Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Short-term effects of human proinsulin on metabolic rates and its long-term action on enzyme induction were studied in primary cultures of rat hepatocytes and in the perfused rat liver, and compared with the effects of bovine insulin. In the perfused rat liver, proinsulin decreased the glucagon-dependent increase of glycogenolysis. The action of 0.5 nM glucagon was almost completely suppressed by 100 nM proinsulin. Proinsulin and insulin showed similar potency. In cultured rat hepatocytes, proinsulin stimulated glycolysis up to fivefold with a half-maximal effective dose of 30 nM. Proinsulin induced the key glycolytic enzymes glucokinase and pyruvate kinase by twofold and antagonized the glucagon-dependent induction of phosphoenolpyruvate carboxykinase with a half-maximal effective dose at 3 nM. For the effects in cultured hepatocytes, about 100-fold higher concentrations of proinsulin than of insulin were required.
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PMID:Insulin-like action of proinsulin on rat liver carbohydrate metabolism in vitro. 388 57

We used a nuclear RNA transcript elongation assay to show that cAMP analogs and dexamethasone cause a selective increase of transcription of the P-enolpyruvate carboxykinase gene in H4IIE hepatoma cells. 8-(4-chlorophenylthio)-cAMP increased transcription within 5 min and the maximal rate, generally 10-15-fold above the basal rate, was attained by 30 min. This increase was of sufficient magnitude to account for the effect on mRNAPEPCK (for example, where PEPCK is phosphoenolpyruvate carboxykinase) accumulation. After the initial increase, and with continued presence of cAMP, transcription of this gene declined to a new steady-state level which was 2-3 times the basal value. The effect of cAMP analogs on P-enolpyruvate carboxykinase gene transcription was obtained in the absence of protein synthesis. This, and the rapidity of the response, indicates that the effect of cAMP is exerted directly on the P-enolpyruvate carboxykinase gene. Dexamethasone results in a specific, 6-fold increase of transcription, sufficient to account for the increase of mRNAPEPCK which follows treatment of H4IIE cells with this glucocorticoid. When 1 nM insulin was added to either untreated H4IIE cells, or cells first treated with a cAMP analog or dexamethasone, there was a marked reduction of cytoplasmic mRNAPEPCK. The inhibitory effect of insulin was readily reversible, as cells regained the basal level of mRNAPEPCK and full responsiveness to cAMP within 1 h after removing insulin. The transcript elongation assay was used to show that insulin inhibits transcription of the gene coding for mRNAPEPCK. The concentration of insulin required for 50% inhibition was 2-5 pM, whereas approximately 200 pM of proinsulin was required to achieve the same inhibition of transcription. This effect was specific, since insulin did not affect the synthesis of total RNA; it was rapid, as 5 nM insulin decreased the rate of P-enolpyruvate carboxykinase gene transcription by 50% within 15 min; and it also does not require ongoing protein synthesis. The magnitude and kinetics of the response suggest that the primary action of insulin in the regulation of P-enolpyruvate carboxykinase synthesis is exerted at the level of mRNAPEPCK transcription. The insulin-mediated inhibition of mRNAPEPCK transcription was noted in untreated cells and in cells first treated with 8-(4-chlorophenylthio)-cAMP, dexamethasone, or both of these agents. Hence, among these compounds, insulin is the dominant regulatory molecule.
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PMID:Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin. 609 65

The mRNA that codes for phosphoenolpyruvate carboxykinase accounts for approximately 0.2% of the protein synthesized in H4IIEC3 hepatoma cells maintained for 24 h in serum-free medium containing N6,O2'-dibutyryl cAMP and theophylline. This value decreases to 0.04% within 3 h after the addition of insulin. Maximal effects are produced by 10(-10) M insulin, and half-maximal deinduction of both the relative rate of synthesis of P-enolpyruvate carboxykinase and mRNA coding for P-enolpyruvate carboxykinase activity occurs at approximately 2 X 10(-12) M insulin. Porcine proinsulin is 4% as potent as porcine insulin since half-maximal deinduction of mRNA coding for P-enolpyruvate carboxykinase occurs at 5 X 10(-11) M. The concentration of proinsulin required to inhibit 125I-insulin binding by 50% is 2 X 10(-7) M, as compared to 6 X 10(-9) M for insulin; thus, the decreased sensitivity of this deinduction to proinsulin parallels the decreased binding affinity H4IIEC3 cells have for proinsulin as compared to insulin. These data indicate that insulin regulates P-enolpyruvate carboxykinase synthesis through a receptor-mediated process, that the effect occurs when less than 2% of the insulin receptors are occupied, and that this effect is exerted prior to the level of mRNA translation.
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PMID:Insulin decreases phosphoenolpyruvate carboxykinase (GTP) mRNA activity by a receptor-mediated process. 627 36

We engineered a hepatoma cell line that produces an up-regulation of insulin in response to cAMP, dexamethasone, and retinoic acid, and a down-regulation in response to insulin. We devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). To assess the ability to regulate insulin secretion, we used the rat hepatoma cell line, H4IIE. The H4IIE cells secreted immunoreactive insulin (IRI) constantly at a level of 1-3 fmol/10(6) cells/h. IRI increased approximately two-fold upon stimulation with 0.5 mM cAMP and five-fold with the addition of the cAMP-dependent phosphodiesterase inhibitor IBMX, as compared to baseline IRI secretion. IRI increased 18-fold by 1-500 nM dexamethasone together with cAMP and IBMX. Addition of exogenous insulin to the culture medium significantly decreased insulin mRNA expression on Northern blot.
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PMID:Regulatable production of mature insulin from a hepatocyte cell line: insulin production is up-regulated by cAMP and glucocorticoids, and down-regulated by insulin. 898 Jan 15

To utilize hepatocytes for insulin-producing surrogate cells, we devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). The expression of PEPCK is down-regulated by insulin, and up-regulated by cAMP and glucagon. To express insulin in hepatocytes, we constructed an adenoviral insulin expression system. After infection, the hepatocytes secreted immunoreactive insulin (IRI) at an increasing rate. IRI secretion increased over four-fold upon stimulation with 300 microM cAMP and 500 microM of the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This increase was also observed with glucagon and IBMX. Production was augmented two-fold by the addition of wortmannin, phosphatidylinositol (PI)-3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. Addition of exogenous insulin to the culture decreased insulin mRNA expression remarkably on Northern blot. Thus, by using a PEPCK promoter for insulin expression, we were able to up-regulate insulin production from hepatocytes with cAMP and glucagon, and down-regulate with insulin itself.
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PMID:Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin. 981 59

Type 1 diabetic patients depend dramatically on insulin replacement therapy, which involves the administration of intermediate- or long-acting insulin, together with short-acting insulin to mimic physiological insulin profiles. However, the delayed-action preparations available are not generally able to produce smooth background levels of insulin. Muscle cells were tested for long-term delivery of active human insulin as an approach to achieve a constant basal level of insulin. Thus, C2C12 mouse myoblast cells were stably transfected with a chimeric gene obtained by linking the myosin-light chain 1 (MLC1) promoter to the human proinsulin gene, containing genetically engineered furin endoprotease cleavage sites (MLC1/Insm). When differentiated, C2C12Insm myotube cells expressed high levels of insulin mRNA and protein, whereas no insulin was detected in myoblast cells. HPLC fractionation of culture medium and cell extracts from differentiated C2C12Insm cells revealed that about 90% of the proinsulin was processed to mature insulin. In addition, these cells released significant levels (about 100 microU/10(6) cells/hr) of mature insulin to the medium. The hormone was biologically active since it increased glucose consumption and utilization by the differentiated C2C12Insm cells and was able to block the expression of the endogenous phosphoenolpyruvate carboxykinase (PEPCK) gene in FTO-2B rat hepatoma cells. Furthermore, when C2C12Insm myoblast cells were transplanted into diabetic mice an increase in insulinemia and a decrease in hyperglycemia were observed. Thus, our results suggest that the use of engineered myotube cells continuously secreting a defined level of insulin might be a useful approach to improve the efficacy of insulin injection treatment.
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PMID:Insulin production by engineered muscle cells. 1034 May 52

