Foxo1 links insulin signaling to C/EBPalpha and regulates gluconeogenesis during liver development.
ABSTRACT: C/EBPalpha is a key transcription factor indispensable for the onset of gluconeogenesis in perinatal liver. However, C/EBPalpha was already expressed in fetal liver, suggesting that the expression of C/EBPalpha alone does not account for the dramatic increase of the expression of metabolic genes, and hence an additional factor(s) is expected to function cooperatively with C/EBPalpha in perinatal liver. We show here that expression of Foxo1 was sharply increased in the perinatal liver and augmented C/EBPalpha-dependent transcription. Foxo1 bound C/EBPalpha via its forkhead domain, and Foxo1 bound to the promoter of a gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK), in a C/EBPalpha-dependent manner in vivo. Insulin inhibited the expression of PEPCK in a culture of fetal liver cells, and also the C/EBPalpha-dependent transcription enhanced by Foxo1. These results indicate that Foxo1 regulates gluconeogenesis cooperatively with C/EBPalpha, and also links insulin signaling to C/EBPalpha during liver development.
Project description:SHP (short heterodimer partner) is an orphan nuclear receptor that plays an important role in regulating glucose and lipid metabolism. A variety of transcription factors are known to regulate transcription of the PEPCK (phosphoenolpyruvate carboxykinase) gene, which encodes a rate-determining enzyme in hepatic gluconeogenesis. Previous reports identified glucocorticoid receptor and Foxo1 as novel downstream targets regulating SHP inhibition [Borgius, Steffensen, Gustafsson and Treuter (2002) J. Biol. Chem. 277, 49761-49796; Yamagata, Daitoku, Shimamoto, Matsuzaki, Hirota, Ishida and Fukamizu (2004) J. Biol. Chem. 279, 23158-23165]. In the present paper, we show a new molecular mechanism of SHP-mediated inhibition of PEPCK transcription. We also show that the CRE1 (cAMP regulatory element 1; -99 to -76 bp relative to the transcription start site) of the PEPCK promoter is also required for the inhibitory regulation by SHP. SHP repressed C/EBPalpha (CCAAT/enhancer-binding protein alpha)-driven transcription of PEPCK through direct interaction with C/EBPalpha protein both in vitro and in vivo. The formation of an active transcriptional complex of C/EBPalpha and its binding to DNA was inhibited by SHP, resulting in the inhibition of PEPCK gene transcription. Taken together, these results suggest that SHP might regulate a level of hepatic gluconeogenesis driven by C/EBPalpha activation.
Project description:The C/EBPalpha transcription factor regulates hepatic nitrogen, glucose, lipid and iron metabolism. However, how it is able to independently control these processes is not known. Here, we use mouse knock-in mutagenesis to identify C/EBPalpha domains that specifically regulate hepatic gluconeogenesis and lipogenesis. In vivo deletion of a proline-histidine rich domain (PHR), dephosphorylated at S193 by insulin signaling, dysregulated genes involved in the generation of acetyl-CoA and NADPH for triglyceride synthesis and led to increased hepatic lipogenesis. These promoters bound SREBP-1 as well as C/EBPalpha, and the PHR was required for C/EBPalpha-SREBP transcriptional synergy. In contrast, the highly conserved C/EBPalpha CR4 domain was found to undergo liver-specific dephosphorylation of residues T222 and T226 upon fasting, and alanine mutation of these residues upregulated the hepatic expression of the gluconeogenic G6Pase and PEPCK mRNAs, but not PGC-1alpha, leading to glucose intolerance. Our results show that pathway-specific metabolic regulation can be achieved through a single transcription factor containing context-sensitive regulatory domains, and indicate C/EBPalpha phosphorylation as a PGC-1alpha-independent mechanism for regulating hepatic gluconeogenesis.
Project description:In the present study, we observed superstimulated levels of cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in cells infected with wild-type adenovirus expressing 12 S and 13 S E1a proteins, or in cells expressing 13 S E1a alone. cAMP-stimulated transcription was inhibited in cells expressing only 12 S E1a, but slightly elevated in cells expressing E1a proteins with mutations in conserved regions 1 or 2, leading us to conclude that the superstimulation was mediated by conserved region 3 of 13 S E1a. E1a failed to enhance cAMP-stimulated transcription from promoters containing mutations that abolish binding by cAMP response element binding protein (CREB) or CCAAT/enhancer binding proteins (C/EBPs). This result was supported by experiments in which expression of dominant-negative CREB and/or C/EBP proteins repressed E1a- and cAMP-stimulated transcription from the PEPCK gene promoter. In reconstitution experiments using a Gal4-responsive promoter, E1a enhanced cAMP-stimulated transcription when chimaeric Gal4-CREB and Gal4-C/EBPalpha were co-expressed. Phosphorylation of CREB on serine-133 was stimulated in cells treated with dibutyryl cAMP, whereas phosphorylation of C/EBPalpha was increased by E1a expression. Our data support a model in which cAMP agonists increase CREB activity and stimulate PEPCK gene transcription, a process that is enhanced by E1a through the phosphorylation of C/EBPalpha.
