京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22510号 / 農博第2414号 / 新制||農||1078(附属図書館) / 学位論文||R2||N5290(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 佐々木 努, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Bei der vorliegenden Arbeit handelt es sich um eine experimentelle Untersuchung zum Einfluss von zwei Ko-Transmittern des autonomen Nervensystems, Neuropeptid Y und dem Pituitary Adenylate Cyclase-activating Polypeptide (PACAP), auf den intrazellulären Kalziumspiegel und die Insulinsensitivität von 3T3-L1 Adipozyten. Mittels Polymerasekettenreaktion und Western Blot Analyse konnte die Expression des NPY-1 (Y1) Rezeptors als auch die der PACAP Rezeptoren PAC1 und VPAC2 nachgewiesen werden. Die Aktivierung des Y1 oder des PAC1 Rezeptors durch ihre Agonisten führte zur Erhöhung des intrazellulären Kalziumspiegels. Im Weiteren führte NPY nach Ko-Applikation mit Insulin zu einer abgeschwächten Insulinsensitivität der Adipozyten, da sowohl die insulin-stimulierte Translokation von Glukosetransporter 4 zur Zelloberfläche als auch die Glukoseaufnahme durch NPY abgeschwächt wurde. Dieser Effekt konnte als Y1 spezifisch beschrieben werden. Diese Ergebnisse gewähren somit neue Einblicke über den peptidergen Einfluss auf den Adipozytenstoffwechsel und erlauben Rückschlüsse über die Rolle des autonomen Nervensystems in der Entwicklung von Adipositas und Diabetes mellitus Typ 2.

For many years, adipose tissue (AT) was thought to be a tissue primarily responsible for cushioning and insulating organs. However, significant advances in knowledge have shown that AT is necessary for maintaining an optimal metabolic balance through paracrine and endocrine mechanisms. Because AT dysfunction is related with illnesses such as obesity and diabetes, it is vital to understand the mechanisms behind these pathologies to restore metabolic health. Beige AT is a unique form of fat that generates heat through uncoupling protein 1 (UCP1), has a dense neurovascular network, and is associated with enhanced metabolic health. Hence, particular emphasis has been made on unraveling the processes behind thermogenic activation and maintenance, as increasing thermogenic activity offers considerable potential for treating metabolic disorders. Activation of beige AT is dependent on norepinephrine release from sympathetic neurons upon physiological cues such as cold exposure. Studies have revealed a major role of monoamine oxidase a (MAOA)-mediated norepinephrine clearance in the maintenance of thermogenic AT. However, major limitations are still present with regards to the mechanisms of neurotransmitter clearance and their role in thermogenic regulation. The initial objective of this thesis is to evaluate the effect of human white and thermogenic adipocytes on the formation of a neurovascular network in order to maintain thermogenesis and whether MAOA plays a direct role in thermogenic control. We demonstrate that implanted human thermogenic adipocytes generate a more vascularized and innervated AT than non-thermogenic adipocytes. Additional findings revealed that MAOA is expressed in human adipocytes and that inhibiting MAOA promotes thermogenesis. The second objective of this thesis is to determine if hAdipoGel (hAG) - a decellularized AT matrix – enhances mesenchymal stem cell (MSC) proliferation and differentiation, as well as human adipocyte engraftment in vivo. We show that MSC can proliferate in hAG and differentiate effectively into white and thermogenic adipocytes. Additionally, when white adipocytes are implanted with hAG, they differentiate into a fully functioning fat graft capable of integrating with the host. Understanding the thermogenic processes of human AT, in combination with the use of a suitable decellularized matrix, can aid in the development of therapeutic treatments that boost thermogenic activity and hence metabolic health.

