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Showing papers on "Pyruvate kinase published in 2014"


Journal ArticleDOI
TL;DR: Evidence is provided to support a novel role for the pyruvate kinase M2 (PKM2)-mediated Warburg effect, namely aerobic glycolysis, in the regulation of high mobility group box 1 (HMGB1) release and shed light on a novel mechanism for metabolic control of inflammation by regulating HMGB1 release.
Abstract: Increasing evidence suggests the important role of metabolic reprogramming in the regulation of the innate inflammatory response, but the underlying mechanism remains unclear. Here, we provide evidence to support a novel role for the pyruvate kinase M2 (PKM2)-mediated Warburg effect, namely aerobic glycolysis, in the regulation of high mobility group box 1 (HMGB1) release. PKM2 interacts with hypoxia-inducible factor 1α (HIF1α) and activates the HIF-1α-dependent transcription of enzymes necessary for aerobic glycolysis in macrophages. Knockdown of PKM2, HIF1α, and glycolysis-related genes uniformly decreases lactate production and HMGB1 release. Similarly, a potential PKM2 inhibitor, shikonin, reduces serum lactate and HMGB1 levels and protects mice from lethal endotoxemia and sepsis. Collectively, these findings shed light on a novel mechanism for metabolic control of inflammation by regulating HMGB1 release and highlight the importance of targeting aerobic glycolysis in the treatment of sepsis and other inflammatory diseases.

320 citations


Journal ArticleDOI
11 Sep 2014-Cell
TL;DR: It is found that a deficiency in the M2 pyruvate kinase isoform (PKM2) reduces the levels of metabolic intermediates important for biosynthesis and impairs progenitor function without perturbing hematopoietic stem cells (HSCs), whereas lactate dehydrogenase A (LDHA) deletion significantly inhibits the function of both HSCs and progenitors during hematoiesis.

282 citations


Journal ArticleDOI
TL;DR: The findings suggest that the early induction of HIF1α targets may be instrumental in iPSC derivation via the activation of a glycolytic program, and implicate the Hif1α pathway as an enabling regulator of cellular reprogramming.
Abstract: Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor one alpha (HIF1α), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1α function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1α significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to upregulate the HIF1α target pyruvate dehydrogenase kinase (PDK) one and set in motion the glycolytic shift. However, additional HIF1α activation appears critical in the early upregulation of other HIF1α-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1α targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1α pathway as an enabling regulator of cellular reprogramming.

225 citations


Journal ArticleDOI
TL;DR: In this article, a Jumonji C domain-containing dioxygenase, called JMJD5, is shown to be upregulated by hypoxia and is crucial for hypoxiosis-induced cell proliferation.
Abstract: JMJD5, a Jumonji C domain-containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxia-induced cell proliferation. JMJD5 interacts directly with pyruvate kinase muscle isozyme (PKM)2 to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes hypoxia-inducible factor (HIF)-1α–mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2–HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α, is recruited to the hypoxia response element site in the lactate dehydrogenase A and PKM2 loci and mediates the recruitment of the latter two proteins. Our data uncover a mechanism whereby PKM2 can be regulated by factor-binding–induced homo/heterooligomeric restructuring, paving the way to cell metabolic reprogram.

216 citations


Journal ArticleDOI
TL;DR: It is suggested that inhibition of glycolysis may be a potentially effective strategy to target BCSCs, and functional validation of proteomic and metabolic data provide evidences for increased activities of key enzymes of anaerobic glucose fate in cancer stem cells as well as different redox status.
Abstract: A number of studies suggest that cancer stem cells are essential for tumour growth, and failure to target these cells can result in tumour relapse. As this population of cells has been shown to be resistant to radiation and chemotherapy, it is essential to understand their biology and identify new therapeutic approaches. Targeting cancer metabolism is a potential alternative strategy to counteract tumour growth and recurrence. Here we applied a proteomic and targeted metabolomic analysis in order to point out the main metabolic differences between breast cancer cells grown as spheres and thus enriched in cancer stem cells were compared with the same cells grown in adherent differentiating conditions. This integrated approach allowed us to identify a metabolic phenotype associated with the stem-like condition and shows that breast cancer stem cells (BCSCs) shift from mitochondrial oxidative phosphorylation towards fermentative glycolysis. Functional validation of proteomic and metabolic data provide evidences for increased activities of key enzymes of anaerobic glucose fate such as pyruvate kinase M2 isoform, lactate dehydrogenase and glucose 6-phopshate dehydrogenase in cancer stem cells as well as different redox status. Moreover, we show that treatment with 2-deoxyglucose, a well known inhibitor of glycolysis, inhibits BCSC proliferation when used alone and shows a synergic effect when used in combination with doxorubicin. In conclusion, we suggest that inhibition of glycolysis may be a potentially effective strategy to target BCSCs.

