Analysis of regulation of gene expression of an insulin-inducible transcription factor and its action mechanism

我们在大鼠肝脏中发现了一种基本的螺旋-环-螺旋蛋白，即分裂和毛发相关蛋白2 （SHARP-2）的增强子，作为胰岛素诱导的转录抑制因子。在本研究中，我们检测了夏普-2转录抑制因子对胰岛素可调节的大鼠磷酸烯醇丙酮酸羧激酶（PEPCK）基因表达的影响。腺病毒介导的胰岛素应答中SHARP-2的过表达。H4IIE大鼠肝癌细胞和原代培养大鼠肝细胞导致PEPCK mRNA水平降低。此外，当SHARP-2表达质粒与多种报告质粒瞬时转染到MH_1C_1细胞时，PEPCK报告质粒的启动子活性特异性降低。基于这些发现，我们得出结论，SHARP-2是PEPCK基因表达的潜在抑制因子。然后，为了研究大鼠夏普-2基因启动子的胰岛素反应元件（IRE），将夏普-2/萤火虫荧光素酶报告质粒转染到H4IIE细胞中。在存在或不存在胰岛素的情况下，测定并比较它们的启动子活性。然而，在该基因转录起始位点上游10 kb以内没有IREs。另一方面，脂肪细胞分泌的脂联素通过amp激活的蛋白激酶（AMPK）途径转录抑制肝脏PEPCK基因，从而降低血糖水平。为了确定AMPK是否会影响大鼠夏普-2基因的表达，我们在使用AICAR（一种化学AMPK激活剂）处理的H4IIE细胞中检测了夏普-2 mRNA的水平。胰岛素治疗后，血糖水平迅速而短暂地升高。由此可见，AMPK可诱导大鼠SHARP-2基因的表达。

We identified a basic helix-loop-helix protein, enhancer of split-and hairy-related protein-2 (SHARP-2), as an insulin-inducible transcriptional repressor in the rat liver. In this study, the influence of the SHARP-2 transcriptional repressor on the expression of an insulin-regulatable rat phosphoenolpyruvate carboxykinase (PEPCK) gene, was examined. The adenovirus-mediated overexpression of SHARP-2 in insulin responsive. H4IIE rat hepatoma cells and primary cultured rat hepatocytes led to a decrease in the levels of PEPCK mRNA. In addition, when a SHARP-2 expression plasmid was transiently transfected with various reporter plasmids into MH_1C_1 cells, the promoter activity of a PEPCK reporter plasmid was specifically decreased. Based on these findings, we concluded that SHARP-2 is a potential repressor of PEPCK gene expression. Then, to investigate the insulin responsive element (IRE) of the rat SHARP-2 gene promoter, SHARP-2/ firefly luciferase reporter plasmids were transfected into H4IIE cells. In the presence or absence of insulin, their promoter activities were determined and compared. However, there was no IREs up to 10 kb upstream of the transcription initiation sites of this gene. On the other hand, adiponectin secreted from adipocytes lower blood glucose level by transcriptional repression of hepatic PEPCK gene via an AMP-activated protein kinase (AMPK) pathway. To determine an issue of whether AMPK affects the expression of the rat SHARP-2 gene, the levels of SHARP-2 mRNA were determined in H4IIE cells treated with AICAR, a chemical AMPK activator. The levels were rapidly and temporally increased as well as insulin treatment. Thus, the result suggests that AMPK induces the expression of the rat SHARP-2 gene.