Figure 8

Overexpression of GSK-3β reduces insulin regulation of the IGFBP-1 TIRE. H4IIE cells were infected with adenovirus expressing either β-Galactosidase (control), GSK-3β (GSK-3wt), or insulin-insensitive GSK-3β (GSK-3S9A). A) Cells were incubated for 16 hours before transfection with 10 μg of BP-1 WT as described under the Methods section. After 24 hours at 37°C +/- insulin (10 nM) cells were lysed and either luciferase assays performed (upper panel) or GSK-3 levels determined by Western Blot (lower panel). Results in the upper panel are presented as % insulin repression of luciferase expression and are the means +/- S.E.M. of at least five experiments performed in duplicate or triplicate. Basal luciferase expression is 3-fold higher in WT and S9A-GSK-3 infected cells compared with control. The lower panels provide a representative analysis of expression of GSK-3 (in triplicate) in each treatment. There was a significant reduction in the effect of insulin on BP-1 WT when either GSK-3 WT (***p < 0.001, control vs WT) or GSK-3 S9A (***p < 0.001, control vs S9A) was overexpressed. There is no significant difference between the WT and S9A data sets (p = 0.160). B) After infection cells were incubated for 24 hr prior to a 3 hr incubation with hormones as indicated. Cells were lysed and IGFBP-1 and cyclophilin mRNA levels assessed by RNase Protection Assay. A representative experiment is shown (lower panel), while relative mRNA levels ± SEM are presented for two experiments performed in duplicate in the upper panel.