Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical to those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.

The glycolytic system is selected for ATP synthesis not only in tumor cells but also in differentiated cells. Differentiated osteoblasts also switch the dominant metabolic pathway to aerobic glycolysis. We found that primary osteoblasts increased expressions of glycolysis-related enzymes such as Glut1, hexokinase 1 and 2, lactate dehydrogenase A and pyruvate kinase M2 during their differentiation. Osteoblast differentiation decreased expression of tumor suppressor p53, which negatively regulates Glut1 expression, and enhanced phosphorylation of AKT, which is regulated by phosphoinositol-3 kinase (PI3K). An inhibitor of PI3K enhanced p53 expression and repressed Glut1 expression. Luciferase reporter assay showed that p53 negatively regulated transcriptional activity of solute carrier family 2 member 1 gene promoter region. Inhibition of glycolysis in osteoblasts reduced ATP contents more significantly than inhibition of oxidative phosphorylation by carbonyl cyanide m-chlorophenyl hydrazine. These results have indicated that osteoblasts increase Glut1 expression through the down-regulation of p53 to switch their metabolic pathway to glycolysis during differentiation.

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to N ε -amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei , the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei , expanding the factors responsible for regulating important pathways in this parasite.

GCN5L1 regulates protein acetylation and mitochondrial energy metabolism in diverse cell types. In the heart, loss of GCN5L1 sensitizes the myocardium to injury from exposure to nutritional excess and ischemia/reperfusion injury. This phenotype is associated with the reduced acetylation of metabolic enzymes and elevated mitochondrial reactive oxygen species (ROS) generation, although the direct molecular targets of GCN5L1 remain largely unknown. In this study, we sought to determine the mechanism by which GCN5L1 impacts energy substrate utilization and mitochondrial health. We find that hypoxia and reoxygenation (H/R) leads to a reduction in cell viability and Akt phosphorylation in GCN5L1 knockdown AC16 cardiomyocytes, in parallel with elevated glucose utilization and impaired fatty acid use. We demonstrate that glycolysis is uncoupled from glucose oxidation under normoxic conditions in GCN5L1-depleted cells. We show that GCN5L1 directly binds to the Akt-activating mTORC2 component Rictor, and that loss of Rictor acetylation is evident in GCN5L1 knockdown cells. Finally, we show that restoring Rictor acetylation in GCN5L1-depleted cells reduces mitochondrial ROS generation and increases cell survival in response to H/R. These studies suggest that GCN5L1 may play a central role in energy substrate metabolism and cell survival via the regulation of Akt/mTORC2 signaling.

The most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pH e ) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H + shuttle system in CA IX to facilitate proton efflux through MCT4.

Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III)-driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose-rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae . In a mutant compromised in RNAP III activity, there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flux.

Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a K i value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

Until recently, prebiotic precursors to metabolic pathways were not known. In parallel, chemistry achieved the synthesis of amino acids and nucleotides only in reaction sequences that do not resemble metabolic pathways, and by using condition step changes, incompatible with enzyme evolution. As a consequence, it was frequently assumed that the topological organisation of the metabolic pathway has formed in a Darwinian process. The situation changed with the discovery of a non-enzymatic glycolysis and pentose phosphate pathway. The suite of metabolism-like reactions is promoted by a metal cation, (Fe(II)), abundant in Archean sediment, and requires no condition step changes. Knowledge about metabolism-like reaction topologies has accumulated since, and supports non-enzymatic origins of gluconeogenesis, the S -adenosylmethionine pathway, the Krebs cycle, as well as CO 2 fixation. It now feels that it is only a question of time until essential parts of metabolism can be replicated non-enzymatically. Here, I review the ‘accidents’ that led to the discovery of the non-enzymatic glycolysis, and on the example of a chemical network based on hydrogen cyanide, I provide reasoning why metabolism-like non-enzymatic reaction topologies may have been missed for a long time. Finally, I discuss that, on the basis of non-enzymatic metabolism-like networks, one can elaborate stepwise scenarios for the origin of metabolic pathways, a situation that increasingly renders the origins of metabolism a tangible problem.

In demyelinating nervous system disorders, myelin basic protein (MBP), a major component of the myelin sheath, is proteolyzed and its fragments are released in the neural environment. Here, we demonstrated that, in contrast with MBP, the cellular uptake of the cryptic 84–104 epitope (MBP84-104) did not involve the low-density lipoprotein receptor-related protein-1, a scavenger receptor. Our pull-down assay, mass spectrometry and molecular modeling studies suggested that, similar with many other unfolded and aberrant proteins and peptides, the internalized MBP84-104 was capable of binding to the voltage-dependent anion-selective channel-1 (VDAC-1), a mitochondrial porin. Molecular modeling suggested that MBP84-104 directly binds to the N-terminal α-helix located midway inside the 19 β-blade barrel of VDAC-1. These interactions may have affected the mitochondrial functions and energy metabolism in multiple cell types. Notably, MBP84-104 caused neither cell apoptosis nor affected the total cellular ATP levels, but repressed the aerobic glycolysis (lactic acid fermentation) and decreased the l -lactate/ d -glucose ratio (also termed as the Warburg effect) in normal and cancer cells. Overall, our findings implied that because of its interactions with VDAC-1, the cryptic MBP84-104 peptide invoked reprogramming of the cellular energy metabolism that favored enhanced cellular activity, rather than apoptotic cell death. We concluded that the released MBP84-104 peptide, internalized by the cells, contributes to the reprogramming of the energy-generating pathways in multiple cell types.

