Figure 1.

Deletion of the G6Pase-promoter sequence between −231 and −159 abolishes the inhibitory effect of insulin on basal G6Pase-CAT fusion gene expression. HepG2 cells were transiently cotransfected, as described (20, 30), with various G6Pase-CAT fusion genes (15 μg) containing distinct lengths of promoter sequence, as indicated by the 5′ deletion endpoint, and an expression vector (5 μg) encoding the insulin receptor. After transfection, cells were incubated for 18–20 hr in serum-free medium in the presence or absence of 100 nM insulin. The cells were then harvested and CAT activity and protein concentration assayed as described in Materials and Methods. Results are presented as the ratio of CAT activities in insulin-treated vs. control cells (expressed as percent control) and represent the mean ± SEM of 9–12 experiments in which each construct was assayed in duplicate. Similar basal G6Pase-CAT expression is obtained with the constructs with 5′ endpoints of −271, −198 and −158 (20) and −231 (data not shown) in the absence of insulin.