GLUCOKINASE GENE EXPRESSION IN THE PANCREATIC BETA CELL

胰腺β细胞的电、离子和分泌反应 葡萄糖是由糖的代谢介导的。 葡萄糖激酶（GK） 催化葡萄糖利用中的初始和限速步骤 在生理葡萄糖浓度下被β细胞吸收， 在调节胰岛素分泌中起重要作用。 研究 GK在β细胞中的功能和调节 从肝脏和胰岛素瘤组织中鉴定编码GK的cDNA 并绘制了两种组织中的转录单位。 我们 研究表明在单个GK基因中有两种不同的启动子。 一种启动子在肝脏中特异性表达， 受胰岛素调节，而另一个启动子位于更上游， 只在β细胞中活跃 我们建议研究 GK在胰岛素瘤细胞系中的功能和调节（Aim 1）， 使用融合基因分析检查GK β细胞启动子（Aim 2）。 在 目标1互补方法将用于测试GK的功能， β细胞“葡萄糖传感器”。 通过改变GK在两个细胞中的表达， 不同的胰岛素瘤细胞系，我们希望改变“设定点”， 葡萄糖刺激的胰岛素分泌。 RINm 5 F细胞具有正常葡萄糖 摄取，但似乎缺乏高Km葡萄糖磷酸化活性， 对葡萄糖没有反应而分泌胰岛素。 另一种细胞系β-TC 显示高Km葡萄糖使用，但刺激分泌胰岛素较少 比正常的葡萄糖浓度。 我们将确定 在RINm 5 f细胞中表达GK以及表达GK的作用 反义RNA在β-TC细胞中的表达。 在目标2中，对β细胞的分析 GK启动子将使用融合基因方法进行。 两个5' 缺失分析和内部缺失突变体的使用应该能够 我们鉴定β细胞特异性表达所必需的元件， GK。 对于GK的细胞类型表达重要的元件可能是 结合对β细胞特异性基因表达重要的因子。 这些因素将进一步研究使用迁移率变化和甲基化 干扰测定。 努力纯化和克隆这些因子之一， 被启动。

The electrical, ionic, and secretory responses of pancreatic beta cells to glucose are mediated by the metabolism of the sugar. Glucokinase (GK) catalyzes the initial and rate limiting step in the utilization of glucose by the beta cell at physiologic glucose concentrations and is thought to play an important role in modulating insulin secretion. To study the function and regulation of GK in the beta cell further, we have characterized cDNAs encoding GK from both the liver and insulinoma tissue and have mapped the mapped the transcription units in both tissues. Our studies have shown there are two different promoters in a single GK gene. One promoter is expressed specifically in the liver and appears to be regulated by insulin while another promoter, located further upstream, appears active only in the beta cell. We are proposing to study the function and regulation of GK in insulinoma cell lines (Aim 1) and to examine the GK beta cell promoter using a fusion gene analysis (Aim 2). In Aim 1 complimentary approaches will be used to test the function of GK as the beta cell "glucose sensor". By altering the expression of GK in two different insulinoma cell lines we hope to alter the "set point" of glucose-stimulated insulin secretion. RINm5F cells have normal glucose uptake but appear to lack a high-Km glucose phosphorylating activity and do not secrete insulin in response to glucose. Another cell line, beta-TC, shows high-Km glucose usage but is stimulated to secrete insulin by less than normal glucose concentrations. We will determine the effects of expressing GK in the RINm5f cells and the effects of expressing GK antisense RNA in the beta-TC cells. In Aim 2 an analysis of the beta cell GK promoter will be undertaken using a fusion gene approach. Both a 5' deletional analysis and the use of internal deletion mutants should enable us to identify elements necessary for the beta cell-specific expression of GK. Elements important for the cell type expression of GK are likely to bind factors which are important for beta cell-specific gene expression. These factors will be studied further using mobility shift and methylation interference assays. Efforts to purify and clone one of these factors will be initiated.