Accumulating molecular data increasingly support an active role for adipose tissue (AT) in the development of obesity and related metabolic diseases. While a number of AT-derived signaling factors have been identified, the mechanisms by which these factors regulate AT formation remain unclear. In vivo observations in rodents have suggested that enlarged (hypertrophic) fat cells (adipocytes) induce proliferation and recruitment of new adipocytes (hyperplasia) from locally resident precursor cells. In the absence of biochemical details, this phenomenon is still controversial, especially in humans. The dual goals of this project are: (a) to investigate a hypothesized link between AT metabolism and development;and (b) to engineer an advanced AT model that will support the investigation of cell-cell signaling events in a well-defined, yet physiologically relevant experimental setting. The envisioned 3-dimensional (3D) model is a hydrogel-based construct of adipocytes, preadipocytes and vascular endothelial cells. For enhanced micro-environmental control, the 3D construct will be loaded into a micro-fluidic gradient chamber (μ-Gradient Chamber) supporting spatially defined chemical settings (on cellular length scales). The dual goals will be addressed through the following four specific aims.
Aim 1 is to develop and characterize the 3D co-culture model.
Aim 2 is to generate fluorescent reporter cells for profiling the dynamics of adipocyte- and endothelial cell-derived signaling factors.
Aim 3 utilizes siRNAmediated knockdowns to characterize the effects of metabolic enzyme inhibitions on adipocyte endocrine signaling activity. The initial knockdown targets are: glucose transporter GLUT4;lipogenic enzymes ACC, FAS and AWAT;and lipolysis enzyme lipase. Selection of these targets is based on an earlier study linking adipocyte hypertrophy with metabolic flux changes (our work). A more recent study with chemical inhibitors demonstrated that down-regulating specific steps in glycolysis or fatty acid synthesis could reduce net lipid storage (our work).
Aim 4 will study the inhibitors? effects on paracrine interactions between adipocytes and neighboring endothelial cells. In vitro model and reporter system development (Aims #1 and #2) and enzyme inhibition experiments (Aims #3 and #4) will proceed along parallel tracks.
While Aims #3 and #4 will ideally leverage the developments of Aims #1 and #2, the research design permits the use of currently available model systems and assay methods as backup. Throughout this project, special emphasis will be placed on comprehensively evaluating a broad range of adipocyte functions through quantitative metabolic analysis tools and advanced imaging techniques. The technical outcomes of this project should provide a broadly useful platform for controlled studies on AT intrinsic biochemical events related to the signaling functions of the tissue. The results of the planned experiments should shed new insights on the relationship between the metabolic and signaling functions of AT. Prospectively, these insights could lead to novel metabolic targets or nutritional strategies to control diseases and disorders resulting from or related to excessive AT expansion, including obesity and type 2 diabetes.
The long-term biomedical objective of this research is to elucidate the molecular events underlying new AT formation, especially during obesity and related disease states. As a chronic condition, obesity increases the risks of developing serious diseases and disorders, including type 2 diabetes, cardiovascular disease, hypertension and some forms of cancer. Obesity occurs following a sustained imbalance between caloric intake and energy expenditure. Treatments for obesity, therefore, generally seek to decrease energy intake, increase energy output, or both. Lifestyle modification based on restricting caloric intake and increasing physical activity is able to produce weight loss with improvements to many obesity-related conditions. However, the long-term success of this approach has been disappointing. Gastric bypass surgery can induce long-term weight loss, but is appropriate only for patients with a BMI greater than 40 or those with BMI over 35 plus other obesity-related medical conditions. Thus, there exists a critical need for safe, specific therapeutic strategies that result in sustained weight loss. Based on long-standing observations correlating adipocyte lipid loading and tissue mass expansion, the proposed work takes an important first step in determining explicit dependencies between the metabolic and signaling functions of the AT. A successful outcome would lead to new therapeutic options for controlling AT expansion, and hence increase in body fat content, for example by targeting one or more metabolic processes in the adipocyte that govern lipid accumulation.
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