FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022894562> ?p ?o ?g. }

• W2022894562 endingPage "22679" @default.
• W2022894562 startingPage "22675" @default.
• W2022894562 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. 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. cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) glucocorticoid regulatory unit accessory factor glucocorticoid receptor hepatic nuclear factor CCAAT enhancer-binding protein glucocorticoid response element insulin regulatory sequence wild type cAMP-response element-binding protein sterol regulatory element-binding protein 1c The cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C)1 (GTP) (4.1.1.32) is a critical enzyme in hepatic gluconeogenesis and glyceroneogenesis (1Hanson R.W. Patel Y.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 69: 203-281PubMed Google Scholar) and is induced during diabetes, where it is involved in the increased rate of hepatic glucose output and triglyceride synthesis characteristic of this disease. Transcription of the gene for PEPCK-C is induced by cAMP (2Lamers W.H. Hanson R.W. Meisner H.M. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5137-5141Crossref PubMed Scopus (279) Google Scholar, 3Liu J. Park E.A. Gurney A.L. Roesler W.J. Hanson R.W. J. Biol. Chem. 1991; 266: 19095-19102Abstract Full Text PDF PubMed Google Scholar, 4Sasaki K. Cripe T.P. Koch S.R. Andreone T.L. Peterson D.D. Beale E.G. Granner D.K. J. Biol. Chem. 1984; 259: 15242-15251Abstract Full Text PDF PubMed Google Scholar) and glucocorticoids (5Imai E. Stromstedt P.E. Quinn P.G. Carlstedt-Duke J. Gustaffson J.A. Granner D.K. Mol. Cell. Biol. 1990; 10: 4712-4719Crossref PubMed Scopus (243) Google Scholar) and inhibited by insulin (6Granner D.K. Andreone T. Sasaki K. Beale E. Nature. 1983; 305: 549-551Crossref PubMed Scopus (246) Google Scholar, 7Forest C.D. O'Brien R.M. Lucas P.C. Magnuson M.A. Granner D.K. Mol. Endocrinol. 1990; 4: 1302-1310Crossref PubMed Scopus (95) Google Scholar, 8Scott D.K. O'Doherty R.M. Stafford J.M. Newgard C.B. Granner D.K. J. Biol. Chem. 1998; 273: 24145-24151Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and high concentrations of glucose (4Sasaki K. Cripe T.P. Koch S.R. Andreone T.L. Peterson D.D. Beale E.G. Granner D.K. J. Biol. Chem. 1984; 259: 15242-15251Abstract Full Text PDF PubMed Google Scholar, 9Short J.M. Wynshaw-Boris A. Short H.P. Hanson R.W. J. Biol. Chem. 1986; 261: 9721-9726Abstract Full Text PDF PubMed Google Scholar). There are three major factors involved in the induction of PEPCK-C during diabetes. First, the concentration of insulin in the blood is dramatically reduced resulting in the removal of the dominant negative regulator of PEPCK-C gene transcription. Second, there is an increase in the levels of glucagon and a subsequent elevation in the concentration of hepatic cAMP; this stimulates PEPCK-C gene transcription (10Hanson R.W. Reshef L. Annu. Rev. Biochem. 1997; 66: 581-611Crossref PubMed Scopus (633) Google Scholar). Third, glucocorticoids are required to ensure the elevated expression of the PEPCK-C gene and the resultant physiological alterations, including increased gluconeogenesis (11Gunn J.M. Hanson R.W. Meyuhas O. Reshef L. Ballard F.J. Biochem. J. 1975; 150: 195-203Crossref PubMed Scopus (59) Google Scholar) (12Friedman J.E. Yun J.S. Patel Y.M. McGrane M.M. Hanson R.W. J. Biol. Chem. 1993; 268: 12952-12957Abstract Full Text PDF PubMed Google Scholar). The mechanism(s) responsible for the induction of PEPCK-C gene transcription during diabetes has been studied in some detail (13Granner D.K. O'Brien R.M. Diabetes Care. 1992; 15: 369-395Crossref PubMed Scopus (130) Google Scholar). The effect of cAMP is exerted through two regions in the PEPCK-C gene promoter; the cAMP-response element, which maps from −90 to −83, and an up-stream regulatory element termed P3(I), which spans bases −250 to −234 in the promoter (14Liu J.