The overall goal of this proposal is to understand the regulation of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) in adipose tissue. The regulation of PEPCK is unique in its tissue specificity. We are particularly interested in the manner by which glucocorticoids alter expression of the gene for the enzyme: in liver and kidney glucocorticoids induce the enzyme yet in adipose tissue the hormone causes a marked de-induction of the enzyme. Specific goals of this proposal include: 1) Determination of the mechanism of action of glucocorticoids on rat epididymal adipose tissue and on 3T3-L1 adipocytes in culture; are transcriptional and/or post-transcriptional processes involved? We will measure synthesis of PEPCK mRNA in vitro using isolated adipocyte nuclei and use RNA-blot analysis and S-1 nuclease protection experiments to measure the steady-state and kinetic properties of PEPCK mRNA. 2) Fusion genes consisting of the rat liver PEPCK gene or portions of the PEPCK gene linked to the neomycin resistance will be introduced into the 3T3-L1 cells by DNA-mediated gene transfer. Transfection of a series of fusion genes bearing deletions through the rat liver PEPCK gene will allow us to identify the sequence elements which are responsible for the glucocorticoid mediated inhibition of PEPCK in adipose tissue. 3) The structure of the PEPCK gene and mRNA in adipose tissue will be determined. We plan on sequencing the promoter- regulatory region of the PEPCK gene in adipose tissue in order to identify if a rearrangement has occurred in adipose tissue. Tissue specific alterations in splicing or the transcriptional start site will be assessed by primer extension from adipocyte PEPCK mRNA. We will define structural regions of the PEPCK gene which are altered by glucocorticoid treatment by assessing nuclease sensitivity in nuclei and by in vivo footprinting techniques. 4) Factors which mediate glucocorticoid inhibition in adipose tissue will be identified using DNA-protein binding assays. These factors will be purified using a classical biochemical approach. The long term objective of this research is to broaden our understanding of how hormones elicit differential and tissue specific responses.
