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: 8371557
• 负责人: Christopher J Easley
• 金额: $ 32.09万
• 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/8371557
• 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. PUBLIC HEALTH RELEVANCE: Nearly two-thirds of the US population is considered overweight or obese by current criteria, and with diabetes incidence on the rise, lipid-lowering drugs are now widely used to treat hyperlipidemia and related effects. Emerging evidence shows that many of these drugs modulate the synthesis and secretion of adipose-tissue secreted proteins (adipokines), which can have significant effects on insulin resistance; yet there is limited understanding of adipokine secretion dynamics and how insulin resistance is affected by these pharmacological treatments. This project is relevant to the mission of the NIDDK due to its direct relevance to the biochemistry and pharmacology of diabetes, obesity, and nutrition-related disorders.

描述（由申请人提供）：现在人们了解到，脂肪组织（脂肪）及其细胞单位（脂肪细胞）不仅仅是甘油三酯的储存场所，而是充当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着重要作用。最近的研究发现，脂肪组织具有将脂联素快速分泌到血液中的机制。然而，由于没有足够的方法来灵敏地检测小体积的脂联素，因此对脂联素分泌动力学和对药物治疗的反应的了解有限。特别是，显然需要更特异且易于使用的脂联素测定。这项研究的长期目标是了解脂联素急性分泌的分子机制，以帮助揭示其与胰岛素抵抗、肥胖和代谢疾病状态的关系。该提案的目的是确定急性脂联素分泌的动态以及常用降脂药物对这些动态的影响。为了填补这一知识空白，将开发均质、灵敏的测定法 (pFRET)，用于仅从几微升的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，以最大限度地减少稀释并以高时间分辨率测量分泌动态。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的、尚未解决的问题。该提案的目标 1 旨在开发均质蛋白检测 (pFRET)，该检测已被证明可用于胰岛素和凝血酶检测，以及脂联素多聚体的直接荧光检测。目标 2 旨在确定急性脂联素分泌的动态，假设急性（<10 分钟）脂联素分泌源自囊泡储备池，并且不依赖于内质网到高尔基体囊泡的运输。微流控分泌物采样与小体积 pFRET 相结合，将有助于检查急性分泌物。在目标 3 中，PI 将研究常用降脂药物对脂联素急性分泌的影响，检验他汀类药物急剧（<10 分钟）分泌的脂联素多聚体的假设。该提案意义重大，因为它将通过填补当前方法中的空白，克服在理解急性脂联素分泌方面取得进展的关键障碍。该提案具有创新性，基于两种新型生物分析方法（邻近分析和被动微流体采样）的开发和整合，用于研究最近发现的急性脂联素分泌事件，而如果没有该技术，则无法获得这些事件。初步证据有力地支持了这些建议的可行性。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用药时机，并允许未来开展揭示急性脂肪因子分泌的分子和生理机制的工作。 公共健康相关性：按照目前的标准，近三分之二的美国人口被认为超重或肥胖，并且随着糖尿病发病率的上升，降脂药物现在被广泛用于治疗高脂血症和相关影响。新的证据表明，其中许多药物可调节脂肪组织分泌蛋白（脂肪因子）的合成和分泌，这对胰岛素抵抗有显着影响；然而，对脂肪因子分泌动力学以及这些药物治疗如何影响胰岛素抵抗的了解有限。该项目与 NIDDK 的使命相关，因为它与糖尿病、肥胖和营养相关疾病的生物化学和药理学直接相关。