Oscillations and trigger phenomena in fructose-2,6-bis-phosphate metabolism. A mathematical model

A mathematical model describing metabolism of fructose-2,6-bisphosphate (F2, 6P2), which is a powerful mediator in glycolysis, is investigated. The model takes into account inhibitory effect of F2, 6P2 and fructose-6-phosphate (F6P) on protein kinase, which phosphorylates the bifunctional enzyme fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase. Such a mechanism of enzyme chemical modification in the presence of F2, 6P outflow from the F6P in equilibrium with F2, 6P2 cycle, caused by nonspecific phosphatases, can display trigger phenomena and sustained oscillations in F2, 6P2 metabolism and in the whole glycolytic system. The results obtained suggest that earlier models of the generation of glycolytic oscillations should be revised.     

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The role of surface loop basic residues in substrate binding to the fructose-2,6-bisphosphatase domain.

Lys-356 has been implicated as a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Li, L., Lin, K., Correia, J., and Pilkis, S. J. (1992) J. Biol. Chem. 267, 16669-16675). To ascertain whether the three other basic residues (Arg-352, Arg-358, and Arg-360), which are located in a surface loop (residues 331-362) which contains Lys-356, are important in substrate binding, these arginyl residues were mutated to Ala, and each arginyl mutant was expressed in Escherichia coli and purified to homogeneity. The far UV circular dichroism spectra of the mutants were identical to that of the wild-type enzyme. The kinetic parameters of 6-phosphofructo-2-kinase of the mutants revealed only small changes. However, the Km for fructose 2,6-bisphosphate, Ki for fructose 6-phosphate, and Ka for inorganic phosphate of fructose-2,6-bisphosphatase for Arg352Ala were, respectively, 2,800-, 4,500-, and 1,500-fold higher than those for the wild-type enzyme, whereas there was no change in the maximal velocity or the Ki for inorganic phosphate. The Km for fructose 2,6-bisphosphate and Ki for inorganic phosphate of Arg360Ala were 10- and 12-fold higher, respectively, than those of the wild-type enzyme, whereas the maximal velocity and Ki for fructose 6-phosphate were unchanged. In addition, substrate inhibition was not observed with Arg352Ala and greatly reduced with Arg360Ala. The properties of the Arg358Ala mutant were identical to those of the wild-type enzyme. The results demonstrate that in addition to Lys-356, Arg-352 is another critical residue in fructose-2,6-bisphosphatase for binding the C-6 phospho group of fructose 2,6-bisphosphate and that Arg-360 binds the C-2 phospho group of fructose 2,6-bisphosphate in the phosphoenzyme.fructose 2,6-bisphosphate complex. The results also provide support for Arg-352, Lys-356, and Arg-360 constituting a specificity pocket for fructose-2,6-bisphosphatase.     

Cyclic organization of the carbohydrate metabolism in Sinorhizobium meliloti.

The pathways of polysaccharide biosynthesis were investigated in cells of Sinorhizobium meliloti (strain Su47) using a stable isotope approach. The isotopic labeling of the periplasmic beta-1,2-glucans synthesized from glucose labeled at various positions evidenced the involvement of catabolic pathways, namely the pentose-phosphate and Entner-Doudoroff pathways, into the early steps of polysaccharide synthesis. The exopolysaccharides produced at the same time had a labeling pattern similar to that of the beta-glucans, indicating similar early steps for both polysaccharides. The results emphasized a cyclic organization of the carbohydrate metabolism in S. meliloti, in which the carbons of the initial hexose were allowed to re-enter the catabolic pathways many times. The metabolic incidences of such metabolic topology are discussed.     

A role for fructose 2,6-bisphosphate in regulating carbohydrate metabolism in guard cells

Fructose 2,6-bisphosphate (Fru2,6P₂) appears to function as a regulator metabolite in glycolysis and gluconeogenesis in animal tissues, yeast, and the photosynthetic cells of leaves. We have investigated the role of Fru2,6P₂ in guard-cell protoplasts from Vicia faba L. and Pisum sativum L. (Argenteum mutant), and in epidermal strips purified by sonication from all cells except for the guard cells. Guard-cell protoplasts were separated into fractions enriched in cytosol and in chloroplasts by passing them through a nylon net, followed by silicone oil centrifugation. The cytosol contained a pyrophosphate: fructose 6-phosphate phosphotransferase (involved in glycolysis) which was strongly stimulated by Fru2,6P₂. A cytosolic fructose 1,6-bisphosphatase (a catalyst of gluconeogenesis) was inhibited by Fru2,6P₂. There was virtually no fructose 1,6-bisphosphatase activity in guard-cell chloroplasts of V. faba. It is therefore unlikely that the starch formed in these chloroplasts originates from imported triose phosphates or phosphoglycerate.

The level of Fru2,6P₂ in guard-cell protoplasts and epidermal strips was about 0.1 to 1 attomole per guard cell in the dark (corresponding to 0.05 to 0.5 nanomole per milligram chlorophyll) and increased three- to tenfold within 15 minutes in the light. Within the same time span, hexose phosphate levels in guard-cell protoplasts declined to approximately one-half, indicating that acceleration of glycolysis involved stimulation of reactions using hexose phosphates. The level of Fru2,6P₂ in guard cells appears to determine the direction in which carbohydrate metabolism proceeds.

