Computational modeling to determine key regulators of hypoxia effects on the lactate production in the glycolysis pathway.

In solid tumors, hypoxia can trigger aberrant expression of transcription factors and genes, resulting in abnormal biological functions such as altered energetic pathways in cancer cells. Glucose metabolism is an important part of this phenomenon, which is associated with changes in the functional expression of transporters and enzymes involved in the glycolysis pathway. The latter phenomenon can finally lead to the lactate accumulation and pH dysregulation in the tumor microenvironment and subsequently further invasion and metastasis of cancer cells. Having capitalized on the computational modeling, in this study, for the first time, we aimed to investigate the effects of hypoxia-induced factor-1 (HIF-1) mediated hypoxia on the magnitude of functional expression of all the enzymes and transporters involved in the glycolysis process. The main objective was to establish a quantitative relationship between the hypoxia intensity and the intracellular lactate levels and determine the key regulators of the glycolysis pathway. This model clearly showed an increase in the lactate concentration during the oxygen depletion. The proposed model also predicted that the phosphofructokinase-1 and phosphoglucomutase enzymes might play the most important roles in the regulation of the lactate production.

Antibiotic overproduction in Streptomyces coelicolor A3 2 mediated by phosphofructokinase deletion.

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris.

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources.

Allosteric regulation of 6-phosphofructo-1-kinase activity of fat body and flight muscle from the bloodsucking bug Rhodnius prolixus.

6-Phosphofructo-1-kinase (phosphofructokinase; PFK) activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues - flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 +/- 1.4 U/mg) than in fat body extracts (3.6+/-0.4 U/mg), although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5) and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.

Allosteric regulation of 6-phosphofructo-1-kinase activity of fat body and flight muscle from the bloodsucking bug Rhodnius prolixus

6-phosphofructo-1-kinase (phosphofructokinase; PFK) activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues - flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 ± 1.4 U/mg) than in fat body extracts (3.6±0.4 U/mg), although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5) and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.

A atividade da fosfofrutocinase (PFK) de Rodnius prolixus, um inseto hematófago, o qual vôa somente pequenas distâncias, foi medida e comparada em dois tecidos metabolicamente ativos - músculo de asa e corpo gorduroso. A atividade desta importante enzima glicolítica regulatória foi muito mais pronunciada em músculo de asa (15,1 ±1,4 U/mg) do que em extrato de corpo gorduroso (3,6 ±0,4 U/mg) embora este último tenha apresentado níveis mais altos da enzima por quantidade de proteína, como medido por western-blotting. Extratos de músculo foram mais responsivos do que corpo gorduroso para ATP e frutose-6-fosfato, ambos substratos da PFK. A regulação alostérica exercida por diferentes efetores tais como ADP, AMP, frutose-2,6-bisfosfato apresentou um padrão singular para cada tecido. O pH ótimo (8,0-8,5) e a sensibilidade a variações de pH, foram muito similares e o citrato foi incapaz de inibir a atividade da PFK em ambos os extratos. Nossos resultados sugerem a existência de uma atividade particular da PFK para cada tecido com padrões regulatórios que são consistentes com suas funções fisiológicas.

Effects of the Na(+)/H(+) exchanger monensin on intracellular pH in astroglia.

Stimulation of astroglial glucose utilization by the Na(+)/H(+) exchanger monensin is only partially blocked by ouabain. The present studies show that monensin also raises intracellular pH in astroglia. Because increased pH stimulates phosphofructokinase activity, the ouabain-insensitive portion of the stimulation of cerebral glucose utilization (CMR(glc)) appears to be due to stimulation of glycolysis by intracellular alkalinization.

Altered brain glucose metabolism in transgenic-PFKL mice with elevated L-phosphofructokinase: in vivo NMR studies.

The gene for the liver-type subunit of phosphofructokinase (PFKL) resides on chromosome 21 and is overexpressed in Down syndrome (DS) patients. Transgenic PFKL (Tg-PFKL) mice with elevated levels of PFKL were used to determine whether, as in DS, overexpression of PFKL was also associated with altered sugar metabolism. We found that Tg-PFKL mice had an abnormal glucose metabolism with reduced clearance rate from blood and enhanced metabolic rate in brain. Transgenic-PFKL mice exhibited elevated activity of phosphofructokinase in both blood and brain, as compared to control non-transgenic (ntg) mice. Following glucose infusion, the rate of glucose clearance from the blood of Tg-PFKL mice was significantly slower than that of control ntg mice, although the basal blood glucose levels were similar. However, unlike the slower rate of glucose metabolism in blood, the initial rate of glucose utilization in the brain of the transgenic mice, was 58% faster than in control ntg mice. This was determined by infusion of [1-13C]-glucose followed by in vivo nuclear magnetic resonance (NMR) measurements of brain glucose metabolism. The faster utilization of glucose in Tg-PFKL brain is similar to the increased rate of cerebral glucose metabolism found in the brain of young adult DS patients, which may play a role in the etiology of their cognitive disabilities.

