The analysis of transcriptional regulation in a series of glucose responsive genes.

我们已分离到人胃抑制多肽（GIP）基因、肝型葡萄糖转运蛋白2（GLUT 2）基因和生长抑素受体（SSTR 2）基因。这些基因的表达受葡萄糖浓度的调节，但其机制尚不清楚。在本研究中，我们分离了人GIP基因、人GLUT 2基因和小鼠SSTR 2基因的5 '启动子区域，并产生了各种缺失突变体。将这些缺失突变体克隆到氯霉素乙酰转移酶（CAT）报告质粒或荧光素酶报告质粒中，转染HIT-T15细胞，检测其启动子活性。在人GIP基因中，两个环AMP反应元件（CRE）位于转录起始位点的上游，并且-180和+14之间的序列对其启动子活性是必需的。此外，钙补充剂通过两个克雷斯增加人GIP启动子活性，并且这种现象被高葡萄糖浓度增强。对于人GLUT 2基因，缺失分析表明转录起始位点下游（+126-+308）的序列对于基础启动子活性是不可缺少的，并且+29和+308之间的序列有助于葡萄糖诱导的转录增强。在原代培养的大鼠肝细胞中也观察到类似的现象。我们用PCR-SSCP方法检测了糖尿病人群中GLUT 2基因葡萄糖敏感序列的突变情况，但在所研究的糖尿病患者中未发现突变。我们还检测了小鼠SSTR 2基因5 '侧翼区的启动子活性。瞬时表达研究表明，没有显着的葡萄糖诱导的启动子活性增强，但删除分析显示两个可能的转录起始位点。

We previously isolated human gastirc inhibitory polypeptide (GIP) gene, liver type glucose transporter (GLUT2) gene and somatostatin receptor (SSTR2) gene. The expression of these genes are regulated by concentration of glucose, but the mechanisms are unknown. In this study, we isolated the 5'-promoter regions of human GIP gene, human GLUT2 gene and mouse SSTR2 gene and generated various deletion mutants. After cloned into chloramphenicol acetyl-transferase (CAT) reporter plasmid or luciferase reporter plasmid, these deletion mutants were transfected to HIT-T15 cells, and the promoter activities were assayd. In case of human GIP gene, tow cyclic-AMP respnse elements (CRE) are located upstream of the transcription initiation site and sequences between -180 and +14 are essential to its promoter activity. Furthermore, calcium supplement increases the human GIP promoter activity via two CREs and this phenomenon is augmented by high glucose concentration. As to human GLUT2 gene, deletion analysis shows that sequences downstream of the transcription initiation site (+126-+308) are indispensable for the basal promoter activity and the sequences between +29 and +308 contributed to glucose induced transcription enhancement. Similar phenomenon is cbserved in primary cultured rat hepatocytes. We performed PCR-SSCP analysis to detect the mutation in these glucose responsive sequences of the human GLUT2 gene in diabetic polulation, but no mutation was found in diabetics studied. We also examined the promoter activity of the mouse SSTR2 gene 5'-flanking region. Transient expression studies showed no significant glucose-induced augmentation of the promoter activity, but deletion analyzes revealed two possible transcription initiation sites.