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CONTROL OF LIPOGENIC ENZYME SYNTHESIS IN ADIPOCYTES

脂肪细胞中脂肪生成酶合成的控制

基本信息

批准号：
3152267
负责人：
BRUCE M. SPIEGELMAN
金额：
$ 13.72万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-07-01 至 1987-06-30
项目状态：
已结题

项目摘要

Experiments are proposed which examine the molecular mechanisms which control lipogenic enzyme synthesis during the differentiation of mammalian adipocytes. The control of lipogenic enzyme development in adipocytes is higly relevant to human obesity, diabetes and cancer of the adipocyte (liposarcoma), the most common sarcoma in humans. The cell types used are cloned preadipocyte lines derived from mouse 3T3 cells used previously to examine the control of lipogenic enzyme syntheiss, predominantly at the protein level. The initial step is the isolation of cDNA clones containing sequences for important and abundant lipogenic enzymes such as glycerophosphate dehydrogenase, malic enzyme and fatty acid synthetase from an adipocyte cDNA library. This has been done first by comparing hybridization of recombinant colonies to cDNA synthesized from preadipocyte or adipocyte mRNA. The identity of particular inserted sequences will be determined by hybridization-selection from adipocyte mRNA and translation in vitro. The regulation of mRNA content for lipogenic enzymes during differentiation is to be examined by measurement of transcription and turnover rates with cloned cDNA probes. Of particular interest is a comparison of a new gene product expressed in the adipocyte (glycerophosphate dehydrogenase) with one of several enzymes quantitatively modulated during differentiation. Also to be examined are mechanisms whereby insulin and cyclic AMP agents, key hormonal influences in vivo, control the levels of enzymes involved in lipid metabolism. The relationship between methylation of genes for lipogenic enzymes, their expression and cellular commitment to the preadipocyte phenotype will be studied with methylation-sensitive restriction enzymes. Whether changes in DNA emthylation correlate with the developmental programming of these genes or with their actual expression will be determined by comparing methylation patterns in cell lines which differ in susceptibility to adipose differentiation. Methylation will also be examined in ells committed to adipocyte differentiation by exposure to 5-azacytidine. The reversibility of differentiation will be determined by replating adipocytes which have had lipid accumulation blocked and retain adhesiveness to the substratum. Subsequent enzyme expression will be monitored at protein, RNA and DNA levels.

实验提出了研究的分子机制，在哺乳动物分化过程中控制脂肪生成酶的合成脂肪细胞控制脂肪细胞中脂肪生成酶的发育是与人类肥胖、糖尿病和脂肪细胞癌高度相关（脂肪肉瘤），人类最常见的肉瘤。使用的细胞类型是来自小鼠3 T3细胞的克隆前脂肪细胞系，检查脂肪生成酶合成的控制，主要是在蛋白质水平第一步是分离cDNA克隆，重要和丰富的脂肪生成酶的序列，甘油磷酸脱氢酶、苹果酸酶和脂肪酸合成酶，脂肪细胞cDNA文库。首先，我们比较了重组集落与前脂肪细胞合成cDNA杂交或脂肪细胞mRNA。特定插入序列的同一性将是通过从脂肪细胞mRNA和翻译的杂交选择确定体外脂肪形成过程中脂肪生成酶mRNA含量的调节通过测量转录来检查分化，周转率与克隆cDNA探针。特别令人感兴趣的是脂肪细胞中表达的新基因产物的比较（甘油磷酸脱氢酶）与几种酶之一定量在分化过程中被调节。还将审查机制其中胰岛素和环AMP试剂，体内关键的激素影响，控制参与脂质代谢的酶的水平。的脂肪生成酶基因的甲基化，表达和细胞定型为前脂肪细胞表型，用甲基化敏感的限制性内切酶研究。的变化是否 DNA甲基化与这些基因的发育程序有关或与它们的实际表达的关系将通过比较甲基化在对脂肪的敏感性不同的细胞系中的模式分化甲基化也将在致力于通过暴露于5-氮杂胞苷进行脂肪细胞分化。可逆性将通过重新接种脂肪细胞来确定分化的程度，阻止了脂质积聚，并将脂质保留在基质中。随后的酶表达将在蛋白质、RNA和DNA水平进行监测程度.