In the past 30 years the prevalence of obesity has almost trebled resulting in an increased incidence of type 2 diabetes mellitus (T2DM) and other co-morbidities. Visceral adipose tissue is believed to play a vital role in these conditions, but underlying mechanisms remain unclear. A close association exists between obesity, diabetes and oxidative stress, resulting in increased reactive oxygen species formation. The experiments in this thesis address this by searching for possible biochemical changes which may be specific for the onset of obesity related T2DM, as well as looking for genetic alterations at molecular and gene expression levels. This thesis also explored various techniques such as polymerase chain reaction (PCR), colorimetric assays and real-time RT-PCR. The aim was to investigate the role of adipose tissue in obesity and T2DM, focusing on markers of oxidative stress and gene expression in human visceral adipose tissue from subjects categorised as lean, obese and obese with T2DM. This cross-sectional study measured two markers of oxidative stress, two markers of DNA damage, gene expression analysis and identification of genes associated with T2DM and obesity. Specific gene sequencing was carried out on the glutathione reductase gene to determine possible gene variants. Results showed a paradoxical decrease in adipose markers of oxidative stress in subjects with obesity and T2DM. There appeared to be a protective mechanism in these subjects, displaying reduced levels of oxidative stress compared to other groups. This could be due to a significant proportion of these subjects being on ACE inhibitor and statin therapy, which may be confounding results and minimising the effects of the oxidative burden. Additionally, the same subjects showed an increased expression of the glutathione reductase gene. It is difficult to conclude if the decreased levels of oxidative stress in these subjects were a result of the increased glutathione reductase expression in the visceral adipose tissue or if there remains an unseen factor influencing the dramatic expression change seen in this group of subjects. No glutathione reductase gene variants were identified in these samples. This analysis highlighted that within this sample set, the impact of oxidative stress is in fact reversible as the antioxidant capacity in these subjects is evident, and in combination with correct drug therapy it may be possible to combat oxidative burden and reduce the subsequent damage inflicted upon the cells.

Adipose tissue (AT) expands through hypertrophy and hyperplasia. Hyperplasic AT expansion requires an adequate number of adipose progenitor cells. This study investigates the influence of depot origin on the susceptibility of adipose progenitors to cell death, and measures the effect of macrophage-secreted factors on adipose progenitor survival. Using serum deprivation alone or in the presence of TNFα, omental (OM) versus subcutaneous (SC) adipose progenitors, obtained from human AT, displayed a 3- and 1.7-fold-increase in apoptosis, respectively, as assessed by Hoechst staining, (p<0.05). Similar results were observed with cell enumeration. The ratio of OM/SC cell death from serum deprivation positively correlated with body mass index (BMI). The depot-specific difference in cell death was lost when TNFα and cycloheximide (CHX) were used. Monocyte-derived macrophages (MD-macrophages), isolated from human blood, did not have an effect on apoptosis. Depot-related differences in adipose progenitor apoptosis may influence AT remodeling and alter metabolic functionality in obesity.

