Showing papers on "Pyruvate kinase published in 2004"

Carbohydrate response element binding protein directly promotes lipogenic enzyme gene transcription

Abstract: Carbohydrate response element (ChRE)-binding protein (ChREBP) is a recently discovered transcription factor that is activated in response to high glucose concentrations in liver independently of insulin. ChREBP was first identified by its ability to bind the ChRE of the liver pyruvate kinase (LPK) gene. We recently reported that the increase in expression of multiple liver lipogenic enzyme mRNAs elicited by feeding a high-carbohydrate diet as well as that of LPK mRNA is markedly reduced in mice lacking ChREBP gene expression (ChREBP-/-) in comparison to WT mice. The present study provides evidence for a direct and dominant role of ChREBP in the glucose regulation of two key liver lipogenic enzymes, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). ACC, FAS, and LPK mRNA levels were higher in WT hepatocytes cultured with high (25 mM) rather than low (5.5 mM) glucose medium, but there was no effect of glucose concentration on these mRNA levels in ChREBP-/- hepatocytes. Similarly, reporter constructs containing ACC, FAS, or LPK gene ChREs were responsive to glucose when transfected into WT but not ChREBP-/- hepatocytes, and glucose transactivation of the constructs in ChREBP-/- hepatocytes was restored by cotransfection with a ChREBP expression plasmid. ChREBP binding to ACC, FAS, and LPK ChRE sequences in vitro was demonstrated by electrophoretic mobility super shift assays. In vivo binding of ChREBP to ACC, FAS, and LPK gene promoters in intact liver nuclei from rats fed a high-carbohydrate diet was demonstrated by using a formaldehyde crosslinking and chromatin immunoprecipitation procedure.

Protein Kinase B-α Inhibits Human Pyruvate Dehydrogenase Kinase-4 Gene Induction by Dexamethasone Through Inactivation of FOXO Transcription Factors

Abstract: Starvation and diabetes increase pyruvate dehydrogenase kinase-4 (PDK4) expression, which conserves gluconeogenic substrates by inactivating the pyruvate dehydrogenase complex. Mechanisms that regulate PDK4 gene expression, previously established to be increased by glucocorticoids and decreased by insulin, were studied. Treatment of HepG2 cells with dexamethasone increases the relative abundance of PDK4 mRNA, and insulin blocks this effect. Dexamethasone also increases human PDK4 (hPDK4) promoter activity in HepG2 cells, and insulin partially inhibits this effect. Expression of constitutively active PKBα abrogates dexamethasone stimulation of hPDK4 promoter activity, while coexpression of constitutively active FOXO1a or FOXO3a, which are mutated to alanine at the three phosphorylation sites for protein kinase B (PKB), disrupts the ability of PKBα to inhibit promoter activity. A glucocorticoid response element for glucocorticoid receptor (GR) binding and three insulin response sequences (IRSs) that bind FOXO1a and FOXO3a are identified in the hPDK4 promoter. Mutation of the IRSs reduces the ability of glucocorticoids to stimulate PDK4 transcription. Transfection studies with E1A, which binds to and inactivates p300/CBP, suggest that interactions between p300/CBP and GR as well as FOXO factors are important for glucocorticoid-stimulated hPDK4 expression. Insulin suppresses the hPDK4 induction by glucocorticoids through inactivation of the FOXO factors.

Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations

Abstract: Metabolic flux analysis based on 13 C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykFmutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6- phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykFmutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracel- lular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.

The effect of pfl gene knockout on the metabolism for optically pure d-lactate production by Escherichia coli

Abstract: The effect of gene knockout on metabolism in the pflA−, pflB−, pflC−, and pflD− mutants of Escherichia coli was investigated. Batch cultivations of the pfl− mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA− and pflB− mutants, but not pflC− and pflD− mutants, produced large amounts of d-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD+ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA− and pflB− mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD+ ratio in pfl− mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.

Analysis of in vivo kinetics of glycolysis in aerobic Saccharomyces cerevisiae by application of glucose and ethanol pulses.

Abstract: This article presents the dynamic responses of several intra- and extracellular components of an aerobic, glucose-limited chemostat culture of Saccharomyces cerevisiae to glucose and ethanol pulses within a time window of 75 sec. Even though the ethanol pulse cannot perturb the glycolytic pathway directly, a distinct response of the metabolites at the lower part of glycolysis was found. We suggest that this response is an indirect effect, caused by perturbation of the NAD/NADH ratio, which is a direct consequence of the conversion of ethanol into acetaldehyde. This effect of the NAD/NADH ratio on glycolysis might serve as an additional explanation for the observed decrease of 3PG, 2PG, and PEP during a glucose pulse. The responses measured during the ethanol pulse were used to evaluate the allosteric regulation of glycolysis. Our results confirm that FBP stimulates pyruvate kinase and suggest that this effect is pronounced. Furthermore, it appears that PEP does not play an important role in the allosteric regulation of phosphofructo kinase.

Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting

Abstract: Fasting forces adaptive changes in whole body and skeletal muscle metabolism that increase fat oxidation and decrease the oxidation of carbohydrate. We tested the hypothesis that 40 h of fasting would decrease pyruvate dehydrogenase (PDH) activity and increase PDH kinase (PDK) isoform mRNA expression in human skeletal muscle. The putative transcriptional activators of PDK isozymes, peroxisome proliferator-activated receptor-alpha (PPAR-alpha) protein, and forkhead homolog in rhabdomyosarcoma (FKHR) mRNA were also measured. Eleven healthy adults fasted after a standard meal (25% fat, 60% carbohydrate, 15% protein) with blood and skeletal muscle samples taken at 3, 15, and 40 h postprandial. Fasting increased plasma free fatty acid, glycerol, and beta-hydroxybutyrate concentrations and decreased glucose and insulin concentrations. PDH activity decreased from 0.88 +/- 0.11 mmol acetyl-CoA. min(-1). kg wet muscle wt(-1) at 3 h to 0.62 +/- 0.10 (P = not significant) and 0.39 +/- 0.06 (P < 0.05) mmol. min(-1). kg wet mass(-1) after 15 and 40 h of fasting. Although all four PDK isoforms were expressed in human skeletal muscle, PDK-2 and -4 mRNA were the most abundant. PDK-1 and -3 mRNA abundance was approximately 1 and 15% of the PDK-2 and -4 levels, respectively. The 40-h fast had no effect on PDK-1, -2, and -3 mRNA expression. PDK-4 mRNA was significantly increased approximately 3-fold after 15 h and approximately 14-fold after 40 h of fasting. Skeletal muscle PPAR-alpha protein and FKHR mRNA abundance were unaffected by the fast. The results suggest that decreased PDH activation after 40 h of fasting may have been a function of the large increase in PDK-4 mRNA expression and possible subsequent increase in PDK protein and activity. The changes in PDK-4 expression and PDH activity did not coincide with increases in the transcriptional activators PPAR-alpha and FKHR.

Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli.

Abstract: The metabolic regulation of Escherichia coli lacking a functional pykF gene was investigated based on gene expressions, enzyme activities, intracellular metabolite concentrations and the metabolic flux distribution obtained based on 13C-labeling experiments. RT-PCR revealed that the glycolytic genes such as glk, pgi, pfkA and tpiA were down regulated, that ppc, pckA, maeB and mdh genes were strongly up-regulated, and that the oxidative pentose phosphate pathway genes such as zwf and gnd were significantly up-regulated in the pykF mutant. The catabolite repressor/activator gene fruR was up-regulated in the pykF mutant, but the adenylate cyclase gene cyaA was down-regulated indicating a decreased rate of glucose uptake. This was also ascertained by the degradation of ptsG mRNA, the gene for which was down-regulated in the pykF mutant. In general, the changes in enzyme activities more or less correlated with ratios of gene expression, while the changes in metabolic fluxes did not correlate with enzyme activities. For example, high flux ratios were obtained through the oxidative pentose phosphate pathway due to an increased concentration of glucose-6-phosphate rather than to favorable enzyme activity ratios. In contrast, due to decreased availability of pyruvate (and acetyl coenzyme A) in the pykF mutant compared with the wild type, low flux ratios were found through lactate and acetate forming pathways.

Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle.

Abstract: Recent studies have revealed the structural and functional interactions between mitochondria, myofibrils and sarcoplasmic reticulum in cardiac cells. Direct channeling of adenosine phosphates between organelles identified in the experiments indicates that diffusion of adenosine phosphates is limited in cardiac cells due to very specific intracellular structural organization. However, the mode of diffusion restrictions and nature of the intracellular structures in creating the diffusion barriers is still unclear, and, therefore, a subject of active research. The aim of this work is to analyze the possible role of two principally different modes of restriction distribution for adenosine phosphates (a) the uniform diffusion restriction and (b) the localized diffusion limitation in the vicinity of mitochondria, by fitting the experimental data with the mathematical model. The reaction-diffusion model of compartmentalized energy transfer was used to analyze the data obtained from the experiments with the skinned muscle fibers, which described the following processes: mitochondrial respiration rate dependency on exogenous ADP and ATP concentrations; inhibition of endogenous ADP-stimulated respiration by pyruvate kinase (PK) and phosphoenolpyruvate (PEP) system; kinetics of oxygen consumption stabilization after addition of 2 mM MgATP or MgADP; ATPase activity with inhibited mitochondrial respiration; and buildup of MgADP concentration in the medium after addition of MgATP. The analysis revealed that only the second mechanism considered – localization of diffusion restrictions – is able to account for the experimental data. In the case of uniform diffusion restrictions, the model solution was in agreement only with two measurements: the respiration rate as a function of ADP or ATP concentrations and inhibition of respiration by PK + PEP. It was concluded that intracellular diffusion restrictions for adenosine phosphates are not distributed uniformly, but rather are localized in certain compartments of the cardiac cells.

Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis.

Abstract: In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction. However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated. We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source. Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium. A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated. DNA microarray experiments revealed increased expression of the malic enzyme gene malE. The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis. Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate. The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate. Whereas during growth of C. glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.

Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines.

Abstract: Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5-fluorouracil (5-FU) were established from human gastric carcinoma cell lines SNU-638 and SNU-620. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and matrix-associated laser desorption ionization-mass spectroscopy (MALDI-MS) was performed on protein extracts from these parental and drug-resistant derivative lines to screen drug resistance-related proteins. Pyruvate kinase M2 (PK-M2) was identified as a protein showing lower expression in cisplatin-resistant cells compared to parental cells. Consistent with this finding, PK-M2 activity was also lower in cisplatin-resistant cells. Suppression of PK-M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU-638 cells. Furthermore, PK-M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK-M2 protein and activity levels were lower in cisplatin-resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK-M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK-M2 and cisplatin resistance mechanisms. © 2003 Wiley-Liss, Inc.

Differential lowering by manganese treatment of activities of glycolytic and tricarboxylic acid (TCA) cycle enzymes investigated in neuroblastoma and astrocytoma cells is associated with manganese-induced cell death.

Abstract: Manganese (Mn) is a trace metal required for normal growth and development. Manganese neurotoxicity is rare and usually associated with occupational exposures. However, the cellular and molecular mechanisms underlying Mn toxicity are still elusive. In rats chronically exposed to Mn, their brain regional Mn levels increase in a dose-related manner. Brain Mn preferentially accumulates in mitochondria; this accumulation is further enhanced with Mn treatment in vivo. Exposure of mitochondria to Mn in vitro leads to uncoupling of oxidative phosphorylation. These observations prompted us to investigate the hypothesis that Mn induces alterations in energy metabolism in neural cells by interfering with the activities of various glycolytic and TCA cycle enzymes using human neuroblastoma (SK-N-SH) and astrocytoma (U87) cells. Treatments of SK-N-SH and U87 cells with MnCl2 induced cell death in these cells, in a concentration- and time-dependent manner, as determined by MTT assays. In parallel with the Mn-induced, dose-dependent decrease in cell survival, treatment of these cells with 0.01 to 4.0 mM MnCl2 for 48 h also induced dose-related decreases in their activities of hexokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, and malate dehydrogenase. Hexokinase in SK-N-SH cells was the most affected by Mn treatments, even at the lower range of concentrations. Mn treatment of SK-N-SH cells affected pyruvate kinase and citrate synthase to a lesser extent as compared to its effect on other enzymes investigated. However, citrate synthase and pyruvate kinase in U87 cells were more vulnerable than other enzymes investigated to the effects of Mn. The results suggest the two cell types exhibited differential susceptibility toward the Mn-induced effects. Additionally, the results may have significant implications in flux control because HK is the first and highly regulated enzyme in brain glycolysis. Thus these results are consistent with our hypothesis and may have pathophysiological implications in the mechanisms underlying Mn neurotoxicity.

Developmental analysis of carbohydrate metabolism in tomato (Lycopersicon esculentum cv. Micro-Tom) fruits

Abstract: Carbohydrate metabolism during the development of fruits of the tomato cultivar Micro-Tom was studied. The metabolism of the pericarp and placental tissues was found to be different. Starch was degraded more slowly in the placenta in comparison with the pericarp, whereas soluble sugars accumulated to a greater extent in the pericarp. The activities of glycolytic enzymes tended to peak at 40 days after flowering. Two of these, phosphoenolpyruvate phosphatase and pyruvate kinase, showed a dramatic increase in activity just before this peak, possibly indicating a role in up-regulating glycolysis to generate increased ATP that would be used during climacteric respiration. The expression of plastidial transporters was studied. Both the TPT and Glu6P transporter were expressed greatest in green fruits, before declining. The expression of the triose-phosphate transporter was greater than that of the glucose 6-phosphate transporter. The ATP/ADP transporter was expressed to a low level throughout fruit development.

Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis

Abstract: Lactococcus lactis strain NZ9000(pNZpyk), which overproduces pyruvate kinase (PK), was constructed. The pNZpyk plasmid carries the P nisA –pyk transcriptional fusion, and the overexpression of its pyk gene was accomplished by using the nisin-inducible expression system of the NZ9000 strain. In vivo 13C- and 31P-NMR spectroscopy was used to evaluate the effect of this modification on the metabolism of glucose in non-growing cells. A detailed description of the kinetics of glucose, end products, glycolytic intermediates, NAD+ and NADH was obtained. A 15-fold increase in the level of PK did not increase the overall glycolytic flux, which, on the contrary, was slightly reduced. Significant differences were observed in (i) the level of 3-phosphoglycerate (3-PGA) and phosphoenolpyruvate (PEP), metabolites associated with starvation; (ii) the rate of fructose 1,6-bisphosphate (FBP) depletion upon glucose exhaustion; and (iii) the NAD+/NADH ratio during glucose catabolism. In the mutant, the rate of FBP consumption after glucose depletion was notably accelerated under anaerobic conditions, whereas 3-PGA and PEP decreased to undetectable levels. Furthermore, the level of NAD+ decreased steadily during the utilization of glucose, probably due to the unanticipated reduction in the lactate dehydrogenase activity in comparison with the control strain, NZ9000(pNZ8020). The results show that PK is an important bottleneck to carbon flux only when glucose becomes limiting; in the overproducer this constriction was no longer present, as evidenced by the faster FBP consumption and lack of accumulation of 3-PGA and PEP in anaerobic as well as aerobic conditions. Despite these clear changes, the PK-overproducing strain showed typical homolactic metabolism under anaerobic conditions, as did the strain harbouring the vector plasmid without the pyk insert. However, under an oxygen atmosphere, there was increased channelling of carbon to the production of acetate and acetoin, to the detriment of lactate production.

Hexachlorobenzene impairs glucose metabolism in a rat model of porphyria cutanea tarda: a mechanistic approach.

