PHOSPHOENOLPYYRUVATE CARBOXYKINASE IN ADIPOSE TISSUE

脂肪组织中的磷酸酚丙酮酸羧激酶

• 批准号: 3462918
• 负责人: DAVID S LOOSE
• 金额: $ 8.58万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3462918
• 关键词: DNA footprinting; RNA biosynthesis; RNA splicing; adipocytes; adipose tissue; carbon carbon lyase; chromatin; dexamethasone; enzyme induction /repression; fusion gene; gel electrophoresis; gene deletion mutation; gene expression; genetic promoter element; genetic regulation; genetic transcription; hormone regulation /control mechanism; laboratory rat; liver; messenger RNA; nucleic acid hybridization; nucleic acid sequence; posttranscriptional RNA processing; tissue /cell culture; transfection

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.

本提案的总体目标是了解 磷酸烯醇式丙酮酸羧激酶（GTP）（PEPCK）基因 在脂肪组织中。 PEPCK的调节在其组织中是独特的 的特异性 我们特别感兴趣的是， 哪些糖皮质激素改变了基因的表达， 酶：在肝脏和肾脏糖皮质激素诱导酶尚未 在脂肪组织中，该激素引起脂肪细胞的显著去诱导， 酵素 该提案的具体目标包括： 1)糖皮质激素作用机制的确定 对大鼠附睾脂肪组织和3 T3-L1脂肪细胞的影响 是转录和/或转录后过程 牵涉其中？ 我们将在体外测定PEPCK mRNA的合成 使用分离的脂肪细胞核，并使用RNA印迹分析和S-1 核酸酶保护实验，以测量稳态和 PEPCK mRNA的动力学性质。 2)由大鼠肝脏PEPCK基因或 与新霉素抗性相关的PEPCK基因部分 将其通过DNA介导的基因导入3 T3-L1细胞 转移 一系列融合基因的转染 通过大鼠肝脏PEPCK基因的缺失将使我们能够 识别负责以下事件的序列元素： 糖皮质激素介导的脂肪组织中PEPCK的抑制。 3)脂肪组织中PEPCK基因及其mRNA的结构 将被确定。 我们计划对启动子进行测序- 脂肪组织中PEPCK基因的调节区， 确定脂肪组织中是否发生了重排。 组织 剪接或转录起始位点的特定改变将 通过从脂肪细胞PEPCK mRNA的引物延伸来评估。 我们将定义PEPCK基因的结构区域， 通过评估核酸酶改变糖皮质激素治疗 细胞核的灵敏度和体内足迹技术。 4)脂肪中糖皮质激素抑制的介导因素 将使用DNA-蛋白质结合测定法鉴定组织。 这些 将使用经典的生物化学方法纯化因子。 这项研究的长期目标是扩大我们的 了解激素如何引起分化和组织 具体的回答。

项目成果

Extracellular matrix regulates proliferation and phospholipid turnover in glomerular epithelial cells.

细胞外基质调节肾小球上皮细胞的增殖和磷脂周转。

• DOI: 10.1152/ajprenal.1990.259.2.f326
• 发表时间: 1990
• 期刊: The American journal of physiology
• 作者: Cybulsky,AV;Bonventre,JV;Quigg,RJ;Wolfe,LS;Salant,DJ
• 通讯作者: Salant,DJ

Sequence of a 1.4-kb region in the 3'-flanking region of the murine c-fos proto-oncogene which contains an estrogen-response element.

鼠 c-fos 原癌基因 3 侧翼区域中 1.4 kb 区域的序列，其中包含雌激素反应元件。

• DOI: 10.1016/0378-1119(91)90164-7
• 发表时间: 1991
• 期刊: Gene
• 影响因子: 3.5
• 作者: Hyder,SM;Cram,LF;Loose-Mitchell,DS
• 通讯作者: Loose-Mitchell,DS

Regulation of the uterine epidermal growth factor receptor by estrogen.

雌激素对子宫表皮生长因子受体的调节。

• 发表时间: 1990
• 期刊: Progress in clinical and biological research
• 作者: Stancel,GM;Chiapetta,C;Gardner,RM;Kirkland,JL;Lin,TH;Lingham,RB;Loose-Mitchell,DS;Mukku,VR;Orengo,CA
• 通讯作者: Orengo,CA

Estrogen regulation of c-fos messenger ribonucleic acid.

c-fos 信使核糖核酸的雌激素调节。

• DOI: 10.1210/mend-2-10-946
• 发表时间: 1988
• 期刊: Molecular endocrinology (Baltimore, Md.)
• 作者: Loose-Mitchell,DS;Chiappetta,C;Stancel,GM
• 通讯作者: Stancel,GM