Protein kinase B lies "downstream" of phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate many of the intracellular actions of insulin and other growth factors. Here we show that FKHR, a human homologue of the DAF16 transcription factor in Caenorhabditis elegans, is rapidly phosphorylated by human protein kinase B␣ (PKB␣) at Thr-24, Ser-256, and Ser-319 in vitro and at a much faster rate than BAD, which is thought to be a physiological substrate for PKB. The same three sites, which all lie in the canonical PKB consensus sequences (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was cotransfected with either PKB or PDK1 (an upstream activator of PKB). All three residues became phosphorylated when 293 cells were stimulated with insulin-like growth factor 1 (IGF-1). The IGF-1-induced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated protein kinase cascade) or by rapamycin. These results indicate that FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known to be a component of a signaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 therefore suggests that DAF16 is also a direct substrate for PKB in C. elegans.In recent years evidence has accumulated that many of the metabolic actions of insulin may be mediated by a protein kinase cascade that lies "downstream" of phosphatidylinositide (PtdIns) 1 3-kinase and the second messengers PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 (reviewed in Refs. 1 and 2). A central player in this cascade is protein kinase B (PKB, also called c-Akt).This enzyme is activated when it becomes phosphorylated at Thr-308 and Ser-473 (3) by 3-phosphoinositide-dependent protein kinases 1 and 2 (PDK1, PDK2), respectively (4 -7). The activation of PKB by PDK1 in vitro has an absolute requirement for PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 (4), and these mediators facilitate activation by binding to the pleckstrin homology domains of both PKB (5, 7) and PDK1 (8). Consistent with these observations, the phosphorylation of PKB at Thr-308, induced by either insulin or insulin-like growth factor 1 (IGF-1) is prevented by inhibitors of PtdIns 3-kinase (3). PDK2 has not yet been characterized although, like the phosphorylation of Thr-308, the insulin or IGF-1-induced phosphorylation of Ser-473 is prevented by inhibitors of PtdIns 3-kinase (3).PKB mediates the metabolic actions of insulin by phosphorylating regulatory proteins at serine or threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs (9), of which the best characterized are the cardiac isoform of 6-phosphofructo-2-kinase (PFK2) (2, 10), the protein kinase glycogen synthase kinase 3 (GSK3) (11,12), and the mammalian target of rapamycin (mTOR) (13), as well as the proapo...

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 resistance is a major underlying mechanism for the “metabolic syndrome”, which is also known as insulin resistance syndrome. Metabolic syndrome is increasing at an alarming rate, becoming a major public and clinical problem worldwide. Metabolic syndrome is represented by a group of interrelated disorders, including obesity, hyperglycemia, hyperlipidemia, and hypertension. It is also a significant risk factor for cardiovascular disease and increased morbidity and mortality. Animal studies demonstrate that insulin and its signaling cascade normally control cell growth, metabolism and survival through activation of mitogen-activated protein kinases (MAPKs) and phosphotidylinositide-3-kinase (PI3K), of which activation of PI-3K-associated with insulin receptor substrate-1 and -2 (IRS1, 2) and subsequent Akt→Foxo1 phosphorylation cascade has a central role in control of nutrient homeostasis and organ survival. Inactivation of Akt and activation of Foxo1, through suppression IRS1 and IRS2 in different organs following hyperinsulinemia, metabolic inflammation, and over nutrition may provide the underlying mechanisms for metabolic syndrome in humans. Targeting the IRS→Akt→Foxo1 signaling cascade will likely provide a strategy for therapeutic intervention in the treatment of type 2 diabetes and its complications. This review discusses the basis of insulin signaling, insulin resistance in different mouse models, and how a deficiency of insulin signaling components in different organs contributes to the feature of the metabolic syndrome. Emphasis will be placed on the role of IRS1, IRS2, and associated signaling pathways that couple to Akt and the forkhead/winged helix transcription factor Foxo1.