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

使用高分辨率微流体采样揭示脂肪组织中脂肪分解动力学的机制

• 批准号: 10442627
• 负责人: Christopher J Easley
• 金额: $ 41.82万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-08 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10442627
• 关键词: Adenylate Cyclase; Adipocytes; Adipose tissue; Alzheimer' s Disease; Automobile Driving; Biochemistry; Biological; Biological Assay; Biology; Biosensing Techniques; Bypass; Cells; Chronic; Complex; Coupling; Custom; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Devices; Diabetes Mellitus; Disease; Electrodes; Endocrine; Endocrine Glands; Endocrine system; Fatty Acids; Fatty acid glycerol esters; Fluorescence; Funding; Gap Junctions; Glycerol; Goals; Health; Hormones; Hour; Human; Hydrolase; Hydrolysis; Image; Immunity; Incidence; Insulin; Intervention; Lead; Ligands; Link; Lipase; Lipolysis; Metabolism; Methodology; Methods; Microfluidics; Mission; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Nutrient; Obesity; Output; Overweight; Pathway interactions; Pharmacology; Phosphorylation; Physiology; Population; Protein Dynamics; Proteins; Research; Resolution; Role; Sampling; Signal Pathway; Signal Transduction; Sodium Chloride; System; Techniques; Tissue Expansion; Tissues; Triglycerides; Tube; Water; Work; adipokines; analog; base; body system; design; dietary; digital; drug discovery; experimental study; improved; in vitro Model; in vivo; innovation; insight; insulin signaling; islet; lens; novel; nutrition; perilipin; protein protein interaction; protein transport; response; sensor; temporal measurement; tool; tool development; uptake

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.

虽然传统上认为脂肪组织（脂肪）仅对能量储存很重要，但现在人们认识到它 成为一个复杂的多细胞内分泌器官，具有深远的全身影响，改变几乎所有的功能 其他器官系统。尽管它很重要，但缺乏关于脂肪分解动态性质的信息， 脂肪因子分泌和营养吸收，突出了方法学中的一些未满足的需求。技巧很少 存在是为了询问少量的脂肪组织，以及我们对脂肪动态功能的理解 组织特别有限，可能是由于对其内分泌性质的迟来认识以及附加的 来自细胞浮力的培养和采样挑战。显然，更好的脂肪定制工具是 为此目的需要。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 使用标准技术。我们的长期目标是利用内分泌生物学方面的专家见解（Granneman、Judd） 推动定制生物分析工具（Easley）和内分泌系统体外模型的开发 用于营养、代谢和药物发现方面的应用。我们的短期目标是完善并进一步 开发微流体和生物传感方法来回答紧迫的问题，例如脂解动力学通过 ABHD5/PLIN1相互作用途径，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 脂肪分解流出和蛋白质动力学。我们期望这些关于脂肪功能的首创结果能够更好地 告知人类生理学。因此，该提案在技术和生物学方法上具有创新性。 该提案的目标 1 将多重定量甘油和非酯化脂肪酸 (NEFA) 通过集成基于液滴的微流体模拟来实现高时间分辨率（<5秒）的脂肪组织 与盐水电极合并的数字电路（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 设备将在 ABHD5 下以高分辨率量化分泌物 配体处理。混合读取荧光测定将被定制用于快速（芯片外）定量 PLIN1 和 HSL 磷酸化，以及 cAMP 水平。目标 3 将重点关注使用这些新颖的工具 白色脂肪细胞中底物和蛋白质流出的机制分析。使用高分辨率微流体 与基因编码的荧光蛋白传感器一起，将蛋白质运输和相互作用与 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 成像过程中的刺激。这项研究的基本原理是定制工具开发将提供新颖的 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的改善 干预措施。因此，该提案在技术和生物学方法方面具有创新性。