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Richard W Hanson - One of the best experts on this subject based on the ideXlab platform.

  • HORMONAL REGULATION OF PHOSPHOENOLPYRYVATE Carboxykinase (GTP) SYNTHESIS
    Regulatory Mechanisms of Carbohydrate Metabolism, 2014
    Co-Authors: Patrick B. Iynedjian, Dimitris Kioussis, Josefa P. Garcia Ruiz, Richard W Hanson
    Abstract:

    ABSTRACT The level of the gluconeogenic enzyme Phosphoenolpyruvate Carboxykinase (GTP) (E.C.4.1.1.32) in the cytosol of rat tissues is controlled by hormones and related factors which cause changes in the synthesis rate of the enzyme. In studies aimed at elucidating the mechanisms of the hormonal regulation of Phosphoenolpyruvate Carboxykinase synthesis, the level of mRNA coding for the enzyme was measured in liver and kidney after various dietary and hormonal manipulations. 1. The level of functional Phosphoenolpyruvate Carboxykinase mRNA in the liver falls precipitously when fasted rats are refed carbohydrate. 2. Functional enzyme mRNA increases rapidly during induction of hepatic Phosphoenolpyruvate Carboxykinase by cyclic AMP. 3. Functional enzyme mRNA increases in the kidney during induction by glucocorticoids. In all cases, changes in mRNA level are commensurate with the changes in rate of enzyme synthesis in vivo. These findings are discussed in reference to “transcriptional” and “translational” theories of hormonal regulation of genetic expression.

  • SREBP-1c and Sp1 interact to regulate transcription of the gene for Phosphoenolpyruvate Carboxykinase (GTP) in the liver.
    The Journal of biological chemistry, 2004
    Co-Authors: Kaushik Chakravarty, David Samols, Cheng Ming Chiang, Richard W Hanson
    Abstract:

    The sterol regulatory element-binding protein-1c (SREBP-1c), as well as SREBP-1a and SREBP-2, inhibit transcription of the gene encoding the cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (PEPCK-C). There are two SREBP regulatory elements (SREs) in the PEPCK-C gene promoter (-322 to -313 and -590 to -581). The SRE at -590 overlaps an Sp1 site on the opposite strand of the DNA. These SREs bound SREBP-1a and SREBP-1c with low affinity but the addition of purified upstream stimulatory activity enhanced the binding of SREBP-1 to both of these sites. Mutating these SREs increased both unstimulated (5-fold) and protein kinase A-stimulated transcription (8-27-fold) from the PEPCK-C gene promoter; this was lost when both SREs were mutated. The SRE at -590 differs by a single base pair from the SRE in the low density lipoprotein (LDL) receptor gene (T in the PEPCK-C gene promoter at -582, compared with an A in the SRE of the gene for the LDL receptor promoter). Introduction of the LDL receptor SRE into the PEPCK-C gene promoter increased SREBP-1c binding and caused a 10-fold enhancement of basal transcription from the promoter, rather than an inhibition as observed with the SRE in the PEPCK-C gene promoter. The T/A change does not alter the binding of Sp1 to its site on the opposite strand of the DNA. Sp1 bound to the promoter independently of SREBP-1c but competed with SREBP-1c for binding. Sp1 does not bind to the SRE at -322. Chromatin immunoprecipitation analysis, using rat hepatocytes, demonstrated that SREBP-1 and Sp1 were associated in vivo with putative regulatory regions corresponding to the SREs in the PEPCK-C gene promoter. We propose that insulin represses transcription of the gene for PEPCK-C by inducing SREBP-1c production in the liver, which interferes with the stimulatory effect of Sp1 at -590 of the PEPCK-C gene promoter.

  • Glucocorticoids Repress Transcription of Phosphoenolpyruvate Carboxykinase (GTP) Gene in Adipocytes by Inhibiting Its C/EBP-mediated Activation
    The Journal of biological chemistry, 2003
    Co-Authors: Yael Olswang, Richard W Hanson, Hannah Cohen, Hanoch Cassuto, Barak Blum, Yael Biberman, Lea Reshef
    Abstract:

    Abstract 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/EBPα 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/EBPα 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/EBPα or C/EBPβ but not by the adipocyte-specific peroxisome proliferator-activated receptor γ2. 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/EBPα-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.