What does it take to convert a living organism into a truly productive biofactory? Apart from optimizing biosynthesis pathways as standalone units, a successful bioengineering approach must bend the endogenous metabolic network of the host, and especially its central metabolism, to support the bioproduction process. In practice, this usually involves three complementary strategies which include tuning-down or abolishing competing metabolic pathways, increasing the availability of precursors of the desired biosynthesis pathway, and ensuring high availability of energetic resources such as ATP and NADPH. In this review, we explore these strategies, focusing on key metabolic pathways and processes, such as glycolysis, anaplerosis, the TCA (tricarboxylic acid) cycle, and NADPH production. We show that only a holistic approach for bioengineering — considering the metabolic network of the host organism as a whole, rather than focusing on the production pathway alone — can truly mold microorganisms into efficient biofactories.

Although ancillary pathways of glucose metabolism are critical for synthesizing cellular building blocks and modulating stress responses, how they are regulated remains unclear. In the present study, we used radiometric glycolysis assays, [ 13 C 6 ]-glucose isotope tracing, and extracellular flux analysis to understand how phosphofructokinase (PFK)-mediated changes in glycolysis regulate glucose carbon partitioning into catabolic and anabolic pathways. Expression of kinase-deficient or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat neonatal cardiomyocytes co-ordinately regulated glycolytic rate and lactate production. Nevertheless, in all groups, >40% of glucose consumed by the cells was unaccounted for via catabolism to pyruvate, which suggests entry of glucose carbons into ancillary pathways branching from metabolites formed in the preparatory phase of glycolysis. Analysis of 13 C fractional enrichment patterns suggests that PFK activity regulates glucose carbon incorporation directly into the ribose and the glycerol moieties of purines and phospholipids, respectively. Pyrimidines, UDP- N -acetylhexosamine, and the fatty acyl chains of phosphatidylinositol and triglycerides showed lower 13 C incorporation under conditions of high PFK activity; the isotopologue 13 C enrichment pattern of each metabolite indicated limitations in mitochondria-engendered aspartate, acetyl CoA and fatty acids. Consistent with this notion, high glycolytic rate diminished mitochondrial activity and the coupling of glycolysis to glucose oxidation. These findings suggest that a major portion of intracellular glucose in cardiac myocytes is apportioned for ancillary biosynthetic reactions and that PFK co-ordinates the activities of the pentose phosphate, hexosamine biosynthetic, and glycerolipid synthesis pathways by directly modulating glycolytic intermediate entry into auxiliary glucose metabolism pathways and by indirectly regulating mitochondrial cataplerosis.

Phosphofructokinase-1 (Pfk) acts as the main control point of flux through glycolysis. It is involved in complex allosteric regulation and Pfk mutations have been linked to cancer development. Whereas the 3D structure and structural basis of allosteric regulation of prokaryotic Pfk has been studied in great detail, our knowledge about the molecular basis of the allosteric behaviour of the more complex mammalian Pfk is still very limited. To characterize the structural basis of allosteric regulation, the subunit interfaces and the functional consequences of modifications in Tarui's disease and cancer, we analysed the physiological homotetramer of human platelet Pfk at up to 2.67 Å resolution in two crystal forms. The crystallized enzyme is permanently activated by a deletion of the 22 C-terminal residues. Complex structures with ADP and fructose-6-phosphate (F6P) and with ATP suggest a role of three aspartates in the deprotonation of the OH-nucleophile of F6P and in the co-ordination of the catalytic magnesium ion. Changes at the dimer interface, including an asymmetry observed in both crystal forms, are the primary mechanism of allosteric regulation of Pfk by influencing the F6P-binding site. Whereas the nature of this conformational switch appears to be largely conserved in bacterial, yeast and mammalian Pfk, initiation of these changes differs significantly in eukaryotic Pfk.

Using mouse embryonic fibroblasts (MEFs) from DJ1-knockout mice, in the present study, we show that DJ1, by binding with Foxo3a (forkhead box O3a), transcriptionally activates pink1 (phosphatase and tensin homologue deleted on chromosome 10-induced protein kinase-1) gene. Moreover, we demonstrate that, by promoting pink1 expression, DJ1 represses the rate of glycolysis and cell proliferation.

Oestrogen receptor α (ERα+) breast tumours rely on mitochondria (mt) to generate ATP. The goal of the present study was to determine how oestradiol (E 2 ) and 4-hydroxytamoxifen (4-OHT) affect cellular bioenergetic function in MCF-7 and T47D ERα+ breast cancer cells in serum-replete compared with dextran-coated charcoal (DCC)-stripped foetal bovine serum (FBS)-containing medium (‘serum-starved’). Serum-starvation reduced oxygen consumption rate (OCR), extracellular acidification rate (ECAR), ATP-linked OCR and maximum mt capacity, reflecting lower ATP demand and mt respiration. Cellular respiratory state apparent was unchanged by serum deprivation. 4-OHT reduced OCR independent of serum status. Despite having a higher mt DNA/nuclear DNA ratio than MCF-7 cells, T47D cells have a lower OCR and ATP levels and higher proton leak. T47D express higher nuclear respiratory factor-1 (NRF-1) and NRF-1-regulated, nuclear-encoded mitochondrial transcription factor TFAM and cytochrome c , but lower levels of cytochrome c oxidase, subunit IV, isoform 1 (COX4, COX4I1 ). Mitochondrial reserve capacity, reflecting tolerance to cellular stress, was higher in serum-starved T47D cells and was increased by 4-OHT, but was decreased by 4-OHT in MCF-7 cells. These data demonstrate critical differences in cellular energetics and responses to 4-OHT in these two ERα+ cell lines, likely reflecting cancer cell avoidance of apoptosis.

