Metabolic Signals Regulating GLUT4 Expression in Vivo

描述（由申请人提供）：GLUT4是主要负责哺乳动物组织中胰岛素介导的葡萄糖摄取的异构体。葡萄糖稳态对GLUT4水平的变化很敏感。因此，调节GLUT4水平是胰岛素抵抗状态（包括糖尿病）治疗干预的一个有吸引力的分子靶点。提高GLUT4表达的一种直接方法是提高该基因的转录率。GLUT4基因的表达在胰岛素缺乏和运动等生理状态下受到转录调控，很可能通过药物干预来增强GLUT4基因的转录。为了实现这一目标，我们必须首先了解GLUT4基因转录调控的分子基础。利用转基因小鼠，我们已经发现调节人类Glut4启动子的顺式元件位于转录起始位点的895 bp内5'处。该区域包含两个主要的调控结构域，称为结构域I和MEF2结构域。这些元件协同作用，支持GLUT4的调控表达。MEF2结构域结合肌细胞增强因子2 （MEF2）家族转录因子的异构体，而结构域I结合GEF （GLUT4增强因子），这是我们实验室鉴定和克隆的转录激活因子。MEF2异构体和GEF在体内形成蛋白质复合物；然而，这种复合物在调节基因转录中的功能尚不清楚。保守的肝脏受体X （LXRE）结构域紧邻MEF2位点。这一元素的功能作用尚未确定。我们假设GLUT4基因的组织特异性和激素和/或代谢调节都是通过这些调节结构域及其同源结合蛋白进行的。本研究的主要目的是了解GLUT4基因转录的组织特异性和激素/代谢调控的分子机制。为了实现这些目标，我们提出以下具体目标：1)确定GEF在GLUT4转录激活中的功能作用；2)了解HDAC功能在GLUT4表达调控中的作用3)确定GLUT4 LXRE介导GLUT4启动子代谢信号的作用。这些目标的完成将告诉我们在胰岛素缺乏或胰岛素抵抗的各种代谢条件下GLUT4表达的信号变化。这些情况都会导致GLUT4表达下调，但尚不清楚这是否由单一机制引起。这项研究的完成将有助于我们更多地了解GLUT4启动子以及胰岛素缺乏和胰岛素抵抗期间的细胞内信号传导。

DESCRIPTION (provided by applicant): GLUT4 is the isoform principally responsible for insulin-mediated glucose uptake in mammalian tissues. Glucose homeostasis is sensitive to changes in GLUT4 levels. Modulation of GLUT4 levels is therefore an attractive molecular target for therapeutic intervention in insulin-resistant states, including diabetes mellitus. A straightforward approach to enhance GLUT4 expression is to increase the transcription rate of the gene. GLUT4 gene expression is transcriptionally regulated in physiologic states such as insulin-deficiency and exercise, and it is likely that a pharmacological intervention can be developed to enhance Glut4 gene transcription. To reach this goal, we must first understand the molecular basis for transcriptional regulation of the GLUT4 gene. Using transgenic mice, we have shown that cis-elements regulating the human Glut4 promoter are located within 895 bp immediately 5' of the transcription initiation site. This region contains two major regulatory domains, referred to as Domain I and the MEF2 domain. These elements function cooperatively to support regulated expression of GLUT4. The MEF2 domain binds isoforms of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors, while Domain I binds GEF (GLUT4 Enhancer Factor), a transcriptional activator identified and cloned in our laboratory. MEF2 isoforms and GEF form a protein complex in vivo; however, the function of this complex in regulating gene transcription is not known. A conserved Liver Receptor X (LXRE) domain is located immediately adjacent to the MEF2 site. The functional role of this element has not been determined. We hypothesize that both the tissue-specific and the hormonal and/or metabolic regulation of the GLUT4 gene are carried out through these regulatory domains and their cognate binding proteins. The primary goal of this proposal is to understand the molecular mechanisms of the tissue-specific and hormonal/metabolic regulation of GLUT4 gene transcription. To achieve these goals, we propose the following specific aims: 1) To determine the functional role of GEF in GLUT4 transcriptional activation; 2) To understand the role of HDAC function in regulation of GLUT4 expression 3) ) To determine the contribution of the GLUT4 LXRE in mediating metabolic signals to the GLUT4 promoter. Completion of these aims will inform us what signals changes in GLUT4 expression in a variety of altered metabolic conditions of insulin- deficiency or insulin resistance. These conditions each result in down-regulation of GLUT4 expression, but it is unclear if this is by a single mechanism. Completion of the aims of this proposal will help us to understand more about the GLUT4 promoter and more about the intracellular signaling during insulin- deficiency and insulin-resistance.