Microfluidic devices for determining dynamics of islets of Langerhans

描述（由申请人提供）：在了解胰腺中的众多胰岛如何同步产生振荡胰岛素分泌方面存在差距。这种间隙的持续存在代表了一个重要的问题，因为这些振荡对于肝脏和外周组织的适当葡萄糖摄取是必不可少的，并且II型糖尿病患者具有扰动的振荡。罗珀实验室的长期目标是通过开发新的分析技术来破译胰岛通讯。本提案的目的是确定胰岛同步化的机制，并确定其对胰岛生理的影响。核心假设是胰腺和外周组织之间的反馈回路导致使胰岛同步的振荡葡萄糖水平，并且这些振荡葡萄糖水平通过限制胰岛内活性氧（ROS）的产生而对胰岛功能至关重要。这一假设是根据申请人实验室的初步数据提出的。这一提议背后的基本原理是，更好地理解胰岛同步化和保护免受ROS损伤的机制将指导II型糖尿病新治疗方法的开发。在强有力的初步数据的指导下，将通过追求三个具体目标来测试该假设：1）确定胰岛同步化的机制; 2）确定同步化的胰岛行为对ROS产生的影响; 3）确定振荡葡萄糖水平对O-连接的N-乙酰葡糖胺（O-GlcNAc）的蛋白质修饰的影响。在第一个目标下，将使用闭环微流体系统，其中将在葡萄糖摄取模型中使用来自胰岛的肽的分泌水平来计算待递送到胰岛的细胞外葡萄糖浓度。在第二个目标下，葡萄糖波形对ROS产生的振幅和频率依赖性将使用微流体装置测量，该装置允许在测量胰岛内ROS水平的同时同时测试一系列波形振幅。在第三个目标下，将使用化学酶检测方法比较振荡和静态葡萄糖水平下产生的O-GlcNAc修饰的蛋白质的量。该方法是创新的，因为它利用新型微流体系统来确定胰岛同步化的机制，同时还测试了胰岛生理学中的新范式，即振荡动力学在胰岛生理学中的保护作用。这项研究意义重大，因为II型糖尿病患者扰乱了胰岛素振荡，因此了解胰岛同步化机制和保护葡萄糖稳态至关重要。最终，这些知识有可能指导治疗方法的开发，以减少II型糖尿病患者中与葡萄糖毒性相关的问题。

DESCRIPTION (provided by applicant): There is a gap in understanding how the numerous islets in the pancreas synchronize to produce oscillatory insulin secretion. Continued existence of this gap represents an important problem because these oscillations are essential for proper glucose uptake by the liver and peripheral tissues and type II diabetics have perturbed oscillations. The long-term goal of the Roper laboratory is to decipher islet communication by developing new analytical techniques. The objective of this proposal is to identify the mechanism of islet synchronization, and identify the effects it has on islet physiology. The central hypothesis is that a feedback loop between the pancreas and peripheral tissues result in oscillatory glucose levels that synchronize islets, and that these oscillatory glucose levels are essential to islet function by limiting the generation of reactive oxygen species (ROS) within islets. This hypothesis was formulated on the basis of preliminary data formulated in the applicants' laboratories. The rationale behind this proposal is that a better mechanistic understanding of islet synchronization and protection from ROS damage will guide the development of new treatments for type II diabetes. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the mechanism of islet synchronization; 2) Identify the effects of synchronized islet behavior on ROS production; 3) Determine the effect of oscillatory glucose levels on protein modification by O-linked N- acetylglucosamine (O-GlcNAc). Under the first aim, a closed-loop microfluidic system will be used where secretory levels of peptides from islets will be used in a model of glucose uptake to calculate the extracellular glucose concentration to be delivered to the islets. Under the second aim, the amplitude and frequency dependence of glucose waveforms on ROS generation will be measured using a microfluidic device that allows simultaneous testing of a range of waveform amplitudes while measuring intraislet ROS levels. Under the third aim, the amount of O-GlcNAc-modified proteins produced under oscillatory and static glucose levels will be compared using a chemoenzymatic detection method. The approach is innovative because it utilizes novel microfluidic systems for determining the mechanism of islet synchronization while also testing a new paradigm in islet physiology, namely the protective role of oscillatory dynamics in islet physiology. The proposed research is significant because type II diabetics have perturbed insulin oscillations, making it critical to understand the mechanism of islet synchronization and protection for proper glucose homeostasis. Ultimately, such knowledge has the potential to guide the development of therapeutics for reducing the problems associated with glucose toxicity in type II diabetics.