http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_1 As the result of its apparent structural and histological simplicity, adipose tissue (AT) functions initially were limited to energy storage, insulation, and thermoregulation. Only decades later was the extraordinarily dynamic role of AT recognized, revealing its participation in a broad range of physiological processes, including reproduction, apoptosis, inflammation, angiogenesis, blood pressure, atherogenesis, coagulation, fibrinolysis, immunity and vascular homeostasis with either direct or indirect implications in the regulation of proliferation. The functional pleiotropism of AT relies on its ability to synthesize and, in some cases, secrete a large number of enzymes, hormones, growth factors, cytokines, complement factors, and matrix and membrane proteins, collectively termed adipokines. At the same time, white AT expresses receptors for most of these factors, warranting a wide cross-talk at both local and systemic levels in response to metabolic changes or other external stimuli. In this chapter, mounting evidence on the specific characteristics of AT from different depots is outlined in relation to fat distribution and comorbidity development. The current knowledge in this field is reviewed with a broad perspective ranging from classification, structure, and distribution to the key functional roles of AT with a particular focus on the role of adipokines and their involvement in the metabolic disorders accompanying obesity. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_10 Determination of the complex secretory proteome of adipocytes and its metabolic changes induced by drug treatment such as insulin or rosiglitazone is possible with the advanced proteomics technologies described herein. To study the secreted proteins of adipocytes, a 2D- liquid chromatography/mass spectrometry/mass spectrometry protocol has been established. With the use of reversed-phase high-performance liquid chromatography, intact proteins were separated in the first dimension into eight fractions, then digested with Lys-C and trypsin. Comparative differences after drug treatment were assessed using 18O proteolytic labeling strategies. With the advent of more and more sophisticated instrumentation and data analysis tools, protocols like this one will likely become standard tools for scientists in the research fields of endocrinology, obesity, and diabetes. These protocols enable researchers to study the dynamic drug-induced changes in a comprehensive and systematic manner that was inconceivable just a few years ago. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_11 Adipose tissue is increasingly recognized as a metabolically active endocrine organ with multiple functions beyond its lipid storage capability. Various constituents of the tissue, such as mature adipocytes and stromal vascular cells, have distinct functions. For example, they express and secrete different kinds of bioactive molecules collectively called adipokines. Altered adipokine secretion patterns characterize obesity and insulin resistance, which are major risk factors for type 2 diabetes mellitus. The contribution of dysregulated adipokine expression to these diseases may be assembled from transcriptomic profiles of the tissue and/or its cellular constituents. The gene expression profiles may also complement genetic approaches to identify disease susceptibility genes. Here, we describe an application of gene expression profiling using DNA microarrays to study human adipose tissue, adipocytes, and stromal vascular cells. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_12 The broad definition of a stem cell is a cell that has the ability to self-renew and differentiate into one or more specialized terminally differentiated cell types. It has become evident that stem cells persist in, and can be isolated from, many adult tissues. Adipose tissue has been shown to contain a population of cells that retain a high proliferation capacity in vitro and the ability to undergo extensive differentiation into multiple cell lineages. These cells are referred to as adipose stem cells and are biologically similar, although not identical, to mesenchymal stem cells derived from the bone marrow. Differentiation causes stem cells to adopt the pheno-typic, biochemical, and functional properties of more terminally differentiated cells. This chapter will provide investigators with some background on stem cells derived from adipose tissue and then provide details on adipose stem cell multilineage differentiation along osteogenic, adipogenic, chondrogenic, and neurogenic lineages. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_13 The mouse embryo fibroblast cell lines 3T3-L1 and C3H10T1/2 differentiate to adipocytes that exhibit similar insulin regulation of lipogenesis. These cell lines, however, differ appreciably in the processes that produce the major regulator PPARγ. Each line is stimulated by a mixture of insulin, dexamethasone, and methylisobutylxanthine (IDM). In the first 24 h, IDM activates each cell type to produce similar regulatory changes and cell contraction. However, the increase in PPARγ is delayed by 24 h in typical 3T3-L1 cells compared with C3H10T1/2 cells. This delay is caused by the need for one or two rounds of cell division (clonal expansion) for PPARγ synthesis in 3T3-L1 cells. This expansion also occurs in C3H10T1/2 cells, but is not needed for PPARγ synthesis and differentiation. Other 3T3-L1 sublines have been described that follow the C3H10T1/2 pattern of differentiation. Culture conditions and inhibitors are described here that remove clonal expansion in C3H10T1/2 cells. With these constraints the cells retain full commitment to differentiation. This distinction is significant because many agents suppress differentiation in 3T3-L1 cells through inhibition of clonal expansion. Other effects on differentiation may be seen in C3H10T1/2 cells that are obscured in 3T3-L1 cells due to this inhibition of proliferation. Human preadipocytes do not need clonal expansion for adipogenesis, thus paralleling C3H10T1/2 cells. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_14 Obesity is characterized by increased adiposity of visceral and subcutaneous depots as well as other organs, including the vasculature. These fat depots secrete various hormone-like proteins implicated in metabolic homeostasis (e.g., adiponectin, resistin), the central control of appetite (e.g., leptin) and the increased production of cytokines. These molecules act either in a paracrine or endocrine manner, contributing to the metabolic and cardiovascular complications of obesity. Explant cultures of white adipose tissue are an important step in analyzing the secretory mechanisms of adipose tissue by preserving the physiological in vivo cross-talk between the various types of cells. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_15 Much of the research devoted to understanding adipose tissue development is currently performed in vitro. Several cell culture models, including preadipocyte cell lines and primary culture of adipose-derived stromal vascular precursor cells, are commonly used to study molecular and cellular events and regulatory influences on preadipocyte proliferation and differentiation. Primary preadipocyte culture systems have several distinct advantages over preadipose cell lines. Because they have not been passaged continuously in culture, primary cultures of adipose derived stromal-vascular (SV) cells more closely reflect the in vivo characteristics of the tissue from which they are derived. In addition, primary cells can be obtained from various adipose tissue depots and from animals at different stages of development, from early postnatal life through advanced age. Cells can also be obtained from genetic rodent models of obesity or from rats and/or mice subjected to nutritional or hormonal manipulation. In each case, specific adipose tissue depots are dissected and the SV cells obtained after collagenase digestion. To examine the effect of tissue source or in vivo or in vitro treatment on preadipocyte proliferation, SV cells are labeled by thymidine incorporation during the exponential growth phase and maintained in culture until sufficiently lipid-filled to allow separation by density. Regulatory influences on various stages of preadipocyte differentiation can be examined in rat SV cultures in a controlled environment featuring chemically defined serum-free medium; whereas, more temperamental mouse SV cultures require the presence of serum for optimal differentiation. Alternatively, preadipocytes differentiated in vitro may be used for examining adipocyte metabolic or secretory responses. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_16 Adipose tissue contains cell types other than adipocytes that may contribute to complications linked to obesity. For example, macrophages have been shown to infiltrate adipose tissue in response to a high-fat diet. Isolation of the stromal-vascular fraction of adipose tissue allows one to use flow cytometry to analyze cell surface markers on leukocytes. Here, we present a technical approach to identify subsets of leukocytes that differentially express cell surface markers. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_17 Chromatin, long thought to be no more than a scaffold supporting DNA compaction inside the cell nucleus, has emerged in the last few years as a major regulatory element involved in the control of gene expression both acutely during interphase and programmatically throughout complex processes of development and differentiation. Adipogenesis is the result of an intertwined network of transcription factors and coregulators with chromatin-modifying activities and offers an excellent model for the study of transcriptional regulation. In this regard, electrophoretic mobility shift assay and immunoprecipitation of chromatin are complementary methods that can be used to study the binding of nuclear proteins to DNA and to characterize how these proteins interact with and modify chromatin to regulate gene expression and, more globally, cell differentiation. This chapter provides some strategies to perform these two assays using 3T3-L1 cells and rodent primary preadipocytes and adipocytes. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_18 RNA interference (RNAi) is a powerful, quick, and easy technique to reduce the expression of a particular gene. However, investigators need to consider several steps for the experiments, including the design of the siRNA, an efficient delivery method, and a means for monitoring the biological effects of the siRNA introduced. Adipocytes have long been recognized as one of the most difficult cell types in which to perform gene delivery. In this chapter, three distinct transfec-tion methods to obtain high efficiency of gene delivery and gene knockdown are described. The transfection efficiency of siRNA and shRNA is also compared. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_19 Adipose tissue has emerged as a major endocrine organ producing a wide spectrum of hormones and factors that play crucial roles in regulating cell turnover and function, not only locally within the adipose tissue but also in the brain and other key metabolic organ systems. It is known that gene activity is controlled at both transcriptional and post-transcriptional levels. Consequently, one of the most important means by which the activity of a gene is assessed is through the determination of levels of the corresponding messenger ribonucleic acid (mRNA). This process involves the isolation of total cellular RNA and subsequent analysis of the mRNA of interest. Given the unique nature of adipose tissue and adipocytes (i.e., containing high amounts of lipid), special RNA isolation techniques that have been tested in both white adipose tissue and isolated mature adipocytes from rats and mice will be presented. Although several methods are available for mRNA quan-titation, we will describe a real-time quantitative reverse transcription polymerase chain reaction protocol because of its superior sensitivity and reliability. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_2 The importance and the role of adipose tissues are now largely expanded not only because the very high occurrence of obesity but also because the emerging view that adipose tissue could be a reservoir of therapeutic cells. A critical examination of the adipose tissue features according to their location shows that sampling is not as easy as previously thought and needs special attention to heterogeneity and differences. We discussed here these different points and give precise protocols to sample the different adipose tissues and manipulate them. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_20 Adipose tissue synthesizes factors involved in the body's homeostasis. Thus, measurements of messenger ribonucleic acid (mRNA) concentrations are important to study the involvement of adipose tissue in various physiological and pathophysiological conditions, in particular in obesity. Because adipose tissue is highly heterogeneous, containing both a stromal and an adipocyte compartment, each one having different cellular composition and functional capacities, in situ hybridization is a powerful tool to analyze the discrete expression of the mRNAs coding for the various factors synthesized within this tissue. Presented here is a detailed protocol for in situ hybridization of mRNAs in adipose tissue using 35S-labeled single-stranded probes with sufficient details for the readers unfamiliar with histologic techniques. Included are details of tissue sectioning and preparation, probe synthesis, hybridization reaction, and macro- and microscopic signal detection. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_21 When isolated from tissue, white adipose cells are round, and their interior is filled with a large (80–120 μm) droplet of stored triglyceride, leaving a thin (1–2-μm) layer of cytoplasm between the lipid droplet and the plasma membrane. Their three-dimensional architecture, together with the fact that these cells ordinarily float in medium, have created major challenges when one attempts to perform microscopy techniques with these cells. Adipocytes serve as the principal energy reservoir in the body, and it is essential to overcome these difficulties to be able to study hormone-mediated responses in real adipose cells, which convey physiological significance that cannot be readily duplicated by the use of cultured model adipocytes. This chapter focuses on the use of confocal microscopy optical sectioning and computer-assisted image reconstruction in the whole adipose cell in the study of insulin-regulated protein trafficking. In addition, we illustrate the possibility to image whole-mount preparations of living adipose tissue, opening new ways to probe adipose cells in situ without disrupting their cellular interactions within living adipose tissue. Confocal microscopy constitutes an effective morphological approach to investigating adipose cell physiology and pathophysiology. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_22 Lipolysis involves the sequential breakdown of triglycerides into free fatty acids and glycerol. The extent of lipolysis is therefore a key determinant of the energy status of an individual and also dictates insulin resistance. Here, we describe a protocol for estimating lipolysis in murine adipocytes. Glycerol released during the lipolytic reaction is estimated radiometrically to determine the extent of lipolysis within the cell and the data are normalized to cell number. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_23 Glucose is the main metabolic fuel in mammalian cells. Glucose entry into cells is facilitated by a family of ubiquitously expressed glucose transporter proteins. Typically, glucose transporters are localized on the plasma membrane. One notable exception is the glucose transporter isoform 4 (Glut4), which is specifically expressed in insulin sensitive tissues, i.e., skeletal muscle, heart muscle, and fat, and is responsible for the insulin effect on blood glucose clearance (1). Under basal conditions, Glut4 is compartmentalized in intracellular membrane vesicles and thus has no access to the extracellular space. Upon insulin administration, Glut4-containing vesicles fuse with the plasma membrane and deliver the transporter to its site of action. As a result, Glut4 content on the plasma membrane is increased, and glucose uptake in the cell is significantly elevated. Here, we describe two complementary techniques. The first one uses tritiated 2-deoxyglucose and is designed to measure insulin-stimulated glucose transport into cultured adipose cells. The second allows one to quantify the degree of Glut4 translocation from an intracellular compartment to the plasma membrane. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_24 Adipogenesis is an important component of adipose tissue development and growth. Alterations in adipogenesis may promote adipose tissue insulin resistance and inflammation. The ability of preadipocytes to differentiate into mature adipocytes depends on the activation of phosphoinositide 3-kinase (PI3K). This chapter describes the methodology used to measure the cellular accumulation of phosphoinositide products of PI3K. This approach involves labeling the cells with myo-[2—3H]-inositol, extraction and deacylation of the phosphoinositides, and HPLC separation of the deacylated derivatives. The assay of PI3K activity itself is also described in detail. The ability to analyze PI3K and its phosphoinositide products is a useful tool for ongoing endeavours to understand adipogenesis and adipose tissue dysfunction. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_3 In vivo adipose tissue quantification is an important tool to characterize phenotypes of obesity, especially in the human. The amount and distribution of adipose tissue is associated with many of the adverse consequences of obesity. Recent studies suggest that adipose tissue is not a single homogeneous compartment. Regional adipose tissue depots vary in biological functions and individual adipose tissue compartments have stronger associations with metabolic conditions than does total adipose tissue mass. Currently there is intense and increasing interest in regional adipose tissue compartments. Computed tomography and magnetic resonance imaging often are used to quantify adipose tissue volumes or cross-sectional adipose tissue areas. Other modalities, including dual-energy absorpti-ometry and magnetic resonance spectroscopy, provide whole-body or regional fat measures instead of adipose tissue mass quantification. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_4 Adipose tissue plays a critical role in energy homeostasis, not only in storing triglycerides, but also in responding to nutrient, neural, and hormonal signals, and secreting adipokines that control feeding, thermogenesis, immunity, and neuroendocrine function. It is conceivable that adipose tissue-specific gene expression would influence the aforementioned functions. A feasible approach to prepare adipose tissue-specific transgenic mouse models is necessary for such studies. Here, we report the preparation of adipose tissue-specific cholesteryl ester transfer protein transgenic mice. The general principle might apply to the establishment of other adipose tissue-specific transgenic mice models. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_5 Angiogenesis is required for the growth and expansion of both healthy and pathological tissues. The plasticity of the adipose tissue is reflected by its remarkable ability to expand or to reduce in size throughout the adult lifespan. We, and others, have recently shown that expansion of fat mass is dependent on angiogenesis, and suppression of angiogenesis might provide a novel therapeutic approach for prevention and treatment of obesity. Here, we outline two technical procedures for assessment of angiogenesis in adipose tissues. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_6 Brown and white adipose tissue have recently gained prominence as key players in obesity and related health problems, such as type-2 diabetes and cardiovascular disease. Brown adipose tissue-dependent nonshivering thermogenesis significantly affects the body's energy balance. Originally considered as a passive store of lipids, white adipose tissue has recently been found to secrete a number of hormones and cytokines and to be thus involved in the control of body metabolism and energy balance at multiple sites. These findings have renewed the interest in adipose organ biology, including its innervation by the autonomic nervous system and sensory nerves. Here, we describe our protocols for detecting different types of adipose tissue nerves by light microscopy using peroxidase immunostaining and by laser scanning confocal microscopy using immunofluorescence. With these techniques, the presence, distribution, and colocalization of autonomic and sensory nerves can be effectively investigated in subcutaneous and visceral adipose depots of normal and obese animals. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_7 Regulation of blood flow in tissues such as skeletal muscle, liver, and adipose tissue is needed to meet the changing local metabolic and physiological demands under varying conditions. In healthy individuals, adipose tissue blood flow (ATBF) is remarkably responsive to meal ingestion, but changes in ATBF in response to other physiological stimuli, such as stress and physical exercise, have also been noted. The ATBF response to nutrient intake may be of particular importance in the regulation of metabolism by facilitating transport of nutrients as well as signaling between adipose tissue and other metabolically active tissues. A reduction in both fasting and postprandial ATBF has been observed in obesity; this impairment is associated with insulin resistance. A better understanding of the physiological basis for (nutritional) regulation of ATBF may therefore give insight to the relationship between disturbances in ATBF and the metabolic disturbances observed in response to insulin resistance. In this chapter, we describe some different approaches to quantify human ATBF, with a particular emphasis on the 133xenon wash-out technique and a method by which regulatory properties of subcutaneous ATBF can be studied by pharmacological micromanipulation (microinfusion). 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_8 Brown and white adipose tissues in mammals have a number of similar properties, such as lipid storage and adipokine production, but also distinctive properties. The energy-storing white adipose tissue has few mitochondria and low oxidative capacity. The heat-producing brown adipose tissue has a high density of mitochondria and high oxidative capacity. Mitochondrial function can be investigated in cells and organelles isolated from both brown and white adipose tissues. This chapter describes methods for successful isolation of suitable preparations of adipose tissues and their subsequent use. Questions concerning thermogenic capacity of the tissues, their potential influence on whole body metabolism, and specific properties of the mitochondria and their mode of function may be addressed using these methods. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-245-8_9 Role of specific reactive lipids as well as amino acids in control of insulin signalling in adipose tissue is well recognized. Since it is practically impossible to measure the levels of all metabolites in the biological sample simultaneously with a single analytical platform, we utilize multiple platforms to study the lipids and metabolites of relevance to adipose tissue metabolism and insulin signalling. Two screening platforms cover a broad range of lipid molecular species (UPLC/MS based lipidomics platform) as well as organic acids and sterols (GCxGC-TOF platform). A targeted platform for amino acids (UPLC) is also applied. 2008-06-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-066-9_1 The method of spectral karyotyping (SKY) is based on a combination of the technologies of charge-coupled device imaging and spectrometry. The engineering feasibility has been realized in the SpectraCube system from Applied Spectral Imaging Inc., and it allows the simultaneous identification of all 24 human chromosomes. This is performed by characterizing the spectral signature of every image pixel in relation to a fluorochrome combinatorial library translating the image and spectral information into chromosome classification. Applications for SKY include pre- and postnatal characterization of certain numerical and structural rearrangements and complex karyotypes and highly informative analysis of sample materials with only single or few cells available for investigation. 2008-05-09T04:00:00Z