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Laryngeal squamous cell carcinoma (LSCC) with a high incidence and mortality rate, has a serious impact worldwide. Phosphofructokinase-1 liver type (PFKL) is a major enzyme in glycolysis progress, but its role in modulating tumorigenesis and cisplatin (DDP) chemosensitivity of LSCC was still unclear. The mRNA and protein levels of PFKL were detected by qRT-PCR and immunohistochemical assay. Cell Counting Kit-8 assay and flow cytometry were carried out to observe cell viability, as well as apoptosis and mitochondrial reactive oxygen species (mito-ROS). Extracellular acidification rate and lactate content were measured using extracellular flux analysis and lactate assay kit. Immunofluorescent staining was used to evaluate the expression of γ-H2AX foci. DNA damage was detected via single-cell gel electrophoresis. Western blotting was introduced to evaluate the protein level of PFKL, LDHA, γ-H2AX, cleaved PARP, H3K27ac, and H3K9ac. Mice xenograft model of LSCC was built for in vivo validation. The PFKL expression was significantly increased in LSCC and associated with poor survival of LSCC patients. Knockdown of PFKL in LSCC cells significantly inhibited cell viability, ECAR, lactate content, and LDHA expression, but promoted mito-ROS level. Furthermore, knockdown of PFKL enhanced response of LSCC cells to DDP by increasing DDP-induced apoptosis, promoting DDP-induced mito-ROS level, γ-H2AX foci, tail DNA, and the expression of γ-H2AX and cleaved PARP. However, the overexpression of PFKL in LSCC cells had opposite experimental results. Nude mice tumor formation experiment proved that downregulation of PFKL significantly enhanced response of cells to DDP, demonstrated by reduced tumor volume, weight and increased TUNEL-positive cells. Suppression of CBP/EP300-mediated PFKL transcription inhibited cell viability and glycolysis and promoted mito-ROS in LSCC. PFKL promotes cell viability and DNA damage repair in DDP-treated LSCC through regulation of glycolysis pathway., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare that are relevant to the content of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Most cancer cells exhibit high glycolysis rates under conditions of abundant oxygen. Maintaining a stable glycolytic rate is critical for cancer cell growth as it ensures sufficient conversion of glucose carbons to energy, biosynthesis, and redox balance. Here we deciphered the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway. Knocking down or knocking out PKM2 induced a thermodynamic equilibration in the glycolytic pathway, characterized by the reciprocal changes of the Gibbs free energy (ΔG) of the reactions catalyzed by PFK1 and PK, leading to a less exergonic PFK1-catalyzed reaction and a more exergonic PK-catalyzed reaction. The changes in the ΔGs of the two reactions cause the accumulation of intermediates, including the substrate PEP (the substrate of PK), in the segment between PFK1 and PK. The increased concentration of PEP in turn increased PK activity in the glycolytic pathway. Thus, the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway maintains the reciprocal relationship between PK concentration and its substrate PEP concentration, by which, PK activity in the glycolytic pathway can be stabilized and effectively counteracts the effect of PKM2 KD or KO on glycolytic rate. In line with our previous reports, this study further validates the roles of the thermodynamics of the glycolytic pathway in stabilizing glycolysis in cancer cells. Deciphering the interaction between glycolytic enzymes and the thermodynamics of the glycolytic pathway will promote a better understanding of the flux control of glycolysis in cancer cells., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1β than Pfkl S775A/S775A after LPS treatment. In an in vivo inflammation model, Pfkl S775A/S775A mice show reduced levels of MCP-1 and IL-1β. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis., (© 2024. The Author(s).)

Aerobic glycolysis is an emerging hallmark of many human cancers, as cancer cells are defined as a "metabolically abnormal system". Carbohydrates are metabolically reprogrammed by its metabolizing and catabolizing enzymes in such abnormal cancer cells. Normal cells acquire their energy from oxidative phosphorylation, while cancer cells acquire their energy from oxidative glycolysis, known as the "Warburg effect". Energy-metabolic differences are easily found in the growth, invasion, immune escape and anti-tumor drug resistance of cancer cells. The glycolysis pathway is carried out in multiple enzymatic steps and yields two pyruvate molecules from one glucose (Glc) molecule by orchestral reaction of enzymes. Uncontrolled glycolysis or abnormally activated glycolysis is easily observed in the metabolism of cancer cells with enhanced levels of glycolytic proteins and enzymatic activities. In the "Warburg effect", tumor cells utilize energy supplied from lactic acid-based fermentative glycolysis operated by glycolysis-specific enzymes of hexokinase (HK), keto-HK-A, Glc-6-phosphate isomerase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, phosphofructokinase (PFK), phosphor-Glc isomerase (PGI), fructose-bisphosphate aldolase, phosphoglycerate (PG) kinase (PGK)1, triose phosphate isomerase, PG mutase (PGAM), glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase isozyme type M2 (PKM2), pyruvate dehydrogenase (PDH), PDH kinase and lactate dehydrogenase. They are related to glycolytic flux. The key enzymes involved in glycolysis are directly linked to oncogenesis and drug resistance. Among the metabolic enzymes, PKM2, PGK1, HK, keto-HK-A and nucleoside diphosphate kinase also have protein kinase activities. Because glycolysis-generated energy is not enough, the cancer cell-favored glycolysis to produce low ATP level seems to be non-efficient for cancer growth and self-protection. Thus, the Warburg effect is still an attractive phenomenon to understand the metabolic glycolysis favored in cancer. If the basic properties of the Warburg effect, including genetic mutations and signaling shifts are considered, anti-cancer therapeutic targets can be raised. Specific therapeutics targeting metabolic enzymes in aerobic glycolysis and hypoxic microenvironments have been developed to kill tumor cells. The present review deals with the tumor-specific Warburg effect with the revisited viewpoint of recent progress.

While aerobic glycolysis, or the Warburg effect, has for a long time been considered a hallmark of tumor metabolism, recent studies have revealed a far more complex picture. Tumor cells exhibit widespread metabolic heterogeneity, not only in their presentation of the Warburg effect but also in the nutrients and the metabolic pathways they are dependent on. Moreover, tumor cells can switch between different metabolic phenotypes in response to environmental cues and therapeutic interventions. A framework to analyze the observed metabolic heterogeneity and plasticity is, however, lacking. Using a mechanistic model that includes the key metabolic pathways active in tumor cells, we show that the inhibition of phosphofructokinase by excess ATP in the cytoplasm can drive a preference for aerobic glycolysis in fast-proliferating tumor cells. The differing rates of ATP utilization by tumor cells can therefore drive heterogeneity with respect to the presentation of the Warburg effect. Building upon this idea, we couple the metabolic phenotype of tumor cells to their migratory phenotype, and show that our model predictions are in agreement with previous experiments. Next, we report that the reliance of proliferating cells on different anaplerotic pathways depends on the relative availability of glucose and glutamine, and can further drive metabolic heterogeneity. Finally, using treatment of melanoma cells with a BRAF inhibitor as an example, we show that our model can be used to predict the metabolic and gene expression changes in cancer cells in response to drug treatment. By making predictions that are far more generalizable and interpretable as compared to previous tumor metabolism modeling approaches, our framework identifies key principles that govern tumor cell metabolism, and the reported heterogeneity and plasticity. These principles could be key to targeting the metabolic vulnerabilities of cancer., Competing Interests: The authors have declared that no competing interests exist.

