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D K Granner

Publications and source records attributed to D K Granner.

At least 127 records · Page 7Linked to original sources
PubMed1988

Reciprocal regulation of gene transcription by insulin. Inhibition of the phosphoenolpyruvate carboxykinase gene and stimulation of gene 33 in a single cell type.

Two H4IIE hepatoma cell genes, phosphoenolpyruvate carboxykinase (PEPCK) and gene 33 (g33), are reciprocally regulated by insulin. Quantitation of mRNAPEPCK and mRNAg33 in total RNA isolated from cells treated with insulin showed a 7-fold increase in mRNAg33 amount and a 3-fold decrease of mRNAPEPCK. The cAMP analog 8-(4-chlorophenylthio)-cAMP induced mRNAPEPCK but had no effect on mRNAg33. The responses to various insulins and related molecules showed that the insulin receptor mediates the effects of physiologic concentrations of insulin on each of these genes. This inverse pattern of regulation by insulin was further characterized by determining the transcription rates of both genes in nuclei isolated at various times after the addition of insulin and 8-(4-chlorophenylthio)-cAMP to H4IIE cells. Insulin increased the rate of synthesis of mRNAg33 from 35 to 354 ppm and decreased the synthesis of mRNAPEPCK from 1175 to 109 ppm. These effects of insulin occurred rapidly and reached their maxima by 60 min. In both cases, greater effects were observed as insulin concentrations were increased from 10(-12) to 10(-8) M. Although the effects of insulin were concentration-dependent for both genes, the PEPCK gene was significantly more sensitive to low concentrations of insulin than was gene 33. The reciprocal effects of insulin on the synthesis of mRNAPEPCK and mRNAg33 in H4IIE cells provide a means of investigating how a hormone can exert opposing effects on two genes in the same cell.

Animals↗
PubMed1988

3-Aminobenzamide inhibits poly(ADP ribose) synthetase activity and induces phosphoenolpyruvate carboxykinase (GTP) in H4IIE hepatoma cells.

The purpose of this study was to determine whether changes in ADP-ribosylation affect expression of the gene encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in H4IIE hepatoma cells. Treatment with 3-aminobenzamide, a specific inhibitor of poly(ADP ribose) synthetase, caused an 89% decrease of ADP-ribosylation in isolated nuclei, and resulted in a two- to threefold induction of immunoassayable PEPCK in cultured cells. In contrast, the structurally related compound p-aminobenzoic acid had no significant effect on either process. In vivo labeling of proteins with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed that the induction of immunoreactive PEPCK by 3-aminobenzamide was due to a selective increase in the synthesis of the protein. The specific induction of PEPCK synthesis by 3-aminobenzamide was accounted for by a twofold increase of mRNAPEPCK which reached its maximal value 4 h after the addition of 3-aminobenzamide and returned to the basal level by 10 h. A possible role of ADP-ribosylation in cAMP or glucocorticoid induction of PEPCK was investigated in experiments in which H4IIE cells were treated with combinations of 3-aminobenzamide and either dexamethasone or a cAMP analog. In each case the effects on PEPCK induction were additive, indicating that glucocorticoids and cAMP induce PEPCK by pathways different from that used by 3-aminobenzamide.

Adenosine Diphosphate Ribose↗
PubMed1988

Regulation of phosphoenolpyruvate carboxykinase gene transcription by insulin and cAMP: reciprocal actions on initiation and elongation.

Nuclei isolated from H4IIE rat hepatoma cells were used in an in vitro run-on assay, with probes directed against various regions of the phosphoenolpyruvate carboxykinase [GTP: oxaloacetate carboxy-lyase (transphosphorylating); EC 4.1.1.32] gene, to analyze whether transcription proceeds uniformly across this gene in response to insulin and cAMP treatment. Fewer polymerase II complexes were associated with the phosphoenolpyruvate carboxykinase gene after insulin treatment, as compared with cAMP-treated cells, but they were distributed uniformly, so insulin does not block transcription at a discrete site, nor does it cause gradual, but progressive, premature termination. The phosphoenolpyruvate carboxykinase primary transcript was synthesized at a rate of about 2500 nucleotides per min in cAMP-treated cells and about 1000 nucleotides per min in insulin-treated cells. Thus insulin retards transcript elongation in comparison with cAMP, but this action does not account for the total effect insulin has on transcription. After insulin treatment, few, if any, nascent transcripts are associated with the first 69 nucleotides of the gene, whereas in cAMP-treated cells the opposite is true. These observations lead us to suggest that both insulin and cAMP exert their primary effects directly at the level of transcription initiation, but in opposite ways.

Animals↗
PubMed1988

Location and characterization of two widely separated glucocorticoid response elements in the phosphoenolpyruvate carboxykinase gene.

Chimeric genes were constructed by fusion of various regions of the 5'-flanking sequence from the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene to the chloramphenicol acetyltransferase-coding sequence and to simian virus 40 splice and polyadenylation sequences. These were used to demonstrate that two glucocorticoid regulatory elements (GREs) combine to confer glucocorticoid responsiveness upon the PEPCK gene in H4IIE hepatoma cells. Both elements, a distal one whose 5' boundary is located between -1264 and -1111 base pairs and a proximal one located between -468 and -420 base pairs relative to the transcription initiation site, act independently, in various positions and orientations, and upon the heterologous thymidine kinase promoter. Each element accounts for half of the maximal response of the chimeric genes. Therefore, two widely separated enhancerlike elements contribute equally to the response of the PEPCK gene to glucocorticoid hormones. Neither of the PEPCK GREs contains the TGTTCT consensus sequence associated with most other GREs.

Acetyltransferases↗
PubMed1988

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.

Animals↗
PubMed1988

Separation of topoisomerase I activity from the regulatory subunit of type II cyclic adenosine monophosphate-dependent protein kinase.

The cAMP-containing phosphoform of the regulatory subunit (RII) of type II cAMP-dependent protein kinase from rat liver has been reported to have intrinsic DNA topoisomerase I activity. We found that highly purified RII preparations from eight different sources, including rat liver, contained no detectable topoisomerase I activity. Topoisomerase I exhibited an overlapping peak of activity with RII when rat liver extracts were fractionated by diethylaminoethyl-cellulose chromatography. Topoisomerase I activity was separated from RII by subsequent cAMP affinity chromatography. The results indicate that the regulatory subunit of cAMP-dependent protein kinase does not contain intrinsic topoisomerase I activity.

Adenosine Triphosphate↗
PubMed1987

Multihormonal regulation of phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase fusion genes. Insulin's effects oppose those of cAMP and dexamethasone.

The multihormonal regulation of phosphoenolpyruvate carboxykinase (PEPCK) was studied using chimeric genes composed of various regions of the PEPCK gene promoter region fused to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. These constructions, transfected into H4IIE hepatoma cells, are regulated like the endogenous PEPCK gene: dexamethasone and cAMP both stimulate PEPCK-CAT gene expression and their effects are additive; insulin inhibits the individual or combined effects of these stimulatory agents; and insulin inhibits dexamethasone-stimulated PEPCK-CAT fusion gene expression in a concentration-dependent fashion that is half-maximal at 10(-11) M. The induction by dexamethasone and the inhibition by insulin is specific for the DNA sequences that flank the 5' end of the PEPCK gene because similar effects were not observed for a plasmid in which the promoter and enhancer sequences of simian virus 40 (SV40) are fused to CAT. These results imply that the DNA adjacent to the transcription start site of the PEPCK gene contains the cis-acting hormone response elements responsible for the multihormonal regulation of this gene, including the insulin response.

Acetyltransferases↗
PubMed1987

The inhibition of phosphoenolpyruvate carboxykinase (guanosine triphosphate) gene expression by insulin is not mediated by protein kinase C.

The role protein kinase C plays in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin and phorbol esters was studied in H4IIE hepatoma cells (ATCC CRL 1548). The combined effects of phorbol 12-myristate 13-acetate (PMA) and insulin on the suppression of mRNA coding for PEPCK (mRNAPEPCK) synthesis were additive. A potent inhibitor of both cyclic nucleotide-dependent protein kinases and protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, inhibited the cAMP and PMA-mediated regulation of mRNAPEPCK synthesis, but did not affect the action of insulin. Desensitization of the protein kinase C pathway by exposure to PMA for 16 h abolished the subsequent action of the phorbol ester, but did not affect insulin- or cAMP-mediated regulation of PEPCK gene expression. We conclude that insulin suppresses PEPCK gene expression independently from the protein kinase C-mediated pathway used by phorbol esters.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

Regulation of phosphoenolpyruvate carboxykinase gene transcription in H4IIE hepatoma cells: evidence for a primary role of the catalytic subunit of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

The purpose of these studies was to determine whether the catalytic subunit of cAMP-dependent protein kinase is involved in the regulation of P-enolpyruvate carboxykinase (PEPCK) gene transcription. Cyclic AMP analog pairs that preferentially stimulate either type I or type II protein kinase in a synergistic manner were used to compare regulation of mRNAPEPCK synthesis in H4IIE rat hepatoma cells with protein kinase activation in vitro. Type II protein kinase is predominant in H4IIE cells and analog pairs directed toward this isozyme resulted in a synergistic increase of mRNAPEPCK that was due to a corresponding enhancement of PEPCK gene transcription. When compared to a single analog the addition of a type II-directed analog pair reduced the total analog concentration required for maximal induction of transcription by about 30-fold. H4IIE cells have a small amount of type I kinase; pairs specific for this form of the enzyme were also effective, but to a lesser extent than those for the type II kinase. (Rp)-cAMPS, a cyclic nucleotide-dependent protein kinase antagonist, inhibited the agonist-induced increase of mRNAPEPCK in a concentration-dependent manner. The results indicate that the activation of PEPCK gene transcription by cAMP in H4IIE cells is mediated by cAMP-dependent protein kinase. Although the type II isozyme is primarily responsible, type I is also effective. These isozymes have identical catalytic subunits, hence this component presumably mediates the cAMP effect.

