Insulin stimulates glucose disposal through enhancing glucose transporter GLUT4 translocation from intracellular storage to the cell surface where GLUT4 moves glucose into the cells for metabolism. Under both insulin deficiency and insulin resistance states, the lack of insulin action leads to the impairment of glucose metabolism and the development of diabetes mellitus. Therefore, factors that improve the glucose transporter system could be potential leads for the development of therapeutics for diabetes. Recently, we developed a sensitive method using an expression vector encoding myc-GLUT4-eGFP fusion protein as a tool for quantitative measurement of GLUT4 translocation to the cell surface. This myc-GLUT4-eGFP translocation assay has proven to be a sensitive functional analysis for factors involved in the regulation of insulin sensitivity and glucose transport system. In this project, we propose to develop a high content screening (HCS) approach based on the novel myc-GLUT4-eGFP translocation assay using insulin responsive and free fatty acid-induced insulin resistant CHO-T cells stably expressing the GLUT4 fusion protein. As a proof-of-principle, the primary HCS assay will be used for screening of small molecule collection to identify positive regulators of GLUT4 transporter system and insulin sensitizers. In addition, a standard 2-deoxyglucose uptake-based secondary screening assay will be used to validate positive hits from the pilot HCS. Counter screening assays will also be implemented to eliminate non-specific activators of exocytic pathways. Together, successful completion of the proposed project would potentially generate new molecular probes for the investigation of GLUT4 functional machinery, and provide new insight for therapeutic drugs for diabetes mellitus.
Insulin regulates glucose metabolism primarily through the activation of glucose transport system that is impaired under insulin resistant state in humans. We propose to develop a high throughput screening assay to identify potential small molecules that activate glucose transport system and improve insulin sensitivity, which may promote new avenues for treatment of diabetes.
