GLUCOKINASE GENE EXPRESSION IN THE PANCREATIC BETA CELL

胰腺β细胞中的葡萄糖激酶基因表达

• 批准号: 3243753
• 负责人: MARK A MAGNUSON
• 金额: $ 11.77万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-06-01 至 1995-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3243753
• 关键词: DNA footprinting; antisense nucleic acid; enzyme mechanism; enzyme structure; fusion gene; gene deletion mutation; gene expression; genetic promoter element; glucokinase; glucose metabolism; molecular cloning; neoplastic cell culture for noncancer research; pancreatic islet function; polymerase chain reaction; protein purification; protein structure function; transcription factor; transfection; transposon /insertion element

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.

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) 催化葡萄糖利用的初始步骤和限速步骤 由 β 细胞在生理葡萄糖浓度下产生，并被认为 在调节胰岛素分泌中发挥重要作用。 为了研究 进一步研究GK在β细胞中的功能和调节，我们有 来自肝脏和胰岛素瘤组织的编码 GK 的 cDNA 特征 并绘制了两种组织中转录单位的图谱。 我们的 研究表明，单个 GK 基因中有两个不同的启动子。 一种启动子在肝脏中特异性表达，似乎是 受胰岛素调节，而位于更上游的另一个启动子， 仅在β细胞中表现出活性。 我们建议研究 GK 在胰岛素瘤细胞系中的功能和调节（目标 1） 使用融合基因分析检查 GK β 细胞启动子（目标 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 的表达在两个 我们希望改变不同胰岛素瘤细胞系的“设定点” 葡萄糖刺激胰岛素分泌。 RINm5F 细胞葡萄糖正常 摄取但似乎缺乏高 Km 葡萄糖磷酸化活性，并且确实 不响应葡萄糖而分泌胰岛素。 另一种细胞系，β-TC， 显示高 Km 葡萄糖消耗，但刺激分泌胰岛素的量较少 高于正常葡萄糖浓度。 我们将确定影响 在 RINm5f 细胞中表达 GK 以及表达 GK 的效果 β-TC 细胞中的反义 RNA。 在目标 2 中，对 β 细胞进行分析 GK 启动子将使用融合基因方法进行。 两者都是 5' 缺失分析和内部缺失突变体的使用应该能够 我们确定β细胞特异性表达所需的元素 GK。 对于 GK 的细胞类型表达很重要的元素可能是 结合对于β细胞特异性基因表达很重要的因子。 将使用迁移率变化和甲基化进一步研究这些因素 干扰测定。 纯化和克隆这些因子之一的努力将 被发起。