Abstract
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.
Highlights
The survival and function of the pancreatic beta-cell is critical in the pathogenesis of all forms of diabetes mellitus [1,2,3,4,5]
We simultaneously examined four parameters for each compound, including insulin promoter activity, pdx1 promoter activity, nuclear morphology, and cell number
We identify novel compounds that can significantly increase or decrease insulin gene expression
Summary
The survival and function of the pancreatic beta-cell is critical in the pathogenesis of all forms of diabetes mellitus [1,2,3,4,5]. Increased beta-cell apoptosis and decreased beta-cell function are major complicating factors in clinical islet transplantation [6,7,8,9,10]. Efforts to generate new beta-cells or increase the function and survival of beta-cells are critical. High throughput screens for novel compounds affecting beta cell function have relied on single reporters typically transfected in non beta-cell lines [17,18]. We simultaneously examined four parameters for each compound, including insulin promoter activity, pdx promoter activity, nuclear morphology, and cell number. Pdx was chosen because it is widely considered to be a master beta-cell transcription factor, controlling both the survival and function of this cell type [14,23,24,25]. Nuclear morphology and cell number were convenient indices of apoptosis and cell viability [14]
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