Fructose 2,6-bisphosphate in human platelets: its possible role in the control of basal and thrombin-stimulated glycolysis

Human platelets contain fructose 2,6-bisphosphate, 6-phosphofructo-l-kinase (ATP: D-Fructose-6-phosphate-1-phosphotransferase, E.C.2.7. 1.11), the rate-limiting enzyme in platelet glycolysis appear to be significantly activated by physiological concentration of the compound, suggesting for fructose 2,6-bisphosphate a key regulatory role in the control of the glycolytic flux. Incubation of human platelets with thrombin results in a parallel and rapid increase of fructose-2,6-bisphosphate levels and glycolytic flux, suggesting that the compound may also be involved in the enhancement of glycolysis elicited by the stimulating agent.     

Mechanism of thrombin-induced rise in platelet fructose 2,6-bisphosphate content. Studies using phorbol myristate acetate, dioctanoylglycerol and ionophore A23187.

The mechanism by which thrombin increases platelet fructose 2,6-bisphosphate content was investigated. The action of thrombin was mimicked by phorbol 12 myristate 13-acetate and 1,2-dioctanoylglycerol. Ca2+ with A23187 potentiated the action of both these compounds. The action of thrombin required mobilization of intracellular and extracellular Ca2+ and was not decreased by indomethacin. This study suggests that protein kinase C activation and Ca2+ mobilization are both involved in the activation of glycolysis by thrombin.     

ACTH stimulates fructose 2,6-bisphosphate synthesis and glycolysis in Y-1 adrenal tumor cells

The effect of ACTH on glycolysis has been studied in Y-1 tumor adrenal cells. ACTH caused a sustained increase in the liberation of lactate as well as a stimulation of both basal and glucose-induced fructose 2,6-bisphosphate content. ACTH produces changes also in the activities of phosphofructokinase-1 and phosphofructokinase-2. The addition of Ca2+ or dibutyryl cyclic AMP did not modify neither lactate production nor fructose 2,6-bisphosphate levels. The results suggest that fructose 2,6-bisphosphate regulates ACTH-induced glycolysis at the phosphofructokinase-1 step, although the biochemical mechanism of phosphofructokinase-2 activation remains elusive.     

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.     

Fructose 2,6-bisphosphate in rat skeletal muscle during contraction.

Fructose 2,6-bisphosphate and several glycolytic intermediates were measured in two rat muscles, extensor digitorum longus and gastrocnemius, which were electrically stimulated in situ. Both the duration and the frequency of stimulation were varied to obtain different rates of glycolysis. There was no relationship between fructose 2,6-bisphosphate content and the increase in tissue lactate in contracting muscle. However, in gastrocnemius stimulated at low frequencies (less than or equal to 5 Hz), there was a 2-fold increase in fructose 2,6-bisphosphate at 10s, followed by a return to basal values, whereas lactate increased only after 1 min of contraction. The concentrations of hexose 6-phosphates, fructose 1,6-bisphosphate and triose phosphates were all increased during the 3 min stimulation. During tetanus (frequencies greater than or equal to 10 Hz) fructose 2,6-bisphosphate was not increased, whereas glycolysis was maximally stimulated and resulted in an accumulation of tissue lactate, mostly from glycogen. The concentrations of hexose 6-phosphate increased continuously during the 1 min tetanus, whereas fructose 1,6-bisphosphate was increased at 10s and then decreased progressively. It therefore appears that fructose 2,6-bisphosphate does not play a role in the stimulation of glycolysis during tetanus; it may, however, be involved in the control of glycolysis when the muscles are stimulated at low frequencies for short periods of time.     

Cyclic AMP regulation of fructose metabolism in germinatingPilobolus longipes spores

DormantPilobolus longipes spores metabolized fructose primarily to ethanol, CO₂, and trehalose. Cyclic AMP-induced spore activation was accompanied by a large stimulation of glycolytic activity. Mobilization of reserves, which was cyclic AMP dependent, accounted for a portion of the glycolytic product. The remaining product was derived from exogenous fructose. Increases in both fructose transport activity and hexose 6-phosphate levels were associated with 6-deoxyglucose-induced spore activation. Phosphofructokinase-1 activity in spore extracts was almost totally dependent upon fructose, 2,6-bisphosphate. High fructose 2,6-bisphosphate levels were correlated with rapid fructose metabolism. However, fructose alone caused a rise in fructose 2,6-bisphosphate content (sufficient to fully stimulate phosphofructokinase-1 activity) but there was no concurrent stimulation of glycolysis. These results suggest that glycolytic rates are determined mainly by hexose 6-phosphate levels and that cyclic AMP regulation of transport is an important determinant of hexose 6-phosphate concentration.     

Role of fructose 2,6-bisphosphate in the control by glucagon of gluconeogenesis from various precursors in isolated rat hepatocytes.

Hepatocytes from overnight-starved rats were incubated with 1-20 mM-fructose, -dihydroxyacetone, -glycerol, -alanine or -lactate and -pyruvate with or without 0.1 microM-glucagon. The production of glucose and lactate was measured, as was the content of fructose 2,6-bisphosphate. The concentrations of fructose (below 5 mM) and dihydroxyacetone (above 1 mM) that gave rise to an increase in fructose 2,6-bisphosphate were those at which a glucagon effect on the production of glucose and lactate could be observed. Glycerol had no effect on fructose 2,6-bisphosphate content or on production of lactate, and glucagon did not stimulate the production of glucose from this precursor. With alanine or lactate/pyruvate as substrates, glucagon stimulated glucose production whether the concentration of fructose 2,6-bisphosphate was increased or not. The extent of inactivation of pyruvate kinase by glucagon was not affected by the presence of the various gluconeogenic precursors. The role of fructose 2,6-bisphosphate in the effect of glucagon on gluconeogenesis from precursors entering the pathway at the level of triose phosphates or pyruvate is discussed.     

Changes in glucose 1,6-bisphosphate content in rat skeletal muscle during contraction.

Glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glycogen, lactate and other glycolytic metabolites were measured in rat gastrocnemius muscle, which was electrically stimulated in situ via the sciatic nerve. Both the frequency and the duration of stimulation were varied to obtain different rates of glycolysis. There was no apparent relationship between fructose 2,6-bisphosphate content and lactate accumulation in contracting muscle. In contrast, glucose 1,6-bisphosphate content increased with lactate concentration during contraction. It is suggested that the increase in glucose 1,6-bisphosphate could play a role in phosphofructokinase stimulation and in the activation of the glycolytic flux during muscle contraction.     

Hormonal regulation of fructose 2,6-bisphosphate levels in epididymal adipose tissue of rat

The participation of fructose 2,6-bisphosphate on glycolysis stimulated by insulin and adrenaline in incubated white adipose tissue of rat was investigated. Adrenaline addition to incubated fat-pads strongly decreased the intracellular levels of fructose 2,6-bisphosphate. When the tissue was preincubated with glucose, the presence of insulin in the incubation medium increased fructose 2,6-bisphosphate levels 2-fold. These variations were related to changes in the substrates, ATP and fructose 6-phosphate. It therefore appears that fructose 2,6-bisphosphate may be involved in the control of insulin-induced glycolysis, but it does not seem to play a role in the stimulation of glucolysis by adrenaline.     

