Insulin and insulin-like growth factor-I regulate hepatic insulin-like growth factor binding protein-3 by different mechanisms

Insulin-dependent diabetes mellitus is associated with decreased levels of circulating insulin-like growth factor binding protein-3 (IGFBP-3), which are restored toward normal by treatment with insulin and/or infusion of insulin-like growth factor-I (IGF-I). To understand underlying mechanisms, we studied IGFBP-3 production in cocultures of parenchymal and nonparenchymal cells from the livers of normal rats. Release of IGFBP-3 was measured by ligand blotting and was increased 1.9- and 15-fold by 10(-8) and 10(-8) M Insulin compared with 10(-10) M (P < 0.05 for 10(-6) vs. 10(-10) M). Expression of IGFBP-3 mRNA was increased concomitantly by 23 and 226% (P < 0.05 for 10(-6) M vs. 10(-10) M), consistent with regulation in part at pretranslational levels. To evaluate mRNA stability, transcription was inhibited with 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB): IGFBP-3 mRNA t1/2 was estimated at 13 h and 17 h with addition of 10(-5) M and 10(-10) M insulin, respectively, ruling out regulation at the level of mRNA turnover. IGFBP-3 gene transcription rates were evaluated by nuclear run-on assays and were increased 2,9-fold with the addition of 10(-6) M insulin, as compared with 10(-10) M insulin, comparable to stimulation of expression. Addition of IGF-I at 2.6 x 10(-8) M and 5.3 x 10(-8) M increased IGFBP-3 release by 5.2- and 8.2-fold (both P < 0.05 vs. no IGF-I), with concomitant increase in IGFBP-3 mRNA expression by 14- and 29-fold (both P < 0.05 vs. no IGF-I), suggesting regulation at a pretranslational level. Further studies showed that IGF-I did not have a significant effect on transcription initiation rates but prolonged the apparent half-life of IGFBP-3 mRNA about 2-fold. Stimulation of IGFBP-3 via type 1 IGF-I receptors was evaluated by studies with [QAYL] IGF-I; the analog increased IGFBP-3 mRNA expression 220 +/- 27% above the level obtained without IGF-I (vs. 133 +/- 9% with wild type IGF-I, P < 0.05), suggesting involvement of receptor-mediated synthesis. Insulin stimulates IGFBP-3 gene transcription but provides proportionally greater increases in IGFBP-3 release, consistent with regulation at both transcriptional and posttranslational levels; in contrast, IGF-I alters IGFBP-3 expression by decreasing IGFBP-3 mRNA degradation, consistent with regulation at pretranslational and posttranscriptional levels. Decreased IGFBP-3 levels in conditions of diabetes mellitus may be due to decreased hepatic IGFBP-3 release, and secondary both to de