Activation of phosphofructokinase 2 by insulin in cultured hepatocytes without accompanying changes of effector levels or cAMP-stimulated protein kinase activity ratios

Activation of glycolysis by insulin in cultured adult rat hepatocytes is accompanied by an activation of phosphofructokinase 2 (PFK 2). PFK 2 activation might be caused by insulin-dependent changes of (a) metabolite levels, (b) basal and (c) Br8cAMP-stimulated cAMP-dependent protein kinase activity; this problem was investigated. 1. Cells cultured with 0.1 nM insulin for 48 h exhibited a low glycolytic rate and low fructose 2,6-bisphosphate [Fru(2,6)P2] levels. Addition of insulin increased Fru(2,6)P2 and Fru(1,6)P2 levels sequentially which points to PFK 2 as first target enzyme of insulin action. 2. Concentrations of Glc6P, Fru6P, phosphoenolpyruvate, glycerol 3-phosphate and citrate, which modulate PFK 2/fructose 2,6-bisphosphatase 2 activity, were not altered by insulin. 3. Activation of PFK 2 by insulin occurred without changes in the levels of total and protein-bound cAMP. Bound cAMP amounted to about 14% of total cAMP. 4. Insulin neither decreased the basal dissociation state of the cAMP-dependent protein kinase nor lowered the sensitivity of the kinase towards cAMP in cell extracts. 5. Addition of the phosphodiesterase-resistant Br8cAMP to the cultures increased cAMP levels 3-4-fold, elevated the protein kinase activity ratio from 0.14 to 0.6 and decreased the Fru(2,6)P2 level and the rate of glycolysis. When Br8cAMP and insulin were given together, insulin was capable of counteracting Br8cAMP in that it activated glycolysis and PFK 2 and elevated the Fru(2,6)P2 level; however, it did not decrease the elevated protein kinase activity ratio. It is concluded that insulin presumably does not activate PFK 2 through changes in cAMP and effector levels or through inhibition of cAMP-dependent protein kinase dissociation. The data support the hypothesis that insulin may act via activation of PFK 2 phosphatase.     

Activation of glycolysis by insulin with a sequential increase of the 6-phosphofructo-2-kinase activity, fructose-2,6-bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes

The involvement of 6-phosphofructo-2-kinase, fructose 2,6-bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin-dependent short-term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short-term influence of insulin on these parameters was dependent on the insulin concentration used for the long-term culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as 'insulin cells' and 'control cells', respectively. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two-fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6-phosphofructo-2-kinase remained constant. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6-phosphofructo-2-kinase directly after the medium change. However, both parameters increased during a 1-2-h incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value. Addition of insulin to control cells led to a 5-8-fold stimulation of glycolysis but only after a 30-90-min lag phase. During this lag period insulin strongly increased sequentially the 6-phosphofructo-2-kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin-stimulated glycolysis. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin-independently, led to an immediate increase in glycolysis without a lag phase. It is concluded that in this insulin-sensitive cell system: the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin-dependent stimulation of glycolysis in insulin cells; activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level. The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.     

Regulation of glycolysis during acclimation of scallops (Patinopecten yessoensis Jay) to anaerobiosis

Some glycolytic metabolites in the adductor muscle were measured after transfer of scallops from aerobic to anaerobic saltwater for 12 h. The level of octopine increased gradually during the initial 3 h incubation, and thereafter the level increased rapidly up to 12 h. The ATP level also did not show any significant change for the initial 3 h, and then decreased rapidly. The fructose 2,6-biphosphate (Fru 2,6-BP) level increased drastically during the initial 3 h incubation, but thereafter the level did not show any significant change up to 12 h. In the short-term effects of anaerobiosis for 90 min, the level of fructose 6-phosphate (Fru 6-P) increased just after transfer to anaerobiosis, and then its level decreased. In contrast, the fructose 1,6-biphosphate (Fru 1,6-BP) level increased greatly, at the time when both glucose 6-phosphate (Glc 6-P) and Fru 6-P decreased. The Fru 2,6-BP level did not any significant change during the initial 15 min incubation, but thereafter the level increased gradually up to 90 min. Scallop 6-phosphofructo 1-kinase (EC 2.7.1.11) (PFK1) was strongly activated by 1 microM Fru 2,6-BP when 0.2 mM Fru 6-P was used as a substrate, but the activity was not affected at 5 mM Fru 6-P. In view of these results, the regulation mechanism of glycolysis is discussed.     

Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP.

We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK). We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35%. The response is hyperbolic with Ks[Fru6P] of 20 microM and Ks[ADP] of 13 microM. The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response. AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response. PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity. However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP. Taken together, these results suggest that, in the absence of effectors or ligands, E. coli PFK exists in a state with high affinity for Fru6P ("R" state). This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.     

Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters.

Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms.     

Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation

The kinetic properties of phosphofructokinase from muscle of the giant cirripede Austromegabalanus psittacus were characterized, after partial purification by ion exchange chromatography on DEAE-cellulose. This enzyme showed differences regarding PFKs from other marine invertebrates: the affinity for fructose 6-phosphate (Fru 6-P) was very low, with an S(0.5) of 22.6+/-1.4 mM (mean+/-S.D., n=3), and a high cooperativity (n(H) of 2.90+/-0.21; mean+/-S.D., n=3). The barnacle PFK showed hyperbolic saturation kinetics for ATP (apparent K(m ATP)=70 microM, at 5 mM Fru 6-P, in the presence of 2 mM ammonium sulfate). ATP concentrations higher than 1 mM inhibited the enzyme. Ammonium sulfate activated the PFK several folds, increasing the affinity of the enzyme for Fru 6-P and V(max). 5'-AMP (0.2 mM) increased the affinity for Fru 6-P (S(0.5) of 6.2 mM). Fructose 2,6-bisphosphate activated the PFK, with a maximal activation at concentrations higher than 2 microM. Citrate reverted the activation of PFK produced by 0.2 mM 5'-AMP (IC(50 citrate)=2.0 mM), producing a higher inhibition than that exerted on other invertebrate PFKs. Barnacle muscular PFK was activated in vitro after exposure to exogenous cyclic-AMP (0.1 mM) as well as by phosphatidylserine (50 microg/ml), indicating a possible control by protein kinase A and a phospholipid dependent protein kinase (PKC). The results suggest a highly regulated enzyme in vivo, by allosteric mechanisms and also by protein phosphorylation.     

Glucose: a more powerful modulator of fructose 2,6-bisphosphate levels than insulin in human hepatocytes

This study provides the first experimental evidence of the short-term control of fructose 2,6-bisphosphate (Fru(2,6)P2) levels in adult human hepatocytes. (1) In hepatocytes whose metabolic status resembles the fed state (glycogen-rich), exposure to glucagon (10(-8) M) caused a drastic decrease in the levels of this effector and a significant fall in lactate production rate. Adrenaline, isoprenaline (a beta-adrenergic agonist) and lactate exerted a similar action decreasing Fru(2,6)P2 concentration. (2) In glucagon pre-treated, glycogen- and Fru(2,6)P2-depleted cells (a situation that mimics the fasted state), Fru(2,6)P2 re-synthesis was strictly dependent on glucose availability. (3) Insulin did not seem to exert a direct action on the control of Fru(2,6)P2 in human hepatocytes. The hormone--which failed to enhance Fru(2,6)P2 in glucose-starved cells--did not further increase Fru(2,6)P2 content nor its time-course evolution as compared to hepatocytes incubated with glucose alone. (4) Lactate caused a significant delay in the glucose-induced increase in Fru(2,6)P2 content that could not be prevented by insulin. (5) Data indicate that in human hepatocytes glucose is a more powerful modulator of Fru(2,6)P2 than insulin, and that variations in blood lactate concentration may also play a role in the control of hepatic Fru(2,6)P2 levels during the fasted-to-fed transition in humans.     

Phosphofructo-2-kinase/fructose-2,6-bisphosphatase modulates oscillations of pancreatic islet metabolism

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. It has been proposed that a positive feedback circuit exists within the glycolytic pathway, the autocatalytic activation of phosphofructokinase-1 (PFK1), which endows pancreatic beta-cells with the ability to generate oscillations in metabolism. Flux through PFK1 is controlled by the bifunctional enzyme PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) in two ways: via (1) production/degradation of fructose-2,6-bisphosphate (Fru2,6-BP), a potent allosteric activator of PFK1, as well as (2) direct activation of glucokinase due to a protein-protein interaction. In this study, we used a combination of live-cell imaging and mathematical modeling to examine the effects of inducibly-expressed PFK2/FBPase2 mutants on glucose-induced Ca(2+) pulsatility in mouse islets. Irrespective of the ability to bind glucokinase, mutants of PFK2/FBPase2 that increased the kinase:phosphatase ratio reduced the period and amplitude of Ca(2+) oscillations. Mutants which reduced the kinase:phosphatase ratio had the opposite effect. These results indicate that the main effect of the bifunctional enzyme on islet pulsatility is due to Fru2,6-BP alteration of the threshold for autocatalytic activation of PFK1 by Fru1,6-BP. Using computational models based on PFK1-generated islet oscillations, we then illustrated how moderate elevation of Fru-2,6-BP can increase the frequency of glycolytic oscillations while reducing their amplitude, with sufficiently high activation resulting in termination of slow oscillations. The concordance we observed between PFK2/FBPase2-induced modulation of islet oscillations and the models of PFK1-driven oscillations furthermore suggests that metabolic oscillations, like those found in yeast and skeletal muscle, are shaped early in glycolysis.     

Effects of 5-hydroxytryptamine, cyclic AMP, AMP, and fructose 2,6-bisphosphate on phosphofructokinase activity in Hymenolepis diminuta

1. 5-HT (10(-4) M) had no effect on the activity of phosphofructokinase in Hymenolepis diminuta. Concentrations of ATP above 33 microM inhibited PFK activity; AMP and cyclic AMP relieved this inhibition. 2. Local levels of cyclic AMP may be indirectly modulated by NaF, guanylyl imidophosphate, or 5-HT in the presence of GTP, which stimulates adenylyl cyclase activity x2 in H. diminuta homogenates. 3. Fructose 2,6-bisphosphate (F2BP), a physiological regulator of PFK activity in rat liver, also relieved ATP-induced inhibition of PFK. F2BP was present in supernatants from the worms at about 20 mumol/g wet wt. 4. 5-HT may cause an increase in the rate of glycolysis in H. diminuta by elevating either cyclic AMP and/or AMP levels; these nucleotides can in turn increase PFK activity.     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

cDNA sequence and kinetic properties of human lung fructose(1, 6)bisphosphatase

A cDNA encoding fructose(1,6)bisphosphatase was isolated from total human lung RNA. The cDNA contained an open reading frame encoding 337 amino acids. The determined nucleotide sequence of the lung cDNA was significantly different from muscle cDNA and slightly differed from human liver cDNA in a single mutation (Gly-336 for Ala-336) and a T for C substitution in position 648. The human lung fructose(1, 6)bisphosphatase [Fru(1,6)Pase] was isolated and its kinetic parameters were compared with liver and muscle isoenzymes. Values of kcat for the lung Fru(1,6)Pase were lower than for the liver and muscle enzyme. Like the liver isoenzyme, lung Fru(1,6)Pase is significantly less inhibited by AMP than the muscle enzyme. The values of I0.5 were 9.5, 9.8, and 0.3 microM for the liver, lung, and muscle enzyme, respectively. The lung enzyme was slightly more sensitive to fructose(2,6)bisphosphate [Fru(2,6)P2] inhibition than the liver enzyme. Ki was 75 microM for the lung and 96 microM for the liver enzyme. The synergistic effect of AMP and Fru(2,6)P2 on the lung and liver Fru(1,6)Pase was also observed. In the presence of AMP the corresponding values of Ki for Fru(2,6)P2 were 16 microM for the lung and 10 microM for the liver enzyme.     

Molecular cloning, sequence analysis, and expression of a human liver cDNA coding for fructose-6-P,2-kinase:fructose-2,6-bisphosphatase

A cDNA coding for 378 amino acids from the C-terminus of the human liver bifunctional enzyme, Fructose-6-phosphate,2-kinase:Fructose-2,6-bisphosphatase was isolated, sequenced, and expressed in E. coli K38. The expressed protein, identified by specific immunoassay, showed Fru 2,6-bisphosphatase activity but no Fru 6-P,2-kinase activity, demonstrating directly that the Fru 2,6-bisphosphatase activity resides in the C-terminal region. The Km for Fru 2,6-P2 was 4.3 microM. Fru 6-P was a noncompetitive inhibitor (Ki = 2.9 microM), and formed a phosphorylated intermediate when incubated with Fru 2,6[2-32P]P2. The subunit Mr of the enzyme was 36,600, and the active enzyme showed Mr = 37,000 by gel filtration.     

Sequence of insulin effects on cytoskeletal and cytosolic phosphofructokinase, mitochondrial hexokinase, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels, and the antagonistic action of calmodulin inhibitors, in diaphragm muscle.

1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.     

Characterization of yeast fructose-2,6-bisphosphate 6-phosphatase

To obtain information on the biological significance of yeast fructose-2,6-bisphosphate 6-phosphatase, kinetic data of the purified enzyme [(1987) Eur. J. Biochem. 164, 27-30] have been measured. Maximal activity was found between pH 6 and 7, the apparent Michaelis constant with fructose 2,6-bisphosphate was 7.2 microM at pH 6.0 and 79 microM at pH 7.0. Concentrations required for 50% inhibition of the enzyme at pH 6.0 were 8 microM Fru2P, 45 microM G1c6P, 80 microM Fru6P and 200 microM inorganic phosphate. The known intracellular steady-state level of about 10 microM fructose 2,6-bisphosphate in the presence of glucose is likely to be the result of a balance between the rapid synthesis of fructose 2,6-bisphosphate catalyzed by 6-phosphofructose 2-kinase and a fructose 2,6-bisphosphate degrading activity. The biological function of fructose-2,6-bisphosphate 6-phosphatase with an apparent Michaelis constant between 7 and 79 microM fructose 2,6-bisphosphate at pH 6-7 is therefore suggested to participate in the maintenance of a steady-state level of fructose 2,6-bisphosphate in a concentration range that fits well with the Michaelis constant of the enzyme.     

Control of fructose 2,6-bisphosphate levels in rat macrophages by glucose and phorbol ester

The presence of fructose 2,6-bisphosphate (Fru 2,6-P2) in elicited peritoneal macrophages of rat was examined. These cells possess an active phosphofructokinase-2 which is diminished by citrate and only slightly inhibited by glycerol 3-phosphate. Phosphofructokinase-1 submaximal activity was increased 26-fold by the addition of 1 microM Fru 2,6-P2. Incubation of cells without glucose decreased the amount of Fru 2,6-P2 to zero, but further addition of 5 mM glucose increased the levels of the sugar ester 20-fold. In addition, the presence of phorbol ester potentiated the synthesis of Fru 2,6-P2. By contrast phenylisopropyladenosine or prostaglandin F2 alpha inhibited the production of Fru 2,6-P2.     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

Effect of recombinant cytokines on glycolysis and fructose 2,6-bisphosphate in rheumatoid synovial cells in vitro.

Recombinant-derived human interleukin 1 (IL1) alpha and beta and interferon gamma (IFN-gamma) each produced similar increases in rheumatoid synovial cell (RSC) glycolysis, as judged by increased values for glucose uptake, lactate production and cellular fructose 2,6-bisphosphate [Fru(2,6)P2]. Measurement of Fru(2,6)P2 proved to be the most sensitive parameter for an assessment of glycolysis: IL1 alpha, IL1 beta and IFN-gamma all produced a 3-6-fold increase in this metabolite whereas tumour necrosis factor (TNF alpha) was far less effective. Prostaglandin E production was stimulated predominantly by IL1 alpha and IL1 beta rather than by IFN-gamma or TNF alpha. When combinations of cytokines were examined the addition of IFN-gamma with either IL1 alpha, IL1 beta or murine IL1 produced a synergistic increase in cellular Fru(2,6)P2. The three forms of IL1 increased Fru(2,6)P2 via the same pathway, whereas IFN-gamma acted via a different mechanism. The increase in Fru(2,6)P2 in subcultured RSC produced by addition of medium from a primary culture exceeded the maximal effects of any of the single cytokines studied, suggesting the presence of a mixture of cytokines in the primary RSC culture medium.     

Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells

Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.     

Different modes of activating phosphofructokinase,a key regulatory enzyme of glycolysis,in working vertebrate muscle

Glycolytic flux in white muscle can be increased several-hundredfold by exercise. Phosphofructokinase (PFK; EC 2.7.1.11) is a key regulatory enzyme of glycolysis, but how its activity in muscle is controlled is not fully understood. In order not to neglect integrative aspects of metabolic regulation, we have studied in frogs (Rana temporaria) a physiological form of muscle work (swimming) that can be triggered like a reflex. We analysed swimming to fatigue in well rested frogs, recovery from exercise, and repeated exercise after 2 h of recovery. At various times, gastrocnemius muscles were tested for glycolytic intermediates and effectors of PFK. All metabolites responded similarly to the two periods of exercise, with the notable exception of fructose 2,6-bisphosphate (F2,6P(2)), which we proved to be a most potent activator of frog muscle PFK. The first bout of exercise triggered a more than 10-fold increase in F2,6P(2); PFK activity and the content of F2,6P(2) in muscle were well correlated. F2,6P(2) decreased to pre-exercise levels in fatigued frogs and it virtually disappeared during recovery. Varying by a factor of 70, F2,6P(2) was the most dynamic of all metabolites in muscle. Even more surprisingly, F2,6P(2) did not respond at all to a second bout of exercise. Other activators of PFK, such as Pi, AMP and ADP, are increased as a consequence of increased ATP turnover in contracting muscle cells. This does not apply to F2,6P(2) which is likely to respond to extracellular signals and could be involved in mechanisms by which muscle metabolism is integrated into the metabolism of the whole body. Whether this phenomenon exists in vertebrates other than the frog, and maybe even in humans, and how the content of F2,6P(2)in muscle is controlled are intriguing open questions.     

Contributions of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 to the elevated glycolysis in hepatocytes from Zucker fa/fa rats

The insulin-resistant Zucker fa/fa rat has elevated hepatic glycolysis and activities of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 (PFK2). The latter catalyzes the formation and degradation of fructose-2,6-bisphosphate (fructose-2,6-P(2)) and is a glucokinase-binding protein. The contributions of glucokinase and PFK2 to the elevated glycolysis in fa/fa hepatocytes were determined by overexpressing these enzymes individually or in combination. Metabolic control analysis was used to determine enzyme coefficients on glycolysis and metabolite concentrations. Glucokinase had a high control coefficient on glycolysis in all hormonal conditions tested, whereas PFK2 had significant control only in the presence of glucagon, which phosphorylates PFK2 and suppresses glycolysis. Despite the high control strength of glucokinase, the elevated glycolysis in fa/fa hepatocytes could not be explained by the elevated glucokinase activity alone. In hepatocytes from fa/fa rats, glucokinase translocation between the nucleus and the cytoplasm was refractory to glucose but responsive to glucagon. Expression of a kinase-active PFK2 variant reversed the glucagon effect on glucokinase translocation and glucose phosphorylation, confirming the role for PFK2 in sequestering glucokinase in the cytoplasm. Glucokinase had a high control on glucose-6-phosphate content; however, like PFK2, it had a relative modest effect on the fructose-2,6-P(2) content. However, combined overexpression of glucokinase and PFK2 had a synergistic effect on fructose-2,6-P(2) levels, suggesting that interaction of these enzymes may be a prerequisite for formation of fructose-2,6-P(2). Cumulatively, this study provides support for coordinate roles for glucokinase and PFK2 in the elevated hepatic glycolysis in fa/fa rats.