Phosphoenolpyruvate Carboxykinase Transcription Research Articles

11-Deoxycortisol is a stress responsive and gluconeogenic hormone in a jawless vertebrate, the sea lamprey (Petromyzon marinus).

Although corticosteroid-mediated hepatic gluconeogenic activity in response to stress has been extensively studied in fishes and other vertebrates, there is little information on the stress response in basal vertebrates. In sea lamprey (Petromyzon marinus), a representative member of the most basal extant vertebrate group Agnatha, 11-deoxycortisol and deoxycorticosterone are the major circulating corticosteroids. The present study examined changes in circulating glucose and 11-deoxycortisol concentrations in response to a physical stressor. Furthermore, the gluconeogenic actions of 11-deoxycortisol and deoxycorticosterone were examined. Within 6 h of exposure of larval and juvenile sea lamprey to an acute handling stress, plasma 11-deoxycortisol levels increased 15- and 6-fold, respectively, and plasma glucose increased 3- and 4-fold, respectively. Radiometric receptor binding studies revealed that a corticosteroid receptor (CR) is present in the liver at lower abundance than in other tissues (gill and anterior intestine) and that the binding affinity of the liver CR was similar for 11-deoxycortisol and deoxycorticosterone. Transcriptional tissue profiles indicate a wide distribution of cr transcription, kidney-specific transcription of steroidogenic acute regulatory protein (star) and liver-specific transcription of phosphoenolpyruvate carboxykinase (pepck). Ex vivo incubation of liver tissue with 11-deoxycortisol resulted in dose-dependent increases in pepck mRNA levels. Finally, intraperitoneal administration of 11-deoxycortisol and deoxycorticosterone demonstrated that only 11-deoxycortisol resulted in an increase in plasma glucose. Together, these results provide the first direct evidence for the gluconeogenic activity of 11-deoxycortisol in an agnathan, indicating that corticosteroid regulation of plasma glucose is a basal trait among vertebrates.

PEPCK1 Antisense Oligonucleotide Prevents Adiposity and Impairs Hepatic Glycogen Synthesis in High-Fat Male Fed Rats.

The increased hepatic gluconeogenesis in type 2 diabetes mellitus has often been ascribed to increased transcription of phosphoenolpyruvate carboxykinase 1, cystolic form (PEPCK1), although recent evidence has questioned this attribution. To assess the metabolic role of PEPCK1, we treated regular chow fed and high-fat fed (HFF) male Sprague-Dawley rats with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) against PEPCK1 and compared them with control ASO-treated rats. PEPCK1 ASO effectively decreased PEPCK1 expression in the liver and white adipose tissue. In chow fed rats, PEPCK1 ASO did not alter adiposity, plasma glucose, or insulin. In contrast, PEPCK1 ASO decreased the white adipose tissue mass in HFF rats but without altering basal rates of lipolysis, de novo lipogenesis, or glyceroneogenesis in vivo. Despite the protection from adiposity, hepatic insulin sensitivity was impaired in HFF PEPCK1 ASO-treated rats. PEPCK1 ASO worsened hepatic steatosis, although without additional impairments in hepatic insulin signaling or activation of inflammatory signals in the liver. Instead, the development of hepatic insulin resistance and the decrease in hepatic glycogen synthesis during a hyperglycemic clamp was attributed to a decrease in hepatic glucokinase (GCK) expression and decreased synthesis of glycogen via the direct pathway. The decrease in GCK expression was associated with increased expression of activating transcription factor 3, a negative regulator of GCK transcription. These studies have demonstrated that PEPCK1 is integral to coordinating cellular metabolism in the liver and adipose tissue, although it does not directly effect hepatic glucose production or adipose glyceroneogenesis.

Increased expression of phosphoenolpyruvate carboxykinase cytoplasmic isoform by hepatitis B virus X protein affects hepatitis B virusreplication.