HCC has remained one of the challenging cancers to treat, owing to the paucity of drugs targeting the critical survival pathways. Considering the cancer cells are deficient in DNase activity, the increase of an autonomous apoptisis endonuclease should be a reasonable choice for cancer treatment. In this study, we investigated whether DNASE1L3, an endonuclease implicated in apoptosis, could inhibit the progress of HCC. We found DNASE1L3 was down-regulated in HCC tissues, whereas its high expression was positively associated with the favorable prognosis of patients with HCC. Besides, serum DNASE1L3 levels were lower in HCC patients than in healthy individuals. Functionally, we found that DNASE1L3 inhibited the proliferation of tumor cells by inducing G0/G1 cell cycle arrest and cell apoptosis in vitro . Additionally, DNASE1L3 overexpression suppressed tumor growth in vivo . Furthermore, we found that DNASE1L3 overexpression weakened glycolysis in HCC cells and tissues via inactivating the rate-limiting enzymes involved in PTPN2-HK2 and CEBPβ-p53-PFK1 pathways. Finally, we identified the HBx to inhibit DNASE1L3 expression by up-regulating the expression of ZNF384. Collectively, our findings demonstrated that DNASE1L3 could inhibit the HCC progression through inducing cell apoptosis and weakening glycolysis. We believe DNASE1L3 could be considered as a promising prognostic biomarker and therapeutic target for HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Rheumatoid arthritis (RA) is a classic autoimmune disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. The specific pathogenesis of RA, a chronic inflammatory disease, remains unclear. However, both key glycolysis rate-limiting enzymes, hexokinase-II (HK-II), phosphofructokinase-1 (PFK-1), and pyruvate kinase M2 (PKM2), as well as indirect rate-limiting enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), are thought to participate in the pathogenesis of RA. In here, we review the latest literature on the pathogenesis of RA, introduce the pathophysiological characteristics of HK-II, PFK-1/PFKFB3, and PKM2 and their expression characteristics in this autoimmune disease, and systematically assess the association between the glycolytic rate-limiting enzymes and RA from a molecular level. Moreover, we highlight HK-II, PFK-1/PFKFB3, and PKM2 as potential targets for the clinical treatment of RA. There is great potential to develop new anti-rheumatic therapies through safe inhibition or overexpression of glycolysis rate-limiting enzymes., 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 Zuo, Tang, Lu, Zhou, Li, Tian, Liu, Gao, Liu and Shao.)

We propose that fructose-1,6-bisphosphate (F-1,6-BP) promotes a feedback loop between phosphofructokinase-1 (PFK1), phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), and PFK2/PFKFB3, which enhances aerobic glycolysis and sustains effector T (T eff ) cell activation, while oxidative metabolism is concomitantly downregulated. This regulation, promoted by low citrate and mitochondrial ATP synthesis, also sustains the Warburg effect in cancer cells., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Temporal order in living matters reflects the self-organizing nature of dynamical processes driven out of thermodynamic equilibrium. Because of functional reasons, the period of a biochemical oscillation must be tuned to a specific value with precision; however, according to the thermodynamic uncertainty relation (TUR), the precision of the oscillatory period is constrained by the thermodynamic cost of generating it. After reviewing the basics of chemical oscillations using the Brusselator as a model system, we study the glycolytic oscillation generated by octameric phosphofructokinase (PFK), which is known to display a period of several minutes. By exploring the phase space of glycolytic oscillations, we find that the glycolytic oscillation under the cellular condition is realized in a cost-effective manner. Specifically, over the biologically relevant range of parameter values of glycolysis and octameric PFK, the entropy production from the glycolytic oscillation is minimal when the oscillation period is (5-10) min. Furthermore, the glycolytic oscillation is found at work near the phase boundary of limit cycles, suggesting that a moderate increase of glucose injection rate leads to the loss of oscillatory dynamics, which is reminiscent of the loss of pulsatile insulin release resulting from elevated blood glucose level.

We monitored changes that took place in glycolytic enzymes, the pyruvate end product of glycolysis, tumor necrosis factor α (TNFα), and toll-like receptors (TLRs) both at the transcriptional and translational levels upon direct interaction between PR8-H1N1 and the human monocytes U937 in vitro system. U937 were first treated with H1N1 infectious viral particles or phorbol-12-myristate-13-acetate (PMA) or left untreated and later infected with the H1N1 virus. Levels of phosphofructokinase 1 (PFK1) and pyruvate were biochemically quantified. In addition, levels of TNFα, TLR3, and TLR7 were measured by ELISA. The transcriptional profiles of PFKs, inflammatory cytokines, TLR3 and TLR7 were relatively quantified by qRT-PCR. The results generally revealed significant changes in both the transcriptional and translational profiles of the studied biochemical and immunological parameters upon influenza infection in a time-dependent manner. In conclusion, H1N1 infection triggers transcriptional and translational changes in immortalized human monocytes, which might serve as markers for infection subject for further validation for their specificities., (© 2020 International Union of Biochemistry and Molecular Biology.)

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3- 3 H]glucose relative to [2- 3 H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2- 13 C 2 ]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, P i , and glycerol 3-phosphate., (© 2020 Moonira et al.)

