Orientador: Dora Maria Grassi-Kassisse / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T06:56:08Z (GMT). No. of bitstreams: 1 Crege_DaniloRobertoXavierdeOliveira_M.pdf: 755904 bytes, checksum: b0b863508794d698b250441e9a8d3d1b (MD5) Previous issue date: 2007 / Resumo: O estudo da célula adiposa como entidade funcional do tecido adiposo iniciou-se quando Rodbell em 1964 descreveu a técnica de isolamento de adipócitos de tecido adiposo de ratos e apresentou os efeitos de hormônios no metabolismo de glicose e na lipólise destas células. A partir de então, numerosos são os artigos descrevendo a quantificação de diferentes receptores, bem como, os mensageiros envolvidos com a ativação dos mesmos em célula adiposa. A maioria dos estudos é baseada nesta primeira descrição de Rodbell, entretanto, estes apresentam algumas modificações. Para ensaios provenientes de adipócitos de humanos, a citação também se refere a Rodbell com modificações. O estudo do funcionamento da célula adiposa nas diferentes espécies é emergente, devido à presença na população ocidental da obesidade como pandemia. Além disto, o ensaio de adipócitos isolados de tecido adiposo humano é uma importante ferramenta para estudos farmacológicos de novos fármacos...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The study of adipose cell as a functional unit of the adipose tissue, began when Rodbell in 1964 described the adipocytes isolation procedure of rat adipose tissue, and showed the effects of hormones in the metabolism of glucose and in lipolysis of these cells. Since that, a great number of researches describing the number of adrenoceptors as well the molecular mechanisms involved with the activation of these receptors has been published. Almost all studies use this first description of Rodbell, however with some modifications. The isolation procedure to human adipocytes also refer the Rodbell's original procedure with modifications. The functional study of adipose tissue in different species is emerging due the presence of obesity as a pandemic problem in occidental population. Beside this, the isolation of adipocytes from the adipose tissue is an important tool to new pharmacological studies of novel drugs...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

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Adipose tissue is a key metabolic organ responsible for maintaining energy homeostasis throughout the body. Healthy adipocytes respond to physiological changes and perform a variety of important functions to regulate glucose and lipid metabolism. Dysregulation of adipose tissue function, on the other hand, is strongly associated with the development of metabolic diseases. Peroxisome Proliferator Activated Receptor gamma (PPARγ) is a key transcription factor that regulates various activities in adipocytes as well as other cell types. A growing body of evidence indicates a more complex role for PPARγ beyond its classical ligand-dependent activity, including the exploration of posttranslational modifications and associated target proteins in non-canonical adipogenic reservoirs and adipocyte-associated cells. The first part of the thesis describes our study identifying Adipsin as a downstream target of PPARγ deacetylation and further uncovers its function within the bone marrow niche. Unlike peripheral adipose tissues, marrow adipose tissue has been shown to be uniquely responsive to nutrient fluctuations, hormonal changes, and metabolic disturbances such as obesity and diabetes mellitus. Expansion of marrow adipose tissue has also been strongly associated with bone loss in mice and humans. However, the regulation of bone marrow plasticity remains poorly understood, as does the mechanism that links changes in marrow adiposity with bone remodeling. We show that Adipsin was robustly induced in the bone marrow during bone loss in mouse and humans, in a manner dependent on PPARγ acetylation. Ablation of Adipsin inhibited marrow adipose expansion and improved skeletal health in bone loss conditions of calorie restriction, thiazolidinedione treatment for insulin resistance, and aging. These effects were mediated by Adipsin’s downstream effector, Complement Component 3, to prime common progenitor cells toward adipogenesis rather than osteoblastogenesis through the inhibition of Wnt/β-catenin signaling. Together, our findings reveal an unknown function of Adipsin, mediated by PPARγ acetylation, to promote adiposity and affect skeletal remodeling in the bone marrow niche. The second part of the thesis addresses another novel role for PPARγ, through acetylation in macrophages, to promote adipose tissue inflammation. Chronic, low-grade inflammation characteristic of obesity and metabolic dysfunction is partially driven by macrophage infiltration of adipose tissue and associated inflammatory signaling. PPARγ plays a critical role in regulating anti-inflammatory, M2 polarization of macrophages. However, the involvement of post-translational modifications, such as acetylation, in macrophages is unknown. Here we generated a macrophage specific, PPARγ constitutive acetylation-mimetic mouse line (K293Qflox/flox;LysMcre, mK293Q) to dissect its role. Upon stimulating macrophage infiltration into adipose tissue by high-fat diet feeding, we assessed the overall metabolic profile and tissue-specific phenotype of the mutant mice. We found that the mK293Q mutant promotes pro-inflammatory macrophage infiltration and subsequent fibrosis specifically in epididymal but not subcutaneous white adipose tissue, driving an impaired metabolic response including decreased energy expenditure, insulin sensitivity, glucose tolerance, and adipose tissue function. These detriments are driven by suppressed anti-inflammatory activation of macrophages. Furthermore, mK293Q mice are resistant to improvements in adipose remodeling by Rosiglitazone treatment. Our study reveals acetylation as a new layer of PPARγ regulation in macrophage activation. These findings highlight the importance of post-translational modifications in determining the function of PPARγ when regulating metabolism and promote the discovery of anti-inflammatory associated therapeutics.

