Abstract
Insulin inhibits glucagon gene transcription, and insulin deficiency is associated with hyperglucagonemia that contributes to hyperglycemia in diabetes mellitus. However, the insulin signaling pathway to the glucagon gene is unknown. Protein kinase B (PKB) is a key regulator of insulin signaling and glucose homeostasis. Impaired PKB function leads to insulin resistance and diabetes mellitus. Therefore, the role of PKB in the regulation of glucagon gene transcription was investigated. After transient transfections of glucagon promoter-reporter genes into a glucagon-producing islet cell line, the use of kinase inhibitors indicated that the inhibition of glucagon gene transcription by insulin depends on phosphatidylinositol (PI) 3-kinase. Furthermore, insulin caused a PI 3-kinase-dependent phosphorylation and activation of PKB in this cell line as revealed by phospho-immunoblotting and kinase assays. Overexpression of constitutively active PKB mimicked the effect of insulin on glucagon gene transcription. Both insulin and PKB responsiveness of the glucagon promoter were abolished when the binding sites for the transcription factor Pax6 within the G1 and G3 promoter elements were mutated. Recruitment of Pax6 or its potential coactivator, the CREB-binding protein (CBP), to G1 and G3 by using the GAL4 system restored both insulin and PKB responsiveness. These data suggest that insulin inhibits glucagon gene transcription by signaling via PI 3-kinase and PKB, with the transcription factor Pax6 and its potential coactivator CBP being critical components of the targeted promoter-specific nucleoprotein complex. The present data emphasize the importance of PKB in insulin signaling and glucose homeostasis by defining the glucagon gene as a novel target gene for PKB.