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.
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