Hepatitis B virus X protein (HBx) can stimulate the transcription of phosphoenolpyruvate carboxykinase (PEPCK), a rate-determining enzyme in gluconeogenic pathway. Two isoforms of PEPCK exist, a cytoplasmic form (PCK1) and a mitochondrial isoform (PCK2). The current study investigated the direct effect of HBx-stimulated PEPCK on hepatitis B virus (HBV) replication. We showed that PCK1 rather than PCK2 was upregulated by HBx. We also demonstrated that overexpression of PCK1 decreased HBV replication, whereas inhibition of PCK1-enhanced HBV replication. Furthermore, we found overexpression of PCK1 led to reduced expression of peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α) and peroxisome proliferator-activated receptor γ (PPAR-γ), whereas knocking down PCK1 resulted in an increased expression of PGC-1α and PPAR-γ. When PPAR-γ was inhibited, knocking down PCK1 could not induce the apparent enhanced HBV replication. Our data suggested that PCK1 induced by HBx led to decreased HBV replication through the downregulation of PGC-1α and PPAR-γ. Thus, our study demonstrates a negative-feedback loop involving PCK1 and HBV may provide a balanced cell environment for HBV persistent infection.

Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), larvae.

Winter mortality is a major factor regulating population size of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Glycerol is the major cryoprotectant in this freeze intolerant insect. We report findings from a gene expression study on an overwintering mountain pine beetle population over the course of 35 weeks. mRNA transcript levels suggest glycerol production in the mountain pine beetle occurs through glycogenolytic, gluconeogenic and potentially glyceroneogenic pathways, but not from metabolism of lipids. A two-week lag period between fall glycogen phosphorylase transcript and phosphoenolpyruvate carboxykinase transcript up-regulation suggests that gluconeogenesis serves as a secondary glycerol-production process, subsequent to exhaustion of the primary glycogenolytic source. These results provide a first look at the details of seasonal gene expression related to the production of glycerol in the mountain pine beetle.

Ginsenoside Rg1 Inhibits Glucagon-Induced Hepatic Gluconeogenesis through Akt-FoxO1 Interaction.

Rationale: Glucagon is involved in hepatic gluconeogenesis, playing a key role in type 2 diabetes. Ginsenosides are reported to have antidiabetic activities. Ginsenoside Rg1 is a major propanaxatriol-type saponin in ginseng. This study aims to investigate the regulatory effects of Rg1 on glucagon-induced hepatic glucose production.Methods: The effects of Rg1 were investigated in high-fat-diet (HFD)-fed mice and glucagon-challenged C57BL/6J mice. Glucose metabolism was evaluated by oral glucose tolerance test and pyruvate tolerance test. The role of Rg1 on the regulation of Akt-FoxO1 interaction was performed using immunofluorescence, immunoprecipitation, siRNA silencing, pharmacological inhibitor and active-site mutant in primary hepatocytes or HepG2 cells.Results: Abnormally elevated fasting glucagon levels were observed in HFD-fed mice, contributing significantly to increased fasting plasma glucose levels. Inappropriate fasting glucagon secretion inactivated Akt and promoted hepatic glucose production via upregulation of FoxO1 activity. Rg1 preserved glucagon-impaired Akt activation partly by binding to Akt at Ser473 site. Rg1 also promoted Akt binding to FoxO1 and inactivated FoxO1 by phosphorylation. Consequently, Rg1 decreased the hepatic glucose production through a decrease in transcription of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). Both siRNA silencing of Akt and Akt inhibitor triciribine attenuated the effects of Rg1 in response to fasting hormone glucagon.Conclusion: Akt phosphorylation at Ser473 by ginsenoside Rg1 is critical for its gluconeogenesis-lowering effect, suggesting a potential for pharmaceutical intervention in response to fasting hormone glucagon.

Knockdown of phosphoenolpyruvate carboxykinase increases carbon flux to lipid synthesis in Phaeodactylum tricornutum

The tricarboxylic acid (TCA) cycle is a key metabolic pathway that unifies carbohydrate, lipid and protein metabolism. In Phaeodactylum tricornutum, accumulation of triacylglycerols (TAGs) under nitrogen stress is a consequence of re-allocation of carbon mainly from the intermediates of TCA cycle. Oxaloacetate, an intermediate of the TCA cycle, can flux out of the cycle by the decarboxylation enzyme phosphoenolpyruvate carboxykinase (PEPCK), thus decreasing the carbon flow directed to fatty acid biosynthesis. We assayed the expression of genes encoding mitochondrially-localized decarboxylation enzymes, essential for putative C4 metabolism, in P. tricornutum. Targeted knockdown of PEPCK was performed using RNA-interference and the photosynthetic characteristics and TAG contents of knockdown lines were characterized. PEPCK was significantly upregulated at both the mRNA and protein levels during TAG accumulation in P. tricornutum, and both PEPCK transcript abundance and enzyme activity were reduced significantly in two RNAi lines. The mutants exhibited lower photosynthetic activity, while TAG content was markedly increased. Meanwhile, cell density was comparable with that of wild-type. We propose that carbon from decarboxylation of C4 metabolites in the mitochondria plays a minor role in carbon concentration in this diatom. Downregulated decarboxylation succeeded in increasing lipid accumulation by decreasing the loss of carbon skeletons without compromising cell biomass.