-K. DiPersio C. Zaret K.S. Mol. Cell. Biol. 1991; 11: 773-784Crossref PubMed Scopus (140) Google Scholar). The regulation of PEPCK-C gene expression by glucocorticoids is accomplished through a glucocorticoid regulatory unit (GRU) in the promoter located between −451 and −353 (15Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 261: 31909-31914Abstract Full Text Full Text PDF Scopus (69) Google Scholar). The GRU includes a linear array of three accessory factor-binding sites, AF1 (−451 to −439), AF2 (−416 to −407), and AF3 (−326 to −321), and two glucocorticoid receptor-binding sites, GR1 and GR2 (−386 to −353) (15Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 261: 31909-31914Abstract Full Text Full Text PDF Scopus (69) Google Scholar). The GRU can bind a variety of transcription factors. The AF1 site binds the chicken ovalbumin up-stream promoter transcription factor and hepatic nuclear factor 4 (HNF-4). Both the CCAAT enhancer-binding protein (C/EBP) family members C/EBPα and C/EBPβ and HNF-3 bind to the AF2 site, but it has been shown that HNF-3 mediates PEPCK-C gene transcription (16Wang J. Stromstedt P. O'Brien R. Granner D. Mol Endocrinol. 1996; 10: 794-800PubMed Google Scholar). The AF2-binding site also contains an overlapping glucocorticoid response element (GRE) and an insulin regulatory sequence (IRS), which regulate the glucocorticoid and insulin responses. A third accessory site has been characterized by Scottet al. (15Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 261: 31909-31914Abstract Full Text Full Text PDF Scopus (69) Google Scholar) and shown to bind chicken ovalbumin upstream promoter transcription factor and up-stream stimulatory factor. A single mutation in any one of these three AF elements results in a 60% decrease of the glucocorticoid response in hepatocytes, whereas a mutation in any two AF elements completely eliminates the glucocorticoid response of the PEPCK-C gene promoter mediated by the GRU (15Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 261: 31909-31914Abstract Full Text Full Text PDF Scopus (69) Google Scholar). As suggested by the overlapping GRE and IRS within the AF2 element, the AF1, AF2, and AF3 are also required for other hormone responses. Both the AF1 (17Hall R.K. Sladek F.M. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 412-416Crossref PubMed Scopus (199) Google Scholar) and AF3 contain GRE and retinoic acid response elements (15Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 261: 31909-31914Abstract Full Text Full Text PDF Scopus (69) Google Scholar) and are necessary for the response of the PEPCK-C gene promoter to retinoic acid. The AF2 element contains an IRS (18O'Brien R.M. Lucas P.C. Forest C.D. Magnunson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google Scholar) that has been proposed to mediate part of the negative response of the gene to insulin (19O'Brien R.M. Noison E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.-C. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar). The present study focuses on use of transgenic animals carrying a mutated AF2 element (in the PEPCK-C gene promoter −2000 to +73) linked to the human growth hormone structural gene (hGx), which lacks a receptor-binding site and thus is biologically inactive. We have analyzed the transcriptional response of the transgene to diabetes, insulin replacement, administration of glucocorticoids, and alterations in dietary protein and carbohydrate. The results suggest that the AF2 element of the GRU is necessary for induction of PEPCK-C mRNA by glucocorticoids during diabetes and that the effect of insulin on this site is modest at best. Theophylline, Bt2cAMP, streptozotocin, and dexamethasone were purchased from Sigma. Strip-EZ DNA probe synthesis kit was purchased from Ambion (Austin, TX). QuickPrep total RNA extraction kit was purchased from Amersham Pharmacia Biotech. GeneScreen Plus and [α-32P]dATP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences. Restriction enzymes and proteinase K were purchased from Roche Molecular Biochemicals. The synthetic diets (20McGrane M.M. deVente J. Yun J. Bloom J. Park E.A. Wynshaw-Boris A. Wagner T. Rottman F.M. Hanson R.W. J. Biol. Chem. 1988; 263: 11443-11451Abstract Full Text PDF PubMed Google Scholar) used in these studies were purchased from Nutritional Biochemical Corp. (Cleveland, OH). Human recombinant insulin was obtained from Calbiochem. The PEPCK-C probe used was a 1.1-kilobase PstI fragment from the 3′-end of the PEPCK-C cDNA, as described previously (20McGrane M.M. deVente J. Yun J. Bloom J. Park E.A. Wynshaw-Boris A. Wagner T. Rottman F.M. Hanson R.W. J. Biol. Chem. 1988; 263: 11443-11451Abstract Full Text PDF PubMed Google Scholar). RNA was normalized with a 752-nt SacI fragment from the mouse 18 S rRNA cDNA as described by Oberbaumer (21Oberbaumer I. Nucleic Acids. 