Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein-1 gene expression by insulin - The role of winged helix/forkhead proteins

Winged helix/forkhead (Fox) transcription factors have been implicated in t he regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insul in-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Previous mutational analyses of the PEPCK and IGFBP-1 IREs revealed mutations which do not affect the bindi ng of HNF3 proteins to these elements but do eliminate the ability of the I REs to mediate an insulin response. This dissociation of binding and functi on provided compelling evidence that HNF3 proteins, per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFB P-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcript ion from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There i s a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FRHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions wher e FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL 1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by ins ulin when this protein is overexpressed, but at the normal cellular concent ration of FKHRL1 the insulin response mediated by these elements must invol ve another protein.