Phosphoenolpyruvate Carboxykinase, a Key Enzyme That Controls Blood Glucose, Is a Target of Retinoic Acid Receptor-Related Orphan Receptor α.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a committed and rate-limiting step in hepatic gluconeogenesis, and its activity is tightly regulated to maintain blood glucose levels within normal limits. PEPCK activity is primarily regulated through hormonal control of gene transcription. Transcription is additionally regulated via a cAMP response unit, which includes a cAMP response element and four binding sites for CCAAT/enhancer-binding protein (C/EBP). Notably, the cAMP response unit also contains a putative response element for retinoic acid receptor-related orphan receptor α (RORα). In this paper, we characterize the effect of the RORα response element on cAMP-induced transcription. Electrophoresis mobility shift assay indicates that RORα binds this response element in a sequence-specific manner. Furthermore, luciferase reporter assays indicate that RORα interacts with C/EBP at the PEPCK promoter to synergistically enhance transcription. We also found that cAMP-induced transcription depends in part on RORα and its response element. In addition, we show that suppression of RORα by siRNA significantly decreased PEPCK transcription. Finally, we found that a RORα antagonist inhibits hepatic gluconeogenesis in an in vitro glucose production assay. Taken together, the data strongly suggest that PEPCK is a direct RORα target. These results define possible new roles for RORα in hepatic gluconeogenesis.

Localization of enzymes relating to C4 organic acid metabolisms in the marine diatom, Thalassiosira pseudonana

In the genome of the marine diatom-Thalassiosira pseudonana, there are several putative genes encoding enzymes potentially constitute a classical C4 type biochemical CO2-concentrating mechanism. Two genes encode a carboxylation enzyme phosphoenolpyruvate carboxylase (PEPC)1 and PEPC2; and another two encode decarboxylation enzymes, NAD(+)-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). These genes were tagged by the enhanced-green fluorescence protein, egfp, ligated in the transformation vector, and transformed into the cells of T. pseudonana for localization of GFP fusion products. The PEPC1:GFP fusion was localized at the matrix of the periplastidal compartment, while the PEPC2:GFP fusion was localized at the mitochondria. The NAD-ME:GFP fusion was localized in the cytosol and the PEPCK:GFP fusion at the mitochondria. The transcripts level of NAD-ME was extremely low, and PEPCK transcript was significantly induced under the dark, suggesting that PEPCK is involved in the dark metabolism such as respiration and amino acid metabolism in the mitochondria. Treatments of low-CO2grown T. pseudonana cells with inhibitors for PEPCK and PEPC efficiently dissipated the maximum rate of photosynthesis while these treatments did not affect high-affinity photosynthesis. These data strongly suggest that classical C4 enzymes play little role in the CCM in T. pseudonana.

Aryl Hydrocarbon Receptor Activation by Dioxin Targets Phosphoenolpyruvate Carboxykinase (PEPCK) for ADP-ribosylation via 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible Poly(ADP-ribose) Polymerase (TiPARP)

Effects of the environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) include a wasting syndrome associated with decreased gluconeogenesis. TCDD is a potent activator of the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The relationship between gene activation by the AHR and TCDD toxicities is not well understood. We recently identified a pathway by which the AHR target gene TiPARP (TCDD-inducible poly(ADP-ribose) polymerase) contributes to TCDD suppression of transcription of phosphoenolpyruvate carboxykinase (PEPCK), a key regulator of gluconeogenesis, by consuming NAD(+) and decreasing Sirtuin 1 activation of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a transcriptional activator of PEPCK. We report here that TCDD-induced TiPARP also targets PEPCK for ADP-ribosylation. Both cytosolic and mitochondrial forms of PEPCK were found to undergo ADP-ribosylation. Unexpectedly, AHR suppression also enhanced ADP-ribosylation and did so by a poly(ADP-ribose) polymerase-independent mechanism. This report 1) identifies ADP-ribosylation as a new posttranslational modification for PEPCK, 2) describes a pathway by which transcriptional induction of TiPARP by the AHR can lead to a downstream posttranslational change in a TCDD target protein (PEPCK), and 3) reveals that the AHR exerts complex, previously unidentified modulatory effects on ADP-ribosylation.

