Interrogating Dynamics of Acute Secretion of Adiponectin Multimers from Adipose T

探究脂肪 T 中脂联素多聚体急性分泌的动力学

• 批准号: 8485601
• 负责人: Christopher J Easley
• 金额: $ 30.85万
• 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 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8485601
• 关键词: Acute; Address; Adipocytes; Adipose tissue; Affect; Aftercare; Biochemistry; Biological Assay; Blood Circulation; Collaborations; Detection; Development; Diabetes Mellitus; Disease; Endocrine; Endocrine Glands; Enzyme-Linked Immunosorbent Assay; Event; Fatty acid glycerol esters; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Future; Gel Chromatography; Goals; Golgi Apparatus; Heart Diseases; Hyperlipidemia; Incidence; Insulin; Insulin Resistance; Islets of Langerhans; Knowledge; Ligation; Link; Lipids; Measures; Metabolic; Metabolic Diseases; Metabolism; Methodology; Methods; Microfluidics; Mission; Molecular; Molecular Weight; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Nicotinic Acids; Obesity; Overweight; Patients; Pharmaceutical Preparations; Pharmacological Treatment; Pharmacology; Physiological; Play; Population; Proteins; Publications; Research; Resolution; Role; Sampling; Site; Solid; Stimulus; Technology; Testing; Thrombin; Time; Triglycerides; Vesicle; Western Blotting; Work; adipokines; adiponectin; base; extracellular; gel electrophoresis; in vivo; innovation; instrumentation; insulin sensitivity; novel; novel strategies; nutrition; receptor; response; sedimentation velocity; trafficking; vascular inflammation

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.

描述（由申请人提供）：而不是仅仅用于甘油三酸酯的存储位点，而是据了解，脂肪组织（脂肪）及其细胞单位脂肪细胞是对细胞外刺激反应的内分泌器官。脂联素是一种由脂肪细胞分泌的多聚体蛋白，是体内最丰富分泌的蛋白质之一，在胰岛素敏感性，中间代谢和血管炎症中起主要作用。最近的研究发现，脂肪组织具有将脂联素急性分泌到血液中的机械。然而，如果没有足够的方法来敏感地检测小体积的脂联素，则对脂联素分泌动力学的了解和对药理治疗的反应有限。特别是，显然需要更具体而易于使用的脂联素分析。这项研究的长期目标是了解急性脂联素分泌的分子机制，以帮助发现其与胰岛素抵抗，肥胖和代谢性疾病状态的关系。该建议的目的是确定急性脂联素分泌的动力学以及常用降低脂质药物对这些动力学的影响。为了填补这一知识的空白，将开发均质，敏感测定（PFRET），以定量仅几种样品的脂联素多聚体。 PFRET分析的简化读数将与初级脂肪细胞的微流体采样相结合，以最大程度地减少稀释度并在高时间分辨率下测量分泌动力学。这些研究不仅将消除脂肪细胞研究进展的关键障碍，而且还将解决有关急性脂联素分泌的重要，未解决的问题。该提案的目标1旨在开发均质蛋白质测定（PFRET），该测定法被证明是胰岛素和凝血酶检测，以直接荧光检测脂联素多聚体。 AIM 2试图确定急性脂联素分泌的动力学，其中假设急性（<10分钟）脂联素分泌来自囊泡的储备库，并且不依赖于ER到Golgi囊泡的运输。微流体分泌抽样，结合小体积Pfret，将有助于审问急性分泌。在AIM 3中，PI将研究常用的降脂药对急性脂联素分泌的影响，以检验汀类药物急性（<10分钟）分泌的脂联素多聚体的假设。该提案很重要，因为它将克服通过填补当前方法中的空白来理解急性脂联素分泌方面的关键障碍。该提案是基于两种新型生物分析方法的开发和整合，邻近测定和被动微流体采样的创新性，用于调查最近发现的没有该技术的急性脂联素分泌事件，这些事件是无法访问的。初步证据强烈支持这些建议的可行性。这些发现可以更好地告知药物管理的时机，目前正在服用脂质下脂药物的许多患者，并允许未来在发现急性脂肪因子分泌的分子和生理机制上进行未来的工作。