Unmasking mechanisms of lipolytic dynamics in adipose tissue using high-resolution microfluidic sampling

虽然脂肪组织（脂肪）传统上被认为是重要的只是为了储存能量，它现在被认为是 是一个复杂的，多细胞的，内分泌器官，具有深刻的全身效应，改变了几乎所有的功能， 其他器官系统。尽管其重要性，但缺乏关于脂解的动态性质的信息， 脂肪因子分泌和营养摄取，突出了方法学中几个未满足的需求。一些技巧 存在于询问少量的脂肪组织，我们对脂肪组织的动态功能的理解 组织是特别有限的，也许是由于迟来的观点，其内分泌性质和增加的 培养和取样面临来自细胞浮力的挑战。很明显，更好的，脂肪定制的工具是 需要为此目的。正如我们前两个资助期所示，我们建议我们的微流体 系统是满足这些持续需求的理想选择，允许以不可能的方式动态询问组织， 用标准的技术。我们的长期目标是利用内分泌生物学专家的见解（Granneman，Judd） 推动定制生物分析工具（伊斯利）和内分泌系统体外模型的开发 用于营养、代谢和药物发现。我们的短期目标是完善和进一步 开发微流体和生物传感方法，以回答紧迫的问题，例如，通过 ABHD 5/PLIN 1相互作用途径，目前的方法无法回答的问题。前提是 我们的基于液滴的微流体系统的无与伦比的时间分辨率提供了独特的透镜， 脂肪分解流出和蛋白质动力学。我们希望这些关于脂肪功能的第一个结果能更好地 告知人类生理学。因此，该提案在技术和生物方法方面具有创新性。 本提案的目的1将对来自于以下的甘油和非酯化脂肪酸（NEFA）进行多重定量： 脂肪组织在高时间分辨率（<5秒），通过整合基于液滴的微流体模拟， 带盐水电极合并器的数字电路（µADC）。在目标2中，我们将定制生物分析工具， 脂肪组织信号通路。µADC器件将在ABHD 5下以高分辨率量化分泌物 配体处理混合和读取荧光测定将被定制用于快速（芯片外）定量 PLIN 1和HSL磷酸化以及cAMP水平。Aim 3将专注于使用这些新工具， 白色脂肪细胞中底物和蛋白流出的机制分析。高分辨率微流控技术， 与基因编码的荧光蛋白传感器，将相关的蛋白质运输和相互作用， 分泌输出。改进的微流体数模电路（µDAC）也将被设计用于快速组织 在成像过程中刺激。这项研究的基本原理是，自定义工具的开发将提供新的 关于脂肪组织动力学的信息，我们已经发现了重要的，以前未知的 组织中的动态功能。进一步的研究应该会导致人类饮食或药理学方面的改善。 干预措施。因此，该提案在技术和生物方法方面具有创新性。

While adipose tissue (fat) was traditionally considered important only for energy storage, it is now recognized to be a complex, multicellular, endocrine organ with profound systemic effects, altering function in nearly all other organ systems. Despite its importance, there is a lack of information on the dynamic nature of lipolysis, adipokine secretion, and nutrient uptake, highlighting several unmet needs in methodology. Few techniques exist to interrogate small amounts of adipose tissue, and our understanding of dynamic function in adipose tissue is particularly limited, perhaps due to the belated perspective on its endocrine nature and the added culture and sampling challenges from cell buoyancy. It is clear that better, adipose-customized tools are needed for this purpose. As shown in our previous two funding periods, we propose that our microfluidic systems are ideal to meet these ongoing needs, permitting dynamic interrogation of tissue in ways not possible with standard techniques. Our long-term goal is to use expert insights in endocrine biology (Granneman, Judd) to drive the development of customized bioanalytical tools (Easley) and in vitro models of the endocrine system for applications in nutrition, metabolism, and drug discovery. Our short-term objective is to refine and further develop microfluidic and biosensing methods to answer pressing questions, e.g. lipolytic dynamics via the ABHD5/PLIN1 interaction pathway, questions that cannot be answered with current methods. The premise is that unmatched temporal resolution of our droplet-based microfluidic systems provide unique lenses into lipolytic efflux and protein dynamics. We expect these first-of-their-kind results on adipose function to better inform human physiology. Thus, the proposal is innovative in its technological and its biological approaches. Aim 1 of this proposal will multiplex quantification of both glycerol and non-esterified fatty acids (NEFA) from adipose tissue at high temporal resolution (<5 sec), achieved by integrating droplet-based microfluidic analog- to-digital circuits (µADC) with salt-water electrode mergers. In Aim 2, we will customize bioanalytical tools for adipose tissue signaling pathways. µADC devices will quantify secretions at high resolution under ABHD5 ligand treatment. Mix-and-read fluorescence assays will be customized for rapid (off-chip) quantification of PLIN1 and HSL phosphorylation, and for cAMP levels. Aim 3 will focus on using these novel tools for mechanistic analysis of substrate and protein efflux in white adipocytes. High-resolution microfluidics, used with genetically-encoded fluorescent protein sensors, will correlate protein trafficking and interactions with secretory output. Improved microfluidic digital-to-analog circuits (µDAC) will also be designed for rapid tissue stimulation during imaging. The rationale for this research is that custom tool development will provide novel information on adipose tissue dynamics, and we have already uncovered significant, previously unknown dynamic function in the tissue. Further study should lead to improvements in human dietary or pharmacological interventions. The proposal is thus innovative in its technological and its biological approaches.