Hydrodynamic delivery of adiponectin and adiponectin receptor 2 gene blocks high-fat diet-induced obesity and insulin resistance

Adiponectin and its receptors are inversely related to the degree of obesity and have been identified as potential therapeutic targets for the treatment of obesity. In this study, we evaluated the effect of hydrodynamic delivery of adiponectin and/or its receptor 2 (adipoR2) genes on controlling the development of obesity and insulin resistance in AKR/J mice fed a high-fat diet. An increase in adiponectin and adipoR2 gene expression by hydrodynamic gene delivery prevented diet-induced weight gain, reduced fat accumulation in liver and adipose tissue, and improved insulin sensitivity. Beneficial effects were seen with reduced gluconeogenesis in the liver and lipogenesis in the liver, white adipose tissue and skeletal muscle. Real-time PCR analysis demonstrated overexpression of adiponectin and adipoR2 significantly suppressed transcription of phosphoenolpyruvate carboxykinase (pepck), glucose-6-phosphatase (g6pase), stearoyl CoA desaturase 1 (scd-1), and fatty acid synthase (fas) gene. Inhibition effects were mediated by activating the AMP-activated protein kinase (AMPK). These results prove that elevation of adiponectin and/or adipoR2 expression via gene transfer is an effective approach in managing obesity epidemics.

Homology Modeling and characterization of Phosphoenolpyruvate Carboxykinase (PEPCK) from Schistosoma japonicum

Phosphoenolpyruvate carboxykinase (PEPCK), a carboxylase enzyme is present in all living organisms. It catalyzes metal-nucleotide coupled reversible decarboxylation and phosphorylation between phosphoenolpyruvate (PEP) and oxaloacetate (OAA) depending on the system and the availability of the intermediates. In fungi, plants and in most bacteria, production of PEP from OAA by PEPCK is the key step during gluconeogenesis. In healthy human cytosolic PEPCK enzyme is present only during glucose starvation; cytosolic PEPCK rapidly disappears on the replenishment of glucose due to hormonal control of the transcription of the cytosolic PEPCK- gene. In some parasitic helminthes like Ascaris suum, nematodes such as Haemonchus contortus, PEPCK carry out the reverse reaction to produce OAA from PEP. In Trypanosoma cruzi and all species of the genus Leishmania, this enzyme is very active even in the presence of high level of carbohydrate. There is a significant functional difference between parasitic PEPCK and mammalian host PEPCK. These differences between the mammalian host and the parasite enzyme strongly support the belief that PEPCK should be further investigated as a possible target for selective chemotherapeutic agents. The experimental 3D structure of PEPCK of Schistosoma japonicum is not available in protein data bank. Therefore, based on the knowledge of the best template (3DT7), model of 3D structure of Schistosoma japonicum PEPCK was prepared using Modeller v9.10 software and processed in to energy minimization, Ramachandran plot analysis, quality assessment and characterization.

Effect of Iron-Mediated Oxidative Stress on Insulin Resistance Through the Forkhead Box-Containing Protein O Subfamily-1 (FOXO-1) Pathway in Chronic Hepatitis C

