Correlation between FOXO1a (FKHR) and FOXO3a (FKHRL1) binding and the inhibition of basal glucose-6-phosphatase catalytic subunit gene transcription by insulin

Insulin inhibits transcription of the genes encoding the glucose-6-phosphatase catalytic subunit (G6Pase), phosphoenolpyruvate carboxykinase, and IGF binding protein-1 through insulin response sequences (IRSs) that share the same core sequence, T(G/A) TTTT(G/T). The transcription factors FOXO1a and FOXO3a have been shown to bind these elements, but there are conflicting reports as to whether this binding correlates with the action of insulin on gene transcription. Some researchers concluded, from overexpression experiments using FOXO1a, that binding correlated with the insulin response, whereas others concluded, mainly from gel retardation competition experiments using FOXO3a, that it did not. We show here that, although these factors can differentially activate gene transcription in a context-dependent manner, these conflicting data are not explained by a difference in FOXO1a and FOXO3a binding specificity. Instead, we find that gel retardation competition and binding experiments give different results; the latter reveal a correlation between FOXO1a/3a binding and the inhibition of basal G6Pase gene transcription by insulin. In addition, these data show that the binding of FOXO1a/3a to two adjacent IRSs in the G6Pase promoter is cooperative and that promoter context alters the specific IRS base requirements for FOXO1a-stimulated fusion gene expression. Surprisingly, an analysis of insulin action mediated through the G6Pase and IGF binding protein-1 IRSs in the context of a heterologous thymidine kinase promoter reveals that signaling through the latter does not support the accepted model for insulin-stimulated FOXO nuclear exclusion.