Phosphoenolpyruvate Carboxykinase Gene Transcription Research Articles

Identification of promoter response elements that mediate propionate induction of bovine cytosolic phosphoenolpyruvate carboxykinase (PCK1) gene transcription

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis that is positively regulated by propionate in bovines at the transcription level. The specific elements that determine propionate responsiveness within the bovine PCK1 promoter are unknown. In silico promoter analysis of the bovine PCK1 gene revealed several clusters of transcription factor binding sites. In the present study, we determined the essentiality of the putative cyclic AMP response element (CRE) at -94 through -87 bp and the 2 putative hepatic nuclear factor 4α (HNF4α) binding elements at +68 through +72 and -1,078 through -1,074, respectively, in mediating bovine PCK1 promoter responses to propionate and other regulators, including butyrate, cyclic AMP (cAMP), and glucocorticoids. The wild-type bovine PCK1 promoter [PCK1(WT)] was ligated to a luciferase reporter gene and transfected into rat hepatoma (H4IIE) cells. Activities of PCK1(WT) were induced by approximately 2-, 2-, 4-, 8-, 9-, 18-, and 16-fold respectively when exposed to cAMP (as 1.0 mM 8-Br-cAMP), 5.0 μM dexamethasone, cAMP + dexamethasone, 2.5 mM propionate, cAMP + propionate, cAMP + dexamethasone + propionate, and 2.5 mM butyrate. Seven mutants lacking either one single site, 2 of the 3 sites, or all 3 sites, generated by site-directed mutagenesis, were tested. Responses to propionate and all other treatments were completely abolished when CRE at -94 through -87 bp and HNF4α at +68 through +72 bp were both deleted. Our data indicate that these 2 regulatory elements act synergistically to mediate the bovine PCK1 promoter responses to propionate as well as butyrate, cAMP, and dexamethasone. The activation of PCK1 through these regulatory elements serves to activate the metabolic potential of bovine toward gluconeogenesis when the primary substrate for gluconeogenesis, propionate, is also present.

Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

Abstract MP75: Assessment of the Genetic Role of Potential Metabolic Therapeutic Targets Along Insulin Signaling and Adipogenesis Pathways

Recent studies in rat hepatoma cells identified MAP kinase phosphatase 3 (MKP3) as the key player in antagonizing repression of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by insulin, indicating that MAP kinase phosphatase can crosstalk with metabolic components of the insulin signaling pathways. The family of histone deacetylases (HDACs) is another set of metabolic therapeutic targets of importance as HDAC1 has recently been shown to be involved in regulating preadipocyte differentiation. We therefore investigated the roles of MKP3 (DUSP6 in human), PEPCK (PCK2), HDAC1&2 single nucleotide polymorphisms (SNPs) in relation to obesity and diabetes risk among 7,287 African American (AA), 3,258 Hispanic American (HA), and 3711 Caucasian American (CA) women participating in the national Women’s Health Initiative (WHI) SNP Health Association Resource (SHARe) and the Genomics and Randomized Trials Network (GARNET). We found significant association between 43 SNPs in the DUSP6 gene region with risk of obesity (risk variants: range of OR=1.08-1.87; protective variants: 0.72-0.93; all P<0.05) and diabetes (risk variants: range of OR=1.08-1.43; protective variants: 0.84-0.93; all P<0.05). Two PCK2 SNPs were associated with obesity risk (risk variant: OR=1.09, P=0.008; protective variant: OR=0.84, P=0.04). Six HDAC2 SNPs were also found to have association with risk of obesity (range of OR: 0.82-0.90, P<0.05) and diabetes (risk variant: OR=1.16; protective variant: 0.77-0.86; all P<0.05). The rs10506969 (C/T) SNP in the DUSP6 gene identified among AA (reference allele=C, OR=0.86, P=0.02) and HA (OR=0.87, P=0.05) was also confirmed in the CARDIoGRAMplusC4D meta-analysis studies of 63,746 coronary artery disease cases and 130,681 controls (reference allele: T, OR=1.05, P=0.003). Our candidate gene study of American women of three ethnicities identified germline mutations in DUSP6, PCK2, and HDAC2 in relation to obesity and diabetes. These human genetic evidence is comparable to data from animal models that support the notion that MAP kinase phosphatase 3, phosphoenolpyruvate carboxykinase, and histone deacetylase 2 may serve as important metabolic therapeutic targets in lowering risk of obesity and diabetes.

