PHOSPHOENOLPYYRUVATE CARBOXYKINASE IN ADIPOSE TISSUE

这项提案的总体目标是了解监管 磷酸烯醇式丙酮酸羧激酶基因(PEPCK) 在脂肪组织中。PEPCK的调节在其组织中是独一无二的 专一性。我们特别感兴趣的是 哪种糖皮质激素会改变基因的表达 酶：在肝脏和肾脏中，糖皮质激素还能诱导这种酶 在脂肪组织中，这种激素导致明显的去诱导性 酵素。 该提案的具体目标包括： 1)糖皮质激素作用机制的测定 对大鼠附睾脂肪组织和3T3-L1脂肪细胞的影响 培养；是转录和/或转录后过程 有牵连吗？我们将在体外测量PEPCK mRNA的合成 利用分离的脂肪细胞核并使用核糖核酸印迹分析和S-1 核酸酶保护实验测量稳态和 PEPCK基因表达的动力学特性。 2)由大鼠肝脏PEPCK基因或 PEPCK基因与新霉素抗性相关的部分序列 将DNA介导的基因导入3T3-L1细胞 调职。一系列融合基因转染法的研究 通过大鼠肝脏PEPCK基因的缺失将使我们能够 确定负责的序列元素 糖皮质激素对脂肪组织中PEPCK的抑制作用 3)脂肪组织中PEPCK基因和mRNA的结构 将会被确定。我们计划对启动子进行测序- 脂肪组织中PEPCK基因的调控区 确定脂肪组织中是否发生了重排。组织 剪接或转录起始点的特定改变将 通过从脂肪细胞PEPCK mRNA中扩增的引物进行鉴定。 我们将定义PEPCK基因的结构区域，它们是 通过评估核酸酶改变糖皮质激素治疗 通过细胞核和活体足迹技术的敏感性。 4)脂肪中介导糖皮质激素抑制的因素 组织将使用DNA-蛋白质结合分析进行鉴定。这些 因子将使用经典的生化方法进行提纯。 这项研究的长期目标是扩大我们的 对激素如何引起分化和组织的理解 具体的回应。

The overall goal of this proposal is to understand the regulation of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) in adipose tissue. The regulation of PEPCK is unique in its tissue specificity. We are particularly interested in the manner by which glucocorticoids alter expression of the gene for the enzyme: in liver and kidney glucocorticoids induce the enzyme yet in adipose tissue the hormone causes a marked de-induction of the enzyme. Specific goals of this proposal include: 1) Determination of the mechanism of action of glucocorticoids on rat epididymal adipose tissue and on 3T3-L1 adipocytes in culture; are transcriptional and/or post-transcriptional processes involved? We will measure synthesis of PEPCK mRNA in vitro using isolated adipocyte nuclei and use RNA-blot analysis and S-1 nuclease protection experiments to measure the steady-state and kinetic properties of PEPCK mRNA. 2) Fusion genes consisting of the rat liver PEPCK gene or portions of the PEPCK gene linked to the neomycin resistance will be introduced into the 3T3-L1 cells by DNA-mediated gene transfer. Transfection of a series of fusion genes bearing deletions through the rat liver PEPCK gene will allow us to identify the sequence elements which are responsible for the glucocorticoid mediated inhibition of PEPCK in adipose tissue. 3) The structure of the PEPCK gene and mRNA in adipose tissue will be determined. We plan on sequencing the promoter- regulatory region of the PEPCK gene in adipose tissue in order to identify if a rearrangement has occurred in adipose tissue. Tissue specific alterations in splicing or the transcriptional start site will be assessed by primer extension from adipocyte PEPCK mRNA. We will define structural regions of the PEPCK gene which are altered by glucocorticoid treatment by assessing nuclease sensitivity in nuclei and by in vivo footprinting techniques. 4) Factors which mediate glucocorticoid inhibition in adipose tissue will be identified using DNA-protein binding assays. These factors will be purified using a classical biochemical approach. The long term objective of this research is to broaden our understanding of how hormones elicit differential and tissue specific responses.