Increased glycolytic metabolism recently emerged as an essential process driving host defense against Mycobacterium tuberculosis (Mtb), but little is known about how this process is regulated during infection. Here, we observe repression of host glycolysis in Mtb-infected macrophages, which is dependent on sustained upregulation of anti-inflammatory microRNA-21 (miR-21) by proliferating mycobacteria. The dampening of glycolysis by miR-21 is mediated through targeting of phosphofructokinase muscle (PFK-M) isoform at the committed step of glycolysis, which facilitates bacterial growth by limiting pro-inflammatory mediators, chiefly interleukin-1β (IL-1β). Unlike other glycolytic genes, PFK-M expression and activity is repressed during Mtb infection through miR-21-mediated regulation, while other less-active isoenzymes dominate. Notably, interferon-γ (IFN-γ), which drives Mtb host defense, inhibits miR-21 expression, forcing an isoenzyme switch in the PFK complex, augmenting PFK-M expression and macrophage glycolysis. These findings place the targeting of PFK-M by miR-21 as a key node controlling macrophage immunometabolic function., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: Enhanced aerobic glycolysis is an essential hallmark of malignant cancer. Blocking the glycolytic pathway has been suggested as a therapeutic strategy to impair the proliferation of tumor cells. Metformin, a widely used anti-diabetes drug, exhibits anti-tumor properties. However, the underlying molecular mechanism of its action linking glucose metabolism with the suppression of proliferation has not been fully clarified., Main Methods: Stable isotope tracing technology and gas chromatography-mass spectrometry method were utilized to analyze the effect of metformin on glycolytic flux in HCC cells. Western blot and immunohistochemistry were utilized to analyze the expression of phosphofructokinase-1 (PFK1) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in HCC cells or xenograft tumor tissues. Lactate measurement and glucose uptake assay were used to analyze the level of lactate and glucose in the presence of frucose-2,6-diphosphate (F2,6BP) in HCC cells treated with metformin., Key Findings: We found that metformin significantly impaired hepatoma cell proliferation by inhibiting the glycolytic flux via PFK1 blockade. Interestingly, activation of PFK1 by F2,6BP reverses the inhibitory effect of metformin on hepatoma cell proliferation and glycolysis. Mechanistically, PFKFB3，a potent allosteric activator of PFK1, was markedly suppressed through inhibiting hypoxia-induced factor 1 (HIF-1α) accumulation mediated by metformin., Significance: Taken together these data indicate that HIF-1α/PFKFB3/PFK1 regulatory axis is a vital determinant of glucose metabolic reprogramming in hepatocellular carcinoma, which gives new insights into the action of metformin in combatting liver cancer., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Iodine-125 ( 125 I) irradiation has been widely applied in the treatment of advanced multiple malignant tumors. However, the underlying mechanism of 125 I exerted an anti-tumor effect on hepatocellular carcinoma (HCC) was largely unknown., Methods: In both HCCLM3 and SMMC-7721 cells, the effect of 125 I irradiation on the glycolysis was detected. The mRNA in HCC tissues and cell lines were detected by RT-qPCR. Cell proliferation, invasion and migration, and apoptosis were examined by CCK-8, Transwell, wound healing assay and flow cytometry assay, respectively. The interaction between miR-338 and PFKL (6-phosphofructokinase) were verified by dual-luciferase reporter gene assay. Western blotting was used to detect the expression of glycolysis-related proteins. We also evaluated the effect of 125 I seed implantation on the tumor growth and Warburg effect in vivo., Results: 125 I irradiation significantly decreased the Warburg effect, cell proliferation, invasion and migration, and induced apoptosis of HCCLM3 and SMMC-7721 cells. miR-338 was upregulated in HCC cells treated with 125 I irradiation, which was a negative correlation with tumor size, tumor metastasis, and tumor development. Moreover, miR-338 directly interacted with PFKL and suppressed its expression. Mechanistically, 125 I irradiation significantly decreased the Warburg effect and exhibited anti-tumorigenesis function through upregulating the inhibitory effect of miR-338 on PFKL expression., Conclusion: 125 I irradiation upregulated the suppression of miR-338 on PFKL to downregulate the Warburg effect and anti-tumorigenesis in HCC and provided a new potential strategy for HCC clinical treatment., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The Warburg effect is a prominent metabolic feature associated with neoplastic diseases; however, the underlying mechanism remains incompletely understood. TAp73, a structural homolog of the tumor suppressor p53, is frequently overexpressed in human tumors, indicating a proliferative advantage that it can confer to tumor cells. Here we show that TAp73 stimulates the expression of phosphofructokinase-1, liver type (PFKL), which catalyzes the committed step in glycolysis. Through this regulation, TAp73 enhances glucose consumption and lactate excretion, promoting the Warburg effect. By activating PFKL, TAp73 also increases ATP production and bolsters anti-oxidant defense. TAp73 deficiency results in a pronounced reduction in tumorigenic potential, which can be rescued by forced PFKL expression. These findings establish TAp73 as a critical regulator of glycolysis and reveal a mechanism by which tumor cells achieve the Warburg effect to enable oncogenic growth.

Allosteric regulation has traditionally been described by mathematically-complex allosteric rate laws in the form of ratios of polynomials derived from the application of simplifying kinetic assumptions. Alternatively, an approach that explicitly describes all known ligand-binding events requires no simplifying assumptions while allowing for the computation of enzymatic states. Here, we employ such a modeling approach to examine the "catalytic potential" of an enzyme-an enzyme's capacity to catalyze a biochemical reaction. The catalytic potential is the fundamental result of multiple ligand-binding events that represents a "tug of war" among the various regulators and substrates within the network. This formalism allows for the assessment of interacting allosteric enzymes and development of a network-level understanding of regulation. We first define the catalytic potential and use it to characterize the response of three key kinases (hexokinase, phosphofructokinase, and pyruvate kinase) in human red blood cell glycolysis to perturbations in ATP utilization. Next, we examine the sensitivity of the catalytic potential by using existing personalized models, finding that the catalytic potential allows for the identification of subtle but important differences in how individuals respond to such perturbations. Finally, we explore how the catalytic potential can help to elucidate how enzymes work in tandem to maintain a homeostatic state. Taken together, this work provides an interpretation and visualization of the dynamic interactions and network-level effects of interacting allosteric enzymes., Competing Interests: The authors have declared that no competing interests exist.

Altered glycolysis is a hallmark of diseases including diabetes and cancer. Despite intensive study of the contributions of individual glycolytic enzymes, systems-level analyses of flux control through glycolysis remain limited. Here, we overexpress in two mammalian cell lines the individual enzymes catalyzing each of the 12 steps linking extracellular glucose to excreted lactate, and find substantial flux control at four steps: glucose import, hexokinase, phosphofructokinase, and lactate export (and not at any steps of lower glycolysis). The four flux-controlling steps are specifically upregulated by the Ras oncogene: optogenetic Ras activation rapidly induces the transcription of isozymes catalyzing these four steps and enhances glycolysis. At least one isozyme catalyzing each of these four steps is consistently elevated in human tumors. Thus, in the studied contexts, flux control in glycolysis is concentrated in four key enzymatic steps. Upregulation of these steps in tumors likely underlies the Warburg effect., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glucose catabolism, also known as glycolysis, is important for energy generation and involves a sequence of enzymatic reactions that convert a glucose molecule into two pyruvate molecules. The glycolysis process generates adenosine triphosphate as a byproduct. In this study, we investigated whether glycolysis plays a role in maintaining neuronal functions in the Drosophila mushroom bodies (MBs), which are generally accepted to be an olfactory learning and memory center. Our data showed that individual knockdown of glycolytic enzymes in the MBs, including hexokinase (HexA), phosphofructokinase (Pfk), or pyruvate kinase (PyK), disrupts olfactory memory. Whole-mount brain immunostaining indicated that pyruvate kinase is strongly expressed in the MB αβ, α'β', and γ neuron subsets. We conclude that HexA, Pfk, and PyK are required in each MB neuron subset for olfactory memory formation. Our data therefore indicates that glucose catabolism in the MBs is important for olfactory memory formation in Drosophila., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Disorders of glucose homeostasis are common in chronic kidney disease (CKD) and are associated with increased mortality, but the mechanisms of impaired insulin secretion in this disease remain unclear. Here, we tested the hypothesis that defective insulin secretion in CKD is caused by a direct effect of urea on pancreatic β cells. In a murine model in which CKD is induced by 5/6 nephrectomy (CKD mice), we observed defects in glucose-stimulated insulin secretion in vivo and in isolated islets. Similarly, insulin secretion was impaired in normal mouse and human islets that were cultured with disease-relevant concentrations of urea and in islets from normal mice treated orally with urea for 3 weeks. In CKD mouse islets as well as urea-exposed normal islets, we observed an increase in oxidative stress and protein O-GlcNAcylation. Protein O-GlcNAcylation was also observed in pancreatic sections from CKD patients. Impairment of insulin secretion in both CKD mouse and urea-exposed islets was associated with reduced glucose utilization and activity of phosphofructokinase 1 (PFK-1), which could be reversed by inhibiting O-GlcNAcylation. Inhibition of O-GlcNAcylation also restored insulin secretion in both mouse models. These results suggest that insulin secretory defects associated with CKD arise from elevated circulating levels of urea that increase islet protein O-GlcNAcylation and impair glycolysis.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

