CREB REGULATES ADIPOCYTE DIFFERENTIATION

肥胖症困扰着中国约25%的人口 美国和其他发达国家，并一再表明 会导致心血管疾病、高血压、糖尿病， 肺功能障碍，以及其他医疗问题。在手机上 在这个水平上，肥胖一直被视为储存过多的热能 作为脂肪组织细胞中的三酰甘油导致 他们的大小，或肥大。然而，这一理论正在逐渐被 放弃，因为最近的数据表明，这两个脂肪细胞的数量，以及 随着体型的增加，肥胖率也会增加。新的脂肪细胞可能起源于前 存在的前脂肪细胞群体，或通过脱分化 从脂肪细胞到前脂肪细胞的增殖和再分化为 成熟的脂肪细胞。显然，完全了解这些因素和 脂肪细胞增殖和分化的调控机制 对于了解、治疗和预防肥胖症及其 相关的医疗问题。 转录因子cAMP反应元件结合蛋白(CREB) 已经被证明可以刺激控制细胞的基因的表达 生长因子对细胞增殖和分化的影响 激活。最近，胰岛素和二丁酰cAMP这两种药物 诱导脂肪分化，被证明能刺激CREB活性 在3T3-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分化。 为了证明CREB在脂肪分化过程中被激活， 初步实验将评估CREB的表达和磷酸化 前脂肪细胞与3T3-L1细胞分化的蛋白质印迹分析 从32P-正磷酸盐标记细胞中回收32P-CREB。 分化过程中CREB转录活性的变化将是 在瞬时转基因实验中进行评估，通过测量 Gal4反应启动子的转录(荧光素酶的产生) 在存在和不存在由Gal4组成的嵌合蛋白的情况下 DNA结合域和CREB反式激活域。这个 CREB在脂肪分化中的作用将被研究 通过引入构成积极和显性否定形式的CREB (分别为VP16-CREB和KCREB)稳定转化3T3-L1前脂肪细胞 转染法。如果CREB确实在脂肪细胞中发挥主要作用 分化过程中，KCREB应抑制，VP16-CREB应抑制 促进稳定表达细胞的分化。最后， CREB结合和调节转录的能力 已知的参与脂肪形成的基因的启动子将是 已评估。总而言之，这一综合研究方案将界定 分化诱导对3T3-L1细胞CREB表达的调节 代理人，直接证明CREB发起或参与 脂肪细胞分化程序，并演示CREB调节 已知的脂肪细胞特异性基因。 这些研究应该会为以下机制提供新的见解 调节和产生脂肪细胞的表型，因此 对全面了解肥胖的发展至关重要。 细胞水平。它们也对我们基本理解 控制细胞增殖的生化和分子过程 和差异化，特别是应该突出重要性 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.