MondoA is a basic helix–loop–helix (bHLH)/leucine zipper (ZIP) transcription factor that is expressed predominantly in skeletal muscle. Studies in vitro suggest that the Max-like protein X (MondoA:Mlx) heterodimer senses the intracellular energy status and directly targets the promoter region of thioredoxin interacting protein (Txnip) and possibly glycolytic enzymes. We generated MondoA-inactivated (MondoA −/− ) mice by gene targeting. MondoA −/− mice had normal body weight at birth, exhibited normal growth and appeared to be healthy. However, they exhibited unique metabolic characteristics. MondoA −/− mice built up serum lactate and alanine levels and utilized fatty acids for fuel during exercise. Gene expression and promoter analysis suggested that MondoA functionally represses peroxisome-proliferator-activated receptor γ co-activator-1α (PGC-1α)–mediated activation of pyruvate dehydrogenase kinase 4 (PDK-4) transcription. PDK4 normally down-regulates the activity of pyruvate dehydrogenase, an enzyme complex that catalyses the decarboxylation of pyruvate to acetyl-CoA for entry into the Krebs cycle; in the absence of MondoA, pyruvate is diverted towards lactate and alanine, both products of glycolysis. Dynamic testing revealed that MondoA −/− mice excel in sprinting as their skeletal muscles display an enhanced glycolytic capacity. Our studies uncover a hitherto unappreciated function of MondoA in fuel selection in vivo . Lack of MondoA results in enhanced exercise capacity with sprinting.

The p53-induced protein TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator] is considered to be a F26BPase (fructose-2,6-bisphosphatase) with an important role in cancer cell metabolism. The reported catalytic efficiency of TIGAR as an F26BPase is several orders of magnitude lower than that of the F26BPase component of liver or muscle PFK2 (phosphofructokinase 2), suggesting that F26BP (fructose 2,6-bisphosphate) might not be the physiological substrate of TIGAR. We therefore set out to re-evaluate the biochemical function of TIGAR. Phosphatase activity of recombinant human TIGAR protein was tested on a series of physiological phosphate esters. The best substrate was 23BPG (2,3-bisphosphoglycerate), followed by 2PG (2-phosphoglycerate), 2-phosphoglycolate and PEP (phosphoenolpyruvate). In contrast the catalytic efficiency for F26BP was approximately 400-fold lower than that for 23BPG. Using genetic and shRNA-based cell culture models, we show that loss of TIGAR consistently leads to an up to 5-fold increase in the levels of 23BPG. Increases in F26BP levels were also observed, albeit in a more limited and cell-type dependent manner. The results of the present study challenge the concept that TIGAR acts primarily on F26BP. This has significant implications for our understanding of the metabolic changes downstream of p53 as well as for cancer cell metabolism in general. It also suggests that 23BPG might play an unrecognized function in metabolic control.

TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator] protein is known for its ability to inhibit glycolysis, shifting glucose consumption towards the pentose phosphate pathway to promote antioxidant protection of cancer cells. According to sequence homology and activity analyses, TIGAR was initially considered to be a fructose-2,6-bisphosphatase; it has thus received much attention in cancer cell metabolism, given its dependence on p53 and the key role of F26BP (fructose 2,6-bisphosphate) at modulating glycolysis and gluconeogenesis. However, in a rigorous study published in this issue of the Biochemical Journal , Gerin and colleagues report that recombinant TIGAR is a 23BPG (2,3-bisphosphoglycerate) phosphatase, although it also dephosphorylates other carboxylic acid-phosphate esters and, weakly, F26BP. As such, inhibition of endogenous TIGAR leads to a dramatic increase in cellular 23BPG, influencing F26BP to a lower extent that depends on the cellular context. These results challenge the currently held notion that TIGAR modulates glycolysis through decreasing F26BP, and opens a yet unrecognized function(s) for TIGAR-mediated 23BPG control of cellular metabolism in health and disease.

In Saccharomyces cerevisiae , synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1 , which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H + -ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1 , in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica , nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H + -ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism.

Besides the necessary changes in the expression of cell cycle-related proteins, cancer cells undergo a profound series of metabolic adaptations focused to satisfy their excessive demand for biomass. An essential metabolic transformation of these cells is increased glycolysis, which is currently the focus of anticancer therapies. Several key players have been identified, so far, that adapt glycolysis to allow an increased proliferation in cancer. In this issue of the Biochemical Journal , Novellasdemunt and colleagues elegantly identify a novel mechanism by which MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2], a key component of the MAPK pathway, up-regulates glycolysis in response to stress in cancer cells. The authors found that, by phosphorylating specific substrate residues, MK2 promotes both increased the gene transcription and allosteric activation of PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), a key glycolysis-promoting enzyme. These results reveal a novel pathway through which MK2 co-ordinates metabolic adaptation to cell proliferation in cancer and highlight PFKFB3 as a potential therapeutic target in this devastating disease.