     

Quasi-stationary concentrations of fructose-2,6-bisphosphate in the phosphofructokinase-2/fructose-2,6-bisphosphatase cycle

The cooperation of phosphofructokinase-2 and fructose-2,6-bisphosphatase is investigated. Experimentally derived rate laws of the kinase and bisphosphatase activities introduced into the respective differential equations permitted to describe the time evolution of fructose-2,6-bisphosphate to quasi-stationary levels. The two enzyme activities were found to exert strong temperature dependence. The quasi-stationary levels of fructose-2,6-bisphosphate, however, are independent on temperature.     

Differences in fructose-2,6-bisphosphate metabolism between sections of developing barley leaves

In order to study the regulation of carbohydrate metabolism in leaf tissue the activity of fructose-6-phosphate,2-kinase was determined in individual sections of developing primary leaves of barley. Activity was about 25-fold higher in the leaf tip than in the leaf sheath when measured on a fresh weight basis. There was a gradual increase in enzyme activity from the leaf base to the leaf tip. The higher activity of fructose-6-phosphate,2-kinase in the apical parts of the leaf was associated with higher levels of fructose-2,6-bisphosphate. This was especially pronounced when isolated leaf segments were treated with vanadate and kept in darkness. As compared to the kinase, little difference was observed in the fructose-2,6-bisphospatase activity among leaf sections. The significance of these patterns for regulation of carbohydrate metabolism in different tissues is discussed.     

The role of leptin in the regulation of carbohydrate metabolism

The hormone leptin is secreted from white adipocytes, and serum levels of leptin correlate with adipose tissue mass. Leptin was first described as acting on the satiety centre in the hypothalamus through specific receptors (ob-R) to restrict food intake and enhance energy expenditure. Leptin plays a crucial role in the maintenance of body weight and glucose homeostasis hrough central and peripheral pathways, including regulation of insulin secretion by pancreatic b cells. Leptin may also directly affect the metabolism and function of peripheral tissues. Leptin has been implicated in causing peripheral insulin resistance by attenuating insulin action, and perhaps insulin signalling, in various insulin-responsive cell types. Research has demonstrated a significant relationship between leptin and insulin, but the mechanisms underlying the changes of leptin induced by insulin, and vice versa, remain to be studied in more detail. Recent data provides convincing evidence that leptin has beneficial effects on glucose homeostasis in mouse models of insulin-deficient type 1 diabetes mellitus. Our study suggests that leptin could be used as an adjunct of insulin therapy in insulin-deficient diabetes, thereby providing an insight into the therapeutic properties of leptin as an anti-diabetic agent. Safety evaluation should include a careful assessment of the effects of this combination therapy on the counterregulatory response to hypoglycaemia. The role of leptin in alpha-cell function has not been studied in detail. Extensive studies will be needed to determine the long-term safety and efficacy of this therapy.     

Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain.

Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure. The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine. The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity. Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme. However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values. However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes. Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme. Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme. Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value. The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.     

Regulation of fructose-2,6-bisphosphate content in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. Regulation of fructose-2,6-bisphosphatase activity.

Fructose-2,6-bisphosphatase (FBPase-2) from the mantle tissue of the mussel Mytilus galloprovincialis shows a hyperbolic kinetic with a Km value (0.40 mM) for its substrate, that suggest that the "in vivo" Fru-2,6-P2 concentration is not a limiting factor for activity. The enzyme possesses an optimum pH for activity between 6 and 7 units, similar to the reached in mussel mantle during physiological hypoxia. The modulation of activity by the pH, and in addition, the positive effect of ATP are in keeping with the little decrease in concentration of the Fru-2,6-P2 that occurs during the first hour of hypoxia due to the valve closure.     

Increase of the glycolytic rate in human resting fibroblasts following serum stimulation. The possible role of the fructose-2,6-bisphosphate

We report that glycolysis in human quiescent fibroblasts stimulated by serum addition is increased, and that the changes of the metabolic route reflect the activity of the phosphofructokinase. A possible role of fructose-2,6-bisphosphate as a positive modulator of the key enzyme is proposed.     

Caveolae and the organization of carbohydrate metabolism in vascular smooth muscle

We have previously found that glycolysis and gluconeogenesis occur in separate "compartments" of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [1999] Am J Physiol Cell Physiol. 277:C1250-C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [2000] Am J Physiol Cell Physiol. 278:C803-C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane-associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non-caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with beta methyl-cyclodextrin (CD) and examined the metabolism of [2-(13)C]glucose (a glycolytic substrate) and [1-(13)C]fructose 1,6-bisphosphate (FBP, a gluconeogenic substrate in PCMV) using (13)C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2-(13)C]glucose to [2-(13)C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1-(13)C]FBP to [3-(13)C]lactate was increased by CD treatment. Alternatively, the increased [3-(13)C]lactate production may reflect changes in FBP uptake, since conversion of [1-(13)C]FBP to [3-(13)C]glucose was also elevated in CD-treated cells. Thus, a link between caveolar organization and metabolic organization may exist.     

