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.     

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.     

Development of gluconeogenesis from different substrates in newborn rabbit hepatocytes

The rates of glucose production from various substrates entering gluconeogenesis at different steps were investigated in hepatocytes isolated from term-fetus and newborn rabbits fasted during the first 2 days of life. The data were compared to the rate of glucose production measured in hepatocytes from young rabbits (50-60 days) starved for 48 h. The net production of glucose from substrates (lactate, pyruvate, propionate, alanine) entering gluconeogenesis below phosphoenolpyruvate was very low at birth and increased during the first day of life, in relation with an increased cytosolic phosphoenolpyruvate carboxykinase activity. The net production of glucose from precursors entering gluconeogenesis at the level of triose phosphates (dihydroxyacetone, fructose) was low at birth but a maximal capacity for gluconeogenesis was reached within 6 h after birth. This enhanced gluconeogenic capacity was associated with a fall in hepatic fructose 2,6-bisphosphate concentration and a reduced glycolytic flux. In contrast, a high glucose production from galactose was already present at birth and did not rise at 24 or 48 h after delivery. These results suggest that the development of gluconeogenic capacity in hepatocytes isolated from newborn rabbit is dependent upon two factors, a decrease in the F2,6-P2 concentration which reduces the glycolytic flux and an increase in the activity of cytosolic phosphoenolpyruvate carboxykinase.     

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.     

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate levels in erythrocytes with high and low 2,3-bisphosphoglycerate content during postnatal development

In rabbit and sheep erythrocytes the concentrations of 2,3-bisphosphoglycerate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3-bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios.     

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.     

Phosphofructokinase in rat lung during perinatal development: characterization of subunit composition and regulation by fructose 2,6-bisphosphate and glucose 1,6-bisphosphate

The subunit composition of phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) was studied in rat lung during perinatal development. No change in subunit composition during this period was observed. The three subunits of phosphofructokinase (L, M and C) were present in a ratio of approx. 65:25:10, respectively. In addition the levels of two effectors of phosphofructokinase were determined in rat lung during perinatal development: glucose 1,6-bisphosphate and fructose 2,6-bisphosphate. Until day 20 of gestation (term is 22 days) the glucose 1,6-bisphosphate level remains relatively constant (approx. 0.55 mumol/g protein), decreases before birth and increases sharply up to 1.04 mumol/g protein 2 days after birth. The amount of fructose 2,6-bisphosphate in rat lung shows a different developmental profile. A small peak is shown at day 17 of gestation whereas a larger peak up to 36.4 nmol/g protein is shown at days 20 and 21 of gestation. The time of maximal fructose 2,6-bisphosphate content corresponds with the time of glycogen breakdown and acceleration of surfactant synthesis in prenatal rat lung. Both glucose 1,6-bisphosphate and fructose 2,6-bisphosphate stimulate lung phosphofructokinase. Half maximal stimulations occur in the range of 24.1-70.9 microM glucose 1,6-bisphosphate and 0.17-0.34 microM fructose 2,6-bisphosphate.     

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.     

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.     

Changes in key regulatory enzymes of hepatic carbohydrate metabolism after glucose loading of starved rats

When glucose was given to starved rats there was an increase in both 6-phosphofructo 2-kinase and pyruvate kinase activity and a decrease in fructose 2,6-bisphosphatase activity 30 min and 60 min later. These changes were accompanied by an increase in glycogen deposition and by modest, but significant increases in fructose 2,6-bisphosphate levels at the same time. Metabolite measurements indicated that flux through 6-phosphofructo 1-kinase and pyruvate kinase were increased. These results suggest that although glycogen deposition may occur via the gluconeogenic pathway, glycolysis is activated at the same time by changes in the phosphorylation state of key regulatory enzymes as well as by the small rise in fructose 2,6-bisphosphate.     

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.     

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.     

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

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

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.     

Fructose 2,6-bisphosphate in livers of genetically obese rats.

Livers of genetically obese (fa/fa) rats, starved for 24 h, contained more fructose 2,6-bisphosphate, glucose 6-phosphate, fructose 6-phosphate and glycogen, and more pyruvate kinase and phosphofructokinase 2 activities, than livers of control lean rats. These changes may be explained in terms of cyclic AMP concentration, which was decreased in livers of obese starved rats.     

On the mechanism of the glucose-induced ATP catabolism in ascites tumour cells and its reversal by pyruvate.

Addition of glucose to Ehrlich-Landschütz ascites tumour cells preincubated for 30-60 min in phosphate-buffered Krebs-Ringer salt solution ("starved cells") resulted within 1-2 min in an approx. 90% decline of their ATP content and a massive accumulation of fructose 1,6-bisphosphate. These alterations, which took place under both aerobic and anaerobic conditions, were followed by a gradual spontaneous recovery with restoration of normal ATP and fructose 1,6-bisphosphate values. The transient derangement of the energy metabolism after glucose addition to starved ascites tumour cells by preventable by simultaneous addition of pyruvate or 2-oxobutyrate, or by preincubating the cells in the presence of glucose. The protective effect of pyruvate was duplicated by addition of phenazine methosulphate or NAD+ to the incubation medium. The data seem to warrant the conclusion that the glucose-induced ATP depletion is determined by a blockade of glycolysis at the stage of glyceraldehyde phosphate dehydrogenase caused by the failure of the cells to oxidize the NADH produced in the same reaction. The continued unrestrained action of 6-phosphofructokinase results in accumulation of fructose 1,6-bisphosphate, which constitutes a trap for the high-energy phosphate bonds of ATP. The primary metabolic disturbance appears to consist of a transient inhibition of pyruvate kinase with the resultant inability of the cells to maintain an unimpaired supply of pyruvate, as required for the lactate dehydrogenase-mediated oxidation of NADH. The regulatory mechanism underlying this phenomenon is discussed.     

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 inhibition of phosphoglucomutase

Fructose 2,6-bisphosphate inhibits phosphoglucomutase noncompetitively with respect to the cofactor glucose 1,6-bisphosphate. Previous studies from our laboratory had shown that phosphoglucomutase was activated by fructose 2,6-bisphosphate in the absence of added glucose 1,6-bisphosphate. The fructose 2,6-bisphosphate activation previously reported was due to the presence of glucose 1,6-bisphosphate in the commercial preparation of fructose 2,6-bisphosphate.     

Effects of fructose 2,6-bisphosphate on phosphoglucomutase from plants

Fructose 2,6-bisphosphate affects phosphoglucomutase from plant and animal sources in a similar way. As previously found with rabbit muscle phosphoglucomutase, fructose 2,6-bisphosphate cannot substitute for glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by phosphoglucomutase from potato tubers, pea seeds, and string-beans. In the presence of glucose 1,6-bisphosphate, fructose 2,6-bisphosphate inhibits phosphoglucomutase from potato tubers. Activation of phosphoglucomutase from plant sources by fructose 2,6-bisphosphate reported by others was probably due to contamination of the commercial preparation of fructose 2,6-bisphosphate by glucose 1,6-bisphosphate.