Villafuerte B C, Zhang W N, Phillips L S
Division of Endocrinology and Metabolism, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Mol Endocrinol. 1996 Jun;10(6):622-30. doi: 10.1210/mend.10.6.8776722.
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 decreased gene transcription (caused by insulin deficiency), as well as to decreased IGFBP-3 mRNA half-life (caused by low levels of IGF-I).
胰岛素依赖型糖尿病与循环中胰岛素样生长因子结合蛋白-3（IGFBP-3）水平降低有关，通过胰岛素治疗和/或输注胰岛素样生长因子-I（IGF-I）可使其恢复正常。为了解其潜在机制，我们研究了正常大鼠肝脏实质细胞和非实质细胞共培养体系中IGFBP-3的产生情况。通过配体印迹法检测IGFBP-3的释放，与10⁻¹⁰ M相比，10⁻⁸ M和10⁻⁶ M胰岛素使IGFBP-3释放分别增加了1.9倍和15倍（10⁻⁶ M与10⁻¹⁰ M相比，P < 0.05）。IGFBP-3 mRNA的表达也相应增加了23%和226%（10⁻⁶ M与10⁻¹⁰ M相比，P < 0.05），这与部分在转录前水平的调控一致。为评估mRNA稳定性，用5,6-二氯-1-β-D-呋喃核糖基苯并咪唑（DRB）抑制转录：分别加入10⁻⁵ M和10⁻¹⁰ M胰岛素时，IGFBP-3 mRNA的半衰期估计为13小时和17小时，排除了在mRNA周转水平的调控。通过细胞核连续转录分析评估IGFBP-3基因转录率，与10⁻¹⁰ M胰岛素相比，加入10⁻⁶ M胰岛素时转录率增加了2.9倍，与表达刺激情况相当。加入2.6×10⁻⁸ M和5.3×10⁻⁸ M的IGF-I使IGFBP-3释放分别增加了5.2倍和8.2倍（两者与未加IGF-I相比，P < 0.05），同时IGFBP-3 mRNA表达分别增加了14倍和29倍（两者与未加IGF-I相比，P < 0.05），表明在转录前水平存在调控。进一步研究表明，IGF-I对转录起始率没有显著影响，但使IGFBP-3 mRNA的表观半衰期延长了约2倍。通过用[QAYL]IGF-I进行研究评估了通过1型IGF-I受体对IGFBP-3的刺激作用；该类似物使IGFBP-3 mRNA表达比未加IGF-I时增加了220±27%（与野生型IGF-I时的133±9%相比，P < 0.05），表明受体介导的合成参与其中。
胰岛素刺激IGFBP-3基因转录，但使IGFBP-3释放增加的比例更大，这与在转录和翻译后水平的调控一致；相比之下，IGF-I通过减少IGFBP-3 mRNA降解来改变IGFBP-3表达，这与在转录前和转录后水平的调控一致。糖尿病状态下IGFBP-3水平降低可能是由于肝脏IGFBP-3释放减少，这继发于基因转录减少（由胰岛素缺乏引起）以及IGFBP-3 mRNA半衰期缩短（由低水平的IGF-I引起）。