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P 2 (fructose 2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is involved in cell proliferation owing to its role in carbohydrate metabolism. In the present study we analysed the mechanism of regulation of PFKFB3 as an immediate early gene controlled by stress stimuli that activates the p38/MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2] pathway. We report that exposure of HeLa and T98G cells to different stress stimuli (NaCl, H 2 O 2 , UV radiation and anisomycin) leads to a rapid increase (15–30 min) in PFKFB3 mRNA levels. The use of specific inhibitors in combination with MK2-deficient cells implicate control by the protein kinase MK2. Transient transfection of HeLa cells with deleted gene promoter constructs allowed us to identify an SRE (serum-response element) to which SRF (serum-response factor) binds and thus transactivates PFKFB3 gene transcription. Direct binding of phospho-SRF to the SRE sequence (−918 nt) was confirmed by ChIP (chromatin immunoprecipiation) assays. Moreover, PFKFB3 isoenzyme phosphorylation at Ser 461 by MK2 increases PFK-2 activity. Taken together, the results of the present study suggest a multimodal mechanism of stress stimuli affecting PFKFB3 transcriptional regulation and kinase activation by protein phosphorylation, resulting in an increase in Fru-2,6-P 2 concentration and stimulation of glycolysis in cancer cells.

Signals derived from the BCR (B-cell antigen receptor) control survival, development and antigenic responses. One mechanism by which BCR signals may mediate these responses is by regulating cell metabolism. Indeed, the bioenergetic demands of naïve B-cells increase following BCR engagement and are characterized by a metabolic switch to aerobic glycolysis; however, the signalling pathways involved in this metabolic reprogramming are poorly defined. The PKC (protein kinase C) family plays an integral role in B-cell survival and antigenic responses. Using pharmacological inhibition and mice deficient in PKCβ, we demonstrate an essential role of PKCβ in BCR-induced glycolysis in B-cells. In contrast, mice deficient in PKCδ exhibit glycolytic rates comparable with those of wild-type B-cells following BCR cross-linking. The induction of several glycolytic genes following BCR engagement is impaired in PKCβ-deficient B-cells. Moreover, blocking glycolysis results in decreased survival of B-cells despite BCR engagement. The results establish a definitive role for PKCβ in the metabolic switch to glycolysis following BCR engagement of naïve B-cells.

Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliana , cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cys 149 . Catalytic Cys 149 is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (p K a =5.7). This property makes GapC1 sensitive to oxidation by H 2 O 2 , which appears to inhibit enzyme activity by converting the thiolate of Cys 149 (–S − ) into irreversible oxidized forms (–SO 2 − and –SO 3 − ) via a labile sulfenate intermediate (–SO − ). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys 149 sulfenates to give rise to a mixed disulfide (Cys 149 –SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys 149 .

Eukaryotic PFK (phosphofructokinase), a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the Fru-2,6-P 2 (fructose 2,6-bisphosphate) allosteric site are formed by opposing N- and C-termini of subunits orientated antiparallel in a dimer. In contrast, we show in the present study that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in PFK-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro . The C-terminus is not catalytic, but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild-type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.

GKAs (glucokinase activators) are promising agents for the therapy of Type 2 diabetes, but little is known about their effects on hepatic intermediary metabolism. We monitored the fate of 13 C-labelled glucose in both a liver perfusion system and isolated hepatocytes. MS and NMR spectroscopy were deployed to measure isotopic enrichment. The results demonstrate that the stimulation of glycolysis by GKA led to numerous changes in hepatic metabolism: (i) augmented flux through the TCA (tricarboxylic acid) cycle, as evidenced by greater incorporation of 13 C into the cycle (anaplerosis) and increased generation of 13 C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) lowering of hepatic [P i ] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) stimulation of glycogen synthesis from glucose, but inhibition of glycogen synthesis from 3-carbon precursors; (iv) increased synthesis of N -acetylglutamate and consequently augmented ureagenesis; (v) increased synthesis of glutamine, alanine, serine and glycine; and (vi) increased production and outflow of lactate. The present study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis, but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.

Reprogramming of energetic metabolism is a phenotypic trait of cancer in which mitochondrial dysfunction represents a key event in tumour progression. In the present study, we show that the acquisition of the tumour-promoting phenotype in colon cancer HCT116 cells treated with oligomycin to inhibit ATP synthase is exerted by repression of the synthesis of nuclear-encoded mitochondrial proteins in a process that is regulated at the level of translation. Remarkably, the synthesis of glycolytic proteins is not affected in this situation. Changes in translational control of mitochondrial proteins are signalled by the activation of AMPK (AMP-activated protein kinase) and the GCN2 (general control non-derepressible 2) kinase, leading also to the activation of autophagy. Changes in the bioenergetic function of mitochondria are mimicked by the activation of AMPK and the silencing of ATF4 (activating transcription factor 4). These findings emphasize the relevance of translational control for normal mitochondrial function and for the progression of cancer. Moreover, they demonstrate that glycolysis and oxidative phosphorylation are controlled at different levels of gene expression, offering the cell a mechanistic safeguard strategy for metabolic adaptation under stressful conditions.

Oxidative and nitrosative stress underlie the pathogenesis of a broad range of human diseases, in particular neurodegenerative disorders. Within the brain, neurons are the cells most vulnerable to excess reactive oxygen and nitrogen species; their survival relies on the antioxidant protection promoted by neighbouring astrocytes. However, neurons are also intrinsically equipped with a biochemical mechanism that links glucose metabolism to antioxidant defence. Neurons actively metabolize glucose through the pentose phosphate pathway, which maintains the antioxidant glutathione in its reduced state, hence exerting neuroprotection. This process is tightly controlled by a key glycolysis-promoting enzyme and is dependent on an appropriate supply of energy substrates from astrocytes. Thus brain bioenergetic and antioxidant defence is coupled between neurons and astrocytes. A better understanding of the regulation of this intercellular coupling should be important for identifying novel targets for future therapeutic interventions.

