Figure 1. Screen of a library of known drugs for those that reverse insulin promoter repression by fatty acids.

A) Palmitate inhibited insulin promoter-GFP activity in T6PNE cells. T6PNE cells were cultured in the presence of 1µM tamoxifen and the indicated concentration of palmitate. GFP-positive cells, indicating expression from the human insulin promoter-GFP transgene introduced by lentivirus-mediated gene transfer, were visualized by fluorescence microscopy after two days. Cells were then harvested for RNA isolation and determination of insulin mRNA level by quantitative RT-PCR³⁰. Palmitate induced a dose-dependent decrease in the number of GFP-positive cells. Scale bars=200µm. B) Alverine and Benfluorex were hits in a lipotoxicity reversal screen. T6PNE cells were plated in 384 well plates in the presence of 1µM tamoxifen, 0.03 mM palmitate and compounds from the NIH/JDRF library of known drugs^4c and was conducted in triplicate because of substantial assay variability. After 48 hours, cells were fixed and read on a high throughput microscopy system. Effects of compounds on the exogenous insulin promoter in T6PNE is reported as percent GFP+ cells, as determined by imaging the green channel and normalizing to the total number of cells per well. The drugs benfluorex (green dot) and alverine (orange dot) were consistent at activating insulin promoter activity. Positive controls (orange triangle) were 0mM palmitate and negative controls were vehicle (DMSO, magenta rectangle) with no compound.