Microfluidic devices for determining dynamics of islets of Langerhans

描述(由申请者提供)：在理解胰腺中众多的胰岛如何同步产生振荡的胰岛素分泌方面存在空白。这种间隙的持续存在是一个重要的问题，因为这些振荡对于肝脏和外周组织正常的葡萄糖吸收是必不可少的，而II型糖尿病患者有扰动的振荡。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。本研究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。中心假说是，胰腺和外周组织之间的反馈环路导致与胰岛同步的血糖水平振荡，这些振荡血糖水平通过限制胰岛内活性氧物种(ROS)的产生而对胰岛功能至关重要。这一假设是在申请者实验室形成的初步数据基础上提出的。这一建议背后的理论基础是，对胰岛同步和保护免受ROS损伤的更好的机械性理解将指导II型糖尿病新治疗方法的开发。在强大的初步数据的指导下，这一假说将通过追求三个具体目标来验证：1)确定胰岛同步的机制；2)确定同步化的胰岛行为对ROS产生的影响；3)确定葡萄糖水平波动对O-连接N-乙酰氨基葡萄糖(O-GlcNAc)修饰蛋白质的影响。在第一个目标下，将使用一个闭环微流控系统，其中来自胰岛的多肽的分泌水平将被用于葡萄糖摄取模型，以计算将被输送到胰岛的胞外葡萄糖浓度。在第二个目标下，将使用微流控装置测量葡萄糖波形对ROS产生的幅度和频率的依赖性，该装置允许同时测试一系列波形幅度，同时测量胰岛内ROS水平。在第三个目标下，将使用化学酶检测方法比较在振荡和静态葡萄糖水平下产生的O-GlcNAc修饰蛋白质的量。这种方法是创新的，因为它利用了新的微流控系统来确定胰岛同步的机制，同时也测试了胰岛生理学中的一个新范式，即振荡动力学在胰岛生理学中的保护作用。这项拟议的研究意义重大，因为II型糖尿病患者的胰岛素振荡受到干扰，这使得了解胰岛同步的机制和保护适当的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发展，以减少与II型糖尿病患者的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.