Hu M, Robertson D G, Murphy L J
Department of Internal Medicine, University of Manitoba, Winnipeg, Canada.
Endocrinology. 1996 Sep;137(9):3702-9. doi: 10.1210/endo.137.9.8756536.
Hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1) is enhanced in hypophysectomized (hypox) rats and can be rapidly down-regulated by GH administration. Here we examined the effect of insulin on IGFBP-1 messenger RNA abundance in hypox rats and the effects of insulin and GH on IGFBP-1/chloramphenicol acetyltransferase (CAT) reporter plasmids transiently transfected into isolated hepatocytes from pituitary-intact and hypox rats. Unlike GH, administration of insulin to hypox rats in doses of 10 or 50 micrograms/100 g BW had no effect on hepatic IGFBP-1 messenger RNA abundance. Insulin at 10(-7) M resulted in a 42.1 +/- 9.8% suppression of CAT activity in hepatocytes from pituitary-intact animals transfected with a CAT reporter plasmid containing 1671 bp of the 5'-flanking region of the rat IGFBP-1 gene. In the same assay, GH at a concentration of 2.3 x 10(-8) M significantly reduced CAT activity. In contrast, insulin had no effect on CAT activity in hepatocytes from hypox rats, whereas GH resulted in comparable suppression of CAT activity in hepatocytes from hypox rats and pituitary-intact rats, 13.6 +/- 2.3% vs. 18.2 +/- 3.2%. Deletional analysis and mobility shift assays were used to identify the GH-responsive regions in the IGFBP-1 gene. GH suppression of CAT activity was lost when the IGFBP-1 5'-flanking region was deleted down to -277 bp, whereas insulin suppression was retained for all but the smallest fragment of the IGFBP-1 gene. Mobility shift assays were used to compare nuclear extracts from sham-operated, hypox, and GH-treated hypox rats. When hepatic nuclear extracts from hypox rats were incubated with the -277 to -82 and the -556 to -368 bp fragments, retarded bands were apparent that were not present in the extracts from sham-operated rats. GH treatment of hypox rats 15 or 30 min before death completely normalized the retardation pattern seen with the -277 to -82 bp fragment, but did not affect the pattern seen with the -556 to -368 bp fragment. A 20-bp fragment corresponding to the previously identified insulin response element, -108 to -89 bp, was also analyzed. An additional retarded band, not seen with nuclear extracts from sham-operated rats, was apparent when nuclear extracts of hypox rats or GH-treated hypox rats were used. These data provide the first in vitro evidence that GH directly regulates transcription of IGFBP-1 expression. In addition, our findings suggest that GH modulates insulin regulation of IGFBP-1 transcription, possibly by altering the milieu of trans-acting factors that interact with both the insulin response element and distinct upstream sites.
胰岛素样生长因子结合蛋白-1（IGFBP-1）在垂体切除的（hypox）大鼠肝脏转录增强，而生长激素（GH）给药可使其迅速下调。在此，我们研究了胰岛素对hypox大鼠IGFBP-1信使核糖核酸丰度的影响，以及胰岛素和GH对瞬时转染到来自垂体完整和hypox大鼠的分离肝细胞中的IGFBP-1/氯霉素乙酰转移酶（CAT）报告质粒的影响。与GH不同，以10或50微克/100克体重的剂量给hypox大鼠注射胰岛素对肝脏IGFBP-1信使核糖核酸丰度无影响。10⁻⁷M的胰岛素导致转染了含大鼠IGFBP-1基因5'侧翼区1671 bp的CAT报告质粒的垂体完整动物肝细胞中CAT活性受到42.1±9.8%的抑制。在同一实验中，浓度为2.3×10⁻⁸M的GH显著降低了CAT活性。相反，胰岛素对hypox大鼠肝细胞中的CAT活性无影响，而GH导致hypox大鼠和垂体完整大鼠肝细胞中的CAT活性受到类似抑制，分别为13.6±2.3%和18.2±3.2%。采用缺失分析和迁移率变动分析来鉴定IGFBP-1基因中的GH反应区域。当IGFBP-1 5'侧翼区缺失至-277 bp时，GH对CAT活性的抑制作用消失，而胰岛素抑制作用在除IGFBP-1基因最小片段外的所有片段中均保留。迁移率变动分析用于比较假手术、hypox和GH处理的hypox大鼠的核提取物。当hypox大鼠的肝脏核提取物与-277至-82和-556至-368 bp片段孵育时，出现了滞后条带，而在假手术大鼠的提取物中不存在。在处死前15或30分钟用GH处理hypox大鼠可使-277至-82 bp片段的滞后模式完全正常化，但不影响-556至-368 bp片段的模式。还分析了与先前鉴定的胰岛素反应元件-108至-89 bp相对应的20 bp片段。当使用hypox大鼠或GH处理的hypox大鼠的核提取物时，出现了一条额外的滞后条带，而在假手术大鼠的核提取物中未见到。这些数据首次提供了体外证据表明GH直接调节IGFBP-1表达的转录。此外，我们的研究结果表明，GH可能通过改变与胰岛素反应元件和不同上游位点相互作用的反式作用因子的环境来调节胰岛素对IGFBP-1转录的调节。