Abstract: Hexachlobenzene (HCB), one of the most persistent environmental pollutants, induces porphyria cutanea tarda (PCT). The aim of this work was to analyze the effect of HCB on some aspects of glucose metabolism, particularly those related to its neosynthesis in vivo. For this purpose, a time-course study on gluconeogenic enzymes, pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G-6-Pase) and on pyruvate kinase (PK), a glycolytic enzyme, was carried out. Plasma glucose and insulin levels, hepatic glycogen, tryptophan contents, and the pancreatic insulin secretion pattern stimulated by glucose were investigated. Oxidative stress and heme pathway parameters were also evaluated. HCB treatment decreased PC, PEPCK, and G-6-Pase activities. The effect was observed at an early time point and grew as the treatment progressed. Loss of 60, 56, and 37%, respectively, was noted at the end of the treatment when a considerable amount of porphyrins had accumulated in the liver as a result of drastic blockage of uroporphyrinogen decarboxylase (URO-D) (95% inhibition). The plasma glucose level was reduced (one-third loss), while storage of hepatic glucose was stimulated in a time-dependent way by HCB treatment. A decay in the normal plasma insulin level was observed as fungicide intoxication progressed (twice to four times lower). However, normal insulin secretion of perifused pancreatic Langerhans islets stimulated by glucose during the 3rd and 6th weeks of treatment did not prove to be significantly affected. HCB promoted a time-dependent increase in urinary chemiluminiscence (fourfold) and hepatic malondialdehide (MDA) content (fivefold), while the liver tryptophan level was only raised at the longest intoxication times. These results would suggest that HCB treatment does not cause a primary alteration in the mechanism of pancreatic insulin secretion and that the changes induced by the fungicide on insulin levels would be an adaptative response of the organism to stimulate gluconeogenesis. They showed for the first time that HCB causes impairment of the gluconeogenic pathway. Therefore, the reduced levels of glucose would thus be the consequence of decreased gluconeogenesis, enhanced glucose storage, and unaffected glycolysis. The impairment of gluconeogenesis (especially for PEPCK) and the related variation in glucose levels caused by HCB treatment could be a consequence of the oxidative stress produced by the fungicide. Tryptophan adds its effect to this decrease in the higher phases of HCB intoxication, where its levels overcome the control values possibly owing to the drastic decline of URO-D. This derangement of carbohydrates leads porphyric hepatocytes to have lower levels of free glucose. These results contribute to our understanding of the protective and modulatory effect that diets rich in carbohydrates have in hepatic porphyria disease.

Cadmium induces both pyruvate kinase and Na+/H+ exchanger activity through protein kinase C mediated signal transduction, in isolated digestive gland cells of Mytilus galloprovincialis (L.).

Abstract: SUMMARY The present study investigates the transduction pathway mediated by cadmium in isolated digestive gland cells of mussel Mytilus galloprovincialis. The effects of cadmium treatment on a key glycolytic enzyme, pyruvate kinase (PK), and on Na+/H+ exchanger activity were examined. Cadmium (50 μmol l–1) caused a significant elevation of intracellular pH (pHi) and a rise (176%) of Na influx relative to control values. The amiloride analogue, EIPA (20 nmol l–1), a Na+/H+ exchanger blocker, together with cadmium, significantly reduced the effect of treatment by cadmium alone on both Na+ influx and pHi. In addition, PK activity was significantly increased after treatment with cadmium. PK activity was inhibited after treatment of cells with amiloride or EIPA together with cadmium. Moreover, phorbol-ester (PMA), a potent activator of protein kinase C (PKC), caused a significant rise in both pHi and PK activity, while staurosporine or calphostin C reversed both events. Adrenaline, isoprenaline and phenylephrine alone or together with cadmium also significantly increased the pHi and PK activity of isolated digestive gland cells. The latter effectors in combination with cadmium showed a synergistic effect on pHi and PK. These responses seem to be blocked by propranolol, metoprolol and prazosin. Our findings suggest a hormone-like effect of cadmium on digestive gland cells. The signal transduction pathway induced by cadmium involves the stimulation of PK, PKC and Na+/H+ exchanger in isolated digestive gland cells of Mytilus galloprovincialis.

Phenotypic expression of pyruvate kinase deficiency and protection against malaria in a mouse model

Abstract: The recombinant congenic mouse strains AcB55 and AcB61 are extremely resistant to malaria (Plasmodium chabaudi AS) despite the presence of susceptibility alleles at the known Char1/Char2 resistance loci. Resistance in AcB55 and AcB61 is controlled by a locus on chromosome 3 (Char4) shown to be allelic with or tightly linked to a loss-of-function mutation in pyruvate kinase (Pklr). AcB55 and AcB61 show important splenomegaly prior to infection caused by the expansion of the red pulp, and display histological signs of extramedullary erythropoiesis in the liver. Examination of splenic cell populations by flow cytometry demonstrates elevated numbers of TER119-positive erythroid precursor cells (>30% of total spleen cells), while RNA expression studies show elevated expression of erythrocyte-specific transcripts such as globin, transferrin receptor, and Nramp2/Slc11a2 in the spleen of both strains. Hematological profiling in both strains is consistent with the presence of anemia as evidenced by low total erythrocyte counts, decreased hemoglobin, as well as abnormally high numbers of circulating reticulocytes (15–20%). These results strongly suggest that the mutant Pklr allele (Pklr269A) of AcB55/61 strains causes hemolytic anemia compensated by constitutive erythropoiesis, which in turn protects the mice against P. chabaudi infection. The possible molecular basis of the Pklr protective effect is discussed and is under current investigation in these two strains.

Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise

Abstract: Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entr...

Erythrocyte changes in canine diabetes mellitus: in vitro effects of hyperglycaemia and ketoacidosis

Abstract: Diabetes mellitus (DM) is a common metabolic disorder in dogs, which occurs often in association with some complications, including haematological problems. Some abnormalities of erythrocyte metabolism have been described both in human patients and in dogs affected by DM. The aim of this work was to test in vitro the direct effects of hyperglycaemia and ketoacidosis on canine erythrocytes (RBCs). RBCs from healthy dogs were incubated in normoglycaemic, mild hyperglycaemic, severe hyperglycaemic and severe hyperglycaemic + ketoacidotic media. The following parameters were examined: osmotic fragility (OF), the concentrations of 2,3-diphosphoglycerate (2,3DPG), reduced glutathione (GSH) and adenosine triphosphate (ATP), the activities of glutathione peroxidase (GPX), glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK), sodium (Na+) and potassium (K+) intracellular concentrations, production of lactate. In comparison with t0, incubated cells showed higher OF (P<0.001) and lower 2,3DPG (P<0.001) and ATP (P<0.001) concentrations. In comparison with normoglycaemic conditions, hyperglycaemia induced minimal changes, such as increased OF (P<0.001) and 2,3DPG (P<0.01) concentrations. Major changes were induced by hyperglycaemic + ketoacidotic media, such as increases in mean corpuscular volume (MCV), OF, PK activity and ATP concentration; this suggested that oxidative stress had occurred. In hyperglycaemic media, lactate production was not associated with glucose concentration, but the higher consumption of glucose suggests the activation of metabolic pathways other than glycolysis. These alterations could be partly responsible for some hypoxic complications during DM.

A novel method for determination of ATP, ADP, and AMP contents of a single pancreatic islet before transplantation

Abstract: Assessment of islet viability before transplantation is mandatory for successful clinical transplantation. ATP content or energy charge (EC) of islets may represent a good parameter to assess viability. We have introduced a novel bioluminescent enzymatic cycling assay using synthetic firefly luciferase, pyruvate kinase) (PK), and pyruvate orthophosphate dikinase (PPDK) to determine adenine nucleotides (AN) in isolated islets. A complete assay requires several minutes. The ATP contents of 1, 3, 10, 30 or 100 islets each with a diameter of 150 μm were 9.95 ± 0.03, 28.3 ± 1.18, 89.5 ± 1.48, 282 ± 10.2, and 673 ± 27.1 pmol, respectively, showing a relatively constant ATP content per 30 islets (9.95, 9.42, 8.95, and 9.41 pmol/IEQ). ECs of each group were 0.74 ± 0.02, 0.74 ± 0.02, 0.75 ± 0.02, 0.74 ± 0.02, and 0.65 ± 0.01, respectively, a value that was quite constant up to 30 islets. The quantity of ATP and EC in a single islet can be measured quickly and reproducibly, offering a new method to determine viability of isolated islets prior to transplantation.

Ex vivo analysis of aberrant splicing induced by two donor site mutations in PKLR of a patient with severe pyruvate kinase deficiency

Abstract: Two single-nucleotide substitutions in PKLR constituted the molecular basis underlying pyruvate kinase (PK) deficiency in a patient with severe haemolytic anaemia. One novel mutation, IVS5+1G>A, abolished the intron 5 donor splice site. The other mutation, c.1436G>A, altered the intron 10 donor splice site consensus sequence and, moreover, encoded an R479H substitution. We studied the effects on PKLR pre-mRNA processing, using ex vivo-produced nucleated erythroid cells from the patient. Abolition of the intron 5 splice site initiated two events in the majority of transcripts: skipping of exon 5 or, surprisingly, simultaneous skipping of exon 5 and 6 (Delta5,6). Subcellular localization of transcripts suggested that no functional protein was produced by the IVS5+1A allele. The unusual Delta5,6 transcript suggests that efficient inclusion of exon 6 in wild-type PKLR mRNA depends on the presence of splice-enhancing elements in exon 5. The c.1436G>A mutation caused skipping of exon 10 but was mainly associated with a severe reduction in transcripts although these were, in general, normally processed. Accordingly, low amounts of PK were detected in nucleated erythroid cells of the patient, thus correlating with the patient's PK-deficient phenotype. Finally, several low-abundant transcripts were detected that represent the first examples of "leaky-splicing" in PKLR.

Proton donor in yeast pyruvate kinase: chemical and kinetic properties of the active site Thr 298 to Cys mutant.

Abstract: The active site T298 residue of yeast pyruvate kinase (YPK), located in a position to serve potentially as the proton donor, was mutated to cysteine. T298C YPK was isolated and purified, and its enzymatic properties were characterized. Fluorescence and CD spectra indicate minor structural perturbations. A kinetic analysis of the Mg 2 + -activated enzyme demonstrates no catalytic activity in the absence of the heterotropic activator fructose 1,6-bisphosphate (FBP). In the presence of Mg 2 + and FBP, T298C has approximately 20% of the activity of wild-type (wt) YPK. The activator constant for FBP increases by 1 order of magnitude compared to this constant with the wt enzyme. T298C shows positive cooperativity by FBP with a Hill coefficient of 2.6 (wt, n H , F B P = 1). Mn 2 + -activated T298C behaves like Mn 2 + -activated wt YPK with a V m a x that is 20% of that for the wt enzyme with or without FBP. A pH-rate profile of T298C relative to that for wt YPK shows that pK a , 2 has shifted from 6.4 in wt to 5.5, indicating that the thiol group elicits an acidic pK shift. Inactivation of both wt and T298C by iodoacetate elicits a pseudo-first-order loss of activity with T298C being inactivated from 8 to 100 times faster than wt YPK. A pH dependence of the inactivation rate constant for T298C gives a value of 8.2, consistent with the pK for a thiol. Changes in fluorescence indicate that the T298C-Mg 2 + complex binds PEP, ADP, and both ligands together. This demonstrates that the lack of activity is not due to the loss of substrate binding but to the lack of ability to induce the proper conformational change. The mutation also induces changes in binding of FBP to all the relevant complexes. Binding of the metal and binding of PEP to the enzyme complexes are also differentially altered. Solvent isotope effects are observed for both wt and T298C. Proton inventory studies indicate that k c a t is affected by a proton from water in the transition state and the effects are metal ion-dependent. The results are consistent with water being the active site proton donor. Active site residue T298 is not critical for activity but plays a role in the activation of the water and affects the pK that modulates catalytic activity.

Possible involvement of pyruvate kinase in acquisition of tolerance to hypoxic stress in glial cells

Abstract: Neurons are highly vulnerable to ischemic/hypoxic stress, while glial cells show tolerance to such stress. However, the mechanisms for tolerance acquisition in glial cells have yet to be established. We attempted to isolate and identify a stress protein that is upregulated in response to hypoxia in human astrocytoma CCF-STTG1 cells. In particular, pyruvate kinase (PK) was upregulated by hypoxia in CCF-STTG1 cells. Hypoxia-inducible factor 1 (HIF-1), the primary transcription factor that is responsible for multiple gene activation under hypoxia, plays a critical role in PK expression during hypoxic challenge. To determine whether newly synthesized PK is involved in tolerance to hypoxic stress, we established the PK-overexpressing neuronal cells. Overexpression of the wild-type, but not the kinase-negative mutant, resulted in attenuation of the loss of cell viability and the typical apoptotic features by hypoxia or oxidative stress in SK-N-MC cells. These findings suggest that upregulation of PK may result in acquisition of tolerance against hypoxic stress, and that the antioxidant effect may be involved in the protective effect of PK.