Insulin stimulates glucose uptake in adipose and muscle cells via translocation of the intracellular vesicles containing GLUT4. It was largely unknown whether and/or how the signaling molecules such as PI 3-kinase and Akt regulate the mechanical movements of the GLUT4-containing vesicles. Hence, this study was performed to test the hypothesis that actin and microtubules function in translocating GLUT4 vesicles. Treatments of insulin as well as endothelin-1 (ET-1), an insulin-mimicking peptide which does not act through PI 3-kinase, induced polymerization of actin without affecting the microtubular network. By mass spectrometry, the tyrosine kinase PYK2 was identified to be tyrosine phosphorylated specifically by ET-1 but not by insulin. Expression of the carboxyl-terminal fragment (CRNK) PYK2, but not wild type nor kinase-deficient PYK2 mutants, inhibited ET-1-stimulated actin polymerization while expression of all three PYK2 constructs had no effect on insulin-stimulated actin polymerization. More importantly, expression of CRNK, but not wild type nor kinase-deficient PYK2 constructs, blocked ET-1- but not insulin-stimulated GLUT4 translocation to the plasma membrane. These suggest that ET-1 and insulin stimulate actin polymerization via distinct signaling pathways, and that the actin polymerization is required for GLUT4 vesicle translocation. In order to test the possible involvement of microtubule in GLUT4 vesicle translocation, time lapse imaging of 3T3-L1 adipocytes expressing GLUT4-YFP and tubulin-CFP was performed. GLUT4-YFP vesicles move long-range bi-directionally on microtubules, which suggests the presence of molecular motors on the vesicles. Moreover, insulin increased the number of vesicle movements on microtubules without changing the velocities. Interestingly, the stimulatory action of insulin appears to be independent of PI 3-kinase activation. Conventional kinesin was identified as a highly expressed kinesin isotype in adipocytes. Notably, expression of dominant negative mutants but not wild type kinesin inhibited insulin-stimulated long-range GLUT4 vesicle movements and GLUT4 translocation to the plasma membrane in live and fixed cells, respectively. These data indicate that insulin signaling induces the movement of GLUT4 vesicles on microtubule which is mediated by conventional kinesin. Overall, the data presented here provide evidence supporting the hypothesis that actin and microtubule cytoskeletons are required for insulin to mobilize GLUT4 vesicles in adipocytes.

Obesity has in recent times become a more serious health issue and was estimated to affect over 650 million people world-wide in 2016. Furthermore, the list of obesity-associated diseases is countless, many of which have severe consequences. Type 2 diabetes (T2D) is such a disease, and it was estimated to be over 1.5 million new cases in America alone in 2015. It is thought that insulin resistance development which causes T2D is associated with a low-level chronic inflammation in the adipose tissue. The inflammatory state is caused by the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) which is secreted by macrophages. To further understand the complexity of the underlying mechanisms of both the adipocytes as well as the macrophages, mathematical models are being developed in the fields of systems biology. However, as of now, no mathematical model has been developed which can explain the association between chronic inflammation and the development of insulin resistance. Because of this, a first model will be presented which is able to describe the mechanisms of insulin resistance development caused by chronic inflammation. The model was fitted to data from intraperitoneal glucose tolerance test in mice and yielded a cost below the threshold of chi-square test, which suggests that the model cannot be rejected. Furthermore, the model was expanded, introducing more complexity in the intracellular cascade reaction of an activated macrophage. Once again, the model was fitted to the same data and yielded a cost below the threshold of chi-square test. Uncertainty tests were made to further validate the models and showed a low uncertainty for both models. These results increase the understanding regarding the association between adipocytes and macrophages, in the role of insulin resistance caused by chronic inflammation. This increased knowledge can help, for instance, in the development of new drugs which are able to prevent the development of insulin resistance and T2D.

No description available.

