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

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

Type II Diabetes Mellitus (T2DM) is a multifactorial metabolic disorder that affects millions of people worldwide. Although there is not one single cause for it, obesity currently remains the largest risk factor. Obesity is characterized by excessive body fat, or adipose tissue. Over time, hypertrophy of white adipose tissue (WAT) leads to the altered secretion of adipocyte cytokines and hormones, or “adipokines”. This is one proposed hypothesis for the development of insulin resistance and glucose intolerance in obese patients. Additionally, the expression of hormones involved in regulating satiety is altered under obese conditions, which also contributes to insulin resistance. This phenomenon of obesity induced diabetes is termed “diabesity”. While the precise molecular mechanism of diabesity remains unclear, it is evident that adipose tissue plays an important and major role in modulating glucose and lipid metabolism by functioning as an endocrine organ. Thus, obese patients with T2DM are encouraged to implement lifestyle changes such as dieting and exercising to reduce their body fat and to help improve their insulin sensitivity. Aside from this, there are other treatment options available that should also be considered. However, many of them have associated risks and undesirable effects such as weight gain. Therefore, T2DM patients should carefully weigh the risks and benefits of all treatment options before deciding on which ones are most suitable for them. The current clinical management of T2DM is a rapidly growing area of research, and as the prevalence of this metabolic disease continues to increase worldwide, the development of future treatment options is expected.

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Because excessive body weight is a major health issue, there is an urgent need to understand all physiological mechanisms relating to control of fat deposition/mobilization. Here we investigated the linkage between polyamine metabolism and fat homeostasis that we recently discovered to operate in mice. Our previous data show that the expression level of spermine/spermidine acetyltransferase (SSAT), a polyamine catabolic enzyme, potently modulates body fat content of mice. In particular, our data indicated that SSAT overexpressing mice (SSAT-Tg) have reduced acetyl CoA levels and are lean while SSAT null mice (SSAT-ko) are obese. Since the acetyl CoA/malonyl CoA levels are critical for control of free fatty acid synthesis and oxidation via malonyl CoA regulation of CPT-1 (carnitine palmitoyltransferase-1), we hypothesized that genetic manipulation of SSAT alters body fat accumulation by activating of AMP-activated protein kinase pathway and thus has a global effect on energy metabolism. To test this hypothesis, we performed a combination of proteomics and antibody based expression studies in white adipose tissue (WAT) of SSAT-ko, SSAT-wt and SSAT-tg: We identified 9 proteins in WAT that show an increasing gradient across SSAT-ko, SSAT-wt and SSAT-tg, all of which have a connection with acetyl-CoA consumption. These include: a) glycolytic enzymes (aldolase, enolase, pyruvate dehydrogenase); b) TCA cycle enzymes (aconitate hydratase, malate dehydrogenase); c) fatty acid lipolysis and beta oxidation enzymes (hormone-sensitive lipase, monoglyceride lipase, 3-hydroxyacyl CoA dehydrogenase). Additional expression studies using Western blots indicated that acetyl CoA regulates metabolism by AMP-activated protein kinase pathway. Furthermore, to determine how tissue-specific changes in SSAT expression will impact fat accumulation and the precise role of SSAT expression status in fat homeostasis and obesity, we generated adipose-specific SAT1 knockout (FSAT1KO) mice using the Cre-Lox method. On 27-week-old, FSAT1KO mice have higher average body weight than wild type mice (WT: 45.13 ± 2.23 g vs. FSAT1KO: 52.28 ± 1.62 g, p&lt;0.05) when fed a high-fat diet. Larger lipid droplets and lipid accumulation were present in FSAT1KO mouse livers compared to the control WT mice. Several proteins involved in fat metabolism were found to be up-regulated in FSAT1KO mice using GeLC-MS proteomics approach. These data indicated that the lack of SSAT activity in adipose tissue, but not liver or muscle, drives the phenotypic changes in SSAT-ko obese mice. Our interpretation of these results is that genetic modulation of SSAT causes a combination of changes in WAT that involve lipolysis, energy metabolism and calorie loss resulting from polyamine export. In summary, the data indicate that modulation of SSAT activity affects fat metabolism and calorie balance. / Biochemistry