A Glucose Nanosensor for Continuous Glucose Monitoring within Living Cells

描述（由申请人提供）：帮助理解糖尿病和肥胖症病理生理学的关键问题之一是葡萄糖在各种细胞和组织中的转运和代谢。这两个过程都依赖于葡萄糖的局部浓度。研究人员可以很容易地使用各种方法来测量血糖，特别是在动物研究中。然而，由于缺乏对细胞无损的方法，在活细胞内连续监测葡萄糖一直是糖尿病研究中的挑战。我们在此提出建立一种新的技术，使用荧光纳米传感器可视化细胞内的葡萄糖，并进一步证明该技术在确定骨骼肌细胞中的葡萄糖摄取中的实用性。骨骼肌中胰岛素刺激的葡萄糖处置受损先于并促成2型糖尿病以及肥胖症的发展。这项新技术的建立不仅可以量化肌肉葡萄糖代谢的两个初始步骤：葡萄糖转运和磷酸化;还有助于了解胰岛素抵抗受试者中肥胖和2型糖尿病的发展。我们从大肠杆菌中分离到一种葡萄糖结合蛋白（GBP）。杆菌利用这种蛋白质，我们设计了一种“葡萄糖指示蛋白”（GIP），它显示荧光强度随葡萄糖浓度的变化。我们还开发了一种构建组合文库的方法，以便我们可以筛选对葡萄糖具有不同亲和力的GBP。我们假设一类具有不同葡萄糖响应范围的GIP可以使用选自组合文库并且对葡萄糖具有不同亲和力的各种GBP突变体进行工程化。这些GIP的基因将被引入细胞中，使得细胞内荧光纳米传感器可以被生物合成并保留在细胞内，用于通过寿命FRET（福斯特共振能量转移）显微镜观察葡萄糖。提出了三个具体目标，包括：i）通过寿命FRET显微镜观察活细胞内的葡萄糖; ii）通过诱变来操纵GBP的葡萄糖结合亲和力; iii）证明GIP在确定骨骼肌细胞中葡萄糖摄取中的效用。这项新技术将使连续监测活细胞内的葡萄糖浓度成为可能，为研究胰岛素抵抗受试者中2型糖尿病和肥胖的发展提供关键数据。长期目标是引入这种技术用于连续监测葡萄糖，并开发闭环控制的胰岛素输送系统，以更好地控制糖尿病患者的血糖。

DESCRIPTION (provided by applicant): One of the key issues to assist the understanding of the pathophysiology of diabetes and obesity is the transport and metabolism of glucose in various cells and tissues. Both of these processes are dependent upon the local concentration of glucose. A variety of methods for measuring blood glucose are readily available to researchers, particularly for animal studies. Nevertheless, the continuous monitoring of glucose within living cells has been a challenge in diabetes researches due to the lack of a methodology that is nondestructive to the cells. We herein propose to establish a novel technology to visualize glucose inside the cells using a fluorescence nanosensor and further to demonstrate the utility of this technology in determining the glucose uptake in skeletal muscle cells. Impaired insulin-stimulated glucose disposal in skeletal muscle precedes and contributes to the development of type 2 diabetes as well as the obesity. The establishing of this new technology will not only allow quantifying the two initial steps of muscular glucose metabolism: glucose transport and phosphorylation; but also help understand the development of obesity and type 2 diabetes in insulin-resistant subjects. We have isolated a glucose binding protein (GBP) from E. coli. With this protein, we engineered a "glucose indicator protein" (GIP) that displays a change in fluorescence intensity as a function of glucose concentration. We have also developed an approach to construct a combinatorial library so that we can screen for GBPs with varied affinities for the glucose. We hypothesize that a class of GIPs that possess different glucose response ranges can be engineered using various GBP's mutants that are selected from a combinatorial library and have varied affinities for the glucose. The gene of these GIPs will be introduced into cells so that an intracellular fluorescence nanosensors can be biosynthesized and remain inside the cells for visualizing the glucose through the lifetime FRET (Forster resonance energy transfer) microscopy. Three specific aims are proposed including: i) to visualize glucose within living cells through the lifetime FRET microscopy; ii) to manipulate the glucose binding affinity of GBP by mutagenesis; iii) to demonstrate the utility of GIP in determining the glucose uptake in skeletal muscle cells. This new technology will make it possible to continuously monitor glucose concentrations inside living cells, providing the key data for studying the development of type 2 diabetes and obesity in insulin resistant subjects. A long-term goal is to introduce this technology for continuous monitoring glucose and developing a close loop controlled insulin delivery system for a better blood glucose control in diabetics.