1992; 20: 671-677Crossref PubMed Scopus (43) Google Scholar). The 700-base pair SmaI cDNA hGx fragment (22Screpanti I. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. Sellitto C. Scarpa S. Bellavia D. Lattanzio G. Bistoni F. Frati L. Cortese R. Gulino A. Ciliberto G. Costantini F. Poli V. EMBO. 1995; 14: 1932-1941Crossref PubMed Scopus (375) Google Scholar) was used to screen the hGx transgenic mice by Southern blotting. All probes were synthesized using [α32P]dATP and a Strip-EZ DNA random-primed StripAble DNA probe synthesis and removal kit from Ambion (Austin, Texas). A mutation was introduced into the specific protein-binding domains of the PEPCK-C gene promoter (−490 to +72) or (−2000 to +72) using a variation of the method of Kunkel (23Kunkel T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar) as described by Liu et al. (24Liu J. Roesler W.J. Hanson R.W. Biotechniques. 1990; 9: 738-741PubMed Google Scholar). The AF2 mutation replaced the entire sequence of the AF2 element (−414 to −405) from the wild type sequence of 5′-ACCGATATCA-3′ to a nonspecific sequence of 5′-TGGTGTTTTG-3′. The mutated AF2 or the WT promoter fragment was used to construct the transgene for AF2 or WT control lines, respectively. Briefly, either the −2000-base pairSacI-BglII or the −490XbaI-BglII region of the cytosolic form of the PEPCK-C gene promoter from the rat was ligated to a 2.2-kilobaseBamHI-EcoRV fragment containing the human growth hormone (hGx) reporter gene. The hGx reporter gene has a frameshift mutation in exon 5 that affects the 55 carboxyl-terminal amino acids and thus does not produce active growth hormone (25Idzerda R.L. Behringer R.R. Theisen M. Huggenvik J.I. McKnight G.S. Brinster R.L. Mol. Cell. Biol. 1989; 9: 5154-5162Crossref PubMed Scopus (50) Google Scholar). Fig. 1 presents a diagram of the genes used in this study and the location of the mutated AF2 element. Recombinant DNA used for microinjection was separated from plasmid sequences by digestion withXbaI for WT-2000/hGx and AF2–2000/hGx orXbaI-BglII for WT-490/hGx and AF2–490/hGx, followed by electrophoresis. DNA was electroeluted from the agarose gel and then used for microinjection into fertilized mouse eggs. The procedure for the generation of transgenic mice has been described previously (26Patel Y.M. Yun J.S. Liu J. McGrane M.M. Hanson R.W. J. Biol. Chem. 1994; 269: 5619-5628Abstract Full Text PDF PubMed Google Scholar). Briefly, fertilized eggs were flushed from the oviducts of super-ovulated C57BL/6× SJL mice. 2 μl of DNA solution (2 ng/μl) was injected into the male pronuclei of the fertilized mouse eggs. Viable embryos were re-implanted into the oviducts of pseudo-pregnant mice as described previously (26Patel Y.M. Yun J.S. Liu J. McGrane M.M. Hanson R.W. J. Biol. Chem. 1994; 269: 5619-5628Abstract Full Text PDF PubMed Google Scholar). Transgenic mice were identified by genomic DNA analysis of tail samples taken at 4 weeks of age. Briefly, small segments of mouse tail were placed in a lysis buffer containing 50 mm KCl, 10 mm Tris-HCL, pH 8.3, 2.5 mm MgCl2, 0.01% gelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 24 μg of proteinase K overnight at 55 °C. 1 μl of this solution, containing the lysed tail cells, was used in a polymerase chain reaction to amplify the hGx gene. The primers used were hGH1, 5′-TTGCAGCTAGGTGAGCT-3′; and hGH2, 5′-CTTTGGCCGTGGGGAGTG-3′. Six lines were generated for AF2 2000, four lines were generated for each of WT 390 and AF2 490 transgenes, and one line was generated for the WT 2000. All lines of transgenic mice demonstrated the tissue-specific pattern of expression characteristic of the endogenous PEPCK-C gene, with highest expression levels in liver and expression in the kidney ∼20% that of hepatic expression. The transgenic mice were normal in body weight and showed no evidence of hepatomegaly or other disorders. Male mice heterozygous for the transgene were back-crossed to C57BL/6× SJL WT females. Only mice heterozygous for the transgene were used for analysis. There were no gender differences noted in the level of expression of hGx mRNA, so both male and female mice heterozygous for the transgene were used. Adult animals were 2–4 months of age at the time of study; they were given free access to water and were fed standard laboratory chow, unless otherwise specified. Adult animals were killed between 9:00 and 11:00 