PPAR gamma 2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene.

Phosphoenolpyruvate carboxykinase (PEPCK) is expressed at high levels in liver, kidney, and adipose tissue. This enzyme catalyzes the rate-limiting step in hepatic and renal gluconeogenesis and adipose glyceroneogenesis. The regulatory factors important for adipose expression of the PEPCK gene are not well defined. Previous studies with transgenic mice established that the region between bp -2086 and -888 is required for expression in adipose tissue but not for expression in liver or kidney tissue. We show here that a DNA fragment containing this region can function as an enhancer and direct differentiation-dependent expression of a chloramphenicol acetyltransferase gene from a heterologous promoter in cultured 3T3-F442A preadipocytes and adipocytes. We further demonstrate that the adipocyte-specific transcription factor PPAR gamma 2, previously identified as a regulator of the adipocyte P2 enhancer, binds in a heterodimeric complex with RXR alpha to the PEPCK 5'-flanking region at two sites, termed PCK1 (bp -451 to -439) and PCK2 (bp -999 to -987). Forced expression of PPAR gamma 2 and RXR alpha activates the PEPCK enhancer in non-adipose cells. This activation is potentiated by peroxisome proliferators and fatty acids but not by 9-cis retinoic acid. Mutation of the PPAR gamma 2 binding site (PCK2) abolishes both the activity of the enhancer in adipocytes and its ability to be activated by PPAR gamma 2 and RXR alpha. These results establish a role for PPAR gamma 2 in the adipose expression of the PEPCK gene and suggest that this factor functions as a coordinate regulator of multiple adipocyte-specific genes.

Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene.

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)

Culture at high density increases phosphoenolpyruvate carboxykinase messenger RNA in H4IIEC3 hepatoma cells.

We report that the concentration of phosphoenolpyruvate carboxykinase (PEPCK) mRNA increased 5- to 10-fold when H4IIEC3 rat hepatoma cells were cultured at high compared to low density. The magnitude and direction of this response were mRNA specific, as the mRNAs encoding tyrosine aminotransferase and albumin increased approximately 20%, whereas the mRNAs encoding beta-actin and alpha-tubulin decreased 40% and 20%, respectively. Paracrine or autocrine mechanisms were not responsible for the density effect, since conditioned medium or frequent medium changes had only a modest effect on the abundance of PEPCK mRNA. Culture of H4IIEC3 cells at low density or on collagen promoted a flattened morphology and low PEPCK mRNA levels. At high density, cells assumed a cuboidal shape on both plastic and collagen and expressed high PEPCK mRNA levels. Induction of PEPCK mRNA by high density culture did not involve increased intracellular cAMP, since treatment with 8-(4-chlorophenylthio)-cAMP was synergistic with density. High cell density increased PEPCK run-on transcription approximately 3-fold, while PEPCK mRNA increased more than 6-fold. These observations suggest that high culture density increases PEPCK mRNA by increasing its transcription and possibly stabilizing PEPCK mRNA. The response could be coupled to the regulation of cell shape, as a close relationship between cell shape and gene expression has previously been shown to be important in the development and maintenance of tissues and organs. The PEPCK gene in H4IIEC3 cells could provide a useful model in which to study the poorly understood mechanisms involved in coordinating form and function.

Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins.

We previously used gel shift assays, Southwestern blots, and UV cross-linking to identify four proteins that bind to the 203-bp 5'-flanking region (-194/ +9) of the rabbit uteroglobin gene. Here we report cloning, by recognition site screening, the cDNAs for two of the uteroglobin promoter-binding proteins (95 kDa and 113 kDa). Their presumptive nucleotide-binding motifs share 61% identity with the SWI2/SNF2 helicase superfamily, and each protein has the novel C3HC4 (RING) zinc-finger signature near its C terminus. RUSH-1 alpha, the 113-kDa protein, is the rabbit homolog of human HIP116, a protein that binds to the human immunodeficiency virus-1 promoter. RUSH-1 beta is a 95-kDa truncated version of RUSH-1 alpha that results from alternative splicing of a 57-bp exon as confirmed by genomic cloning. Northern analysis showed mRNA expression (5.2 kb) was induced by progesterone +/- PRL and antagonized by estrogen. However, because the two proteins result from alternative splicing of a 57-bp exon, the small difference in their mRNA sizes could not be detected by Northern analysis. Therefore, competitive RT-PCR and HPLC were used to quantify differences in the ratios of their mRNAs. Progesterone +/- PRL treatment increased (P < 0.005) the ratio of message for RUSH-1 alpha compared with RUSH-1 beta. Western analysis showed the RUSH-1 alpha protein is increased in response to progesterone +/- PRL and decreased in response to estrogen. The antiserum used for immunoblotting specifically supershifts uteroglobin promoter-protein complexes in gel shift experiments. Because RUSH-1 alpha and beta messages were detected in lung, liver, and HRE-H9 cells, these proteins may regulate genes in numerous cell types.

Characterization of the corticosteroid-binding globulin messenger ribonucleic acid response in the pregnant hamster.

In this study we measured corticosteroid-binding globulin (CBG) mRNA levels in liver and various nonhepatic tissues of pregnant and nonpregnant hamsters. The N-terminal amino acid sequence (37 residues) of hamster CBG was determined and compared with published cDNA-deduced sequence information for rat and human CBG. Hamster CBG showed considerable sequence homology with both rat (70%) and human (59%) CBG. Because of the high level of homology, we were able to use a cRNA prepared from a rat CBG cDNA as a probe in Northern blot and solution hybridization analyses. Northern blots of hamster and rat liver RNA extracts revealed that the rat CBG cDNA probe hybridized to RNAs that were the same size in rats and hamsters. Further, the Northern blot showed that pregnant hamster liver contained substantially more CBG mRNA than nonpregnant hamster liver. The relative amounts of CBG mRNA in pregnant and nonpregnant hamster livers were compared using a solution hybridization assay. Slope-ratio analysis of the hybridization data revealed that pregnant hamster liver (day 14) contained 40-fold more CBG mRNA than nonpregnant hamster liver. When other tissues (kidney, spleen, small intestine, and decidual tissue) were assayed for CBG mRNA, a small amount of hybridization was detected by solution hybridization. However, Northern blot analysis of RNA extracts from nonhepatic tissues showed that the hybridizable sequences did not migrate at the same position as mature CBG mRNA. These results indicate that the observed increase in serum CBG during hamster pregnancy is largely attributable to an increase in hepatic CBG mRNA.

A single element in the phosphoenolpyruvate carboxykinase gene mediates thiazolidinedione action specifically in adipocytes.

Phosphoenolpyruvate carboxykinase (PEPCK) is the key enzyme in glyceroneogenesis, an important metabolic pathway that functions to restrain the release of non-esterified fatty acids (NEFAs) from adipocytes. The antidiabetic drugs known as thiazolidinediones (TZDs) are thought to achieve some of their benefits by lowering elevated plasma NEFAs. Moreover, peroxisome proliferator activated receptor gamma (PPARgamma) mediates the antidiabetic effects of TZDs, though many TZD responses appear to occur via PPARgamma-independent pathways. PPARgamma is required for adipocyte PEPCK expression, hence PEPCK could be a major target gene for the antidiabetic actions of TZDs. Here we used tissue culture and transfection assays to confirm that the TZD, rosiglitazone, stimulates PEPCK gene transcription specifically in adipocytes. We made the novel observation that this effect was by far the most rapid and robust among several other genes expressed in adipocytes. Adipocytes were transfected with a PEPCK/chloramphenicol acetyltransferase chimeric gene, in which either of the two previously discovered PPARgamma/retinoid X receptor alpha response elements, PCK2 and gAF1/PCK1, had been inactivated by mutagenesis. We demonstrate that PCK2 alone is a bona fide thiazolidinedione response element. We show also that the regulation of PEPCK by PPARs is cell-specific and isotype-specific since rosiglitazone induces PEPCK gene expression selectively in adipocytes, and PPARalpha- and PPARbeta-specific activators are inefficient. Hence, TZDs could lower plasma NEFAs via PPARgamma and PEPCK by enhancing adipocyte glyceroneogenesis.

A computer program for modeling the kinetics of gene expression.

Enzyme induction may be modeled on the basis of four, quantifiable processes that control the rates at which specific gene products accumulate and decay. These processes include synthesis of functional mRNA, translation and degradation of mRNA, and degradation of the protein product. We present a simple computer program that permits mathematical simulation of gene expression on the basis of experimentally determined rates of synthesis and degradation. The program was implemented as a spreadsheet using Microsoft Excel for Macintosh and MS-DOS operating systems and also was adapted for HyperCard on the Macintosh. It contains a formula to account for growth of tissue or cell populations. The program predicts amounts of individual mRNAs and proteins (or enzyme activities) in cells as a function of time after a stimulus alters their rates of synthesis or degradation.

A rapid and simple chemiluminescent assay for Escherichia coli beta-galactosidase.

We describe a chemiluminescent assay for E. coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate. Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer. Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay. Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay. As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay.

Acute and selective regulation of glyceroneogenesis and cytosolic phosphoenolpyruvate carboxykinase in adipose tissue by thiazolidinediones in type 2 diabetes.

AIMS/HYPOTHESIS: Regulation of glyceroneogenesis and its key enzyme cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays a major role in the control of fatty acid release from adipose tissue. Here we investigate the effect of rosiglitazone on the expression of genes involved in fatty acid metabolism and the resulting metabolic consequences. MATERIALS AND METHODS: Rosiglitazone was administered to Zucker fa/fa rats for 4 days and to 24 diabetic patients for 12 weeks, then mRNA expression for the genes encoding PEPCK-C, mitochondrial PEPCK, adipocyte lipid-binding protein, glycerol kinase, lipoprotein lipase and glycerol-3-phosphate dehydrogenase was examined in s.c. adipose tissue by real-time RT-PCR. Glyceroneogenesis was determined using [1-(14)C]pyruvate incorporation into lipids. Cultured adipose tissue explants from overweight women undergoing plastic surgery were incubated with rosiglitazone for various times before mRNA determination and analysis of PEPCK-C protein, activity and glyceroneogenesis. RESULTS: Rosiglitazone administration to rats induced the expression of the gene encoding PEPCK-C mRNA (PCK1) and PEPCK-C activity in adipose tissue with a resulting 2.5-fold increase in glyceroneogenesis. This was accompanied by an improvement in dyslipidaemia as demonstrated by the decrease in plasma NEFAs and triacylglycerol. In rosiglitazone-treated diabetic patients, PCK1 mRNA was raised 2.5-fold in s.c. adipose tissue. Rosiglitazone treatment of adipose tissue explants from overweight women caused a selective augmentation in PCK1 mRNA which reached a maximum of 9-fold at 14 h, while mRNA for other genes remained unaffected. Experiments with inhibitors showed a direct and transcription-only effect, which was followed by an increase in PEPCK-C protein, enzyme activity and glyceroneogenesis. CONCLUSIONS/INTERPRETATION: These results favour adipocyte glyceroneogenesis as the initial thiazolidinedione-responsive pathway leading to improvement in dyslipidaemia.

Expression and regulation of cytosolic phosphoenolpyruvate carboxykinase in 3T3-L1 adipocytes.

Animal studies have shown that the gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is controlled by unique mechanisms in fat. For example, a unique cis-acting DNA sequence located 1-2 kilobase pairs upstream of the promoter is required for PEPCK gene expression in adipocytes but not in other cell types. Moreover, glucocorticoids repress PEPCK gene transcription in fat whereas these steroids induce the same gene in liver and kidney. An in vitro system of cultured adipocytes would greatly facilitate studies of PEPCK gene regulation in fat cells. In this study, we report that cultured 3T3-L1 cells activate the PEPCK gene upon differentiation from fibroblasts to adipocytes. In addition, we report that cAMP induces and insulin and dexamethasone repress PEPCK mRNA in 3T3-L1 adipocytes. Thus these cells may provide a useful model system for future studies.

Regulation of phosphoenolpyruvate carboxykinase mRNA in mouse liver, kidney, and fat tissues by fasting, diabetes, and insulin.

Previous investigations of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been conducted using rats. In a recent comparative study, we investigated, for the first time, the effects of fasting, refeeding, alloxan-induced diabetes, and insulin treatment on the levels of PEPCK mRNA in mouse liver, kidney, and adipose tissues. As in rats, fasting and diabetes induced, while insulin repressed, hepatic PEPCK mRNA. In contrast, the response of renal PEPCK mRNA to fasting, refeeding, and diabetes in mice differed quantitatively with that in rats: fasting caused a twofold increase in mice and a fourfold increase in rats. Moreover, diabetes, which induces renal PEPCK mRNA indirectly by causing acidosis in rats, was without effect in mice. In adipose tissue, the results of previous studies in both rats and mice have shown that the amount of PEPCK protein and its rate of synthesis are increased by fasting and diabetes and decreased by refeeding and insulin treatment. Thus, it was surprising to find that fasting, refeeding, alloxan-induced diabetes, and insulin treatment had no effect on adipose tissue PEPCK mRNA in either rats or mice.

Regulation of rat liver phosphoenolpyruvate carboxykinase (GTP) messenger ribonucleic acid activity by N6, O2'-dibutyryladenosine 3',5'-phosphate.

N6,O2'-Dibutyryladenosine 3',5'-phosphate (Bt2cAMP) induces the synthesis of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.32), in rat liver by increasing the activity of messenger ribonucleic acid (mRNA) coding for this enzyme (mRNAPEPCK) more than 20-fold (from less than 0.01% to greater than 0.20% of total mRNA activity) as determined by using in vitro translation systems which measure only active mRNAPEPCK. The increase in mRNAPEPCK activity could result from increased synthesis, increased processing, or decreased inactivation rates. Actinomycin D and cordycepin inhibit mRNAPEPCK induction by 89% and 70%, respectively, a result that indicates a requirement for ongoing RNA synthesis but that does not distinguish which of these steps is regulated by cAMP. We have employed a kinetic approach, not involving RNA synthesis inhibitors, to determine the half-life of mRNAPEPCK both during a period of deinduction following glucose feeding and during a subsequent induction by Bt2cAMP. An estimated half-life of 20 +/-5 min during both of these periods indicates that Bt2cAMP has no effect on the rate of inactivation of mRNAPEPCK. We conclude that Bt2cAMP effects the increase in activity of mRNAPEPCK by promoting its synthesis or processing.

N6,O2'-dibutyryl cycle AMP and glucose regulate the amount of messenger RNA coding for hepatic phosphoenolpyruvate carboxykinase (GTP).

Rat liver phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA was purified to 25% of total mRNA activity (greater than 50-fold enrichment) by polysome immunoprecipitation. This preparation was used as template for the synthesis of cDNA that was subsequently cloned in Escherichia coli. The resulting clones were screened by in situ hybridization and by hybrid-selected translation of phosphoenolpyruvate carboxykinase mRNA. The cDNA insert of one plasmid, pPC2, was complementary to phosphoenolpyruvate carboxykinase mRNA as determined by these screening procedures. pPC2 cDNA was 760 base pairs in length and a partial restriction enzyme map was constructed. pPC2 was labeled with 32P by nick translation and was used as a hybridization probe to quantitate phosphoenolpyruvate carboxykinase mRNA following N6,O2'-dibutyryl cAMP (Bt2cAMP) injection or glucose feeding. Bt2cAMP increased whereas glucose decreased the level of hybridizable phosphoenolpyruvate carboxykinase mRNA and in all cases the changes were proportional to the in vitro translational activities measured in a reticulocyte lysate system. The half-life of phosphoenolpyruvate carboxykinase mRNA sequences was measured by an indirect procedure involving their quantitation, by hybridization assay, during deinduction and induction. The half-life was approximately 10-40 min during deinduction by glucose or during induction stimulated by Bt2cAMP. Our data indicate that cAMP enhances some step in the generation of phosphoenolpyruvate carboxykinase mRNA.

