High content screening assay for activators of glucose transporter GLUT4

葡萄糖转运蛋白 GLUT4 激活剂的高内涵筛选试验

• 批准号: 7993032
• 负责人: Zhen Yue Jiang
• 金额: $ 19.1万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993032
• 关键词: 1-Phosphatidylinositol 3-Kinase; Biological Assay; Cell Line; Cell membrane; Cell surface; Cells; Chemicals; Chimeric Proteins; Collection; Deoxyglucose; Development; Diabetes Mellitus; Eligibility Determination; Epitopes; Factor Analysis; Fatty acid glycerol esters; Figs - dietary; GLUT4 gene; Genes; Glucose; Glucose Transporter; Hormones; Human; Human body; Immunoglobulin G; Impairment; Insulin; Insulin Resistance; Intracellular translocation; Investigation; Lead; Measurement; Measures; Membrane Transport Proteins; Metabolism; Methods; Molecular Probes; Mus; NRG1 gene; Nonesterified Fatty Acids; Palmitates; Pathway interactions; Pharmaceutical Preparations; Phosphatidylinositols; Regulation; Resistance; Screening procedure; Serum; Signal Pathway; Signal Transduction; Skeletal Muscle; Starvation; Surface; System; T-Lymphocyte; Therapeutic; Vesicle; Zeocin; antibody conjugate; base; blood glucose regulation; expression vector; extracellular; glucose disposal; glucose metabolism; glucose transport; high standard; high throughput screening; improved; insight; insulin sensitivity; insulin sensitizing drugs; novel; phosphatidylinositol 3,4,5-triphosphate; public health relevance; small molecule; stable cell line; therapeutic development; tool; uptake; vector

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.

描述（由申请人提供）：胰岛素通过增强葡萄糖转运蛋白GLUT4的易位从细胞内存储到细胞表面，刺激葡萄糖处置，在该储存中，GLUT4将葡萄糖移入细胞中以形成新陈代谢。在胰岛素缺乏症和胰岛素抵抗状态下，缺乏胰岛素作用会导致葡萄糖代谢损害和糖尿病的发展。因此，改善葡萄糖转运蛋白系统的因素可能是开发糖尿病治疗剂的潜在铅。最近，我们使用编码MYC-GLUT4-EGFP融合蛋白的表达矢量开发了一种灵敏的方法，作为定量测量GLUT4转运到细胞表面的工具。该MYC-GLUT4-EGFP易位测定法已被证明是对调节胰岛素敏感性和葡萄糖转运系统调节的因素的敏感功能分析。在这个项目中，我们建议使用新型的MyC-Glut4-EGFP易位测定方法开发高含量筛选（HCS）方法，并使用胰岛素响应和游离脂肪酸诱导的胰岛素耐胰岛素耐药蛋白CHO-T细胞稳定地表达GLUT4融合蛋白。作为原则证明，主要的HCS分析将用于筛选小分子收集，以鉴定GLUT4转运蛋白系统和胰岛素敏化剂的阳性调节剂。此外，将使用标准的2-脱氧葡萄糖摄取次级筛选测定法来验证Pilot HCS的正命中。还将实施反筛查测定法以消除非特异性活化剂。共同完成该项目的成功完成将有可能生成新的分子探针，以研究GLUT4功能机械，并为糖尿病的治疗药物提供新的见解。 公共卫生相关性：胰岛素主要通过在人类抗胰岛素耐药状态下受损的葡萄糖传输系统的激活来调节葡萄糖代谢。我们建议开发高吞吐量筛选测定法，以鉴定激活葡萄糖转运系统并提高胰岛素敏感性的潜在小分子，这可能会促进治疗糖尿病的新途径。