DNA damage induces down-regulation of <italic>PEPCK</italic> and <italic>G6P</italic> gene expression through degradation of PGC-1α

Hepatic gluconeogenesis plays a crucial role in glucose homeostasis. Although it is well established that various cellular processes are modulated by DNA damage, whether the DNA damage signaling pathway regulates gluconeogenesis has not yet been studied. In this study, we found that mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P), key enzymes for gluconeogenesis, were dramatically decreased upon IR- and UV-irradiation. PEPCK and G6P promoter activities were also suppressed by IR- and UV-irradiation, suggesting that PEPCK and G6P gene transcription are down-regulated upon DNA damage. We also found that the protein level of PGC-1α, which is a critical transcription factor for PEPCK gene expression, is decreased upon UV-irradiation. The decreased PGC-1α protein level was abolished by MG132, a potent proteasome inhibitor, suggesting that PGC-1α is degraded through the ubiquitin-proteasome pathway upon UV-irradiation. These results reveal a novel link between glucose metabolism and the DNA damage signaling pathway and suggest a possible role for PEPCK and G6P in the DNA damage response.

Rubratoxin B induces signs of fatty acid oxidation disorders (FAODs) in mice

Rubratoxin B is a mycotoxin that causes hypoglycemia and fatty liver. We investigated the effect of rubratoxin B on hepatic glycogen content and regulation, because blood glucose levels are associated with hepatic glycogen storage. Mice were treated with 1.5mg/kg rubratoxin B for 24h. Stomachs of treated mice became extremely swollen, and the contents were significantly heavier than those of controls. Hypoglycemia stimulates appetite; therefore, rubratoxin B may perturb satiation. Rubratoxin B evidently depleted hepatic glycogen stores. Phosphoenolpyruvate carboxykinase (PEPCK) activity and mRNA levels in treated mice were reduced, indicating that rubratoxin B caused hepatic glycogen depletion by inhibiting PEPCK. PEPCK activity and mRNA levels were reduced to similar degrees; it appears that PEPCK activity is regulated transcriptionally. Levels of the PEPCK gene trans-activators phospho-CREB (active form) and C/EBPα were significantly reduced in the livers of treated mice, suggesting that these factors are important for PEPCK gene transcription. Rubratoxicosis and fatty acid oxidation disorders (FAODs) share characteristic signs, such as robust appetite, hypoglycemia, hepatic glycogen depletion, and fatty liver. Although FAODs are generally considered genetic deficiencies, our results indicate that a chemical can also cause FAOD-like signs in mice.

Pharmacological evaluation of insulin mimetic novel suppressors of PEPCK gene transcription from Paeoniae Rubra Radix

