Insulin inhibits the expression of multiple genes in the liver containing an insulin response sequence (IRS) (CAAAA(C/T)AA), and we have reported that protein kinase B (PKB) mediates this effect of insulin. Genetic studies in Caenorhabditis elegans indicate that daf-16, a forkhead/winged-helix transcription factor, is a major target of the insulin receptor-PKB signaling pathway. FKHR, a human homologue of daf-16, contains three PKB sites and is expressed in the liver. Reporter gene studies in HepG2 hepatoma cells show that FKHR stimulates insulin-like growth factor-binding protein-1 promoter activity through an IRS, and introduction of IRSs confers this effect on a heterologous promoter. Insulin disrupts IRS-dependent transactivation by FKHR, and phosphorylation of Ser-256 by PKB is necessary and sufficient to mediate this effect. Antisense studies indicate that FKHR contributes to basal promoter function and is required to mediate effects of insulin and PKB on promoter activity via an IRS. To our knowledge, these results provide the first report that FKHR stimulates promoter activity through an IRS and that phosphorylation of FKHR by PKB mediates effects of insulin on gene expression. Signaling to FKHR-related forkhead proteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression.Insulin exerts important effects on gene expression in multiple tissues (1). In the liver, insulin suppresses the expression of a number of genes that contain a conserved insulin response sequence (IRS) 1 (CAAAA(C/T)AA), including insulin-like growth factor-binding protein-1 (IGFBP-1), apolipoprotein CIII (apoCIII), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (2-6). This observation suggests that insulin may regulate the expression of multiple hepatic genes through a common mechanism. Insulin rapidly suppresses the expression of IGFBP-1 and PEPCK at the transcriptional level, and this effect is not disrupted by pretreatment with cycloheximide (7, 8), indicating that it is mediated by post-translational modification of pre-existing factors, perhaps by their phosphorylation. Specific factors that mediate the inhibitory effects of insulin on hepatic gene expression through a conserved IRS remain to be identified.Recent studies indicate that protein kinase B (PKB) functions downstream from phosphatidylinositol 3Ј-kinase (PI3K) in the insulin signaling pathway (9, 10) and that it plays an important role in mediating effects of insulin and related growth factors on glucose and amino acid transport, glycogen and protein synthesis, and cell survival (11)(12)(13)(14)(15)(16)(17)(18)(19). Following its activation, PKB is translocated to the nucleus where it may exert effects on gene expression (20,21). Activated PKB increases the expression of leptin and fatty acid synthase in adipocytes (22, 23) and suppresses PEPCK mRNA levels in liver-derived cells stimulated by cAMP and glucocorticoids (24), mimicking the effects of insulin. Based on studies using pha...

Insulin regulates the expression of multiple hepatic genes through a conserved insulin response sequence (IRS) (CAAAAC/TAA) by an as yet undetermined mechanism. Protein kinase B/Akt (PKB/Akt), a member of the PKA/PKC serine/threonine kinase family, functions downstream from phosphatidylinositol 3-kinase (PI3K) in mediating effects of insulin on glucose transport and glycogen synthesis. We asked whether PKB/Akt mediates sequence-specific effects of insulin on hepatic gene expression using the model of the insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Insulin lowers IGFBP-1 mRNA levels, inhibits IGFBP-1 promoter activity, and activates PKB/Akt in HepG2 hepatoma cells through a PI3K-dependent, rapamycin-insensitive mechanism. Constitutively active PI3K and PKB/ Akt are each sufficient to mediate effects of insulin on the IGFBP-1 promoter in a nonadditive fashion. Dominant negative K179 PKB/Akt disrupts the ability of insulin and PI3K to activate PKB/Akt and to inhibit promoter activity. The IGFBP-1 promoter contains two IRSs each of which is sufficient to mediate sequencespecific effects of insulin, PI3K, and PKB/Akt on promoter activity. Highly related IRSs from the phosphoenolpyruvate carboxykinase and apolipoprotein CIII genes also are effective in this setting. These results indicate that PKB/Akt functions downstream from PI3K in mediating sequence-specific effects of insulin on the expression of IGFBP-1 and perhaps multiple hepatic genes through a conserved IRS.The binding of insulin to its cell surface receptor results in the activation of multiple signaling pathways (1). 1 plays an important role in mediating cellular effects of insulin (2, 3). Rapamycin-sensitive pathways mediate PI3K-dependent effects of insulin on p70S6 kinase activity and cellular proliferation (4) and hexokinase II expression (5). Protein kinase B/Akt (PKB/Akt), a PKA/PKC serine/threonine kinase family member (6), is activated through a PI3K-dependent mechanism that is not blocked by rapamycin (7) and mediates effects of growth factors on cell survival (8 -10). PKB/Akt is thought to mediate the phosphorylation and inactivation of glycogen synthase kinase-3 by insulin (11) and stimulates glucose transporter translocation in 3T3-L1 adipocytes (12). The role of PKB/Akt in mediating sequence-specific effects of insulin on hepatic gene expression has not been determined. Insulin regulates gene expression in a variety of tissues (13). Hepatic expression of insulin-like growth factor binding protein-1 (IGFBP-1) is rapidly suppressed by insulin at the level of gene transcription (14 -16) through two insulin response sequences located ϳ100 bp 5Ј to the RNA cap site (17,18). Each IRS is sufficient to mediate effects of insulin on promoter activity (18, 19) and closely resembles IRSs, which mediate effects of insulin on several other genes in the liver, including phosphoenolpyruvate carboxykinase (PEPCK) (20) and apolipoprotein CIII (apoCIII) (21). These observations have suggested that a common, but as yet undetermined mechani...

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