The LONG-TERM GOALS are: 1) to understand the molecular mechanisms that control the expression of adipocyte-specific genes both in differentiating preadipocytes and in mature adipocytes and 2) to define the role of insulin in adipocyte gene activation and repression. We plan to continue our studies on the regulatory mechanisms that operate during the differentiation and maturation of murine 3T3-L1 preadipocytes in culture. In this model system differentiation is accompanied by the activation of a large number of adipocyte-specific genes and the development of increased responsiveness to insulin. In the terminally differentiated cell the expression of certain adipocyte-specific genes is reciprocally regulated by insulin, consistent with the role of the hormone in the activation of lipogenesis and the inhibition of lipolysis. We have cloned and partially characterized two adipocyte genes (the 422 and 122 genes), both of which are transcriptionally activated during differentiation of 3T3-L1 preadipocytes into "adipocytes," and are also reciprocally regulated by insulin in the mature adipocyte. Considerable background information with the 3T3-L1 cell system has already been obtained on the biochemistry of the adipose differentiation program and on insulin action. The SPECIFIC AIMS will be: 1) to determine the genetic organization and structures of the 422 and 122 genes which represent two classes of genes having energy storage and mobilizing functions, respectively, 2) to identify and sequence the genetic elements which control transcription of the 422 and 122 genes during preadipocyte differentiation and insulin stimulation, 3) to identify and characterize the regulatory proteins that interact with control elements of the 422 and 122 genes and to establish how this interaction alters transcription, and 4) (to determine the site(s) at which insulin acts to increase and decrease, respectively, the cellular levels of the 422 and 122 mRNAs, i.e. whether by altering gene transcription or by altering the rate of specific mRNA turnover. The molecular mechanisms by which this regulation is exerted will be investigated.