CREB REGULATES ADIPOCYTE DIFFERENTIATION

肥胖困扰着大约25%的人口， 美国和其他发达国家，并一再表明， 导致心血管疾病、高血压、糖尿病、 肺功能障碍和其他医学问题。 在细胞 在这个水平上，肥胖被认为是储存了多余的热量 作为脂肪组织细胞中的三酰基甘油， 他们的大小，或肥大。 然而，这一理论正逐渐被 最近的数据表明，脂肪细胞数量，以及 随着体型的增大，肥胖症的增加。 新的脂肪细胞可能产生于前- 现有的前脂肪细胞群体，或通过去分化 脂肪细胞转化为前脂肪细胞， 成熟脂肪细胞 显然，对这些因素的全面了解， 调节脂肪细胞增殖和分化的机制 对于理解、治疗和预防肥胖及其 相关的医疗问题。 cAMP反应元件结合蛋白（CREB） 已经显示出刺激控制细胞的基因的表达 对生长因子应答的增殖和分化 activation. 最近，胰岛素和二丁酰-cAMP，两种药物， 诱导脂肪分化，显示刺激CREB活性 在3 T3-L1前脂肪细胞中。 本申请中提出的研究将 进一步研究CREB在启动和/或维持 前脂肪细胞向脂肪细胞的分化。 为了证明CREB在脂肪分化过程中被激活， 最初的实验将评估CREB的表达和磷酸化， Western blot分析前脂肪细胞和分化的3 T3-L1细胞 从~（32）P-正磷酸标记的细胞中回收~（32）P-CREB。 分化期间CREB转录活性的变化将是 在瞬时转染实验中，通过测量 从Gal 4响应性启动子的转录（荧光素酶产生） 在存在和不存在由Gal 4组成的嵌合蛋白的情况下， DNA结合域和CREB反式激活域。 的 将研究CREB在脂肪分化中的参与 通过引入CREB的组成性活性和显性负性形式， （分别为VP 16-CREB和KCREB）转染3 T3-L1前脂肪细胞， 转染 如果CREB在脂肪细胞中起主要作用， 分化过程中，那么KCREB应该抑制，VP 16-CREB应该 增强稳定转染细胞的分化。 最后， CREB结合并调节转录的能力， 已知参与脂肪形成的基因的启动子将被 评估。 总之，这项综合研究计划将确定 诱导分化对3 T3-L1细胞CREB表达调控 代理，直接证明CREB启动或参与 脂肪细胞分化程序，并证明CREB调节 已知的脂肪细胞特异性基因。 这些研究应该提供新的见解的机制， 调节和产生脂肪细胞表型，因此它们是 这对全面了解肥胖的发展至关重要， 细胞水平。 它们对我们基本理解 控制细胞增殖的生化和分子过程 和差异化，特别是应强调的重要性， CREB在细胞周期调控中的作用。

Obesity afflicts approximately 25 percent of the population of the United States and other developed nations, and has repeatedly been shown to contribute to cardiovascular disease and hypertension, diabetes, pulmonary dysfunction, and other medical problems. At the cellular level, obesity has been viewed as the storage of excess caloric energy as triacylglycerols in adipose tissue cells leading to an increase in their size, or hypertrophy. However, this theory is gradually being abandoned as recent data indicate that both adipose cell number, as well as size, increase in obesity. The new adipocytes may arise from pre- existing population of preadipocytes, or through the dedifferentiation of adipocytes to preadipocytes that proliferate and redifferentiate to mature adipocytes. Clearly, a complete understanding of the factors and mechanisms that regulate adipose cell proliferation and differentiation is crucial to understanding, treating, and preventing obesity and its related medical problems. The transcription factor, cAMP-response element binding protein (CREB) has been shown to stimulate the expression of genes that control cell proliferation and differentiation in response to growth factor activation. Recently, insulin and dibutyryl-cAMP, two agents that induce adipose differentiation, were shown to stimulate CREB activity in 3T3-L1 preadipocytes. The research proposed in this application will further investigate the role of CREB in initiating and/or sustaining the differentiation of preadipocytes to adipocytes. To demonstrate the CREB is activated during adipose differentiation, initial experiments will assess CREB expression and phosphorylation in preadipocytes and differentiating 3T3-L1 cells by Western blot analysis and by the recovery of 32P-CREB from 32P-orthophosphate labeled cells. Changes in CREB transcriptional activity during differentiation will be assessed in transient transfection experiments, by measuring transcription (luciferase production) from a Gal4-responsive promoter in the presence and absence of a chimeric protein composed of the Gal4 DNA binding domain and the CREB transactivation domain. The participation of CREB in adipose differentiation will be investigated by introducing constitutively active and dominant negative forms of CREB (VP16-CREB and KCREB, respectively) into 3T3-L1 preadipocytes by stable transfection. If CREB does play a major role in adipocyte differentiation process, then KCREB should inhibit, and VP16-CREB should enhance the differentiation of the stably transfected cells. Finally, the ability of CREB to bind to and regulate transcription from the promoters of genes known to participate in adipogenesis will be evaluated. In summary, this comprehensive research program will define the regulation of CREB in 3T3-L1 cells by differentiation-inducing agents, directly demonstrate the CREB initiates or participates in the adipocyte differentiation program, and demonstrate the CREB regulates known, adipocyte-specific genes. These studies should provide new insights into the mechanisms that regulate and generate the adipocyte phenotype, and they are therefore crucial to a complete understanding of the development of obesity at the cellular level. They are also important to our basic understanding of the biochemical and molecular processes that control cell proliferation and differentiation, and in particular, should highlight the importance of CREB in cell cycle regulation.