Paeoniae Rubra Radix (root of Paeonia lactiflora) has been frequently employed in Traditional Chinese Medicine (TCM) as and anti-diabetic therapy to enhance blood circulation and dissipate stasis. Previously, we identified a novel hypoglycemic action of a crude extract from Paeoniae Rubra Radix, which also suppressed phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Therefore, the current investigation intended to elucidate potential active bio-constituents of this herb and mechanisms of action. Glucocorticoid receptor (GR) nuclear localization, the PEPCK messenger (m)RNA level, pregnane X receptor (PXR) mRNA expression, cAMP-responsive element-binding protein (CREB) serine phosphorylation and DNA binding were evaluated in dexamethasone (Dex) and 8-bromo-cAMP (CA)-stimulated H4IIE cells, while efficacy of agents was assessed in a stable cell line containing a green fluorescent protein (GFP) reporter driven by the PEPCK promoter. HPLC profiling, colorimetric assays, and NMR analysis were employed for chemical characterization purpose. An extract of Paeoniae Rubra Radix lacking the insulin mimetic compound, 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG), and termed the non-PGG fraction (NPF), consisting of tannin polymers, suppressed PEPCK expression in the presence of an insulin receptor antagonist (HNMPA-AM(3)), suggesting the action of this fraction is independent of the insulin receptor. Furthermore, Dex-stimulated GR nuclear localization and transactivation were prevented by the NPF. Similarly, CA-stimulated CREB serine phosphorylation and DNA binding were also inhibited by the NPF in H4IIE cells. Hence NPF antagonizes both signaling pathways that induce PEPCK gene transcription. In conclusion, the current study proposes that the potent suppressive activity on PEPCK gene transcription observed with Paeoniae Rubra Radix extract, can be attributed to at least two distinct components, namely PGG and NPF.

Maternal protein restriction during pregnancy induces CCAAT/enhancer-binding protein (C/EBPβ) expression through the regulation of histone modification at its promoter region in female offspring rat skeletal muscle

Maternal nutrition during pregnancy is an important intrauterine environmental factor that can cause persistent alterations of the offspring genome and is associated with potential disease risk later in life. In the present study, we investigated the impact of a maternal low protein diet (LP) on the expression of the transcription factor CCAAT/enhancer-binding protein (C/EBPβ) in offspring skeletal muscle. C/EBPβ belongs to a family of transcription factors that regulates the expression of genes involved in energy homeostasis and muscle development. We investigated C/EBPβ transcriptional regulation from an epigenetic aspect. We observed sex-dependent differences in C/EBPβ expression in offspring skeletal muscle subjected to a maternal protein-restricted diet. In female offspring skeletal muscle, both C/EBPβ mRNA and protein levels were increased by maternal protein restriction. However, C/EBPβ expression was not altered in other tissues or male offspring. Analysis of transcriptional and epigenetic regulation showed acetylated histone 3 and acetylated histone 4 at significantly increased levels at the C/EBPβ promoter region in female LP pup's muscle. Phosphoenolpyruvate carboxykinase (PEPCK) gene transcription was also up-regulated in female LP pups through the increased binding of C/EBPβ at its promoter. The induction of C/EBPβ expression in female offspring skeletal muscle by maternal protein restriction during pregnancy may indicate C/EBPβ involvement in signaling response in energy metabolism to a low maternal protein diet.

AMPK-dependent Repression of Hepatic Gluconeogenesis via Disruption of CREB·CRTC2 Complex by Orphan Nuclear Receptor Small Heterodimer Partner

Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of gluconeogenic enzyme gene expression. Here, we show that SHP inhibited protein kinase A-mediated transcriptional activity of cAMP-response element-binding protein (CREB), a major regulator of glucose metabolism, to modulate hepatic gluconeogenic gene expression. Deletion analysis of phosphoenolpyruvate carboxykinase (PEPCK) promoter demonstrated that SHP inhibited forskolin-mediated induction of PEPCK gene transcription via inhibition of CREB transcriptional activity. In vivo imaging demonstrated that SHP inhibited CREB-regulated transcription coactivator 2 (CRTC2)-mediated cAMP-response element-driven promoter activity. Furthermore, overexpression of SHP using adenovirus SHP decreased CRTC2-dependent elevations in blood glucose levels and PEPCK or glucose-6-phosphatase (G6Pase) expression in mice. SHP and CREB physically interacted and were co-localized in vivo. Importantly, SHP inhibited both wild type CRTC2 and S171A (constitutively active form of CRTC2) coactivator activity and disrupted CRTC2 recruitment on the PEPCK gene promoter. In addition, metformin or overexpression of a constitutively active form of AMPK (Ad-CA-AMPK) inhibited S171A-mediated PEPCK and G6Pase gene expression, and hepatic glucose production and knockdown of SHP partially relieved the metformin- and Ad-CA-AMPK-mediated repression of hepatic gluconeogenic enzyme gene expression in primary rat hepatocytes. In conclusion, our results suggest that a delayed effect of metformin-mediated induction of SHP gene expression inhibits CREB-dependent hepatic gluconeogenesis.