204 citations


Journal ArticleDOI
TL;DR: Constant evidence suggests a critical role played by the low‐activity‐dimeric PKM2 in tumor progression, supported by the identification of mutations which result in the down‐regulation of its activity and tumorigenesis in a nude mouse model.

164 citations


Journal ArticleDOI
TL;DR: It is reported that stimulation of epithelial–mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT.
Abstract: Pyruvate kinase M2 (PKM2) is an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells. PKM2 alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect (also referred to as aerobic glycolysis). Although previous reports suggest that PKM2 functions in nonmetabolic transcriptional regulation, its significance in cancer biology remains elusive. Here we report that stimulation of epithelial–mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT. Immunoprecipitation and LC-electrospray ionized TOF MS analyses revealed that EMT stimulation causes direct interaction of PKM2 in the nucleus with TGF-β–induced factor homeobox 2 (TGIF2), a transcriptional cofactor repressor of TGF-β signaling. The binding of PKM2 with TGIF2 recruits histone deacetylase 3 to the E-cadherin promoter sequence, with subsequent deacetylation of histone H3 and suppression of E-cadherin transcription. This previously unidentified finding of the molecular interaction of PKM2 in the nucleus sheds light on the significance of PKM2 expression in cancer cells.

152 citations


Journal ArticleDOI
TL;DR: It is shown that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2.
Abstract: Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by “designer microbes.” The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make “designer microbes” includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made. To improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme’s affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization. An efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.

141 citations


Journal ArticleDOI
TL;DR: This study suggests that PKM2 overexpression promotes CRC cell migration and cell adhesion by regulating STAT3-associated signalling and thatPKM2 may serve as a therapeutic target for CRC metastasis.

118 citations


Journal ArticleDOI
23 Jan 2014-PLOS ONE
TL;DR: The regulatory role of miR-122 on PKM2 in HCC is demonstrated, having an implication of therapeutic intervention targeting cancer metabolic pathways.
Abstract: In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg's effect), with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122) is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC). Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK) gene showed the strongest anti-correlation coefficient (Pearson r = -0.6938, p = <0.0001). In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2) is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.

117 citations


Journal ArticleDOI
TL;DR: It is shown that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln but the metabolic cost of these deficiencies is that they impair the Müller cell’s ability to metabolize Glc.
Abstract: Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Muller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Muller cell’s ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Muller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Muller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1.

Journal ArticleDOI
01 Jul 2014-Mbio
TL;DR: It is demonstrated for the first time that cell division is intimately linked to central carbon metabolism in the model Gram-positive bacterium Bacillus subtilis and pyruvate, the final product of glycolysis, plays an important role in maintaining normal division.
Abstract: Cell division in bacteria is driven by a cytoskeletal ring structure, the Z ring, composed of polymers of the tubulin-like protein FtsZ. Z-ring formation must be tightly regulated to ensure faithful cell division, and several mechanisms that influence the positioning and timing of Z-ring assembly have been described. Another important but as yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. In this study, we demonstrated for the first time that cell division is intimately linked to central carbon metabolism in the model Gram-positive bacterium Bacillus subtilis. We showed that a deletion of the gene encoding pyruvate kinase ( pyk ), which produces pyruvate in the final reaction of glycolysis, rescues the assembly defect of a temperature-sensitive ftsZ mutant and has significant effects on Z-ring formation in wild-type B. subtilis cells. Addition of exogenous pyruvate restores normal division in the absence of the pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the coordination of bacterial growth and division. Our results support a model in which pyruvate levels are coupled to Z-ring assembly via an enzyme that actually metabolizes pyruvate, the E1α subunit of pyruvate dehydrogenase. We have shown that this protein localizes over the nucleoid in a pyruvate-dependent manner and may stimulate more efficient Z-ring formation at the cell center under nutrient-rich conditions, when cells must divide more frequently. IMPORTANCE How bacteria coordinate cell cycle processes with nutrient availability and growth is a fundamental yet unresolved question in microbiology. Recent breakthroughs have revealed that nutritional information can be transmitted directly from metabolic pathways to the cell cycle machinery and that this can serve as a mechanism for fine-tuning cell cycle processes in response to changes in environmental conditions. Here we identified a novel link between glycolysis and cell division in Bacillus subtilis. We showed that pyruvate, the final product of glycolysis, plays an important role in maintaining normal division. Nutrient-dependent changes in pyruvate levels affect the function of the cell division protein FtsZ, most likely by modifying the activity of an enzyme that metabolizes pyruvate, namely, pyruvate dehydrogenase E1α. Ultimately this system may help to coordinate bacterial division with nutritional conditions to ensure the survival of newborn cells.

Journal ArticleDOI
TL;DR: HMGB1 released during tumorigenesis recruits muscle to supply glutamine to cancer cells as an energy source, suggesting that cancer energy production and host muscle are linked.
Abstract: Cancer cells produce energy through aerobic glycolysis, but contributions of host tissues to cancer energy metabolism are unclear. In this study, we aimed to elucidate the cancer-host energy production relationship, in particular, between cancer energy production and host muscle. During the development and progression of colorectal cancer, expression of the secreted autophagy-inducing stress protein HMGB1 increased in the muscle of tumor-bearing animals. This effect was associated with decreased expression of pyruvate kinase PKM1 and pyruvate kinase activity in muscle via the HMGB1 receptor for advanced glycation endproducts (RAGE). However, muscle mitochondrial energy production was maintained. In contrast, HMGB1 addition to colorectal cancer cells increased lactate fermentation. In the muscle, HMGB1 addition induced autophagy by decreasing levels of active mTOR and increasing autophagy-associated proteins, plasma glutamate, and (13)C-glutamine incorporation into acetyl-CoA. In a mouse model of colon carcinogenesis, a temporal increase in HMGB1 occurred in serum and colonic mucosa with an increase in autophagy associated with altered plasma free amino acid levels, increased glutamine, and decreased PKM1 levels. These differences were abolished by administration of an HMGB1 neutralizing antibody. Similar results were obtained in a mouse xenograft model of human colorectal cancer. Taken together, our findings suggest that HMGB1 released during tumorigenesis recruits muscle to supply glutamine to cancer cells as an energy source.

Patent
13 Mar 2014
TL;DR: In this article, compounds and compositions comprising compounds that modulate pyruvate kinase M2 (PKM2) are described and methods of using these compounds in the treatment of cancer.
Abstract: Compounds and compositions comprising compounds that modulate pyruvate kinase M2 (PKM2) are described herein. Also described herein are methods of using the compounds that modulate PKM2 in the treatment of cancer.

Journal ArticleDOI
TL;DR: A model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress is supported.
Abstract: The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 A resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.

Journal ArticleDOI
TL;DR: It is demonstrated that the period ranging from the polyculture to the external feeding stage is an important window for potential modification of the long-term physiological functions of adult zebrafish through early nutritional programming.
Abstract: The aim of the present study was to determine the potential long-term metabolic effects of early nutritional programming on carbohydrate utilisation in adult zebrafish (Danio rerio). High-carbohydrate diets were fed to fish during four ontogenetic stages: from the first-feeding stage to the end of the yolk-sac larval stage; from the first-feeding stage to 2 d after yolk-sac exhaustion; after yolk-sac exhaustion for 3 or 5 d. The carbohydrate stimuli significantly increased the body weight of the first-feeding groups in the short term. The expression of genes was differentially regulated by the early dietary intervention. The high-carbohydrate diets resulted in decreased plasma glucose levels in the adult fish. The mRNA levels and enzyme activities of glucokinase, pyruvate kinase, α-amylase and sodium-dependent glucose co-transporter 1 were up-regulated in the first-feeding groups. There was no significant change in the mRNA levels of glucose-6-phosphatase (G6Pase) in any experimental group, and the activity of G6Pase enzyme in the FF-5 (first feeding to 2 d after yolk-sac exhaustion) group was significantly different from that of the other groups. The expression of phosphoenolpyruvate carboxykinase gene in all the groups was significantly decreased. In the examined early programming range, growth performance was not affected. Taken together, data reported herein indicate that the period ranging from the polyculture to the external feeding stage is an important window for potential modification of the long-term physiological functions. In conclusion, the present study demonstrates that it is possible to permanently modify carbohydrate digestion, transport and metabolism of adult zebrafish through early nutritional programming.

Journal ArticleDOI
TL;DR: It is demonstrated that PKM2 in the blood facilitates tumor growth by promoting tumor angiogenesis by increasing endothelial cell proliferation, migration, and cell-ECM adhesion, which is consistent with the observations that PKm2 in circulation of cancer patients is a dimer form.

Journal ArticleDOI
TL;DR: Results showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by MRI methods as an early sensor of TMZ therapeutic response.
Abstract: Recent findings show that exposure to temozolomide (TMZ), a DNA damaging drug used to treat glioblastoma, can suppress the conversion of pyruvate to lactate. To understand the mechanistic basis for this effect and its potential utility as a TMZ response biomarker, we compared the response of isogenic glioblastoma cell populations differing only in expression of the DNA repair protein MGMT, a TMZ-sensitivity determinant, after exposure to TMZ in vitro and in vivo. Hyperpolarized [1-(13)C]-pyruvate-based magnetic resonance imaging was used to monitor temporal effects on pyruvate metabolism in parallel with DNA damage responses and tumor cell growth. TMZ exposure decreased conversion of pyruvate to lactate only in MGMT-deficient cells. This effect coincided temporally with TMZ-induced increases in levels of the DNA damage response protein pChk1. Changes in pyruvate to lactate conversion triggered by TMZ preceded tumor growth suppression and were not associated with changes in levels of NADH or lactate dehydrogenase activity in tumors. Instead, they were associated with a TMZ-induced decrease in the expression and activity of pyruvate kinase PKM2, a glycolytic enzyme that indirectly controls pyruvate metabolism. PKM2 silencing decreased pyruvate kinase activity, intracellular lactate levels, and conversion of pyruvate to lactate in the same manner as TMZ, and Chk1 silencing blocked the TMZ-induced decrease in PKM2 expression. Overall, our findings showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by magnetic resonance imaging methods as an early sensor of TMZ therapeutic response.

Journal ArticleDOI
09 Jun 2014-PLOS ONE
TL;DR: Using a mathematical model based on reported mechanisms for the allosteric regulations of the enzymes, glycolysis exhibits multiple steady state behavior segregating glucose metabolism into high flux and low flux states, revealing new means of metabolic intervention in the treatment of cancer and other metabolic disorders through suppression of glyCOlysis.
Abstract: The flux of glycolysis is tightly controlled by feed-back and feed-forward allosteric regulations to maintain the body's glucose homeostasis and to respond to cell's growth and energetic needs. Using a mathematical model based on reported mechanisms for the allosteric regulations of the enzymes, we demonstrate that glycolysis exhibits multiple steady state behavior segregating glucose metabolism into high flux and low flux states. Two regulatory loops centering on phosphofructokinase and on pyruvate kinase each gives rise to the bistable behavior, and together impose more complex flux control. Steady state multiplicity endows glycolysis with a robust switch to transit between the two flux states. Under physiological glucose concentrations the glycolysis flux does not move between the states easily without an external stimulus such as hormonal, signaling or oncogenic cues. Distinct combination of isozymes in glycolysis gives different cell types the versatility in their response to different biosynthetic and energetic needs. Insights from the switch behavior of glycolysis may reveal new means of metabolic intervention in the treatment of cancer and other metabolic disorders through suppression of glycolysis.

Journal ArticleDOI
TL;DR: In this paper, the effects of B-deficiency on gas exchange, carbohydrates, organic acids, amino acids, total soluble proteins and phenolics, and the activities of key enzymes involved in organic acid and amino acid metabolism in "Xuegan" [Citrus sinensis (L.) Osbeck] leaves and roots were investigated.
Abstract: Boron (B) deficiency is a widespread problem in many crops, including Citrus. The effects of B-deficiency on gas exchange, carbohydrates, organic acids, amino acids, total soluble proteins and phenolics, and the activities of key enzymes involved in organic acid and amino acid metabolism in ‘Xuegan’ [Citrus sinensis (L.) Osbeck] leaves and roots were investigated. Borondeficient leaves displayed excessive accumulation of nonstructural carbohydrates and much lower CO 2 assimilation, demonstrating feedback inhibition of photosynthesis. Dark respiration, concentrations of most organic acids [i.e., malate, citrate, oxaloacetate (OAA), pyruvate and phosphoenolpyruvate] and activities of enzymes [i.e., phosphoenolpyruvate carboxylase (PEPC), NAD-malate dehydrogenase, NAD-malic enzyme (NAD-ME), NADP-ME, pyruvate kinase (PK), phosphoenolpyruvate phosphatase (PEPP), citrate synthase (CS), aconitase (ACO), NADP-isocitrate dehydrogenase (NADP-IDH) and hexokinase] involved in glycolysis, the tricarboxylic acid (TCA) cycle and the anapleurotic reaction were higher in B-deficient leaves than in controls. Also, total free amino acid (TFAA) concentration and related enzyme [i.e., NADH-dependent glutamate 2- o xoglutarate aminotransferase (NADH-GOGAT) and glutamate OAA transaminase (GOT)] activities were enhanced in B-deficient leaves. By contrast, respiration, concentrations of nonstructural carbohydrates and three organic acids (malate, citrate and pyruvate), and activities of most enzymes [i.e., PEPC, NADP-ME, PK, PEPP, CS, ACO, NAD-isocitrate dehydrogenase, NADP-IDH and hexokinase] involved in glycolysis, the TCA cycle and the anapleurotic reaction, as well as concentration of TFAA and activities of related enzymes (i.e., nitrate reductase, NADH-GOGAT, glutamate pyruvate transaminase and glutamine synthetase) were lower in B-deficient roots than in controls. Interestingly, leaf and root concentration of total phenolics increased, whereas that of total soluble protein decreased, in response to B-deficiency. In conclusion, respiration, organic acid (i.e., glycolysis and the TCA cycle) metabolism, the anapleurotic pathway and amino acid biosynthesis were upregulated in B-deficient leaves with excessive accumulation of carbohydrates to ‘consume’ the excessive carbon available, but downregulated in B-deficient roots with less accumulation of carbohydrates to maintain the net carbon balance.

Journal ArticleDOI
TL;DR: The results indicated that cancer glycolysis should be inhibited at multiple controlling sites, regardless of external glucose levels, to effectively block the pathway.
Abstract: The effect of hypoglycemia on the contents of glycolytic proteins, activities of enzymes/transporters and flux of HeLa and MCF-7 tumor cells was experimentally analyzed and modeled in silico. After 24 h hypoglycemia (2.5 mm initial glucose), significant increases in the protein levels of glucose transporters 1 and 3 (GLUT 1 and 3) (3.4 and 2.1-fold, respectively) and hexokinase I (HKI) (2.3-fold) were observed compared to the hyperglycemic standard cell culture condition (25 mm initial glucose). However, these changes did not bring about a significant increase in the total activities (Vmax) of GLUT and HK; instead, the affinity of these proteins for glucose increased, which may explain the twofold increased glycolytic flux under hypoglycemia. Thus, an increase in more catalytically efficient isoforms for two of the main controlling steps was sufficient to induce increased flux. Further, a previous kinetic model of tumor glycolysis was updated by including the ratios of GLUT and HK isoforms, modified pyruvate kinase kinetics and an oxidative phosphorylation reaction. The updated model was robust in terms of simulating most of the metabolite levels and fluxes of the cells exposed to various glycemic conditions. Model simulations indicated that the main controlling steps were glycogen degradation > HK > hexosephosphate isomerase under hyper- and normoglycemia, and GLUT > HK > glycogen degradation under hypoglycemia. These predictions were experimentally evaluated: the glycolytic flux of hypoglycemic cells was more sensitive to cytochalasin B (a GLUT inhibitor) than that of hyperglycemic cells. The results indicated that cancer glycolysis should be inhibited at multiple controlling sites, regardless of external glucose levels, to effectively block the pathway. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/database/achcar/index.html. [Database section added 21 July 2014 after original online publication]