  • Sterol regulatory element-binding protein-1c mimics the negative effect of insulin on Phosphoenolpyruvate Carboxykinase (GTP) gene transcription.
    The Journal of biological chemistry, 2001
    Co-Authors: Kaushik Chakravarty, Patrick Leahy, Parvin Hakimi, Dominique Bécard, Marc Foretz, Pascal Ferré, Fabienne Foufelle, Richard W Hanson
    Abstract:

    Abstract We have assessed the potential role of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of the gene for the cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C). SREBP-1c introduced into primary hepatocytes with an adenovirus vector caused a total loss of PEPCK-C mRNA and a marked induction of fatty acid synthase mRNA that directly coincided with the appearance of SREBP-1c in the hepatocytes. It also blocked the induction of PEPCK-C mRNA by cAMP and dexamethasone in these cells. In contrast, a dominant negative form of SREBP-1c (dnSREBP-1c) stimulated the accumulation of PEPCK-C mRNA in these cells. SREBP-1c completely blocked the induction of PEPCK-C gene transcription by the catalytic subunit of protein kinase A (PKA), and increasing concentrations of dnSREBP-1c reversed the negative effect of insulin on transcription from the PEPCK-C gene promoter in WT-IR cells. The more than 10-fold induction of PKA-stimulated PEPCK-C gene transcription caused by the co-activator CBP, was also blocked by SREBP-1c. In addition, dnSREBP-1c reversed the strong negative effect of E1A and NF1 on PKA-stimulated transcription from the PEPCK-C gene promoter. An analysis of the possible site of action of SREBP-1c using stepwise truncations of the PEPCK-C gene promoter indicated that the negative effect of SREBP-1c on transcription is exerted at a site between −355 and −277. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver and acts by blocking the stimulatory effect cAMP that is mediated via an interaction with cAMP-binding protein.

  • The Use of Transgenic Mice to Analyze the Role of Accessory Factor Two in the Regulation of Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription during Diabetes
    The Journal of biological chemistry, 2001
    Co-Authors: Pamela S. Lechner, Jeung S. Yun, Parvin Hakimi, Colleen M. Croniger, Carrie A. Millward, Christina Fekter, Richard W Hanson
    Abstract:

    Abstract The regulation of transcription of the gene for the cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (PEPCK-C) (4.1.1.32) during diabetes is a complex process that involves a number of regulatory elements in the PEPCK-C gene promoter. The accessory factor 2 (AF2)-binding region that is contained within the glucocorticoid regulatory unit of the PEPCK-C gene promoter (−451 to −353) has been implicated in the action of both insulin and glucocorticoids on PEPCK-C gene transcription. To determine the role of AF2 in these processes, we have generated a mouse model bearing a transgene that contains the PEPCK-C gene promoter with a mutation in the AF2-binding region. This promoter is linked to the structural gene for human growth hormone that is biologically inactive (AF2–2000/hGx). In the absence of the AF2 regulatory element, the transcription of the transgene in the liver is not induced by diabetes but is inhibited by the administration of insulin. There is also a marked reduction in the response of the AF2–2000/hGx gene in the kidney to the administration of glucocorticoids. The AF2–2000/hGx gene in the liver responds normally to a high carbohydrate diet with a marked decrease in gene transcription. This suggests that insulin is not exerting its usual negative effect on the PEPCK-C gene promoter through the AF2 site. In contrast, the response of this transgene to a high fat/carbohydrate-free diet is severely blunted. Our results support a role for the AF2 site in the PEPCK-C gene promoter in the effect of glucocorticoids, but not insulin, on PEPCK-C gene transcription in the liver.

Parvin Hakimi - One of the best experts on this subject based on the ideXlab platform.

