Role of Early B Cell Factor-1 (EBF-1) in Adipocyte Biology

描述(由申请人提供)：拟议研究的广泛目标是通过创造性的发现和创新的实验方法，为我们的脂肪细胞分化如何工作的知识库做出贡献。为此，将研究脂肪代谢酶硬脂酰辅酶A去饱和酶-1(SCD-1)在脂肪细胞分化中的可能调节作用。尽管SCD-1在脂肪细胞代谢中的消极作用已被认识多年，但越来越多的新证据表明，这种酶有助于控制脂肪细胞的发育。具体地说，靶向缺失SCD-1的小鼠的脂肪细胞较小，并增加了转录因子PPAR-伽马的水平，PPAR-伽马是脂肪形成的主要调节因子。此外，利用shRNA技术去除3T3-L1脂肪细胞中的SCD-1既可促进脂肪生成，又可上调PPAR-γ的表达/活性。然而，这些变化的机制(S)仍不清楚。本研究的主要目的是：1)证实SCD-1缺失促进了SCD-1-/-小鼠成纤维细胞的成脂作用；2)检测了SCD-1缺失对脂肪细胞可能带来的基因和代谢变化；3)通过寻找SCD-1的新的蛋白质相互作用伙伴或该酶产生(或去除)的活性代谢物，确定了SCD-1缺失对脂肪形成影响的分子机制。本研究分为观测臂和机械臂两部分。在观察性ARM中，将分别使用微阵列和脂质组学分析来深入了解SCD-1缺乏引起的转录和代谢物变化。在机械臂中，将利用“死亡”突变蛋白进行互补研究，以确定SCD-1的酶活性是否是该基因在脂肪形成中所必需的。然后，将使用串联亲和纯化或高压液相色谱和质谱仪分别鉴定SCD-1的蛋白质伙伴或脂代谢产物，它们是导致在脂肪细胞中观察到的现象的原因。与公共健康相关：肥胖及其医学后果是我们国家目前最大的健康问题之一，传统观点认为，阻止新脂肪细胞(脂肪细胞)的生长将是对抗这一问题的有效方法。然而，有证据表明，一个人不能产生新的脂肪细胞可能是导致疾病的主要原因，这种疾病通常是由超重引起的。了解脂肪细胞发育的机制，包括硬脂酰辅酶A脱饱和酶等脂代谢基因的作用，是实现减轻与肥胖有关的负面健康影响的目标的重要一步。

DESCRIPTION (provided by applicant): The broad goal of the proposed research is to contribute to our knowledge base of how adipocyte differentiation works through both creative discovery and innovative experimental approaches. To do this, a possible regulatory role for the lipid metabolic enzyme Stearoyl-CoA Desaturase-1 (SCD-1) in the differentiation of adipocytes will be examined. Although a passive function for SCD-1 in adipocyte metabolism has been recognized for years, new evidence is accumulating that this enzyme helps to control fat cell development. Specifically, adipocytes from mice with a targeted deletion of SCD-1 are smaller and have increased levels of the transcription factor PPAR-gamma, the master regulator of adipogenesis. In addition, depletion of SCD-1 in 3T3-L1 adipocytes with shRNA technology both enhances adipogenesis and up-regulates PPAR-gamma expression/activity. However, the mechanism(s) for these remains unknown. The major aims of this research are 1) to confirm that lack of SCD-1 enhances adipogenesis in fibroblasts derived from SCD-1 -/- mice; 2) to examine the putative gene and metabolic changes brought about by SCD-1 deficiency in adipocytes; and 3) to determine the molecular mechanism for the observed effect of loss of SCD-1 on adipogenesis, either by identifying a novel protein interaction partner for SCD-1 or an active metabolite produced (or removed) by this enzyme. The proposed research is divided into two parts, an observational arm and a mechanistic arm. In the observational arm, microarray and lipidomics analyses will be used to gain insight into the transcriptional and metabolite changes incurred by SCD-1 deficiency, respectively. In the mechanistic arm, complementation studies using "dead" mutant proteins will be employed to determine whether the enzymatic activity of SCD-1 is required for the effect of this gene on adipogenesis. Then, tandem affinity purification or high-pressure liquid chromatography and mass spectrometry will be used to either identify a protein partner for SCD-1 or a lipid metabolic product, respectively, that is responsible for the observed phenomenon in adipocytes. PUBLIC HEALTH RELEVANCE: Obesity and its medical consequences is one of the biggest health issues in our nation right now, and conventional wisdom dictates that preventing the growth of new adipocytes (fat cells) would be an effective way to combat this. However, evidence suggests that the failure of a person to generate new adipocytes may be a primary cause of the morbidities that often result from excess weight. Understanding the mechanisms that govern adipocyte development, including the roles of lipid metabolism genes such as Stearoyl-CoA Desaturase, is an important step towards reaching our goal of alleviating the negative health effects linked with obesity.