Aims: Chronic hepatitis C virus (HCV) infection is often associated with glucose metabolic disorders and iron overload. It has recently been shown that reactive oxygen species (ROS) increase gluconeogenesis in hepatocytes through the forkhead box-containing protein O subfamily-1 (FOXO1)-dependent pathway. The aim of this study is proving a cause-and-effect relationship between iron-mediated ROS production and insulin resistance (IR) in chronic hepatitis C (CH-C) patients. Methods: The study included 42 patients with CH-C (22 males and 20 females, median age 53 years). Homeostasis model assessment of insulin resistance (HOMA-IR) value was assessed for each patient at entry. Gene expression levels in the biopsied liver tissues were determined by quantitative reverse transcription-polymerase chain reaction (RT- PCR). In addition, the effect of ROS on gluconeogenesis was assessed using HepG2 cells treated with a well-known ROS generator, diethylmaleate (DEM). Results: The serum ferritin levels were significantly correlated with the serum aspartate aminotransferase level, alanine aminotransferase level, HOMA-IR value, grade of fatty accumulation, total hepatic iron score, and 8-OH-deoxy-2’-guanosine (8-OHdG)-positive cell count. FOXO1 expression was correlated with 8-OHdG-positive cell count, phosphoenolpyruvate carboxykinase (PEPCK) expression, and HOMA-IR. In HepG2 cells, the gene transcription of FOXO1 and PEPCK was increased by DEM treatment, which was associated with an increase in non-phosphorylated FOXO1 protein in the nuclear fraction. Conclusions: Iron-mediated ROS production enhances gluconeogenesis through the FOXO1-mediated pathway and is an affecting factor to IR in patients with CH-C.

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs). Obesity is a metabolic disorder caused by an imbalance between energy uptake and expenditure, resulting in excess stored body fat. Recent studies have shown that GSL levels are increased in obese rodents and that pharmacologically reducing GSL levels by inhibiting GlcCer synthesis improves adipocyte function. However, the molecular mechanism underlying these processes is still not clearly understood. Using Drosophila as a model animal, we report that GlcT-1 expression in the fat body, which is equivalent to mammalian adipose tissue, regulates energy metabolism. Overexpression of GlcT-1 increases stored nutrition (triacylglycerol and carbohydrate) levels. Conversely, reduced expression of GlcT-1 in the fat body causes a reduction of fat storage. This regulation occurs, at least in part, through the activation of p38-ATF2 signaling. Furthermore, we found that GlcCer is the sole GSL of the fat body, indicating that regulation of GlcCer synthesis by GlcT-1 in the fat body is responsible for regulating energy homeostasis. Both GlcT-1 and p38-ATF2 signaling are evolutionarily conserved, leading us to propose an evolutionary perspective in which GlcT-1 appears to be one of the key factors that control fat metabolism.

Identification of the Aryl Hydrocarbon Receptor Target Gene TiPARP as a Mediator of Suppression of Hepatic Gluconeogenesis by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and of Nicotinamide as a Corrective Agent for This Effect

The environmental toxin TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin) produces diverse toxic effects including a lethal wasting syndrome whose hallmark is suppressed hepatic gluconeogenesis. All TCDD toxicities require activation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor. Whereas the mechanism for AHR induction of target genes is well understood, it is not known how AHR activation produces any TCDD toxicity. This report identifies for the first time an AHR target gene, TiPARP (TCDD-inducible poly(ADP-ribose) polymerase, PARP7) that can mediate a TCDD toxicity, i.e. suppression of hepatic gluconeogenesis. TCDD suppressed hepatic glucose production, expression of key gluconeogenic genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), and NAD(+) levels, and increased PARP activity and TiPARP expression. TCDD also increased acetylation and ubiquitin-dependent proteosomal degradation of the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α), a coactivator of PEPCK and G6Pase transcription. TiPARP overexpression reproduced TCDD effects on glucose output and NAD(+) levels whereas TiPARP silencing diminished them. TiPARP overexpression also increased PGC1α acetylation and decreased PGC1α levels. In contrast, silencing of cytochromes P450 (CYP) 1A, main AHR-induced genes, did not alter TCDD suppression of gluconeogenesis. The vitamin B3 constituent, nicotinamide (NAM), prevented TCDD suppression of glucose output, NAD(+), and gluconeogenic genes and stabilized PGC1α. The corrective effects of NAM could be attributed to increased NAD(+) levels and suppression of AHR target gene induction. The results reveal that TiPARP can mediate a TCDD effect, that the AHR is linked to PGC1α function and stability and that NAM has novel AHR antagonist activity.

