京都大学 / 新制・課程博士 / 博士(医学) / 甲第24519号 / 医博第4961号 / 新制||医||1065(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 辻川 明孝, 教授 大鶴 繁 / 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・課程博士 / 博士(医学) / 甲第24200号 / 医博第4894号 / 新制||医||1061(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 長船 健二, 教授 中本 裕士, 教授 江木 盛時 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

PI3K signaling in pancreatic β cells has been shown to be important in modulating β cell mass and function under basal condition. Evidence suggests that a specific group of insulin promoter-expressing neurons also modulates glucose metabolism and energy homeostasis through their PI3K signaling. Thus we hypothesize that PI3K activation via PTEN deletion under the control of rat insulin promoter (RIP) in pancreatic β cells and RIP-expressing neurons will protect against hyperglycemia and diabetes in experimentally induced mouse models of type 2 diabetes. In Chapter IV, we showed that RIP-mediated PTEN deletion in pancreatic β cells led to PI3K activation and subsequent increased β cell mass and function, thus protected the mice from high fat diet (HFD)-induced diabetes. Furthermore in the absence of global leptin signaling, β cell-specific PTEN deletion maintained β cell function in the setting of severe insulin resistance, therefore prevented diabetes development. Interestingly, RIP-mediated PTEN deletion also resulted in increased peripheral insulin sensitivity due to PI3K activation in central nervous system. In Chapter V, we showed this increased insulin sensitivity was maintained after HFD feeding, which also contributed to the protection against diabetes. These mice also showed increased visceral adipogenesis and subcutaneous adiposity on HFD, which were dramatically attenuated in the absence of leptin signaling, indicated the essential role of peripheral leptin action in mediating the insulin sensitive phenotype from neuronal RIP PTEN deletion. Finally, we demonstrated that the insulin sensitizing phenotype in these mice was not mediated through ventromedial hypothalamic nuclei (VMH), such that VMH-specific PTEN deletion did not alter energy homeostasis or glucose metabolism. Together, the data from this thesis points to an inhibitory role of PTEN in both central nervous system and pancreatic β cells in glycemic control. Therefore, PTEN may represent a potential target for diabetes prevention and treatment.

PI3K signaling in pancreatic β cells has been shown to be important in modulating β cell mass and function under basal condition. Evidence suggests that a specific group of insulin promoter-expressing neurons also modulates glucose metabolism and energy homeostasis through their PI3K signaling. Thus we hypothesize that PI3K activation via PTEN deletion under the control of rat insulin promoter (RIP) in pancreatic β cells and RIP-expressing neurons will protect against hyperglycemia and diabetes in experimentally induced mouse models of type 2 diabetes. In Chapter IV, we showed that RIP-mediated PTEN deletion in pancreatic β cells led to PI3K activation and subsequent increased β cell mass and function, thus protected the mice from high fat diet (HFD)-induced diabetes. Furthermore in the absence of global leptin signaling, β cell-specific PTEN deletion maintained β cell function in the setting of severe insulin resistance, therefore prevented diabetes development. Interestingly, RIP-mediated PTEN deletion also resulted in increased peripheral insulin sensitivity due to PI3K activation in central nervous system. In Chapter V, we showed this increased insulin sensitivity was maintained after HFD feeding, which also contributed to the protection against diabetes. These mice also showed increased visceral adipogenesis and subcutaneous adiposity on HFD, which were dramatically attenuated in the absence of leptin signaling, indicated the essential role of peripheral leptin action in mediating the insulin sensitive phenotype from neuronal RIP PTEN deletion. Finally, we demonstrated that the insulin sensitizing phenotype in these mice was not mediated through ventromedial hypothalamic nuclei (VMH), such that VMH-specific PTEN deletion did not alter energy homeostasis or glucose metabolism. Together, the data from this thesis points to an inhibitory role of PTEN in both central nervous system and pancreatic β cells in glycemic control. Therefore, PTEN may represent a potential target for diabetes prevention and treatment.