Impaired glucose-induced insulin response in transgenic mice overexpressing the L-phosphofructokinase gene.

The selective impairment of glucose-induced insulin secretion in NIDDM can be attributed to defects in the glucose-signaling system. An alteration in the activity of phosphofructokinase (PFK), a key enzyme in the glycolytic pathway, may play a role in the abnormal glucose-induced insulin secretion. In this study, we evaluated insulin secretion in transgenic (Tg) mice overexpressing the liver-type subunit of phosphofructokinase (PFKL). Three independently derived Tg-PFKL lines showed random and postprandial hyperglycemia with diminished acute insulin response following intravenous glucose tolerance load. Isolated islets of Tg-PFKL mice exhibited a shift to the right of the glucose insulin dose curve. However, the maximal insulin secretory capacity, as well as the potentiation effect by arginine, were retained. PFK activity in Tg-PFKL islets was increased by 30-70%, because of the overexpression of PFKL. Conceivably, a selective overexpression of the PFKL isoform in Tg-PFKL mice altered the enzymatic properties of the tetrameric PFK and thereby affected glucose metabolism. A similar phenomenon was previously observed in transfected PC12-PFKL cells. The data show that overexpression of PFKL in transgenic mice was associated with diminished glucose-induced insulin response and suggest a mechanism to explain the role of beta-cell PFK activity in glucose-induced insulin secretion.

Glycolytic enzyme binding during entrance to daily torpor in deer mice (Peromyscus maniculatus).

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

6-fosfofructo-1-quinasa (PFK-1) / 6-phosphofructo-1-kinase

El objetivo de la presente revisión es describir los efectos que tienen algunos moduladores descritos recientemente en la literatura, sobre la actividad de la 6-fosfofructo-1-quinasa. Esta enzima es clave del proceso glicolitico y su control se realiza mediante fosforilación/defosforilación, en los distintos tejidos estudiados

[Lipid metabolism and insulin resistance--clinical aspects and pathobiochemistry]. / Fettstoffwechsel und Insulinresistenz--Klinik und Pathobiochemie.

About 3 decades ago insulin resistance has been described as the pathogenetic factor leading from abnormal fat metabolism to diabetes mellitus. Within the metabolic syndrome insulin resistance is related to the upper body (android) type of obesity, hypertriglyceridaemia, hypertension, and diabetes mellitus ("deadly quartet"). It precedes the development of arterial hypertension and the metabolic disorders. The pathomechanisms leading from obesity and hypertriglyceridaemia to insulin resistance may be described by the glucose fatty acid cycle of Randle et al. According to their metabolic scheme increased supply of fatty acids results in reduced glucose oxidation. Concomittantly hepatic glucose production is increased. On the other hand insulin resistance combined with hyperinsulinaemia may lead to an elevation of VLDL-triglycerides and to a decrease of HDL-cholesterol in blood, thus creating a vicious cycle, in which elevated VLDL-triglycerides reinforce insulin resistance via the glucose fatty acid cycle. Interventions to improve insulin sensitivity and thereby lower plasma insulin should reduce obesity and hypertriglyceridaemia by dietary treatment. They usually improve promptly diabetic metabolism. New developments in pharmacological inhibition of fatty acid oxidation are discussed.

Functional and biochemical effects of a K(+)-ionophore on the isolated perfused rat heart.

Valinomycin, a K(+)-specific ionophore, influenced function and metabolism of isolated perfused rat hearts in a dose-dependent fashion. At a concentration of 0.05 micrograms ml-1 in perfusion fluid a 50% reduction of heart rate (HR) and a 90% reduction in max dP/dt were observed. These effects were paralleled by a substantial decrease of myocardial energy charge from about 0.80 to 0.20. A 2.5 fold increase in tissue lactate concentration indicated an increased rate of glycolytic activity. Low ATP combined with high ADP and AMP levels as found in these valinomycin-treated hearts is known to promote phosphofructokinase activity and may explain the elevated lactate levels. A significant increase in the concentrations of adenosine, IMP and inosine was observed as well.

Decrease of phosphofructokinase activity in relation to the pathogenesis of triorthocresyl-phosphate-induced delayed neuropathy.

The in vivo effect of a single dose of the neuropathic compound triorthocresyl-phosphate (TOCP) on phosphofructokinase (PFC, E.C. 2.7.1.11) and its relation with the initiation step (inhibition and aging of neuropathy target esterase, NTE) in the TOCP-induced delayed neuropathy have been studied. Hens were treated with a neurotoxic dose of TOCP (500 mg/kg, p.o.) and with a protective compound (Phenylmethanesulfonyl fluoride, PMSF, 30 mg/kg s.c.) in different combinations: TOCP, TOCP + PMSF, PMSF + TOCP and PMSF. PFK activity was determined in brain and sciatic nerve 1, 3, 7 and 15 days after treatment. PFK activity decreased in sciatic nerve 15 days after dosing with TOCP or TOCP + PMSF. When animals were dosed with the protective agent (PMSF) alone or before administering the neurotoxic compound, PFK activity was unaltered and clinical signs of neuropathy were absent. The data presented here suggest that phosphofructokinase is involved in the pathogenesis of the neuropathy induced by TOCP.

Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells.

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

Regulation of muscle carbohydrate metabolism during exercise.

This review examines the mechanisms that regulate muscle carbohydrate metabolism during exercise. Muscle carbohydrate utilization is regulated primarily by two factors, namely, delivery of substrate to the glycolytic pathway either from glycogenolysis or from transport of extracellular glucose into the fibers, and formation of triosephosphate by phosphofructokinase. The regulation involves the integration of the glycolytic controls with other metabolic controls and the needs of the whole muscle in meeting the physiological demand. The controls operating in the glycolytic sequence in vivo appear to couple glycolytic recruitment to signals from the rate of energy demand, the TCA cycle state, and the mitochondrial redox state so as to satisfy the major regulatory goal of maintaining the supply of ATP for tension development.

Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan.

The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.

Properties of the interaction between phosphofructokinase and actin.

The interaction of rabbit skeletal muscle phosphofructokinase (PFK) with actin is characterized in terms of the binding of PFK to actin in the presence and absence of tropomyosin and troponin, the effect of PFK on actin polymerization, and the involvement of adenylates in the binding of PFK to actin. The thin filament proteins, tropomyosin and troponin, are associated with skeletal muscle actin and reduce the binding of PFK to actin, thus influencing the probable distribution of PFK in skeletal muscle. The binding of PFK to actin is inhibited by ATP and ADP but not by fructose 6-phosphate or fructose 2,6-bisphosphate. This specific inhibition, plus evidence from fluorescence quenching and photoaffinity labeling, suggests that actin binds at the adenosine activation sites of PFK. Light scattering measurements used to monitor actin polymerization indicate that PFK dramatically increases the level of light scattering produced by the polymerization of actin, indicative of a superaggregate of PFK and actin. PFK inhibits the polymerization of actin when polymerization is induced by low concentrations of added salts. Although PFK binds to actin with high affinity, it seems to have little effect on the high shear viscosity of actin filaments.

Regulation of pathways of glucose metabolism in the kidney. The activity of the pentose phosphate pathway, glycolytic route and the regulation of phosphofructokinase in the kidney of lean and genetically obese (ob/ob) mice; comparison with effects of diabetes.

The activities of enzymes of the glycolytic route, the pentose phosphate pathway and NADPH-linked enzymes have been measured in the kidneys of genetically obese (ob/ob) mice and their lean litter mates. The renal content of glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (Fru-1,6-P2) and fructose 2,6-bisphosphate (Fru-2,6-P2) were also measured. Increases were found in hexokinase and enolase with an upward trend in pyruvate kinase in the ob/ob mouse kidney; a significant decline in malic enzyme was also seen. The renal content of G6P and Fru-1,6-P2 increased. There was no renal hypertrophy despite a degree of hyperglycaemia, which was, however, considerably below that observed in experimental diabetes. Comparison of the renal changes in the hyperglycaemic-hyperinsulinaemic ob/ob mice with the hyperglycaemic-hypoinsulinaemic diabetic group showed two distinct groupings. Firstly, changes which were similar in the two groups included: increases in hexokinase, G6P and Fru-1,6-P2, and a decrease in malic enzyme. Secondly, opposite changes were seen in enolase and in enzymes at the G6P crossroads, phosphoglucose isomerase and phosphoglucomutase. The elevated hexokinase and G6P in both ob/ob and diabetic groups may be involved in the eventual accumulation of basement membrane material in the glomerulus which is a common feature of the two conditions.

Modulation of phosphofructokinase action by macromolecular interactions. Quantitative analysis of the phosphofructokinase-aldolase-calmodulin system.

The simultaneous effect of calmodulin and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) on the concentration-dependent behaviour of muscle phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been analysed by means of a covalently attached fluorescent probe, gel penetration experiments, and using a kinetic approach. We found that calmodulin-induced inactivation of phosphofructokinase is suspended by addition of an equimolar amount of aldolase. This effect was attributed to an apparent competition of calmodulin and aldolase for the dimeric forms of kinase. Moreover, the direct binding of aldolase to calmodulin has also been demonstrated, which resulted in a significant decrease in the kcat value of the enzyme. The quantitative analysis of these interactions in the system phosphofructokinase-calmodulin-aldolase is presented. A possible molecular model for the modulation of phosphofructokinase action by macromolecular interactions is envisaged.