Lors de l’obésité, les défauts d’expansion du tissu adipeux blanc sont à l’origine des désordres métaboliques. Lorsque les adipocytes atteignent leurs capacités maximales de stockage de triglycérides, des dépôts ectopiques de lipides apparaissent dans le foie et le muscle conduisant à la résistance à l’insuline. De plus, les adipocytes dysfonctionnels sécrètent des facteurs d’alertes établissant une inflammation dans le tissu adipeux. Cette inflammation est médiée par les communications entre les adipocytes et les cellules immunitaires qui sont contrôlées par l’endocytose et le trafic intracellulaire. L’endocytose et les protéines Rab gouvernant ce processus pourraient être des éléments clés de l’expansion du tissu adipeux. L’équipe a démontré que l’expression de Rab4b était diminuée dans le tissu adipeux de patients obèses diabétiques et de souris obèses. Nos travaux montrent que l’expression de Rab4b est diminuée dans les lymphocytes T du tissu adipeux de souris et de patients obèses. L’invalidation de Rab4b dans les lymphocytes T in vivo induit une résistance à l’insuline et une accumulation d’acides gras dans le foie et dans le muscle sous régime normal. Ces défauts sont dus à une inhibition de l’adipogenèse par l’IL-6 et l’IL-17, limitant l’expansion du tissu adipeux. L’augmentation de ces cytokines pro-inflammatoires est une conséquence de l’augmentation du nombre de lymphocytes Th17 et une diminution des lymphocytes T régulateurs. Nous décrivons un nouveau mécanisme par lequel l’expression de Rab4b dans les lymphocytes T régule les complications métaboliques de l’obésité en changeant les sous-populations de cellules immunitaires dans le tissu adipeux. / Expendability defect of adipose tissue during obesity is at the basis of obesity-related metabolic complications. Indeed, when adipocytes reach their maximal triglyceride storage capacity, ectopic lipid depots are appearing in liver and muscles, leading to insulin resistance. Moreover, dysfunctional adipocytes secrete alarming factors leading to adipose tissue inflammation. This inflammation is sustained by adipocytes and immune cells communications that are controlled by endocytosis and intracellular trafficking. Endocytosis and its governing proteins, the Rab GTPases, could be pivotal in the regulation of adipose tissue expandability. Our team has demonstrated that Rab4b expression is reduced in obese diabetic patients and mice adipose tissues. The present work demonstrates that Rab4b is decreased in adipose tissue T cells in both obese patient and mice. The depletion of Rab4b in T cells in vivo leads to insulin resistance and lipid accumulation in liver and muscles under normal diet. These defects are due to adipogenesis inhibition by IL-6 and IL-17, which limits adipose tissue expansion. These pro-inflammatory cytokines are increased in adipose tissue of the mice depleted for Rab4b in T cells because the number of Th17 is increased at the expense of the number of regulatory T cells. We describe here a new mechanism in which Rab4b expression in T cells control obesity-related metabolic complications by tuning T cells subpopulations in adipose tissue.

archives@tulane.edu / Hydrogels serve as three-dimensional scaffolds whose composition can be customized to allow the attachment and proliferation of several different cell types. Decellularized tissue-derived scaffolds are considered close replicates of the tissue microenvironment. Decellularized adipose tissue (DAT) hydrogel has proven to be a useful tool for tissue engineering applications in pre-clinical models. The first aim of the present study was to characterize the biochemical composition of DAT hydrogel. The DAT hydrogel was prepared by processing adipose tissue acquired from three female human donors, and subsequently quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS). The enriched and depleted proteins were determined in DAT hydrogel and further analyzed by gene ontology (GO) analysis. Extracellular matrix proteins were found to be enriched, while cellular proteins were depleted relative to native adipose tissue. Furthermore, GO analysis identified that the enriched proteins could affect various biological processes via the regulation of a range of cellular pathways. The second aim was focused on the analysis of the effect of adipose-derived stromal/stem cells (ASCs) and DAT hydrogel interaction on cell morphology, proliferation, differentiation, and hydrogel microstructure. The ASCs seeded in DAT hydrogel remained viable and displayed proliferation. The adipogenic and osteogenic differentiation of ASCs seeded in DAT hydrogel was confirmed by marker gene expression and histochemical staining. Moreover, ASC attachment and differentiation altered the fibril arrangement, which indicated remodeling of the DAT hydrogel. The third aim was to analyze the regenerative potential of DAT hydrogel in a critical-sized mouse femoral defect model. The DAT hydrogel alone, or its composites with ASCs, osteo-induced ASCs (OIASC), and hydroxyapatite were tested for the ability to mediate repair of the femoral defect. The data indicated that DAT hydrogel promoted bone regeneration alone, while the regeneration was enhanced in the presence of OIASCs and hydroxyapatite. In summary, the current findings confirm that DAT hydrogel is a cytocompatible and bio-active scaffold, with potential utility as an off-the-shelf product for tissue engineering applications. In future, the analysis of DAT hydrogel using a wider range of donors representing different body mass index, age, gender, and ethnicity will provide a more comprehensive characterization. / 0 / Omair A. Mohiuddin

