Marie S, Diaz-Guerra M J, Miquerol L, Kahn A, Iynedjian P B
Division de Biochimie Clinique, University of Geneva School of Medicine, Switzerland.
J Biol Chem. 1993 Nov 15;268(32):23881-90.
The insulinoma beta-cell line INS-1 expresses the L-type pyruvate kinase gene at high level and responds to a rise in extracellular glucose by strong induction of gene expression. Following the addition of glucose to the culture medium in the 3.5-33 mM concentration range, the cellular level of L-type pyruvate kinase mRNA increases within 2 h and reaches a maximum 15-fold above basal in 8-12 h. By run-on nuclear assay, the relative transcription rate of the pyruvate kinase gene is shown to increase 4-fold at maximal stimulation, suggesting that both transcriptional and post-transcriptional effects contribute to mRNA accumulation. The glucose effect is totally suppressed by the hexokinase inhibitor mannoheptulose, indicating a requirement for glucose phosphorylation. The mRNA induction is not inhibited in glutamine-free culture medium or by azaserine, suggesting that the hexosamine biosynthetic pathway is not involved. Moreover, metabolism along the glycolytic pathway does not appear to be an absolute requisite, since 2-deoxyglucose partly mimics the inductive effect of glucose. The glucose effect on the pyruvate kinase gene is reversibly antagonized by agents increasing intracellular cAMP. In addition, the effect is highly specific to the pyruvate kinase gene. Neither proinsulin I mRNA nor glucokinase mRNA are increased in glucose-stimulated INS-1 cells. Short term transfection with CAT plasmids driven by the pyruvate kinase L promoter reveals specific glucose-inducible reporter activity with the 183-base pair promoter region upstream of the cap site. Within this region, the previously described L4 cis-acting element is crucial for glucose responsiveness, as demonstrated by the fact that a plasmid with a mutation in this element does not elicit glucose-inducible CAT activity. Induction of L-type pyruvate kinase mRNA occurs in the islets of rats subjected to fasting and carbohydrate refeeding. In conclusion, the L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene in the endocrine beta-cell type.
胰岛素瘤β细胞系INS-1高水平表达L型丙酮酸激酶基因，并通过强烈诱导基因表达来响应细胞外葡萄糖浓度的升高。在培养基中添加浓度范围为3.5 - 33 mM的葡萄糖后，L型丙酮酸激酶mRNA的细胞水平在2小时内增加，并在8 - 12小时内达到比基础水平高15倍的最大值。通过核转录活性分析，丙酮酸激酶基因的相对转录速率在最大刺激时增加4倍，表明转录和转录后效应都有助于mRNA的积累。己糖激酶抑制剂甘露庚酮糖完全抑制了葡萄糖效应，表明葡萄糖磷酸化是必需的。在无谷氨酰胺的培养基中或用重氮丝氨酸处理时，mRNA的诱导不受抑制，这表明己糖胺生物合成途径不参与其中。此外，沿糖酵解途径的代谢似乎不是绝对必需的，因为2-脱氧葡萄糖部分模拟了葡萄糖的诱导作用。增加细胞内cAMP的试剂可可逆地拮抗葡萄糖对丙酮酸激酶基因的作用。此外，该效应对丙酮酸激酶基因具有高度特异性。在葡萄糖刺激的INS-1细胞中，胰岛素原I mRNA和葡萄糖激酶mRNA均未增加。用丙酮酸激酶L启动子驱动的CAT质粒进行短期转染，发现在帽位点上游183个碱基对的启动子区域具有特异性的葡萄糖诱导型报告基因活性。在该区域内，先前描述的L4顺式作用元件对于葡萄糖反应性至关重要，这一事实证明，该元件发生突变的质粒不会引发葡萄糖诱导型CAT活性。在禁食和碳水化合物再喂养的大鼠胰岛中也会发生L型丙酮酸激酶mRNA的诱导。总之，L型丙酮酸激酶基因提供了一个在内分泌β细胞类型中葡萄糖调节基因的有趣模型。
