Parsa R, Decaux J F, Bossard P, Robey B R, Magnuson M A, Granner D K, Girard J
Centre de Recherche sur l'Endocrinologie Moléculaire et le Développement, CNRS, Meudon, France.
Eur J Biochem. 1996 Feb 15;236(1):214-21. doi: 10.1111/j.1432-1033.1996.00214.x.
Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.
此前的体内实验表明，适宜的激素环境（高血浆胰岛素、低血浆胰高血糖素）无法诱导足月胎鼠肝脏中葡萄糖激酶mRNA的积累，而在新生大鼠中则非常有效。我们在本研究中证实，胰岛素可诱导1日龄新生大鼠培养的肝细胞中葡萄糖激酶mRNA的积累，但不能诱导21日龄胎儿培养的肝细胞中该mRNA的积累。为了确定参与胰岛素反应的葡萄糖激酶基因的调控区域，我们在体内构象下扫描了葡萄糖激酶基因座的DNase I超敏位点。我们证实该基因5'侧翼区域存在四个肝脏特异性DNase I超敏位点。此外，在帽位点上游2.5 kb和3.5 kb处发现了另外两个超敏位点，但这些新位点均未表现出对胰岛素的诱导性。最后，如先前体内研究中所观察到的，超敏位点1和超敏位点2对DNase I敏感性的增加与胰岛素诱导1日龄大鼠培养肝细胞中葡萄糖激酶基因表达的能力相关。这表明，无论是预先暴露于胰岛素，还是在培养中激素存在的情况下胎儿细胞的简单老化，都无助于葡萄糖激酶基因对胰岛素新生儿反应所必需的两个近端位点的完全DNase-I超敏性。靠近肝脏转录起始位点的近端超敏位点1与一个序列（命名为IRSL）重合，该序列与磷酸烯醇丙酮酸羧激酶IRS有80%的同源性，并且与一个已鉴定的与该序列重叠的DNase-I足迹重合。然而，对该序列的功能分析表明，单独的胰岛素反应序列不太可能足以介导胰岛素对肝脏葡萄糖激酶基因的转录作用。