C/EBPβ is AMP kinase sensitive and up-regulates PEPCK in response to ER stress in hepatoma cells

Diabetes and obesity are associated with activation of endoplasmic reticulum (ER) stress; however a direct link between ER stress and increased hepatic gluconeogenesis remains unclear. Here we show that ER stress triggers a significant increase in expression of CCAAT/enhancer-binding protein (C/EBPβ) and phosphorylated CREB together with reduced phospho-AMP-activated protein kinase (pAMPK) in hepatoma cells. ER stress contributed to transcriptional activation of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK) promoter in Huh7 and HepG2 cells via cAMP binding motif (CRE site). Chromatin immunoprecipitation assays demonstrate that C/EBPβ is recruited to the PEPCK promoter during ER stress and is reversed by pre-treatment with a JNK inhibitor that relieves ER stress. C/EBPβ but not pCREB was suppressed by the AMPK-activator AICAR or constitutively active AMPK, while dominant negative AMPK increased C/EBPβ expression. These data suggest that ER stress triggers suppression of AMPK while increasing C/EBPβ and pCREB expression which activates PEPCK gene transcription. Understanding how ER stress suppresses AMPK activation and increases C/EBPβ expression could lead to a potentially novel pathway for treatment of diabetes.

Aspects of the Control of Phosphoenolpyruvate Carboxykinase Gene Transcription

Abstract This article reviews our current understanding of factors that regulate transcription of the gene for PEPCK-C, focusing on the effect of methylation on gene expression. It also provides insight into the evolutionary status of the PEPCK-C gene.

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.

Insulin Represses Phosphoenolpyruvate Carboxykinase Gene Transcription by Causing the Rapid Disruption of an Active Transcription Complex: A Potential Epigenetic Effect

Insulin represses gluconeogenesis, in part, by inhibiting the transcription of genes that encode rate-determining enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase). Glucocorticoids stimulate expression of the PEPCK gene but the repressive action of insulin is dominant. Here, we show that treatment of H4IIE hepatoma cells with the synthetic glucocorticoid, dexamethasone (dex), induces the accumulation of glucocorticoid receptor, as well as many transcription factors, coregulators, and RNA polymerase II, on the PEPCK gene promoter. The addition of insulin to dex-treated cells causes the rapid dissociation of glucocorticoid receptor, polymerase II, and several key transcriptional regulators from the PEPCK gene promoter. These changes are temporally related to the reduced rate of PEPCK gene transcription. A similar disruption of the G-6-Pase gene transcription complex was observed. Additionally, insulin causes the rapid demethylation of arginine-17 on histone H3 of both genes. This rapid, insulin-induced, histone demethylation is temporally related to the disruption of the PEPCK and G-6-Pase gene transcription complex, and may be causally related to the mechanism by which insulin represses transcription of these genes.

CCAAT/Enhancer-binding Protein α Mediates Induction of Hepatic Phosphoenolpyruvate Carboxykinase by p38 Mitogen-activated Protein Kinase

Excessive hepatic gluconeogenesis and glucose production are important contributors to hyperglycemia in both type 1 and type 2 diabetes. In diabetic humans and animal models, elevated levels of p38 mitogen-activated protein kinase (p38) are observed in several tissues. Our study shows that activity of p38 is significantly elevated in livers of db/db or streptozocin-induced type 1 diabetic mice. Using cultured hepatoma cells, we find that activation of p38 enhances expression of hepatic gluconeogenic gene phosphoenolpyruvate carboxykinase (PEPCK). Furthermore, our studies demonstrate that activation of p38 stimulates phosphorylation of CCAAT/enhancer-binding protein alpha (C/EBPalpha) at serine 21 and increases its transactivation activity in the context of PEPCK gene transcription. Our results indicate that C/EBPalpha mediates p38-stimulated PEPCK transcription in liver cells.

