GLUCOSE TRANSPORTER EXPRESSION IN TRANSGENIC MICE

本研究的总体目标是检查组织特异性 以及GLUT 4促进葡萄糖的激素/代谢调节 脂肪组织、心脏和骨骼肌中的转运蛋白基因。 先前的研究已经确定，GLUT 4基因表达是 在胰岛素缺乏状态下转录调节， 禁食和链脲佐菌素（STZ）诱导的糖尿病。 最近的证据 表明GLUT 4的过表达增强了胰岛素敏感性， 在正常和遗传性糖尿病转基因小鼠中。使用 转基因小鼠，我们也已经证明， 组织特异性和激素/代谢 调节位于主要转录起始点的1 kb内 人GLUT 4基因的一个位点。通过缺失分析， 转基因小鼠，我们已经表明， 赋予骨骼肌特异性表达不与 负责脂肪组织和心脏特异性 表情此外，负责监管的区域 在STZ诱导的糖尿病中的表达与假定的 骨骼肌特有的元素。 在推定的骨骼中 肌肉特异性元件，我们已经确定了一个真实的MEF 2结合 可能部分负责基因表达位点。我们 建议进一步定义调控区和DNA结合 负责GLUT 4基因表达的转录因子 来确定组织的分子机制 特异性和激素/代谢调节。完成这些 为了实现这些目标，将实现以下具体目标：1.进一步 定义人GLUT 4启动子内的调控区 区域负责调控基因表达，我们将 产生携带各种基因之间融合的转基因小鼠品系， 人GLUT 4启动子区和CAT报告基因的缺失 基因2.为了开始确定转录因子 对于GLUT 4基因表达，我们将首先关注以下因素的作用： MEF 2.我们将分析脂肪组织、心脏和 骨骼肌的MEF 2结合活性，并确定组织- MEF 2同种型的特异性表达。3. 我们将使用隔离 从转基因小鼠的脂肪细胞中， GLUT 4时间依赖性降低的潜在机制 在这些细胞中发生的转录速率。理解这一 机制将提供有用的洞察机制， GLUT 4基因在体内表达。

The overall goal of this research is to examine the tissue-specific and hormonal/metabolic regulation of the GLUT4 facilitative glucose transporter gene in adipose tissue, heart and skeletal muscle. Previous studies have established that GLUT4 gene expression is transcriptionally regulated in insulin-deficient states such as fasting and streptozotocin (STZ)-induced diabetes. Recent evidence suggests that overexpression of GLUT4 enhances insulin sensitivity in both normal and genetically diabetic transgenic mice. Using transgenic mice, we have also demonstrated that the regulatory sequences required for tissue-specific and hormonal/metabolic regulation are located within 1 kb of the major transcription start site of the human GLUT4 gene. By deletion analysis carried out in transgenic mice, we have shown that the regulatory regions conferring skeletal muscle-specific expression do not overlap with regions responsible for adipose tissue- and cardiac specific expression. Further, the regions responsible for regulated expression in STZ-induced diabetes do not coincide with the putative skeletal muscle-specific elements. Within the putative skeletal muscle-specific element we have identified an authentic MEF2 binding site which may be responsible in part for gene expression. We propose to further define the regulatory region(s) and DNA binding transcription factors responsible for GLUT4 gene expression in order to determine the molecular mechanisms responsible for tissue specificity and hormonal/metabolic regulation. To accomplish these goals, the following specific aims will be addressed: 1 To further define the regulatory regions within the human GLUT4 promoter regions are responsible for regulated gene expression, we will generate lines of transgenic mice carrying fusions between various deletions of the human GLUT4 promoter regions and a CAT reporter gene. 2. To begin determining the transcription factors responsible for GLUT4 gene expression, we will initially focus on the role of MEF2. We will assay nuclear extracts from adipose tissue, heart and skeletal muscle for MEF2 binding activity and identify the tissue- specific expression of MEF2 isoforms. 3. We will used isolated adipocytes from transgenic mice to determine the molecular mechanisms underlying the time dependent decrease in GLUT4 transcription rate that occurs in these cells. Understanding this mechanism will provide useful insight into the mechanisms by which GLUT4 gene expression occurs in vivo.