Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain.
Mar Biotechnol (NY). 2012 Feb;14(1):46-62. doi: 10.1007/s10126-011-9386-3. Epub 2011 May 24.
Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5'-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L: -alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.
丙氨酸氨基转移酶（ALT）在氨基酸代谢和糖异生中起着重要作用。肉食性鱼类优先选择蛋白质氨基酸而不是碳水化合物作为能量来源，这促使我们研究线粒体丙氨酸氨基转移酶（mALT）基因的转录调控，并研究不同营养和激素条件下，真鲷肾脏中 mALT 基因的酶动力学和表达调控。在瞬时转染的 HEK293 细胞中对 mALT 启动子进行 5'缺失分析、定点突变和电泳迁移率变动分析，使我们能够鉴定出 HNF4α 是参与 mALT 表达转录调控的新因子。定量 RT-PCR 分析表明，饥饿和链脲佐菌素（STZ）处理降低了真鲷肾脏中的 HNF4α 水平，导致 mALT 转录下调。组织分布分析表明，肾脏、肝脏和肠道是 mALT 和 HNF4α 表达较高的组织。动力学分析表明，mALT 酶更有效地催化 L: -丙氨酸向丙酮酸的转化，而不是相反的反应。根据这些结果，我们得出结论，HNF4α 反式激活 mALT 启动子，而饥饿和 STZ 处理的鱼肾脏中发现的 mALT 表达水平较低是由于 HNF4α 表达减少所致。我们的研究结果表明，mALT 同工酶在氧化膳食氨基酸方面起着主要作用，并指出 ALT 是一种生物技术作用的靶点，可以节约蛋白质并优化鱼类养殖中膳食营养素的利用。
