Terry G. Unterman

Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B.

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 Bα (PKBα) at Thr-24, Ser-256, and Ser-319in 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.

Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence.

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 inCaenorhabditis elegans indicate that daf-16, aforkhead/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 forkheadproteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression.

FoxO1 Regulates Multiple Metabolic Pathways in the Liver EFFECTS ON GLUCONEOGENIC, GLYCOLYTIC, AND LIPOGENIC GENE EXPRESSION

FoxO transcription factors are important targets of insulin action. To better understand the role of FoxO proteins in the liver, we created transgenic mice expressing constitutively active FoxO1 in the liver using the α1-antitrypsin promoter. Fasting glucose levels are increased, and glucose tolerance is impaired in transgenic (TGN) versus wild type (WT) mice. Interestingly, fasting triglyceride and cholesterol levels are reduced despite hyperinsulinemia, and post-prandial changes in triglyceride levels are markedly suppressed in TGN versus WT mice. Activation of pro-lipogenic signaling pathways (atypical protein kinase C and protein kinase B) and the ability to suppress β-hydroxybutyrate levels are not impaired in TGN. In contrast, de novo lipogenesis measured with 3H2O is suppressed by ∼70% in the liver of TGN versus WT mice after refeeding. Gene-array studies reveal that the expression of genes involved in gluconeogenesis, glycerol transport, and amino acid catabolism is increased, whereas genes involved in glucose utilization by glycolysis, the pentose phosphate shunt, lipogenesis, and sterol synthesis pathways are suppressed in TGN versus WT. Studies with adenoviral vectors in isolated hepatocytes confirm that FoxO1 stimulates expression of gluconeogenic genes and suppresses expression of genes involved in glycolysis, the shunt pathway, and lipogenesis, including glucokinase and SREBP-1c. Together, these results indicate that FoxO proteins promote hepatic glucose production through multiple mechanisms and contribute to the regulation of other metabolic pathways important in the adaptation to fasting and feeding in the liver, including glycolysis, the pentose phosphate shunt, and lipogenic and sterol synthetic pathways.

Regulation of Glucose-6-phosphatase Gene Expression by Protein Kinase Bα and the Forkhead Transcription Factor FKHR EVIDENCE FOR INSULIN RESPONSE UNIT-DEPENDENT AND -INDEPENDENT EFFECTS OF INSULIN ON PROMOTER ACTIVITY

Glucose-6-phosphatase plays an important role in the regulation of hepatic glucose production, and insulin suppresses glucose-6-phosphatase gene expression. Recent studies indicate that protein kinase B and Forkhead proteins contribute to insulin-regulated gene expression in the liver. Here, we examined the role of protein kinase B and Forkhead proteins in mediating effects of insulin on glucose-6-phosphatasepromoter activity. Transient transfection studies with reporter gene constructs demonstrate that insulin suppresses both basal and dexamethasone/cAMP-induced activity of the glucose-6-phosphatasepromoter in H4IIE hepatoma cells. Both effects are partially mimicked by coexpression of protein kinase Bα. Coexpression of the Forkhead transcription factor FKHR stimulates the glucose-6-phosphatase promoter activity via interaction with an insulin response unit (IRU), and this activation is suppressed by protein kinase B. Coexpression of a mutated form of FKHR that cannot be phosphorylated by protein kinase B abolishes the regulation of theglucose-6-phosphatase promoter by protein kinase B and disrupts the ability of insulin to regulate theglucose-6-phosphatase promoter via the IRU. Mutation of the insulin response unit of the glucose-6-phosphatase promoter also prevents the regulation of promoter activity by FKHR and protein kinase B but only partially impairs the ability of insulin to suppress both basal and dexamethasone/cAMP-stimulated promoter function. Taken together, these results indicate that signaling by protein kinase B to Forkhead proteins can account for the ability of insulin to regulateglucose-6-phosphatase promoter activity via the IRU and that other mechanisms that are independent of the IRU, protein kinase B, and Forkhead proteins also are important in mediating effects of in insulin on glucose-6-phosphatase gene expression.

Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 binding, transactivation and nuclear targetting.

