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)

A gluconeogenic strain of renal epithelial cells (LLC-PK1-F+) was used to characterize the effect of pH and bicarbonate concentration on the levels of phosphoenolpyruvate carboxykinase (PCK) and glutaminase (GA) mRNAs. The levels of both mRNAs are markedly dependent upon medium glucose concentration. The level of PCK mRNA is increased with increasing glucose concentration from 0 to 40 mM, whereas the level of GA mRNA is maximal between 3 and 5 mM glucose. When LLC-PK1-F+ cells are grown with 5 mM glucose and then subjected to an acute decrease in pH (from 7.4 to 6.9) and bicarbonate concentration (from 25 to 10 mM), the level of PCK mRNA exhibits a biphasic response. The PCK mRNA is initially increased 4-fold within 3 h, then decreases slightly and subsequently increases between 10 and 20 h to a level that is 17-fold greater than normal. Only the initial increase parallels the changes observed in vivo. In contrast, after onset of acidosis, the level of GA mRNA initially remains unchanged, is then increased 8-fold between 10 and 16 h, and then decreases slightly. This response closely mimics the results obtained in vivo. A decrease in media pH at constant bicarbonate causes a marked increase in both mRNAs. However, the levels of the two mRNAs are also elevated by decreasing bicarbonate at a constant pH. Thus, both parameters independently affect the level of the two mRNAs. The use of actinomycin D to measure the half-lives of PCK and GA mRNAs at pH 7.4 and 6.9 indicates that stabilization may fully account for the induction of GA mRNA and contributes to the inductive effects of decreased pH and/or bicarbonate on PCK mRNA. Following recovery from acidic conditions, the two mRNAs exhibit a rapid and coordinate decrease (t1/2 approximately 20 min). Dexamethasone had no effect on the level of either mRNA, whereas cAMP increased only PCK mRNA. The latter effect was additive with the increase caused by decreased pH and/or bicarbonate and was reversed by incubating in alkalotic media. Thus, the induction of PCK and GA mRNAs during acidosis is initiated in direct response to a decrease in extracellular pH and/or bicarbonate.
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PMID:Effect of pH and bicarbonate on phosphoenolpyruvate carboxykinase and glutaminase mRNA levels in cultured renal epithelial cells. 185 45

LLC-PK1 cells, derived from pig kidney, retain several properties of the proximal tubule, but are incapable of gluconeogenesis, due to the lack of fructose-1,6-bisphosphatase (FBPase) [Am. J. Physiol. 248 (Cell Physiol. 17): C181-185, 1985]. Cells incapable of gluconeogenesis require a hexose, pentose, or nucleoside to provide ribose-5-phosphate for RNA biosynthesis. To induce or select cells that express FBPase activity, we cultured LLC-PK1 cells in glucose-free medium. We obtained cells (designated LLC-PK1-FBPase+) that express FBPase activity and are capable of growing in the complete absence of sugars or nucleosides. The cells have apical membrane enzyme activities that differ from those of wildtype cells. Tests of metabolic flow through the gluconeogenic pathway, using 3-mercaptopicolinic acid, a specific inhibitor of phosphoenolpyruvate carboxykinase, confirmed that the cells are gluconeogenic. LLC-PK1-FBPase+ cells grown in medium containing 5 mM glucose for five weekly passages continued to express FBPase activity and apical membrane enzyme activities characteristic of the FBPase+ strain. When switched back to glucose-free medium, they proliferated well. The strain appears to be stable. It should provide a model for studying the relationship between gluconeogenesis and other proximal tubule functions. An incidental finding is that in both strains, the activity of lactate dehydrogenase varied directly with the concentration of glucose in the growth medium, indicating that the expression of lactate dehydrogenase may be regulated by glucose or a metabolite of glucose.
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PMID:Isolation, growth, and characterization of a gluconeogenic strain of renal cells. 303 Jan 22

The two gluconeogenic substrains of renal epithelial cells, LLC-PK1-FBPase+ and OKGNG+, have been shown to differ markedly in their metabolism of lactate and pyruvate. OKGNG+ cells consumed lactate as well as pyruvate at high rates in contrast to LLC-PK1-FBPase+ cells, which failed to take up or utilize lactate. (Aminooxy)acetate (AOA), an inhibitor of transamination reactions, was used to further delineate these differences. Lactate consumption of OKGNG+ cells was significantly inhibited by AOA, whereas pyruvate consumption by LLC-PK1-FBPase+ cells was slightly stimulated. Growth of OKGNG+ cultures, however, could be achieved on lactate in the presence of AOA. From these results it was concluded that the cell strains might differ in the subcellular distribution of phosphoenolpyruvate carboxykinase (PEPCK). LLC-PK1-FBPase+ cells may express both mitochondrial and cytosolic PEPCK isoenzymes, whereas OKGNG+ cells express only the mitochondrial isoenzyme. This was tested by directly assaying PEPCK activity in subcellular fractions of the cells. In OKGNG+ cells PEPCK activity fractionated with the mitochondrial marker glutamate dehydrogenase; however, in LLC-PK1-FBPase+ cells two-thirds of PEPCK activity was found in the cytosol. In LLC-PK1-FBPase+ cells, PEPCK activity increased twofold on incubation in acidic culture medium (pH 6.9) for 18 h, in contrast to the PEPCK activity in OKGNG+ cells. Northern blot analysis using cDNA probes specific for the mitochondrial and cytosolic PEPCK mRNAs confirmed the enzyme activity data. In LLC-PK1-FBPase+ cells strong expression of cytosolic PEPCK mRNA was observed, whereas in OKGNG+ cells only very low levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subcellular localization of PEPCK and metabolism of gluconeogenic substrains of renal cell lines. 786 84

Rat kidney expresses two forms of glutaminase (GA) mRNA which probably result from the use of alternative polyadenylation signals. The two mRNAs are increased coordinately in response to metabolic acidosis via a mechanism that apparently does not involve transcriptional or translational regulation. A 956-bp fragment that contains the 3'-nontranslated sequence of the smaller GA cDNA was cloned into an expression vector (p beta G) that encodes a chimeric beta-globin growth hormone mRNA. Both the parent and the derived construct (p beta G-GA) were transfected into LLC-PK1-F+ cells. Stable transfectants express sixfold lower levels of beta G-GA mRNA than that of the parent beta G mRNA. However, only the beta G-GA mRNA is increased 2.5-fold by growth in acidic medium (pH 6.9, 10 mM HCO3-). The apparent half-life of the beta G mRNA (> 24 h) is unaffected by the pH of the growth media. In contrast, the apparent half-life of the beta G-GA mRNA is increased from 4.5 h to approximately 24 h when cells are transferred to acidic medium for 8 h. The observed pH response is not reproduced when the beta G-GA construct is stably transfected into COS-7 cells or when a beta-globin-phosphoenolpyruvate carboxykinase chimeric gene is expressed in LLC-PK1-F+ cells. Thus the 3'-nontranslated region of the GA mRNA contains a pH-responsive stability element.
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PMID:The 3'-nontranslated region of rat renal glutaminase mRNA contains a pH-responsive stability element. 876 Feb 53

The onset of metabolic acidosis causes an increased transcription of the renal phosphoenolpyruvate carboxykinase (PCK) gene. When transgenic mice carrying a bovine growth hormone (bGH) gene driven by the -460 to +73 segment of the PCK promoter were made chronically acidotic, the bGH mRNA was increased twofold after 4 days. Confluent and well-differentiated cultures of LLC-PK1-F+ cells exhibit a 2.5-fold increase in PCK mRNA when transferred to acidic media (pH 6.9, 10 mM HCO3-) for 16 h. Confluent cultures transfected with PCK-490 CAT exhibit an increase (3.5-fold) in chloramphenicol acetyltransferase (CAT) activity when shifted to acidic medium for 48 h. Mutation or deletion of the P2 element causes a four- to fivefold decrease in basal CAT activity but does not affect the pH response. In contrast, mutations of the P3(II) element or the CRE-1 cAMP-response element have little effect on basal activity but cause a 50% decrease in the pH response. Other deletions or mutations have little effect on either activity. Thus changes in the activity or levels of the protein(s) in the renal proximal tubule that binds to the P3(II) and CRE-1 elements may mediate increased transcription of the PCK gene during metabolic acidosis.
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PMID:Promoter elements that mediate the pH response of PCK mRNA in LLC-PK1-F+ cells. 877 Jan 65

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+ cells caused a rapid decrease in the level of phosphoenolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM HCO-3) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the alpha-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life (t1/2 = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription (t1/2 = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+ cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+ cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.
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PMID:PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+ cells. 972 8

Type 2 diabetes is characterized by the inability of insulin to suppress glucose production in the liver and kidney. Insulin inhibits glucose production by indirect and direct mechanisms. The latter result in transcriptional suppression of key gluconeogenetic and glycogenolytic enzymes, phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6p). The transcription factors required for this effect are incompletely characterized. We report that in glucogenetic kidney epithelial cells, Pepck and G6p expression are induced by dexamethasone (dex) and cAMP, but fail to be inhibited by insulin. The inability to respond to insulin is associated with reduced expression of the forkhead transcription factor Foxo1, a substrate of the Akt kinase that is inhibited by insulin through phosphorylation. Transduction of kidney cells with recombinant adenovirus encoding Foxo1 results in insulin inhibition of dex/cAMP-induced G6p expression. Moreover, expression of dominant negative Foxo1 mutant results in partial inhibition of dex/cAMP-induced G6p and Pepck expression in primary cultures of mouse hepatocyes and kidney LLC-PK1-FBPase(+) cells. These findings are consistent with the possibility that Foxo1 is involved in insulin regulation of glucose production by mediating the ability of insulin to decrease the glucocorticoid/cAMP response of G6p.
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PMID:The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. 1169 81

The increase in intracellular pH (pHi) associated with various tumour cells triggers changes in gene expression. Similar adaptations also occur as part of the physiological response to changes in acid base balance. For example, during metabolic acidosis, increased renal ammoniagenesis and bicarbonate synthesis are sustained by the increased expression of various transport proteins and key enzymes of glutamine metabolism. In rat kidney, increased expression of the mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) results from stabilization of their respective mRNAs. The 3'-untranslated region (UTR) of the GA mRNA contains a direct repeat of an 8-base AU sequence that functions as a pH-response element. This sequence exhibits a high affinity and specificity for z-crystallin. The same protein binds to two separate, but homologous, 8-base AU sequences within the 3'-UTR of the GDH mRNA. The apparent binding activity of z-crystallin is increased significantly during onset of metabolic acidosis. Thus, increased binding of z-crystallin may initiate the pH-responsive stabilization of the two mRNAs. In contrast, induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene occurs at the transcriptional level. In LLC-PK1-FBPase+ kidney cells, a decrease in pHi leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of ATF-2. This transcription factor binds to the CRE-1 element within the promoter of the PEPCK gene to enhance transcription. Similar mechanisms may contribute to altered gene expression in tumour cells.
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PMID:pH regulation of renal gene expression. 1172 24

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3'-untranslated region (3'-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds zeta-crystallin/NADPH:quinone reductase (zeta-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that zeta-crystallin also binds to the full-length 3'-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind zeta-crystallin with different affinities. Insertion of the 3'-UTR of GDH cDNA into a beta-globin expression vector (pbetaG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+ cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a betaG construct that contained only the single GDH4 ARE and a destabilizing element from phosphoenolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.
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PMID:pH-responsive stabilization of glutamate dehydrogenase mRNA in LLC-PK1-F+ cells. 1268 30

The transcription of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) gene is differentially regulated in each of the several PEPCK-C-expressing tissues. In the kidney, it is regulated by glucocorticoids and acidosis. Previously, we reported that in LLC-PK1 and derived kidney cell lines, mutation of the hepatic nuclear factor 1 (HNF-1) binding site in PEPCK-C gene promoter markedly reduced both the basal activity of the gene promoter and its response to acidic pH. Using the same kidney cell line, we now report that nuclear receptors robustly stimulate transcription from the PEPCK-C gene promoter. This stimulation is markedly reduced by mutation of the accessory factor 1 (AF1) site in the glucocorticoid responsive unit (GRU) residing within the glucocorticoid-responsive domain. The stimulation is likewise reduced by mutation of the HNF-1 site, residing outside the nuclear receptor-responsive domain of the PEPCK-C gene promoter. There is no binding similarity between HNF-1 and AF1 binding sites, as is evident from gel shift assays showing a lack of competition of either site for the binding of renal nuclear proteins to the other. We further assessed that the regulation of PEPCK-C gene transcription by acidosis is not mediated by nuclear receptors. This became evident from studies of transgenic mice harboring a rat PEPCK-C transgene driven by truncated 5' flanking region of the gene, which contains the HNF-1 site but lacks the glucocorticoid responsive domain. The full transcriptional response of this transgene to acidosis establishes that the truncated 5' flanking region (362 bp) of the PEPCK-C gene contains the information required for the acidosis-mediated regulation independent of the glucocorticoid domain. Taking together the previous and present results, it appears that acidosis and nuclear receptors regulate the renal transcription of the PEPCK-C gene via two independent domains in the 5' flanking region of the gene. These two modulations, as well as the basal activity of the gene, require intact HNF-1 binding site in the gene promoter.
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PMID:The transcriptional regulation of phosphoenolpyruvate carboxykinase gene in the kidney requires the HNF-1 binding site of the gene. 1458 10


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