ABSTRACT
Compounds that enhance or inhibit insulin secretion could become therapeutics as well as lead to the identification of requisite β cell regulatory pathways and increase our understanding of pancreatic islet function. Toward this goal, we previously generated an insulin-linked luciferase that is co-secreted with insulin in MIN6 β cells. With this assay we completed a high-throughput natural product screen for chronic effects on glucose-stimulated insulin secretion. Using a distributive phenotypic analysis approach we identified that one of the top natural product hits, chromomycin A2 (CMA2), potently inhibited insulin secretion (EC50=11.8 nM) through at least three mechanisms: disruption of Wnt signaling, interfering with β cell gene expression, and suppression of triggering calcium influx. Chronic treatment with CMA2 largely ablated glucose-stimulated insulin secretion even post-washout, but did not inhibit glucose-stimulated generation of ATP, Ca2+ influx, or ERK1/2 activation. However, experiments using the KATP channel opener diazoxide uncovered defects in the triggering Ca2+ influx which may contribute to the suppressed secretory response. Using the FUSION bioinformatic database, we found that the phenotypic effects of CMA2 clustered with a number of Wnt/GSK3β pathway-related genes. Consistently, CMA2 decreased GSK3β phosphorylation and suppressed activation of a β-catenin activity reporter. CMA2 and a related natural product mithramycin are described to have DNA-interaction properties, possibly abrogating transcription factor binding to critical β cell gene promoters. We observed that CMA2, but not mithramycin, suppressed expression of PDX1 and UCN3. Neither expression of INSI/II or insulin content was affected. We conclude that chronic treatment with CMA2 treatment results in the disruption of signaling pathways and expression of genes that support β cell function that both support Ca2+ influx and are required downstream, independent of insulin abundance. Future applications of CMA2 and similar aureolic acid analogs for disease therapies should consider the potential impacts on pancreatic islet function.
