Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of protein synthesis in the control of phosphoenolpyruvate carboxykinase (PEPCK; 4.1.1.32) mRNA turnover was studied in FTO-2B rat hepatoma cells. A previous study demonstrated that incubation of these cells with cAMP prolongs the half-life of the otherwise short-lived PEPCK mRNA. The decay rate of PEPCK mRNA was also slowed in cells incubated with cycloheximide, but not in cells incubated with other translation inhibitors, such as puromycin or pactamycin, even though protein synthesis was inhibited 85-95% by these agents. No correlation was noted between the rate of L-[3H]valine incorporation into cellular proteins and PEPCK mRNA half-life, suggesting that protein synthesis per se is not required for breakdown of the mRNA. Exposure of cells to the translation initiation inhibitor pactamycin together with cycloheximide abolished the "slowing" effect of cycloheximide, and PEPCK mRNA decayed at the same rate as in cells incubated in the presence of pactamycin alone. In contrast, pactamycin did not reverse the effect of cAMP, and the mRNA decayed at the same slow rate in cells incubated in the presence of either (Bu)2cAMP alone or (Bu)2cAMP together with pactamycin. Since pactamycin promotes polysomes dissociation, these results suggest that cAMP enhances the stability of a polysome-free PEPCK mRNA. Furthermore, these results strongly indicate that neither the rapid decay of PEPCK mRNA nor the cAMP-mediated stabilization of the mRNA requires on-going protein synthesis.
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PMID:The role of protein synthesis in the decay of phosphoenolpyruvate carboxykinase messenger RNA. 133 75

Incubation of isolated hepatocytes from fasted rats with 20 mM LiCl for 1 h decreased glucose production from lactate, pyruvate, and alanine. In addition, phosphoenolpyruvate carboxykinase (PEPCK) gene expression in FTO-2B rat hepatoma cells was inhibited by treatment with LiCl. Lithium was also able to counteract the increased PEPCK mRNA levels caused by both Bt2cAMP and dexamethasone, in a concentration-dependent manner. A chimeric gene containing the PEPCK promoter (-550 to +73) linked to the amino-3-glycosyl phosphotransferase (neo) structural gene was transduced into FTO-2B cells using a Moloney murine leukemia virus-based retrovirus. In these infected cells, 20 mM LiCl decreased both the concentration of neo mRNA transcribed from the PEPCK-neo chimeric gene and mRNA from the endogenous PEPCK gene. Lithium also inhibited the stimulatory effect of Bt2cAMP and dexamethasone on both genes. The stability of neo mRNA was not altered by lithium, since in cells infected with retrovirus containing only the neo gene transcribed via the retroviral 5'-LTR and treated with 20 mM LiCl, no change in neo mRNA levels was observed. The intraperitoneal administration of LiCl to rats caused a decrease in hepatic PEPCK mRNA, indicating that lithium could also modify gene expression in vivo. The effects of lithium were not due to an increase in the concentration of insulin in the blood but were correlated with an increase in hepatic glycogen and fructose 2,6-bisphosphate levels. These results indicate that lithium ions, at concentrations normally used therapeutically for depression in humans, can inhibit glucose synthesis in the liver by a mechanism which can selectively modify the expression of hepatic phosphoenolpyruvate carboxykinase.
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PMID:Lithium inhibits hepatic gluconeogenesis and phosphoenolpyruvate carboxykinase gene expression. 137 Nov 8

The interaction of promoters contained in a Moloney murine leukemia virus (MoMLV)-based retroviral vector was studied after infection of FTO-2B rat hepatoma and NIH 3T3 mouse fibroblast cells. Segments of the phosphoenolpyruvate carboxykinase (PEPCK) promoter-regulatory region, which are known from previous studies to confer responsiveness to hormones, were linked to the structural genes for bovine growth hormone, amino-3'-glycosyl phosphotransferase (neo), and herpes-virus thymidine kinase and inserted into a MoMLV-based retroviral vector. In vectors in which PEPCK was the only internal promoter, it was the major site of gene transcription. This dominant effect was independent of the orientation of the PEPCK promoter relative to the 5' long terminal repeat of the provirus and was noted with as little as -174 base pairs of the 5'-flanking sequence. NIH 3T3 cells, which do not express the endogenous PEPCK gene, transcribed the transduced PEPCK-chimeric genes at the same high levels as was observed in hepatoma cells. When two promoters were present in the provirus, the expression of chimeric structural genes depended on the relative position and orientation of these genes as well as the type of cell infected by the retrovirus. Differential responses of proviral promoters in infected cells were also observed in the presence of hormones. Dibutyryl cyclic AMP increased the expression of genes linked to the PEPCK promoter in FTO-2B and NIH 3T3 cells, whereas glucocorticoids stimulated transcription from both the PEPCK promoter and the long terminal repeat in FTO-2B cells. The effect of these hormones on transcription of proviral promoters depended on their position relative to the 5' long terminal repeat. In contrast, insulin uniformly inhibited transcription from the PEPCK promoter in a position-independent manner but only in hepatoma cells and not in fibroblasts. In clonally isolated FTO-2B cells infected with a retrovirus, the site of proviral integration was also a major factor determining the expression and hormonal regulation from the internal promoters. The data suggest that the hormonal regulation of the expression of genes contained in retroviral vectors depends on the type and position of the regulatory elements present in the provirus and the lineage of the infected cell.
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PMID:Hormonal control of interacting promoters introduced into cells by retroviruses. 202 56

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.
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PMID:Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells. 216 40

It is now well established that cAMP induces the transcription rate of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that this induction is dependent on a nucleotide domain located within the promoter-regulatory region of the gene (Short, J. M., Wynshaw-Boris, A., Short, H. P., and Hanson, R. W. (1986) J. Biol. Chem. 261, 9721-9726). We report here that cAMP also stabilizes phosphoenolpyruvate carboxykinase mRNA against degradation. Using two independent experimental approaches, we show that the half-life of the mRNA for phosphoenolpyruvate carboxykinase is extended when FTO-2B rat hepatoma cells are exposed to dibutyryl cyclic AMP (Bt2cAMP). In the first experiment, the rate of decay of phosphoenolpyruvate carboxykinase mRNA was determined in cells incubated in the presence of insulin, which has been shown to block the transcription rate of the gene for the enzyme. Under these conditions, the half-life of phosphoenolpyruvate carboxykinase mRNA was 30 min. However, in cells incubated in the presence of Bt2cAMP, the mRNA decayed with a half-life of 150 min. In the other experiment, mRNA stability was measured under steady state conditions, utilizing a "pulse-chase" approach. The apparent half-life of phosphoenolpyruvate carboxykinase mRNA increased from 40 min to over 250 min in Bt2cAMP-treated cells. No significant change in the stability of total cellular RNA was noted. Other experiments have shown that the transcription rate of the gene for phosphoenolpyruvate carboxykinase peaks within the first 20 min after exposing the cells to Bt2cAMP and then levels off, while the abundance of the mRNA reaches a maximum at about 90 min and remains at this level thereafter. Thus, the long term effect of cAMP on the expression of the gene coding for phosphoenolpyruvate carboxykinase occurs at least in part, through an alteration in the degradation rate of the mRNA for this enzyme.
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PMID:Cyclic AMP stabilizes the mRNA for phosphoenolpyruvate carboxykinase (GTP) against degradation. 283 95

The effect of insulin on the abundance of mRNAs coding for tyrosine aminotransferase (TAT; EC 2.6.1.5), tryptophan oxygenase (TO; EC 1.13.1.12), and P-enolpyruvate carboxykinase(GTP) (PEPCK; EC 4.1.1.32) was examined in primary cultures of adult rat hepatocytes and in FTO-2B rat hepatoma cells by Northern blot analysis using RNA probes made from SP6-cDNAs. Insulin (10(-11)-10(-7) M), which has been reported to induce TAT and decrease the activity of TO, did not change the levels of TAT mRNA and TO mRNA in hepatocytes regardless of the presence of other inducers. In the same cells, dexamethasone increased TAT mRNA up to 19-fold and TO mRNA up to 15-fold, and 8pClPhS-cAMP (CPT-cAMP) raised the level of TAT mRNA up to 36-fold. The abundance of TO mRNA was not altered by CPT-cAMP. In contrast to TAT mRNA and TO mRNA, the level of PEPCK mRNA was dramatically decreased by insulin in the same hepatocytes. The sensitivity to this inhibitory effect of insulin was enhanced by dexamethasone and reduced by CPT-cAMP. FTO-2B hepatoma cells, which do not express detectable levels of TO mRNA, showed responses similar to those of hepatocytes, except that insulin caused a moderate reduction in TAT mRNA, but only in the presence of CPT-cAMP. The PEPCK mRNA in FTO-2B cells was suppressed by insulin in a manner closely resembling the effects in hepatocytes in the present study and in H4IIE hepatoma cells previously reported.
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PMID:Regulation of gene expression in rat hepatocytes and hepatoma cells by insulin: quantitation of messenger ribonucleic acid's coding for tyrosine aminotransferase, tryptophan oxygenase, and phosphoenolpyruvate carboxykinase. 287 68

Transcription of the gene for cytosolic Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from rat liver is increased by cAMP and glucocorticoids and decreased by insulin. A PEPCK-thymidine kinase (TK) chimeric gene was transfected into FTO-2B rat hepatoma cells, which were TK-deficient. Previous studies showed that a cAMP regulatory element is located at the 5' end of the PEPCK gene. In this report, we demonstrate that the 5' end of the gene also contains a glucocorticoid regulatory element, but not one for insulin. Regions of the PEPCK gene that contain these regulatory elements were attached to the Herpes simplex virus TK structural gene containing its own promoter. The hormone regulatory elements within the 5' flanking region of the PEPCK gene conferred cAMP and glucocorticoid responsiveness on the TK gene after transfection into FTO-2B cells. Like viral enhancer elements, these regulatory elements functioned properly when placed in either orientation at various positions 5' or 3' to TK. The presence of the SV40 enhancer element upstream from the PEPCK-TK gene had little effect on the basal level of expression or hormonal regulation of the chimeric gene.
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PMID:Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. I. Multiple hormone regulatory elements and the effects of enhancers. 301 2

Hormonal regulatory elements within the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) promoter region were mapped using a series of 5' deletions linked to the amino-3'-glycosyl phosphotransferase structural gene. These deletion mutants were stably transfected into the genome of FTO-2B hepatoma cells. A 47-base pair region of the PEPCK promoter was identified which was essential for stimulation by dibutyryl cAMP. A 12-base pair core sequence (CTTACGTCAGAG) within this region shows significant homology with sequences in four other cAMP-regulated genes. There are two glucocorticoid regulatory elements within the promoter, as well as an inhibitory element which depresses the level of basal gene transcription. The deletion of this inhibitory sequence prevents the induction of the chimeric gene by dexamethasone. The existence of the hormone regulatory domains within the PEPCK promoter was confirmed by attaching these elements upstream of the heterologous Herpes simplex virus thymidine kinase structural gene, containing its own promoter.
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PMID:Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. II. Identification of cAMP and glucocorticoid regulatory domains. 301 3

cAMP stimulates the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in rat liver. We have investigated the nucleotide sequences required for regulation of PEPCK gene expression by cAMP. A chimeric gene was constructed in which a 620-base pair fragment of the 5'-end of the PEPCK gene (including 547 base pairs of 5'-flanking sequence) was ligated to the herpes simplex virus thymidine kinase (TK) structural gene. The PEPCK promoter fragment was introduced either in the proper orientation for transcription of the TK gene or in the opposite orientation. These fusion genes and the parent vector, pOPF, which contains the intact TK gene, were transfected individually into TK-deficient FTO-2B rat hepatoma cells. FTO-2B cells contain an active endogenous PEPCK gene which is stimulated by cAMP. Cells were selected in HAT medium and grown either as mass cell cultures or as individual clones. Dibutyryl cyclic AMP (Bt2cAMP) plus theophylline (16 h) stimulated TK activity 1.6-6.1-fold in cell lines transfected with the PEPCK-TK fusion gene containing the PEPCK promoter fragment in the correct orientation. However, the intact TK gene was not induced by Bt2cAMP after transfection, nor was there any expression of the PEPCK-TK fusion gene in cells which contained the PEPCK promoter fragment in the wrong transcriptional orientation. Bt2cAMP also increased the levels of TK mRNA in cells transfected with the PEPCK-TK fusion gene, but not in cells transfected with the intact TK gene. The chimeric PEPCK-TK mRNA initiated at the PEPCK start site, as determined by S1 nuclease mapping. There was no relationship between the number of copies of the PEPCK-TK gene integrated in the various cell lines and either the basal level of TK activity or its inducibility of Bt2cAMP.
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PMID:Identification of a cAMP regulatory region in the gene for rat cytosolic phosphoenolpyruvate carboxykinase (GTP). Use of chimeric genes transfected into hepatoma cells. 609 Apr 58

The role of the GTP-binding regulatory protein (G-protein) Gi alpha 2 in vivo was explored using transgenic mice in which the alpha-subunit of Gi alpha 2 was suppressed by antisense RNA. Rat hepatoma FTO-2B cells provide an ideal test system for constructs employing the expression vector pPCK-AS, designed to express antisense RNA at birth under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Cells transfected with the expression vector containing a sequence antisense to Gi alpha 2 (pPCK-ASGi alpha 2) displayed expression of RNA antisense to Gi alpha 2 that, like transcription of the PEPCK gene, was inducible by cyclic AMP. Expression of RNA antisense to Gi alpha 2 and suppression of the expression of Gi alpha 2, but not Gsa and Gi alpha 3, was observed in the transfected FTO-2B cells. BDF1 mice carrying the transgene displayed suppression of Gi alpha 2 in liver and fat, two targets for tissue-specific expression of the PEPCK gene. The loss of Gi alpha 2 in white adipocytes of transgenic mice resulted in 3.1-fold elevation of basal cyclic AMP accumulation. Cyclic AMP accumulation in response to stimulation by epinephrine (10 microM) was normal in adipocytes of transgenic mice, demonstrating no alteration in the stimulatory adenylylcyclase capacity in the Gi alpha 2-deficient cells. The inhibitory adenylylcyclase pathway, in sharp contrast, was severely blunted in response to challenge by the inhibitory A1-purinergic agonist, (-)R-N6-phenylisopropyladenosine. These studies illuminate a critical role of Gi alpha 2 in the inhibitory adenylylcyclase signaling pathway in vivo.
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PMID:Gi alpha 2 mediates the inhibitory regulation of adenylylcyclase in vivo: analysis in transgenic mice with Gi alpha 2 suppressed by inducible antisense RNA. 769 86


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