Fig. 5.

Rat ASBT promoter activity is decreased by insulin. A: intestinal Caco-2 cells were plated on Transwell inserts and were exposed to 50 nM insulin for 24 h from the basolateral compartment. Total RNA was extracted, and ASBT mRNA expression was assessed by real-time RT-PCR normalized to actin mRNA level (internal control). Control cells were treated with vehicle only. B: Caco-2 cells were transiently cotransfected by electroporation (Amexa) with rat ASBT or the promoter or human SLC26A3 transporter along with expression vector of human insulin receptor and CMV β for β-galactosidase to normalize for the transfection efficiency. Cells were plated on Transwell, treated with 100 nM insulin for 24 h, and harvested for firefly luciferase and β-galactosidase assays to assess the promoter activity. Control cells are cells treated with vehicle only. C: Caco-2 cells were transfected with rat ASBT promoter along with different amounts of the expression vector of human insulin receptor. Cells were plated on Transwell, treated with 100 nM insulin from 24 h, and harvested for firefly luciferase and β-galactosidase assays. Control cells are transfected with rat ASBT promoter and β-galactosidase vectors only without insulin receptor expression vector. Results are presented as % of control and represent means ± SE for 6–9 determinations performed on 3 separate occasions. *P ≤ 0.05 compared with control.