A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression
| Autor: | Dan S. Luciani, Timothy J. Kieffer, James D. Johnson, Michael J. Riedel, Kevin Tsai, Ziliang Ao, Corinne A. Hoesli, James M. Piret, Tatyana B. Kalynyak, Marta Szabat, David E. Williams, Yu Hsuan Carol Yang, Raymond J. Andersen, Garth L. Warnock, Blair K. Gage, Jessica A. Hill |
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| Rok vydání: | 2010 |
| Předmět: |
Cell Survival
Science medicine.medical_treatment High-throughput screening Drug Evaluation Preclinical 030209 endocrinology & metabolism Biology Cell Line 03 medical and health sciences Biological Factors Islets of Langerhans Mice 0302 clinical medicine Insulin-Secreting Cells Gene expression Diabetes and Endocrinology/Endocrinology medicine Animals Humans Insulin Diabetes and Endocrinology/Type 2 Diabetes Gene 030304 developmental biology Regulation of gene expression Homeodomain Proteins 0303 health sciences Multidisciplinary Pancreatic islets Molecular biology Invertebrates 3. Good health Cell biology High-Throughput Screening Assays medicine.anatomical_structure Gene Expression Regulation Trans-Activators Medicine PDX1 Diabetes and Endocrinology/Type 1 Diabetes Genetic screen Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 5, Iss 9, p e12958 (2010) |
| ISSN: | 1932-6203 |
| Popis: | Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements. |
| Databáze: | OpenAIRE |
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