Adipose tissue is a crucial organ that sits at the nexus of organismal metabolism. Evolutionary systems seemingly developed to regulate weight such that the risk of starvation accompanying low weight was balanced against the risk of predation accompanying high weight, but the molecular underpinnings of these systems have not been fully elucidated. The modern obesogenic diet has led these processes to become dysregulated, resulting in increased rates of obesity and associated metabolic disorders, making a full understanding of the mechanisms underlying weight regulation even more important. Parabiosis experiments support the existence of an uncharacterized anorectic factor that opposes weight gain. A previously established system of murine overfeeding recapitulates the defense of body weight against rapid weight gain and uncovers a non-inflammatory adipose tissue environment in the setting of obesity. Building on this past work, this thesis sets out to characterize the protein secretome of adipose tissue from overfed mice in order to provide insight into possible candidate anorectic factors and better understand the physiology of adipose tissue in this experimental form of obesity.In doing so, we uncover a previously unappreciated phenomenon of mitochondrial secretion from adipose tissue depending on metabolic state. We find that mitochondria are secreted in greater number from overfed adipose tissue and that these mitochondria are enriched for enzymes related to de novo lipogenesis. We also demonstrate that mitochondria are released intact. We find that some of these phenotypes are shared in genetically obese db/db mice, pointing toward potential physiological roots. We also characterize the plasma proteomes of overfed mice, finding that in overfed mice, inflammatory pathways are increased in the absence of induction of canonical inflammatory cytokines and in the absence of inflammation in the adipose tissue. Collectively, this work demonstrates the utility and importance of using experimental models in order to better disentangle phenomena of feeding, obesity, and inflammation. It offers direction for future studies that can positively identify an adipocyte-secreted anorectic factor peptide and work to define the manner in which local and systemic inflammation can be uncoupled from adipose tissue hypertrophy.

Indiana University-Purdue University Indianapolis (IUPUI) / Literature has demonstrated the ability of human adipose tissue to generate large amounts of lactate. However, it is not understood why adipose tissue produces lactate, how the production of lactate is regulated, and what potential benefit this has to the adipocyte or the organism. We first characterized a human model of adipogenic differentiation with minimal donor to donor variability to assess metabolic changes associated with mature adipocytes compared to their precursors. Indeed, similar to what was observed in human clinical studies, the differentiated adipocytes demonstrated increased lactate production. However, the differentiated adipocytes compared to their precursors (preadipocytes or ASCs) demonstrate an aerobic glycolysis-like (also called Warburg effect-like) increase in glycolysis characterized by a 5.2 fold increase in lactate production in normoxic conditions (atmospheric oxygen tension). Remarkably, this increase in lactate occurred even though the differentiated adipocytes simultaneously demonstrate an increase in oxidative capacity. This low fraction of oxidative capacity coupled with increased lactate production indicated regulation of oxidative rates most likely at the point of pyruvate conversion to either acetyl-CoA (oxidative metabolism) or lactate (glycolytic metabolism). To investigate the potential regulation of this metabolic phenotype, PDK isoform expression was assessed and we found PDK 1 and 4 transcript and protein elevated in the differentiated cells. Non-selective pharmacologic inhibition of the PDKs resulted in decreased lactate production, supporting a regulatory role for PDK in modulation of the observed Warburg effect. PDK inhibition also resulted in increased ROS production in the adipocytes after several hours of treatment and a decrease in cell viability when PDK inhibition was carried out over 36 hours. The resulting loss in viability could be rescued by antioxidant (Tempol) treatment, indicating the decrease in viability was ROS mediated. Similar to what is seen in cancer cells, our data demonstrate that differentiation of human adipocytes is accompanied by a PDK-dependent increase in glycolytic metabolism (Warburg effect) that not only leads to lactate production, but also seems to protect the cells from increased and detrimental generation of ROS.