Fructose 2,6-bisphosphate. Hormonal regulation and mechanism of its formation in liver

Vasopressin, phenylephrine, and A23187 cause an accumulation of fructose 2,6-bisphosphate in hepatocytes from fed rats, but not in Ca2+-depleted hepatocytes from fed rats or in phosphorylase kinase-deficient hepatocytes from (gsd/gsd) rats. The effect of vasopressin and phenylephrine is not found in hepatocytes from overnight-starved rats. Thus, the accumulation of fructose 2,6-bisphosphate by these agents may depend on the stimulation of glycogenolysis and on the resulting accumulation of hexose 6-phosphate. In support of this hypothesis, conditions are described for the enzymatic synthesis of fructose 2,6-bisphosphate from fructose 6-phosphate and Mg-ATP in liver extracts. Half-maximal activity (0.8 nmol/min.g) is obtained with about 60 microM fructose 6-phosphate, and the activity can be separated fom phosphofructokinase by ammonium sulfate fractionation. Treatment of rats or isolated hepatocytes with glucagon results in a 4-5-fold decrease in the maximal activity of this enzyme.     

Stereotaxic Administration of 1-Methyl-4-Phenylpyridinium Ion (MPP+) Decreases Striatal Fructose 2,6-Bisphosphate in Rats

Abstract: The Stereotaxic administration of 1-methyl-4-phenylpyridinium ion (MPP⁺) into the neostriatum of male rats caused a lesion that resulted in a large dose-dependent loss of striatal fructose 2,6-bisphosphate; initial values were restored 5 days after the treatment. This effect was not protected by systemic administration of MK-801 or by nitroarginine. The content of hexose 6-phosphates and ATP was also reduced by MPP⁺ treatment, whereas lactate was increased. Biochemical and histological results suggested that MPP⁺ caused a nonselective cell death, followed by a pronounced astroglial response, parallel to fructose 2, 6-bisphosphate recovery. The Stereotaxic administration of rotenone showed a different time effect on fructose 2,6-bisphosphate cerebral content, with a significantly faster recovery. These results indicate that cerebral fructose 2,6-bisphosphate may be a sensitive metabolite related to brain damage caused by potent neurotoxins such as MPP⁺. On the other hand, they show that MPP⁺ acts in the brain through a quick, strong cytotoxic mechanism, which probably involves mechanisms other than mitochondria! chain blockage     

Inhibition of phosphoglucomutase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate inhibits phosphoglucomutase. The inhibition is mixed with respect to glucose 1,6-bisphosphate and non-competitive with respect to glucose 1-phosphate. In contrast with fructose 1,6-bisphosphate and glycerate 1,3-bisphosphate, which also possess inhibitory effect, fructose 2,6-bisphosphate does not phosphorylate phosphoglucomutase. Fructose 2,6-bisphosphate preparations contain contaminants which can explain artefactual results previously reported.     

Adenylate cyclase stimulating agents and mitogens raise fructose 2,6-bisphosphate levels in human fibroblasts. Evidence for a dual control of the metabolite

Fructose 2,6-bisphosphate, the most potent activator of 6-phosphofructo-1-kinase, has been demonstrated to mediate the increase of glycolytic flux induced by mitogens human fibroblasts. In the present work the molecular basis of transmembrane control of fructose 2,6-bisphosphate has been investigated. Prostacyclin and isoprenaline, known to activate adenylate cyclase, are able to increase fructose 2,6-bisphosphate levels, indicating that in human fibroblasts cyclic AMP plays a positive role in the control of the metabolite concentration, opposite to that exerted in hepatocytes. Substances known to activate protein kinase C such as phorbol 12-myristate 13-acetate, or to stimulate phosphoinositide turnover such as thrombin and bradykinin are also effective in raising fructose 2,6-bisphosphate. Therefore, we conclude that cyclic AMP and protein kinase C are likely involved in the control of fructose 2,6-bisphosphate levels in human fibroblasts.     

Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.     

The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase

The activation of monocytes involves a stimulation of glycolysis, release of potent inflammatory mediators, and alterations in gene expression. All of these processes are known to be further increased under hypoxic conditions. The activated monocytes express inducible 6-phosphofructo-2-kinase (iPFK-2), which synthesizes fructose 2,6-bisphosphate, a stimulator of glycolysis. During ischemia, AMP-activated protein kinase (AMPK) activates the homologous heart 6-phosphofructo-2-kinase isoform by phosphorylating its Ser-466. Here, we studied the involvement of AMPK and iPFK-2 in the stimulation of glycolysis in activated monocytes under hypoxia. iPFK-2 was phosphorylated on the homologous serine (Ser-461) and activated by AMPK in vitro. The activation of human monocytes by lipopolysaccharide induced iPFK-2 expression and increased fructose 2,6-bisphosphate content and glycolysis. The incubation of activated monocytes with oligomycin, an inhibitor of oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, fructose 2,6-bisphosphate content, and glycolysis. In cultured human embryonic kidney 293 cells, the expression of a dominant-negative AMPK prevented both the activation and phosphorylation of co-transfected iPFK-2 by oligomycin. It is concluded that the stimulation of glycolysis by hypoxia in activated monocytes requires the phosphorylation and activation of iPFK-2 by AMPK.     

Signal transduction in isolated fat body from the cockroach Blaptica dubia exposed to hypertrehalosaemic neuropeptide

Hypertrehalosaemic hormones stimulate trehalogenesis while inhibiting glycolysis in cockroach fat body. Signal transduction of the hypertrehalosaemic peptide Bld HrTH was examined in isolated fat body of the Argentine cockroach Blaptica dubia with respect to its effects on the increase in trehalose production and decrease in the content of the glycolytic activator fructose 2,6-bisphosphate in the tissue. Cyclic AMP does not seem to be involved in these processes as the cAMP analogue cpt-cAMP and the phosphodiesterase inhibitor IBMX, which both permeate cell membranes, had no effect on either parameter. Octopamine at physiological concentrations (10⁻⁷ mol · l⁻¹) was also ineffective, but at 10⁻⁵ mol · l⁻¹ or above, octopamine stimulated trehalose production although the content of fructose 2,6-bisphosphate in fat body was not affected. Both calcium entry and the release of Ca²⁺ from intracellular stores seem to be involved in the action of the hormone. If Ca²⁺ was omitted from the incubation medium, the hormone stimulated trehalose production less, though still significantly, whereas the hormone effect on fructose 2,6-bisphosphate was completely abolished in the absence of extracellular Ca²⁺. With Ca²⁺ present in the medium, the effect of the hormone on fructose 2,6-bisphosphate could be fully mimicked by the calcium ionophore A23187, suggesting that calcium entry is a␣decisive step in this signalling pathway. Trehalose production, on the other hand, was increased by thimerosal and thapsigargin which increase cytosolic Ca²⁺ from intracellular stores, whereas thimerosal in the absence of extracellular Ca²⁺ increased rather than decreased the content of fructose 2,6-bisphosphate, thus dissociating the two effects, which are normally coordinated by the hormone. Trehalose production and the content of fructose 2,6-bisphosphate were not significantly affected by mepacrine and mellitin, which are known to inhibit, respectively stimulate, phospholipase A₂. Our data suggest that the effects of Bld HrTH on the stimulation of trehalose production and reduction of fructose 2,6-bisphosphate content in fat body are mediated by Ca²⁺, but that different signalling pathways are involved, suggesting that the two processes, although they are functionally linked, could be regulated separately. Accepted: 10 November 1997     

Fructose 2,6-bisphosphate in Dictyostelium discoideum. Independence of cyclic AMP production and inhibition of fructose-1,6-bisphosphatase

The occurrence of fructose 2,6-bisphosphate was detected in Dictyostelium discoideum. The levels of this compound were compared with those of cyclic AMP and several glycolytic intermediates during the early stages of development. Removal of the growth medium and resuspension of the organism in the differentiation medium decreased the content of fructose 2,6-bisphosphate to about 20% within 1 h, remaining low when starvation-induced development was followed for 8 h. The content of cyclic AMP exhibited a transient increase that did not correlate with the change in fructose 2,6-bisphosphate. If after 1 h of development 2% glucose was added to the differentiation medium, fructose 2,6-bisphosphate rapidly rose to similar levels to those found in the vegetative state, while the increase in cyclic AMP was prevented. The contents of hexose 6-phosphates, fructose 1,6-bisphosphate and triose phosphates changed in a way that was parallel to that of fructose 2,6-bisphosphate, and addition of sugar resulted in a large increase in the levels of these metabolites. The content of fructose 2,6-bisphosphate was not significantly modified by the addition of the 8-bromo or dibutyryl derivatives of cyclic AMP to the differentiation medium. These results provide evidence that the changes in fructose 2,6-bisphosphate levels in D. discoideum development are not related to a cyclic-AMP-dependent mechanism but to the availability of substrate. Fructose 2,6-bisphosphate was found to inhibit fructose-1,6-bisphosphatase activity of this organism at nanomolar concentrations, while it does not affect the activity of phosphofructokinase in the micromolar range. The possible physiological implications of these phenomena are discussed.     

Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6-bisphosphate

The activity of pyruvate kinase present in a crude extract of the bloodstream form of Trypanosoma brucei was greatly increased by fructose 2,6-bisphosphate, which converted the saturation curve for phosphoenolpyruvate from a sigmoid into a hyperbola with no change in V. Phosphate and arsenate had an effect opposite to that of fructose 2,6-bisphosphate and the apparent Ka for fructose 2,6-bisphosphate was shifted from 75 nM to 1.5 microM by the presence of 5 mM phosphate. Fructose 1,6-bisphosphate had effects similar to those of fructose 2,6-bisphosphate but at approximately 4000-fold higher concentrations. Pyruvate kinases of Crithidia luciliae and of Leishmania major, two trypanosomatids which are like T. brucei in containing glycosomes, were also stimulated by fructose 2,6-bisphosphate and inhibited by phosphate.     

The ability of adenosine to decrease the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. A cyclic AMP-mediated effect.

The presence of adenosine (25-250 microM) or of 2-chloroadenosine (2.5-100 microM) in the incubation medium caused a marked decrease in the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. This effect was accompanied by an increase in the concentration of cyclic AMP, an activation of phosphorylase and of fructose 2,6-bisphosphatase, and an inactivation of pyruvate kinase and of 6-phosphofructo-2-kinase. As a rule, the changes in the fructose 2,6-bisphosphate-modifying system were slower but more persistent than those in the activities of phosphorylase and pyruvate kinase. The effect of the nucleoside to decrease the concentration of fructose 2,6-bisphosphate was not affected by an inhibitor of adenosine transport and could not be obtained in a liver high-speed supernatant. These data indicate that the effect of adenosine to decrease the concentration of fructose 2,6-bisphosphate is mediated by the stimulation of adenylate cyclase, secondary to the binding of adenosine to membranous receptors. Like glucagon, 2-chloroadenosine stimulated gluconeogenesis in isolated hepatocytes, whereas adenosine had an opposite effect.     

Effect of TPA on fructose 2,6-bisphosphate levels and protein kinase C activity in B-chronic lymphocytic leukemia (B-CLL).

Normal B lymphocytes and peripheral mononuclear blood cells from B-chronic lymphocytic leukemia (B-CLL) patients were incubated in the presence of the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In normal B lymphocytes and lymphocytes from five patients with B-CLL, TPA stimulation increased lymphocyte fructose 2,6-bisphosphate (fructose 2,6-P2) content and activity of 6-phosphofructo 2-kinase (PFK-2), which is the enzyme that catalyzes the synthesis of fructose 2,6-P2. This effect was evident after 6 h and maximal after 12-24 h of TPA exposure. In three patients, lymphocytes seemed to be refractory to TPA stimulation in the conditions described here. Lymphocyte stimulation by TPA was associated with the translocation of protein kinase C (PKC) from the soluble to the particulate membrane fraction, except in B-CLL lymphocytes refractory to the TPA effect. These results give further support to the existence within B-CLL of subsets of cells which are refractory to the stimulation by TPA and demonstrate that the tumor promoter TPA induces important metabolic changes in lymphocytes of some patients with B-CLL.