To employ hepatocytes as surrogate beta-cells for gene therapy of diabetes, a regulatory system was devised in this study by placing the human insulin cDNA under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, followed by the cytomegalovirus immediate early promoter-driven enhanced-green-fluorescent-protein open reading frame. The expression cassette was inserted into the adeno-associated virus vector between two inverted terminal repeats, and used to produce recombinant adeno-associated virus (rAAV). HepG2 human hepatoma cells were transduced by rAAV at the desired multiplicity of infection, followed by treatment with various concentrations of retinoic acid, dexamethasone, dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX). The cell-culture media were collected at 8, 16 and 24 h later. Proinsulin/insulin levels were determined with human proinsulin/insulin radioimmunoassay kits. Transduction of HepG2 cells by rAAV showed that green fluorescence was produced as early as 12 h after rAAV infection. Flow-cytometrical analysis demonstrated that transduction efficiency increased with the numbers of transducing rAAV particles used. The transduced hepatocytes were shown to secrete immunoreactive proinsulin/insulin, which were stimulated by the concentrations of retinoic acid, dexamethasone and dbcAMP in the culture medium. High conversion from proinsulin into insulin occurred when these cells were treated with dexamethasone and dbcAMP. The presence of IBMX enhanced the secretion of proinsulin/insulin from the dbcAMP-treated cells. We conclude that rAAV is a promising vector for gene therapy of diabetes. Regulated secretion of proinsulin/insulin can be obtained in the rAAV-transduced HepG2 cells conferred with the PEPCK promoter via rAAV-mediated gene transfer.
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PMID:Regulated secretion of proinsulin/insulin from human hepatoma cells transduced by recombinant adeno-associated virus. 1127 67

We engineered an artificial beta cell line that produces an up-regulation of insulin in response to dexamethasone, and a down-regulation in response to insulin. A regulatory secretion system was devised by placing proinsulin cDNA containing genetically engineered furin endoprotease cleavage sites and a regulatory promoter for phosphoenolpyruvate carboxykinase (PEPCK), and an insulin expressing retrovirus vector (pN-PEPCK-mINS) was constructed and transfected into Hepa1-6 cells. The levels of insulin in culture medium and expression of insulin gene was estimated by radioimmunoassay and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The clone (Hepa1-6/INS21), which secreted the highest level of insulin (10.79 microIU/106 cells per day), was selected for the regulation experiment. Compared with the non-treated Hepa1-6/INS21 cells, insulin production was augmented 3.6-fold by the addition of 10-7 M of dexamethasone. Addition of exogenous insulin to the culture medium decreased insulin mRNA expression remarkably on RT-PCR results, while dexamethasone increased insulin gene expression at the transcriptional level. The data indicated that genetically engineered Hepa1-6 cells could synthesize process and secrete insulin in a physiological manner.
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PMID:Regulated production of mature insulin in rat hepatoma cells: insulin production is up-regulated by dexamethasone and down-regulated by insulin. 1647 99

This study was conducted to determine whether dietary Se deficiency precluded overproduction of glutathione peroxidase-1 (GPX1) activity in mice overexpressing (OE) this gene and thus rescued their type 2 diabetes-like phenotypes. A total of 20 male OE and wild-type (WT) mice were fed an Se-deficient (<0.02 mg/kg) diet or an Se-supplemented (0.3 mg/kg as sodium selenite) diet from 1 to 5 mo of age. Dietary Se deficiency eliminated or attenuated (P < 0.05) genotype differences in concentrations of blood glucose, plasma insulin, and/or hepatic lipids, insulin sensitivity, and glucose-stimulated insulin secretion at the end of the study. Dietary Se deficiency decreased (P < 0.05) OE islet mRNA levels of 2 key transcriptional activators (Beta2 and Foxa2) and removed genotype differences in islet mRNA levels of 7 genes (Beta2, Cfos, Foxa2, Pregluc, Ins1, p53, and Sur1) related to insulin synthesis and secretion. Compared with those of the Se-adequate OE mice, the Se-deficient OE mice had lower (P < 0.05) hepatic mRNA levels of 2 key rate-limiting enzymes for lipogenesis (Acc1) and glycolysis (Gk1), along with lower (P < 0.05) activities of hepatic glucokinase and muscle phosphoenolpyruvate carboxykinase. Dietary Se deficiency also decreased (P < 0.05) blood glucose and hepatic lipid concentrations in the WT mice. In conclusion, dietary Se deficiency precluded the overproduction of GPX1 in full-fed OE mice and partially rescued their metabolic syndromes. This alleviation resulted from modulating the expression and/or function of proinsulin genes, lipogenesis rate-limiting enzyme genes, and key glycolysis and gluconeogenesis enzymes in islets, liver, and muscle.
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PMID:Dietary selenium deficiency partially rescues type 2 diabetes-like phenotypes of glutathione peroxidase-1-overexpressing male mice. 2301 91

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