a.m. Mice were injected intraperitoneally with 35 mg of Bt2cAMP per kg of body weight and 30 mg of theophylline per kg of body weight in 150 μl of saline or not injected (controls). The animals were killed 2 h later, and their livers and kidneys were isolated, frozen in liquid nitrogen, and stored for analysis at −70 °C. Animals were made diabetic by a single intraperitoneal injection of streptozotocin (200 mg/kg body weight). Mice were fasted overnight prior to injection and fed 2 h later. Diabetes was confirmed by measuring the concentration of blood glucose (tail vein) using glucose dipsticks and an ENCORE® glucometer (VWR Scientific). Animals treated with insulin received 1 unit per 30 grams of body weight. All diabetic animals had blood glucose concentrations of more than 300 mg/dl. To determine the effect of glucocorticoids on the PEPCK-C gene promoter in the transgenic mice, the animals were injected with 1 μm dexamethasone, and their livers and kidneys were removed 5 h later. The tissues were frozen in liquid nitrogen and stored at −70 °C. For dietary studies, the mice were fed either a high carbohydrate diet that contained 81.5% glucose, 12.2% casein, 0.3%dl-methionine, 4% cottonseed oil, 2% brewers' yeast, and 1% mineral mix with vitamins for 1 week or a high fat/carbohydrate-free diet that contained 64% casein, 22% nutritive fiber, 11% vegetable oil, 2% brewers' yeast, and 1% mineral mix plus vitamins. (27McGrane M.M. Yun J.S. Moorman A.F. Lamers W.H. Hendrick G.K. Arafah B.M. Park E.A. Wagner T.E. Hanson R.W. J. Biol. Chem. 1990; 265: 22371-22379Abstract Full Text PDF PubMed Google Scholar). As controls, mice were fed normal laboratory chowad libitum. The animals were anesthetized, and the liver and kidneys were removed, frozen in liquid nitrogen, and stored at −70 °C for subsequent analysis. Total RNA was isolated from the liver using a QuickPrep total RNA extraction kit from Amersham Pharmacia Biotech.Total RNA was isolated from the kidneys using the modified acid-phenol guanidine thiocyanate procedure described previously (28Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). Northern blot analysis was performed as described previously (29Eisenberger C.L. Nechushtan H. Cohen H. Shanti M. Reshef L. Mol. Cell. Biol. 1992; 12: 1396-1403Crossref PubMed Scopus (48) Google Scholar) using 20 μg of total RNA. Gels were blotted to GeneScreen Plus and hybridized with PEPCK-C cDNA or hGx cDNA probes at 42 °C in hybridization buffer containing 50% formamide (deionized), 1% SDS, 1 msodium chloride, 110 μg/ml denatured salmon sperm, and 10% dextran sulfate. The filters were washed at 55 °C in 2× SSC and 0.1% SDS for 20 min and 1× SSC, 0.1% SDS for 20 min. The relative radioactivity was determined using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The concentration of PEPCK-C mRNA and hGx mRNA were expressed relative to the concentration of 18 S ribosomal RNA to control for differences in loading of the RNA. Male mice heterozygous for the transgene were back-crossed to C57BL/6× SJL non-transgenic females. On day 19 of gestation, the livers and kidneys were removed from fetal mice, and the RNA was extracted and analyzed by Northern analysis using an hGx cDNA probe. The concentration of hGx mRNA was expressed relative to 18 S ribosomal RNA. The gene for PEPCK-C is not expressed in the liver before birth (30Ballard F.J. Hanson R.W. Biochem. J. 1967; 104: 866-871Crossref PubMed Scopus (304) Google Scholar), partly because of repression by insulin. Because the AF2 site has been implicated in the insulin regulation of PEPCK-C gene transcription (18O'Brien R.M. Lucas P.C. Forest C.D. Magnunson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google Scholar), we analyzed the role of the AF2 element in the developmental expression of the PEPCK-C gene in the liver and kidney. For this purpose, chimeric genes were constructed that contained either the wild type promoter (WT) or a gene with a deletion for AF2 element (AF2) in the −2000 or −490 region of the PEPCK-C gene promoter that was ligated to the hGx structural gene (Fig.1). These chimeric genes were used to generate transgenic animals. Heterozygous, transgenic males were then mated to C57BL/6× SJL non-transgenic females. Each litter consisted of non-transgenic controls and heterozygous transgenic littermates. The livers and kidneys of 19-day-old fetuses were collected from each