High content screening assay for activators of glucose transporter GLUT4

描述（由申请方提供）：胰岛素通过增强葡萄糖转运蛋白GLUT 4从细胞内储存转运至细胞表面（GLUT 4将葡萄糖移入细胞进行代谢）来刺激葡萄糖处置。在胰岛素缺乏和胰岛素抵抗状态下，胰岛素作用的缺乏导致糖代谢受损和糖尿病的发展。因此，改善葡萄糖转运系统的因素可能是开发糖尿病治疗药物的潜在线索。最近，我们开发了一种灵敏的方法，使用编码myc-GLUT 4-eGFP融合蛋白的表达载体作为定量测量GLUT 4易位到细胞表面的工具。这种myc-GLUT 4-eGFP易位测定已被证明是对参与调节胰岛素敏感性和葡萄糖转运系统的因素的敏感功能分析。在这个项目中，我们建议开发一个高内容筛选（HCS）的基础上，新的myc-GLUT 4-eGFP易位试验使用胰岛素响应和游离脂肪酸诱导的胰岛素抵抗CHO-T细胞稳定表达GLUT 4融合蛋白的方法。作为原理验证，主要HCS试验将用于筛选小分子集合，以鉴定GLUT 4转运蛋白系统的阳性调节剂和胰岛素增敏剂。此外，将使用标准的基于2-脱氧葡萄糖摄取的二次筛选试验来验证中试HCS的阳性命中。还将实施计数器筛选测定以消除胞吐途径的非特异性激活剂。总之，该项目的成功完成将有可能产生新的分子探针，用于研究GLUT 4功能机制，并为糖尿病治疗药物提供新的见解。 公共卫生相关性：胰岛素主要通过激活人体胰岛素抵抗状态下受损的葡萄糖转运系统来调节葡萄糖代谢。我们建议开发一种高通量筛选方法，以确定潜在的小分子，激活葡萄糖转运系统和改善胰岛素敏感性，这可能会促进新的途径，治疗糖尿病。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.