CONTROL OF LIPOGENIC ENZYME SYNTHESIS IN ADIPOCYTES

该项目的长期目标是提高我们的 了解脂肪细胞如何调节脂肪生成， 脂肪分解通过控制特定的基因表达， 相应的蛋白质产品。 通过提高我们对 在这些过程中，我们最终可能会提高我们的能力， 为肥胖相关糖尿病提供治疗， 相关心血管疾病。 在此前的融资中， 期间，我们已经分离和表征了3个基因， 在小鼠脂肪细胞 分化：甘油磷酸脱氢酶，脂肪细胞P2 和adipsin。 激活这些细胞所需的DNA序列 首先通过瞬时转染表达来探测基因， 比色法 在证明了适当的细胞类型特异性后， 表达，如脂肪细胞P2基因所做的那样，这些 DNA片段将被进一步解剖以确定（1） 序列元件在其自身启动子的激活中起作用， (2)它是否可以指导其他启动子的转录，以及 (3)它是否能像增强子一样发挥作用。 最大 注意力将集中在遗传因素上，这些因素似乎 代表了不同细胞活化途径中的共同步骤， 脂肪特异性基因 同样在研究中的还有 要求得到适当的反应，以2激素的关键 脂肪生成：环AMP和肿瘤坏死因子（cachectin）。 将进行实验以分离和表征那些 核因子结合并可调节脂肪细胞特异性 启动子/增强子/应答元件。 包括在这里 将是FSE 2结合蛋白， 发育调节并以序列特异性方式结合 与至少两个参与脂肪细胞的基因中发现的元素 分化 最终的目标是在体外 细胞类型特异性转录模式的重建，使用 DNA模板 除了转录调控外，我们还将分析 丝氨酸蛋白酶adipsin的催化和生理功能 由脂肪细胞产生和分泌的同源物。 这将 通过克隆的cDNA在大肠杆菌中的大规模表达来完成。 杆状病毒载体和随后的蛋白水解研究 这种酶对多种细胞外 在脂肪生理学中具有潜在重要性的底物。 生物活性也将通过中和脂蛋白酶进行检查 与单特异性抗体的活性和随后的 对脂肪细胞分化和脂肪生成的影响。

The long range goals of this project are to improve our understanding of how the adipocyte regulates lipogenesis and lipolysis through the control of specific gene expression and the corresponding protein products. By improving our understanding of these processes, we may ultimately improve our ability to provide therapy for obesity obesity-linked diabetes and correlative cardiovascular disorders. In the previous funding period, we have isolated and characterized 3 genes which are transcriptionally activated during mouse adipocyte differentiation: glycerophosphate dehydrogenase, adipocyte P2 and adipsin. The DNA sequences necessary to activate these genes will be probed first by a transient transfection-expression assay. After demonstrating proper cell type specificity of expression, as has been done with the adipocyte P2 gene, these DNA segments will be further dissected to determine (1) which sequence elements play a role in activation of its own promoter, (2) whether it can direct transcription from other promoters, and (3) whether it can function in an enhancer-like fashion. Greatest attention will be focused on genetic elements which appear to represent common steps in the pathway of activation of different fat-specific genes. Also under study will be the sequence requirements to get proper responses to 2 hormones key in lipogenesis: cyclic AMP and tumor necrosis factor (cachectin). Experiments will be carried out to isolate and characterize those nuclear factors which bind to and may regulate adipocyte-specific promoters/enhancers/hormone-response elements. Included here will be the FSE2 binding protein which appears to be developmentally regulated and binds in sequence-specific fashion to an element found in at least 2 genes participating in adipocyte differentiation. The ultimate goal will be an in vitro reconstruction of a cell type-specific transcription pattern, using isolated DNA templates. In addition to transcriptional regulation, we will analyze the catalytic and physiological function of adipsin, the serine protease homologue which is produced and secreted by fat cells. This will be done by large scale expression of the cloned cDNA in a baculovirus vector and subsequent study of the proteolytic activity of this enzyme toward an variety of extracellular substrates of potential importance in adipose physiology. Biological activity will also be examined by neutralizing adipsin activity with monospecific antibodies and following subsequent effects on adipocyte differentiation and lipogenesis.