The transcription factor, forkhead in rhabdomyosarcoma (FKHR), is phosphorylated at three amino acid residues (Thr-24, Ser-256 and Ser-319) by protein kinase B (PKB)alpha. In the present study, mutagenesis has been used to study the roles of these phosphorylation events in regulating FKHR function in transfected HEK-293 cells. We find that the overexpression of FKHR[S256A] (where Ser-256-->Ala) blocks PKB activity in cells, preventing phosphorylation of the endogenous substrates FKHRL1 and glycogen synthase kinase-3. Thus some reported effects of overexpression of this and other mutants may be indirect, and result from suppression of the phosphorylation of other sites on FKHR and/or other PKB substrates. For example, we have shown that Thr-24 phosphorylation alone is critical for interaction with 14-3-3 proteins, and that the substitution of Ser-256 with an alanine residue indirectly blocks 14-3-3 protein binding by preventing the phosphorylation of Thr-24. We also found that insulin-like growth factor (IGF)-1 and serum-induced nuclear exclusion of FKHR[S256A] depends on the degree of overexpression of this mutant. Our results indicated that the interaction of FKHR with 14-3-3 proteins was not required for IGF-1-stimulated exclusion of FKHR from the nucleus. We present evidence in support of another mechanism, which depends on the phosphorylation of Ser-256 and may involve the masking of a nuclear localization signal. Finally, we have demonstrated that the failure of IGF-1 to suppress transactivation by FKHR[S256A] is not explained entirely by its failure to bind 14-3-3 proteins or to undergo nuclear exclusion. This result suggests that Ser-256 phosphorylation may also suppress transactivation by FKHR by yet another mechanism, perhaps by disrupting the interaction of FKHR with target DNA binding sites and/or the function of the transactivation domain.

Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion.

FKHR is phosphorylated by protein kinase B (PKB) at Thr24, Ser256 and Ser319 in response to growth factors, stimulating the nuclear exit and inactivation of this transcription factor. Here we identify two further residues, Ser322 and Ser325, that become phosphorylated in insulin‐like growth factor‐1 (IGF‐1)‐stimulated cells and which are mediated by the phosphatidylinositol 3‐kinase‐dependent PKB‐catalysed phosphorylation of Ser319. Phosphorylation of Ser319 forms a consensus sequence for phosphorylation by CK1, allowing it to phosphorylate Ser322, which in turn primes the CK1‐catalysed phosphorylation of Ser325. IGF‐1 stimulates the phosphorylation of Thr24, Ser256, Ser319, Ser322 and Ser325 in embryonic stem (ES) cells, but not in PDK1−/− ES cells, providing genetic evidence that PDK1 (the upstream activator of PKB) is required for the phosphorylation of FKHR in mammalian cells. In contrast, the phosphorylation of Ser329 is unaffected by IGF‐1 and the phosphorylation of this site is not decreased in PDK1−/− ES cells. The cluster of phosphorylation sites at Ser319, Ser322, Ser325 and Ser329 appears to accelerate nuclear export by controlling the interaction of FKHR with the Ran‐containing protein complex that mediates this process.

Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration

Fatty liver disease is associated with obesity and type 2 diabetes, and hepatic lipid accumulation may contribute to insulin resistance. Histone deacetylase 3 (Hdac3) controls the circadian rhythm of hepatic lipogenesis. Here we show that, despite severe hepatosteatosis, mice with liver-specific depletion of Hdac3 have higher insulin sensitivity without any changes in insulin signaling or body weight compared to wild-type mice. Hdac3 depletion reroutes metabolic precursors towards lipid synthesis and storage within lipid droplets and away from hepatic glucose production. Perilipin 2, which coats lipid droplets, is markedly induced upon Hdac3 depletion and contributes to the development of both steatosis and improved tolerance to glucose. These findings suggest that the sequestration of hepatic lipids in perilipin 2–coated droplets ameliorates insulin resistance and establish Hdac3 as a pivotal epigenomic modifier that integrates signals from the circadian clock in the regulation of hepatic intermediary metabolism.

Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action.

Hepatic insulin resistance directly promotes formation of cholesterol gallstones

Despite the well-documented association between gallstones and the metabolic syndrome, the mechanistic links between these two disorders remain unknown. Here we show that mice solely with hepatic insulin resistance, created by liver-specific disruption of the insulin receptor (LIRKO mice) are markedly predisposed toward cholesterol gallstone formation due to at least two distinct mechanisms. Disinhibition of the forkhead transcription factor FoxO1, increases expression of the biliary cholesterol transporters Abcg5 and Abcg8, resulting in an increase in biliary cholesterol secretion. Hepatic insulin resistance also decreases expression of the bile acid synthetic enzymes, particularly Cyp7b1, and produces partial resistance to the farnesoid X receptor, leading to a lithogenic bile salt profile. As a result, after twelve weeks on a lithogenic diet, all of the LIRKO mice develop gallstones. Thus, hepatic insulin resistance provides a crucial link between the metabolic syndrome and increased cholesterol gallstone susceptibility.

Protein Kinase B/Akt Mediates Effects of Insulin on Hepatic Insulin-like Growth Factor-binding Protein-1 Gene Expression through a Conserved Insulin Response Sequence

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 sequence-specific 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.