Palmitate inhibits liver glycolysis. Involvement of fructose 2,6-bisphosphate in the glucose/fatty acid cycle.

In hepatocytes from overnight-fasted rats incubated with glucose, palmitate decreased the production of lactate, the detritiation of [2-3H]- and [3-3H]-glucose, and the concentration of fructose 2,6-bisphosphate. Similarly, perfusion of hearts from fed rats with beta-hydroxybutyrate resulted in an inhibition of the detritiation of [3-3H]glucose and a fall in fructose 2,6-bisphosphate concentration. This fall could result from an increase in citrate (hepatocytes and heart) and sn-glycerol 3-bisphosphate concentration. It is suggested that a fall in fructose 2,6-bisphosphate concentration participates in the inhibition of glycolysis by fatty acids and ketone bodies.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

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.     

Fructose 2,6-bisphosphate and insulin stimulation of glycolysis in 3T3-L1 adipocytes

1. Insulin is able to stimulate lactate production and to enhance fructose 2,6-bisphosphate (Fru-2,6-P2) content in 3T3-L1 adipocytes. 2. Phorbol 12-myristate 13-acetate is more efficacious than insulin in rising Fru-2,6-P2 content and less effective in the stimulation of glycolysis. 3. 3T3-L1 adipocyte 6-phosphofructo-l-kinase appears to be very sensitive to exogenous Fru-2,6-P2. 4. Insulin treatment does not affect the maximum activity of 6-phosphofructo-1-kinase whereas it markedly increases the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The role of Fru-2,6-P2 in the insulin induced enhancement of glycolytic flux is discussed.     

Expression and regulation of 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase isozymes in white adipose tissue.

The aim of this work was to identify the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isozyme(s) present in white adipose tissue. Ion-exchange chromatography of PFK-2 from rat epididymal fat pads yielded an elution pattern compatible with the presence of both the L (liver) and M (muscle) isozymes. This was consistent with a study of the phosphorylation of the purified adipose tissue enzyme by cAMP-dependent protein kinase, by specific labelling of the preparation with [2-32P]fructose 2,6-bisphosphate and by reaction with antibodies. Characterization of the PFK-2/FBPase-2 mRNAs showed that mature adipocytes express the mRNA that codes for the L isozyme and the two mRNAs that code for the M isozyme. Preadipocytes expressed mRNA that codes for the M isozyme. Incubation of rat epididymal fat pads with adrenaline stimulated glycolysis but decreased fructose 2,6-bisphosphate concentrations without significant inactivation of PFK-2. These results support previous findings showing that fructose 2,6-bisphosphate is not involved in the adrenaline-induced stimulation of glycolysis in white adipose tissue.     

On the mechanism by which noradrenaline increases the activity of phosphofructokinase in isolated rat adipocytes.

We confirmed that, as reported by Sooranna & Saggerson [(1982) Biochem. J. 202, 753-758], the affinity of 6-phosphofructo-1-kinase (PFK) for fructose 6-phosphate in an adipocyte extract was increased after incubation of the cells in the presence of noradrenaline. The participation of fructose 2,6-bisphosphate in this kinetic modification could be excluded, because the noradrenaline effect persisted after extensive gel filtration of the extracts and also because the treatment did not cause any change in the concentration of fructose 2,6-bisphosphate in the adipocytes. Oleic acid was found to be another potent positive effector of PFK in an adipocyte extract, with a Ka of 10 microM. Its effect was synergistic with that of fructose 2,6-bisphosphate and AMP, and was counteracted by serum albumin. Palmitic acid had a similar effect. We conclude that the large increase in fatty acid concentration caused by noradrenaline treatment is an explanation for the activation of phosphofructokinase at low fructose 6-phosphate concentrations in an adipocyte extract.     

Fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase during liver regeneration.

