Showing papers on "Pyruvate kinase published in 2009"
TL;DR: Overall, data strongly suggest that the liver of most fish species is apparently capable of regulating glucose storage, and the persistent high level of endogenous glucose production independent of carbohydrate intake level may lead to a putative competition between exogenous glucose and endogenous glucose as the source of energy.
Abstract: Glucose plays a key role as energy source in the majority of mammals, but its importance in fish appears limited. Until now, the physiological basis for such apparent glucose intolerance in fish has not been fully understood. A distinct regulation of hepatic glucose utilization (glycolysis) and production (gluconeogenesis) may be advanced to explain the relative inability of fish to efficiently utilize dietary glucose. We summarize here information regarding the nutritional regulation of key enzymes involved in glycolysis (hexokinases, 6-phosphofructo-1-kinase and pyruvate kinase) and gluconeogenesis (phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase) pathways as well as that of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The effect of dietary carbohydrate level and source on the activities and gene expression of the mentioned key enzymes is also discussed. Overall, data strongly suggest that the liver of most fish species is apparently capable of regulating glucose storage. The persistent high level of endogenous glucose production independent of carbohydrate intake level may lead to a putative competition between exogenous (dietary) glucose and endogenous glucose as the source of energy, which may explain the poor dietary carbohydrate utilization in fish.
400 citations
TL;DR: regulation of enzymes at the level of transcripts and proteins is likely to play an important role in plant adaptation to K deficiency by maintaining carbon flux into amino acids and proteins, decreasing negative metabolic charge, and increasing the nitrogen-carbon ratio in amino acids.
Abstract: Potassium (K) is required in large quantities by growing crops, but faced with high fertilizer prices, farmers often neglect K application in favor of nitrogen and phosphorus. As a result, large areas of farmland are now depleted of K. K deficiency affects the metabolite content of crops with negative consequences for nutritional quality, mechanical stability, and pathogen/pest resistance. Known functions of K in solute transport, protein synthesis, and enzyme activation point to a close relationship between K and metabolism, but it is unclear which of these are the most critical ones and should be targeted in biotechnological efforts to improve K usage efficiency. To identify metabolic targets and signaling components of K stress, we adopted a multilevel approach combining transcript profiles with enzyme activities and metabolite profiles of Arabidopsis (Arabidopsis thaliana) plants subjected to low K and K resupply. Roots and shoots were analyzed separately. Our results show that regulation of enzymes at the level of transcripts and proteins is likely to play an important role in plant adaptation to K deficiency by (1) maintaining carbon flux into amino acids and proteins, (2) decreasing negative metabolic charge, and (3) increasing the nitrogen-carbon ratio in amino acids. However, changes in transcripts and enzyme activities do not explain the strong and reversible depletion of pyruvate and accumulation of sugars observed in the roots of low-K plants. We propose that the primary cause of metabolic disorders in low-K plants resides in the direct inhibition of pyruvate kinase activity by low cytoplasmic K in root cells.
285 citations
TL;DR: Flux analysis in cultured embryos revealed changes in nutrient uptakes and fluxes into biomass as well as an increase in tricarboxylic acid cycle activity for both mutations.
Abstract: Major storage reserves of Arabidopsis (Arabidopsis thaliana) seeds are triacylglycerols (seed oils) and proteins. Seed oil content is severely reduced for the regulatory mutant wrinkled1 (wri1-1; At3g54320) and for a double mutant in two isoforms of plastidic pyruvate kinase (pkpβ1pkpα; At5g52920 and At3g22960). Both already biochemically well-characterized mutants were now studied by 13C metabolic flux analysis of cultured developing embryos based on comparison with their respective genetic wild-type backgrounds. For both mutations, in seeds as well as in cultured embryos, the oil fraction was strongly reduced while the fractions of proteins and free metabolites increased. Flux analysis in cultured embryos revealed changes in nutrient uptakes and fluxes into biomass as well as an increase in tricarboxylic acid cycle activity for both mutations. While in both wild types plastidic pyruvate kinase (PKp) provides most of the pyruvate for plastidic fatty acid synthesis, the flux through PKp is reduced in pkpβ1pkpα by 43% of the wild-type value. In wri1-1, PKp flux is even more reduced (by 82%), although the genes PKpβ1 and PKpα are still expressed. Along a common paradigm of metabolic control theory, it is hypothesized that a large reduction in PKp enzyme activity in pkpβ1pkpα has less effect on PKp flux than multiple smaller reductions in glycolytic enzymes in wri1-1. In addition, only in the wri1-1 mutant is the large reduction in PKp flux compensated in part by an increased import of cytosolic pyruvate and by plastidic malic enzyme. No such limited compensatory bypass could be observed in pkpβ1pkpα.