Animals↗
PubMed1986

The effect of phorbol esters and diacylglycerol on expression of the phosphoenolpyruvate carboxykinase (GTP) gene in rat hepatoma H4IIE cells.

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on expression of the P-enolpyruvate carboxykinase gene was studied in rat hepatoma H4IIE cells. Like insulin, PMA provokes a concentration and time-dependent decrease of mRNA coding for that enzyme that is due to an inhibition of P-enolpyruvate carboxykinase gene transcription. This effect of PMA is rapid, reversible, specific for phorbol esters known to be active in other systems, and it does not require on-going protein synthesis. PMA overrides the stimulatory effects cAMP and glucocorticoid analogs have on the transcription of this gene. A synthetic diacylglycerol, sn-1,2-dioctanoylglycerol, also inhibits P-enolpyruvate carboxykinase gene transcription. These effects of PMA and synthetic diacylglycerol are specific, since neither affected total mRNA synthesis. We conclude that diacylglycerol and phorbol esters, specific stimulators of protein kinase C, inhibit the transcription of P-enolpyruvate carboxykinase gene in H4IIE cells. The findings support the hypothesis that diacylglycerols generated in the plasma membrane can act as an intracellular signal that regulates specific gene expression.

Animals↗
PubMed1986

Insulin decreases H4IIE cell PEPCK mRNA by a mechanism that does not involve cAMP.

Insulin is thought to influence some metabolic events by decreasing the intracellular concentration of cyclic AMP (cAMP). To test whether this explains the repression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) by insulin we measured intracellular cAMP, cAMP-dependent protein kinase, mRNAPEPCK, and PEPCK gene transcription in cultured Reuber H4IIE hepatoma cells treated with forskolin with and without insulin. In untreated cells, the concentration of cAMP was 2.9 pmol/mg of protein. Forskolin at 1, 10, and 50 microM increased the level of cAMP to 9.2, 35.8, and 115 pmol/mg of protein, respectively; 5 nM insulin had no significant effect on these cAMP concentrations. In untreated cells, the activity ratio of cAMP-dependent protein kinase was 0.43, and 50 microM forskolin increased this to 0.96; insulin had no effect on this ratio at times from 15-180 min. In untreated cells mRNAPEPCK bound 15 cpm of a 32P-labeled cDNA probe per microgram of total cellular RNA. Forskolin, at 1, 10, and 50 microM increased this to 48, 96, and 115 cpm/microgram RNA. Insulin (5 nM), in combination with 0, 1, 10, and 50 microM forskolin, decreased the concentration of mRNAPEPCK to 5, 8, 23, and 29 cpm/micrograms RNA, respectively. Finally, the rate of transcription of the PEPCK gene was 85, 168, 630, 823, and 884 parts per million (ppm) in H4IIE cells treated for 30 min with 0, 1, 5, 10, and 50 microM forskolin, respectively, while the corresponding rates in the presence of 5 nM insulin were 49, 45, 84, 85, and 136 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene.

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.

Amino Acid Sequence↗
PubMed1985

Iodine-induced thyrotoxicosis in a woman with a multinodular goiter taking levothyroxine.

In a 63-year-old woman with a multinodular goiter who was receiving suppressive therapy with levothyroxine sodium, iodine-induced thyrotoxicosis developed after povidone-iodine was applied to the surface of a granulating hip wound. Signs and symptoms of apathetic thyrotoxicosis developed on two occasions, once within a week after exposure of the wound to povidone-iodine soaks and again following repeated Hubbard tank debridement with added povidone-iodine. Iodine-induced thyrotoxicosis was confirmed by markedly elevated serum thyroxine and serum and urine iodine levels. On eliminating the sources of exogenous iodine and inhibiting thyroxine biosynthesis with propylthiouracil, the process was gradually controlled. A year later the patient was taking no medication and was clinically and chemically euthyroid. Apparently, iodine-induced thyrotoxicosis can result from passive diffusion of iodine into autonomous thyroid tissue. Iodine-containing preparations given to patients with multinodular goiters may result in thyrotoxicosis even if thyrotropin is suppressed with exogenous thyroxine.

Female↗