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.


CONCLUSIONS
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 decreased gene transcription (caused by insulin deficiency), as well as to decreased IGFBP-3 mRNA half-life (caused by low levels of IGF-I).
Villafuerte
B C
BC
Division of Endocrinology and Metabolism, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Zhang
W N
WN
Phillips
L S
LS
D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D005786:Gene Expression Regulation; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D000078790:Insulin Secretion; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D007328:Insulin; D018972:Insulin-Like Growth Factor Binding Protein 3; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I