MULTIHORMONAL REGULATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-1 IN RAT H4IIE HEPATOMA-CELLS - THE DOMINANT ROLE OF INSULIN

Circulating levels of insulin-like growth factor-binding protein-1 (IGFBP-1) and the abundance of hepatic IGFBP-1 mRNA are increased in streptozotocin-diabetic rats and are regulated in accordance with insulin and metabolic status. We recently purified rat IGFBP-1 from medium conditioned by well differentiated rat H4IIE hepatoma cells. Since this cell line provides a useful model for examining the effects of hormones on hepatocellular function, we used H4IIE cells to examine the relative role that insulin and other factors may play in the regulation of IGFBP-1 production. H4IIE cells were stabilized in serum-free medium, then treated with specific hormones. The availability of IGFBPs in conditioned medium was estimated by [I-125]IGF-I binding assay, and specific BPs were assessed by Western ligand and immunoblot analyses. The abundance of IGFBP-1 mRNA was determined by Northern and slot blot analysis. Initial studies revealed that [I-125]IGF-I-binding activity in conditioned medium was reduced after 24-h incubation with 100 nM insulin (52 +/- 4% of control; P < 0.001). In contrast, binding activity was increased after only 4 h of incubation with 75-mu-M 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) or 1-mu-M dexamethasone (P < 0.001 vs. control for each), but these effects were prevented by insulin. Ligand and immunoblotting demonstrated that insulin decreased the production of 32K and 34K forms of IGFBP-1, while both 8-CPT-cAMP and dexamethasone increased the production of IGFBP-1; again, insulin prevented the effects of 8-CPT-cAMP and dexamethasone. Of note, 1-mu-M rat GH, testosterone, progesterone, or 17-beta-estradiol had no effect on either IGF-binding activity or IGFBP-1 production. Northern and slot blot analyses revealed that 100 nM insulin profoundly lowered the abundance of IGFBP-1 mRNA in H4IIE cells (4 +/- 0.6% of control at 4 h; P < 0.001), while IGFBP-1 mRNA was increased 2-fold during incubation with 75-mu-M 8-CPT-cAMP (P < 0.001) and 9-fold with 1-mu-M dexamethasone (P < 0.001). Once again, the effect of insulin was dominant; insulin both prevented and reversed the effects of maximally effective concentrations of 8-CPT-cAMP and dexamethasone. To determine whether this effect of insulin reflected altered generation or stability of IGFBP-1 mRNA, H4IIE cells were incubated with 2.5-mu-g/ml actinomycin-D with or without insulin, and mRNA was quantitated by Northern blot. The apparent half-life of IGFBP-1 mRNA was approximately 1.3 h, and this was not reduced by insulin. We conclude that insulin rapidly lowers the abundance of IGFBP-1 mRNA and reduces the production of IGFBP-1 in H4IIE hepatoma cells, while dexamethasone and 8-CPT-cAMP augment IGFBP-1 production and mRNA. In combination with 8-CPT-cAMP, insulin exerts a dominant effect without reducing the stability of IGFBP-1 mRNA. These results support the concept that insulin plays a critical role in regulation of the production of IGFBP-1 and suggest that the major effect of insulin may occur at the level of IGFBP-1 gene transcription and/or processing of IGFBP-1 mRNA.