreas morphology, fate and on the function of the pancreatic islet by comparative analysis between pancreas in young and middle-aged rats, as well as the role of pro-senescent endothelial MPs on islet function and their premature senescence. Our in vivo data show that the pancreas is an early sensitive organ to oxidative stress accumulating with age and leading to overexpression of the procoagulant and senescence markers without appearance of apoptosis. In vitro, MPs of senescent endothelial cells have a pro-senescent effect on pancreatic islets isolated from young rats with characteristic SA-β-galactosidase activity, overexpression of p53, p21 and p16 markers and reducing the ability of insulin secretion in response to glucose. Altogether, our in vivo and in vitro data indicate the contribution of endothelial senescence as a possible contributor to graft dysfunction.

<p>Type 1 diabetes is caused by immuno-mediated β-cell destruction leading to insulin deficiency and hyperglycaemia. The decline in β-cell function and the clinical course after diagnosis vary. Whether the process of destruction of the β-cells is associated with markers of a non-specific inflammatory response is unknown. The aims of these studies were to identify factors of importance for clinical remission (low insulin need and normoglycaemia) and long-term β-cell function and estimate the degree of non-inflammatory response in type 1 diabetes in young adults. Clinical remission and β-cell function eight years after diagnosis were assessed and related to clinical, biochemical and immunological variables at diagnosis, including islet autoantibodies [ICA, GADA, IA-2A]. Markers of low-grade inflammation in plasma [CRP and IL-6] were estimated and the concentrations were related β-cell function [plasma C-peptide], glycaemic control and autoimmunity at diagnosis and the first year thereafter. The results showed that clinical remission occurred in about half of the patients with newly diagnosed type 1 diabetes. Preserved β-cell function eight years after diagnosis was observed in 16% of the patients classified at diagnosis as having autoimmune type 1 diabetes. Duration of remission was dependent on BMI, degree of metabolic derangement and presence of GADA at diagnosis. BMI at diagnosis was also of importance for preserved β-cell function after eight years of the disease, as were the amount of islet antibodies and presence of ICA. Elevated CRP levels were noted in the majority of cases at diagnosis and both CRP and IL-6 concentrations were stable the first year after clinical diagnosis. High concentrations of CRP and IL-6 did not relate to β-cell destruction or the degree of autoimmunity. CRP concentrations were higher in islet antibody negative than in positive patients. CRP also correlated positively to BMI, C-peptide at 12 months and to increasing HbA1c between six and 12 months. In general, females had shorter remissions, lower concentrations of serum bicarbonate and higher levels and prevalence of GADA at diagnosis, compared to males. Females also had higher HbA1c and CRP values the first year after diagnosis. In summary, BMI at diagnosis is a strong predictor of duration of remission and preservation of β-cell function. Elevated CRP concentrations are correlated to factors linked rather to insulin resistance than to β-cell destruction. Females appear to have a more acute onset and a more severe course of the disease than males.</p>