  • Activation of SIRT1 by Resveratrol Represses Transcription of the Gene for the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) by Deacetylating Hepatic Nuclear Factor 4α
    The Journal of biological chemistry, 2009
    Co-Authors: Jianqi Yang, Parvin Hakimi, David Samols, Xiaoying Kong, Maria Emilia S Martins-santos, Gabriela Aleman, Ernestine Chaco, George E. Liu, Cheng Ming Chiang
    Abstract:

    The SIRT1 activators isonicotinamide (IsoNAM), resveratrol, fisetin, and butein repressed transcription of the gene for the cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (PEPCK-C). An evolutionarily conserved binding site for hepatic nuclear factor (HNF) 4α (−272/−252) was identified, which was required for transcriptional repression of the PEPCK-C gene promoter caused by these compounds. This site contains an overlapping AP-1 binding site and is adjacent to the C/EBP binding element (−248/−234); the latter is necessary for hepatic transcription of PEPCK-C. AP-1 competed with HNF4α for binding to this site and also decreased HNF4α stimulation of transcription from the PEPCK-C gene promoter. Chromatin immunoprecipitation experiments demonstrated that HNF4α and AP-1, but not C/EBPβ, reciprocally bound to this site prior to and after treating HepG2 cells with IsoNAM. IsoNAM treatment resulted in deacetylation of HNF4α, which decreased its binding affinity to the PEPCK-C gene promoter. In HNF4α-null Chinese hamster ovary cells, IsoNAM and resveratrol failed to repress transcription from the PEPCK-C gene promoter; overexpression of HNF4α in Chinese hamster ovary cells re-established transcriptional inhibition. Exogenous SIRT1 expression repressed transcription, whereas knockdown of SIRT1 by RNA interference reversed this effect. IsoNAM decreased the level of mRNA for PEPCK-C but had no effect on mRNA for glucose-6-phosphatase in AML12 mouse hepatocytes. We conclude that SIRT1 activation inhibited transcription of the gene for PEPCK-C in part by deacetylation of HNF4α. However, SIRT1 deacetylation of other key regulatory proteins that control PEPCK-C gene transcription also likely contributed to the inhibitory effect.

  • Sterol regulatory element-binding protein-1c mimics the negative effect of insulin on Phosphoenolpyruvate Carboxykinase (GTP) gene transcription.
    The Journal of biological chemistry, 2001
    Co-Authors: Kaushik Chakravarty, Patrick Leahy, Parvin Hakimi, Dominique Bécard, Marc Foretz, Pascal Ferré, Fabienne Foufelle, Richard W Hanson
    Abstract:

    Abstract We have assessed the potential role of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of the gene for the cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C). SREBP-1c introduced into primary hepatocytes with an adenovirus vector caused a total loss of PEPCK-C mRNA and a marked induction of fatty acid synthase mRNA that directly coincided with the appearance of SREBP-1c in the hepatocytes. It also blocked the induction of PEPCK-C mRNA by cAMP and dexamethasone in these cells. In contrast, a dominant negative form of SREBP-1c (dnSREBP-1c) stimulated the accumulation of PEPCK-C mRNA in these cells. SREBP-1c completely blocked the induction of PEPCK-C gene transcription by the catalytic subunit of protein kinase A (PKA), and increasing concentrations of dnSREBP-1c reversed the negative effect of insulin on transcription from the PEPCK-C gene promoter in WT-IR cells. The more than 10-fold induction of PKA-stimulated PEPCK-C gene transcription caused by the co-activator CBP, was also blocked by SREBP-1c. In addition, dnSREBP-1c reversed the strong negative effect of E1A and NF1 on PKA-stimulated transcription from the PEPCK-C gene promoter. An analysis of the possible site of action of SREBP-1c using stepwise truncations of the PEPCK-C gene promoter indicated that the negative effect of SREBP-1c on transcription is exerted at a site between −355 and −277. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver and acts by blocking the stimulatory effect cAMP that is mediated via an interaction with cAMP-binding protein.