Glycogen and fructose 2,6-bisphosphate levels in rat liver decreased quickly after partial hepatectomy. After 7 days the glycogen level was normalized and fructose 2,6-bisphosphate concentration still remained low. The 'active' (non-phosphorylated) form of 6-phosphofructo-2-kinase varied in parallel with fructose 2,6-bisphosphate levels, whereas the 'total' activity of the enzyme decreased only after 24 h, similarly to glucokinase. The response of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from hepatectomized rats (96 h) to sn-glycerol 3-phosphate and to cyclic AMP-dependent protein kinase was different from that of the enzyme from control animals and similar to that of the foetal isoenzyme.     

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.     

Phorbol 12,13-dibutyrate and mitogens increase fructose 2,6-bisphosphate in lymphocytes. Comparison of lymphocyte and rat-liver 6-phosphofructo-2-kinase

The influence of tumour promoters and growth factors on glycolysis and on fructose-2,6-bisphosphate concentration was studied in isolated mouse spleen lymphocytes and in purified B-cells. The intracellular concentration of fructose 2,6-bisphosphate and the rate of lactate release were increased 2-3-fold in spleen lymphocytes exposed to active phorbol esters, mitogenic lectins, interleukin 4 or lipopolysaccharide. The maximal effect was observed after 1 h of exposure. In these cells hexose 6-phosphates increased 2-fold and 6-phosphofructo-2-kinase activity remained unchanged after treatment with phorbol 12,13-dibutyrate or with lectins. Exposure of B-cells to phorbol 12,13-dibutyrate, interleukin 4 or lipopolysaccharide increased the glycolytic flux and the concentration of fructose 2,6-bisphosphate without relation to their mitogenic activity. Lymphocytes and rat liver 6-phosphofructo-2-kinase were partially purified using the same procedure. The lymphocyte enzyme was not inhibited by sn-glycerol 3-phosphate in contrast to the potent inhibition observed in liver. Treatment of both enzymes with the catalytic subunit of the cyclic-AMP-dependent protein kinase failed to inactivate 6-phosphofructo-2-kinase from lymphocytes. These differences suggest that lymphocytes and liver contain different forms of this enzyme.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of glucose and glucagon on the fructose 2,6-bisphosphate content of pancreatic islets and purified pancreatic B-cells. A comparison with isolated hepatocytes.

Glucose caused a sustained and dose-related increase in the fructose 2,6-bisphosphate content of isolated pancreatic islets, as well as of purified pancreatic B-cells. With isolated B-cells, the glucose saturation curve was sigmoidal and superimposable on that obtained with hepatocytes isolated from unfed rats. However, the response to glucose was notably faster in purified B-cells than in isolated hepatocytes. In contrast again with the situation prevailing in the liver, glucagon failed to decrease significantly the concentration of fructose 2,6-bisphosphate in either islets or purified B-cells. It is proposed that, in the process of glucose-stimulated insulin secretion, an early increase in fructose 2,6-bisphosphate formation may, by causing activation of 6-phosphofructo-1-kinase, allow glycolysis to keep pace with the rate of glucose phosphorylation.     

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.     

Fructose 2,6-bisphosphate and its phosphorothioate analogue. Comparison of their hydrolysis and action on glycolytic and gluconeogenic enzymes.

Purified chicken liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated either from fructose 2,6-bis[2-32P]phosphate or fructose 2-phosphoro[35S]thioate 6-phosphate. The turnover of the thiophosphorylated enzyme intermediate as well as the overall phosphatase reaction was four times faster than with authentic fructose 2,6-bisphosphate. Fructose 2-phosphorothioate 6-phosphate was 10-100-fold less potent than authentic fructose 2,6-bisphosphate in stimulating 6-phosphofructo-1-kinase and pyrophosphate:fructose 6-phosphate phosphotransferase, but about 10 times more potent in inhibiting fructose 1,6-bisphosphatase. The analogue was twice as effective as authentic fructose 2,6-bisphosphate in stimulating pyruvate kinase from trypanosomes.     

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.     

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.