Obesity is a major global health concern due to its steadily increasing rates and significant contribution to numerous diseases, including cancer. Ovarian cancer specifically, is associated with a 30% increased risk with obesity, although the mechanisms for this are unknown. Waist-to-hip ratio has been especially associated with ovarian cancer, suggesting that visceral fat may be the greatest contributor. Here, we investigated individual visceral fat depots as independent contributors to cancer progression, specifically focusing on adipose tissue-derived stem and progenitor cells, which have previously been shown to be recruited by cancer cells and participate in cancer progression. We confirmed that ovarian cancer tumor burden was indeed significantly increased in mice on a high fat as compared to low fat diet. To further investigate mechanisms, we examined changes in progenitor populations that occurred in intra-abdominal parametrial (pmWAT), retroperitoneal (rpWAT), and omental (omWAT) white adipose tissue (WAT) depots with cancer presence. The greatest tumor burden was evident in omWAT, which also displayed an increase in CD45- cells but a decrease in adipose progenitor cells (APC) and endothelial progenitor cells, suggesting that there was an increase in stromal cells, but that the stem cells were pushed towards differentiation. PmWAT and rpWAT showed remarkably stable progenitor populations. However, a tumor from pmWAT had a significant presence of CD45- cells, actually matching that of its surrounding tissue and differing from the omWAT tumors, indicating that microenvironment has a major influence on tumor stromal cells. We also found that with high fat diet, many cancer-associated changes were exacerbated, such as an increased inflammatory response in all tissues and further decreases in APCs in omWAT. In vitro studies further confirmed that ovarian cancer cells and SVF cells were able to directly interact. Additionally, SVF cells were able to increase the proliferation, mobility, and invasiveness of cancer cells. Conversely, co-culturing also enhanced the proliferation and mobility of SVF cells, providing further evidence that SVF cells may be recruited by cancer cells and that their relationship may be bilateral. Thus, this study provides a good foundation for examining the cellular contributions of adipose tissue to cancer. By further characterizing the mechanism for the association between obesity and cancer development, we could find novel targets to decrease the progress of cancer development in at-risk obese individuals. / Ph. D.

Hypothalamus and adipose tissue are essential central and peripheral sites regulating energy homeostasis. Disruption of energy homeostasis can lead to diseases like anorexia and obesity in humans and reduced productivity in animals. Therefore, integrating knowledge in hypothalamic appetite regulation and adipose tissue metabolism is essential to maintain homeostasis. The aim of this dissertation was to elucidate molecular and cellular mechanisms of energy homeostasis in birds. We determined adipose tissue physiological changes during the first two weeks post-hatch in chickens from lines selected for low (LWS) and high (HWS) body weight. LWS was more dependent on yolk and subcutaneous fat mobilization for growth from hatch to day 4 post-hatch, with hyperplasia-predominated replenishment of the reservoir. In contrast, HWS was more dependent on feed for growth and maintained depot mass through hyperplasia and hypertrophy. From day 4 to 14 post-hatch, compared to maintenance of depot weight and adipocyte size in LWS, HWS accumulated clavicular and abdominal fat with minimal lipolysis. There was greater expression of precursor and proliferation markers in LWS with more apoptotic cells in the abdominal stromal vascular fraction on day 14 post-hatch, suggesting that apoptosis contributed to lower adipogenic potential and lack of abdominal fat in LWS. Exposure to thermal and nutritional stressors at hatch impaired growth by reducing yolk utilization and lowering body weight, lean and fat masses in LWS. Stress exposure resulted in increased global DNA methylation and DNA methyltransferase activity in the arcuate nucleus of the hypothalamus in LWS. Moreover, there was decreased binding to methyl-CpG-binding domain protein 2 in the promoter of corticotropin-releasing factor (CRF) because of hypomethylation in one CpG site at its core binding site in stressed LWS, which explains the increased CRF expression in the paraventricular nucleus of the hypothalamus. We next determined effects of nutritional status on adipose tissue physiology in Japanese quail, a less-intensively selected avian species. Six-hour fasting promoted lipolysis and gene expression changes in 7-day old quail with some changes restored to original levels within 1 hour of refeeding. Overall, our results reveal novel cellular and molecular mechanisms regulating appetite