Evidence for the short-term regulation of glycolytic flux in the isolated perfused ventricle of the land snail Helix lucorum (L.) after treatment with serotonin (5-hydroxytryptamine)

The glycolytic flux and the regulation of phosphofructokinase (PFK) activity by fructose 2,6-bisphosphate and covalent modification was investigated in isolated ventricles of land snail Helix lucorum perfused with or without serotonin. Serotonin evoked a significant increase in the level of glycolytic intermediates and a threefold increase of glycolytic flux. Studies of saturation curves of PFK for the substrate fructose 6-phosphate at pH similar to intracellular pH of heart muscle showed that serotonin increases enzyme sensitivity to activation by fructose 6-phosphate. Moreover, PFK preparations from ventricles perfused with serotonin exhibited lower K _a values for the activators AMP and fructose 2,6-bisphosphate, compared with the enzyme preparations from serotonin-untreated ventricles. The results suggest that PFK was converted to a more active form when exposed to serotonin. In vitro experiments of PFK phosphorylation showed that the conversion of the enzyme to a more active form was possibly due to its phosphorylation by an endogenous cyclic-AMP-dependent protein kinase. The concentration of fructose 2,6-bisphosphate increased in serotonin-treated ventricles and it exerted a synergistic effect with AMP on the activation of PFK. The bound fraction of glycolytic enzymes increased in the serotonin-treated ventricles only after the 4th min of perfusion. The results suggest that the stimulation of glycolytic flux in the ventricles of H. lucorum in the first minutes of perfusion with serotonin was partly due to the activation of PFK via enzyme molecule covalent modification and to increase of fructose 2,6-bisphosphate. Accepted: 8 April 1997     

Regulation of fructose 2,6-bisphosphate concentration in white adipose tissue.

Injection of insulin to fed rats diminished the concentration of fructose 2,6-bisphosphate in white adipose tissue. Incubation of epididymal fat-pads or adipocytes with insulin stimulated lactate release and sugar detritiation and also decreased fructose 2,6-bisphosphate concentration. Such a decrease was, however, not observed in fat-pads from starved or alloxan-diabetic rats. Incubation of adipocytes from fed rats with various concentrations of glucose or fructose led to a dose-dependent rise in fructose 2,6-bisphosphate which correlated with lactate output and detritiation of 3-3H-labelled sugar. In adipocytes from fed rats, palmitate stimulated the detritiation of [3-3H]glucose without affecting lactate production and fructose 2,6-bisphosphate concentration. Incubation of epididymal fat-pads from fed rats in the presence of antimycin stimulated lactate output but decreased fructose 2,6-bisphosphate concentration. Changes in lipolytic rates brought about by noradrenaline, insulin, adenosine and corticotropin in adipocytes from fed rats were not related to changes in fructose 2,6-bisphosphate or to rates of lactate output. In fed rats, the activity of 6-phosphofructo-2-kinase was not changed after treatment of adipocytes with insulin, noradrenaline or adenosine. It is suggested that the decrease in fructose 2,6-bisphosphate concentration observed after insulin treatment can be explained by the increase in sn-glycerol 3-phosphate, an inhibitor of 6-phosphofructo-2-kinase.     

The mechanism by which ethanol decreases the concentration of fructose 2,6-bisphosphate in the liver.

