Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15, Mexico.
Am J Physiol Endocrinol Metab. 2011 Jun;300(6):E1092-102. doi: 10.1152/ajpendo.00459.2010. Epub 2011 Mar 8.
Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5' regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at -354 and -48 bp. Our results, using deletion analysis of the 5' regulatory region of the SNAT2 gene, revealed that the CRE site located at -48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.
小分子中性氨基酸转运蛋白 2（SNAT2）是带电荷的短链氨基酸最丰富和无处不在的转运蛋白。这种氨基酸转运蛋白的活性在体内或体外受到胰高血糖素或 cAMP 类似物的刺激。然而，目前尚不清楚蛋白质水平活性的增加是否是由于 SNAT2 基因转录的增加。因此，本研究的目的是研究 cAMP 是否能够刺激 SNAT2 基因表达，并定位和表征控制大鼠 SNAT2 基因表达的启动子中 cAMP 反应元件 (CRE) 的存在。我们发现，高蛋白饮食增加血清胰高血糖素浓度或给予胰高血糖素或用 forskolin 孵育肝细胞均可增加 SNAT2 mRNA 水平。然后，我们分离了 SNAT2 基因的 5'调控区，并确定转录起始位点位于翻译起始密码子上游 970bp 处。我们鉴定了两个位于 -354 和 -48bp 的潜在 CRE 位点。使用 SNAT2 基因 5'调控区的缺失分析，我们的结果表明，位于 -48bp 的 CRE 位点完全负责 cAMP 对 SNAT2 的调节。该证据得到了 CRE 位点突变和 EMSA 和 ChIP 分析的有力支持。大鼠、小鼠和人类序列的比对表明，该 CRE 位点在物种间高度保守，表明其在 SNAT2 基因表达调控中的重要作用。