PFKFB (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P 2 (fructose-2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is extensively involved in cell proliferation owing to its key role in carbohydrate metabolism. In the present study we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or norgestrel) leads to a rapid increase in Fru-2,6-P 2 concentration. Our Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a PRE (progesterone-response element) to which PR (progesterone receptor) binds and thus transactivates PFKFB3 gene transcription. PR expression in the PR-negative cell line MDA-MB-231 induces endogenous PFKFB3 expression in response to norgestrel. Direct binding of PR to the PRE box (−3490 nt) was confirmed by ChIP (chromatin immunoprecipiation) experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK (extracellular-signal-regulated kinase)/RSK (ribosomal S6 kinase) pathway leading to phosphorylation of PFKFB3 on Ser 461 is followed by activation of mRNA transcription via cis -acting sequences on the PFKFB3 promoter.

PP i is a critical element of cellular metabolism as both an energy donor and as an allosteric regulator of several metabolic pathways. The apicomplexan parasite Toxoplasma gondii uses PP i in place of ATP as an energy donor in at least two reactions: the glycolytic PP i -dependent PFK (phosphofructokinase) and V-H + -PPase [vacuolar H + -translocating PPase (pyrophosphatase)]. In the present study, we report the cloning, expression and characterization of cytosolic TgPPase ( T. gondii soluble PPase). Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a family I soluble PPase. Overexpression of the enzyme in extracellular tachyzoites led to a 6-fold decrease in the cytosolic concentration of PP i relative to wild-type strain RH tachyzoites. Unexpectedly, this subsequent reduction in PP i was associated with a higher glycolytic flux in the overexpressing mutants, as evidenced by higher rates of proton and lactate extrusion. In addition to elevated glycolytic flux, TgPPase-overexpressing tachyzoites also possessed higher ATP concentrations relative to wild-type RH parasites. These results implicate PP i as having a significant regulatory role in glycolysis and, potentially, other downstream processes that regulate growth and cell division.

During cardiac remodelling, the heart generates higher levels of reactive species; yet an intermediate ‘compensatory’ stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischaemia or in the failing heart. For this, we measured energetics in control and PE (phenylephrine)-treated NRCMs (neonatal rat cardiomyocytes) under basal conditions and when stressed with HNE (4-hydroxynonenal). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a 2-fold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homoeostasis under conditions of increased oxidative stress.

Up-regulation of lipogenesis by androgen is one of the most characteristic metabolic features of LNCaP prostate cancer cells. The present study revealed that androgen increases glucose utilization for de novo lipogenesis in LNCaP cells through the activation of HK2 (hexokinase 2) and activation of the cardiac isoform of PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase). Activation of PKA (cAMP-dependent protein kinase) by androgen increased phosphorylation of CREB [CRE (cAMP-response element)-binding protein], which in turn bound to CRE on the promoter of the HK2 gene resulting in transcriptional activation of the HK2 gene. Up-regulation of PFKFB2 expression was mediated by the direct binding of ligand-activated androgen receptor to the PFKFB2 promoter. The activated PI3K (phosphoinositide 3-kinase)/Akt signalling pathway in LNCaP cells contributes to the phosphorylation of PFKFB2 at Ser 466 and Ser 483 , resulting in the constitutive activation of PFK-2 (6-phosphofructo-2-kinase) activity. Glucose uptake and lipogenesis were severely blocked by knocking-down of PFKFB2 using siRNA (small interfering RNA) or by inhibition of PFK-2 activity with LY294002 treatment. Taken together, our results suggest that the induction of de novo lipid synthesis by androgen requires the transcriptional up-regulation of HK2 and PFKFB2, and phosphorylation of PFKFB2 generated by the PI3K/Akt signalling pathway to supply the source for lipogenesis from glucose in prostate cancer cells.

On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [γ- 32 P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser 466 and Ser 483 . However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBβ-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBα/β siRNA or PKBα siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBα.

Mitochondria play a critical role in mediating the cellular response to oxidants formed during acute and chronic cardiac dysfunction. It is widely assumed that, as cells are subjected to stress, mitochondria are capable of drawing upon a ‘reserve capacity’ which is available to serve the increased energy demands for maintenance of organ function, cellular repair or detoxification of reactive species. This hypothesis further implies that impairment or depletion of this putative reserve capacity ultimately leads to excessive protein damage and cell death. However, it has been difficult to fully evaluate this hypothesis since much of our information about the response of the mitochondrion to oxidative stress derives from studies on mitochondria isolated from their cellular context. Therefore the goal of the present study was to determine whether ‘bioenergetic reserve capacity’ does indeed exist in the intact myocyte and whether it is utilized in response to stress induced by the pathologically relevant reactive lipid species HNE (4-hydroxynonenal). We found that intact rat neonatal ventricular myocytes exhibit a substantial bioenergetic reserve capacity under basal conditions; however, on exposure to pathologically relevant concentrations of HNE, oxygen consumption was increased until this reserve capacity was depleted. Exhaustion of the reserve capacity by HNE treatment resulted in inhibition of respiration concomitant with protein modification and cell death. These data suggest that oxidized lipids could contribute to myocyte injury by decreasing the bioenergetic reserve capacity. Furthermore, these studies demonstrate the utility of measuring the bioenergetic reserve capacity for assessing or predicting the response of cells to stress.