Journal ArticleDOI
13 Mar 2014-PLOS ONE
TL;DR: It is found that Oleanolic acid (OA) induced a switch from PKM2 to PKM1, and consistently, abrogated Warburg effect in cancer cells, and there is potential thatPKM2 may be developed as an important target in aerobic glycolysis pathway for developing novel anticancer agents.
Abstract: Warburg effect, one of the hallmarks for cancer cells, is characterized by metabolic switch from mitochondrial oxidative phosphorylation to aerobic glycolysis. In recent years, increased expression level of pyruvate kinase M2 (PKM2) has been found to be the culprit of enhanced aerobic glycolysis in cancer cells. However, there is no agent inhibiting aerobic glycolysis by targeting PKM2. In this study, we found that Oleanolic acid (OA) induced a switch from PKM2 to PKM1, and consistently, abrogated Warburg effect in cancer cells. Suppression of aerobic glycolysis by OA is mediated by PKM2/PKM1 switch. Furthermore, mTOR signaling was found to be inactivated in OA-treated cancer cells, and mTOR inhibition is required for the effect of OA on PKM2/PKM1 switch. Decreased expression of c-Myc-dependent hnRNPA1 and hnRNPA1 was responsible for OA-induced switch between PKM isoforms. Collectively, we identified that OA is an antitumor compound that suppresses aerobic glycolysis in cancer cells and there is potential that PKM2 may be developed as an important target in aerobic glycolysis pathway for developing novel anticancer agents.

Journal ArticleDOI
TL;DR: Glycogenolysis versus gluconeogenesis correlates with metabolism of hyperpolarized dihydroxyacetone, and a ratio of glycolytic to gluconeogenic products distinguished the gluc oneogenic from glycogenolytic state in these functioning livers.

Journal ArticleDOI
TL;DR: It is argued that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid.
Abstract: Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady-state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of (13) C into metabolic intermediates and end products when cells were labelled with [1-(13) C], [2-(13) C], or [(13) C6 ]glucose, in combination with (14) C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid.

Journal ArticleDOI
TL;DR: HIF-1α knockdown can inhibit the prolife ratio and promote apoptosis of pancreatic cancerous BxPC-3 cells in vitro.
Abstract: Objectives: The aim of this study is to explore the possible role of HIF-1α in glucose metabolism, proliferation and apoptosis of pancreatic cancerous cells. Method: The pancreatic cancerous BxPC-3 cells were cultured in normoxia or hypoxia (3% O2), respectively. Cell proliferation was determined by MTT assay, apoptosis was determined by Annexin V/PI staining. Expression of Pyruvate dehydrogenase kinase (PDK1), Lactate dehydrogenase (LDHA), pyruvate kinase M2 (PKM2) and citrate synthase (CS) was determined by Western-blot and Real-time PCR. Results: Under hypoxia, the expression of HIF-1α and the lactate production were increased. The expression of glucose metabolic enzymes PDK1, LDHA, PKM2 was also increased compared with that under aerobic condition. Hypoxia treatment had little effect on expression of CS. Under hypoxia, knockdown of HIF-1α inhibited the production of lactate and the expression of PDK1, LDHA and PKM2. Knockdown of HIF-1α repressed the growth of pancreatic cancer BxPC-3 cells and induced apoptosis of the cells under hypoxia. Conclusion: Under hypoxia, the expression of HIF-1α is induced, leading to the increase of glycolysis in BxPC-3 cells possibly through upregulation of the enzymes related to glycolysis. HIF-1α knockdown can inhibit the prolife ratio and promote apoptosis of pancreatic cancerous BxPC-3 cells in vitro.

Journal ArticleDOI
TL;DR: This work identifies the pyruvate-TCA cycle node in the carbon core metabolism of Y. pseudotuberculosis as a focal point for controlling the host colonization and virulence of Yersinia.