  • The Use of Transgenic Mice to Analyze the Role of Accessory Factor Two in the Regulation of Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription during Diabetes
    The Journal of biological chemistry, 2001
    Co-Authors: Pamela S. Lechner, Jeung S. Yun, Parvin Hakimi, Colleen M. Croniger, Carrie A. Millward, Christina Fekter, Richard W Hanson
    Abstract:

    Abstract The regulation of transcription of the gene for the cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (PEPCK-C) (4.1.1.32) during diabetes is a complex process that involves a number of regulatory elements in the PEPCK-C gene promoter. The accessory factor 2 (AF2)-binding region that is contained within the glucocorticoid regulatory unit of the PEPCK-C gene promoter (−451 to −353) has been implicated in the action of both insulin and glucocorticoids on PEPCK-C gene transcription. To determine the role of AF2 in these processes, we have generated a mouse model bearing a transgene that contains the PEPCK-C gene promoter with a mutation in the AF2-binding region. This promoter is linked to the structural gene for human growth hormone that is biologically inactive (AF2–2000/hGx). In the absence of the AF2 regulatory element, the transcription of the transgene in the liver is not induced by diabetes but is inhibited by the administration of insulin. There is also a marked reduction in the response of the AF2–2000/hGx gene in the kidney to the administration of glucocorticoids. The AF2–2000/hGx gene in the liver responds normally to a high carbohydrate diet with a marked decrease in gene transcription. This suggests that insulin is not exerting its usual negative effect on the PEPCK-C gene promoter through the AF2 site. In contrast, the response of this transgene to a high fat/carbohydrate-free diet is severely blunted. Our results support a role for the AF2 site in the PEPCK-C gene promoter in the effect of glucocorticoids, but not insulin, on PEPCK-C gene transcription in the liver.

  • Nuclear Factor I Regulates Expression of the Gene for Phosphoenolpyruvate Carboxykinase (GTP)
    The Journal of biological chemistry, 1998
    Co-Authors: Deborah R. Crawford, Patrick Leahy, Gregory Grossman, Richard M. Gronostajski, Ali Z. Chaudhry, Jason Woods, Parvin Hakimi, William J. Roesler, Richard W Hanson
    Abstract:

    Abstract Nuclear factor-I (NFI) binds to the Phosphoenolpyruvate Carboxykinase (GTP) (PEPCK) gene promoter immediately 5′ to the cAMP regulatory element (CRE). This suggests an interaction between NFI and factors that bind the CRE. Of the four NFI isoforms expressed in mammalian tissues, NFI-A and -B stimulate basal transcription from the PEPCK gene promoter in HepG2 cells, while NFI-C and -X are slightly inhibitory. All four NFI isoforms abrogate the 20-fold protein kinase Ac (PKAc)-mediated induction of transcription from the PEPCK gene promoter. Normal PKAc-mediated induction was noted when the CRE was moved 10 base pairs 3′ of its original location. However if the CRE was moved 5 base pairs 3′, placing it out of phase with the other elements in the promoter, or moved 5′ to −285 (the P3(I) site in the promoter), some PKA-mediated stimulation was lost. The NFI-C isoform effectively inhibited PKAc induction regardless of the relative positions of the CRE and the NFI binding sites. NFI-C also abrogated cAMP regulatory element-binding protein (CREB)-induced activity of wild type and mutant PEPCK promoters. There was some cooperativity in the binding of CREB and NFI to their respective binding sites but this did not appear to be physiologically important.

  • Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription and Hyperglycemia Are Regulated by Glucocorticoids in Genetically Obesedb/db Transgenic Mice
    The Journal of biological chemistry, 1997
    Co-Authors: Jacob E. Friedman, Parvin Hakimi, Pamela S. Lechner, Yang Sun, Tatsuya Ishizuka, Craig J. Farrell, Shana E. Mccormack, Lisa M. Herron, Jeung S. Yun
    Abstract:

    Abstract The molecular mechanisms underlying increased hepatic Phosphoenolpyruvate Carboxykinase (PEPCK) gene transcription and gluconeogenesis in type II diabetes are largely unknown. To examine the involvement of glucocorticoids and thecis-acting insulin response sequence (IRS, −416/−407) in the genetically obese db/db mouse model, we generated crosses between C57BL/KsJ-db/+ mice and transgenic mice that express −460 or −2000 base pairs of the rat PEPCK gene promoter containing an intact or mutated IRS, linked to a reporter gene. Transgenic mice expressing the intact PEPCK(460)-CRP (C-reactive protein) transgene bred to near homozygosity at thedb locus were obese, hyperinsulinemic, and developed fasting hyperglycemia (389 ± 26 mg/100 ml) between 4 and 10 weeks of age. Levels of CRP reporter gene expression were increased 2-fold despite severe hyperinsulinemia compared with non-diabetic non-obese transgenic mice. Reporter gene expression was also increased 2-fold in transgenic obese diabetic db/db mice bearing a mutation in the IRS, −2000(IRS)-hGx, compared with non-obese non-diabetic transgenic 2000(IRS)-hGx mice. Treatment of obese diabeticdb/db transgenic mice with the glucocorticoid receptor blocker RU 486 decreased plasma glucose by 50% and reduced PEPCK, GLUT2, glucose-6-phosphatase, tyrosine aminotransferase, CRP, and hGx reporter gene expression to levels similar to those of non-obese normoglycemic transgenic mice. Taken together, these results establish that −460 bp of 5′-flanking sequence is sufficient to mediate the induction of PEPCK gene transcription in genetically obesedb/db mice during the development of hyperglycemia. The results further demonstrate that the mechanism underlying increased expression of gluconeogenic enzymes in thedb/db mouse requires the action of glucocorticoids and occurs independently of factors acting through the PEPCK IRS (−416/−407) promoter binding site.

Claude Forest - One of the best experts on this subject based on the ideXlab platform.

  • Fatty acid recycling in adipocytes: a role for glyceroneogenesis and Phosphoenolpyruvate Carboxykinase
    Biochemical Society Transactions, 2003
    Co-Authors: Claude Forest, Elmus G. Beale, Joan Tordjman, Martine Glorian, Eric Duplus, Geneviève Chauvet, Joelle Quette, Bénédicte Antoine
    Abstract:

    FA (fatty acid) recycling in adipose tissue appears to be an important pathway for regulating FA release into the blood during fasting. Re-esterification requires G3P (glycerol 3-phosphate), which cannot be synthesized from glucose because glycolysis is much reduced under such circumstances. In addition, G3P can scarcely originate from glycerol since glycerol kinase has a very low activity in white adipose tissue. It was shown about 35 years ago that a metabolic pathway named glyceroneogenesis, which allows G3P synthesis from non-carbohydrate precursors like pyruvate, lactate or amino acids, is activated during fasting. The major enzyme in this pathway was shown to be PEPCK-C [cytosolic Phosphoenolpyruvate Carboxykinase (GTP); EC 4.1.1.32]. The present review analyses the mechanisms by which a series of hormones and nutrients affect PEPCK-C gene transcription and glyceroneogenesis and describes evidence for dysregulation of this pathway in type 2 diabetes.

  • Isolation and characterization of the mouse cytosolic Phosphoenolpyruvate Carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites.
    Molecular and cellular endocrinology, 1999
    Co-Authors: Christine P. Williams, Catherine Postic, Danielle Robin, Pierre Robin, Joseph Parrinello, Richard L. Printz, Mark A. Magnuson, Daryl K. Granner, K Shelton, Claude Forest
    Abstract:

    A 72 kilobase pair DNA fragment that contains the mouse Phosphoenolpyruvate Carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

  • ISOLATION AND CHARACTERIZATION OF THE MOUSE CYTOSOLIC Phosphoenolpyruvate Carboxykinase (GTP) GENE : EVIDENCE FOR TISSUE-SPECIFIC HYPERSENSITIVE SITES
    Molecular and Cellular Endocrinology, 1999
    Co-Authors: Christine P. Williams, Catherine Postic, Danielle Robin, Pierre Robin, Joseph Parrinello, Kathy D. Shelton, Richard L. Printz, Mark A. Magnuson, Daryl K. Granner, Claude Forest
    Abstract:

    Abstract A 72 kilobase pair DNA fragment that contains the mouse Phosphoenolpyruvate Carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from ∼−5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

Lea Reshef - One of the best experts on this subject based on the ideXlab platform.

  • Glucocorticoids Repress Transcription of Phosphoenolpyruvate Carboxykinase (GTP) Gene in Adipocytes by Inhibiting Its C/EBP-mediated Activation
    The Journal of biological chemistry, 2003
    Co-Authors: Yael Olswang, Richard W Hanson, Hannah Cohen, Hanoch Cassuto, Barak Blum, Yael Biberman, Lea Reshef
    Abstract:

    Abstract 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/EBPα 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/EBPα 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/EBPα or C/EBPβ but not by the adipocyte-specific peroxisome proliferator-activated receptor γ2. 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/EBPα-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.

  • Involvement of HNF-1 in the regulation of Phosphoenolpyruvate Carboxykinase gene expression in the kidney
    FEBS letters, 1997
    Co-Authors: Hanoch Cassuto, Richard W Hanson, Hovav Nechushtan, Yael Olswang, Alejandro F Livoff, Lea Reshef
    Abstract:

    The cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (PEPCK) gene is differentially expressed in several tissues. A specific set of regulatory elements in the promoter are responsible for the control of PEPCK gene transcription and, in turn, determine its distinct metabolic role in each tissue. DNase I footprinting analysis of the PEPCK promoter, using nuclear proteins from tissues which express the gene for PEPCK, and transient expression assays in renal cell lines have demonstrated that the HNF-1 recognition motif (P2) in the PEPCK promoter characterizes kidney-specific expression. This site is required also for the response to acidosis. Since the P2 site is not involved in the expression of the PEPCK gene in the liver, we propose that its critical role in the kidney stems from a combination of abundance of HNF-1 together with low concentrations of members of the C/EBP family in this tissue.

  • REGULATION OF Phosphoenolpyruvate Carboxykinase (GTP) GENE EXPRESSION
    Annual review of biochemistry, 1997
    Co-Authors: Richard W Hanson, Lea Reshef
    Abstract:

    Phosphoenolpyruvate Carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is a key enzyme in the synthesis of glucose in the liver and kidney and of glyceride-glycerol in white adipose tissue and the small intestine. The gene for the cytosolic form of PEPCK (PEPCK-C) is acutely regulated by a variety of dietary and hormonal signals, which result in alteration of synthesis of the enzyme. Major factors that increase PEPCK-C gene expression include cyclic AMP, glucocorticoids, and thyroid hormone, whereas insulin inhibits this process. PEPCK-C is absent in fetal liver but appears at birth, concomitant with the capacity for gluconeogenesis. Regulatory elements that control transcription of the PEPCK-C gene in liver, kidney, and adipose tissue have been delineated, and many of the transcription factors that bind to these elements have been identified. Transgenic mice have been especially useful in elucidating the physiological roles of specific sequence elements in the PEPCK-C gene promoter and in demonstrating the key role played at these sites by the isoforms of CAAT/enhancer binding protein in patterning of PEPCK-C gene expression during the perinatal period. The PEPCK-C gene provides a model for the metabolic control of gene transcription.

Austin L. Gurney - One of the best experts on this subject based on the ideXlab platform.

  • The Promoter Regulatory Regions of the Genes for the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) from the Chicken and the Rat Have Different Species-Specific Roles in Gluconeogenesis
    The Journal of nutrition, 1997
    Co-Authors: Summer Savon, Deborah R. Crawford, Parvin Hakimi, Dwight J. Klemm, Austin L. Gurney, Richard W Hanson
    Abstract:

    Hepatic expression of the gene for Phosphoenolpyruvate Carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) in birds occurs prior to birth and decreases to negligible levels before hatching, whereas in mammals the gene for PEPCK-C in the liver is expressed at birth and is active throughout the life of the animal. The administration of cyclic AMP to adult chickens results in the induction of transcription of the gene for PEPCK-C and the transient accumulation of PEPCK-C mRNA in the liver. DNase I footprint analysis of 330 bp of the avian PEPCK-C promoter immediately 5' of the start-site of transcription indicated the presence of several protein binding domains, purified CAAT/enhancer binding protein alpha, cAMP regulatory element binding protein and nuclear factor-1 bound to these regions of the promoter. Sequences corresponding to an hepatic nuclear factor-1 binding domain and to the insulin response sequence, previously identified in the rat PEPCK-C promoter, were also found in the chicken PEPCK-C promoter. Co-transfection of an expression vector for CAAT/enhancer binding protein alpha or CAAT/enhancer binding protein beta markedly stimulated transcription from both the chicken and rat PEPCK-C promoters in human hepatoma cells. Sequences involved in the regulation of gene transcription by cyclic AMP and insulin were found to reside between -210 and +1 of the avian PEPCK-C promoter. In general, transcription from the avian promoter was more sensitive to inhibition by insulin than was noted for the rat PEPCK-C promoter, which may explain in part the lack of expression of the gene for PEPCK-C in the livers of adult birds.

  • Relative roles of CCAAT/enhancer-binding protein beta and cAMP regulatory element-binding protein in controlling transcription of the gene for Phosphoenolpyruvate Carboxykinase (GTP).
    The Journal of biological chemistry, 1993
    Co-Authors: Edwards A. Park, Parvin Hakimi, Austin L. Gurney, Antoon F M Moorman, Steven E. Nizielski, Zhodan Cao, Richard W Hanson
    Abstract:

    Abstract The gene for Phosphoenolpyruvate Carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is expressed in a tissue-specific manner in the liver, kidney, and adipose tissue and is regulated by hormones including cAMP and insulin. Previous studies have shown that the CCAAT/enhancer-binding protein alpha (C/EBP alpha) binds to several sites on the PEPCK promoter and activates transcription from the promoter in hepatoma cells. Here, we report that a second member of the C/EBP family, C/EBP beta, bound to the same sites on the PEPCK promoter. However, C/EBP beta stimulated transcription primarily through the cAMP-responsive element (CRE), which maps between positions -77 to -94, but not at the more 5'-binding sites. In addition, the nuclear factor-1 site, which is immediately adjacent to the CRE in the PEPCK promoter, was also required for the full response of the promoter to cotransfected C/EBP beta. In gel mobility assays, antibodies to both C/EBP beta and the cAMP regulatory element-binding protein (CREB), but not to C/EBP alpha, "supershifted" DNA-protein complexes formed between a synthetic CRE oligomer and proteins prepared from rat liver nuclei. C/EBP beta mRNA was expressed at low levels in both the periportal and pericentral regions of the liver lobule, whereas expression of the gene for C/EBP alpha was confined to the pericentral region of the liver lobule. PEPCK gene transcription is greatest in the periportal region of the liver. CREB also bound to the CRE and stimulated transcription of a PEPCK-CAT vector in the presence of an expression vector for the catalytic subunit of protein kinase A. C/EBP beta and CREB bound to the CRE with similar affinities, both of which were greater than the affinity of C/EBP alpha. Within 90 min after the administration of dibutyryl cAMP to rats, there was a marked increase in the hepatic concentration of C/EBP beta mRNA and a decrease in the level of mRNA for C/EBP alpha. These studies indicate that C/EBP beta can regulate PEPCK gene transcription by acting through the CRE and that C/EBP beta, together with CREB, may contribute to the cAMP responsiveness of the PEPCK promoter.

  • relative roles of ccaat enhancer binding protein beta and camp regulatory element binding protein in controlling transcription of the gene for Phosphoenolpyruvate Carboxykinase GTP
    Journal of Biological Chemistry, 1993
    Co-Authors: Edwards A. Park, Parvin Hakimi, Austin L. Gurney, Steven E. Nizielski, Zhodan Cao, Antoon Moorman, Richard W Hanson
    Abstract:

    Abstract The gene for Phosphoenolpyruvate Carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is expressed in a tissue-specific manner in the liver, kidney, and adipose tissue and is regulated by hormones including cAMP and insulin. Previous studies have shown that the CCAAT/enhancer-binding protein alpha (C/EBP alpha) binds to several sites on the PEPCK promoter and activates transcription from the promoter in hepatoma cells. Here, we report that a second member of the C/EBP family, C/EBP beta, bound to the same sites on the PEPCK promoter. However, C/EBP beta stimulated transcription primarily through the cAMP-responsive element (CRE), which maps between positions -77 to -94, but not at the more 5'-binding sites. In addition, the nuclear factor-1 site, which is immediately adjacent to the CRE in the PEPCK promoter, was also required for the full response of the promoter to cotransfected C/EBP beta. In gel mobility assays, antibodies to both C/EBP beta and the cAMP regulatory element-binding protein (CREB), but not to C/EBP alpha, "supershifted" DNA-protein complexes formed between a synthetic CRE oligomer and proteins prepared from rat liver nuclei. C/EBP beta mRNA was expressed at low levels in both the periportal and pericentral regions of the liver lobule, whereas expression of the gene for C/EBP alpha was confined to the pericentral region of the liver lobule. PEPCK gene transcription is greatest in the periportal region of the liver. CREB also bound to the CRE and stimulated transcription of a PEPCK-CAT vector in the presence of an expression vector for the catalytic subunit of protein kinase A. C/EBP beta and CREB bound to the CRE with similar affinities, both of which were greater than the affinity of C/EBP alpha. Within 90 min after the administration of dibutyryl cAMP to rats, there was a marked increase in the hepatic concentration of C/EBP beta mRNA and a decrease in the level of mRNA for C/EBP alpha. These studies indicate that C/EBP beta can regulate PEPCK gene transcription by acting through the CRE and that C/EBP beta, together with CREB, may contribute to the cAMP responsiveness of the PEPCK promoter.

  • Opposing actions of Fos and Jun on transcription of the Phosphoenolpyruvate Carboxykinase (GTP) gene. Dominant negative regulation by Fos.
    The Journal of biological chemistry, 1992
    Co-Authors: Austin L. Gurney, Jinsong Liu, Edwards A. Park, Marta Giralt, Richard W Hanson
    Abstract:

    Jun homodimers and Fos/Jun heterodimers bind to the gene for Phosphoenolpyruvate Carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) at three sites within the first 350 base pairs of the promoter. These include CRE-1 (-82 to -90), and P3(II) and P4 (-252 to -258 and -268 to -285, respectively). Over-expression of Jun in HepG2 cells resulted in a 10-15-fold increase in the level of transcription of a chimeric PEPCK (-490 to +73)-CAT gene, while expression of Fos decreased transcription and blocked the induction of transcription from the PEPCK promoter by Jun. The action of Fos and Jun on PEPCK gene transcription involved each of the Fos/Jun-binding sites and was modulated by additional transcriptional regulatory elements within the PEPCK promoter. The ability of Fos to inhibit PEPCK transcription was dependent upon P3(I), a region of the promoter which does not bind Fos/Jun heterodimers, but does bind members of the C/EBP family of transcription factors. Stimulation of PEPCK transcription by 8-Br-cAMP or by overexpression of the catalytic subunit of protein kinase A was inhibited by Fos expression. The inhibitory effects of phorbol esters and protein kinase C on PEPCK gene expression may be mediated through the action of Fos and Jun.

  • identification of a thyroid hormone response element in the Phosphoenolpyruvate Carboxykinase GTP gene evidence for synergistic interaction between thyroid hormone and camp cis regulatory elements
    Journal of Biological Chemistry, 1991
    Co-Authors: Marta Giralt, Parvin Hakimi, Edwards A. Park, Jinsong Liu, Austin L. Gurney, Richard W Hanson
    Abstract:

    Transcription of the gene for the cytosolic form of Phosphoenolpyruvate Carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in the liver is regulated by many hormones including thyroid hormone (T3). In order to identify the elements in the promoter which are required for transcriptional induction by T3, we cotransfected a T3 receptor expression vector with a PEPCK-CAT reporter gene into HepG2 cells. Using vectors with deletions in the PEPCK promoter, we identified a single T3 response element (TRE) between positions -332 and -308. This element binds [125I]T3-labeled T3 receptor contained in nuclear extracts prepared from rat liver. Furthermore, the P3(I) element (-250 to -234), a previously described cis-sequence involved in mediating the induction of PEPCK gene transcription by cAMP, is also required for the T3 responsiveness of the promoter. In the absence of either the TRE or the P3(I) binding sites, no stimulation of transcription from the PEPCK promoter by T3 was observed, indicating that both elements are required for the T3 transcriptional regulation. Finally, a synergistic induction of PEPCK gene transcription by T3 and cAMP is described. This interaction requires both T3- and cAMP-responsive cis-acting elements.