Difference in glucose sensitivity of liver glycolysis and glycogen synthesis. Relationship between lactate production and fructose 2,6-bisphosphate concentration.

Incubation of isolated rat hepatocytes from fasted rats with 0-6 mM-glucose caused an increase in [fructose 2,6-bisphosphate] (0.2 to about 5 nmol/g) without net lactate production. A release of 3H2O from [3-3H]glucose was, however, detectable, indicating that phosphofructokinase was active and that cycling occurred between fructose 6-phosphate and fructose 1,6-bisphosphate. A relationship between [fructose 2,6-bisphosphate] and lactate production was observed when hepatocytes were incubated with [glucose] greater than 6 mM. Incubation with glucose caused a dose-dependent increase in [hexose 6-phosphates]. The maximal capacity of liver cytosolic proteins to bind fructose 2,6-bisphosphate was 15 nmol/g, with affinity constants of 5 X 10(6) and 0.5 X 10(6) M-1. One can calculate that, at 5 microM, more than 90% of fructose 2,6-bisphosphate is bound to cytosolic proteins. In livers of non-anaesthetized fasted mice, the activation of glycogen synthase was more sensitive to glucose injection than was the increase in [fructose 2,6-bisphosphate], whereas the opposite situation was observed in livers of fed mice. Glucose injection caused no change in the activity of liver phosphofructokinase-2 and decreased the [hexose 6-phosphates] in livers of fed mice.     

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.     

Influence of fructose 2,6-bisphosphate on the phosphofructokinase/fructose 1,6-bisphosphatase cycle

In a reconstituted enzyme system multiple stationary states and oscillatory motions of the substrate cycle catalyzed by phosphofructokinase and fructose 1,6-bisphosphatase are significantly influenced by fructose 2,6-bisphosphate. Depending on the initial conditions, fructose 2,6-bisphosphate was found either to generate or to extinguish oscillatory motions between glycolytic and gluconeogenic states. In general, stable glycolytic modes are favored because of the efficient activation of phosphofructokinase by this effector. The complex effect of fructose 2,6-bisphosphate on the rate of substrate cycling correlates with its synergistic cooperation with AMP in the activation of phosphofructokinase and inhibition of fructose 1,6-bisphosphatase.     

Vanadate raises fructose 2,6-bisphosphate concentrations and activates glycolysis in rat hepatocytes.

In rat hepatocytes, vanadate increases fructose 2,6-bisphosphate (Fru-2,6-P2) in a time- and dose-dependent manner, and counteracts the decrease in this metabolite caused by glucagon, forskolin or exogenous cyclic AMP. Vanadate does not directly modify the activity of 6-phosphofructo-2-kinase, even though it can counteract the inactivation of this enzyme caused by glucagon. Furthermore, vanadate raises the yield of 3H2O from [3-3H]glucose, indicating that it increases the flux through 6-phosphofructo-1-kinase. Moreover, vanadate in hepatocytes incubated in the presence of glucose increases the production of both lactate and CO2. Therefore vanadate has insulin-like effects on the glycolytic pathway in rat hepatocytes. These results clearly contrast with our previous observation that vanadate exerts glycogenolytic non-insulin-like effects on glycogen synthase and phosphorylase.     

Role of fructose 2,6-bisphosphate in the stimulation of glycolysis by anoxia in isolated hepatocytes.

1. Incubation of hepatocytes from fed or starved rats with increasing glucose concentrations caused a stimulation of lactate production, which was further increased under anaerobic conditions. 2. When glycolysis was stimulated by anoxia, [fructose 2,6-bis-phosphate] was decreased, indicating that this ester could not be responsible for the onset of anaerobic glycolysis. In addition, the effect of glucose in increasing [fructose 2,6-bisphosphate] under aerobic conditions was greatly impaired in anoxic hepatocytes. [Fructose 2,6-bisphosphate] was also diminished in ischaemic liver, skeletal muscle and heart. 3. The following changes in metabolite concentration were observed in anaerobic hepatocytes: AMP, ADP, lactate and L-glycerol 3-phosphate were increased; ATP, citrate and pyruvate were decreased: phosphoenolpyruvate and hexose 6-phosphates were little affected. Concentrations of adenine nucleotides were, however, little changed by anoxia when hepatocytes from fed rats were incubated with 50 mM-glucose. 4. The activity of ATP:fructose 6-phosphate 2-phosphotransferase was not affected by anoxia but decreased by cyclic AMP. 5. The role of fructose 2,6-bisphosphate in the regulation of glycolysis is discussed.     