Journal ArticleDOI
TL;DR: It is demonstrated that aerobic glycolysis is a valuable HH-dependent downstream target, since its inhibition significantly counteracts the HH-mediated growth of normal and tumor cells, and pharmacological targeting with the pyruvate kinase inhibitor dichloroacetate efficiently represses MB growth in vitro and in vivo.
Abstract: Aberrant activation of SHH pathway is a major cause of medulloblastoma (MB), the most frequent brain malignancy of the childhood. A few Hedgehog inhibitors, all antagonizing the membrane transducer Smo, have been approved or are under clinical trials for the treatment of human MB. However, the efficacy of these drugs is limited by the occurrence of novel mutations or by activation of downstream or non-canonical Hedgehog components. Thus, the identification of novel druggable downstream pathways represents a critical step to overcome this problem. In the present work we demonstrate that aerobic glycolysis is a valuable HH-dependent downstream target, since its inhibition significantly counteracts the HH-mediated growth of normal and tumor cells. Hedgehog activation induces transcription of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), two key gatekeepers of glycolysis. The process is mediated by the canonical activation of the Gli transcription factors and causes a robust increase of extracellular lactate concentration. We show that inhibition of glycolysis at different levels blocks the Hedgehog-induced proliferation of granule cell progenitors (GCPs), the cells from which medulloblastoma arises. Remarkably, we demonstrate that this glycolytic transcriptional program is also upregulated in SHH-dependent tumors and that pharmacological targeting with the pyruvate kinase inhibitor dichloroacetate (DCA) efficiently represses MB growth in vitro and in vivo. Together, these data illustrate a previously uncharacterized pharmacological strategy to target Hedgehog dependent growth, which can be exploited for the treatment of medulloblastoma patients.

Journal ArticleDOI
TL;DR: The optimization of a weakly active screening hit to a structurally novel series of small molecule 3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as potent PKM2 activators is described.

Journal ArticleDOI
TL;DR: It is demonstrated that PKM2 depletion could provoke glutamine metabolism by enhancing the β-catenin signaling pathway and consequently promoting its downstream c-Myc-mediated glutamines metabolism in colon cancer cells, supply evidence that glutaminolysis plays a compensatory role for cell survival upon glucose metabolism impaired.

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TL;DR: IP6Ks are identified as novel nuclear and cytosolic InsP6- (and InsP5-) dephosphorylating enzymes whose activity is sensitively driven by a decrease in the cellular ATP/ADP ratio, thus suggesting a role for IP6ks as cellular adenylate energy 'sensors'.
Abstract: InsP6 (inositol hexakisphosphate), the most abundant inositol phosphate in metazoa, is pyrophosphorylated to InsP7 [5PP-InsP5 (diphosphoinositol pentakisphosphate)] by cytosolic and nuclear IP6Ks (InsP6 kinases) and to 1PP-InsP5 by another InsP6/InsP7 kinase family. MINPP1 (multiple inositol-polyphosphate phosphatase 1), the only known InsP6 phosphatase, is localized in the ER (endoplasmic reticulum) and lysosome lumina. A mechanism of cytosolic InsP6 dephosphorylation has remained enigmatic so far. In the present study, we demonstrated that IP6Ks change their kinase activity towards InsP6 at a decreasing ATP/ADP ratio to an ADP phosphotransferase activity and dephosphorylate InsP6. Enantio-selective analysis revealed that Ins(2,3,4,5,6) P 5 is the main InsP5 product of the IP6K reaction, whereas the exclusive product of MINPP1 activity is the enantiomer Ins(1,2,4,5,6) P 5. Whereas lentiviral RNAi-based depletion of MINPP1 at falling cellular ATP/ADP ratios had no significant impact on Ins(2,3,4,5,6) P 5 production, the use of the selective IP6K inhibitor TNP [ N 2-(m-trifluorobenzyl), N 6-( p -nitrobenzyl)purine] abolished the production of this enatiomer in different types of cells. Furthermore, by analysis of rat tissue and human blood samples all (main and minor) dephosphorylation products of InsP6 were detected in vivo . In summary, we identified IP6Ks as novel nuclear and cytosolic InsP6- (and InsP5-) dephosphorylating enzymes whose activity is sensitively driven by a decrease in the cellular ATP/ADP ratio, thus suggesting a role for IP6Ks as cellular adenylate energy ‘sensors’. Abbreviations: AMF, adenylate mole fraction; CCCP, carbonyl cyanide m-cholorophenyl hydrazone; CV, column volume; InsP5, inositol pentakisphosphate; InsP6, inositol hexakisphosphate; IP6K, InsP6 kinase; IPMK, inositol polyphosphate multikinase; IPPK, InsP5 2-kinase; MDD, metal-dye detection; MINPP1, multiple inositol-polyphosphate phosphatase 1; PEP, phosphoenol pyruvate; PK, pyruvate kinase; TCA, trichloroacetic acid; TNP, N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine