REGULATION OF BBB GLUT1 GLUCOSE TRANSPORTER

BBB GLUT1 葡萄糖转运蛋白的调节

• 批准号: 6347085
• 负责人: WILLIAM M PARDRIDGE
• 金额: $ 7.63万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6347085
• 关键词: blood brain barrier; cell line; cerebral ischemia /hypoxia; cerebrovascular occlusions; disease /disorder model; gene induction /repression; glucose metabolism; glucose receptor; glucose transport; glucose transporter; glycogenesis; glycogenolysis; immunocytochemistry; posttranscriptional RNA processing; protein structure function; reporter genes; tissue /cell culture; transfection; vascular endothelium; vascular endothelium permeability

DESCRIPTION: (Verbatim from the Applicant's Abstract) The principal glucose transporter at the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, is the Glut1 isoform of the sodium independent glucose transporter gene family. Previous studies have demonstrated an important mode for regulation of BBB Glut1 gene expression is at the post-transcriptional level in pathophysiologic states such as human brain tumors, glucose deprivation/hypoglycemia, development, and cerebral ischemia. Post-transcriptional regulation of mRNA function is mediated via the interaction of cytosolic or polysome proteins with specific cis-elements located within either the 5' - or 3' -untranslated region (UTR) of the Glut1 mRNA. Previous studies have described a polysome protein, designated p44, and a cytosolic protein, designated p88, which interact with two different cis-elements within the 3'-UTR of the glout1 mRNA. The p88 cis-element is associated with an increase in Glut1 gene expression and mediates the stabilization of the Glut1mRNA. The p44 cis-element causes destabilization of the Glut1 transcript, and is associated with an endonuclease/exonuclease activity. The proposed work is comprised of 3 specific aims. Fist, the mechanism of post-transcriptional regulation of Glut1 gene expression will be further defined using a luciferase reporter gene, and cells will also be stably transfected with the Glut1 gene containing selective deletions of th4 putative cis-elements within 3'-UTR. The stably transfected cells will be subjected to hypoxia and glucose deprivation in tissue culture. In parallel, in vivo studies of regional cerebral ischemia using the middle cerebral artery occlusion (MCAO) model as well as global cereal ischemia, using the transient forebrain ischemia (TFI) model, will be performed and correlations will be made between polysome p44, cytosol p88, and Glut1 mRNA levels in whole brain and polysomes, immunoreacitve Glut1 protein, both in brain and at the BBB in vivo. Second, cDNAs encoding the glut1 mRNA UTR-binding proteins will be cloned, sequenced, and analyzed, in parallel with cloning of cDNAs encoding non-glut1 transcripts expressing the cis-acting elements found in the glut1 mRNA 3'-UTR. Third, the secondary structure of the Glut1 mRNA both with and without binding to the p44 and p88 proteins will be determined experimentally using both chemical and enzymatic approaches. These studies will provide insight into molecular mechanisms of regulation of a step crucial to the maintenance of cerebral intermediary metabolism, i.e., the continuous transport of glucose across the blood-brain barrier in vivo.

描述：（逐字摘自申请人的摘要） 脑毛细血管内皮的转运蛋白，形成血脑 体内血脑屏障（BBB），是钠非依赖性葡萄糖的Glut 1亚型 转运蛋白基因家族以前的研究已经证明了一个重要的模式 调节血脑屏障Glut 1基因表达是在转录后 在病理生理状态下的水平，如人类脑肿瘤，葡萄糖 剥夺/低血糖、发育和脑缺血。 mRNA功能的转录后调节是通过 胞质或多核糖体蛋白与特定顺式元件的相互作用 位于Glut 1的5' -或3' -非翻译区（UTR）内 mRNA。以前的研究已经描述了一种多核糖体蛋白，命名为p44，和一种多核糖体蛋白。 胞浆蛋白，命名为p88，与两种不同的 glout 1 mRNA的3 '-UTR内的顺式元件。p88顺式元件是 与Glut 1基因表达增加相关，并介导 Glut 1 mRNA的稳定性。p44顺式元件导致细胞不稳定 Glut 1转录物，并与核酸内切酶/核酸外切酶相关 活动拟议的工作包括三个具体目标。拳头 Glut 1基因表达的转录后调控机制将是 使用荧光素酶报告基因进一步定义，并且细胞也将被稳定地 用Glut 1基因转染，该基因含有选择性缺失的th 4推定的 3 '-UTR内的顺式元件。稳定转染的细胞将经受 缺氧和葡萄糖剥夺。同时，体内研究 大脑中动脉闭塞（MCAO） 模型以及全球谷物缺血，使用短暂的前脑缺血 (TFI)模型，将执行和相关性将多核糖体之间 全脑和多核糖体中的p44、胞浆p88和Glut 1 mRNA水平， 免疫反应性Glut 1蛋白，无论是在大脑和在体内的血脑屏障。第二、 将克隆、测序编码p53 1 mRNA UTR结合蛋白的cDNA， 并与克隆编码非BMP 1转录物的cDNA平行进行分析 表达glut 1 mRNA 3 '-UTR中发现的顺式作用元件。三是 Glut 1 mRNA的二级结构（结合和不结合p44） 和p88蛋白将通过实验使用化学和 酶法。这些研究将提供深入了解分子 调节机制的一个步骤至关重要的维持大脑 中间代谢，即，葡萄糖通过血管的连续运输 体内血脑屏障。