Multihormonal regulation of insulin-like growth factor-binding protein-1 in rat H4IIE hepatoma cells: the dominant role of insulin.
Multihormonal regulation of insulin-like growth factor-binding protein-1 in rat H4IIE hepatoma cells: the dominant role of insulin.
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DOI：
10.1210/endo-128-6-2693
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发表时间：
1991-06
期刊：
影响因子：
4.8
通讯作者：
T. Unterman;D. Oehler;L. Murphy;R. Lacson
中科院分区：
文献类型：
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作者：
T. Unterman;D. Oehler;L. Murphy;R. Lacson
胰岛素样生长因子结合蛋白-1（IGFBP-1）的循环水平和肝脏IGFBP-1 mRNA的丰度在链脲佐菌素糖尿病大鼠中增加，并根据胰岛素和代谢状态进行调节。我们最近从分化良好的大鼠H4 IIE肝癌细胞的条件培养基中纯化了大鼠IGFBP-1。由于该细胞系为检查激素对肝细胞功能的影响提供了有用的模型，因此我们使用H4 IIE细胞来检查胰岛素和其他因素在IGFBP-1产生调节中可能发挥的相对作用。将H4 IIE细胞稳定在无血清培养基中，然后用特定激素处理。通过[125 I]IGF-I结合试验估计条件培养基中IGFBPs的可用性，并通过Western配体和免疫印迹分析评估特异性BPs。北方杂交和狭缝杂交分析IGFBP-1 mRNA的丰度。初步研究显示，在与100 nM胰岛素孵育24小时后，条件培养基中的[125 I]IGF-I结合活性降低（对照的52 +/- 4%; P <0.001）。相反，在与75 μ M 8-（4-氯苯硫基）cAMP（8-CPT-cAMP）或1 μ M地塞米松孵育仅4小时后，结合活性增加（P均小于0.001，与对照相比），但这些作用被胰岛素阻止。配体和免疫印迹表明，胰岛素减少32 K和34 K形式的IGFBP-1的产生，而8-CPT-cAMP和地塞米松增加IGFBP-1的产生;再次，胰岛素阻止8-CPT-cAMP和地塞米松的作用。值得注意的是，1 μ M大鼠GH、睾酮、孕酮或17 β-雌二醇对IGF结合活性或IGFBP-1产生均无影响。北方和狭缝印迹分析显示，100 nM胰岛素显著降低H4 IIE细胞中IGFBP-1 mRNA的丰度（4小时时为对照的4 +/- 0.6%; P小于0.001），而在与75 μ M 8-CPT-cAMP孵育期间，IGFBP-1 mRNA增加2倍（P小于0.001）和9倍的1 μ M地塞米松（P小于0.001）。再次，胰岛素的作用是占主导地位的;胰岛素都防止和逆转8-CPT-cAMP和地塞米松的最大有效浓度的影响。为了确定胰岛素的这种作用是否反映了IGFBP-1 mRNA的产生或稳定性的改变，将H4 IIE细胞与2.5 μ g/ml放线菌素-D（含或不含胰岛素）一起孵育，并通过北方印迹定量mRNA。（400字处截断摘要）
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 [125I]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 [125I]IGF-I-binding activity in conditioned medium was reduced after 24-h incubation with 100 nM insulin (52 +/- 4% of control; P less than 0.001). In contrast, binding activity was increased after only 4 h of incubation with 75 microM 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) or 1 microM dexamethasone (P less than 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 microM 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 less than 0.001), while IGFBP-1 mRNA was increased 2-fold during incubation with 75 microM 8-CPT-cAMP (P less than 0.001) and 9-fold with 1 microM dexamethasone (P less than 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 micrograms/ml actinomycin-D with or without insulin, and mRNA was quantitated by Northern blot.(ABSTRACT TRUNCATED AT 400 WORDS)