Call number: LD2668 .T4 1985 E44 / Master of Science

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

Ng, Wing Chi Linda. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-142). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.iv / Publications --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Stem Cells --- p.1 / Chapter 1.1.1 --- Definition of Stem Cells --- p.1 / Chapter 1.1.2 --- Different Origins of Stem Cells --- p.2 / Chapter 1.1.3 --- Challenges and Importance of Stem Cell Research --- p.5 / Chapter 1.2 --- Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.1 --- Characteristics of Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.2 --- Adipose Tissue as an Alternate Source of MSCs --- p.8 / Chapter 1.2.3 --- Adipose Tissue Versus Bone Marrow as a Source of MSCs --- p.10 / Chapter 1.3 --- Adipose Tissue-derived Stem Cells (ATSCs) --- p.11 / Chapter 1.3.1 --- Cell Surface Marker Characteristic of ATSCs --- p.11 / Chapter 1.3.2 --- Global Gene Expression Profile of ATSCs --- p.14 / Chapter 1.3.3 --- Immunomodulatory Effect of ATSCs --- p.15 / Chapter 1.3.4 --- Proliferation Capacity of ATSCs --- p.17 / Chapter 1.3.5 --- Multilineage Differentiation of ATSCs --- p.18 / Chapter 1.3.5.1 --- Differentiation Capability of ATSCs : Adipogenesis --- p.18 / Chapter 1.3.5.2 --- Osteogenesis --- p.19 / Chapter 1.3.5.3 --- Skeletal and Smooth Muscle Myogenesis --- p.21 / Chapter 1.3.5.4 --- Cardiomyogenesis --- p.23 / Chapter 1.3.5.5 --- Chondrogenesis --- p.24 / Chapter 1.3.5.6 --- Neurogenesis --- p.27 / Chapter 1.4 --- Signaling Pathways in Stem Cells --- p.31 / Chapter 1.4.1 --- Wnt Signaling --- p.31 / Chapter 1.4.2 --- Notch Signaling --- p.33 / Chapter 1.4.3 --- Signaling Pathway of the TGF-β Superfamily --- p.34 / Chapter 1.5 --- Pathways Controlling Chondrogenesis --- p.36 / Chapter 1.6 --- MicroRNA --- p.39 / Chapter 1.6.1 --- MicroRNA - A Novel Gene Regulator --- p.39 / Chapter 1.6.2 --- Biogenesis of MicroRNAs --- p.40 / Chapter 1.6.3 --- Post-transcriptional Repression by MicroRNAs --- p.43 / Chapter 1.6.4 --- Role of MicroRNAs in Development --- p.45 / Chapter 1.6.5 --- MicroRNAs in Stem Cell Differentiation --- p.46 / Chapter 1.6.5.1 --- MicroRNA Expression Profile in ESCs --- p.46 / Chapter 1.6.5.2 --- Lineage Differentiation --- p.47 / Chapter 1.7 --- Project Aims --- p.52 / Chapter 1.8 --- Significance of Study --- p.53 / Chapter Chapter 2 --- Materials and Methods --- p.54 / Chapter 2.1 --- Sample Collection --- p.54 / Chapter 2.2 --- Isolation and Culture of ATSCs --- p.54 / Chapter 2.3 --- Measurement of Cell Growth --- p.55 / Chapter 2.4 --- Effect of Estrogen Treatment on ATSC Proliferation --- p.55 / Chapter 2.5 --- Multilineage Differentiation of ATSCs --- p.55 / Chapter 2.5.1 --- Chondrogenic Differentiation --- p.56 / Chapter 2.5.2 --- Neural Differentiation --- p.56 / Chapter 2.6 --- Immunocytochemical Analysis of Surface Markers and Lineage Specific Markers --- p.57 / Chapter 2.7 --- Alcian Blue Staining --- p.58 / Chapter 2.8 --- RNA Extraction --- p.58 / Chapter 2.9 --- Reverse Transcription --- p.59 / Chapter 2.10 --- Quantitative Real-time Polymerase Chain Reaction --- p.59 / Chapter 2.11 --- Statistical Analysis of Real-time PCR Data --- p.61 / Chapter 2.12 --- MicroRNA Profiling --- p.61 / Chapter 2.12.1 --- Reverse Transcription --- p.62 / Chapter 2.12.2 --- Quantitative Real-time Polymerase Chain Reaction --- p.62 / Chapter 2.13 --- mRNA Target Prediction of MicroRNA --- p.63 / Chapter 2.14 --- MicroRNA Knockdown Assay --- p.63 / Chapter 2.15 --- MicroRNA Over-expression Assay --- p.64 / Chapter 2.15.1 --- Vector Amplification --- p.64 / Chapter 2.15.1.1 --- Transformation --- p.64 / Chapter 2.15.1.2 --- Purification of Plasmid DNA --- p.65 / Chapter 2.15.1.3 --- Confirmation of Construct Insertion --- p.66 / Chapter 2.15.2 --- Transfection of Plasmid and Establishment of MicroRNA Precursor Expressing Cell Lines --- p.66 / Chapter 2.16 --- Gene Expression Microarry --- p.67 / Chapter 2.16.1 --- Preparation of Amplification and Labeling Reaction --- p.67 / Chapter 2.16.2 --- Purification of the Labeled/Amplified RNA --- p.68 / Chapter 2.16.3 --- RNA Fragmentation --- p.68 / Chapter 2.16.4 --- Hybridization --- p.69 / Chapter 2.16.5 --- Array Washing and Scanning --- p.69 / Chapter 2.16.6 --- Statistical Analysis of Microarray Data --- p.69 / Chapter CHAPTER 3 --- RESULTS --- p.71 / Chapter 3.1 --- Isolation and Characterization of ATSCs --- p.71 / Chapter 3.2 --- ATSCs Exhibited Multilineage Differentiation --- p.75 / Chapter 3.2.1 --- Chondrogenic Differentiation --- p.75 / Chapter 3.2.2 --- Expression of Chondrogenic Markers --- p.76 / Chapter 3.2.3 --- Neural Differentiation --- p.80 / Chapter 3.2.4 --- Expression of Neural Markers --- p.83 / Chapter 3.3 --- Effect of Donor's Reproductive Status on the Proliferation and Differentiation Capacity of ATSCs --- p.83 / Chapter 3.3.1 --- Expression of Stem Cell Makers --- p.86 / Chapter 3.3.2 --- Cell Proliferation Assay --- p.86 / Chapter 3.3.3 --- Differentiation Capacity of ATSCs --- p.89 / Chapter 3.4 --- Effect of E2 Treatment on the Proliferation Rate of ATSCs --- p.89 / Chapter 3.5 --- MicroRNA --- p.91 / Chapter 3.5.1 --- MicroRNA Expression Profile of Undifferentiated and Chondrogenic Differentiated ATSCs --- p.91 / Chapter 3.5.2 --- Clustering Analysis Identified MicroRNAs Segregate with ATSCs --- p.91 / Chapter 3.5.3 --- Identification of Differentially Expressed MicroRNAs in Chondrogenic-induced ATSCs --- p.95 / Chapter 3.5.4 --- mRNA Target Prediction for miR-199a --- p.97 / Chapter 3.6 --- Correlating MicroRNA Expression and mRNA Levels: Clues to MicroRNA Function --- p.97 / Chapter 3.6.1 --- Effect ofmiR-199a RNAi in Phenotypic Changes of Chondrogenic-induced ATSCs --- p.97 / Chapter 3.6.2 --- Identification of Potential Target Genes by Microarray Analysis of ATSCs with miR-199a Over-expression and Knockdown --- p.102 / Chapter CHAPTER 4 --- DISCUSSION --- p.104 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.115 / APPENDICES --- p.117 / REFERENCES --- p.120

Abstract not available