The determination and terminal differentiation of adipose cells results in the initiation of the expression of a large number of adipocyte- specific genes, a coordinate rise in their mRNA levels and an increased responsiveness to insulin. The overall objective of this proposed work are: 1) to define the molecular mechanism by which a lipogenic hormone, insulin, controls adipocyte gene activity, and 2) to understand the biochemical changes that occur during the process of preadipocyte cell commitment to the terminally differentiated adipocyte phenotype. This complex series of events involved in adipocyte differentiation will be analyzed using the murine 3T3-L1 cell line and the gene encoding the CCAAT/Enhancer Binding Protein (C/EBP). 3T3-L1 cells differentiate with proper hormonal stimuli from fibroblast-like preadipocyte cells to mature adipocytes. the 3T3-L1 system has a well characterized adipose differentiation program and are highly responsive to insulin, making it an excellent model for analysis of insulin action and preadipose cell commitment. C-EBP has been shown to be involved in the development of the terminally differentiated adipocyte and the regulation of adipocyte- specific gene expression. Previous studies have shown that there are several cis-elements in the 5' flanking sequence of the C-EBP gene that interact with 3T3-L1 nuclear proteins, and have an altered DNaseI sensitivity pattern upon adipocyte differentiation, which may be important in C-EBP gene regulation. Recent data from our laboratory has shown that C/EBP mRNA levels are altered by insulin in 3T3-L1 adipocyte cells. Analysis of earlier events in the regulatory pathways involved in the cascade of preadipocyte to adipocyte cell commitment and the effects of insulin on the lipogenic regulatory factor, C/EBP, will be tested by the following specific aims. First, we will determine hormonal effects on C/EBP mRNA levels in vivo. Nuclear run-on transcription assay will be used to assay alterations in rates of transcription of the C/EBP gene in adipocytes by insulin, diet and other hormones. Second, we will analyze the mechanisms that control C/EBP gene activity using CAT reporter constructs containing the C/EBP promoter in transfection expression assays in 3T3-L1 cells. Third, since C/EBP is expressed only in the differentiated 3T3-L1 adipocytes we will identify the preadipocyte specific DNA-binding protein(s), a putative repressor of gene activity, that interacts with the 5' flanking sequence of the C/EBP gene, using a preadipocyte expression library. The fourth aim of this project is to analyze the elements in the 5' flanking region of the C/EBP gene that interact with the immediate early gene product Zif268, C/EBP gene product, and HLH proteins.