150 citations
TL;DR: These studies show that cancer cells effectively maintain low levels of pyruvate to prevent inhibition of HDAC1/HDAC3 and thereby to evade cell death.
Abstract: Human colon cancer cells and primary colon cancer silence the gene coding for LDH (lactate dehydrogenase)-B and up-regulate the gene coding for LDH-A, resulting in effective conversion of pyruvate into lactate. This is associated with markedly reduced levels of pyruvate in cancer cells compared with non-malignant cells. The silencing of LDH-B in cancer cells occurs via DNA methylation, with involvement of the DNMTs (DNA methyltransferases) DNMT1 and DNMT3b. Colon cancer is also associated with the expression of pyruvate kinase M2, a splice variant with low catalytic activity. We have shown recently that pyruvate is an inhibitor of HDACs (histone deacetylases). Here we show that pyruvate is a specific inhibitor of HDAC1 and HDAC3. Lactate has no effect on any of the HDACs examined. Colon cancer cells exhibit increased HDAC activity compared with non-malignant cells. HDAC1 and HDAC3 are up-regulated in colon cancer cells and in primary colon cancer, and siRNA (small interfering RNA)-mediated silencing of HDAC1 and HDAC3 in colon cancer cells induces apoptosis. Colon cancer cells silence SLC5A8, the gene coding for a Na(+)-coupled pyruvate transporter. Heterologous expression of SLC5A8 in the human colon cancer cell line SW480 leads to inhibition of HDAC activity when cultured in the presence of pyruvate. This process is associated with an increase in intracellular levels of pyruvate, increase in the acetylation status of histone H4, and enhanced cell death. These studies show that cancer cells effectively maintain low levels of pyruvate to prevent inhibition of HDAC1/HDAC3 and thereby to evade cell death.
144 citations
TL;DR: It is concluded that genetic selection for muscle fat content resulted in overactivation of the TOR signaling pathway-associated lipogenesis and probably also improved utilization of glucose.
Abstract: Genetic selection is commonly used in farm animals to manage body fat content. In rainbow trout, divergent selection for low or high muscle fat content leads to differences in utilization of dietary energy sources between the fat muscle line (FL) and the lean muscle line (LL). To establish whether genetic selection on muscle fat content affects the hepatic insulin/nutrient signaling pathway, we analyzed this pathway and the expression of several metabolism-related target genes in the livers of the two divergent lines under fasting and then refeeding conditions. Whereas glycemia returned to basal level 24 h after refeeding in FL trout, it remained elevated in the LL trout. Target of rapamycin (TOR) protein was more abundant in the livers of FL trout than in LL trout, and refeeding activation of the hepatic TOR signaling pathway (TOR, S6K1, and S6) was therefore enhanced. Genes related to glycolysis (glucokinase and pyruvate kinase) and gluconeogenesis (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) were only slightly affected by refeeding and genetic selection. Refeeding stimulated expression of lipogenic genes and the sterol-responsive element binding protein (SREBP1), and expression of fatty acid synthase, glucose-6-phosphate dehydrogenase, and serine dehydratase was predominant in the livers of FL fish compared with LL fish. In agreement with recent findings linking TOR to lipogenesis control, we concluded that genetic selection for muscle fat content resulted in overactivation of the TOR signaling pathway-associated lipogenesis and probably also improved utilization of glucose.
119 citations
TL;DR: It is suggested that mitochondrial ND2 mutation contributes to HIF1α accumulation via increased ROS production, up-regulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in Hif1α stabilization.
Abstract: Purpose: Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1α accumulation and increased cell growth. The purpose of the present study was to examine the pathways relating these alterations. Experimental Design: Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1α, pyruvate dehydrogenease (PDH), phosphorylated PDH, and pyruvate dehydrogenease kinase 2 (PDK2), together with ROS generation, were compared between the mutant and the wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2 and mitochondria-targeted catalase were evaluated on the ND2 mutant transfectants. Results: We determined that ND2 mutant down-regulated PDH expression via up-regulated PDK2, with an increase in phosphorylated PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1α accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1α expression, and introduction of mitochondria-targeted catalase decreased mitochondrial mutation-mediated PDK2 and HIF1α expression and suppressed cell growth. Conclusions: Our findings suggest that mitochondrial ND2 mutation contributes to HIF1α accumulation via increased ROS production, up-regulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1α stabilization. This may provide insight into a potential mechanism, by which mitochondrial mutations contribute to HNSCC development.
99 citations
TL;DR: It is demonstrated that, at normal blood glucose concentrations, peptide aptamer-mediated inhibition of M2-PK induces a significant decrease of the population doubling and cell proliferation rate as well as a increase in cell size, whereas under glucose restriction an increase in PDL and celliferation rates but a decrease incell size was observed.
94 citations
TL;DR: Findings are the first evidence that PKCvarepsilon activity modulates cardiac glucose metabolism and provide a possible explanation for the synergistic effect of PKCdelta and PKCcdelta in cardioprotection.
78 citations
TL;DR: Results show directly that under physiological conditions the major energy carrier from mitochondria into cytoplasm is PCr, produced by mitochondrial creatine kinase (MtCK), which functional coupling to adenine nucleotide translocase is enhanced by selective limitation of permeability of mitochondrial outer membrane within supercomplex ATP Synthasome-Mt CK-VDAC-tubulin, Mitochondrial Interactosome.
Abstract: The aim of this study was to measure energy fluxes from mitochondria in isolated permeabilized cardiomyocytes. Respiration of permeabilized cardiomyocytes and mitochondrial membrane potential were measured in presence of MgATP, pyruvate kinase - phosphoenolpyruvate and creatine. ATP and phosphocreatine concentrations in medium surrounding cardiomyocytes were determined. While ATP concentration did not change in time, mitochondria effectively produced phosphocreatine (PCr) with PCr/O(2) ratio equal to 5.68 +/- 0.14. Addition of heterodimeric tubulin to isolated mitochondria was found to increase apparent Km for exogenous ADP from 11 +/- 2 microM to 330 +/- 47 microM, but creatine again decreased it to 23 +/- 6 microM. These results show directly that under physiological conditions the major energy carrier from mitochondria into cytoplasm is PCr, produced by mitochondrial creatine kinase (MtCK), which functional coupling to adenine nucleotide translocase is enhanced by selective limitation of permeability of mitochondrial outer membrane within supercomplex ATP Synthasome-MtCK-VDAC-tubulin, Mitochondrial Interactosome.
73 citations
TL;DR: Thermal limits of the Mediterranean fish Sparus aurata are identified and the processes providing heat protection during exposure to high temperatures as well as metabolic adjustments are characterized.
Abstract: Tolerance to a changing climate regime and persistence in the natural environment depends on the limited capacity to acclimate to changing temperatures. The present study aimed to identify and characterize thermal limits of the Mediterranean fish Sparus aurata as well as the processes providing heat protection during exposure to high temperatures. Processes studied included heat shock protein expression, protein kinase activity and metabolic adjustments. Molecular responses were addressed through the expression of Hsp70 and Hsp90 and the phosphorylation of stress-activated protein kinases, p38 mitogen-activated protein kinase (p38 MAPK) and cJun-N-terminal kinases (JNKs). Thermal impacts on metabolic capacities were assessed by studying the maximum activities of citrate synthase (CS), malate dehydrogenase (MDH) and 3-hydroxyacyl CoA dehydrogenase (HOAD) as well as pyruvate kinase (PK) and lactate dehydrogenase (L-LDH). The expression of Hsp70 and hsp90 was activated when the fish were exposed to temperatures beyond 20°C. Increased phosphorylation of p38 MAPK and JNKs indicated the parallel activation of MAPK signaling cascades and the potential involvement of MAPKs in the induction of Hsp genes. Exposure to extreme temperatures beyond 24°C caused an increase in the enzymatic activity of PK and LDH indicating an enhanced glycolytic potential.
71 citations
TL;DR: The apparent paradoxical effect of growth signaling through tyrosine phosphorylation, which decreases rather than increases PKM2 activity, stimulates a revised perspective of the Warburg effect, which must now accommodate links among glycolysis, the tricarboxylic acid cycle, and glutamine metabolism in cancer cells.