It has been recognized that the rate-limiting function of pyruvate kinase M2 (PKM2) in glycolysis plays an important role in distributing glycolytic intermediates for anabolic and catabolic purposes in cancer cells. However, after analysis of the catalytic capacity of PKM2 relative to other glycolytic enzymes, the regulation range of PKM2 activity, metabolic flux control, and thermodynamics, we suggest that the PKM2-catalyzed reaction is not a rate-limiting step in cancer cell glycolysis. Hexokinase and phosphofructokinase 1 (PFK1), the first and third enzyme along the pathway, are rate-limiting enzymes that limit the overall glycolytic rate, whereas PKM2 and lactate dehydrogenase, the last two enzymes in the pathway, are for the fast removal of upstream intermediates to prevent the obstruction of the pathway. The argument is in accordance with the catalytic capacity of glycolytic enzymes, regulation range of enzyme activities, metabolic flux control, and thermodynamics., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The optimal aerobic exercise training (AET) protocol for patients following myocardial infarction (MI) has remained under debate. The present study therefore aimed to compare the effects of continuous moderate-intensity training (CMT) and high-intensity interval training (HIT) on cardiac functional recovery, and to investigate the potential associated mechanisms in a post-MI rat model. Female Sprague Dawley rats (8-10 weeks old) undergoing MI or sham surgery were subsequently submitted to CMT or HIT, or kept sedentary for eight weeks. Prior to and following AET, echocardiographic parameters and exercise capacity of the rats were measured. Western blotting was used to evaluate the levels of apoptosis and associated signaling pathway protein expression. The concentrations of biomarkers of oxidative stress were also determined by ELISA assay. Messenger (m)RNA levels and activity of the key enzymes for glycolysis and fatty acid oxidation, as well as the rate of adenosine triphosphate (ATP) synthesis, were also measured. Compared with the MI group, exercise capacity and cardiac function were significantly improved following AET, particularly following HIT. Left ventricular ejection fraction and fraction shortening were further improved in the MI-HIT group in comparison to that of the MI-CMT group. The two forms of AET almost equally attenuated apoptosis of the post-infarction myocardium. CMT and HIT also alleviated oxidative stress by decreasing the concentration of malondialdehyde and increasing the concentration of superoxide dismutase and glutathione peroxidase (GPx). In particular, HIT induced a greater increase in the concentration of GPx than that of CMT. AET, and HIT in particular, significantly increased the levels of mRNA and the maximal activity of phosphofructokinase-1 and carnitine palmitoyl transferase-1, as well as the maximal ratio of ATP synthesis. In addition, compared with the MI group, the expression of signaling proteins PI3K, Akt, p38mapk and AMPK was significantly altered in the MI-CMT and MI-HIT groups. HIT was superior to CMT in its ability to improve cardiac function and exercise capability in a post-MI rat model. HIT was also superior to CMT with regard to attenuating oxidative stress and improving glucolipid metabolism of the post-MI myocardium.

Introduction: Early detection and/or prediction of metastasis provide more prognostic relevance than local recurrence. Direct spread into the peritoneum is frequently found in pancreatic cancer patients, but positron emission tomography (PET) with 2-deoxy-2-fluoro-d-glucose (FDG) is not useful for identifying such metastasis. We investigated a method to enhance FDG accumulation using AsPC-1 human ascites tumor cells., Methods: (14)C-FDG accumulation was assessed under the following conditions: 1) characteristics of (14)C-FDG transport were examined using phloridzin, a Na(+)-free buffer, and various hexoses, and 2) accumulation of (14)C-FDG was measured in cells that were pretreated with hexose for various time periods, and activity of 6-phosphofructo-1-kinase (PFK-1) was assayed., Results: (14)C-FDG transport into AsPC-1 cells was mediated primarily by a Na(+)-independent transport mechanism. Aldohexoses such as d-glucose, D-mannose, and D-galactose inhibited (14)C-FDG transport. Cells pretreated with d-glucose, D-mannose, or D-fructose exhibited augmented (14)C-FDG accumulation. Pretreatment with higher concentrations of D-glucose or D-fructose tended to increase PFK-1 activity., Conclusions: Very little information has been published about the association between PFK-1 and FDG accumulation, and we confirmed the impacts of various hexoses on the activity of PFK-1 and FDG accumulation in AsPC-1 cells. Clarifying the relevance of PFK-1 in FDG accumulation will contribute to developing new features of FDG-PET, because PFK-1 is the main regulator of glycolysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Major efforts have been put in anti-angiogenic treatment for glioblastoma (GBM), an aggressive and highly vascularized brain tumor with dismal prognosis. However clinical outcome with anti-angiogenic agents has been disappointing and tumors quickly develop escape mechanisms. In preclinical GBM models we have recently shown that bevacizumab, a blocking antibody against vascular endothelial growth factor, induces hypoxia in treated tumors, which is accompanied by increased glycolytic activity and tumor invasiveness. Genome-wide transcriptomic analysis of patient derived GBM cells including stem cell lines revealed a strong up-regulation of glycolysis-related genes in response to severe hypoxia. We therefore investigated the importance of glycolytic enzymes in GBM adaptation and survival under hypoxia, both in vitro and in vivo. We found that shRNA-mediated attenuation of glycolytic enzyme expression interfered with GBM growth under normoxic and hypoxic conditions in all cellular models. Using intracranial GBM xenografts we identified seven glycolytic genes whose knockdown led to a dramatic survival benefit in mice. The most drastic effect was observed for PFKP (PFK1, +21.8%) and PDK1 (+20.9%), followed by PGAM1 and ENO1 (+14.5% each), HK2 (+11.8%), ALDOA (+10.9%) and ENO2 (+7.2%). The increase in mouse survival after genetic interference was confirmed using chemical inhibition of PFK1 with clotrimazole. We thus provide a comprehensive analysis on the importance of the glycolytic pathway for GBM growth in vivo and propose PFK1 and PDK1 as the most promising therapeutic targets to address the metabolic escape mechanisms of GBM.

Tp53-induced glycolysis and apoptosis regulator (TIGAR) activation blocks glycolytic ATP synthesis by inhibiting phosphofructokinase-1 activity. Our data indicate that TIGAR is selectively induced and activated in renal outermedullary proximal straight tubules (PSTs) after ischemia-reperfusion injury in a p53-dependent manner. Under severe ischemic conditions, TIGAR expression persisted through 48 h postinjury and induced loss of renal function and histological damage. Furthermore, TIGAR upregulation inhibited phosphofructokinase-1 activity, glucose 6-phosphate dehydrogenase (G6PD) activity, and induced ATP depletion, oxidative stress, autophagy, and apoptosis. Small interfering RNA-mediated TIGAR inhibition prevented the aforementioned malevolent effects and protected the kidneys from functional and histological damage. After mild ischemia, but not severe ischemia, G6PD activity and NADPH levels were restored, suggesting that TIGAR activation may redirect the glycolytic pathway into gluconeogenesis or the pentose phosphate pathway to produce NADPH. The increased level of NADPH maintained the level of GSH to scavenge ROS, resulting in a lower sensitivity of PST cells to injury. Under severe ischemia, G6PD activity and NADPH levels were reduced during reperfusion; however, blockade of TIGAR enhanced their levels and reduced oxidative stress and apoptosis. Collectively, these results demonstrate that inhibition of TIGAR may protect PST cells from energy depletion and apoptotic cell death in the setting of severe ischemia-reperfusion injury. However, under low ischemic burden, TIGAR activation induces the pentose phosphate pathway and autophagy as a protective mechanism., (Copyright © 2015 the American Physiological Society.)

Melatonin co-operates with insulin in the regulation of glucose homeostasis. Within the testis, glucose metabolism in the somatic Sertoli cells (SCs) is pivotal for spermatogenesis. Since the effects of melatonin on male reproductive physiology remain largely unknown, we hypothesized that melatonin may affect spermatogenesis by modulating SC metabolism, interacting with insulin. To test our hypothesis, rat SCs were maintained in culture for 24 h in the presence of insulin, melatonin or both and metabolite production/consumption was determined by proton nuclear magnetic resonance ((1)H-NMR). Protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by western blot. LDH activity was also assessed. SCs treated with melatonin showed an increase in glucose consumption via modulation of GLUT1 levels, but decreased LDH protein expression and activity, which resulted in lower lactate production. Moreover, SCs exposed to melatonin produced and accumulated less acetate than insulin-exposed cells. The combined treatment (insulin plus melatonin) increased acetate production by SCs, but intracellular acetate content remained lower than in insulin exposed cells. Finally, the intracellular redox state, as reflected by intracellular lactate/alanine ratio, was maintained at control levels in SCs by melatonin exposure (i.e. melatonin, alone or with insulin, increased the lactate/alanine ratio versus cells treated with insulin). Furthermore, SCs exposed to insulin plus melatonin produced more lactate and maintained the protein levels of some glycolysis-related enzymes and transporters at control levels. These findings illustrate that melatonin regulates SCs metabolism, and thus may affect spermatogenesis. Since lactate produced by SCs provides nutritional support and has an anti-apoptotic effect in developing germ cells, melatonin supplementation may be an effective therapy for diabetic male individuals facing subfertility/infertility., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: Many health benefits have been attributed to tea (Camellia sinensis (L.)), and tea infusions are used as dietary agent and included in food supplements. Herein, we report the effect of a white tea (WTEA) extract in Sertoli cell (SC) metabolism. The SC is responsible for the nutritional support of the developing germ cells., Methods: An aqueous WTEA extract was prepared and analyzed by (1)H-NMR. Rat SCs were cultured with or without the WTEA extract. mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by qPCR and western blot. LDH activity was assessed and metabolite production/consumption determined by (1)H-NMR., Results: WTEA-exposed SCs presented decreased protein and mRNA levels of GLUT1 and decreased glucose uptake. However, intracellular LDH activity was increased and SC lactate production was stimulated by the presence of the WTEA extract. Interestingly, alanine production was also found to be stimulated in WTEA extract-exposed SCs., Conclusion: WTEA extract altered the glycolytic profile of cultured SCs, stimulating lactate production. Since lactate is used as metabolic substrate and has an anti-apoptotic effect in the developing germ cells, the supplementation with WTEA extract may be advantageous to improve male reproductive health.

Unlabelled: 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]., (© 2014 FEBS.)

The effects of pre-incubation with mercury (Hg(2+)) and cadmium (Cd(2+)) on the activities of individual glycolytic enzymes, on the flux and on internal metabolite concentrations of the upper part of glycolysis were investigated in mouse muscle extracts. In the range of metal concentrations analysed we found that only hexokinase and phosphofructokinase, the enzymes that shared the control of the flux, were inhibited by Hg(2+) and Cd(2+). The concentrations of the internal metabolites glucose-6-phosphate and fructose-6-phosphate did not change significantly when Hg(2+) and Cd(2+) were added. A mathematical model was constructed to explore the mechanisms of inhibition of Hg(2+) and Cd(2+) on hexokinase and phosphofructokinase. Equations derived from detailed mechanistic models for each inhibition were fitted to the experimental data. In a concentration-dependent manner these equations describe the observed inhibition of enzyme activity. Under the conditions analysed, the integral model showed that the simultaneous inhibition of hexokinase and phosphofructokinase explains the observation that the concentrations of glucose-6-phosphate and fructose-6-phosphate did not change as the heavy metals decreased the glycolytic flux.

Cancer cells exhibit profound metabolic alterations, allowing them to fulfill the metabolic needs that come with increased proliferation and additional facets of malignancy. Such a metabolic transformation is orchestrated by the genetic changes that drive tumorigenesis, that is, the activation of oncogenes and/or the loss of oncosuppressor genes, and further shaped by environmental cues, such as oxygen concentration and nutrient availability. Understanding this metabolic rewiring is essential to elucidate the fundamental mechanisms of tumorigenesis as well as to find novel, therapeutically exploitable liabilities of malignant cells. Here, we describe key features of the metabolic transformation of cancer cells, which frequently include the switch to aerobic glycolysis, a profound mitochondrial reprogramming, and the deregulation of lipid metabolism, highlighting the notion that these pathways are not independent but rather cooperate to sustain proliferation. Finally, we hypothesize that only those genetic defects that effectively support anabolism are selected in the course of tumor progression, implying that cancer-associated mutations may undergo a metabolically convergent evolution., (© 2014 Elsevier Inc. All rights reserved.)

Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.