Project description:PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding receptors for M-CSF, G-CSF and GM-CSF, were expressed at low levels in PU.1(-/-) fetal liver compared with wild type. To identify additional myeloid genes regulated by PU.1 and C/EBPalpha, we performed representational difference analysis (RDA), a PCR-based subtractive hybridization using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. By introducing a new modification of RDA, that of tissue-specific gene suppression, we could selectively identify a set of differentially expressed genes specific to myeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. In addition, eight novel genes were identified that were upregulated in expression during myeloid differentiation. These methods provide a general strategy for elucidating the genes affected in murine knockout models.
Project description:Renewed interest in alternative medicine among diabetic individuals prompted us to investigate anti-diabetic effects of Morinda citrifolia (noni) in high-fat diet (HFD)-fed mice. Type 2 diabetes is associated with increased glucose production due to the inability of insulin to suppress hepatic gluconeogenesis and promote glycolysis. Insulin inhibits gluconeogenesis by modulating transcription factors such as forkhead box O (FoxO1). Based on microarray analysis data, we tested the hypothesis that fermented noni fruit juice (fNJ) improves glucose metabolism via FoxO1 phosphorylation. C57BL/6 male mice were fed a HFD and fNJ for 12 weeks. Body weights and food intake were monitored daily. FoxO1 expression was analysed by real-time PCR and Western blotting. Specificity of fNJ-associated FoxO1 regulation of gluconeogenesis was confirmed by small interfering RNA (siRNA) studies using human hepatoma cells, HepG2. Supplementation with fNJ inhibited weight gain and improved glucose and insulin tolerance and fasting glucose in HFD-fed mice. Hypoglycaemic properties of fNJ were associated with the inhibition of hepatic FoxO1 mRNA expression, with a concomitant increase in FoxO1 phosphorylation and nuclear expulsion of the proteins. Gluconeogenic genes, phosphoenolpyruvate C kinase (PEPCK) and glucose-6-phosphatase (G6P), were significantly inhibited in mice fed a HFD+fNJ. HepG2 cells demonstrated more than 80 % inhibition of PEPCK and G6P mRNA expression in cells treated with FoxO1 siRNA and fNJ. These data suggest that fNJ improves glucose metabolism via FoxO1 regulation in HFD-fed mice.
Project description:OBJECTIVE: Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by Pck1) catalyzes the first committed step in gluconeogenesis. Extensive evidence demonstrates a direct correlation between PEPCK-C activity and glycemia control. Therefore, we aimed to evaluate the metabolic impact and their underlying mechanisms of knocking down hepatic PEPCK-C in a type 2 diabetic model. RESEARCH DESIGN AND METHODS: PEPCK-C gene targeting was achieved using adenovirus-transduced RNAi. The study assessed several clinical symptoms of diabetes and insulin signaling in peripheral tissues, in addition to changes in gene expression, protein, and metabolites in the liver. Liver bioenergetics was also evaluated. RESULTS: Treatment resulted in reduced PEPCK-C mRNA and protein. After treatment, improved glycemia and insulinemia, lower triglyceride, and higher total and HDL cholesterol were measured. Unsterified fatty acid accumulation was observed in the liver, in the absence of de novo lipogenesis. Despite hepatic lipidosis, treatment resulted in improved insulin signaling in the liver, muscle, and adipose tissue. O(2) consumption measurements in isolated hepatocytes demonstrated unaltered mitochondrial function and a consequent increased cellular energy charge. Key regulatory factors (FOXO1, hepatocyte nuclear factor-4alpha, and peroxisome proliferator-activated receptor-gamma coactivator [PGC]-1alpha) and enzymes (G6Pase) implicated in gluconeogenesis were downregulated after treatment. Finally, the levels of Sirt1, a redox-state sensor that modulates gluconeogenesis through PGC-1alpha, were diminished. CONCLUSIONS: Our observations indicate that silencing PEPCK-C has direct impact on glycemia control and energy metabolism and provides new insights into the potential significance of the enzyme as a therapeutic target for the treatment of diabetes.