Abstract: New evidence suggests that the receptor tyrosine kinase FGFR1 (fibroblast growth factor receptor 1) directly phosphorylates pyruvate kinase M2 (PKM2), resulting in reduced conversion of phosphoenolpyruvate to pyruvate, which is further catabolized to lactate by lactate dehydrogenase A. Mutation of the critical tyrosine Tyr(105) to Phe rendered PKM2 more active but was associated with decreased cellular lactate production, increased oxygen consumption, and decreased hypoxic cell proliferation relative to wild-type PKM2. The apparent paradoxical effect of growth signaling through tyrosine phosphorylation, which decreases rather than increases PKM2 activity, stimulates a revised perspective of the Warburg effect. This effect, which describes the propensity for cancer cells to convert glucose to lactate at a high rate, must now accommodate links among glycolysis, the tricarboxylic acid cycle, and glutamine metabolism in cancer cells.
TL;DR: Caffeic acid phenethyl ester exhibits a significant potential as an antidiabetic agent by suppressing hepatic glucose output via inducing mRNA expression of glucokinase and pyruvate kinase, whilst inhibiting phoshoenolpyruVate carboxykinase in diabetes.
TL;DR: It is shown that the SUMO‐E3 ligase protein PIAS3 (inhibitor of activated STAT3) physically interacts with M2‐ PK and its isoenzyme M1‐PK and it is demonstrated that endogenoussumO‐1‐M2‐PK conjugates exist in mammalian cells.
Abstract: Pyruvate kinase M2 (M2-PK) controls the rate-limiting step at the end of the glycolytic pathway in normal proliferating and tumor cells. Other functions of M2-PK in addition to its role in glycolysis are little understood. The aim of this study was to identify new cellular interaction partners of M2-PK in order to discover novel links between M2-PK and cellular functions. Here we show that the SUMO-E3 ligase protein PIAS3 (inhibitor of activated STAT3) physically interacts with M2-PK and its isoenzyme M1-PK. Moreover, we demonstrate that endogenous SUMO-1-M2-PK conjugates exist in mammalian cells. Furthermore, we show that transient expression of PIAS3 but not the RING domain mutant PIAS3 (C299S, H301A) is consistent with nuclear localization of M2-PK and PIAS3 and M2-PK partially co-localize in the nucleus of these cells. This study suggests a link between PIAS3 and nuclear pyruvate kinase.
TL;DR: A fluorescence methodology to measure G6P concentrations in cell extracts based on the reaction of glucose-6-phosphate and the diaphorase-resazurin amplifying system is developed, illustrating an in vitro Warburg-like effect on cell metabolism.
TL;DR: There is a direct link between pneumococcal fermentative metabolism and virulence, and Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wildtype group.
Abstract: Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.
TL;DR: It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intrACEllular MgATPases and cytoplasmic glycolytic system.
TL;DR: The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.
Abstract: Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37 degrees C. At 42 degrees C, its plasmid content was ninefold higher than JM101 at 37 degrees C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp(uv), under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.
TL;DR: Results demonstrated that the EEPO possessed notable anti-hypoxic activity, which might be related to promoting the activity of the key enzymes in glycolysis and improving the level of ATP in hypoxic mice.
TL;DR: It is concluded that a decrease in enolase activity affects secondary pathways, such as the shikimate branch of amino acid biosynthesis, but does not inhibit the rate of respiration.
Abstract: Summary
• Enolase catalyses the reversible conversion of 2-phosphoglycerate and phosphoenolpyruvate in glycolysis. Phosphoenolpyruvate constitutes an important branch point in plant metabolism. It is converted to pyruvate by pyruvate kinase and organic acids by phosphoenolpyruvate carboxylase. Phosphoenolpyruvate also acts as a precursor for the synthesis of aromatic amino acids in plastids.
• Tobacco (Nicotiana tabacum) enolase antisense plants were analysed for changes in metabolite composition, respiration and photosynthetic parameters.
• Antisense repression resulted in up to a 95% reduction in total enolase activity. It also resulted in fundamental changes in foliar metabolism. Although 2-phosphoglycerate remained largely unaltered, there was a substantial decrease in phosphoenolpyruvate. The levels of aromatic amino acids and secondary phenylpropanoid metabolites that are derived from these compounds decreased strongly, as did branched chain amino acids. The level of pyruvate was unaltered, as was the rate of respiration. There were substantial increases in tricarboxylic acid cycle intermediates, including a 16-fold increase in isocitrate, an increase in the total free amino acid content, including a 14-fold increase in asparagine and glutamine, and a 50% decrease in free sugars.
• We conclude that a decrease in enolase activity affects secondary pathways, such as the shikimate branch of amino acid biosynthesis, but does not inhibit the rate of respiration.
TL;DR: The results of this study comply with the assumption that in contrast to CM in which oxidative phosphorylation is a predominant provider of ATP and the CK system is a main carrier of energy from mitochondria to ATPases, in HL-1 cells the energy metabolism is based mostly on the glycolytic reactions coupled to oxidativeosphorylation through HK.
