We have previously described the ability of arachidonic acid (AA) to regulate GLUT4 gene expression (Tebbey, P.W., McGowan, K.M., Stephens, J.M., Buttke, T.M., and Pekala, P.H. (1994) J. Biol. Chem. 269, 639-644). Chronic exposure (48 h) of fully differentiated 3T3-L1 cells to AA resulted in an approximately 90% suppression of GLUT4 mRNA accumulation. This decrease was demonstrated to be due to a 50% decrease in GLUT4 gene transcription as well as a destabilization of the GLUT4 message (t1/2 decreased from 8.0 to 4.6 h). In the current study we have identified, at least in part, the mechanism by which AA exerts its effects on GLUT4 expression. Compatible with a cyclooxygenase mediated event, the AA-induced suppression of GLUT4 mRNA was abolished by pretreating the cells with the inhibitor, indomethacin. Consistent with this observation, exposure of the cells to 10 microM PGE2 mimicked the effect of AA, in contrast to products of the lipoxygenase pathway which were unable to suppress GLUT4 mRNA content. Quantification of the conversion of AA to PGE2 demonstrated a 50-fold increase in PGE2 released into the media within 7 h of AA addition. Cyclic AMP levels were also increased 50-fold with AA treatment consistent with PGE2 activation of adenylate cyclase. Various long chain fatty acids, including the nonmetabolizable analog of AA, eicosatetraenoic acid (ETYA), also decreased GLUT4 mRNA levels. The effect of ETYA, a potent inhibitor of both lipo- and cyclooxygenases and a potent activator of peroxisome proliferator activated receptors (PPARs), suggested the presence of a second pathway where non-metabolized fatty acid functioned to suppress GLUT4 mRNA levels. Further support for a PPAR-mediated mechanism was obtained by exposure of the cells to the classic PPAR activator, clofibrate, which resulted in a approximately 75% decrease in GLUT4 mRNA content. Nuclear extracts prepared from the adipocytes contained a protein complex that bound to the PPAR responsive element (PPRE) found in the promoter of the fatty acyl-CoA oxidase gene. When the adipocytes were treated with either AA or ETYA, binding to the PPRE was disrupted, consistent with an ability of these fatty acids to control gene expression by altering the occupation of a PPRE. However, a perfect PPRE has yet to be identified in the GLUT4 promoter, but this does not rule the possibility of a PPAR playing an indirect role in the AA-induced GLUT4 mRNA suppression.