title: A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression.
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.
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Hill
authors.forename: Marta
authors.initials: M
authors.lastname: Szabat
authors.forename: Corinne A
authors.initials: CA
authors.lastname: Hoesli
authors.forename: Blair K
authors.initials: BK
authors.lastname: Gage
authors.forename: Yu Hsuan C
authors.initials: YH
authors.lastname: Yang
authors.forename: David E
authors.initials: DE
authors.lastname: Williams
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Riedel
authors.forename: Dan S
authors.initials: DS
authors.lastname: Luciani
authors.forename: Tatyana B
authors.initials: TB
authors.lastname: Kalynyak
authors.forename: Kevin
authors.initials: K
authors.lastname: Tsai
authors.forename: Ziliang
authors.initials: Z
authors.lastname: Ao
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Andersen
authors.forename: Garth L
authors.initials: GL
authors.lastname: Warnock
authors.forename: James M
authors.initials: JM
authors.lastname: Piret
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kieffer
authors.forename: James D
authors.initials: JD
authors.lastname: Johnson
mesh_terms: D000818:Animals; D001685:Biological Factors / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects*; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007448:Invertebrates / Q000737:chemistry*; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D001685:Biological Factors; D018398:Homeodomain Proteins; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein