Engineering an in vitro model of adipose tissue formation and metabolism

越来越多的分子数据支持脂肪组织(AT)在糖尿病的发生发展中发挥积极作用 肥胖和相关的代谢性疾病。虽然已经识别了多个AT派生的信令因子， 这些因素调节AT形成的机制尚不清楚。啮齿动物的活体观察 已提出扩大(肥大的)脂肪细胞(脂肪细胞)可诱导新的 脂肪细胞(增殖)来自本地居住的前体细胞。在缺乏生化细节的情况下，这 这一现象仍然存在争议，尤其是在人类身上。该项目的双重目标是：(A)调查 假设AT新陈代谢和发育之间的联系；以及(B)设计一个先进的AT模型，该模型 将支持在定义明确但生理上相关的细胞-细胞信号事件的研究 实验环境。设想的3维(3D)模型是基于水凝胶的脂肪细胞结构， 前脂肪细胞和血管内皮细胞。为了加强微环境控制，3D构造将 装载到支持空间定义的化学品的微流控梯度室(-梯度室)中 设置(在蜂窝长度刻度上)。这两个目标将通过以下四个具体目标来实现。 目的1是建立和表征3D共培养模型。目标2是产生荧光报告细胞 用于分析脂肪细胞和内皮细胞衍生的信号因子的动态。AIM 3利用siRNA介导的 基因敲除技术研究代谢酶抑制对脂肪细胞内分泌的影响 信号活动。最初的击倒靶标是：葡萄糖转运蛋白GLUT4，脂酶ACC，Fas 和Awat；以及脂肪分解酶脂肪酶。这些目标的选择是基于一项较早的研究 脂肪细胞肥大与代谢流量变化(我们的工作)。最近一项关于化学抑制剂的研究 证明了下调糖酵解或脂肪酸合成的特定步骤可以减少净脂肪 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(AIMS#1和#2)和酶 抑制实验(目标3和4)将沿着平行轨道进行。而目标#3和#4在理想情况下 利用AIMS#1和#2的发展，研究设计允许使用当前可用的 模型体系和检测方法作为后备。在整个项目中，将特别强调 应用定量代谢分析工具综合评价多种脂肪细胞功能 和先进的成像技术。这个项目的技术成果应该会提供一个广泛有用的 与组织信号功能相关的AT内在生化事件对照研究平台。 计划中的实验结果应该会为新陈代谢之间的关系提供新的见解 和AT的信令功能。展望未来，这些见解可能会带来新的新陈代谢目标或营养 控制由AT过度扩张引起或与之相关的疾病和紊乱的战略，包括 肥胖和2型糖尿病。

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.