Dynamic of Pancreatic Islet Function

胰岛功能动态

• 批准号: 6640202
• 负责人: David W Piston
• 金额: $ 29.26万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6640202
• 关键词: Adenoviridae; NAD(H) phosphate; adenosine diphosphate; adenosine triphosphate; biological signal transduction; confocal scanning microscopy; enzyme activity; enzyme mechanism; fluorescence microscopy; gap junctions; gene expression; gene targeting; genetically modified animals; glucokinase; glucose metabolism; immunofluorescence technique; insulin; intracellular; laboratory mouse; pancreatic islet function; recombinant virus; secretion; tissue /cell culture

DESCRIPTION (provided by applicant): Blood glucose homeostasis depends on glucose-stimulated insulin secretion (GSIS) from b cells in pancreatic islets. Diminished insulin secretion, along with peripheral insulin resistance, is a hallmark of Type II diabetes. Recent work indicates that glucose signaling depends on subcellular localization of molecules. For instance, biochemical and immunolabeling approaches have shown that glucokinase (GK) is associated with insulin granules. However, these data provide only static snapshots of b cell function and do not elucidate the interplay between signaling pathways involved in GSIS. We have now shown that binding of GK to granules is regulated by b cell insulin receptor and that this regulation correlates with GK activity changes. Other work has shown that while glucose stimulates increases in both cytoplasmic and mitochondrial metabolism, certain nutrients can cause secretion through mitochondrial metabolism alone, yet the mechanisms underlying these different processes is not understood. Intercellular synergy also plays a role in GSIS, since b cells within intact islets secrete approximately 10-fold more insulin in response to glucose than do isolated b cells; this functional enhancement can only be examined in intact islets. Using our unique quantitative optical imaging methods, the dynamics of these subcellular events can be followed accurately in living cells within intact islets. We hypothesize that dynamic subcellular localization of glucose signaling, such as binding of GK to secretory vesicles and compartmentalization in organelles, is required for normal regulation of GSIS. We also hypothesize that gap junctional coupling between b cells in the islet accounts for the increased insulin response from intact islets over isolated b cells. The validity and limits of these hypotheses will be determined via three specific aims: 1) To determine the ability of gap junctional coupling to enhance insulin secretion. 2) To determine the mechanism of GK binding to insulin containing vesicles and the role of this binding in the regulation of GK activity. 3) To determine the relative roles of cytoplasmic and mitochondrial metabolism in GSIS, and determine the ability of each compartment to compensate for defects in the normal signaling pathway. Because numerous studies have shown marked differences between isolated b cells and whole islets, the proposed experiments will be performed largely on intact islets, but guided by parallel investigations using tissue culture models. Experiments on intact islets are made possible through a unique combination of quantitative fluorescence imaging methods and will utilize several available transgenic and tissue-specific knock-out mouse models with demonstrated diabetic phenotypes. Further, we propose a knock-in mouse model containing a GK-GFP fusion that will greatly facilitate the proposed experiments. These novel investigations will advance our understanding of the in vivo interplay between biochemical mechanisms that are directly involved in diabetic phenotypes, and bring us closer to our long-term goal of understanding the spatio-temporal dynamics of glucose-stimulated insulin secretion.

描述（由申请人提供）：血糖稳态取决于胰岛中B细胞的葡萄糖刺激的胰岛素分泌（GSI）。胰岛素分泌减少，以及周围胰岛素抵抗，是II型糖尿病的标志。最近的工作表明葡萄糖信号传导取决于分子的亚细胞定位。例如，生化和免疫标记方法表明葡萄糖激酶（GK）与胰岛素颗粒有关。但是，这些数据仅提供B细胞功能的静态快照，并且不会阐明GSIS中涉及的信号通路之间的相互作用。现在，我们已经表明，GK与颗粒的结合受B细胞胰岛素受体的调节，并且该调节与GK活性变化相关。其他工作表明，虽然葡萄糖刺激细胞质和线粒体代谢的增加，但某些营养物质可能仅通过线粒体代谢引起分泌，但尚不了解这些不同过程的机制。细胞间的协同作用在GSI中也起作用，因为完整胰岛内的B细胞对葡萄糖的响应比分离的B细胞分泌大约10倍的胰岛素。这种功能增强只能在完整的胰岛中检查。使用我们独特的定量光学成像方法，可以在完整胰岛内的活细胞中准确遵循这些亚细胞事件的动力学。我们假设葡萄糖信号传导的动态亚细胞定位（例如GK与分泌囊泡的结合和细胞器中的分隔）是GSIS的正常调节所必需的。我们还假设，胰岛中B细胞之间的间隙连接耦合是隔离B细胞中完整胰岛的胰岛素反应增加的增加。这些假设的有效性和限制将通过三个特定目的确定：1）确定间隙连接耦合增强胰岛素分泌的能力。 2）确定GK与含有囊泡的胰岛素结合的机理，以及该结合在GK活性调节中的作用。 3）确定GSI中细胞质和线粒体代谢的相对作用，并确定每个隔室补偿正常信号传导途径中缺陷的能力。由于许多研究表明，分离的B细胞和整个胰岛之间存在明显的差异，因此提出的实验将主要在完整的胰岛上进行，但通过使用组织培养模型进行平行研究。通过定量荧光成像方法的独特组合，可以实现完整胰岛的实验，并将利用几种可用的转基因和组织特异性的敲除小鼠模型，并具有证明的糖尿病表型。此外，我们提出了一个含有GK-GFP融合的敲门小鼠模型，该模型将极大地促进所提出的实验。这些新颖的研究将促进我们对直接参与糖尿病表型的生化机制之间体内相互作用的理解，并使我们更接近我们了解葡萄糖刺激的胰岛素分泌的时空动力学的长期目标。