Type 1 and 2 Diabetes Mellitus are a growing health problem throughout the world. There is an increasing  need for methodologies, which are both reliable and non-invasive to measure the amount of insulin-producing tissue (Beta-cell mass, or BCM), as well as rapidly quantify changes in the BCM due to the onset of disease, beta-cell replacement therapy, or other treatments. Positron Emission Tomography (PET) is a non-invasive, quantitative functional imaging technique which can be used to study dynamical or static processes inside the body. In this thesis, we present a study protocol for in vivo imaging of the most common form of beta- cell replacement therapy; islet transplantation. Islets were labeled with the PET tracer, 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG), and administered intra-portally, while the recipient was monitored by PET/CT. The hepatic distribution of the islets was highly heterogeneous, and around 25% (human) or 50% (porcine) of the administered islets could not be found in the liver after completed transplantation, confirming previous reports of considerable cell injury during the procedure leading to low hepatic engraftment. Native BCM in the pancreas can potentially be quantified using a PET tracer with sufficiently high specificity, but the major obstacle is the relative low amounts of insulin producing tissue (only 1-2% of the pancreatic volume). Two tetrabenazine analogues, [18F]FE-(+)-DTBZ and [18F]FE-(+)-DTBZ-d4, are ligands to VMAT2, which is expressed in islet tissue. Both analogues were investigated and characterized as potential BCM imaging agents both in vitro and in vivo.  Both tracers exhibited high preferential binding to islet tissue compared to exocrine pancreatic tissue. However, the specificity was not high enough to overcome the obscuring exocrine signal in vivo (7-10% of the signal originating from specific islet tracer uptake). This thesis demonstrates that it is possible to quantitatively assess islet transplantation by PET imaging. In vivo determination of native pancreatic BCM is, in theory, possible with both [18F]FE-(+)-DTBZ and [18F]FE-(+)-DTBZ-d4, but tracer analogues with higher islet specificity is needed for quantification of smaller BCM changes with physiological impact.

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 gastrointestinal tract comprises a number of digestive organs including the stomach and pancreas. The stomach is involved in the digestion and short term storage of food while the pancreas is a mixed endocrine and exocrine gland which provides the body with hormones and enzymes essential for nutritional utilisation. The pancreas consists of three different cell lineages, acinar, ductal and endocrine cells. The endocrine cells, organised in the islets of Langerhans, are scattered throughout the exocrine parenchyma and regulate blood glucose levels by production of hormones such as glucagon and insulin. The Nkx family of homeodomain proteins controls numerous processes during development. Previous studies have identified two members belonging to the Nkx6 subfamily of Nkx proteins, Nkx6.1 and Nkx6.2. We have described the cloning and embryonic expression pattern of Nkx6.3. All three members of the Nkx6 gene family were shown to be expressed in partially overlapping domains during the development of the gastrointestinal tract and the central nervous system. Nkx6.2 was also identified as a transient marker for pancreatic exocrine cells. Analysing gene expression patterns and morphological features in tissues and organs is often performed by stereologic sampling which is a labour-intensive two dimensional approach that rely on certain assumptions when calculating e.g. β-cell mass and islet number in the pancreas. By combined improvements in immunohistochemical protocols, computational processing and tomographic scanning, we have developed a methodology based on optical projection tomography (OPT) allowing for 3D visualisation and quantification of specifically labelled objects within intact adult mouse organs. In the pancreas, this technique allows for spatial and quantitative measurements of total islet number and β-cell mass. We have further developed a protocol allowing for high resolution regional analyses based on global OPT assessments of the pancreatic constitution. This methodology is likely to facilitate detailed cellular and molecular analysis of user defined regions of interest in the pancreas, at the same time providing information on the overall disease state of the gland. Type 1 diabetes mellitus (T1D) can occur at any age and is characterized by the marked inability of the pancreas to secrete insulin due to an autoimmune destruction of the insulin producing β-cells. Information on the key cellular and molecular events underlying the recruitment of lymphocytes, their infiltration of the islets of Langerhans and consequent β-cell destruction is essential for understanding the pathogenesis of T1D. Using the developed methodology we have recorded the spatial and quantitative distribution of islet β-cells and infiltrating lymphocytes in the non obese diabetic (NOD) mouse model for T1D. This study shows that the smaller islets, which are predominantly organised in the periphery of the organ, are the first to disappear during the progression of T1D. The larger islets appear more resistant and our data suggest that a compensatory proliferative process is going on side by side with the autoimmune-induced β-cell destruction. Further, the formation of structures resembling tertiary lymphoid organs (TLOs) in areas apparently unaffected by insulitis suggests that local factors may provide cues for the homing of these lymphocytes back to the pancreas.

Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet β cells. However, the role and sequence of β cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of β cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, β cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained β cell volume further declines. These results indicate that dysfunction of persisting β cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.