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: 10298595
• 负责人: Christopher J Easley
• 金额: $ 50.59万
• 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/10298595
• 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; S Phase; 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.

虽然传统上仅认为脂肪组织（脂肪）仅对储能很重要，但现在可以识别 成为一个复杂的多细胞内分泌器官，具有深远的全身作用，几乎改变了功能 其他器官系统。尽管它很重要，但缺乏有关脂解的动态性质的信息， 脂肪因子分泌和营养吸收，突出了方法学上的几种未满足的需求。很少的技术 存在用于询问少量脂肪组织，以及我们对脂肪动态功能的理解 组织特别有限，也许是由于对其内分泌性质的迟来的观点以及添加的观点 细胞浮力引起的培养和抽样挑战。显然，脂肪量化的工具更好 为此目的需要。如前两个资金期间所示，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对组织进行动态询问 使用标准技术。我们的长期目标是在内分泌生物学中使用专家见解（Granneman，Judd） 推动定制生物分析工具（EASLEY）和内分泌系统的体外模型的开发 用于营养，代谢和药物发现中的应用。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压迫问题，例如通过 ABHD5/PLIN1相互作用途径，无法用当前方法回答的问题。前提是 我们基于液滴的微流体系统无与伦比的临时分辨率为您提供了独特的镜头 脂溶解和蛋白质动力学。我们希望这些对脂肪功能的首次效果会更好 告知人类生理学。这是该提案在技术及其生物学方法上具有创新性。 该提案的目标1将对甘油和非酯化脂肪酸（NEFA）的多重定量。 高临时分辨率（<5 sec）的脂肪组织，通过整合基于液滴的微流体类似物来实现 与盐 - 水电极合并的数字电路（µADC）。在AIM 2中，我们将自定义生物分析工具 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 配体治疗。将定制混合荧光测定法，以快速（片）数量的数量 PLIN1和HSL磷酸化，以及营地水平。 AIM 3将专注于使用这些新颖的工具作为 白色脂肪细胞中底物和蛋白质外排的机械分析。高分辨率微流体使用 使用一般编码的荧光蛋白传感器，将使蛋白质运输和相互作用与 分泌输出。改进的微流体数字到分析电路（µDAC）也将用于快速组织 成像过程中的刺激。这项研究的理由是，自定义工具开发将提供新颖 有关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。进一步的研究应导致人类饮食或药理的改善 干预措施。因此，该提案的技术及其生物学方法具有创新性。