Hormonal regulation of assembly of the pepck gene transcription complex

Phosphoenolpyruvate carboxykinase (PEPCK) gene transcription is activated in response to glucocorticoids, such as dexamethasone (Dex). Previous studies, using a variety of techniques, including reporter gene assays, showed that the induction of PEPCK gene transcription by Dex requires a complex array of DNA accessory elements and associated DNA-binding transcription factors. These, in turn, bind a set of transcription coactivators, including CREB binding protein (CBP) and p300. We have now used the chromatin immunoprecipitation assay (ChIP) to assess promoter occupancy of these proteins, in vivo, in the presence and absence of the ligand, Dex. Dex causes a remarkable increase in the binding of glucocorticoid receptor, polymerase II, CBP and p300 to the promoter. However, even though both CBP and p300 are expressed in the nucleus of H4IIE cells, they do not co-exist in the same transcription complex on the PEPCK gene promoter. Furthermore, the selective reduction of either p300 or CBP, using small interfering RNAs (siRNAs), partially abolishes the Dex response of the PEPCK gene. Reduction of both CBP and p300 totally abolishes the Dex response of the PEPCK gene. Although endogenous p300 and CBP are not capable of compensating for the loss of CBP and p300, respectively, the overexpression of either of these proteins compensates for the loss of the other, and both coactivators support the dominant suppression of the gene by insulin.

Estrogen-related Receptor α Is a Repressor of Phosphoenolpyruvate Carboxykinase Gene Transcription

The orphan nuclear receptor estrogen-related receptor (ERR) alpha is a downstream effector of the transcriptional coactivator PGC-1alpha in the regulation of genes important for mitochondrial oxidative capacity. PGC-1alpha is also a potent activator of the transcriptional program required for hepatic gluconeogenesis, and in particular of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). We report here that the regulatory sequences of the PEPCK gene harbor a functional ERRalpha binding site. However, in contrast to the co-stimulating effects of ERRalpha and PGC-1alpha on mitochondrial gene expression, ERRalpha acts as a transcriptional repressor of the PEPCK gene. Suppression of ERRalpha expression by small interfering RNA leads to reduced binding of ERRalpha to the endogenous PEPCK gene, and an increase in promoter occupancy by PGC-1alpha, suggesting that part of the ERRalpha function at this gene is to antagonize the action of PGC-1alpha. In agreement with the in vitro studies, animals that lack ERRalpha show increased expression of gluconeogenic genes, including PEPCK and glycerol kinase, but decreased expression of mitochondrial genes, such as ATP synthase subunit beta and cytochrome c-1. Our findings suggest that ERRalpha has opposing effects on genes important for mitochondrial oxidative capacity and gluconeogenesis. The different functions of ERRalpha in the regulation of these pathways suggest that enhancing ERRalpha activity could have beneficial effects on glucose metabolism in diabetic subjects by two distinct mechanisms: increasing mitochondrial oxidative capacity in peripheral tissues and liver, and suppressing hepatic glucose production.

Insulin Regulation of PEPCK Gene Expression: A Model for Rapid and Reversible Modulation