Long-Term Ritonavir Exposure Increases Fatty Acid and Glycerol Recycling in 3T3-L1 Adipocytes as Compensatory Mechanisms for Increased Triacylglycerol Hydrolysis

Lipodystrophy with high nonesterified fatty acid (FA) efflux is reported in humans receiving highly active antiretroviral therapy (HAART) to treat HIV infection. Ritonavir, a common component of HAART, alters adipocyte FA efflux, but the mechanism for this effect is not established. To investigate ritonavir-induced changes in FA flux and recycling through acylglycerols, we exposed differentiated murine 3T3-L1 adipocytes to ritonavir for 14 d. FA efflux, uptake, and incorporation into acylglycerols were measured. To identify a mediator of FA efflux, we measured adipocyte triacylglycerol lipase (ATGL) transcript and protein. To determine whether ritonavir-treated adipocytes increased glycerol backbone synthesis for FA reesterification, we measured labeled glycerol and pyruvate incorporation into triacylglycerol (TAG). Ritonavir-treated cells had increased FA efflux, uptake, and incorporation into TAG (all P < 0.01). Ritonavir increased FA efflux without consistently increasing glycerol release or changing TAG mass, suggesting increased partial TAG hydrolysis. Ritonavir-treated adipocytes expressed significantly more ATGL mRNA (P < 0.05) and protein (P < 0.05). Ritonavir increased glycerol (P < 0.01) but not pyruvate (P = 0.41), utilization for TAG backbone synthesis. Consistent with this substrate utilization, glycerol kinase transcript (required for glycerol incorporation into TAG backbone) was up-regulated (P < 0.01), whereas phosphoenolpyruvate carboxykinase transcript (required for pyruvate utilization) was down-regulated (P < 0.001). In 3T3-L1 adipocytes, long-term ritonavir exposure perturbs FA metabolism by increasing ATGL-mediated partial TAG hydrolysis, thus increasing FA efflux, and leads to compensatory increases in FA reesterification with glycerol and acylglycerols. These changes in FA metabolism may, in part, explain the increased FA efflux observed in ritonavir-associated lipodystrophy.

In vivo phosphoenolpyruvate carboxykinase promoter mapping identifies disrupted hormonal synergism as a target of inflammation during sepsis in mice†

In mammals, proper maintenance of blood glucose levels within narrow limits is one of the most critical prerequisites for healthy energy homeostasis and body function. Consequently, hyper- and hypoglycemia represent hallmarks of severe metabolic pathologies, including type II diabetes and acute sepsis, respectively. Although the liver plays a crucial role in the control of systemic glucose homeostasis, the molecular mechanisms of aberrant hepatic glucose regulation under metabolic stress conditions remain largely unknown. Here we report the development of a liver-specific adenoviral in vivo system for monitoring promoter activity of the key gluconeogenic enzyme gene phosphoenolpyruvate carboxykinase (PEPCK) in mice. By employing in vivo promoter deletion technology, the glucocorticoid response unit (GRU) and the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) were identified as critical cis-regulatory targets of proinflammatory signaling under septic conditions. In particular, both elements were found to be required for inhibition of PEPCK transcription during sepsis, thereby mediating endotoxic hypoglycemia. Indeed, expression of nuclear receptor cofactor peroxisome proliferator-activator receptor coactivator 1alpha (PGC-1alpha), the molecular mediator of GRU/CRE synergism on the PEPCK promoter, was found to be specifically repressed in septic liver, and restoration of PGC-1alpha in cytokine-exposed hepatocytes blunted the inhibitory effect of proinflammatory signaling on PEPCK gene expression. The dysregulation of hormonal synergism through the repression of PGC-1alpha as identified by in vivo promoter monitoring may provide a molecular rationale for hypoglycemia during sepsis, thereby highlighting the importance of hepatic glucose homeostasis for metabolic dysfunction in these patients.