The intragastric administration of ethanol to fed rats caused in their liver, within about 1 h, a 20-fold decrease in the concentration of fructose 2,6-bisphosphate, an activation of fructose 2,6-bisphosphatase, an inactivation of phosphofructo-2-kinase but no change in the concentration of cyclic AMP. Incubation of isolated hepatocytes in the presence of ethanol caused a rapid increase in the concentration of sn-glycerol 3-phosphate and a slower and continuous decrease in the concentration of fructose 2,6-bisphosphate with no change in that of hexose 6-phosphates. There was also a relatively slow activation of fructose 2,6-bisphosphatase and inactivation of phosphofructo-2-kinase. Glycerol and acetaldehyde had effects similar to those of ethanol on the concentration of phosphoric esters in the isolated liver cells. 4-Methylpyrazole cancelled the effect of ethanol but reinforced those of acetaldehyde. High concentrations of glucose or of dihydroxyacetone caused an increase in the concentration of hexose 6-phosphates and counteracted the effect of ethanol to decrease the concentration of fructose 2,6-bisphosphate. As a rule, hexose 6-phosphates had a positive effect and sn-glycerol 3-phosphate had a negative effect on the concentration of fructose 2,6-bisphosphate in the liver, so that, at a given concentration of hexose 6-phosphates, there was an inverse relationship between the concentration of fructose 2,6-bisphosphate and that of sn-glycerol 3-phosphate. These effects could be explained by the ability of sn-glycerol 3-phosphate to inhibit phosphofructo-2-kinase and to counteract the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate. sn-Glycerol 3-phosphate had also the property to accelerate the inactivation of phosphofructo-2-kinase by cyclic AMP-dependent protein kinase whereas fructose 2,6-bisphosphate had the opposite effect. The changes in the activity of phosphofructo-2-kinase and fructose 2,6-bisphosphatase appear therefore to be the result rather than the cause of the decrease in the concentration of fructose 2,6-bisphosphate.     

Fructose 2,6-bisphosphate in relation with the resumption of metabolic activity in slices of Jerusalem artichoke tubers

When slices of Jerusalem artichoke tubers were incubated at 25°C, their concentration in fructose 2,6-bisphosphate increased up to 250-fold within 2 h. Fructose 2,6-bisphosphate was also formed, although at a slower rate, in slices incubated at 0°C. Its formation could not be explained by an increase in the concentration of fructose 6-phosphate or of ATP either by an activation of phosphofructo-2-kinase. Pyrophosphate—fructose-6-phosphate 1-phosphotransferase was the only enzyme present in a tuber extract which was found to be sensitive to fructose 2,6-bisphosphate. An improved procedure for the assay of fructose 2,6-bisphosphate is also reported.     

Effect of experimental hypothyroidism on the control of 6-phosphofructo-1-kinase activity in rat jejunal mucosa.

Changes in the activity of 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) from the epithelial cells of rat small intestine during experimental hypothyroidism were studied. Hypothyroidism resulted in significant decreases in the plasma concentrations of total tri-iodothyronine, free tri-iodothyronine, total thyroxine, free thyroxine and insulin. These changes were associated with a significant increase in the plasma concentration of thyrotropin. The total activity and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V)) of jejunal PFK of hypothyroid rats were significantly diminished as compared to control rats. PFK of hypothyroid rats was more sensitive to inhibition by ATP. The mucosal enzyme of both control and hypothyroid state was sensitive to stimulation by AMP and fructose 2,6-bisphosphate. It is concluded that during hypothyroidism the rate of glycolytic pathway in the small intestine is reduced as a result of a fall in glucose uptake, and the subsequent kinetic changes of PFK are primarily to maintain the concentrations of fructose 6-phosphate (and glucose 6-phosphate) during the reduced glycolytic flux. These changes in PFK activity may be caused by changes in plasma insulin concentrations, glucose utilization and fructose 2,6-bisphosphate concentrations.     

Stimulation of Spermatid Phosphofructokinase by Fructose 2,6-Bisphosphate from Rat Testes

Effect of fructose 2, 6-bisphosphate on 6-phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) in spermatid extract from rat testes was studied. Fructose 2, 6-bisphosphate stimulated the enzyme greatly by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Fructose 2, 6-bisphosphate (0.8 μM) was required for 50% activation of 6-phosphofructokinase (PFK). In addition, fructose 2, 6-bisphosphate, AMP and fructose 6-phosphate acted cooperatively to stimulate the activity of PFK. This stimulation may play an important role in the regulation of glycolysis in spermatids of rat testes.     