Pyrophosphate and fructose 2,6-bisphosphate effects on glycolysis in pea seed extracts

The participation of pyrophosphate-dependent phosphofructokinase (PPi-PFK) in plant glycolysis was examined using extracts from pea seeds (Pisum sativum L. cv Alaska). Glycolysis starting with fructose 6-phosphate was measured under aerobic conditions as the accumulation of pyruvate. Pyruvate accumulated in a medium containing PPi and adenosine diphosphate at about two-thirds of the rate in a medium containing adenosine diphosphate and adenosine triphosphate (ATP). The PPi-dependent pyruvate accumulation had the same reactant requirements and sensitivity to glycolysis inhibitors, sodium fluoride, and iodoacetamide, as the well-established ATP-dependent glycolysis. Added fructose 2,6-bisphosphate stimulated both the PPi-dependent pyruvate accumulation and PPi-PFK activity whereas this modulator had no effect on ATP-dependent glycolysis or ATP-PFK. Collectively these results demonstrate a PPi-dependent glycolytic pathway in plants which is responsive to fructose 2,6-bisphosphate.     

Perinatal changes of fructose 2,6-bisphosphate in the rat liver

In order to investigate a possible regulatory role of fructose 2,6-bisphosphate in early developmental stages, where profound changes in the carbohydrate metabolism are known to occur, this effector was estimated in fetal and postnatal rat liver.Polyphasic changes of the hepatic fructose 2,6-bisphosphate levels were found, which could be correlated to alterations in the glucose metabolism. A minimum in the hepatic fructose 2,6-bisphosphate level at the ?3rd day coincides with the initiation of glycogen synthesis and its increase two hours after birth concurs with glycogen mobilization.     

Lepidimoic acid increases fructose 2,6-bisphosphate in Amaranthus seedlings

This study examines the influence of the growth promoter, lepidimoic acid, on the level of an important cytosolic signal metabolite, fructose 2,6-bisphosphate (Fru-2,6-P2), which can activate pyrophosphatedependent:phosphofructokinase (PFP, EC 2.7.1.90), and on glycolytic metabolism in Amaranthus caudatus seedlings. Fru-2,6-P2 concentrations were respectively increased by approximately 2-, 3- and 4-fold when the seedlings were treated with 0.3, 3 and 30 mM lepidimoic acid. Exogenous lepidimoic acid also affected levels of glycolytic intermediates in the seedlings. The increase in fructose 1,6-bisphosphate and decreases in fructose 6-phosphate and glucose 6-phosphate were found in response to the elevated concentration of lepidimoic acid. These results suggest that lepidimoic acid may affect glycolytic metabolism in the Amaranthus seedlings by increasing the activity of PFP due to increasing level of Fru-2,6-P2.     

Ribose 1,5-bisphosphate is a putative regulator of fructose 6-phosphate/fructose 1,6-bisphosphate cycle in liver

6-Phosphofructo-1-kinase and fructose-1,6-bisphosphatase are rate-limiting enzymes for glycolysis and gluconeogenesis respectively, in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver. The effect of ribose 1,5-bisphosphate on the enzymes was investigated. Ribose 1,5-bisphosphate synergistically relieved the ATP inhibition and increased the affinity of liver 6-phosphofructo-1-kinase for fructose 6-phosphate in the presence of AMP. Ribose 1,5-bisphosphate synergistically inhibited fructose-1,6-bisphosphatase in the presence of AMP. The activating effect on 6-phosphofructo-1-kinase and the inhibitory effect on fructose-1,6-bisphosphatase suggest ribose 1,5-bisphosphate is a potent regulator of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver.     

Endotoxin increases the liver fructose 2,6-bisphosphate concentration in fasted rats

Following endotoxin administration to fasted rats, the liver fructose 2,6-bisphosphate level is significantly increased within 1 hr, is elevated 2.3-fold by 3 hrs, and remains elevated 2 to 3-fold for at least 24 hrs. This increase in the potent allosteric activator of phosphofructokinase occurs when there is no change in the liver Glc 6-P, glycogen or cAMP concentrations, or in the activities of phosphoenolpyruvate carboxykinase or pyruvate kinase. The increase in fructose 2,6-bisphosphate concentration accounts for the increased phosphofructokinase activity previously observed in hepatocytes isolated 18 hours following endotoxin administration to rats (1). By stimulating the phosphofructokinase/Fru 1,6-bisphosphate cycle in the direction of glycolysis, fructose 2,6-bisphosphate is likely the factor responsible for decreased gluconeogenesis in endotoxemia.     