line, and the level of hepatic and kidney hGx mRNA transcribed from the −2000 and −490 segments of the PEPCK-C gene promoter, with and without the AF2 regulatory element, were measured by Northern hybridization (Fig. 2). Chimeric genes containing the WT-2000, AF2–2000, and WT-490 promoters expressed very little hGx mRNA in the liver and kidney before birth. The AF2–490/hGx gene, however, had an unexpected high level of expression before birth in both the kidney and liver compared with WT (*,p < 0.05). This result demonstrates the importance of the AF2 element in the PEPCK-C gene promoter for the correct developmental expression of this gene before birth. The gene for PEPCK-C is not expressed in the liver before birth (30Ballard F.J. Hanson R.W. Biochem. J. 1967; 104: 866-871Crossref PubMed Scopus (304) Google Scholar). The high levels of glucose from the maternal circulation repress expression of the gene in the fetal liver and the subsequent production of insulin by the fetal pancreas. At birth, there is a decrease in the concentration of glucose in the blood, accompanied by a decrease in secretion of insulin and an increase in hepatic cAMP (31Girard J.R. Cuendet G.S. Marliss E.B. Kervran A. Rieutort M. Assan R. J. Clin. Invest. 1973; 52: 3190-3200Crossref PubMed Scopus (258) Google Scholar); this initiates transcription of the gene for PEPCK-C in the liver. In the absence of the AF2 site, we noted a premature appearance of hGx mRNA in the livers of 19-day-old fetal mice. This occurred only when the AF2 mutation was in the PEPCK-C gene promoter from −490 to +73, suggesting that a region of the promoter between −490 and −2000 contains an element required to suppress the premature expression of PEPCK-C in the fetal liver. More work will be required to identify this region of the PEPCK-C gene promoter. To dissect the role of AF2 in the response of the PEPCK-C gene to diabetes and to the subsequent administration of insulin, the animals were made diabetic by injection of streptozotocin (the blood glucose concentration was in excess of 300 mg/dl in these mice). In all animals, the endogenous gene for PEPCK-C responded to diabetes as expected, i.e. the mRNA increased in diabetes and decreased when insulin was injected (Fig. 3 B). The WT-2000/hGx transgene was induced 1.7-fold by diabetes as shown by an increased hepatic hGx mRNA; insulin reduced its expression by half when administered for 8 h. The difference in the time course of response to insulin may be attributable to differences in half-life between PEPCK-C and hGx mRNAs. Transcription of the AF2–2000/hGx transgene in the liver was not induced by diabetes but was inhibited 50% by insulin treatment (Fig. 3 A) (**, p< 0.05). This finding strongly suggests that AF2 is necessary for the induction of PEPCK-C by diabetes but that it is not involved in the response of the PEPCK-C gene promoter to insulin. The AF2 regulatory element in the PEPCK-C gene promoter contains a glucocorticoid-binding site, as well as an insulin regulatory element. Because glucocorticoids are required for the increase in PEPCK-C gene transcription that occurs during diabetes (12Friedman J.E. Yun J.S. Patel Y.M. McGrane M.M. Hanson R.W. J. Biol. Chem. 1993; 268: 12952-12957Abstract Full Text PDF PubMed Google Scholar), we next determined the response of the AF2–2000/hGx transgene to glucocorticoids. Dexamethasone (1 μm) was administered to both WT-2000/hGx and AF2–2000/hGx transgenic mice; the animals were killed 5 h later. The administration of dexamethasone had no effect on the concentration of mRNA for hGx in the livers of WT-2000/hGx and AF2–2000/hGx transgenic mice (Fig.4 A). The lack of response of the transgenes to dexamethasone was expected, because it is well established that glucocorticoids cause an increase in the secretion of insulin (11Gunn J.M. Hanson R.W. Meyuhas O. Reshef L. Ballard F.J. Biochem. J. 1975; 150: 195-203Crossref PubMed Scopus (59) Google Scholar); this insulin secretion would, in turn, prevent the induction of transcription from the PEPCK-C gene promoter (12Friedman J.E. Yun J.S. Patel Y.M. McGrane M.M. Hanson R.W. J. Biol. Chem. 1993; 268: 12952-12957Abstract Full Text PDF PubMed Google Scholar). For this reason, we determined the response to glucocorticoids of both transgenes in the kidney. There was a 3-fold induction of hGx mRNA in the kidneys of AF2–2000/hGx transgenic mice, accompanied by a 5.5-fold induction of hGx mRNA in the kidneys of WT-2000/hGx mice (Fig. 4 B) (*, p < 0.05). Thus the loss of one accessory factor 2-binding site resulted in a 60% reduction in the glucocorticoid response of the transgene. These data are consistent with the in vitro studies of Scott et al. (15Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 261: 31909-31914Abstract Full Text Full Text PDF Scopus (69) Google Scholar,32Scott D.K. Stromstedt P.