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

虽然脂肪组织传统上被认为只对能量储存很重要，但现在人们认识到 成为一个复杂的、多细胞的内分泌器官，具有深刻的全身影响，几乎所有人的功能都会改变 其他器官系统。尽管它很重要，但关于脂肪分解的动态性质缺乏信息， 脂肪分泌和营养摄取，突出了方法学上几个未得到满足的需求。几种技巧 存在对少量脂肪组织的询问，以及我们对脂肪动态功能的理解 组织尤其有限，可能是因为对其内分泌性质的迟来的看法，以及增加的 细胞浮力对培养和取样的挑战。显然，更好的、针对脂肪的定制工具 为此目的而需要的。正如我们前两个资助期所显示的，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对组织进行动态询问 用标准的技术。我们的长期目标是利用内分泌生物学方面的专家见解(格兰曼，贾德) 推动定制生物分析工具(EASLEY)和内分泌系统体外模型的开发 在营养、新陈代谢和药物研发方面的应用。我们的短期目标是完善和进一步 开发微流控和生物传感方法来回答紧迫的问题，例如通过 ABHD5/PLIN1相互作用途径，这是目前方法无法回答的问题。前提是 我们基于液滴的微流体系统无与伦比的时间分辨率为 脂解外流和蛋白质动力学。我们希望这些首次在脂肪功能方面的结果会更好。 告知人类生理学。因此，该提案在技术和生物方法上都是创新的。 这项提案的目标1将对甘油和非酯化脂肪酸(NEFA)进行多重定量 高时间分辨率(&lt；5秒)的脂肪组织，通过集成基于液滴的微流控模拟- 采用盐水电极合并的数字转换电路(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。？ADC设备将在ABHD5下以高分辨率量化分泌物 配基治疗。将定制混合读取荧光分析，以实现快速(芯片外)定量 PLIN1和HSL的磷酸化，以及cAMP水平。Aim 3将专注于使用这些新工具来 白色脂肪细胞底物和蛋白质外流的机制分析。高分辨率微流控技术，使用 利用基因编码的荧光蛋白传感器，将蛋白质的运输和相互作用与 分泌性输出。改进的微流控数字模拟电路(µDAC)也将设计用于快速组织 成像过程中的刺激。这项研究基本原理是，定制工具开发将提供新的 关于脂肪组织动力学的信息，我们已经发现了以前未知的重要信息 组织中的动态功能。进一步的研究应该会改善人类的饮食或药理 干预措施。因此，该提案在技术和生物方法上都是创新的。

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.