Effects of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate on phosphofructokinase from chicken erythrocytes

1. Phosphofructokinase (EC 2.7.1.11) from chicken erythrocytes is activated by fructose 2,6-bisphosphate, glucose 1,6-bisphosphate and AMP, and it is inhibited by 2,3-bisphosphoglycerate and inositol hexaphosphate. 2. The stimulatory effects produced by the two bisphosphorylated hexoses are additive and the effects produced by fructose 2,6-bisphosphate and by AMP are synergistic. 3. The activatory effect produced by fructose 2,6-bisphosphate is counteracted by fructose 1,6-bisphosphate. 4. The inhibition produced by both 2,3-bisphosphoglycerate and inositol hexaphosphate is released by fructose 2,6-bisphosphate. 5. It is concluded that, like phosphofructokinase from mammalian tissues, the enzyme from chicken erythrocytes can be modulated by the relative concentrations of those metabolites.     

Regulation of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by enzyme phosphorylation and sn-glycerol 3-phosphate

The regulation of the Fru-6-P/Fru-2,6-P2 cycle by the cooperation of allosteric and covalent mechanisms was investigated in a reconstituted enzyme system under in vitro conditions. Phosphorylation of the bifunctional enzyme exerts a much stronger effect than sn-glycerol 3-phosphate in lowering the quasi-stationary concentration of fructose 2,6-bisphosphate and in increasing the critical concentration of the fructose phosphates, respectively. However, sn-glycerol 3-phosphate is able to strongly amplify the decrease of the quasi-stationary concentration of fructose 2,6-bisphosphate due to phosphorylation. The experiments can be described by a mathematical model involving rate equations for the dephosphorylated and the phosphorylated PFD-2 and FBPase-2. The results are compared with data from the literature obtained under in vivo conditions.     

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.     

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.     

The zonation of liver and the distribution of fructose 2,6-bisphosphate in rat liver

Livers of starved rats refed for 2 h were perfused in situ by a modification of the dual digitonin pulse technique of Quistorff and Grunnet (Quistorff, B., and Grunnet, N. (1987) Biochem. J. 243, 87-95). A pulse of digitonin (2 mg/ml) was infused first antegrade through the portal vein followed retrograde through the vena cava, or in reverse order, 13 mg of digitonin per zone. Microscopic examination showed that this procedure permeabilized the periportal and perivenous zones of the liver without overlap, with a narrow unaffected band of hepatocytes between the zones. The distribution pattern between periportal and perivenous zones ratio for alanine transaminase, lactate hydrogenase, fructose-1,6-bisphosphatase, and phosphoenolpyruvate carboxykinase ranged from 1.5 to 3. Glucokinase activity was higher in the perivenous zone (periportal/perivenous ratio of 0.7) and glutamine synthetase was exclusively present in that zone. Fructose 2,6-bisphosphate concentration was nearly equal in the two zones.     

Fructose 2,6-bisphosphate in rat erythrocytes. Inhibition of fructose 2,6-bisphosphate synthesis and measurement by glycerate 2,3-bisphosphate.

The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.     

Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression

The ability to overexpress6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase(PFK-2)/(FBPase-2) or a truncated form of the enzyme with only thebisphosphatase domain allowed us to analyze the relative role of thekinase and the bisphosphatase activities in regulating fructose2,6-bisphosphate (Fru-2,6-P₂) concentration and toelucidate their differential metabolic impact in epithelial Mv1Lucells. The effect of overexpressing PFK-2/FBPase-2 resulted in a smallincrease in the kinase activity and in the activity ratio of thebifunctional enzyme, increasing Fru-2,6-P₂ levels, butthese changes had no major effects on cell metabolism. In contrast,expression of the bisphosphatase domain increased the bisphosphataseactivity, producing a significant decrease in Fru-2,6-P₂ concentration. The fall in the bisphosphorylated metabolite correlated with a decrease in lactate production and ATP concentration, as well asa delay in cell cycle. These results provide support for Fru-2,6-P₂ as a regulator of glycolytic flux and point outthe role of glycolysis in cell cycle progression.

     

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.