Bonny C, Thompson N, Nicod P, Waeber G
Department of Internal Medicine B, University Hospital of Lausanne, Switzerland.
Mol Endocrinol. 1995 Oct;9(10):1413-26. doi: 10.1210/mend.9.10.8544849.
A defect in glucose sensing of the pancreatic beta-cells has been observed in several animal models of type II diabetes and has been correlated with a reduced gene expression of the glucose transporter type 2 (Glut2). In a transgenic mouse model, expression of Glut2 antisense RNA in pancreatic beta-cells has recently been shown to be associated with an impaired glucose-induced insulin secretion and the development of diabetes. To identify factors that may be involved in the specific decrease of Glut2 in the beta-cells of the diabetic animal, an attempt was made to localize the cis-elements and trans-acting factors involved in the control of Glut2 expression in the endocrine pancreas. It was demonstrated by transient transfection studies that only 338 base pairs (bp) of the murine Glut2 proximal promoter are needed for reporter gene expression in pancreatic islet-derived cell lines, whereas no activity was detected in nonpancreatic cells. Three cis-elements, GTI, GTII, and GTIII, have been identified by DNAse I footprinting and gel retardation experiments within these 338 bp. GTI and GTIII bind distinct but ubiquitously expressed trans-acting factors. On the other hand, nuclear proteins specifically expressed in pancreatic cell lines interact with GTII, and their relative abundance correlates with endogenous Glut2 expression. These GTII-binding factors correspond to nuclear proteins of 180 and 90 kilodaltons as defined by Southwestern analysis. The 180-kilodalton factor is present in pancreatic beta-cell lines but not in an alpha-cell line. Mutation of the GTI or GTIII cis-elements decreases transcriptional activity directed by the 338-bp promoter, whereas mutation of GTII increases gene transcription. Thus negative and positive regulatory sequences are identified within the proximal 338 bp of the GLUT2 promoter and may participate in the islet-specific expression of the gene by binding beta-cell specific trans-acting factors.
在几种II型糖尿病动物模型中已观察到胰腺β细胞的葡萄糖感知缺陷，且这与葡萄糖转运蛋白2（Glut2）的基因表达降低相关。在一个转基因小鼠模型中，最近发现胰腺β细胞中Glut2反义RNA的表达与葡萄糖诱导的胰岛素分泌受损及糖尿病的发生有关。为了确定可能参与糖尿病动物β细胞中Glut2特异性减少的因素，人们试图定位参与内分泌胰腺中Glut2表达调控的顺式元件和反式作用因子。瞬时转染研究表明，在胰岛来源的细胞系中，报告基因表达仅需要鼠Glut2近端启动子的338个碱基对（bp），而在非胰腺细胞中未检测到活性。通过DNA酶I足迹法和凝胶阻滞实验在这338 bp内鉴定出了三个顺式元件，即GTI、GTII和GTIII。GTI和GTIII结合不同但普遍表达的反式作用因子。另一方面，在胰腺细胞系中特异性表达的核蛋白与GTII相互作用，它们的相对丰度与内源性Glut2表达相关。这些与GTII结合的因子对应于通过蛋白质印迹分析确定的180和90千道尔顿的核蛋白。180千道尔顿的因子存在于胰腺β细胞系中，而不存在于α细胞系中。GTI或GTIII顺式元件的突变会降低由338 bp启动子指导的转录活性，而GTII的突变会增加基因转录。因此，在GLUT2启动子的近端338 bp内鉴定出了负性和正性调控序列，它们可能通过结合β细胞特异性反式作用因子参与该基因的胰岛特异性表达。
