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Rationale: Fibrosis is a pathologic condition of abnormal accumulation of collagen fibrils. Collagen is a major extracellular matrix (ECM) protein synthesized and secreted by myofibroblasts, composing mainly (Gly-X-Y)n triplet repeats with >30% Gly residue. During fibrosis progression, myofibroblasts must upregulate glycine metabolism to meet the high demands of amino acids for collagen synthesis. Method: Expression of PKM2 in myofibroblasts was analyzed in cultured fibroblasts and fibrosis disease tissues. Functional roles of PKM2 and PKM2 activator in biosynthesis of serine → glycine and production of collagen from glycolysis intermediates were assayed in cultured activated LX-2 and human primary lung fibroblast cells. Mouse models of Liver, lung, and pancreas fibrosis were employed to analyze treatment effects of PKM2 activator in organ tissue fibrosis. Results: We report here that myofibroblast differentiation upregulates pyruvate kinase M2 (PKM2) and promotes dimerization of PKM2. Dimer PKM2 slows the flow rate of glycolysis and channels glycolytic intermediates to de novo glycine synthesis, which facilitates collagen synthesis and secretion in myofibroblasts. Our results show that PKM2 activator that converts PKM2 dimer to tetramer, inhibits fibrosis progression in mouse models of liver, lung, and pancreatic fibrosis. Furthermore, metabolism alteration by dimer PKM2 increases NADPH production, which consequently protects myofibroblasts from apoptosis. Conclusion: Our study uncovers a novel role of PKM2 in tissue/organ fibrosis, suggesting a possible strategy for treatment of fibrotic diseases using PKM2 activator., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Pyruvate kinase M2 (PKM2) is a key glycolysis enzyme, and its effect on macrophages has not been entirely elucidated. Here, we identified that the PKM2 small-molecule agonist TEPP-46 mediated PKM2 activation by inducing the formation of PKM2 tetramer and promoted macrophage endotoxin tolerance. Lipopolysaccharide (LPS)-tolerant mice had higher expression of the PKM2 tetramer, which was associated with a reduced in vivo immune response to LPS. Pretreatment of macrophages with TEPP-46 resulted in tolerance to LPS stimulation, as demonstrated by a significant reduction in the production of TNF-α and IL-6. We found that TEPP-46 induced mitochondrial biogenesis in macrophages. Inhibition of mitochondrial biogenesis by mtTFA knockdown effectively inhibited TEPP-46-mediated macrophage tolerance to endotoxins. We discovered that TEPP-46 promoted the expression of PGC-1α and that PGC-1α was the key regulator of mitochondrial biogenesis in macrophages induced by TEPP-46. PGC-1α was negatively regulated by the PI3K/Akt signaling pathway. Knockdown of PKM2 or PGC-1α uniformly inhibited TEPP-46-mediated endotoxin tolerance by inhibiting mitochondrial biogenesis. In addition, TEPP-46 protected mice from lethal endotoxemia and sepsis. Collectively, these findings reveal novel mechanisms for the metabolic control of inflammation and for the induction of endotoxin tolerance by promoting mitochondrial biogenesis. Targeting PKM2 appears to be a new therapeutic option for the treatment of sepsis and other inflammatory diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yi, Wu, Zhang, Wang, Gong, Cheng and Miao.)

The Pyruvate kinase isozymes M2 (PKM2) protein is a metabolic enzyme that regulates the final step of glycolysis. This enzyme is present in highly proliferating cells and is expressed in the placenta. We recently demonstrated upregulated placental PKM2 during human intrauterine growth restriction (IUGR). Our current objective was to determine PKM2 regulation of trophoblast invasion, trophoblast PKM2 localization as well as mTOR protein expression, and to determine effects of activation of PKM2 during IUGR. Human placental tissues were obtained and analyzed by immunohistochemistry and western blot. Trophoblast cells were cultured in normoxic and hypoxic conditions and real time cell invasion and PKM2 protein were determined during activation (Fructose-6-bisphosphate; FBP6) or inhibition (Shikonin) of PKM2. In vivo studies determined the effects of PKM2 activation on placental and fetal weights. IUGR samples had elevated levels of p-PKM2. Different trophoblast PKM2 localization and expression was observed during normoxia and hypoxia. Decreased trophoblast invasion and PKM2 expression was observed during mTOR inhibition. Protection from decreased placental and fetal weights was observed by PKM2 activation. We conclude that PKM2 regulates trophoblast cell invasion depending on its subcellular location. Our results suggest that PKM2 regulation in trophoblast cells is more directly affected during hypoxia and its expression is regulated by mTOR activity. Additionally, we conclude that activation of PKM2 could reverse and/or rescue the deceased placental and fetal weights observed during IUGR. These results suggest that PKM2 could be a mediator of trophoblast cell invasion and its abundance influences the development of complicated pregnancies like IUGR., Competing Interests: Declaration of competing interest None of the authors have any actual or potential competing financial or other interests with entities possibly interested in the subject matter., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Th17 cell differentiation and pathogenicity depend on metabolic reprogramming inducing shifts toward glycolysis. Here, we show that the pyruvate kinase M2 (PKM2), a glycolytic enzyme required for cancer cell proliferation and tumor progression, is a key factor mediating Th17 cell differentiation and autoimmune inflammation. We found that PKM2 is highly expressed throughout the differentiation of Th17 cells in vitro and during experimental autoimmune encephalomyelitis (EAE) development. Strikingly, PKM2 is not required for the metabolic reprogramming and proliferative capacity of Th17 cells. However, T cell-specific PKM2 deletion impairs Th17 cell differentiation and ameliorates symptoms of EAE by decreasing Th17 cell-mediated inflammation and demyelination. Mechanistically, PKM2 translocates into the nucleus and interacts with STAT3, enhancing its activation and thereby increasing Th17 cell differentiation. Thus, PKM2 acts as a critical nonmetabolic regulator that fine-tunes Th17 cell differentiation and function in autoimmune-mediated inflammation., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Damasceno et al.)

Lymphangioleiomyomatosis (LAM) is a devastating lung disease caused by inactivating gene mutations in either TSC1 or TSC2 that result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). As LAM occurs predominantly in women during their reproductive age and is exacerbated by pregnancy, the female hormonal environment, and in particular estrogen, is implicated in LAM pathogenesis and progression. However, detailed underlying molecular mechanisms are not well understood. In this study, utilizing human pulmonary LAM specimens and cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we tested the hypothesis that estrogen promotes the growth of mTORC1-hyperactive cells through pyruvate kinase M2 (PKM2). Estrogen increased the phosphorylation of PKM2 at Ser37 and induced the nuclear translocation of phospho-PKM2. The estrogen receptor antagonist Faslodex reversed these effects. Restoration of TSC2 inhibited the phosphorylation of PKM2 in an mTORC1 inhibitor-insensitive manner. Finally, accumulation of phosphorylated PKM2 was evident in pulmonary nodule from LAM patients. Together, our data suggest that female predominance of LAM might be at least in part attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. Targeting metabolic regulators of PKM2 might have therapeutic benefits for women with LAM and other female-specific neoplasms., Competing Interests: The authors have declared that no competing interests exist.

Pyruvate Kinase isozymes M2 (PKM2) is a glycolytic enzyme involved in glycolysis that decarboxylates phosphoenolpyruvate to pyruvate and generates ATP. PKM2 also plays a significant role in tumor growth, in cell division, angiogenesis, apoptosis and metastasis. In this study, we have investigated the role of PKM2 in cortical neurons which suffered hypoxic-ischemic encephalopathy (HIE) in newborn rats. Immunohistochemistry and Western blot analysis revealed the protein expression of PKM2 peaking at 24 h after HIE. Double immunofluorescence labeling showed that PKM2 was mainly located in the neurons of the ipsilateral cerebral cortex, not in astrocytes or microglia. The increased level of active caspase-3 and the decreased level of phosphorylated AKT (p-AKT) were consistent with the PKM2 expression. TUNEL staining assay showed that PKM2 may participate in neuronal apoptosis in the rat ipsilateral cerebral cortex. Silencing of PKM2 in primary cultures of cortical neurons using a specific siRNA reduced the expression of active caspase-3 and upregulated p-AKT expression. Taken together, the results indicate that PKM2 may be involved in neuronal apoptosis after HIE by a mechanism dependent on the inactivation of p-AKT.

Bcl-2 family proteins control a decisive apoptotic event: mitochondrial outer membrane permeabilization (MOMP). To discover MOMP-regulating proteins, we expressed a library of intracellular single-chain variable fragments (scFvs) ("intrabodies") and selected for those rescuing cells from apoptosis induced by BimS (the short isoform of Bim). One anti-apoptotic intrabody, intrabody 5 (IB5), recognized pyruvate kinase M2 (PKM2), which is expressed in cancer cells. PKM2 deletion ablated this clonogenic rescue; thus, IB5 activated a latent cytoprotective function of PKM2. This resulted not from pyruvate kinase activity per se but rather from the formation of an active tetrameric conformation of PKM2. A stably tetrameric PKM2 mutant, K422R, promoted cell survival even in the absence of IB5, and IB5 further increased survival. Mitochondria isolated from IB5-expressing cells were relatively resistant to MOMP in vitro. In cells, IB5 expression up-regulated Mitofusin-1 (Mfn1) and increased mitochondrial length. Importantly, Mfn1 deficiency abrogated IB5's cytoprotective effect. PKM2's anti-apoptotic function could help explain its preferential expression in human cancer., Competing Interests: The authors have declared that no competing interests exist.

Pyruvate Kinase M2 (PKM2) mediates metabolic reshuffling and is ubiquitously upregulated in several cancer types. The non-metabolic function of PKM2 as key nuclear kinase and modulator of gene expression is instrumental in cancer progression and tumorigenesis. Here, we attempt to discern the non-canonical function of PKM2 as an epigenetic modulator and the underlying implication of this activity. Using 5'-FAM labelled reconstituted mononucleosome we have shown that PKM2 interacts with the complex through Histone H3 and possibly obstruct the access to DNA binding factors. Subsequently, the interaction negatively impacts the ATP dependent remodeling activity of Chromodomain Helicase DNA binding protein-7 (Chd7). Chd7 remodeling activity is required to ameliorate DNA damage and is crucial to genome stability. Our study shows that PKM2 blocks the Chd7 mediated sliding of nucleosome. It can be conjectured that stalling Chd7 may lead to impaired DNA damage and increased genomic instability. We propose a mechanism in which PKM2 negatively regulate nucleosome repositioning in chromatin and may exacerbate cancer by altering the nucleosome architecture. This research is imperative to our understanding of how altered cancer metabolism can potentially modulate the gene expression and sustain incessant proliferation by tweaking the chromatin topography., Competing Interests: The authors have declared that no competing interests exist.

Starch is the main form of energy storage in higher plants. Although several enzymes and regulators of starch biosynthesis have been defined, the complete molecular machinery remains largely unknown. Screening for irregularities in endosperm formation in rice represents valuable prospect for studying starch synthesis pathway. Here, we identified a novel rice white-core endosperm and defective grain filling mutant, ospk2, which displays significantly lower grain weight, decreased starch content and alteration of starch physicochemical properties when compared to wild-type grains. The normal starch compound granules were drastically reduced and more single granules filled the endosperm cells of ospk2. Meanwhile, the germination rate of ospk2 seeds after 1-year storage was observably reduced compared with wild-type. Map-based cloning of OsPK2 indicated that it encodes a pyruvate kinase (PK, ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which catalyses an irreversible step of glycolysis. OsPK2 has a constitutive expression in rice and its protein localizes in chloroplasts. Enzyme assay showed that the protein product from expressed OsPK2 and the crude protein extracted from tissues of wild-type exhibits strong PK activity; however, the mutant presented reduced protein activity. OsPK2 (PKpα1) and three other putative rice plastidic isozymes, PKpα2, PKpβ1 and PKpβ2, can interact to form heteromer. Moreover, the mutation leads to multiple metabolic disorders. Altogether, these results denote new insights into the role of OsPK2 in plant seed development, especially in starch synthesis, compound granules formation and grain filling, which would be useful for genetic improvement of high yield and rice grain quality., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Pyruvate kinase M2 (PKM2) has been wildly verified to modulate glycolysis in tumor cells. However, the role of PKM2 on the glycolysis of osteoarthritis (OA) chondrocytes is still unclear. In present study, we investigate the function of PKM2 on OA chondrocyte glycolysis and the collagen matrix generation in vitro. Results showed that PKM2 was upregulated in OA chondrocytes compared with healthy control chondrocytes. In OA chondrocytes, ATP expression was lower compared with healthy control chondrocytes. Loss-of-function experiment showed that PKM2 knockdown mediated by lentivirus transfection could significantly suppress the glucose consumption and lactate secretion levels and decrease glucose transporter-1 (Glut-1), lactate dehydrogenase A (LDHA), and hypoxia inducible factor 1-alpha (HIF-1α), indicating the inhibition of PKM2 knockdown on glycolysis. Moreover, Cell Counting Kit-8 (CCK-8), flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay showed that PKM2 knockdown inhibited OA chondrocyte proliferation and promoted the apoptosis. Western blot and immunocytochemical staining showed that PKM2 knockdown downregulated the expression levels of COL2A1 and SOX-9. In summary, our results conclude that PKM2 modulates the glycolysis and extracellular matrix generation, providing the vital role of PKM2 on OA pathogenesis and a novel therapeutic target for OA.

Pyruvate kinase muscle isozymes (PKMs) have crucial roles in regulating metabolic changes during carcinogenesis. A switch from PKM1 to PKM2 isoform was thought to lead to aerobic glycolysis promoting carcinogenesis, and was considered as one of the cancer signatures. However, recent evidence has argued against the existence of PKM isoform switch and related metabolic effects during cancer progression. We compared the effects of PKM1 and PKM2 in cell invasiveness and metastasis of pancreatic ductal adenocarcinoma (PDAC). Both PKM1 and PKM2 expression affected cell migration and invasion abilities of PDAC cells, but only knockdown of PKM2 suppressed metastasis in a xenograft model. By comparing the established PKM2 mutants in the regulation of cell invasion, we found that PKM2 may control cell mobility through its protein kinase and phopho-peptide binding abilities. Further survey for PKM2-associated proteins identified PAK2 as a possible phosphorylation target in PDAC. In vitro binding and kinase assays revealed that PKM2 directly phosphorylated PAK2 at Ser20, Ser141, and Ser192/197. Knockdown of PKM2 decreased PAK2 protein half-life by increasing ubiquitin-dependent proteasomal degradation. Moreover, we identified PAK2 as an HSP90 client protein and the mutation at Ser192/197 of PAK2 reduced PAK2-HSP90 association. Knockdown of PAK2 diminished in vitro cell mobility and in vivo metastatic ability of PKM2 overexpressed PDAC cells. PKM2 and PAK2 protein expression also positively correlated with each other in PDAC tissues. Our findings indicate that PKM2-PAK2 regulation is critical for developing metastasis in PDAC, and suggest that targeting the PKM2/HSP90/PAK2 complex has a potential therapeutic value in this deadly disease.

Human immunodeficiency virus type 1 (HIV-1) recruits diverse cellular factors into viral particles during its morphogenesis, which apparently play roles in modulating its infectivity. In our study, proteomic techniques demonstrated that a key glycolytic protein, pyruvate kinase muscle type 2 (PKM2), is incorporated into viral particles. Here, we show that virion-packaged PKM2 significantly reduces viral infectivity by affecting the incorporation level of a cellular tRNA Lys3 into virions. Enhanced expression of PKM2 in HIV-1-producing cells led to a higher incorporation level of PKM2 into progeny virions without affecting the viral maturation process. Compared with the control virus, the high-level-PKM2-packaging virus showed decreased levels of both reverse transcription products and cellular tRNA Lys3 packaging, suggesting that the shortage of intravirion tRNA Lys3 suppresses reverse transcription efficiency in target cells. Interestingly, the enhanced expression of PKM2 also suppressed the virion recruitment of other nonpriming cellular tRNAs such as tRNA Lys1,2 and tRNA Asn , which are known to be selectively packaged into virions, without affecting the steady level of the cytoplasmic pool of those tRNAs in producer cells, suggesting that PKM2 specifically impedes the selective incorporation of tRNAs into virions. Taken together, our findings indicate that PKM2 is a vital host factor that negatively affects HIV-1 infectivity by targeting the tRNA Lys3 -mediated initiation of reverse transcription in target cells.

Backgrounds: Numerous studies have reported that aberrant pyruvate kinase M2 isoform (PKM2) expressed in cancer, indicating that PKM2 plays a critical role in tumor initiation and progression. Nevertheless, its prognostic value in breast cancer tumor is yet contentious. Therefore, we performed this meta-analysis to evaluate the prognostic significance of PKM2 in breast cancer., Methods: Eligible relevant literatures were retrieved by searching PubMed, the Cochrane Library, Embase through December 2016. Articles that comparing different PKM2 expression levels in human breast cancer tissues and prognostic significance were included. Software RevMan 5.3 and STATA (Review Manager (RevMan): [Computer program]. Version 5.3. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014., Stata: StataCorp. 2011. Stata Statistical Software: Release 12. College Station, TX: StataCorp LP) were applied to analyze the outcomes. Pooled results were presented in hazardous ratios (HRs) of 5-year overall survival (OS), progression-free survival (PFS), and odds ratios (ORs) of clinicopathological features with 95% confidence intervals., Results: Data from 6 involved studies with 895 patients were summarized. Breast cancer patients with high PKM2 had a worse OS (pooled HR = 1.65, 95% CI = 1.31-2.08, P < .001) and PFS (pooled HR = 2.49, 95% CI = 1.84-3.36, P < .00001). High PKM2 expression is related to lymph node metastasis (N1+N2+N3 vs N0, OR = 1.97, 95%CI = 1.39-2.80, P = .0001). The outcome stability was verified via sensitivity analysis. But elevated PKM2 expression was not correlated to tumor stage (T2+T3 vs T1, pooled OR = 0.80, 95% CI = 0.36-1.77, P = .58) and differential grade (G2+G3 vs G1, OR = 2.74, 95%CI = 0.76-9.84, P = .12). No publication bias was found in the included studies for OS (Begg test, P = .260; Egger test, P = .747)., Conclusions: High PKM2 expression denotes worse OS and PFS in breast cancer patients, and correlate with the lymph node metastasis. However, there is no evidence for the impact of PKM2 expression on T stage and tumor differentiation.

Metastasis is one of the main causes of breast cancer (BCa)-related deaths in female. It has been reported that cancer stem cell played an important role in metastasis. Here we first revealed a specific role of pyruvate kinase isozymes M2 (PKM2) in the stemness of breast cancer cells. Breast cancer tissue analysis confirmed the upregulation of PKM2 in breast cancer, and high PKM2 levels were associated with poor prognosis of breast cancer patients. Holoclone assay and colony formation assay significantly elucidated the role of PKM2 in the self-renewal of breast cancer cells. Moreover, PKM2 elevated the proportion of stem cell and the ability of sphere formation in breast cancer cells. PKM2 played its functional role in stemness by regulating β-catenin. Collectively, we identified critical roles of PKM2 in the stemness of breast cancer cells which may elevate the therapeutic effect on breast cancer patients.

Unlabelled: Hormones and their corresponding receptors are vital in controlling metabolism under normal physiologic and pathologic conditions, but less is known about their roles in the metabolism of cancer. Using a small interfering RNA screening approach, we examined the effects of silencing 20 well-known hormone receptors on the Warburg effect, specifically by measuring the production of lactate in four established hepatocellular carcinoma (HCC) cell lines. We found that silencing a variety of hormone receptors had effects on the production of this metabolite. Unexpectedly silencing of mineralocorticoid receptor (MR) significantly increased lactate production in all these HCC cell lines. Subsequent in vitro and in vivo studies showed that gain- and loss-of-function of MR significantly influenced HCC cellular proliferation, cell cycle distribution, and apoptosis. Furthermore, mechanistic studies revealed that MR as a transcriptional factor directly regulated the expression of miR-338-3p, suppressing the Warburg effects of HCC cells by targeting a key enzyme of glycolysis: pyruvate kinase, liver and red blood cells. Moreover, MR expression was significantly down-regulated in 81% of HCC patient tissues, caused by both chromosome deletion and histone deacetylation. Low expression of MR in tumor tissues was associated with poor patient prognosis. The expression level of miR-338-3p was found to positively correlate with the expression of MR in HCC tissues and to inversely correlate with expression of the enzyme pyruvate kinase, liver and red blood cells., Conclusion: MR affects HCC development by modulating the miR-338-3p/pyruvate kinase, liver and red blood cells axis with an ability to suppress the Warburg effect., (© 2015 The Authors. HEPATOLOGY published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Lysine acetylation plays an essential role in metabolism. Five individual studies have identified that a large number of cellular proteins are potentially acetylated. Notably, almost every enzyme involved in central metabolic pathways such as glycolysis, the TCA cycle, fat acid metabolism, urea cycle and glycogen metabolism, is acetylated in response to nutrition fluctuations. Metabolic reprogramming is a critical hallmark during cancer development. Tumor cells preferentially utilize glycolysis instead of oxidative phosphorylation to produce more lactate and metabolic intermediates even under normal oxygen pressure, which was first noted as the "Warburg Effect". This review focuses on recent advances in the acetylation regulation of metabolic enzymes involved in the Warburg effect, the dysfunction of acetylation regulation in tumorigenesis and their potential role in cancer metabolism therapy.

Cancer cells are characterized by reprogramming of energy metabolism. Over the last decade, understanding of the metabolic changes that occur in cancer has increased dramatically, with great interest in targeting metabolism for cancer therapy. Pyruvate kinase isoenzyme type M2 (abbreviations: PKM2, M2-PK) plays a key role in modulating glucose metabolism to support cell proliferation. PKM2, like other PK isoforms, catalyzes the last energy-generating step in glycolysis, but is unique in its capacity to be regulated. PKM2 is regulated at several cellular levels, including gene expression, alternative splicing and post-translational modification. In addition, PKM2 is regulated by key metabolic intermediates and interacts with more than twenty different proteins. Hence, this isoenzyme is an important regulator of glycolysis, and additionally functions in other novel roles that have recently emerged. Recent evidence indicates that intervening with the complex regulatory network of PKM2 has severe consequences on tumor cell proliferation, indicating the potential of this enzyme as a target for tumor therapy.

Background: The early diagnosis of biliary tract cancer (BTC) remains challenging, and there are few effective therapies. This study investigated whether the M2 isotype of pyruvate kinase (M2-PK), which serves as the key regulator of cellular energy metabolism in proliferating cells, could play a role in the diagnosis and therapy of BTC., Methods: Plasma and bile M2-PK concentrations were measured by enzyme-linked immunosorbent assay in 88 patients with BTC, 79 with benign biliary diseases, and 17 healthy controls. M2-PK expression was assayed in a BTC tissue array by immunohistochemistry. The role of M2-PK in tumor growth, invasion, and angiogenesis was evaluated in BTC cell lines by retrovirus-mediated M2-PK transfection and short hairpin RNA silencing techniques., Results: Sensitivity (90.3%) and specificity (84.3%) of bile M2-PK for malignancy were significantly higher than those for plasma M2-PK and serum carbohydrate antigen 19-9. M2-PK expression was specific for cancer cells and correlated with microvessel density. M2-PK positivity was a significant independent prognostic factor by multivariable analysis. Transfection of M2-PK in a negatively expressed cell line (HuCCT-1 cells) increased cell invasion, whereas silencing in an M2-PK-positive cell line (TFK cells) decreased tumor nodule formation and cellular invasion. A significant increase in endothelial tube formation was noted when supernatants from M2-PK-transfected cells were added to an in vitro angiogenesis assay, whereas supernatants from silenced cells negated endothelial tube formation., Conclusions: Bile M2-PK is a novel tumor marker for BTC and correlates with tumor aggressiveness and poor outcome. Short hairpin RNA-mediated inhibition of M2-PK indicates the potential of M2-PK as a therapeutic target., (Copyright © 2012 American Cancer Society.)

In Clostridium thermocellum, a thermophilic anaerobic bacterium able to rapidly ferment cellulose to ethanol, pyruvate kinase (EC 2.7.1.40) is absent based on both the genome sequence and enzymatic assays. Instead, a new pathway converting phosphoenolpyruvate to pyruvate via a three-step pathway involving phosphoenolpyruvate carboxykinase, NADH-linked malate dehydrogenase, and NADP-dependent malic enzyme has been found. We examined the impact of targeted modification of enzymes associated with this pathway, termed the "malate shunt", including expression of the pyruvate kinase gene from Thermoanaerobacterium saccharolyticum, mutation of the phosphoenolpyruvate carboxykinase and deletion of malic enzyme gene. Strain YD01 with exogenous pyruvate kinase, in which phosphoenolpyruvate carboxykinase expression was diminished by modifying the start codon from ATG to GTG, exhibited 3.25-fold higher ethanol yield than the wild-type strain. A second strain, YD02 with exogenous pyruvate kinase, in which the gene for malic enzyme and part of malate dehydrogenase were deleted, had over 3-fold higher ethanol yield than the wild-type strain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate by transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as a phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression of a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate-limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein-protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications.

Tumor aerobic glycolysis, or the Warburg effect, plays important roles in tumor survival, growth, and metastasis. Pyruvate kinase isoenzyme M2 (PKM2) is a key enzyme that regulates aerobic glycolysis in tumor cells. Recent research has shown that PKM2 can be used as a tumor marker for diagnosis and, in particular, as a potential target for cancer therapy. We investigated the effects of combining shRNA targeting PKM2 and docetaxel on human A549 lung carcinoma cells both in vivo and in vitro. We observed that the shRNA can significantly downregulate the expression level of PKM2. The decrease of PKM2 resulted in a decrease in ATP synthesis, which caused intracellular accumulation of docetaxel. Furthermore, the combination of pshRNA-pkm2 and docetaxel inhibited tumor growth and promoted more cancer cell apoptosis both in vivo and in vitro. Our findings suggest that targeting tumor glycolysis can increase the efficacy of chemotherapy. In particular, the targeting of PKM2 could, to some extent, be a new way of reversing chemotherapy resistance to cancer therapy.

The energetic landscape of the allosteric regulatory mechanism of rabbit muscle pyruvate kinase (RMPK) was characterized by isothermal titration calorimetry (ITC). Four novel insights were uncovered. (1) ADP exhibits a dual property. Depending on the temperature, ADP can regulate RMPK activity by switching the enzyme to either the R or T state. (2) The assumption that ligand binding to RMPK is state-dependent is only correct for PEP but not Phe and ADP. (3) The effect of pH on the regulatory behavior of RMPK is partly due to the complex pattern of proton release or absorption linked to the multiple linked equilibria which govern the activity of the enzyme. (4) The R <--> T equilibrium is accompanied by a significant DeltaC(p), rendering RMPK most sensitive to temperature under physiological conditions. To rigorously test the validity of conclusions derived from the ITC data, in this study a fluorescence approach, albeit indirect, that tracks continuous structural perturbations was employed. Intrinsic Trp fluorescence of RMPK in the absence and presence of substrates phosphoenolpyruvate (PEP) and ADP, and the allosteric inhibitor Phe, was measured in the temperature range between 4 and 45 degrees C. For data analysis, the fluorescence data were complemented by ITC experiments to yield an extended data set allowing more complete characterization of the RMPK regulatory mechanism. Twenty-one thermodynamic parameters were derived to define the network of linked interactions involved in regulating the allosteric behavior of RMPK through global analysis of the ITC and fluorescent data sets. In this study, 27 independent curves with more than 1600 experimental points were globally analyzed. Consequently, the consensus results substantiate not only the conclusions derived from the ITC data but also structural information characterizing the transition between the active and inactive states of RMPK and the antagonism between ADP and Phe binding. The latter observation reveals a novel role for ADP in the allosteric regulation of RMPK.

Rabbit muscle pyruvate kinase (RMPK) is an important allosteric enzyme of the glycolytic pathway catalyzing a transfer of the phosphate from phosphoenolpyruvate (PEP) to ADP. The energetic landscape of the allosteric regulatory mechanism of RMPK was characterized by isothermal titration calorimetry (ITC) in the temperature range from 4 to 45 degrees C. ITC data for RMPK binding to substrates PEP and ADP, for the allosteric inhibitor Phe, and for combination of ADP and Phe were globally analyzed. The thermodynamic parameters characterizing the linked-multiple-equilibrium system were extracted. Four novel insights were uncovered. (1) The binding preference of ADP for either the T or R state is temperature-dependent, namely, more favorable to the T and R states at high and low temperatures, respectively. This crossover of affinity toward R and T states implies that ADP plays a complex role in modulating the allosteric behavior of RMPK. Depending on the temperature, binding of ADP can regulate RMPK activity by favoring the enzyme to either the R or T state. (2) The binding of Phe is negatively coupled to that of ADP; i.e., Phe and ADP prefer not to bind to the same subunit of RMPK. (3) The release or absorption of protons linked to the various equilibria is specific to the particular reaction. As a consequence, pH will exert a complex effect on these linked equilibria, resulting in the proton being an allosteric regulatory ligand of RMPK. (4) The R <--> T equilibrium is accompanied by a significant DeltaC(p), rendering RMPK most sensitive to temperature under physiological conditions. During muscle activity, both pH and temperature fluctuations are known to happen; thus, results of this study are physiologically relevant.

Mammalian pyruvate kinase exists in four isoforms with characteristics tuned to specific metabolic requirements of different tissues. All of the isoforms, except the muscle isoform, exhibit typical allosteric behavior. The case of the muscle isoform is a conundrum. It is inhibited by an allosteric inhibitor, Phe, yet it has traditionally not been considered as an allosteric enzyme. In this series of study, an energetic landscape of rabbit muscle pyruvate kinase (RMPK) was established. The phenomenon of inhibition by Phe is shown to be physiological. Furthermore, the thermodynamics for the temperature fluctuation and concomitant pH change as a consequence of muscle activity were elucidated. We have shown that (1) the differential number of protons released or absorbed with regard to the various linked reactions adds another level of control to shift the binding constants and equilibrium of active <--> inactive state changes (the latter controls quantitatively the activity of RMPK); (2) ADP plays a major role in the allosteric mechanism in RMPK under physiological temperatures (depending on the temperature, ADP can assume dual and opposite roles of being an inhibitor by binding preferentially to the inactive form and a substrate); and (3) simulation of the RMPK behavior under physiological conditions shows that the net results of the 21 thermodynamic parameters involved in the regulation are well-tuned to allow the maximal response of the enzyme to even minute changes in temperature and ligand concentration.

To establish an infection, the pathogen Candida albicans must assimilate carbon and grow in its mammalian host. This fungus assimilates six-carbon compounds via the glycolytic pathway, and two-carbon compounds via the glyoxylate cycle and gluconeogenesis. We address a paradox regarding the roles of these central metabolic pathways in C. albicans pathogenesis: the glyoxylate cycle is apparently required for virulence although glyoxylate cycle genes are repressed by glucose at concentrations present in the bloodstream. Using GFP fusions, we confirm that glyoxylate cycle and gluconeogenic genes in C. albicans are repressed by physiologically relevant concentrations of glucose, and show that these genes are inactive in the majority of fungal cells infecting the mouse kidney. However, these pathways are induced following phagocytosis by macrophages or neutrophils. In contrast, glycolytic genes are not induced following phagocytosis and are expressed in infected kidney. Mutations in all three pathways attenuate the virulence of this fungus, highlighting the importance of central carbon metabolism for the establishment of C. albicans infections. We conclude that C. albicans displays a metabolic program whereby the glyoxylate cycle and gluconeogenesis are activated early, when the pathogen is phagocytosed by host cells, while the subsequent progression of systemic disease is dependent upon glycolysis.

Proliferating cells and tumor cells in particular express the pyruvate kinase isoenzyme type M2 (M2-PK). Within the tumor metabolome M2-PK regulates the proportions of glucose carbons that are channelled to synthetic processes (inactive dimeric form) or used for glycolytic energy production (highly active tetrameric form, a component of the glycolytic enzyme complex). In tumor cells, the dimeric form of M2-PK (Tumor M2-PK) is always predominant. The dimerization is caused by direct interaction of M2-PK with certain oncoproteins. The switch between the tetrameric and dimeric form of M2-PK allows tumor cells to survive in environments with varying oxygen und nutrient supply.

Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

A mutation was recovered in the slr0721 gene, which encodes the decarboxylating NADP(+)-dependent malic enzyme in the cyanobacterium Synechocystis sp. strain PCC 6803, yielding the mutant 3WEZ. Under continuous light, 3WEZ exhibits poor photoautotrophic growth while growing photoheterotrophically on glucose at rates nearly indistinguishable from wild-type rates. Interestingly, under diurnal light conditions (12 h of light and 12 h of dark), normal photoautotrophic growth of the mutant is completely restored.

The pyruvate kinases from Genus Bacillus and a few other bacteria have an extra C-terminal sequence with a phosphoenolpyruvate binding motif composed of about 110 amino acids. To elucidate the possible structure and function of this sequence, the enzyme lacking the sequence was prepared and characterized. The N-terminal sequences of the peptides, which were found only in the lysylendopeptidase digest of the wild enzyme and not in that of the truncated enzyme, were determined. All the determined sequences were found in the extra C-terminal sequence deduced from the DNA sequence. The truncated enzyme showed decreased affinity for phosphoenolpyruvate and the allosteric effector ribose 5-phosphate, and had a reduced thermostability. Other properties, such as tetrameric structure, specific activity, and allosteric characteristics were unchanged. A comparison of the CD spectra of the truncated enzyme and the recombinant enzyme indicated that the structure of the C-terminal sequence should be rich in beta-sheet. These findings suggest that the sequence actually exists and that it may form a steady domain interacting with the A-domain and C-domain, which are the catalytic domain and allosteric effector binding domain, respectively.

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.

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.

Pyruvate kinase (PK) is a key enzyme for the glycolytic pathway and carbon metabolism in general. On the basis of the relevance and enormous diverse properties of this enzyme, this paper describes the results of a current and extensive review that determines the sites of conservation and/or difference in PK sequences, and the differences in the functional and regulatory properties of the enzymes. An alignment and analysis of 50 PK sequences from different sources and a phylogenetic tree are presented. This analysis was performed with reference to crystallographically characterized PK principally from E. coli, cat and rabbit muscle. A number of attributes of the enzyme that make it of particular interest in biomedicine and industry are also discussed.

Pyruvate kinase (PK) deficiency is a common cause of hereditary non-spherocytic haemolytic anaemia. It is an autosomal recessive disorder caused by mutations in the gene coding for erythrocyte and liver-type pyruvate kinase (PKLR). So far, more than 130 mutations in this gene have been identified. Clinical symptoms, usually restricted to homozygous and compound-heterozygous individuals, are variable, ranging from neonatal jaundice requiring erythrocyte transfusions to a fully compensated haemolytic anaemia. The exact mechanism of erythrocyte destruction is unknown, however adenosine-triphosphate depletion and an increase in 2,3-disphosphoglycerate are thought to be important. The diagnosis of pyruvate kinase deficiency depends upon the demonstration of low PK enzyme activity. Due to the pitfalls in determining true PK activity, DNA testing is a valuable tool in the diagnosis of pyruvate kinase deficiency. By centralizing the molecular diagnostics of pyruvate kinase deficiency in Utrecht, more care can be provided for the diagnosis, treatment and support of patients.

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling.

Cell proliferation is a process that consumes large amounts of energy. A reduction in the nutrient supply can lead to cell death by ATP depletion, if cell proliferation is not limited. A key sensor for this regulation is the glycolytic enzyme pyruvate kinase, which determines whether glucose carbons are channelled to synthetic processes or used for glycolytic energy production. In unicellular organisms pyruvate kinase is regulated by ATP, ADP and AMP, by ribose 5-P, the precursor of the nucleic acid synthesis, and by the glycolytic intermediate fructose 1,6-P2 (FBP), thereby adapting cell proliferation to nutrient supply. The mammalian pyruvate kinase isoenzyme type M2 (M2-PK) displays the same kinetic properties as the pyruvate kinase enzyme from unicellular organisms. The mammalian M2-PK isoenzyme can switch between a less active dimeric form and a highly active tetrameric form which regulates the channeling of glucose carbons either to synthetic processes (dimeric form) or to glycolytic energy production (tetrameric form). Tumor cells are usually characterized by a high amount of the dimeric form leading to a strong accumulation of all glycolytic phosphometabolites above pyruvate kinase. The tetramer-dimer ratio is regulated by ATP, FBP and serine and by direct interactions with different oncoproteins (pp60v-src, HPV-16 E7). In solid tumors with sufficient oxygen supply pyruvate is supplied by glutaminolysis. Pyruvate produced in glycolysis and glutaminolysis is used for the synthesis of lactate, glutamate and fatty acids thereby releasing the hydrogen produced in the glycolytic glyceraldehyde 3-phosphate dehydrogenase reaction.

Polymyositis (PM) is an inflammatory muscle disease caused by autoimmune dysfunction, considered to be caused by cytotoxic CD8 T cells. To date, no autoantigens have been identified. We attempted to induce an experimental allergic myositis (EAM) in BALB/c mice by inoculating syngeneic dendritic cells (DC) presenting peptides that are expected to match the binding anchor motif of H-2K(d) (BALB/c). We selected peptides that are highly expressed in skeletal muscle. Only when we inoculated syngeneic bone marrow-derived DC presenting pyruvate kinase M1/M2 peptide 464-472 in BALB/c mice, 41.7% of the mice (EAM) developed pathological changes in skeletal muscle compatible to human PM. Under other conditions (when we inoculated DC presenting no synthetic peptides into BALB/c or C57BL/6 mice and DC presenting pyruvate kinase M1/M2 peptide into C57BL/6 mice), there were no necrotizing and inflammatory lesions. Induction of EAM in the same manner as above also induced CTL activity against P815 cells with the same peptide and syngeneic differentiated cultured myotubes without peptides by the chromium release assay. Consistent with the similarity of the binding anchor motif of H-2K(d) (BALB/c) and HLA A*2402, we conclude that pyruvate kinase M1/M2 peptide is a candidate autoantigen not only in BALB/c-EAM but also in human-PM with the HLA A*2402 allele.

Pyruvate kinase deficiency is the most frequent enzyme abnormality of the Embden-Meyerhof pathway causing hereditary non-spherocytic haemolytic anaemia. The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life-threatening neonatal anaemia and jaundice necessitating exchange transfusions. Splenectomy should be reserved for young patients who require regular blood transfusions. The gene encoding for pyruvate kinase (PK-LR) has been localized to the long arm of chromosome I; the cDNA of R-type is 2060 bp long and codes for 574 amino acids. More than 130 different mutations, mostly missense, have so far been described in association with PK deficiency, 1529A and 1456T being considered to be the most common mutations in Caucasians. Analysis of the three-dimensional structure of the enzyme may help in predicting the severity of the molecular defect. Further data on clinical features of homozygous patients are needed, at least for some mutations, to allow a more precise genotype/phenotype correlation.

Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the crystal structure of the first eukaryotic pyruvate kinase in the T-state (the inactive or 'tense' conformation of allosteric enzymes) is described. A comparison of the effector sites of the Leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops, comprising residues 443-453 and 480-489, adopt very different conformations in the two enzymes, and Lys-453 and His-480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These and other differences offer an opportunity for the design of drugs that would exploit regulatory differences between parasite and host.

We have initiated an analysis of protein kinase A (PKA) in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. We have constructed GAL4/UAS-regulated transgenes in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. We present evidence that the wild-type transgenes are active and summarize the phenotypes produced by a number of GAL4 enhancer-detector strains. We compare the effects of transgenes encoding PKI(1-31) with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits.

Properties of mung bean pyruvate kinase were studied and the active site groups were derived. Metabolites like AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-bisphosphate, 3-phospho-glycerate, isocitrate, malate and alpha-ketoglutarate had practically no effect on pyruvate kinase activity. Alanine, serine, glutamine, methionine and GMP had a weak activating effect on the enzyme. Some metabolites such as ATP, GTP, and UMP were found to be weakly inhibitory. Moderate to strong inhibition was observed with citrate, succinate, glutamate and oxalate. Inhibition brought about by ATP and citrate when present together showed synergistic effect. Inhibition by citrate was non-competitive with respect to both PEP and ADP suggesting the presence of a regulatory site. Mung bean pyruvate kinase showed half optimal activity at pH 6.6 and 8.9 at saturating concentrations of PEP, ADP and Mg2+. Small concentrations of the SH specific reagents, namely iodoacetamide (0.1 and 0.2 mM), N-ethylmaleimide(0.05-0.1 mM) and p-chloromercuribenzoate (0.1 mM) inactivated the enzyme; single exponential loss of activity was observed in each case. Photooxidation of the enzyme in the presence of methylene blue (100 and 200 micrograms/ml) and rose bengal (5 and 10 micrograms/ml) also led to a single exponential activity decay. When the enzyme was treated with diethyl pyrocarbonate (DEP), a time dependent exponential decay in its activity was observed with a parallel increase in absorbance at 240 nm. PEP protected the enzyme against inactivation by DEP. Reagents specific for tyrosine (iodine and tetranitromethane) and tryptophan residues (N-bromosuccinimide) residues had no effect. These observations confirm that SH and imidazole groups are vital for the activity of the enzyme.

Associations of glycolytic enzymes with the subcellular particulate fraction of skeletal muscle and heart were examined during entrance to daily torpor in deer mice (Peromyscus maniculatus). In skeletal muscle a significant decrease in enzyme binding occurred during torpor entrance for phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase, with an additional significant decrease for phosphofructokinase and pyruvate kinase during the deepest state of torpor. Reductions in enzyme binding during torpor entrance also occurred in heart; significant changes were observed in hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase binding. Contrary to the finding of additional reductions in enzyme binding seen in skeletal muscle, significant increases in enzyme binding during the deepest torpor state were observed for hexokinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase in heart. These results suggest that a decrease in the binding of glycolytic enzymes to subcellular structures in skeletal muscle and heart may be at least partially responsible for initiating the reduction in metabolic rate during torpor entrance. This decreased binding may continue to mediate the metabolic reduction in skeletal muscle throughout torpor; heart, however, may require the use of different molecular mechanisms. The increased binding in heart during the deepest state of torpor may represent an anticipatory response in preparation for increased activity during arousal.

The Wistar fatty rat is a model of obese non-insulin-dependent diabetes mellitus. Males, but not females, develop hyperglycemia, glucouria and polyuria within 8 weeks of age. The regulation of gene expression by insulin has been shown to be differentially impaired in the liver of the fatty rats. The genes resistant to insulin include glucokinase gene and phosphoenolpyruvate carboxykinase gene. In contrast, L-type pyruvate kinase gene responds to insulin normally, raising the possibility that the signaling pathway from the insulin receptor to the insulin-resistant genes, but not to the insulin-sensitive genes, is defective at a point beyond the receptor kinase in the fatty rats. On the other hand, female fatty rats develop hyperglycemia only when they are given sucrose for several weeks. This treatment causes a decrease in gucokinase while enzymes involved in gluconeogenesis are increased. Chronic feeding of sucrose also leads to hypertriglycemia and visceral fat accumulation, which is more frequently associated with abnormalities in glucose and lipid metabolisms. Fructose is believed to be the responsible component of sucrose for these effects. Hypertriglyceridemic effect of fructose is mainly due to an increase in hepatic production of VLDL. Most enzymes related to lipogenesis in the liver are induced by dietary fructose even in diabetes. L-type pyruvate kinase is one of such enzymes. Cis-acting element named PKL-III in the 5'-flanking region of this gene is shown to be responsive to dietary fructose as well as to dietary glucose. Thus, identification and characterization of a protein bound to this element could help in the further understanding of the molecular mechanism of the fructose actions.

To investigate the effects of estrogen, the hormone responsible for vitellogenesis, on gluconeogenesis, male rainbow trout were implanted with 17 beta-estradiol or given a sham procedure. Plasma glucose concentration in estrogenized fish was 50% of the control fish (6.4 mM). Glucose synthesis from physiological concentrations of alanine was 0.08 mumol.g cells-1 x h-1 compared with 0.20 mumol.g cells-1 x h-1 in control fish; synthesis from physiological concentrations of lactate was reduced by over 50% (0.88 vs. 0.36 mumol.g cells-1 x h-1) in implanted fish. Gluconeogenesis from 5 mM lactate was also significantly depressed in implanted fish. Oxidation of alanine, serine, and lactate was not significantly affected by estrogen implantation. The maximum clearance velocity of a key enzyme negatively regulating gluconeogenesis, pyruvate kinase, was 3.03 mumol.g cells-1 x h-1 in estrogen (E2) implanted fish compared with 7.83 mumol.g cells-1 x h-1 in control fish. No significant differences in plasma insulin or glucagon were found in the two groups. We conclude that estrogen depresses gluconeogenesis and that this reduction contributes to the lower plasma glucose concentration seen in vitellogenic trout.

Key enzymes related to lipogenesis in the liver are induced by a high glucose diet or insulin and suppressed by starvation, diabetes, or glucagon. Most of these enzymes are also induced by dietary fructose, even in diabetic liver. This regulation occurs at the posttranscriptional level as well as at the transcriptional level. We studied extensively the molecular mechanism of induction of L-type pyruvate kinase (LPK). The transcription of the LPK gene in the liver was stimulated by insulin and inhibited by glucagon. This insulin action required ongoing protein synthesis and metabolism of glucose and was enhanced by glucocorticoid. On the other hand, the mechanism of induction of the LPK by dietary fructose depended on plasma insulin levels. Dietary fructose stimulated transcription of the LPK gene in normal rats, whereas it acted mainly at the posttranscriptional level in diabetic rats. These fructose effects were attributable to a common metabolite of fructose and glycerol. The induction of LPK mRNA by dietary glucose was impaired in the liver of Wistar fatty rats, a model of obese non-insulin-dependent diabetes mellitus, but fructose-induced accumulation of the mRNA was not. Studies on transgenic mice indicated that the 5'-flanking region up to -3 kb of the LPK gene contained all cis-acting elements necessary for tissue-specific expression of LPK and its stimulation by diets and insulin. Further analysis using a transient expression assay revealed the presence of three cis-acting elements necessary for expression of LPK in hepatocytes in the region up to -170 kb. However, these elements alone were not sufficient for dietary and hormonal regulation of this enzyme when analyzed in transgenic mice.

As a common characteristic of tumor cells, as well as of normal proliferating cells in the G1-phase of cell cycle, one finds constitutive high levels of all the glycolytic metabolites arising between glucose 6-phosphate and phosphoenolpyruvate. Thus, it is that the phosphometabolites fructose 1,6-bisphosphate, ribose 5-P, P-ribose-PP, NAD, GTP, CTO, UTP, UDP-glucose, glycerol 3-P, glycerol phosphocholine and glycerol phosphoethanolamine are useful in the 31P-nuclear magnetic resonance (NMR) detection of solid tumors in animals and man. This expansion of phosphometabolites is achieved during tumor formation as a result of reductions in levels of enzymes degrading phosphometabolites, owing to the decline in the glycerol 3-P hydrogen shuttle, and as a consequence of alterations in the glycolytic isoenzyme equipment. Tumor cells typically express a particular isoenzyme of pyruvate kinase called type M2 (K) at high levels. This isoenzyme is subject to a complex regulation by amino acids, by fructose 1,6-bisphosphate, and by hormonal- and oncogene-dependent phosphorylation. Pyruvate kinase type M2 is a substrate for the oncogene encoded PP60v-src-tyrosine kinase. A drastic decrease in the affinity for its substrate phosphoenolpyruvate found after transformation by the src-oncogene can be explained as a consequence of the phosphorylation of pyruvate kinase in serine and tyrosine. These phosphorylations induce the breakdown of tetrameric pyruvate kinase to the trimeric and dimeric forms. Unlike the tetrameric form, the dimeric form as a low affinity for phosphoenolpyruvate. Partial inactivation of pyruvate kinase and enolase on the one hand, and a hyperactivation of hexokinase and phosphofructokinase on the other hand, lead to an expansion of all metabolites. Only when these metabolites attain high levels, thereby assuring a sufficient supply of metabolites for RNA, DNA, lipid, and complex carbohydrate synthesis, can cell proliferation proceed. This accumulation of metabolites in the G1-phase cells has been termed a "metabolic budget system" because it senses not only the actual nutrient levels, but also the supply over a period of time. Monoclonal antibodies specific for the dimeric form of pyruvate kinase type M2 can be used for the immunohistological detection of tumor cells. The amount of the dimeric form in tumor cells closely correlates with the degree of malignancy and can be used for a nonspecific detection of tumors based on assays performed with patient's plasma.

Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. The enzyme has an activation energy of 31.9 kJ mol-1. Magnesium and potassium ions are essential for activity. Cobalt or manganese ions replace Mg2+ but this leads to a decrease in maximal velocity. Potassium ions can be substituted by ammonium ions, while sodium ions behave as a competitive inhibitor with respect to both K+ and NH4+. All metal ions studied displayed sigmoidal kinetics. The enzyme is activated, with decreasing efficiency by fructose 2-phosphorothioate 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. They all display hyperbolic kinetics. Glycerate 2,3-bisphosphate, glyceraldehyde 3-phosphate, CoASAc, oxalate, AMP, ADP, and ATP inhibit the enzyme. At substrate saturation PK was activated by Pi up to a concentration of 0.8 mM. At higher Pi concentrations the enzyme is inhibited. The enzyme is unaffected by most amino acids, only phenylalanine stimulates and tyrosine inhibits.