Abstract: Comparative analysis of the bioenergetic parameters of adult rat cardiomyocytes (CM) and HL-1 cells with very different structure but similar cardiac phenotype was carried out with the aim of revealing the importance of the cell structure for regulation of its energy fluxes. Confocal microscopic analysis showed very different mitochondrial arrangement in these cells. The cytochrome content per milligram of cell protein was decreased in HL-1 cells by a factor of 7 compared with CM. In parallel, the respiratory chain complex activities were decreased by 4-8 times in the HL-1 cells. On the contrary, the activities of glycolytic enzymes, hexokinase (HK), and pyruvate kinase (PK) were increased in HL-1 cells, and these cells effectively transformed glucose into lactate. At the same time, the creatine kinase (CK) activity was significantly decreased in HL-1 cells. In conclusion, the results of this study comply with the assumption that in contrast to CM in which oxidative phosphorylation is a predominant provider of ATP and the CK system is a main carrier of energy from mitochondria to ATPases, in HL-1 cells the energy metabolism is based mostly on the glycolytic reactions coupled to oxidative phosphorylation through HK.
TL;DR: The results show how genetic and maternal factors interact to determine the metabolic setpoint of the offspring and indicate an important role for maternal thyroid hormone in the susceptibility to metabolic disorders in adulthood.
Abstract: Mice expressing the mutant thyroid hormone receptor TRα1R384C, which has a 10-fold reduced affinity to the ligand T3, exhibit hypermetabolism due to an overactivation of the sympathetic nervous system. To define the consequences in the liver, we analyzed hepatic metabolism and the regulation of liver genes in the mutant mice. Our results showed that hepatic phosphoenolpyruvate-carboxykinase was up-regulated and pyruvate kinase mRNA down-regulated, contrary to what observed after T3 treatment. In contrast, mice expressing a mutant TRα1L400R specifically in the liver did not show a dysregulation of these genes; however, when the TRα1L400R was expressed ubiquitously, the hepatic phenotype differed from TRα1R384C animals, suggesting that the localization of the mutation plays an important role for its consequences on glucose metabolism. Furthermore, we observed that glycogen stores were completely depleted in TRα1R384C animals, despite increased gluconeogenesis and decreased glycolysis. Exposure of the mutant...
TL;DR: The results fail to support a general correlation between pH dependentChanges in effector affinity and pH dependent changes in the corresponding allosteric response.
TL;DR: It is concluded that cAMP and glucose signaling converge on a complex containing ChREBP, HNF4α, and CBP, and that camp acts by disrupting this transcriptional complex assembled by glucose‐derived signals.
Abstract: Glucose-mediated activation of the L-type pyruvate kinase (L-PK) gene is repressed by cAMP, making this an excellent model for studying the mechanism by which these contrary signals regulate gene expression. Using the 832/13 rat insulinoma cell line, we demonstrate using RNA interference and chromatin immunoprecipitation that carbohydrate response element binding protein (ChREBP), hepatic nuclear factor 4α (HNF4α), and the coactivator CREB binding protein (CBP) are required for the glucose response of the L-PK gene and are recruited to the promoter by glucose. The cAMP agonist forskolin blocked the glucose-mediated induction of the L-PK gene in a PKA-dependent manner and blocked the recruitment of ChREBP, HNF4α, and CBP to the L-PK promoter, while simultaneously recruiting CBP to the cAMP-inducible gene, nuclear receptor subfamily 4, group A, member 2 (NR4A2). Overexpression of CBP, but not ChREBP, reversed the cAMP repression of the L-PK gene. In addition, CBP augmented the glucose response of the L-PK p...
TL;DR: The molecular characterization of 38 PK‐deficient patients from 35 unrelated families and the three‐dimensional structure of recombinant human tetrameric PK were used to evaluate the protein structural context of the affected residues provided a rationale for the observed enzyme deficiency.
Abstract: Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK-deficient patients from 35 unrelated families. Twenty-nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in-frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N-terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three-dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme-linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype-to-phenotype correlation in PK deficiency as well as the enzyme's structure and function. Hum Mutat 0, 1–8, 2008. © 2008 Wiley-Liss, Inc.
TL;DR: In this third minireview, high-resolution crystal structures of mammalian PEPCK are examined to gain insights into the mechanism ofPEPCK catalysis, including the reaction's reversibility and nucleotide specificity.
TL;DR: Calorie restriction reduces MMS toxicity through increased respiration and reduced ROS accumulation, enhancing the survival and recovery of cells, and is dependent on the glucose concentration in the medium.
Abstract: Maintenance and adaptation of energy metabolism could play an important role in the cellular ability to respond to DNA damage. A large number of studies suggest that the sensitivity of cells to oxidants and oxidative stress depends on the activity of cellular metabolism and is dependent on the glucose concentration. In fact, yeast cells that utilize fermentative carbon sources and hence rely mainly on glycolysis for energy appear to be more sensitive to oxidative stress. Here we show that treatment of the yeast Saccharomyces cerevisiae growing on a glucose-rich medium with the DNA alkylating agent methyl methanesulphonate (MMS) triggers a rapid inhibition of respiration and enhances reactive oxygen species (ROS) production, which is accompanied by a strong suppression of glycolysis. Further, diminished activity of pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase upon MMS treatment leads to a diversion of glucose carbon to glycerol, trehalose and glycogen accumulation and an increased flux through the pentose-phosphate pathway. Such conditions finally result in a significant decline in the ATP level and energy charge. These effects are dependent on the glucose concentration in the medium. Our results clearly demonstrate that calorie restriction reduces MMS toxicity through increased respiration and reduced ROS accumulation, enhancing the survival and recovery of cells.
TL;DR: In this article, two patients with mild chronic haemolytic anaemia, a mother and her son, were found to be heterozygous for erythrocyte pyruvate kinase deficiency.
Abstract: Two patients with mild chronic haemolytic anaemia, a mother and her son, were found to be heterozygous for erythrocyte pyruvate kinase deficiency In the red blood cells the enzymatic activity was reduced by about 50% and the residual PK had normal kinetic properties, stability and electrofocusing pattern The PK antigen concentration was also decreased by half, so that the ratio of the enzymatic activity to the immunological reactivity (ie the molecular specific activity) was normal In the son's liver PK enzymatic activity was slightly reduced and, above all, an abnormal active form, more anodic than normal PK, was detected by electrofocusing The propositus's liver PK was also slightly thermo-unstable It is suggested that the patients were heterozygous for an unstable PK variant which is found in liver, nucleated tissue actively synthesizing proteins, but which disappeared from the erythrocytes because of its unstability
TL;DR: Evidence is presented that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate intosynaptic vesicles and the possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed.
Abstract: Glucose metabolism is essential for normal brain function and plays a vital role in synaptic transmission. Recent evidence suggests that ATP synthesized locally by glycolysis, particularly via glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglycerate kinase, is critical for synaptic transmission. We present evidence that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate into synaptic vesicles. Isolated synaptic vesicles incorporated [3H]glutamate in the presence of phosphoenolpyruvate (PEP) and ADP. Pyruvate kinase activators and inhibitors stimulated and reduced PEP/ADP-dependent glutamate uptake, respectively. Membrane potential was also formed in the presence of pyruvate kinase activators. “ATP-trapping” experiments using hexokinase and glucose suggest that ATP produced by vesicle-associated pyruvate kinase is more readily used than exogenously added ATP. Other neurotransmitters such as GABA, dopamine, and serotonin were also taken up into crude synaptic vesicles in a PEP/ADP-dependent manner. The possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed.
Patent•
09 Oct 2009
TL;DR: In this paper, the pyruvate kinase M2 activators are bis sulfonamide piperazinyl and piperidinyl compounds of Formula (I), 2,4-disubstituted 4H- thieno[3,2-c]pyrrole-2-(substitized benzyl)pyridazin- 3 (2H) -ones of formula (II) and 6-(3,4)-dimethylphenylaminosulfonyl)-3, 4-dihydro-1H-
Abstract: Disclosed are pyruvate kinase M2 activators, which are bis sulfonamide piperazinyl and piperidinyl compounds of Formula (I), 2,4-disubstituted 4H- thieno[3,2-c]pyrrole-2-(substituted benzyl)pyridazin- 3 (2H) -ones of Formula (II) and 6-(3,4-dimethylphenylaminosulfonyl)-3,4-dihydro-1H-quinolin-2-one of formula (III), wherein L, R1, R2, R11 to R16, R21 and R22 are as defined herein, that are useful in treating a number of diseases that are treatable by the activation of PKM2, for example, cancer and anemia.
TL;DR: The data suggest that transduction of human RPK cDNA in PKLR mutated HSCs could be an effective strategy in severe cases of PKD.