Obesity is an urgent global public health concern as prevalence rates continue to increase, especially among children. At the cellular level obesity is defined by an increase in adipocyte number (hyperplasia) and size (hypertrophy). Both lead to the dysfunction of adipose tissue, which has been identified as the link between obesity and chronic disease. Bioactive compounds, naturally occurring in fruits and vegetables, hold enormous potential in regulating adipocyte biology. Quercetin, the most commonly consumed dietary flavonoid, is a strong potential anti-obesity agent that has been implicated as an AMP-activated protein kinase (AMPK) activator and shown to ameliorate symptoms of metabolic syndrome in vivo. Here we investigated quercetin’s effect on (1) adipogenesis, the process of increasing adipocyte number, and (2) metabolism of mature adipocytes. In 3T3-L1 preadipocytes, quercetin dose-dependently inhibited adipogenesis, as evidenced by decreased lipid accumulation and expression of adipogenic markers such as peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/ enhancer binding protein (C/EBP) α, adipocyte fatty acid binding protein 2 (aP2), and acetyl-CoA carboxylase (ACC) on mRNA and protein levels. This inhibitory effect was limited to the early stages of adipogenesis (0-36 hours), and quercetin treatment altered the normal expression pattern of cell cycle related genes Cyclin A and p27, indicating quercetin may inhibit adipogenesis through cell cycle events. We next investigated quercetin’s ability to activate AMPK and the metabolic pathways related to AMPK activation: lipolysis and b-oxidation. Quercetin increased phosphorylation of AMPK and its downstream target ACC. Further, quercetin treatment (100μM) increased free fatty acid content in the media through an AMPK-dependent mechanism. Quercetin up-regulated mRNA expression of uncoupling proteins 3 (UCP3) and peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1a), indicating that quercetin may induce mitochondrial oxidative pathways, also through an AMPK-dependent pathway. These findings suggest (1) quercetin inhibits adipogenesis through the regulation of early cell cycle events required for adipogenic differentiation, and (2) quercetin’s activation of AMPK induces lipolytic and oxidative pathways. Taken together, quercetin could be further developed as an anti-obesity agent because of its potential to inhibit both hyperplasia and hypertrophy in vitro.

No description available.

No description available.

Chronic venous insufficiency (CVI) is characterized by valve dysfunction and venous hypertension, leading to erythrocytes’ extravasation into the tissue over time. CVI patients present dermal manifestations as; hyperpigmentation of the leg due to iron accumulation, histological changes regarding the dermal layer and fat (lipodermatosclerosis), and a high risk of developing a leg ulcer. For decades, researchers have studied CVI, and chronic venous ulcer (CVU) and iron have been considered critical pathological factors in this context, especially concerning oxidative stress and ROS formation. However, a clear understanding of the pathogenic effects of iron on the tissue network and the cross-talk of resident immune and skin tissue cells in the course of CVI is still missing. Therefore, in this project, we aim to investigate the pathological effect of iron overload on the cross-talk of resident immune and tissue cells in the skin. In the current thesis work, biopsies from CVI patients were analysed. Immunohistochemistry (IHC) and spectrometric assay confirmed a massive iron accumulation in their dermis and hypodermis. To dissect the different skin cells’ responses to iron overload, in vitro techniques were exploited. Mimic the erythrocyte overload in a macrophage (M2-like) culture revealed a shift in the gene signature towards inflammatory activation states of these cells. Hence, cytokine analysis confirmed an evident pro-inflammatory activation of macrophages (Ma). A mouse model with skin iron overload was generated to investigate the effect of iron overload in a more complex picture. Here, it was confirmed that iron induces an expansion of immune cells and a pro-inflammatory activation in the skin with a shift in resident macrophage subtypes, which was coupled to the adipose layer. Indeed, the dWAT of these mice shirked and showed clear signs of lipolysis. Moreover, IHC and IF staining of iron-mice skin showed increased cellularity of the lower dermis, which was linked to an expansion of the fibroblast population (dermis and stromal vascular fraction of the dWAT). Consistent with in vitro data, ECM genes were downregulated in the dermis, which may explain changes in the skin architecture of these mice. In this thesis, the newly established mouse model made it possible to understand how iron may affect skin cells in CVI patients and can be extremely useful for future research to develop a new therapeutic approach. This work wants to highlight the significance of iron overload in the skin, which affects cellular cross-talk, altering skin homeostasis and possibly leading to an ulcer.