Engineering an in vitro model of adipose tissue formation and metabolism

构建脂肪组织形成和代谢的体外模型

• 批准号: 8038517
• 负责人: KYONGBUM LEE
• 金额: $ 20.53万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-10-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038517
• 关键词: 3-Dimensional; Address; Adipocytes; Adipose tissue; Behavior Therapy; Biochemical; Biological Assay; Biological Models; Body Weight decreased; Body fat; Cardiovascular Diseases; Cells; Chemicals; Chronic; Coculture Techniques; Computer Systems Development; Controlled Study; Data; Dependency; Development; Disease; Endocrine; Endothelial Cells; Energy Intake; Energy Metabolism; Engineering; Enzyme Inhibition; Enzymes; Event; Fatty Acids; GLUT4 gene; Glucose Transporter; Glycolysis; Goals; Human; Hydrogels; Hyperplasia; Hypertension; Hypertrophy; Imaging Techniques; Investigation; Lead; Length; Link; Lipase; Lipids; Lipolysis; Malignant Neoplasms; Medical; Metabolic; Metabolic Diseases; Metabolism; Methods; Modeling; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Nutritional; Obesity; Obesity associated disease; Outcome; Output; Patients; Physical activity; Reporter; Research; Research Design; Risk; Rodent; Role; Signal Transduction; Therapeutic; Tissue Expansion; Tissue Model; Tissues; Vascular Endothelial Cell; Work; bariatric surgery; base; in vitro Model; in vivo; inhibitor/antagonist; insight; intercellular communication; novel; novel therapeutics; obesity treatment; paracrine; precursor cell; research study; success; tool

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.

积累的分子数据越来越多地支持脂肪组织（AT）在肿瘤发生发展中的积极作用。 肥胖和相关代谢疾病。虽然已经鉴定了许多AT衍生的信号传导因子， 这些因子调节AT形成的机制仍不清楚。啮齿类动物体内观察结果 已经表明，扩大（肥大）的脂肪细胞（脂肪细胞）诱导增殖和招募新的 脂肪细胞（增生）从局部驻留的前体细胞。由于缺乏生物化学细节， 这种现象仍然存在争议，尤其是在人类身上。该项目的双重目标是：（a）调查 AT代谢和发育之间假设联系;和（B）设计先进的AT模型， 将支持在一个明确的，但生理相关的细胞间信号事件的调查 实验设置。设想的三维（3D）模型是脂肪细胞的基于水凝胶的构造， 前脂肪细胞和血管内皮细胞。为了增强微环境控制，3D结构将 被装载到微流体梯度室（-梯度室）中，该微流体梯度室支持空间限定的化学物质， 设置（在细胞长度尺度上）。将通过以下四个具体目标实现这两个目标。 目的1是开发和表征3D共培养模型。目的二是制备荧光报告细胞 用于分析脂肪细胞和内皮细胞衍生的信号传导因子的动力学。目的3利用siRNA介导的 敲低以表征代谢酶抑制对脂肪细胞内分泌的影响 信号活动。最初的敲除靶点是：葡萄糖转运蛋白GLUT 4;脂肪生成酶ACC、FAS 和AWAT;和脂解酶脂肪酶。这些目标的选择是基于一项早期的研究， 脂肪细胞肥大与代谢通量的变化（我们的工作）。最近一项关于化学抑制剂的研究 表明下调糖酵解或脂肪酸合成中的特定步骤可以减少净脂质 存储（我们的工作）。目的4将研究抑制剂对脂肪细胞和 邻近的内皮细胞。体外模型和报告系统开发（目的#1和#2）和酶 抑制实验（目标#3和#4）将沿着沿着轨道进行。虽然目标#3和#4将理想地 利用目标#1和#2的发展，研究设计允许使用现有的 模型系统和分析方法作为备份。在整个项目中，将特别强调 通过定量代谢分析工具全面评估广泛的脂肪细胞功能 和先进的成像技术。该项目的技术成果将提供一个广泛有用的 用于对与组织的信号传导功能相关的AT内在生化事件进行对照研究的平台。 计划中的实验的结果应该会对代谢与代谢之间的关系提供新的见解。 以及AT的信令功能。这些见解可能会导致新的代谢目标或营养 控制由AT过度扩张引起或与AT过度扩张相关的疾病和病症的策略，包括 肥胖和2型糖尿病。