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.

Fatty acid recycling in adipocytes: a role for glyceroneogenesis and phosphoenolpyruvate carboxykinase.

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.

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.

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.

Peroxisome proliferator-activated receptor gamma and chicken ovalbumin upstream promoter transcription factor II negatively regulate the phosphoenolpyruvate carboxykinase promoter via a common element.

A heterodimer of peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid X receptor (RXR) is required for adipocyte differentiation. The gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is a PPARgamma/RXR target gene in adipose tissue. Of the two PPARgamma response elements, gAF1/PCK1 and PCK2, only PCK2 is required for PEPCK expression and responsiveness to the PPARgamma agonist, rosiglitazone, in adipose tissue even though both elements bind PPARgamma/RXR in vitro. In contrast, gAF1/PCK1 is essential for glucocorticoid inhibition of PPARgamma-induced PEPCK gene expression in adipocytes. We report that chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is the predominant nuclear receptor bound to gAF1/PCK1 in preadipocytes. COUP-TFII declines during adipogenesis in reciprocal fashion to PPARgamma. In transiently transfected fibroblasts COUP-TFII acts at gAF1/PCK1 to inhibit PPARgamma/RXR activation via PCK2. In contrast COUP-TFs are transcriptional activators of PEPCK in hepatocytes. PPARgamma/RXR occupies gAF1/PCK1 in adipocytes, and mutation of gAF1/PCK1 enhances PEPCK promoter transactivation by PPARgamma/RXR in fibroblasts, suggesting that this element is also a negative PPARgamma response element. These results indicate that gAF1/PCK1 is a pleiotropic element through which COUP-TFII inhibits premature PEPCK expression, and perhaps adipogenesis in general, and PPARgamma/RXR uses this same element in adipocytes to participate in PEPCK modulation by glucocorticoids.

Adipose expression of the phosphoenolpyruvate carboxykinase promoter requires peroxisome proliferator-activated receptor gamma and 9-cis-retinoic acid receptor binding to an adipocyte-specific enhancer in vivo.

A putative adipocyte-specific enhancer has been mapped to approximately 1 kilobase pair upstream of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene. In the present study, we used transgenic mice to identify and characterize the 413-base pair (bp) region between -1242 and -828 bp as a bona fide adipocyte-specific enhancer in vivo. This enhancer functioned most efficiently in the context of the PEPCK promoter. The nuclear receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and 9-cis-retinoic acid receptor (RXR) are required for enhancer function in vivo because: 1) a 3-bp mutation in the PPARgamma-/RXR-binding element centered at -992 bp, PCK2, completely abolished transgene expression in adipose tissue; and 2) electrophoretic mobility supershift experiments with specific antibodies indicated that PPARgamma and RXR are the only factors in adipocyte nuclear extracts which bind PCK2. In contrast, a second PPARgamma/RXR-binding element centered at -446 bp, PCK1, is not involved in adipocyte specificity because inactivation of this site did not affect transgene expression. Moreover, electrophoretic mobility shift experiments indicated that, unlike PCK2, PCK1 is not selective for PPARgamma/RXR binding. To characterize the enhancer further, the rat and human PEPCK 5'-flanking DNA sequences were compared by computer and found to have significant similarities in the enhancer region. This high level of conservation suggests that additional transcription factors are probably involved in enhancer function. A putative human PCK2 element was identified by this sequence comparison. The human and rat PCK2 elements bound PPARgamma/RXR with the same affinities. This work provides the first in vivo evidence that the binding of PPARgamma to its target sequences is absolutely required for adipocyte-specific gene expression.

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.

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.

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.

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.

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.

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.

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.

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.

Serum corticosteroid-binding globulin (CBG) and hepatic CBG mRNA relationships during hamster pregnancy: contribution of decidualization.

We measured serum corticosteroid-binding globulin (CBG) and hepatic CBG mRNA from individual hamsters throughout pregnancy and during decidualization. Serum CBG levels were determined by 3H-cortisol binding assay, and hepatic CBG mRNA levels were measured by Northern blots and solution hybridization assays, using a 32P-labeled cRNA probe derived from a rat CBG cDNA. There was a positive relationship between hepatic CBG mRNA levels and serum CBG levels during pregnancy. Both parameters increased significantly from the time of mating (cycle Day 4) to pregnancy Day 4, showing that CBG synthesis and secretion increased prior to implantation (Day 4). After implantation, serum CBG and hepatic CBG mRNA rose further from pregnancy Day 4 to a peak on Day 14, then decreased before parturition on Day 16. The prepartum decline in hepatic CBG mRNA preceded the fall in serum CBG. Decidualization on pseudopregnancy Day 4 resulted in an increase in serum CBG and hepatic CBG mRNA. Hepatic CBG mRNA increased from Day 5 to Day 7, and serum CBG increased progressively from Day 5 through Day 9 after uterine decidualization in the hamster. The present results demonstrate that the pattern of serum CBG observed in the pregnant hamster follows closely that of hepatic CBG mRNA. A signal emanating from uterine decidual tissue appears to be important in the regulation of hepatic CBG synthesis and secretion during midpregnancy, but other unknown factors appear to be involved in controlling CBG during the early and late stages of pregnancy.

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 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.

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.

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)