Somatic embryogenesis in conifers: The role of carbohydrate metabolism

Summary Somatic embryogenesis represents a promising tool for mass propagation of elite genotypes of conifers. The efficiency of the technique strongly depends on cultivation conditions, with the exogenous saccharide supply being one of the most important factors. Different types and concentrations of saccharides have been empirically evaluated with respect to production of acceptable numbers and quality of somatic embryos for particular conifer species. Only a few recently published papers have focused on deeper studies of carbohydrate metabolism, enabling insight into the physiological background of the crucial effects of carbohydrates. Generally, saccharides are known to serve as carbon and energy sources, osmotic agents, stress protectants, and signal molecules in plants. This review collects and critically discusses the experimental data on exogenous saccharide supplies, resulting endogenous levels, and key enzyme activities obtained from the most thoroughly described genus Picea. In conclusion, it stresses the necessily to broaden the studies and consider the unltiple roles of saccharides during conifer somatic embryogenesis.     

The yeast FBP26 gene codes for a fructose-2,6-bisphosphatase.

Sequencing of an open reading frame 450 bp downstream from the yeast VPS35 gene revealed a putative peptide of 452 amino acids and 52.7 kDa. The predicted amino acid sequence has 45% identity with the 55-kDa subunit of the 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (EC 2.7.1.105/EC 3.1.3.46) from rat liver and 42% identity with 480 amino acids in the center of the recently reported 93.5-kDa subunit of yeast 6-phosphofructo-2-kinase (EC 2.7.1.105). The product of the new yeast gene is similar to the entire sequence of the bifunctional rat liver enzyme and, unlike yeast 6-phosphofructo-2-kinase, has the histidine residue essential for fructose-2,6-bisphosphatase activity. Extracts from a chromosomal null mutant strain, fbp26::HIS3, incubated in the presence of [2-32P]fructose 2,6-P2, lacked in autoradiograms the characteristic 56-kDa labeled band observed in wild-type. The same band was intensified 3-fold over wild-type level with the FBP26 gene introduced on multicopy in the fbp26::HIS3 background. A similar increase was found for fructose-2,6-bisphosphatase activity in the same extracts. The FBP26 gene did not cause detectable increase in 6-phosphofructo-2-kinase activity when introduced on multicopy in a pfk26::LEU2 mutant, indicating that its gene product is predominantly a fructose-2,6-bisphosphatase. Growth on glucose, fructose, galactose, pyruvate, and glycerol/lactate was not impaired in strains carrying the fbp26::HIS3 allele.     

A mathematical model of iron metabolism

A mathematical model of iron metabolism is presented. It comprises the following iron pools within the body: transferrin-bound iron in the plasma, iron in circulating red cells and their bone marrow precursors, iron in mucosal, parenchymal and reticuloendothelial cells. The control exerted by a hormone, called erythropoietin, on bone marrow utilization of iron for hemoglobin synthesis is taken into account. The model so obtained consists of a system of functional differential equations of retarded type. Most model parameters can be estimated from radiotracer experiments, others can be measured and numerical values can be assigned to the remaining ones making few reasonable assumptions according to the available physiological knowledge. Iron metabolism behavior under different therapeutical treatments was simulated. Model predictions were compared to experimental data collected in clinical routine.This work has been partially supported by C.N.R. (Italy) through grants N. 80.01227.07 and N. 81.00888.07     

The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

Critical switch of the metabolic fluxes by phosphofructo-2-kinase:fructose-2,6-bisphosphatase. A kinetic model

A kinetic model for the bifunctional enzyme, phosphofructo-2-kinase:fructose-2,6-bisphosphatase, is analysed by application of the graph-theoretical method, considering comparable levels for all participants. Certain elementary reactions, distributed on the enzyme surface, are considered to be co-ordinated in a single conformational transition (a model of parallel molecular operations). The method allows us to identify in the kinetic scheme its destabilising sub-scheme as a branched cycle of elementary reactions. Under certain conditions this sub-scheme induces critical phenomena (bistability or oscillations). The computer calculations for the estimated parameter values fit well the experimental observations for this system. The model explains the periodic or bistable counterphase changes of the two opposing activities of this enzyme, observed after glucose perfusion of rat hepatic enzyme samples, and predicts drastic critical changes in kinetic behaviour induced by small external signals. The model also shows the necessity of the phosphoryl intermediate in the mechanism of the bisphosphatase for the critical kind of kinetic behaviour.     

Inhibition of key enzymes of carbohydrate metabolism in regenerating mouse liver by ascorbic acid.

The effect of ascorbic acid on the key enzymes of carbohydrate metabolism e.g. hexokinases, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and malic enzyme was determined in regenerating mouse liver. All the enzymes showed a significant increase in the activity during regeneration. Ascorbic acid reduced the activities of the enzymes in regenerating liver. A decrease in liver weight in ascorbic acid treated animals may be correlated with its effect on these enzymes as glycolytic pathway is the main source of energy required by the dividing cells.