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. ? ? ?
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