Hepatic gluconeogenesis is absolutely required for survival during prolonged fasting or starvation, but is inappropriately activated in diabetes mellitus. Glucocorticoids and glucagon have strong gluconeogenic actions on the liver. In contrast, insulin suppresses hepatic gluconeogenesis. Two components known to have important physiological roles in this process are the forkhead transcription factor FOXO1 (also known as FKHR) and peroxisome proliferative activated receptor-gamma co-activator 1 (PGC-1alpha; also known as PPARGC1), a transcriptional co-activator; whether and how these factors collaborate has not been clear. Using wild-type and mutant alleles of FOXO1, here we show that PGC-1alpha binds and co-activates FOXO1 in a manner inhibited by Akt-mediated phosphorylation. Furthermore, FOXO1 function is required for the robust activation of gluconeogenic gene expression in hepatic cells and in mouse liver by PGC-1alpha. Insulin suppresses gluconeogenesis stimulated by PGC-1alpha but co-expression of a mutant allele of FOXO1 insensitive to insulin completely reverses this suppression in hepatocytes or transgenic mice. We conclude that FOXO1 and PGC-1alpha interact in the execution of a programme of powerful, insulin-regulated gluconeogenesis.

An outstanding question in adipocyte biology is how hormonal cues are relayed to the nucleus to activate the transcriptional program that promotes adipogenesis. The forkhead transcription factor Foxo1 is regulated by insulin via Akt-dependent phosphorylation and nuclear exclusion. We show that Foxo1 is induced in the early stages of adipocyte differentiation but that its activation is delayed until the end of the clonal expansion phase. Constitutively active Foxo1 prevents the differentiation of preadipocytes, while dominant-negative Foxo1 restores adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. Further, Foxo1 haploinsufficiency protects from diet-induced diabetes in mice. We propose that Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes adipocyte differentiation.

Diabetes causes pancreatic beta cell failure through hyperglycemia-induced oxidative stress, or "glucose toxicity." We show that the forkhead protein FoxO1 protects beta cells against oxidative stress by forming a complex with the promyelocytic leukemia protein Pml and the NAD-dependent deacetylase Sirt1 to activate expression of NeuroD and MafA, two Insulin2 (Ins2) gene transcription factors. Using acetylation-defective and acetylation-mimicking mutants, we demonstrate that acetylation targets FoxO1 to Pml and prevents ubiquitin-dependent degradation. We show that hyperglycemia suppresses MafA expression in vivo and that MafA inhibition can be prevented by transgenic expression of constitutively nuclear FoxO1 in beta cells. The findings provide a mechanism linking glucose- and growth factor receptor-activated pathways to protect beta cells against oxidative damage via FoxO proteins.

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Leptin controls food intake by regulating the transcription of key neuropeptides in the hypothalamus. The mechanism by which leptin regulates gene expression is unclear, however. Here we show that delivery of adenovirus encoding a constitutively nuclear mutant FoxO1, a transcription factor known to control liver metabolism and pancreatic beta-cell function, to the hypothalamic arcuate nucleus of rodents results in a loss of the ability of leptin to curtail food intake and suppress expression of Agrp. Conversely, a transactivation-deficient FoxO1 mutant prevents induction of Agrp by fasting. We also find that FoxO1 and the transcription factor Stat3 exert opposing actions on the expression of Agrp and Pomc through transcriptional squelching. FoxO1 promotes opposite patterns of coactivator-corepressor exchange at the Pomc and Agrp promoters, resulting in activation of Agrp and inhibition of Pomc. Thus, FoxO1 represents a shared component of pathways integrating food intake and peripheral metabolism.

Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.Akt is a protein serine-threonine kinase that contains a pleckstrin homology domain and whose kinase domain has structural similarity with those of protein kinase C (PKC) isozymes and cyclic AMP (cAMP)-dependent protein kinase (PKA) (9, 21). Thus, Akt has also been termed protein kinase B. Akt was originally shown to be activated by growth factors such as platelet-derived growth factor and insulin, but later the enzyme was also found to be activated by cytokines and ligands for G protein-coupled receptors (21,33,34). Moreover, expression of polyomavirus middle T antigen as well as cellular stresses such as hyperosmolarity, heat shock, and fluid shear stress also induces activation of Akt (17,27,42). However, the mechanisms by which Akt is activated by these diverse stimuli are not fully understood. The activation of Akt by growth factors or cytokines is blocked by pharmacological or molecular biological inhibitors of phosphoinositide (PI) 3-kinase (7,19,24), indicating that PI 3-kinase is an upstream regulator of Akt, although PI 3-kinase-independent stimuli that induce activation of Akt also appear to exist (27,33,38).Akt is a general mediator of cell survival and protection from apoptosis (9, 21). It has also been suggested to participate in meiosis in oocytes (3), in endocytosis elicited by RAS (5), in differentiation of adipocytes (25), and in various metabolic actions of insulin (23,25,44,45). In spite of the potential importance of Akt in such diverse biological activities, only a few proteins have been identified as physiological substrates of this enzyme. The first iden...

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:l-phosphatidyl-lD-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer coIng of regulatory 85-kDa (p85) and catalytic 110-kDa (p11O) subunits. appears to be the subunit that links PI 3-kinase activity in pilO to the tyrosine-phosphorylated proteins.The insulin receptor belongs to the family of structurally related transmembrane growth factor receptors with ligandactivated protein-tyrosine kinase activity (7,8). Insulin treatment of cells has been found to increase PI 3-kinase activity in immunoprecipitates made by using antibody to phosphotyrosine (9, 10). Insulin treatment of various intact cells causes rapid tyrosine phosphorylation of a high molecular weight protein (Mr 160,000-185,000) (11, 12) termed insulin receptor substrate 1 (IRS-1) and its sequence was deduced by cDNA cloning (13). Insulin drives the formation ofa complex between tyrosine-phosphorylated IRS-1 and SH2 domains of several proteins including p85 (14-16). However, the role of the binding of PI 3-kinase to IRS-1 in insulin signal transduction is not clear. To address this issue, we disrupted complex formation between the catalytic p110 subunit of PI 3-kinase and IRS-1 by overexpressing mutant p85a (Ap85), which lacks a binding site for p110. MATERIALS AND METHODSCel Cults and Antibodies. CHO-IR cells were maintained and cultured as described (14). The antibodies used were as follows: monoclonal antibodies (mAbs) against the bovine p85a (F12 and G12) (14); polyclonal antipeptide antibodies against a synthetic C-terminal peptide of bovine p85a (residues 713-724) or bovine p85,B (residues 707-724); a polyclonal anti-pilO antibody against a glutathione S-transferase (GST) fusion protein containing residues 441-605 of bovine p110 (Transduction Laboratory, Lexington, KY); polyclonal anti-IRS-i antibodies against a synthetic rat IRS-1 peptide (pep8o) corresponding to residues 489-507 (13) or a GST fusion protein containing N-terminal residues 1-240 of rat IRS-1 (generously provided by Alan Saltiel, WarnerLambert, Ann Arbor, MI); a mAb against rat IRS-1 (ID6) (17); a mAb (py2O; ICN) and a polyclonal antibody (Upstate Biotechnology) against phosphotyrosine residues.Abbreviations: IRS-1, insulin receptor substrate 1; PtdIns, phosphatidylinositol(s); SH2, Src homology region 2; mAb, monoclonal antibody; PI(3,4,5)P3, PtdIns 3,4,5-trisphosphate; PI(4)P, PtdIns 4-phosphate; PI(4,5)P2, PtdIns 4,5-bisphosphate; PI(3)P, PtdIns 3-phosphate; PI(3,4)P2, PtdIns 3,4-bisphosphate; PI 3-kinase, 1-PtdIns 3-kinase; ATB-BMPA, 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4yloxy)-2-propylamine; PMA, phorbol 12-myristate 13-acetate.'lTo whom reprint requests should be addressed. 7415The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 sol...

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