Does the understanding of the dynamics of biochemical networks in vivo, in terms of the properties of their components determined in vitro, require the latter to be determined all under the same conditions? An in vivo-like assay medium for enzyme activity determination was designed based on the concentrations of the major ionic constituents of the Escherichia coli cytosol: K(+), Na(+), Mg(2+), phosphate, glutamate, sulfate and Cl(-). The maximum capacities (V(max)) of the extracted enzymes of two pathways were determined using both this in vivo-like assay medium and the assay medium specific for each enzyme. The enzyme activities differed between the two assay conditions. Most of the differences could be attributed to unsuspected, pleiotropic effects of K(+) and phosphate. K(+) activated some enzymes (aldolase, enolase and glutamate dehydrogenase) and inhibited others (phosphoglucose isomerase, phosphofructokinase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase), whereas phosphate inhibited all glycolytic enzymes and glutamine synthetase but only activated glutamine 2-oxoglutarate amidotransferase. Neither a high glutamate concentration, nor macromolecular crowding affected the glycolytic or nitrogen assimilation enzymes, other than through the product inhibition of glutamate dehydrogenase by glutamate. This strategy of assessing all pathway enzymes kinetically under the same conditions may be necessary to avoid inadvertent differences between in vivo and in vitro biochemistry. It may also serve to reveal otherwise unnoticed pleiotropic regulation, such as that demonstrated in the present study by K(+) and phosphate., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Prostate cancer (PCa) progresses from an early stage, confined to prostate, to a more aggressive metastasized cancer related with loss of androgen responsiveness. Although, it has been recognized that PCa cells have unique metabolic features, their glycolytic profile in androgen-dependent and androgen-independent stages of disease is much less known. Hence, the main purpose of this study was to compare glucose metabolism in androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) PCa cells. Cell culture medium was collected and differences in glucose consumption and, lactate and alanine production were measured using Proton Nuclear Magnetic Resonance ((1)H NMR) spectra analysis. The mRNA and protein expression of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK1), lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) were determined by real-time PCR and Western Blot, respectively. The obtained results demonstrate that androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) cells consumed similar amounts of glucose, whereas PC3 cells present higher lactate production. This increase in lactate production was concomitant with higher levels of MCT4 protein, increased LDH activity and higher lactate/alanine ratio, also suggesting increased levels of oxidative stress in PC3 cells. However, protein levels of LDH, associated with lactate metabolism, and GLUT3, involved in glucose uptake, were decreased in PC3 comparatively with LNCaP. Androgen-responsive and nonresponsive PCa cells present distinct glycolytic metabolism profiles, which suggest that targeting LDH and MCT4 metabolic pathways may be an important step for the development of new diagnostic and therapeutic strategies in the different stages of PCa., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Rhizoctonia solani Kuhn causes sheath blight disease in rice, and genetic resistance against it is the most desirable characteristic. Current improvement efforts are based on analysis of polygenic quantitative trait loci (QTLs), but interpretation is limited by the lack of information on the changes in metabolic pathways. Our previous studies linked activation of the glycolytic pathway to enhanced generation of lignin in the phenylpropanoid pathway. The current studies investigated the regulation of glycolysis by examining the time course of changes in enzymatic activities and metabolite contents. The results showed that the activities of all glycolytic enzymes as well as fructose-6-phosphate (F-6-P), fructose-1,6-bisphosphate (F-1,6-P(2)), dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), 3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP) and pyruvate contents increased. These results combined with our previous findings that the expression of phosphoglucomutase (PGM), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase and pyruvate kinase (PK) increased after infection suggested that the additional establishment of glycolysis in the cytosol compartment occurred after infection. Further evidence for this was our recent findings that the increase in expression of the 6-phosphofructokinase (PFK) plastid isozyme Os06g05860 was accompanied by an increase in expression of three cytosolic PFK isozymes, i.e. Os01g09570, Os01g53680 and Os04g39420, as well as pyrophosphate-dependent phosphofrucokinase (PFP) isozymes Os08g25720 (α-subunit) and Os06g13810 (β-subunit) in infected rice plants of the resistant line. The results also showed that the reactions catalysed by PFK/PFP, aldolase, GAPDH + phosphoglycerate kinase (PGK) and PK in leaf sheaths of R. solani-infected rice plants were non-equilibrium reactions in vivo. This study showed that PGM, phosphoglucose isomerase (PGI), TPI and phosphoglycerate mutase (PGmu) + enolase could be regulated through coarse control whereas, PFK/PFP, aldolase, GAPDH + PGK and PK could be regulated through coarse and fine controls simultaneously.

The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC₅₀ = 0.74-6.7 μM) was more effective to inhibit 24-72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC₅₀ = 45-100 μM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3-21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 μM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.

6-Phosphofructo-1-kinase (PFK) and aldolase are two sequential glycolytic enzymes that associate forming heterotetramers containing a dimer of each enzyme. Although free PFK dimers present a negligible activity, once associated to aldolase these dimers are as active as the fully active tetrameric conformation of the enzyme. Here we show that aldolase-associated PFK dimers are not inhibited by clotrimazole, an antifungal azole derivative proposed as an antineoplastic drug due to its inhibitory effects on PFK. In the presence of aldolase, PFK is not modulated by its allosteric activators, ADP and fructose-2,6-bisphosphate, but is still inhibited by citrate and lactate. The association between the two enzymes also results on the twofold stimulation of aldolase maximal velocity and affinity for its substrate. These results suggest that the association between PFK and aldolase confers catalytic advantage for both enzymes and may contribute to the channeling of the glycolytic metabolism., (Copyright © 2011 Wiley Periodicals, Inc.)

Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Regulation of glucose metabolism is a crucial aspect of cell physiology in normal and disease conditions. Many regulatory events are involved in determining the metabolic fate of glucose and the pathways into which it is directed. The first reaction that commits glucose to the glycolytic pathway is catalyzed by the enzyme phosphofructokinase-1 (PFK-1) and is tightly regulated. One of the most potent activators of PFK-1 is fructose 2,6 bisphosphate (F2,6BP) and its cellular levels are correlated with glycolytic flux. F2,6BP is synthesized and degraded by a family of bifunctional enzymes-the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB). The interplay among F2,6BP levels, the enzymes that generate and degrade it, and PFK-1 activity has important consequences for several different aspects of cell metabolism as well as for systemic metabolic conditions. TIGAR, a recently identified F2,6 bisphosphatase (F2,6BPase), could also contribute to this complexity and participate in shaping the metabolic profile of the cell.

Cancer cells are characterized by increased aerobic glycolysis, which correlates with a negative prognosis. Although this correlation is well known, the mechanism of the elevated rate of glycolysis in cancer and the role of glycolytic enzymes have yet to be determined. The present work aims to evaluate the activity of the major enzymes that regulate glycolysis in breast cancer cell lines of varying aggressiveness. MCF10A, MCF-7 and MDA-mb-231 are human breast-derived cell lines with non-tumorigenic, tumorigenic and metastatic profiles, respectively. These cell lines have increasing degrees of glycolytic efficiency, i.e., lactate produced per glucose consumed, corresponding to their metastatic potential. Although, there are no differences in phosphofructokinase (PFK) or pyruvate kinase (PK) activities, the activity of hexokinase (HK) activity is higher in both tumorigenic cell lines compared to MCF10A cells. No difference in HK activity is observed between MCF-7 and MDA-mb-231 cells, suggesting that the difference in their glycolytic efficiency could not be attributed to this enzyme. However, we find that expression of the PFK-L isoform directly and strongly correlates with aggressiveness and glycolytic efficiency in these cell lines. Thus, we conclude that glycolytic efficiency, which is important for the survival of cancer cells, depends primarily on the preferential expression of PFK-L over the M and P isoforms., (Copyright 2010 Elsevier Inc. All rights reserved.)

This research aimed to study the effect of distillage recycling on ethanol fermentation, the key glycolytic enzymes and cell composition of the self-flocculating yeast. With the self-flocculating yeast SPSC01 and medium composed of 220 g/L glucose, 8 g/L yeast extract and 6 g/L peptone, continuous ethanol fermentation was carried out at the dilution rate of 0.04 h(-1) with a 1.5 L tank bioreactor. Fermentation broth was collected every 3 days, and ethanol and other volatile byproducts were removed by distillation, but the stillage with high boiling byproducts was recycled to prepare the medium instead of fresh water. The system was run for 20 days, during which ethanol and biomass concentrations in the effluent decreased continuously, indicating the significant inhibition of the high boiling byproducts accumulated within the system. Thus, the activities of the key enzymes of the glycolytic pathway: hexokinase, 6-phosphofructose kinase, and pyruvate kinase were analyzed, and it was observed that all of them were inhibited. Furthermore, the biosynthesis of the stress response metabolites glycerol and trehalose was investigated, and it was found that glycerol production that can protect yeast cells against osmotic pressure stress was enhanced, but trehalose biosynthesis that can protect yeast cells against ethanol inhibition was not improved, correspondingly. And in the meantime, the biosynthesis of the major intracellular components proteins and hydrocarbons was adjusted, correspondingly.

Malaria parasite-infected erythrocytes exhibit enhanced glucose utilisation and 6-phospho-1-fructokinase (PFK) is a key enzyme in glycolysis. Here we present the characterisation of PFK from the human malaria parasite Plasmodium falciparum. Of the two putative PFK genes on chromosome 9 (PfPFK9) and 11 (PfPFK11), only the PfPFK9 gene appeared to possess all the catalytic features appropriate for PFK activity. The deduced PfPFK proteins contain domains homologous to the plant-like pyrophosphate (PPi)-dependent PFK beta and alpha subunits, which are quite different from the human erythrocyte PFK protein. The PfPFK9 gene beta and alpha regions were cloned and expressed as His(6)- and GST-tagged proteins in Escherichia coli. Complementation of PFK-deficient E. coli and activity analysis of purified recombinant proteins confirmed that PfPFK9beta possessed catalytic activity. Monoclonal antibodies against the recombinant beta protein confirmed that the PfPFK9 protein has beta and alpha domains fused into a 200 kDa protein, as opposed to the independent subunits found in plants. Despite an overall structural similarity to plant PPi-PFKs, the recombinant protein and the parasite extract exhibited only ATP-dependent enzyme activity, and none with PPi. Unlike host PFK, the Plasmodium PFK was insensitive to fructose-2,6-bisphosphate (F-2,6-bP), phosphoenolpyruvate (PEP) and citrate. A comparison of the deduced PFK proteins from several protozoan PFK genome databases implicates a unique class of ATP-dependent PFK present amongst the apicomplexan protozoans.

The present work aimed to contribute to the understanding of the adaptation of the glycolytic pathway in tissues of frog Rana ridibunda and land snail species Helix lucorum during seasonal hibernation. Moreover responses of glycolytic enzymes from cold acclimated R. ridibunda and H. lucorum were studied as well. The drop in Po(2) in the blood of hibernated frogs and land snails indicated lower oxygen consumption and a decrease in their metabolic rate. The activities of glycolytic enzymes indicated that hibernation had a differential effect on the glycolyis in the two species studied and also in the tissues of the same species. The activity of l-LDH decreased significantly in the skeletal muscle and heart of hibernated R. ridibunda indicating a low glycolytic potential. Similar biochemical responses were observed in the same tissues during cold acclimation. The continuous increase in the activities of glycolytic enzymes studied, except for HK, might indicate a compensation for the impacts of low temperature on the enzymatic activities. In contrast to R. ridibunda, the activities of the enzymes increased and remained at higher levels than those of the prehibernation controls indicating maintenance of glycolytic potential in the tissues of hibernating land snails.

Besides its essential role at regulating neural functions through cyclic GMP, nitric oxide is emerging as an endogenous physiological modulator of energy conservation for the brain. Thus, nitric oxide inhibits cytochrome c oxidase activity in neurones and glia, resulting in down-regulation of mitochondrial energy production. The subsequent increase in AMP facilitates the activation of 5'-AMP-dependent protein kinase, which rapidly triggers the activation of 6-phosphofructo-1-kinase--the master regulator of the glycolytic pathway--and Glut1 and Glut3--the main glucose transporters in the brain. In addition, nitric oxide activates glucose-6-phosphate dehydrogenase, the first and rate-limiting step of the pentose-phosphate pathway. Here, we review recent evidences suggesting that nitric oxide exerts a fine control of neuronal energy metabolism by tuning the balance of glucose-6-phosphate consumption between glycolysis and pentose-phosphate pathway. This may have important implications for our understanding of the mechanisms controlling neuronal survival during oxidative stress and bioenergetic crisis.

Objective: To investigate the influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia., Methods: The primary passage of cultured myocardial cells from neonatal rats were divided into A group (with hypoxia), B group (with hypoxia and administration of l0 micromol/L colchicine), C group (with hypoxia and administration of 5 micromol/L taxol), D group (with hypoxia and administration of 10 micromol/L taxol), E group (with hypoxia and administration of 15 micromol/L taxol). The morphology of microtubule was observed with laser scanning microscope (LSM). The cell vitality was assayed by cell counting kit (CCK). The activities of hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were assayed with colorimetry., Results: In group B and E, the microtubule structure was damaged heavily, and the cell vitality was decreased significantly [The cell vitality was (89.99 +/- 3.47)% in B group and (84.56 +/- 6.61)% in E group, respectively, at 1.0 post hypoxia hour (PHH), and hoth values were obviously lower than that in A group (97.44 +/- 1.76)%, P < 0.01]. The HK, PK and PFK activities decreased obviously. The activities of HK, PK and PFK in group C were similar to those of the A group. Compared with that in other groups, the degree of damage of microtubule structure in D group was milden. The activities of HK, PK and PFK in D group during 0.5 - 6.0 PHH were significantly higher than those in A group. The activity of LDH in each group was increased after hypoxia., Conclusion: Proper concentration of microtubule-stabilizing drugs can alleviate the damages to microtubule structure, and enhance the activity of glycolytic key enzymes of myocardial cells at early stage of hypoxia.