The kinetics of mammalian gene expression.

When rates of transcription from specific genes change, delays of variable length intervene before the corresponding mRNAs and proteins attain new levels. For most mammalian genes, the time required to complete transcription, processing, and transport of mRNA is much shorter than the period needed to achieve a new, steady-state level of protein. Studies of inducible genes have shown that the period required to attain new levels of individual mRNAs and proteins is related to their unique half-lives. The basis for this is a physical principle that predicts rates of accumulation of particles in compartmental systems. The minimum period required to achieve a new level is directly proportional to product half-lives because rates of decay control the ratio between the rate of synthesis and the concentration of gene products at steady state. This kinetic model suggests that sensitivity of gene products to degradation by ribonucleases and proteinases is an important determinant of the time scale of gene expression.

Cell-specific expression of cytosolic phosphoenolpyruvate carboxykinase in transgenic mice.

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is expressed in multiple cell types in diverse tissues including liver, kidney, intestine, and white and brown adipose tissues. It can thus be considered a model system for examining the regulation of cell-specific transcription. The PEPCK gene is transcribed from a single start site, but studies of transgenic mice have revealed that distinct cis-acting elements (and thus different trans-acting factors) regulate PEPCK expression in hepatocytes, renal proximal tubule epithelial cells, and adipocytes. Hepatocytes require elements between -457 and +69 bp; renal proximal tubule epithelia require elements between -363 and +69 bp; and adipocytes require elements between -2086 and -888 bp. An additional element downstream of +69 bp is required to either attenuate PEPCK mRNA levels in liver and fat or increase renal PEPCK mRNA. We hypothesize that the transcription factors C/EBP and DBP are the principal tissue-specific regulators in liver, and that HNF-1 and perhaps C/EBP are important for kidney-specific PEPCK expression. We propose that the putative downstream element is involved in regulating PEPCK mRNA turnover in liver and fat. Finally, we suggest that the fat-specific element is an enhancer that requires a novel adipogenic regulatory factor, ARF6, to function. The long-term objective will be to fine map the cis-acting elements and identify the cognate trans-acting factors that regulate PEPCK in liver, kidney and fat. This information will help elucidate the combinatorial mechanisms that control the cell-specific expression of this complex gene.

C/EBPbeta interacts with the P-enolpyruvate carboxykinase adipocyte-specific enhancer.

CCAAT/enhancer binding protein (C/EBP) family members are known to transactivate the gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) in hepatocytes via promoter proximal C/EBP response elements. PEPCK is also expressed in adipocytes; however, fibroblasts that are homozygous null for C/EBPbeta cannot express PEPCK when induced to differentiate into adipocytes (Tanaka et al., EMBO J. 16, 7432-7443, 1997). This along with our previous observation that an upstream adipocyte-specific enhancer contains multiple putative C/EBP binding elements suggested the possibility that C/EBPbeta transactivates the PEPCK gene in adipocytes via distal elements. We report here that C/EBPbeta transactivates a PEPCK-luciferase chimera in transient transfection assays. C/EBPbeta acted independently of peroxisome proliferator-activated receptor gamma (PPARgamma) which is required for function of the enhancer. C/EBPbeta in nuclear extracts and recombinant C/EBPbeta bound three of the putative C/EBP-binding elements within the enhancer. C/EBPbeta binding to these three elements was strongly cooperative. However, mutation of all three elements did not affect reporter transactivation by C/EBPbeta suggesting that additional elements participate in PEPCK regulation or that the effects of C/EBPbeta are indirect.

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP.

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.

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.