Regulation of 6-phosphofructo-1-kinase from the epithelial cells of rat small intestine during pregnancy and lactation

The regulation of 6-phosphofructo-1-kinase (PFK) in the epithelial cells of rat small intestine was studied during pregnancy and lactation. The total activities and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (nu 0.5/V] of the partially purified mucosal PFK were found to increase initially in early pregnant rats (11-12 days of gestation) and to fall back to normal in late pregnant rats (19-20 days of gestation). These changes in enzyme activity during pregnancy were associated with similar changes in the circulating levels of progesterone. The maximal activity and activity ratio (nu 0.5/V) were increased in male and female rats injected with progesterone. An increase in the total activity and activity ratio of mucosal PFK was also obtained in lactating rats. However, the enzyme was not strongly activated by inorganic phosphate, fructose 2,6-bisphosphate or glucose 1,6-bisphosphate either in early pregnant or lactating rats. These results indicate that mucosal PFK is already present as an active form during early pregnancy and lactation. Therefore, it is suggested that female sex hormones increase the circulating levels of insulin during early pregnancy which, in turn, positively affect the activity of mucosal PFK which could be also stimulated by the increased levels of fructose 2,6-bisphosphate. The increased activity of PFK in the peak lactating rats could be possible because of an increased demand for lactate production from glucose together with the stimulation of PFK by the increased concentrations of fructose 2,6-bisphosphate which therefore increases the rate of glycolysis.     

Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6-bisphosphate and of glycolytic intermediates in Trypanosoma brucei

Upon differential centrifugation of cell-free extracts of Trypanosoma brucei, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6-phosphofructo-2-kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycerol 3-phosphate. The Km of fructose-2,6-bisphosphatase was 7 microM; this enzyme was inhibited by fructose 1,6-bisphosphate (Ki = 10 microM) and, less potently, by fructose 6-phosphate, phosphoenolpyruvate and glycerol 3-phosphate. Melarsen oxide inhibited 6-phosphofructo-2-kinase (Ki less than 1 microM) and fructose-2,6-bisphosphatase (Ki = 2 microM) much more potently than pyruvate kinase (Ki greater than 100 microM). The intracellular concentrations of fructose 2,6-bisphosphate and hexose 6-phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6-bisphosphate, ATP, hexose 6-phosphate and fructose 1,6-bisphosphate. These studies indicate that the concentration of fructose 2,6-bisphosphate is mainly controlled by the concentration of the substrates of 6-phosphofructo-2-kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6-bisphosphate on pyruvate kinase. At micromolar concentrations, melarsen oxide blocked almost completely the formation of fructose 2,6-bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6-phosphates. At higher concentrations (3-10 microM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolypyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.     

The appearance of a critical input concentration of fructose 6-phosphate in the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase cycle

Stationary states of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle were investigated in relation to the input concentration of fructose 6-phosphate. Below a critical input concentration of fructose 6-phosphate very low levels of fructose 2,6-bisphosphate were obtained. Above this point the fructose 2,6-bisphosphate changes in direct proportion to the input of fructose 6-phosphate. Phosphorylation of the enzyme causes an increase of the critical input concentration of fructose 6-phosphate. The control coefficients for fructose 2,6-bisphosphate have their maximum at the critical input concentration of fructose 6-phosphate.     

Creation of an allosteric phosphofructokinase starting with a nonallosteric enzyme. The case of dictyostelium discoideum phosphofructokinase.

An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n(H) = 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S(0.5) = 4500 microm), strong activation by fructose 2,6-bisphosphate (K(act) = 0.1 microm) and fructose 1,6-bisphosphate (K(act) = 40 microm), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n(H) = 1.0; K(m) = 22 microm) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu(834), as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.     

Effect of ethylene treatment on the concentration of fructose-2,6-bisphosphate and on the activity of phosphofructokinase 2/fructose-2,6-bisphosphatase in banana

Preclimacteric bananas fruits were treated for 12 h with ethylene to induce the climacteric rise in respiration. One day after the end of the hormonal treatment, the two activities of the bifunctional enzyme, phosphofructokinase 2/fructose-2,6-bisphosphatase started to increase to reach fourfold their initial value 6 days later. By contrast, the activities of the pyrophosphate-dependent and of the ATP-dependent 6-phosphofructo-1-kinases remained constant during the whole experimental period, the first one being fourfold greater than the second. The concentrations of fructose 2,6-bisphosphate and of fructose 1,6-bisphosphate increased in parallel during 4 days and then slowly decreased, the second one being always about 100-fold greater than the first. The change in fructose 2,6-bisphosphate concentration can be partly explained by the rise of the bifunctional enzyme, but also by an early increase in the concentration of fructose 6-phosphate, the substrate of all phosphofructokinases, and also by the decrease in the concentration of glycerate 3-phosphate, a potent inhibitor of phosphofructokinase 2. The burst in fructose 2,6-bisphosphate and the activity of the pyrophosphate-dependent phosphofructokinase, which is in banana the only enzyme known to be sensitive to fructose 2,6-bisphosphate, can explain the well-known increase in fructose 1,6-bisphosphate which occurs during ripening.     

Metabolic integration in locust flight: the effect of octopamine on fructose 2,6-bisphosphate content of flight muscle in vivo

The biogenic amine octopamine was injected into the haemolymph of 20-days old male locusts,Locusta migratoria, and the content of fructose 2,6-bisphosphate, a potent activator of glycolysis, was measured in the flight muscle after various time. Octopamine brought about a transient increase in fructose 2,6-bisphosphate. After the injection of 10 l of 10 mmol·l^-1 d, l-octopamine fructose 2,6-bisphosphate was increased by 61% within 2 min. Ten minutes after the injection fructose 2,6-bisphosphate was increased to 6.71±0.89 nmol·g^-1 flight muscle, almost 300% over the control value. Flight caused fructose 2,6-bisphosphate in flight muscle to decrease, but this decrease was counteracted by octopamine injected into the haemolymph of flying locusts. Octopamine and fructose 2,6-bisphosphate may act as signals to stimulate the oxidation of carbohydrate and to integrate muscle performance and metabolism. This mechanism appears particularly significant in the initial stage of flight when carbohydrates are the main fuel.Abbreviations F2,6P₂ fructose 2,6-bisphosphate - F6P fructose 6-phosphate - PFK1 6-phosphofructokinase (EC 2.7.1.11) - P _i inorganic phosphate - PP _i -PFK pyrophosphate dependent fructose 6-phosphate phosphotransferase (EC 2.7.1.90)     

Effect of benzoate on the metabolism of fructose 2,6-bisphosphate in yeast

When benzoate (2 mM, pH 3.5) was added together with glucose (0.1 M) to a suspension of Saccharomyces cerevisiae in the stationary phase, it caused a relative increase in the concentration of glucose 6-phosphate and fructose 6-phosphate and a decrease in the concentration of fructose 1,6-bisphosphate. These effects are in confirmation of similar observations made by Krebs et al. [Biochem. J. 214, 657-663 (1983)] and are indicative of an inhibition of 6-phosphofructo-1-kinase. Benzoate also caused an about fourfold relative decrease in the concentration of fructose 2,6-bisphosphate, an increase in that of cyclic AMP with no change in that of ATP. It also greatly decreased the activation of 6-phosphofructo-2-kinase, but not that of trehalase, both of which normally occur upon addition of glucose to a yeast suspension. When added 10 min after glucose, benzoate caused a rapid (within 2-3 min) decrease in fructose 2,6-bisphosphate concentration and in 6-phosphofructo-2-kinase activity. In the presence of benzoate, there was also a parallel decrease in the concentration of fructose 2,6-bisphosphate and in the rate of ethanol production when the external pH was dropped from 5.0 to 2.5, with minimal change in the concentration of ATP. Purified 6-phosphofructo-2-kinase was inhibited by benzoate and also by an acid pH. Experiments with cell-free extracts did not provide an explanation for the rapid disappearance of fructose-2,6-bisphosphate or the inactivation of 6-phosphofructo-2-kinase in yeast upon addition of benzoate.     

Metabolite-mediated catalyst conversion of PFK and PFP: can PFK really be converted to PFP?

It has been suggested that in spinach leaves an enzyme able to catalyze the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate can exist in two different interconvertible forms which use ATP and pyrophosphate respectively as phosphoryl donors [FEBS Letters 169 (1984) 287-292]. However, the data presented to support this suggestion could also be interpreted without assuming such an unusual type of interconversion. This reinterpretation considers that PFK and PFP are two distinct enzymes which are differentially activated by incubation with various effectors such as UDPG, pyrophosphate, ATP, fructose 6-phosphate and fructose 2,6-bisphosphate.     

The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae. A cyclic-AMP-dependent activation of phosphofructokinase 2

When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate. These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature. Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate. Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange. When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase. Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase. This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km. Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate. From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S. cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme. In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.     

Occurrence and characterization of fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase in Euglena gracilis

1. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase occurred in Euglena gracilis SM-ZK, and is located in cytosol. 2. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase were partially purified, and both enzyme activities were not separated during the partial purification. 3. The pH optimum for fructose 6-phosphate, 2-kinase activity was 7.0. The saturation curve of the enzyme activity for ATP concentration was hyperbolic, and the Km value for the substrate was 0.88 mM. On the other hand, the saturation curve of the enzyme activity for fructose 6-phosphate concentration was sigmoidal, and the K0.5 value for the substrate was 70 microM. 4. The pH optimum for fructose 2,6-bisphosphatase activity was 6.5. The saturation curve for fructose 2,6-bisphosphate concentration was sigmoidal, and the K0.5 value for the substrate was 1.29 microM. Fructose 2,6-bisphosphate showed a substrate inhibition at high concentration over 5 microM, and the enzyme activity was completely inhibited by 20 microM of fructose 2,6-bisphosphate.     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate

Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-PFK) has been purified 150-fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6-phosphate and also for fructose 1,6-bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6-bisphosphate, the affinity for fructose 6-phosphate and for fructose 1,6-bisphosphate were greatly increased and the kinetics became Micha?lian. The effect of fructose 2,6-bisphosphate was increased by the presence of fructose 6-phosphate and decreased by the presence of Pi. Consequently, the Ka for fructose 2,6-bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6-bisphosphate in amounts lower than a picomole, is described.     

Phorbol esters, bombesin and insulin elicit differential responses on the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase system in primary cultures of foetal and adult rat hepatocytes.

The effects of 4 beta-phorbol 12-myristate 13-acetate (PMA), bombesin and insulin on 6-phosphofructo-2-kinase (PFK-2) activity, on fructose 2,6-bisphosphate concentration and on the phosphorylation state of PFK-2 were investigated in primary cultures of hepatocytes from foetal and adult rats. Bombesin stimulated PFK-2 activity and increased hexose phosphate (glucose 6-phosphate and fructose 6-phosphate) and fructose 2,6-bisphosphate content in hepatocytes both in the foetal and adult state. However, PMA-treated foetal cells exhibited a marked stimulation in fructose 2,6-bisphosphate concentration and in PFK-2 activity as well as in the content of hexose phosphates, while no response was found in the case of adult hepatocytes. Moreover, the effect of PMA on foetal hepatocytes was suppressed when cells were incubated with cycloheximide, but not when this effect was elicited by bombesin or insulin. These results, and those obtained on the phosphorylation state of PFK-2, suggest that there are different pathways that modulate fructose 2,6-bisphosphate content and, therefore, the control mechanisms of glycolysis and gluconeogenesis at this regulatory step, both in adult and foetal rat liver.