Saccharomyces cerevisiae shows a marked preference for glucose and fructose, revealed by the repression of genes whose products are involved in processing other carbon sources. This response seems to be driven by sugar phosphorylation in the first steps of glycolysis rather than by the external sugar concentration. To gain a further insight into the role of the internal sugar signalling mechanisms, were measured the levels of upper intracellular glycolytic metabolites and adenine nucleotides in three mutant strains, HXT1, HXT7 and TM6*, with progressively reduced uptake capacities in comparison with the wild type. Reducing the rate of sugar consumption caused an accumulation of hexose phosphates upstream of the phosphofructokinase (PFK) and a reduction of fructose-1,6-bisphosphate levels. Mathematical modelling showed that these effects may be explained by changes in the kinetics of PFK and phosphoglucose isomerase. Moreover, the model indicated a modified sensitivity of the pyruvate dehydrogenase and the trichloroacetic acid cycle enzymes towards the NAD/NADH in the TM6* strain. The activation of the SNF1 sugar signalling pathway, previously observed in the TM6* strain, does not correlate with a reduction of the ATP : AMP ratio as reported in mammals. The mechanisms that may control the glycolytic rate at reduced sugar transport rates are discussed.

Serotonin (5-HT) is a hormone implicated in the regulation of many physiological and pathological events. One of its most intriguing properties is the ability to up-regulate mitosis. Moreover, it has been shown that 5-HT stimulate glucose uptake on skeletal muscle, suggesting that 5-HT may regulate glucose metabolism of peripheric tissues. Here we demonstrate that 5-HT stimulates skeletal muscle 6-phosphofructo-1-kinase (PFK) activity in a dose-response manner, through 5-HT(2A) receptor subtype. Maximal activation of the enzyme (2.5-fold compared to control) is achieved in the presence of 25pM 5-HT, increasing both PFK maximal velocity and affinity for the substrate fructose-6-phosphate. These effects occur due to tyrosine phosphorylation of the enzyme that is 2-fold enhanced upon 5-HT stimulation of skeletal muscles preparation. Once 5-HT-induced tyrosine phosphorylation of PFK is prevented by genistein, a tyrosine kinase inhibitor, the hormone stimulatory effect on PFK is abrogated. Wortmannin, a phosphatidylinositol-3-kinase (PI3K) inhibitor, does not interfere on 5-HT-induced stimulation of PFK, supporting that the observed effects are independent on insulin signaling pathway. Furthermore, 5-HT promotes the association of PFK to the muscle f-actin, suggesting that the hormone alters PFK intracellular distribution, favoring its association to the cytoskeleton. Altogether, our results support evidences that 5-HT augments skeletal muscle glucose consumption through stimulation of glycolysis key regulatory enzyme, PFK, throughout tyrosine phosphorylation and intracellular redistribution of the enzyme.

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.

Pattern formation in glycolysis is studied with a classical reaction-diffusion allosteric enzyme model. It is found that, similar to recent experimental reports in the yeast extracts, a small magnitude local perturbation can induce transient target waves in a two dimensional oscillatory medium. An above threshold stimulation generates target waves which eventually evolve into spatiotemporal chaos upon collisions with the boundary or other wave activities. Detailed simulation studies show that the studied simple glycolytic reaction-diffusion model can support three types of spatiotemporal behaviors which are independent of the boundary conditions: (1) a spatially uniform stable steady state, (2) periodic global oscillations and (3) spatiotemporal chaos.

Although membrane-associated glycolysis has been observed in a variety of cell types, the mechanism of localization of glycolytic enzymes to the plasma membrane is not known. We hypothesized that caveolin-1 (CAV-1) serves as a scaffolding protein for glycolytic enzymes and may play a role in the organization of cell metabolism. To test this hypothesis, we over-expressed CAV-1 in cultured A7r5 (rat aorta vascular smooth muscle; VSM) cells. Confocal immunofluorescence microscopy was used to study the distribution of phosphofructokinase (PFK) and CAV-1 in the transfected cells. Areas of interest (AOI) were analyzed in a central Z-plane across the cell transversing the perinuclear region. To quantify any shift in PFK localization resulting from CAV-1 over-expression, we calculated a periphery to center (PC) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found the PC index to be 1.92 +/- 0.57 (mean +/- SEM, N = 8) for transfected cells and 0.59 +/- 0.05 (mean +/- SEM, N = 11) for control cells. Colocalization analysis demonstrated that the percentage of PFK associated with CAV-1 increased in transfected cells compared to control cells. The localization of aldolase (ALD) was also shifted towards the plasma membrane (and colocalized with PFK) in CAV-1 over-expressing cells. These results demonstrate that CAV-1 creates binding sites for PFK and ALD that may be of higher affinity than those binding sites localized in the cytoplasm. We conclude that CAV-1 functions as a scaffolding protein for PFK, ALD and perhaps other glycolytic enzymes, either through direct interaction or accessory proteins, thus contributing to compartmented metabolism in vascular smooth muscle., (2006 Wiley-Liss, Inc.)

Objective: To investigate the influence of hypoxia induction factor-1alpha (HIF-1alpha) on glycosis of rat myocardial cell under hypoxic condition., Methods: The myocardial cells of the rats were routinely isolated and cultured. The cells were divided into single hypoxia (H) and HIF-1alpha inhibiting (I) groups. The cells in H group were cultured in glucose-free medium with mixed low-oxygen gas [1% O2, 94% N2 and 5% CO2 (v/v)]. While the cells in I group were cultured with low-oxygen gas after the cell model of low expression of HIF-1alpha protein constructed by RNAi technique. The cells in both groups were all observed before hypoxia (routine culture) and at the time points of 1, 3, 6, 12 and 24 hours of hypoxia. The LA (lactate acid ) content in the supernatant of the culture and the activity of the key enzymes in glycolysis such as hexokinase (HK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) of both groups of cells were determined at all the time points., Results: (1) After hypoxia, the HK and PFK activities of the rat myocardial cells in H and I groups were obviously increased at the beginning and decreased thereafter when compared with that before hypoxia. While the activities of HK and PFK in H group at 1, 3 and 6 hours after hypoxia were evidently higher than those in I group (P <0.05 or 0.01), and the peak activity of them in H and I groups was 159 +/- 13 U/g vs 133 +/- 55 U/g, and 298 +/- 44 U/g vs 188 +/- 55 U/g, respectively. (2) Compared with normal control (92 +/- 12 U/g), the LDH activity of the cells in H group after hypoxia increased significantly, reaching the peak at 6 hours after hypoxia (2 568 +/- 125 U/g, P < 0. 01), and it decreased thereafter, while that in I group peaked at 3 hours after hypoxia (2125 +/- 126 U/g, P <0.01). The LA content in the culture supernatant in H group increased significantly after hypoxia with the passage of time, while that in I group increased in smaller magnitude (P <0.01)., Conclusion: High expression of HIF-1alpha in the rat myocardial cells after hypoxia could directly cause continuous enhancement of cell glycolysis, which was beneficial to the protection of myocardial cells under hypoxic condition.