-E. Wang J.-C. Granner D.K. Mol. Endocrinol. 1998; 12: 482-491Crossref PubMed Scopus (65) Google Scholar) showing a similar reduction in PEPCK-C gene transcription when one accessory factor 2 element of the GRU was deleted. We have shown that the AF2-binding site in the PEPCK-C gene promoter is critical for the regulation of PEPCK-C mRNA by glucocorticoids. The AF2-binding site has an overlapping GRE and a putative IRS that can potentially bind C/EBPβ and HNF-4; both can then act as accessory factors for the glucocorticoid response. In animals with a deletion in the gene for C/EBPβ, the glucocorticoid response is totally blocked. 2C. M. Croniger and R. W. Hanson, unpublished results. C/EBPβ binds to the PEPCK-C gene promoter through the cAMP-response element-1 and P3(I) regions, both of which are downstream from the AF2-binding site and GRU. Yamada et al. (33Yamada K. Duong D.T. Scott D.K. Wang J.-C. Granner D.K. J. Biol. Chem. 1999; 274: 5580-5587Google Scholar) have determined that C/EBPβ is an accessory factor for the glucocorticoid response by acting via the cAMP-response element region in the PEPCK-C gene promoter. This suggests a complex mechanism for the regulation of PEPCK-C by glucocorticoids. Recently Leahy et al. (34Leahy P. Crawford D.R. Grossman G. Chaudhry A. Gronostajski R. Hanson R.W. J. Biol. Chem. 1999; 274: 8813-8822Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) have stressed the role of CREB-binding protein in coordinating the regulation of the PEPCK-C promoter. CREB-binding protein can bind both steroids and C/EBPβ, which interact with the promoter. It is likely that the AF2 region interacts with the GR or with a protein that binds the GR. Here we note that the lack of an AF2 site results in a 60% reduction in the increase in hGx mRNA when stimulated with glucocorticoids. Thus, the absence of a binding site for AF2 may disrupt the ability to form the needed complex with CREB-binding protein and C/EBPβ. Feeding mice a diet high in carbohydrate (or a high fat/carbohydrate-free diet) markedly decreases (or increases) the transcription of the gene for PEPCK-C in the liver (20McGrane M.M. deVente J. Yun J. Bloom J. Park E.A. Wynshaw-Boris A. Wagner T. Rottman F.M. Hanson R.W. J. Biol. Chem. 1988; 263: 11443-11451Abstract Full Text PDF PubMed Google Scholar). This effect is presumably because of the secretion of insulin by the pancreas that in turn represses hepatic PEPCK-C gene transcription. To determine whether the AF2 regulatory element in the PEPCK-C gene promoter is involved in the response of the gene to dietary carbohydrate, WT-2000/hGx and AF2–2000/hGx transgenic mice were fed a high carbohydrate diet for 1 week. This diet did not significantly alter the blood glucose concentration of the animals. Control mice fed a normal lab chow diet had a blood glucose level of 124 mg/dl ± 6.4, whereas mice fed a high carbohydrate diet had a blood glucose level of 114 mg/dl ± 12.9. The high carbohydrate diet dramatically decreased the level of hGx mRNA in the livers of both the WT-2000/hGx and AF2–2000/hGx transgenic mice. This demonstrates that the AF2 site in the PEPCK-C gene promoter is not required for the transcriptional response of the promoter to dietary carbohydrate and presumably to insulin (Fig.5). Mice that were fed the high carbohydrate diet for 1 week were then injected with Bt2cAMP and theophylline to stimulate transcription from the PEPCK-C gene promoter. There was no difference in the response to Bt2cAMP between the WT-2000/hGx and AF2–2000/hGx transgenes, indicating that the AF2 site is not required for the effect of cAMP on PEPCK-C gene transcription in the liver. Finally, the mice were fed a diet in which the protein content was not changed, but the carbohydrate was replaced with fat (high fat/carbohydrate-free diet). There was a robust 3.4-fold stimulation in the level of hGx mRNA in the livers of the WT-2000/hGx mice as compared with hGx mRNA in the livers of the same lines of mice fed a control diet (Fig. 5). In contrast, the AF2–2000/hGx transgenic mice had a 50% increase in response to the high fat/carbohydrate-free diet. If a high carbohydrate diet inhibits transcription from the PEPCK-C gene promoter via the action of insulin then the AF2 site is clearly not required for the insulin effect. It is possible that insulin inhibits PEPCK-C gene transcription via another site on the promoter. In this regard, we have recently found that sterol regulatory element-binding protein 1c (SREBP-1c) is an intermediate in the action of insulin on transcription of the PEPCK-C gene in hepatocytes. 3K. Chakravarty, P. Ferre, F. Foufelle, and R. W. Hanson, unpublished data. Hasty et al. (35Hasty A.H. Shimano H. Yahagi N. Amemiya-Kudo M. Perrey S. Yoshikawa T. Osuga J. Okazaki H. Tamura Y. Iizuka Y. Shionoiri F. Ohashi K. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. J. Biol. Chem. 2000; 275: 31069-31077Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) have reported that the addition of 5.5 to 25 mm glucose to hepatocytes in culture increases the concentration of mature SREBP-1c in the nucleus, most likely by inducing the level of mRNA for the transcription factor. In addition, reporter gene analysis demonstrated a glucose-induced increase in transcription from the SREBP-1c gene promoter (35Hasty A.H. Shimano H. Yahagi N. Amemiya-Kudo M. Perrey S. Yoshikawa T. Osuga J. Okazaki H. Tamura Y. Iizuka Y. Shionoiri F. Ohashi K. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. J. Biol. Chem. 2000; 275: 31069-31077Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). It is thus possible that dietary glucose, which induces insulin secretion in the mice, increased the level of SREBP-1c in the liver leading to a repression of PEPCK-C gene transcription. If so, this must occur at a site on the PEPCK-C gene promoter distinct from the AF2-binding site. In the absence of the AF2 site, there is a blunted transcriptional response of the PEPCK-C gene promoter to a high fat/carbohydrate-free diet. This suggests that this site is needed to achieve the full stimulation of transcription when the concentration of insulin is decreased by feeding the mice a high fat/carbohydrate-free diet. This is very similar to the response of the AF2–2000/hGx transgene to diabetes where the stimulatory response is blunted (see Fig. 3). We assume that the increase in transcription of the hepatic AF2–2000/hGx transgene caused by feeding a high fat/carbohydrate-free diet is partly because of the stimulatory effect of glucocorticoids on the PEPCK-C gene promoter. Alternatively, the AF2 site is adjacent to a PPAR-binding domain that maps at −451 to −439 (36Tontonoz P. Hu E. Devine J. Beale E.G. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 351-357Crossref PubMed Google Scholar). A diet high in fat increases the concentration of PPARα in the liver (37Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (858) Google Scholar). Therefore, in the absence of the AF2 site, PPARα might be less effective in inducing PEPCK-C gene transcription. Clearly, further work is needed to establish the exact effect of a high fat/carbohydrate-free diet on the control of PEPCK-C gene transcription. We are indebted to Dr. Ifeanyi J. Arinze for critically reading the manuscript and to Jianqi Yang for help in preparing the figures." @default.
• W2022894562 created "2016-06-24" @default.
• W2022894562 creator A5001449811 @default.
• W2022894562 creator A5018361141 @default.
• W2022894562 creator A5019042963 @default.
• W2022894562 creator A5046453964 @default.
• W2022894562 creator A5057165756 @default.
• W2022894562 creator A5058952343 @default.
• W2022894562 creator A5064905794 @default.
• W2022894562 date "2001-06-01" @default.
• W2022894562 modified "2023-09-27" @default.
• W2022894562 title "The Use of Transgenic Mice to Analyze the Role of Accessory Factor Two in the Regulation of Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription during Diabetes" @default.
• W2022894562 cites W1527396710 @default.
• W2022894562 cites W1553229669 @default.
• W2022894562 cites W1563828822 @default.
• W2022894562 cites W1575011694 @default.
• W2022894562 cites W1586437812 @default.
• W2022894562 cites W1588631487 @default.
• W2022894562 cites W1680099832 @default.
• W2022894562 cites W1915827411 @default.
• W2022894562 cites W1972835861 @default.
• W2022894562 cites W1981071038 @default.
• W2022894562 cites W199241068 @default.
• W2022894562 cites W2013922414 @default.
• W2022894562 cites W2016298359 @default.
• W2022894562 cites W2021791229 @default.
• W2022894562 cites W2036931150 @default.
• W2022894562 cites W2056481567 @default.
• W2022894562 cites W2059911808 @default.
• W2022894562 cites W2061793296 @default.
• W2022894562 cites W2103673771 @default.
• W2022894562 cites W2107749241 @default.
• W2022894562 cites W2120432009 @default.
• W2022894562 cites W2133208346 @default.
• W2022894562 cites W2141771347 @default.
• W2022894562 cites W2146053609 @default.
• W2022894562 cites W2150413922 @default.
• W2022894562 cites W2152043023 @default.
• W2022894562 cites W2170571176 @default.
• W2022894562 cites W321874077 @default.
• W2022894562 cites W4294216491 @default.
• W2022894562 doi "https://doi.org/10.1074/jbc.m102422200" @default.
• W2022894562 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11309401" @default.
• W2022894562 hasPublicationYear "2001" @default.
• W2022894562 type Work @default.
• W2022894562 sameAs 2022894562 @default.
• W2022894562 citedByCount "22" @default.
• W2022894562 countsByYear W20228945622015 @default.
• W2022894562 countsByYear W20228945622016 @default.
• W2022894562 countsByYear W20228945622018 @default.
• W2022894562 countsByYear W20228945622020 @default.
• W2022894562 countsByYear W20228945622021 @default.
• W2022894562 crossrefType "journal-article" @default.
• W2022894562 hasAuthorship W2022894562A5001449811 @default.
• W2022894562 hasAuthorship W2022894562A5018361141 @default.
• W2022894562 hasAuthorship W2022894562A5019042963 @default.
• W2022894562 hasAuthorship W2022894562A5046453964 @default.
• W2022894562 hasAuthorship W2022894562A5057165756 @default.
• W2022894562 hasAuthorship W2022894562A5058952343 @default.
• W2022894562 hasAuthorship W2022894562A5064905794 @default.
• W2022894562 hasBestOaLocation W20228945621 @default.
• W2022894562 hasConcept C102230213 @default.
• W2022894562 hasConcept C104317684 @default.
• W2022894562 hasConcept C118716 @default.
• W2022894562 hasConcept C138885662 @default.
• W2022894562 hasConcept C141035611 @default.
• W2022894562 hasConcept C143937172 @default.
• W2022894562 hasConcept C179926584 @default.
• W2022894562 hasConcept C181199279 @default.
• W2022894562 hasConcept C41895202 @default.
• W2022894562 hasConcept C54355233 @default.
• W2022894562 hasConcept C55493867 @default.
• W2022894562 hasConcept C86339819 @default.
• W2022894562 hasConcept C86803240 @default.
• W2022894562 hasConcept C95444343 @default.
• W2022894562 hasConceptScore W2022894562C102230213 @default.
• W2022894562 hasConceptScore W2022894562C104317684 @default.
• W2022894562 hasConceptScore W2022894562C118716 @default.
• W2022894562 hasConceptScore W2022894562C138885662 @default.
• W2022894562 hasConceptScore W2022894562C141035611 @default.
• W2022894562 hasConceptScore W2022894562C143937172 @default.
• W2022894562 hasConceptScore W2022894562C179926584 @default.
• W2022894562 hasConceptScore W2022894562C181199279 @default.
• W2022894562 hasConceptScore W2022894562C41895202 @default.
• W2022894562 hasConceptScore W2022894562C54355233 @default.
• W2022894562 hasConceptScore W2022894562C55493867 @default.
• W2022894562 hasConceptScore W2022894562C86339819 @default.
• W2022894562 hasConceptScore W2022894562C86803240 @default.
• W2022894562 hasConceptScore W2022894562C95444343 @default.
• W2022894562 hasIssue "25" @default.
• W2022894562 hasLocation W20228945621 @default.
• W2022894562 hasOpenAccess W2022894562 @default.
• W2022894562 hasPrimaryLocation W20228945621 @default.
• W2022894562 hasRelatedWork W1553229669 @default.
• W2022894562 hasRelatedWork W2036244858 @default.
• W2022894562 hasRelatedWork W2042829340 @default.
• W2022894562 hasRelatedWork W2064584857 @default.
• W2022894562 hasRelatedWork W2069518563 @default.

• next