The control of glycolysis in contracting muscle is not fully understood. The aim of the present study was to examine whether activation of glycolysis is mediated by factors related to the energy state or by a direct effect of Ca 2+ on the regulating enzymes. Extensor digitorum longus muscles from rat were isolated, treated with cyanide to inhibit aerobic ATP production and stimulated (0.2 s trains every 4 s) until force was reduced to 70% of initial force (control muscle, referred to as Con). Muscles treated with BTS ( N -benzyl- p -toluene sulfonamide), an inhibitor of cross-bridge cycling without affecting Ca 2+ transients, were stimulated for an equal time period as Con. Energy utilization by the contractile apparatus (estimated from the observed relation between ATP utilization and force–time integral) was 60% of total. In BTS, the force–time integral and ATP utilization were only 38 and 58% of those in Con respectively. Glycolytic rate in BTS was only 51% of that in Con but the relative contribution of ATP derived from PCr (phosphocreatine) and glycolysis and the relation between muscle contents of PCr and Lac (lactate) were not different. Prolonged cyanide incubation of quiescent muscle (low Ca 2+ ) did not change the relation between PCr and Lac. The reduced glycolytic rate in BTS despite maintained Ca 2+ transients, and the unchanged PCr/Lac relation in the absence of Ca 2+ transients, demonstrates that Ca 2+ is not the main trigger of glycogenolysis. Instead the preserved relative contribution of energy delivered from PCr and glycolysis during both conditions suggests that the glycolytic rate is controlled by factors related to energy state.

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 μM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 μM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.

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.

Previous work has shown that GAPDH (glyceraldehyde-3-phosphate dehydrogenase), aldolase, PFK (phosphofructokinase), PK (pyruvate kinase) and LDH (lactate dehydrogenase) assemble into a GE (glycolytic enzyme) complex on the inner surface of the human erythrocyte membrane. In an effort to define the molecular architecture of this complex, we have undertaken to localize the binding sites of these enzymes more accurately. We report that: (i) a major aldolase-binding site on the erythrocyte membrane is located within N-terminal residues 1–23 of band 3 and that both consensus sequences D 6 DYED 10 and E 19 EYED 23 are necessary to form a single enzyme-binding site; (ii) GAPDH has two tandem binding sites on band 3, located in residues 1–11 and residues 12–23 respectively; (iii) a PFK-binding site resides between residues 12 and 23 of band 3; (iv) no GEs bind to the third consensus sequence (residues D 902 EYDE 906 ) at the C-terminus of band 3; and (v) the LDH- and PK-binding sites on the erythrocyte membrane do not reside on band 3. Taken together, these results argue that band 3 provides a nucleation site for the GE complex on the human erythrocyte membrane and that other components near band 3 must also participate in organizing the enzyme complex.

Explicit modelling of metabolic networks relies on well-known mathematical tools and specialized computer programs. However, identifying and estimating the values of the very numerous enzyme parameters inherent to the models remain a tedious and difficult task, and the rate equations of the reactions are usually not known in sufficient detail. A way to circumvent this problem is to use ‘non-mechanistic’ models, which may account for the behaviour of the systems with a limited number of parameters. Working on the first part of glycolysis reconstituted in vitro , we showed how to derive, from titration experiments, values of effective enzyme activity parameters that do not include explicitly any of the classical kinetic constants. With a maximum of only two parameters per enzyme, this approach produced very good estimates for the flux values, and enabled us to determine the optimization conditions of the system, i.e. to calculate the set of enzyme concentrations that maximizes the flux. This fast and easy method should be valuable in the context of integrative biology or for metabolic engineering, where the challenge is to deal with the dramatic increase in the number of parameters when the systems become complex.

The proton-transport activity of UCP1 (uncoupling protein 1) triggers mitochondrial uncoupling and thermogenesis. The exact role of its close homologues, UCP2 and UCP3, is unclear. Mounting evidence associates them with the control of mitochondrial superoxide production. Using CHO (Chinese-hamster ovary) cells stably expressing UCP3 or UCP1, we found no evidence for respiration uncoupling. The explanation lies in the absence of an appropriate activator of UCP protonophoric function. Accordingly, the addition of retinoic acid uncouples the respiration of the UCP1-expressing clone, but not that of the UCP3-expressing ones. In a glucose-containing medium, the extent of the hyperpolarization of mitochondria by oligomycin was close to 22 mV in the five UCP3-expressing clones, contrasting with the variable values observed with the 15 controls. Our observations suggest that, when glycolysis and mitochondria generate ATP, and in the absence of appropriate activators of proton transport, UCPs do not transport protons (uncoupling), but rather other ions of physiological relevance that control mitochondrial activity. A model is proposed using the known passive transport of pyruvate by UCP1.

Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. Two Hungarian compound heterozygote brothers inherited the same TPI mutations (F240L and E145Stop), but only the younger one suffers from neurodegeneration. In the present study, we determined the kinetic parameters of key glycolytic enzymes including the mutant TPI for rational modelling of erythrocyte glycolysis. We found that a low TPI activity in the mutant cells (lower than predicted from the protein level and specific activity of the purified recombinant enzyme) is coupled with an increase in the activities of glycolytic kinases. The modelling rendered it possible to establish the steady-state flux of the glycolysis and metabolite concentrations, which was not possible experimentally due to the inactivation of the mutant TPI and other enzymes during the pre-steady state. Our results showed that the flux was 2.5-fold higher and the concentration of DHAP (dihydroxyacetone phosphate) and fructose 1,6-bisphosphate increased 40- and 5-fold respectively in the erythrocytes of the patient compared with the control. Although the rapid equilibration of triosephosphates is not achieved, the energy state of the cells is not ‘sick’ due to the activation of key regulatory enzymes. In lymphocytes of the two brothers, the TPI activity was also lower (20%) than that of controls; however, the remaining activity was high enough to maintain the rapid equilibration of triosephosphates; consequently, no accumulation of DHAP occurs, as judged by our experimental and computational data. Interestingly, we found significant differences in the mRNA levels of the brothers for TPI and some other, apparently unrelated, proteins. One of them is the prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases. We found that the peptidase activity of the affected brother was reduced by 30% compared with that of his neurologically intact brother.

Recently, we have reported that the inhibition of mitochondrial respiration by nitric oxide (NO) leads to an up-regulation of glycolysis and affords cytoprotection against energy failure through the stimulation of AMPK (5′-AMP-activated protein kinase) [Almeida, Moncada and Bolaños (2004) Nat. Cell Biol. 6 , 45–51]. To determine whether glucose transport contributes specifically to this effect, we have now investigated the possible role of NO in modulating glucose uptake through GLUT3, a facilitative high-affinity glucose carrier that has been suggested to afford cytoprotection against hypoglycaemic episodes. To do so, GLUT3-lacking HEK-293T cells (human embryonic kidney 293T cells) were transformed to express a plasmid construction encoding green fluorescent protein-tagged GLUT3 cDNA. This carrier was preferentially localized to the plasma membrane, was seen to be functionally active and afforded cytoprotection against low glucose-induced apoptotic death. Inhibition of mitochondrial respiration by NO triggered a rapid, cGMP-independent enhancement of GLUT3-mediated glucose uptake through a mechanism that did not involve transporter translocation. Furthermore, the functional disruption of AMPK by the RNA interference strategy rendered cells unable to respond to NO by activating GLUT3-mediated glucose uptake. These results suggest that the inhibition of mitochondrial respiration by NO activates AMPK to stimulate glucose uptake, thereby representing a novel survival pathway during pathophysiological conditions involving transient reductions in the supply of cellular glucose.

Fru-2,6-P 2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure–function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P 2 . The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans , could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.

Recent findings indicate that the expression of the β-catalytic subunit of the mitochondrial H + -ATP synthase (β-F 1 -ATPase) is depressed in liver, kidney and colon carcinomas, providing further a bioenergetic signature of cancer that is associated with patient survival. In the present study, we performed an analysis of mitochondrial and glycolytic protein markers in breast, gastric and prostate adenocarcinomas, and in squamous oesophageal and lung carcinomas. The expression of mitochondrial and glycolytic markers varied significantly in these carcinomas, when compared with paired normal tissues, with the exception of prostate cancer. Overall, the relative expression of β-F 1 -ATPase was significantly reduced in breast and gastric adenocarcinomas, as well as in squamous oesophageal and lung carcinomas, strongly suggesting that alteration of the bioenergetic function of mitochondria is a hallmark of these types of cancer.

Systematic deletions and point mutations in the C-terminal extension of mammalian PFK (phosphofructokinase) led us to identify Leu-767 and Glu-768 of the M-type isoform (PFK-M) as the motifs responsible for the role of this region in inhibition by MgATP. These amino acids are the only residues of the C-terminus that are conserved in all mammalian isoforms, and were found to have a similar function in the C-type isoenzyme. Both residues in PFK-C and Leu-767 in PFK-M were also observed to be critical for inhibition by citrate, which is synergistic with that by MgATP. Binding studies utilizing titration of intrinsic protein fluorescence indicated that the C-terminal part of the enzyme participates in the signal transduction route from the MgATP inhibitory site to the catalytic site, but does not contribute to the binding of this inhibitor, whereas it is essential for the binding of citrate. Mutations of the identified structural motifs did not alter either the action of other allosteric effectors that also interact with MgATP, such as the inhibitor phosphoenolpyruvate and the strong activator fructose 2,6-bisphosphate, or the co-operative effect of fructose 6-phosphate. The latter data provide evidence that activation by fructose 2,6-bisphosphate and fructose 6-phosphate co-operativity are not linked to the same allosteric transition as that mediating inhibition by MgATP.

Cellular senescence is considered a major tumour-suppressor mechanism in mammals, and many oncogenic insults, such as the activation of the ras proto-oncogene, trigger initiation of the senescence programme. Although it was shown that activation of the senescence programme involves the up-regulation of cell-cycle regulators such as the inhibitors of cyclin-dependent kinases p16INK4A and p21CIP-1, the mechanisms underlying the senescence response remain to be resolved. In the case of stress-induced premature senescence, reactive oxygen species are considered important intermediates contributing to the phenotype. Moreover, distinct alterations of the cellular carbohydrate metabolism are known to contribute to oncogenic transformation, as is best documented for the phenomenon of aerobic glycolysis. These findings suggest that metabolic alterations are involved in tumourigenesis and tumour suppression; however, little is known about the metabolic pathways that contribute to these processes. Using the human fibroblast model of in vitro senescence, we analysed age-dependent changes in the cellular carbohydrate metabolism. Here we show that senescent fibroblasts enter into a metabolic imbalance, associated with a strong reduction in the levels of ribonucleotide triphosphates, including ATP, which are required for nucleotide biosynthesis and hence proliferation. ATP depletion in senescent fibroblasts is due to dysregulation of glycolytic enzymes, and finally leads to a drastic increase in cellular AMP, which is shown here to induce premature senescence. These results suggest that metabolic regulation plays an important role during cellular senescence and hence tumour suppression.

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden–Meyerhof and the Entner–Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

In this study, we show that reactive oxygen species production induced by tumour necrosis factor α (TNF-α) in L929 cells was associated with a decrease in the steady-state mRNA levels of the mitochondrial transcript ATPase 6-8. Simultaneously, the transcript levels of two nuclear-encoded glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase, were increased. These changes were associated with decreased protein levels of the ATPase subunit a (encoded by the mitochondrial ATPase 6 gene) and cytochrome c oxidase subunit II, and increased protein levels of phosphofructokinase. Since TNF-α had no effect on the amount of mitochondrial DNA, the results suggested that TNF-α acted at the transcriptional and/or post-transcriptional level. Reactive oxygen species scavengers, such as butylated hydroxianisole and butylated hydroxytoluene, blocked the production of free radicals, prevented the down-regulation of ATPase 6-8 transcripts, preserved the protein levels of ATPase subunit a and cytochrome c oxidase subunit II, and attenuated the cytotoxic response to TNF-α, indicating a direct link between these two phenomena.

In the present study, we demonstrate that E2F is implicated in the regulation of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) during cell division. The expression of this enzyme is induced during the G 1 /S transition of the cell cycle. We identified and monitored the E2F-pocket protein complexes that bind to the E2F site of the F-type promoter during cell-cycle entry, and we analysed their contribution to the phosphatidylinositol 3-kinase (PI 3-kinase)-mediated regulation of the promoter. We found that the predominant E2F complex bound to the F-type promoter in unstimulated/quiescent cells contains E2F4, DP1 and p130 proteins. In serum-stimulated (S-phase) cells, the composition of the complex switched to E2F1/4, DP1 and p107, together with cyclin A and cyclin-dependent kinase 2. Treatment with the PI 3-kinase specific inhibitor LY 294002 prevented the formation of the S-phase complex, suggesting that activation of the PI 3-kinase pathway is essential for the formation of this complex. Further supporting this idea, we obtained results showing that treatment of cycling NIH 3T3 cells with either wortmannin or LY 294002 induces the accumulation of the transcriptionally repressive p130—E2F4—DP1 complex. Using the Rat-1 ER—E2F1 cell line where E2F1 activity can be conditionally induced, we demonstrated that E2F activity is involved in the in vivo transcriptional regulation of the F-type 6PF2K/Fru-2 , 6-BPase gene. Taken together, our results show that the F-type 6PF2K/Fru-2 , 6-BPase is a genuine E2F-regulated gene, and that its regulation by the PI 3-kinase pathway is at least partially mediated through the E2F transcription factor.

Plants, such as Arabidopsis thaliana and Selaginella lepidophylla , contain genes homologous with the trehalose-6-phosphate synthase (TPS) genes of bacteria and fungi. Most plants do not accumulate trehalose with the desert resurrection plant S. lepidophylla , being a notable exception. Overexpression of the plant genes in a Saccharomyces cerevisiae tps1 mutant results in very low TPS-catalytic activity and trehalose accumulation. We show that truncation of the plant-specific N-terminal extension in the A. thaliana AtTPS1 and S. lepidophylla SlTPS1 homologues results in 10–40-fold higher TPS activity and 20–40-fold higher trehalose accumulation on expression in yeast. These results show that the plant TPS enzymes possess a high-potential catalytic activity. The growth defect of the tps1 strain on glucose was restored, however, the proper homoeostasis of glycolytic flux was not restored, indicating that the plant enzymes were unable to substitute for the yeast enzyme in the regulation of hexokinase activity. Further analysis of the N-terminus led to the identification of two conserved residues, which after mutagenesis result in strongly enhanced trehalose accumulation upon expression in yeast. The plant-specific N-terminal region may act as an inhibitory domain allowing modulation of TPS activity.

Glucokinase (GK) is a key enzyme for glucose utilization in liver and shows a higher expression in the perivenous zone. In primary rat hepatocytes, the GK gene expression was activated by HNF (hepatic nuclear factor)-4α via the sequence −52/−39 of the GK promoter. Venous p O 2 enhanced HNF-4 levels and HNF-4 binding to the GK—HNF-4 element. Thus, HNF-4α could play the role of a regulator for zonated GK expression.

Using H9c2 cells derived from rat cardiomyocytes, we investigated the mechanism of cell death during hypoxia in the presence of serum and glucose. Hypoxic cell death is by necrosis and is accompanied by metabolic acidosis. Moreover, hypoxic cell death is inhibited by Hepes buffer as well as by 2-deoxyglucose, an inhibitor of glycolysis, indicating that metabolic acidosis should play an essential role in hypoxic injury. The involvement of phosphoinositide 3-kinase (PI 3-kinase), which is known to activate glucose metabolism, was examined using its inhibitor, LY290042, or adenovirus-mediated gene transfer. Hypoxic cell death was inhibited by LY294002 in a dose-dependent manner. Overexpression of dominant negative PI 3-kinase was found to reduce cell death, whereas wild-type PI 3-kinase enhanced it. Dominant negative PI 3-kinase also reduced glucose consumption and acidosis, but this was stimulated by wild-type PI 3-kinase. The data indicate that PI 3-kinase stimulates cell death by enhancing metabolic acidosis. LY294002 significantly reduced glucose uptake, showing that PI 3-kinase regulates glycolysis at the step of glucose transport. These findings indicate the pivotal role of glucose metabolism in hypoxic cell death, and reveal a novel death-promoting effect of PI 3-kinase during hypoxia, despite this enzyme being considered to be a survival-promoting factor.