Dietary polyenoic (n-6) and (n-3) fatty acids (PUFA) reduces blood VLDL by coordinately: (a) inhibiting fatty acid biosynthesis; (b) reducing fatty acid flux to triglycerides; and (3) enhancing liver and skeletal muscle fatty acid oxidation. In a sense, PUFA are classic fuel partitioners (i.e. direct lipid from storage to oxidation); and in this way may protect against the development of insulin resistance and cardiovascular disease. PUFA regulation of metabolism is often viewed as a simple pleiotropic effect resulting from changes in membrane fatty acid composition. However, fatty acids may also govern gene transcription by functioning as ligand activations for members of the steroid receptor super-family (e.g. PPARs). Although PPARs per se appear not to mediate the PUFA suppression of hepatic lipogenic genes (e.g. fatty acid synthase-FA), the kinetics of the PUFA inhibition are consistent with a ligand mediated event. Thus we hypothesize that, PUFA, or a metabolite, bind to a specific PUFA response factor (RF) which interacts with the PUFA-RE and functions to silence FAS transcription by interfering with the transfactors with the PUFA-RE and functions to silence FAS transcription by interfering with the trans- factors associated with the glucocorticoid (GRE) and/or insulin (IRE) response sequences. Moreover, since PUFA appear to activate trans-factors of the steroid receptor super-family, we propose PUFA are dietary humoral factors when consumed t critical periods of development can """"""""imprint"""""""" genes in a manner that alters their gene expression throughout the life cycle. The rat FAS gene including 15kbp of 5' flanking will be employed to: (a) functionally map the yet unidentified PUFA-RE and the GRE in the 5'-flanking sequences of the FAS gene; (b) characterize the interaction between the IRE and the GRE which leads to amplification pf FAS transcription; and determine how the PUFA-RE interacts to silence the GRE and IRE of FAS; and (c) identify and characterize the trans-factors responsible for the PUFA regulation of FAS transcription.
These Aims will be addressed by a combination of techniques including: transfection analyses with rat liver cells in primary culture; DNAse-I hypersensitivity site mapping and in vivo footprinting of nuclei from rats fed PUFA versus 18:1 (n-9); and in vivo footprinting with hepatocytes monolayuers treated with (n-6)/(n-3) PUFA; UV cross-linking for trans-factor characterization; and yeast one-hybrid system for cloning PUFA-RE trans-factors associated.
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