Figure 4.

The HNF-1-binding site in region A fails to confer an inhibitory effect of insulin on the expression of a heterologous fusion gene. H4IIE cells were transiently transfected, as described (20, 22) with either the basic TKC-VI vector or constructs in which double-stranded oligonucleotides representing either the region A (RA) HNF-1-binding site (G6Pase-promoter sequence from −227 to −190; see Fig. 2A) or the region B (RB) IRS (G6Pase-promoter sequence from −197 to −159; see ref. 20) had been ligated into the BamHI site of the TK promoter in either the correct (Corr.) or inverted (Inv.) orientation, relative to that in the G6Pase promoter. After transfection, cells were incubated for 18–20 hr in serum-free medium in the presence (I) or absence (C) of 10 nM insulin. The cells were then harvested and CAT activity was 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 of ± SEM of four experiments in which each construct was assayed in duplicate.