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(申请人提供)：胰岛素通过促进葡萄糖转运体GLUT4从细胞内存储到细胞表面的移位来刺激葡萄糖的处置，在细胞表面GLUT4将葡萄糖移动到细胞内进行代谢。在胰岛素缺乏和胰岛素抵抗状态下，胰岛素作用的缺失会导致糖代谢障碍和糖尿病的发生。因此，改善葡萄糖转运蛋白系统的因素可能成为糖尿病治疗药物发展的潜在先导。最近，我们开发了一种灵敏的方法，使用编码myc-GLUT4-EGFP融合蛋白的表达载体作为工具来定量测量GLUT4到细胞表面的移位。Myc-GLUT4-EGFP易位试验已被证明是一种灵敏的功能分析，可用于调节胰岛素敏感性和葡萄糖转运系统的因素。在这个项目中，我们提出了一种基于新的myc-GLUT4-EGFP易位试验的高含量筛选(HCS)方法，该方法使用稳定表达GLUT4融合蛋白的胰岛素反应性和游离脂肪酸诱导的胰岛素抵抗CHO-T细胞。作为一项原则证明，初级HCS试验将用于小分子收集的筛选，以确定GLUT4转运蛋白系统的阳性调节因子和胰岛素增敏剂。此外，将使用标准的基于2-脱氧葡萄糖摄取的二次筛查试验来验证试点HCS的阳性结果。还将实施反筛选试验，以消除胞外途径的非特异性激活物。总之，该项目的成功完成可能会为GLUT4功能机制的研究产生新的分子探针，并为糖尿病治疗药物提供新的见解。 与公共健康相关：胰岛素主要通过激活葡萄糖运输系统来调节葡萄糖代谢，在人类胰岛素抵抗状态下，葡萄糖运输系统受到损害。我们建议建立一种高通量筛选方法，以确定激活葡萄糖转运系统和改善胰岛素敏感性的潜在小分子，这可能为糖尿病的治疗开辟新的途径。