Interrogating Dynamics of Acute Secretion of Adiponectin Multimers from Adipose T

描述（由申请人提供）：脂肪组织（脂肪）及其细胞单位（脂肪细胞）不是甘油三酯的单纯储存部位，而是对细胞外刺激作出反应的内分泌器官。脂联素是由脂肪细胞分泌的多聚体蛋白，是体内分泌量最多的蛋白质之一，在胰岛素敏感性、中间代谢和血管炎症中起重要作用。最近的研究发现，脂肪组织具有急性分泌脂联素进入血液的机制，但没有足够的方法来灵敏地检测小体积的脂联素，脂联素分泌动力学和药物治疗的反应的理解有限。特别是，有一个明确的需要更具体和简单易用的脂联素测定。这项研究的长期目标是了解急性脂联素分泌的分子机制，以帮助揭示其与胰岛素抵抗，肥胖和代谢疾病状态的关系。本提案的目的是确定急性脂联素分泌的动力学和常用的降脂药物对这些动力学的影响。为了填补这一知识空白，将开发均相、灵敏的测定法（pFRET），用于仅从几微升样品中定量脂联素多聚体。pFRET测定的简化读数将与原代脂肪细胞的微流体采样相结合，以最小化稀释并以高时间分辨率测量分泌动力学。这些研究不仅将消除脂肪细胞研究进展的关键障碍，而且还将解决关于急性脂联素分泌的重要的未解决的问题。该提案的目的1旨在开发均相蛋白质测定法（pFRET），该方法已被证明可用于胰岛素和凝血酶检测，用于脂联素多聚体的直接荧光检测。目的2试图确定急性脂联素分泌的动力学，其中假设急性（<10 min）脂联素分泌来源于囊泡的储备池，并且不依赖于ER至高尔基体囊泡的运输。微流体分泌物取样，结合小体积pFRET，将有助于询问急性分泌。在目标3中，PI将研究常用降脂药物对急性脂联素分泌的影响，检验他汀类药物急性（<10 min）增加分泌的脂联素多聚体的假设。这一建议是重要的，因为它将克服一个关键的障碍，以了解急性脂联素分泌填补空白，在目前的方法。该提案是创新的基础上开发和整合两种新的生物分析方法，接近测定和被动微流体采样，用于调查最近发现的急性脂联素分泌事件，没有这种技术是无法访问的。初步证据有力地支持这些建议的可行性。这些发现可以更好地为目前正在服用降脂药物的许多患者提供给药时机，并允许未来的工作揭示急性脂肪因子分泌的分子和生理机制。

DESCRIPTION (provided by applicant): Rather than a mere storage site for triglycerides, it is now understood that adipose tissue (fat) and its cellular units, adipocytes, serve as an endocrine organ that responds to extracellular stimuli. Adiponectin, a multimeric protein secreted by adipocytes, is one of the most abundantly secreted proteins in the body and plays a major role in insulin sensitivity, intermediary metabolism, and vascular inflammation. Recent studies have uncovered that adipose tissue has machinery for acutely secreting adiponectin into the bloodstream; yet without adequate approaches for sensitive detection of adiponectin in small volumes, there is limited understanding of adiponectin secretion dynamics and the response to pharmacological treatments. In particular, there is a clear need for more specific and simple-to-use adiponectin assays. The long-term goal of this research is to understand the molecular mechanisms of acute adiponectin secretion to help uncover its relation to insulin resistance, obesity, and metabolic disease states. The objective of this proposal is to determine the dynamics of acute adiponectin secretion and the effects of commonly used lipid-lowering drugs on these dynamics. To fill this gap in knowledge, homogeneous, sensitive assays (pFRET) will be developed for quantitation of adiponectin multimers from only a few microliters of sample. The simplified readout of the pFRET assays will be combined with microfluidic sampling of primary adipocytes to minimize dilution and measure secretion dynamics at high temporal resolution. The studies will not only eliminate a critical barrier to progress in adipocyt research, but will also address important, unresolved questions on acute adiponectin secretion. Aim 1 of the proposal seeks to develop homogeneous protein assays (pFRET), which are proven for insulin and thrombin detection, for direct fluorescence detection of adiponectin multimers. Aim 2 seeks to determine the dynamics of acute adiponectin secretion, where it is hypothesized that acute (<10 min) adiponectin secretion originates from a reserve pool of vesicles and is not dependent on ER-to-Golgi vesicle trafficking. Microfluidic secretion sampling, combined with the small-volume pFRET, will help interrogate acute secretion. In Aim 3, the PI will investigate the effects of a commonly-used lipid-lowering drug on acute adiponectin secretion, testing the hypothesis that statins increase acutely (<10 min) secreted adiponectin multimers. This proposal is significant because it will overcome a critical barrier to progress in understanding acute adiponectin secretion by filling gaps in the current methodology. The proposal is innovative based on the development and integration of two novel bioanalytical approaches, proximity assays and passive microfluidic sampling, for investigating recently discovered acute adiponectin secretion events that are not accessible without this technology. Preliminary evidence strongly supports the feasibility of these proposals. These findings could better inform the timing of drug administration to the many patients currently taking lipid lowerin drugs and permit future work on uncovering molecular and physiological mechanisms of acute adipokine secretion.