Project description:Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. In this way, HCV enhanced the cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor forkhead box O1 (FoxO1). We observed that although neither the mRNA levels nor the protein levels of FoxO1 expression were affected by HCV, the level of phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting in an increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It was unlikely that the decreased level of FoxO1 phosphorylation was mediated through Akt inactivation, as we observed an increased phosphorylation of Akt at Ser473 in HCV-infected cells compared to control cells. By using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in decreased FoxO1 phosphorylation, FoxO1 nuclear accumulation, and, eventually, increased glucose production. We also found that HCV NS5A mediated increased ROS production and JNK activation, which is directly linked with the FoxO1-dependent increased gluconeogenesis. Taken together, these observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.
Project description:The mitochondrial enriched GCN5-like 1 (GCN5L1) protein has been shown to modulate mitochondrial protein acetylation, mitochondrial content and mitochondrial retrograde signaling. Here we show that hepatic GCN5L1 ablation reduces fasting glucose levels and blunts hepatic gluconeogenesis without affecting systemic glucose tolerance. PEPCK and G6Pase transcript levels are downregulated in hepatocytes from GCN5L1 liver specific knockout mice and their upstream regulator, FoxO1 protein levels are decreased via proteasome-dependent degradation and via reactive oxygen species mediated ERK-1/2 phosphorylation. ERK inhibition restores FoxO1, gluconeogenic enzyme expression and glucose production. Reconstitution of mitochondrial-targeted GCN5L1 blunts mitochondrial ROS, ERK activation and increases FoxO1, gluconeogenic enzyme expression and hepatocyte glucose production. We suggest that mitochondrial GCN5L1 modulates post-translational control of FoxO1, regulates gluconeogenesis and controls metabolic pathways via mitochondrial ROS mediated ERK activation. Exploring mechanisms underpinning GCN5L1 mediated ROS signaling may expand our understanding of the role of mitochondria in gluconeogenesis control.Hepatic gluconeogenesis is tightly regulated at transcriptional level and is essential for survival during prolonged fasting. Here Wang et al. show that the mitochondrial enriched GCN5-like 1 protein controls hepatic glucose production by regulating FoxO1 protein levels via proteasome-dependent degradation and, in turn, gluconeogenic gene expression.
Project description:SREBP1c is a key lipogenic transcription factor activated by insulin in the postprandial state. Although SREBP1c appears to be involved in suppression of hepatic gluconeogenesis, the molecular mechanism is not thoroughly understood. Here we show that CRY1 is activated by insulin-induced SREBP1c and decreases hepatic gluconeogenesis through FOXO1 degradation, at least, at specific circadian time points. SREBP1c(-/-) and CRY1(-/-) mice show higher blood glucose than wild-type (WT) mice in pyruvate tolerance tests, accompanied with enhanced expression of PEPCK and G6Pase genes. CRY1 promotes degradation of nuclear FOXO1 by promoting its binding to the ubiquitin E3 ligase MDM2. Although SREBP1c fails to upregulate CRY1 expression in db/db mice, overexpression of CRY1 attenuates hyperglycaemia through reduction of hepatic FOXO1 protein and gluconeogenic gene expression. These data suggest that insulin-activated SREBP1c downregulates gluconeogenesis through CRY1-mediated FOXO1 degradation and that dysregulation of hepatic SREBP1c-CRY1 signalling may contribute to hyperglycaemia in diabetic animals.
Project description:Hepatic glucose production is normally activated at birth but has been observed in response to experimental hypoglycemia in fetal sheep. The cellular basis for this process remains unknown. We determined the impact of 2 wk of fetal hypoglycemia during late gestation on enzymes responsible for hepatic gluconeogenesis, focusing on the insulin-signaling pathway, transcription factors, and coactivators that regulate gluconeogenesis. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNA increased 12-fold and 7-fold, respectively, following chronic hypoglycemia with no change in hepatic glycogen. Chronic hypoglycemia decreased fetal plasma insulin with no change in glucagon but increased plasma cortisol 3.5-fold. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA and phosphorylation of cAMP response element binding protein at Ser(133) were both increased, with no change in Akt, forkhead transcription factor FoxO1, hepatocyte nuclear factor-4alpha, or CCAAT enhancer binding protein-beta. These results demonstrate that chronic fetal hypoglycemia triggers signals that can activate gluconeogenesis in the fetal liver.