Photosynthetic Carbon Assimilation in the Coccolithophorid Emiliania huxleyi (Haptophyta): Evidence for the Predominant Operation of the C3 Cycle and the Contribution of β-Carboxylases to the Active Anaplerotic Reaction

The coccolithophorid Emiliania huxleyi (Haptophyta) is a representative and unique marine phytoplankton species that fixes inorganic carbon by photosynthesis and calci-fication. We examined the initial process of photosynthetic carbon assimilation by analyses of metabolites, enzymes and genes. When the cells were incubated with a radioactive substrate (2.3 mM NaH(14)CO(3)) for 10 s under illumination, 70% of the (14)C was incorporated into the 80% methanol-soluble fraction. Eighty-five and 15% of (14)C in the soluble fraction was incorporated into phosphate esters (P-esters), including the C(3) cycle intermediates and a C(4) compound, aspartate, respectively. A pulse-chase experiment showed that (14)C in P-esters was mainly transferred into lipids, while [(14)C]aspartate, [(14)C]alanine and [(14)C]glutamate levels remained almost constant. These results indicate that the C(3) cycle functions as the initial pathway of carbon assimilation and that beta-carboxylation contributes to the production of amino acids in subsequent metabolism. Transcriptional analysis of beta-carboxylases such as pyruvate carboxylase (PYC), phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK) revealed that PYC and PEPC transcripts were greatly increased under illumination, whereas the PEPCK transcript decreased remarkably. PEPC activity was higher in light-grown cells than in dark-adapted cells. PYC activity was detected in isolated chloroplasts of light-grown cells. According to analysis of their deduced N-terminal sequence, PYC and PEPC are predicted to be located in the chloroplasts and mitochondria, respectively. These results suggest that E. huxleyi possesses unique carbon assimila-tion mechanisms in which beta-carboxylation by both PYC and PEPC plays important roles in different organelles.

DFOXO Regulates Transcription of a Drosophila Acid Lipase

Insulin resistance is a major feature of pathological states such as obesity and diabetes. A consequence of insulin resistance is enhanced lipolysis, which causes excessive release of free fatty acids and deregulates fatty acid homeostasis. The transcription factor FOXO1 has a central role in the regulation of glucose levels by insulin: reduced insulin signaling causes FOXO1 activation, which increases hepatic glucose production by activating transcription of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. Our results suggest an additional role for FOXO transcription factors: the regulation of lipid homeostasis by insulin. Here, we show that in flies, dFOXO regulates lipase 4 (dLip4), a Drosophila homologue of human acid lipases. dFOXO binds and activates the dLip4 promoter, in vitro and in vivo, and regulates dLip4 expression. In addition, dLip4 mRNA expression in flies is dependent on dFOXO. Our results support a model where dFOXO acts as a key modulator of lipid metabolism by insulin signaling and integrates insulin responses to glucose and lipid homeostasis.

Nuclear Corepressor Is Required for Inhibition of Phosphoenolpyruvate Carboxykinase Expression by Tumor Necrosis Factor-α

Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) by TNF-alpha contributes to the pathogenesis of hypoglycemia in endotoxin shock. In this study, the molecular mechanism underlying the inhibition was investigated in hepatoma cells (rat H4IIE and human HepG2). PEPCK expression was induced by cAMP, and the induction was reduced by TNF-alpha at protein and mRNA levels in H4IIE cells. The inhibition was observed in the PEPCK gene promoter in a PEPCK-luciferase reporter. Activation of nuclear factor kappaB (NF-kappaB) pathway was required for the transcriptional inhibition of PEPCK gene. Degradation of NF-kappaB inhibitor (IkappaB) and p65 nuclear translocation were involved in the inhibition. An interaction of histone deacetylase 3 (HDAC3) and silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) with the PEPCK gene promoter was induced by TNF-alpha and observed in a chromatin immunoprecipitation assay. The TNF-induced inhibition was blocked by HDAC inhibitor or HDAC3 knockdown. The blocking effect was also observed in knockdown of corepressor SMRT. Point mutation suggests that cAMP response element (CRE) is required for TNF-induced inhibition of the PEPCK gene promoter. Phosphorylation of cAMP response element-binding protein at Ser133 and expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha were not changed by TNF-alpha in H4IIE cells. The transcriptional activity of CRE-binding protein was inhibited by TNF-alpha in a CRE-luciferase reporter. The data suggests that the nuclear corepressor proteins of HDAC3 and SMRT mediate TNF inhibition of PEPCK transcription. The inhibition mechanism is related to activation of NF-kappaB and inhibition of CRE-binding protein activity by the corepressor. These data suggest a novel activity of nuclear corepressor in the regulation of PEPCK expression by TNF-alpha.

Transcriptional repression of the gluconeogenic gene PEPCK by the orphan nuclear receptor SHP through inhibitory interaction with C/EBPα

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.