Insulin and glucagon regulate the expression and/or activity of a variety of proteins to maintain blood glucose within normal limits. A key target is the gene encoding phosphoenolpyruvate carboxykinase (PEPCK), which catalyzes the first committed step in hepatic gluconeogenesis. Acute regulation of PEPCK is achieved by modulating transcription of the gene, which is tightly regulated by cAMP (the mediator of glucagon and catecholamines), glucocorticoids and insulin. Normally, PEPCK expression is induced by glucagon, catecholamines and glucocorticoids during periods of fasting and in response to stress, but is dominantly inhibited by glucose-induced increases in insulin secretion upon feeding. The incomplete effectiveness of insulin action, whether due to intermittent insulin injection in type I diabetics or insulin resistance in type II diabetics, contributes to hyperglycemia and complications, resulting in damage to the eyes, nerves, kidneys and other organs over time. Thus, defining a molecular mechanism for insulin inhibition of PEPCK gene transcription has been a major goal of research in several labs, because it would allow the development of drugs to prevent episodic increases in circulating glucose in diabetics. Here, we review the main lines of investigation into this complex problem and the likely properties of an inhibitor. Any mechanism must account for the rapidity, specificity and dominance with which insulin is known to act in regulating PEPCK transcription. To date Foxo1 (FKHR) is the only transcription factor for which a complete path from the insulin receptor to gene regulation has been described. While this explains the regulation of some genes, such as IGFBP-1, Foxo1 appears not to play a requisite role in regulating PEPCK transcription. Investigation of cis-acting elements in the PEPCK promoter has shed considerable light on the mechanisms of activation by cAMP and glucocorticoids but has failed to identify a regulatory element that mediates insulin inhibition of transcription. This, together with evidence from analysis of the inducing mechanisms, has prompted us, and others, to investigate the possibility that insulin disrupts activation rather than independently promoting repression. Thus, we hypothesize that insulin-induced modification of a key transcription regulatory protein prevents an essential factor from participating in the induction process, leading to rapid but reversible inhibition, as is seen in animals. The ability to alter the sensitivity of a key transcription factor to improve insulin-regulated control of blood glucose would be a major improvement in the treatment of diabetes, a growing problem in the industrialized world.

The Synergistic Effect of Dexamethasone and All-trans-retinoic Acid on Hepatic Phosphoenolpyruvate Carboxykinase Gene Expression Involves the Coactivator p300

Activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in response to all-trans-retinoic acid (RA) or a glucocorticoid such as dexamethasone (Dex) requires a distinct arrangement of DNA-response elements and their cognate transcription activators on the gene promoter. Two of the accessory factor-binding elements involved in the Dex response (gAF1 and gAF3) coincide with the DNA-response elements involved in the RA response. We demonstrate here that the combination of Dex/RA has a synergistic effect on endogenous PEPCK gene expression in rat hepatocytes and H4IIE hepatoma cells. Reporter gene studies show that the gAF3 element and one of the two glucocorticoid receptor-binding elements (GR1) are most important for this effect. Chromatin immunoprecipitation assays revealed that when H4IIE cells were treated with Dex/RA, ligand-activated retinoic acid receptors (retinoic acid receptor/retinoid X receptor) and glucocorticoid receptors are recruited to this gene promoter, as are the transcription coregulators p300, CREB-binding protein, p/CIP, and SRC-1. Notably, the recruitment of p300 and RNA polymerase II to the PEPCK promoter is increased by the combined Dex/RA treatment compared with Dex or RA treatment alone. The functional importance of p300 in the Dex/RA response is illustrated by the observation that selective reduction of this coactivator, but not that of CREB-binding protein, abolishes the synergistic effect in H4IIE cells.

Glucocorticoids Repress Transcription of Phosphoenolpyruvate Carboxykinase (GTP) Gene in Adipocytes by Inhibiting Its C/EBP-mediated Activation

The cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) gene is selectively expressed in several tissues, primarily in the liver, kidney, and adipose tissue. The transcription of the gene is reciprocally regulated by glucocorticoids in these tissues. It is induced in the liver and kidney but repressed in the white adipose tissue. To elucidate which adipocyte-specific transcription factors participate in the repression of the gene, DNase I footprinting analyses of nuclear proteins from 3T3-F442A adipocytes and transient transfection experiments in NIH3T3 cells were utilized. Glucocorticoid treatment slightly reduced the nuclear C/EBP alpha concentration but prominently diminished the binding of adipocyte-derived nuclear proteins to CCAAT/enhancer-binding protein (C/EBP) recognition sites, without affecting the binding to nuclear receptor sites in the PEPCK-C gene promoter. Of members of the C/EBP family of transcription factors, C/EBP alpha was the strongest trans-activator of the PEPCK-C gene promoter in the NIH3T3 cell line. The glucocorticoid receptor (GR), in the presence of its hormone ligand, inhibited the activation of the PEPCK-C gene promoter by C/EBP alpha or C/EBP beta but not by the adipocyte-specific peroxisome proliferator-activated receptor gamma 2. This inhibition effect was similar using the wild type or mutant GR and did not depend on GR binding to the DNA. The glucocorticoid response unit (GRU) in the PEPCK-C gene promoter (-2000 to +73) restrained C/EBP alpha-mediated trans-activation, because mutation of each single GRU element increased this activation by 3-4-fold. This series of GRU mutations were repressed by wild type GR to the same percent as was the nonmutated PEPCK-C gene promoter. In contrast, the repression by mutant GR depended on the intact AF1 site in the gene promoter, whereby mutation of the AF1 element abolished the repression.

C-Myc Is Required for the Glucose-mediated Induction of Metabolic Enzyme Genes

Glucose exerts powerful effects on hepatocyte gene transcription by mechanisms that are incompletely understood. c-Myc regulates hepatic glucose metabolism by increasing glycolytic enzyme gene transcription while concomitantly decreasing gluconeogenic and ketogenic enzyme gene expression. However, the molecular mechanisms by which c-Myc exerts these effects is not known. In this study, the glucose-mediated induction of L-type pyruvate kinase and glucose-6-phosphatase mRNA levels was diminished by maneuvers involving recombinant adenoviral vectors that interfere with (i) c-Myc protein levels by antisense expression or (ii) c-Myc function through a dominant-negative Max protein. These results were obtained using both HL1C rat hepatoma cells and primary rat hepatocytes. Furthermore, a decrease in c-Myc abundance reduced glucose production in HL1C cells, presumably by decreasing glucose-6-phosphatase activity. The repression of hormone-activated phosphoenolpyruvate carboxykinase gene transcription by glucose was not affected by a reduction in c-Myc levels. The basal mRNA levels for L-pyruvate kinase and glucose-6-phosphatase were not altered to any significant degree by adenoviral treatment. Furthermore, adenoviral overexpression of the c-Myc protein induced glucose-6-phosphatase mRNA in the absence of glucose stimulation. We conclude that multiple mechanisms exist to communicate the glucose-derived signal and that c-Myc has a key role in the hepatic glucose signaling pathway.

Conserved Amino Acids within CCAAT Enhancer-binding Proteins (C/EBPα and β) Regulate Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression

Thyroid hormone and cAMP stimulate transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK). CCAAT enhancer-binding proteins (C/EBP(alpha) and beta) are involved in multiple aspects of the nutritional, developmental and hormonal regulation of PEPCK gene expression. Previously, we have identified a thyroid hormone response element in the PEPCK promoter and demonstrated that C/EBP proteins bound to the P3(I) site are participants in the induction of PEPCK gene expression by thyroid hormone and cAMP. Here, we identify several peptide regions within the transactivation domain of C/EBP(alpha) that enhance the ability of T(3) to stimulate gene transcription. We also demonstrate that several conserved amino acids in the transactivation domain of C/EBP(alpha) and C/EBPbeta are required for the stimulation of basal gene expression and identify amino acids within C/EBPbeta that participate in the cAMP induction of the PEPCK gene. Finally, we show that the CREB-binding protein (CBP) enhanced the induction of PEPCK gene transcription by thyroid hormone and that CBP is associated with the PEPCK gene in vivo. Our results indicate that both C/EBP proteins and CBP participate in the regulation of PEPCK gene transcription by thyroid hormone.