Interrogating Dynamics of Acute Secretion of Adiponectin Multimers from Adipose T

描述(申请人提供)：脂肪组织(FAT)及其细胞单位，脂肪细胞，不再仅仅是甘油三酯的储存场所，而是一个对细胞外刺激做出反应的内分泌器官。脂联素是一种由脂肪细胞分泌的多聚体蛋白，是体内分泌最丰富的蛋白质之一，在胰岛素敏感性、中间代谢和血管炎症中发挥重要作用。最近的研究发现，脂肪组织具有将脂联素迅速分泌到血流中的机制；然而，由于缺乏足够的方法灵敏地检测小容量脂联素，对脂联素分泌动力学和药物治疗的反应的了解有限。特别是，显然需要更具体、更易用的脂联素检测方法。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、肥胖和代谢性疾病状态的关系。这项建议的目的是确定急性脂联素分泌的动力学以及常用降脂药物对这些动力学的影响。为了填补这一知识空白，将开发出均相、灵敏的分析方法(PFRET)，用于仅从几微升样本中定量测定脂联素多聚体。简化的pFRET分析读数将与原代脂肪细胞的微流控采样相结合，以最大限度地减少稀释，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，还将解决有关急性脂联素分泌的重要、悬而未决的问题。该提案的目的1旨在建立均相蛋白分析(PFRET)，该方法已被证明可用于检测胰岛素和凝血酶，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(1t；10min)脂联素分泌来自储备囊泡池，而不依赖于内质网到高尔基体囊泡的运输。微流控分泌物采样，结合小容量pFRET，有助于急性分泌物的讯问。在目标3中，PI将调查一种常用的降脂药物对急性脂联素分泌的影响，验证他汀类药物急剧增加(&lt；10分钟)分泌脂联素多聚体的假设。这一建议意义重大，因为它将通过填补当前方法学中的空白，克服在理解急性脂联素分泌方面取得进展的关键障碍。这项提议是创新的，基于两种新的生物分析方法的开发和整合，即接近分析和被动微流控采样，用于调查最近发现的急性脂联素分泌事件，如果没有这项技术，这些事件是无法获得的。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药物治疗的时机，并使未来的工作能够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.