and adiposity in birds early post-hatch. / Doctor of Philosophy / Hypothalamus and adipose tissue are essential for regulating energy homeostasis in central and peripheral body sites, respectively. Disruption of energy homeostasis can lead to diseases like anorexia and obesity in humans and reduced productivity in animals. Therefore, integrating knowledge in hypothalamic appetite regulation and adipose tissue metabolism is essential to maintain energy homeostasis in both humans and animals. The aim of this dissertation was to elucidate molecular and cellular mechanisms of energy homeostasis in birds. We first determined adipose tissue physiological changes in chickens during the first two weeks post-hatch from lines selected for low (LWS) and high (HWS) body weight. These chickens have been selected for juvenile body weight for over 60 generations. The LWS are lean and anorexic, while HWS eat compulsively and develop obesity and metabolic syndrome. Such characteristics make the body weight line chickens good animal models to study physiological changes under anorexia and obesity. We found that LWS was more dependent on yolk reserves and subcutaneous fat mobilization for growth from hatch to day 4 post-hatch, with replenishment of the fat reservoir by increases in cell number. By contrast, HWS was more dependent on feed for growth and maintained depot mass through increased cell number and cell size. From day 4 to 14 post-hatch, HWS accumulated fat throughout the body, with less fat breakdown as compared to LWS. There was greater expression of cellular precursor and proliferation markers in LWS, with more dying cells in their abdominal fat on day 14 post-hatch, suggesting that programmed cell death is responsible for the lack of fat cell development in LWS. Exposure to thermal and nutritional stressors at hatch impaired growth by reducing yolk utilization and lowering body weight, lean and fat masses in LWS. There were many molecular changes in the hypothalamus, including changes in DNA that led to increased activation of corticotropin-releasing factor (CRF), a signaling molecule that is known to regulate the body's stress and appetite responses. Stress exposure increased global DNA methylation and DNA methyltransferase activity in the arcuate nucleus of the hypothalamus in LWS. Moreover, there was less methylation at the core binding site of methyl-CpG-binding domain protein 2 (MBD2), a protein that binds to methylated DNA to repress gene expression, in the CRF gene, in stressed LWS. In response to stress, there was decreased binding of MBD2 to the promoter region of CRF, which may explain increased expression of CRF in the paraventricular nucleus of LWS. These results demonstrate that early-life stressful events can cause epigenetic changes (like DNA methylation) that lead to alterations in physiology and behavior that persist to later in life. We next determined effects of nutritional status on adipose tissue physiology in Japanese quail, which have undergone less artificial selection than chickens and are more representative of a wilder-type bird. Six-hour fasting promoted lipolysis and gene expression changes in 7-day old quail with some changes restored to original levels within 1 hour of refeeding. Overall, our results provide novel perspectives on cellular and molecular mechanisms regulating appetite and adiposity in birds during early post-hatch development.

Background. Bisphenol A (BPA) and BPS are environmental pollutants that are associated with the development of insulin resistance and type 2 diabetes (T2D). Although skeletal muscle and adipose tissue dysfunction are involved the development of insulin resistance, there are few studies that have investigated the effects of bisphenols on their metabolism. In this study, we investigated the effects of BPA and BPS exposure on skeletal muscle and adipose tissue metabolism to determine how they contribute to the development of T2D. Methods. L6 muscle cells were treated with BPA during the last 24 hours of differentiation, and mitochondrial function and glucose metabolism was measured. Human subcutaneous adipose tissue was incubated for 24 or 72 hours with BPA or BPS, and adipokine gene expression and glucose metabolism was measured in adipose tissue. Results. L6 muscle cells treated with high concentrations of BPA (10⁵ nM) had mitochondrial dysfunction and a compensatory increase in glucose metabolism; however, there were no effects at environmentally-relevant concentrations. Adipose tissue treated with BPA for 24 hours had reduced expression of proinflammatory cytokines and adipokines, and reduced insulin-stimulated glucose uptake. Conclusions. BPA exposure for 24 hours did not alter L6 muscle cell mitochondrial function and glucose metabolism at environmentally-relevant concentrations; however, adipose tissue had altered proinflammatory expression and glucose metabolism at low concentrations. This has important implications in regulatory guidelines in the use of BPA in the manufacturing of consumer products.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21639号 / 医博第4445号 / 新制||医||1034(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 濵﨑 洋子, 教授 小池 薫, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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