CONTROL OF LIPOGENIC ENZYME SYNTHESIS IN ADIPOCYTES

这个项目的长期目标是改善我们的 了解脂肪细胞如何调节脂肪生成和 通过控制特定基因表达和血管内皮细胞的脂肪分解 相应的蛋白质产品。通过提高我们的理解 在这些过程中，我们最终可能会提高我们的能力 为肥胖和与肥胖相关的糖尿病提供治疗 相关的心血管疾病。在之前的资助中 期间，我们已经分离并鉴定了3个基因，它们是 小鼠脂肪细胞转录激活 分化：甘油磷酸脱氢酶、脂肪细胞P2 还有脂肪酶。激活这些基因所需的DNA序列 基因将首先通过瞬时转染-表达来探测 化验。在证明了适当的细胞类型特异性之后 表达，正如脂肪细胞P2基因所做的那样，这些 DNA片段将被进一步解剖以确定(1) 序列元件在自身启动子的激活中发挥作用， (2)是否可以直接从其他启动子转录；以及 (3)能否以增强剂的方式发挥作用。最棒的 人们的注意力将集中在似乎可以 代表了不同的激活途径中的共同步骤 脂肪专一性基因。同样在研究中的还有这个序列 对两种荷尔蒙做出适当反应的要求 脂肪生成：环磷酸腺苷和肿瘤坏死因子(Cachectin)。 将进行实验以分离和鉴定这些细胞 结合并可能调节脂肪细胞特异性的核因子 促进剂/增强剂/激素反应元件。包含在此处 将是FSE2结合蛋白，它似乎是 发育调节并以序列特定的方式结合 与至少2个参与脂肪细胞的基因中发现的一个元件有关 差异化。最终的目标将是体外培养 重建特定细胞类型的转录模式，使用 分离的DNA模板。 除了转录调控外，我们还将分析 丝氨酸蛋白酶--脂酶的催化和生理功能 由脂肪细胞产生和分泌的同系物。这将是 通过在大肠杆菌中大规模表达克隆的cDNAs 杆状病毒载体及其蛋白水解物的后续研究 该酶对多种胞外物质的活性 脂肪生理学中潜在重要的底物。 生物活性也将通过中和脂肪酶来检测。 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.