Claeyssens Sophie, Gangneux Christophe, Brasse-Lagnel Carole, Ruminy Philippe, Aki Toshihiko, Lavoinne Alain, Salier Jean-Philippe
Faculté de Médecine-Pharmacie, 22 Bvd Gambetta, 76183 Rouen cedex, France.
Am J Physiol Gastrointest Liver Physiol. 2003 Nov;285(5):G840-9. doi: 10.1152/ajpgi.00060.2003. Epub 2003 Jul 3.
Glutamine (Gln) is the most potent of the amino acids (AAs) that regulate liver anabolism, and its effect is similar to that of insulin in peripheral tissues. However, the influence of AAs on regulation of metabolic enzyme-encoding genes is not known at the molecular level in liver. We now report that Gln and some essential AAs activate the human GAPDH gene that codes for GAPDH, a central enzyme of glycolysis and a target for insulin regulation. In HepG2 cells, Gln upregulated the GAPDH mRNA level, and this effect was additive to that of insulin. Transient transfection of GAPDH promoter/cat constructs demonstrated that a gene-specific and insulin-independent transcriptional step is involved in the Gln responsiveness of GAPDH. Transfected HepG2 cells challenged with various AAs, Gln metabolites or inhibitors of Gln metabolism showed that the Gln-induced effect is similar to that of some essential AAs and that Gln metabolism is a necessary step for GAPDH activation. Deletion mutants and site-directed mutagenesis of the GAPDH promoter indicated that the Gln responsiveness is mediated by a sequence that is distinct from insulin-responsive elements and from positively acting elements previously described in this promoter. This motif located at -126/-118 clearly differs from AA-responsive elements recently identified in other genes. Electromobility shift assay and supershifts showed that the transcription factors bound to the Gln-responsive element in the GAPDH promoter are C/EBPalpha and -delta. This finding is consistent with the role of C/EBP family members in controlling the hepatic expression of genes involved in nutrient metabolism.
谷氨酰胺（Gln）是调节肝脏合成代谢的氨基酸中最有效的一种，其作用与胰岛素在周围组织中的作用相似。然而，氨基酸对肝脏中代谢酶编码基因调控的影响在分子水平上尚不清楚。我们现在报告，谷氨酰胺和一些必需氨基酸可激活编码甘油醛-3-磷酸脱氢酶（GAPDH）的人类GAPDH基因，GAPDH是糖酵解的关键酶，也是胰岛素调节的靶点。在HepG2细胞中，谷氨酰胺上调了GAPDH mRNA水平，且这种作用与胰岛素的作用具有叠加性。GAPDH启动子/氯霉素乙酰转移酶（cat）构建体的瞬时转染表明，GAPDH对谷氨酰胺的反应性涉及一个基因特异性且不依赖胰岛素的转录步骤。用各种氨基酸、谷氨酰胺代谢产物或谷氨酰胺代谢抑制剂处理转染的HepG2细胞，结果显示谷氨酰胺诱导的效应与一些必需氨基酸相似，且谷氨酰胺代谢是GAPDH激活的必要步骤。GAPDH启动子的缺失突变体和定点诱变表明，谷氨酰胺反应性是由一个与胰岛素反应元件以及该启动子中先前描述的正向作用元件不同的序列介导的。位于-126/-118的这个基序明显不同于最近在其他基因中鉴定出的氨基酸反应元件。电泳迁移率变动分析和超迁移显示，与GAPDH启动子中谷氨酰胺反应元件结合的转录因子是C/EBPα和C/EBPδ。这一发现与C/EBP家族成员在控制参与营养代谢的基因的肝脏表达中的作用一致。
