Overexpression of ubiquitous 6-phosphofructo-2-kinase in the liver of transgenic mice results in weight gain

Fructose 2,6-bisphosphate (Fru-2,6-P₂) is an important metabolite that controls glycolytic and gluconeogenic pathways in several cell types. Its synthesis and degradation are catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2). Four genes, designated Pfkfb1-4, codify the different PFK-2 isozymes. The Pfkfb3 gene product, ubiquitous PFK-2 (uPFK-2), has the highest kinase/bisphosphatase activity ratio and is associated with proliferation and tumor metabolism. A transgenic mouse model that overexpresses uPFK-2 under the control of the phosphoenolpyruvate carboxykinase promoter was designed to promote sustained and elevated Fru-2,6-P₂ levels in the liver. Our results demonstrate that in diet-induced obesity, high Fru-2,6-P₂ levels in transgenic livers caused changes in hepatic gene expression profiles for key gluconeogenic and lipogenic enzymes, as well as an accumulation of lipids in periportal cells, and weight gain.     

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.

     

Effect of thiamin on the content of fructose 2,6-bisphosphate in Euglena

• 1.1. Thiamin deprived Euglena cells contained twice as high a concentration of Fru-2,6-P₂ in sufficient cells and the rate of the uptake of glucose from the medium was twice as great as that found in sufficient cells, indicating that Fru-2,6-P₂ is responsible for the glycolysis.
• 2.2. Moreover, incubation of thiamin deprived cells with increasing thiamin concentrations caused a decrease of Fru-2,6-P₂. The activity of Fru-6-P, 2-kinase was affected by thiamin and the activity of PFK-2 in thiamin deprived cells was 2.3 times greater than that observed in sufficient cells.
• 3.3. Incubating thiamin-deficient cells in a thiamin-containing medium, the activity of Fru-6-P, 2-kinase decreased rapidly and became the same activity as that found in thiamin sufficient cells.
• 4.4. In contrast, the two thiamin dependent 2-OGDC and PNOR activities showed a reverse relationship, being higher in thiamin-sufficient cells and lower in thiamin deficient cells.
• 5.5. We concluded that the carbohydrate metabolism of Euglena is regulated by Fru-2,6-P₂ through an intracellular concentration of thiamin based on the present evidence.

     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

Regulation of the futile cycle of fructose phosphate in sea mussel

Carbohydrate metabolism in mussels shows two phases separated seasonally. During summer and linked to food supply, carbohydrates, mainly glycogen, are accumulated in the mantle tissue. During winter, mantle glycogen decreases concomitantly with an increase in triglyceride synthesis. In spring, after spawning, the animals go in to metabolic rest until the beginning of a new cycle. This cycle is regulated by the futile cycle of fructose phosphate that implicates PFK-1 and FBPase-1 activities. These enzymes and the bifunctional PFK-2/FBPase-2 that regulates the Fru-2,6-P₂ levels, are seasonally modulated by covalent phosphorylation/dephosphorylation mechanisms, as a response to unknown factors. The futile cycle of the fructose phosphates also controls the transition from physiological aerobiosis to hypoxia. The process is independent of the phosphorylation state. In this sense, a pH decrease triggers a small Pasteur effect during the first 24 h of aerial exposure. Variations in the concentration of Fru-2,6-P₂ and AMP are the sole factor responsible for this effect. Longer periods of hypoxia induce a metabolic depression characterized by a decrease in Fru-2,6-P₂ which is hydrolyzed by drop in the pH. In this review, the authors speculate on the two regulation processes.     

Characterization of a new liver- and kidney-specific pfkfb3 isozyme that is downregulated by cell proliferation and dedifferentiation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P₂), a signalling molecule that controls the balance between glycolysis and gluconeogenesis in several cell types. Four genes, designated Pfkfb1-4, code several PFK-2 isozymes that differ in their kinetic properties, molecular masses, and regulation by protein kinases. In rat tissues, Pfkfb3 gene accounts for eight splice variants and two of them, ubiquitous and inducible PFK-2 isozymes, have been extensively studied and related to cell proliferation and tumour metabolism. Here, we characterize a new kidney- and liver-specific Pfkfb3 isozyme, a product of the RB2K3 splice variant, and demonstrate that its expression, in primary cultured hepatocytes, depends on hepatic cell proliferation and dedifferentiation. In parallel, our results provide further evidence that ubiquitous PFK-2 is a crucial isozyme in supporting growing and proliferant cell metabolism.     

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P₂) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P₂ is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P₂ was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P₂ in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P₂, respectively) were sufficient to account for the highest observed rates of Fru-2,6-P₂ metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P₂ level. Variation in the level of Fru-2,6-P₂ in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.     

Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves

Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P₂ase) were identified in leaves of corn (Zea mays), a C₄ plant. Fru-6-P,2K and Fru-2,6-P₂ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C₃ species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P₂ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P₂ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C₄ Fru-6-P,2K and Fru-2,6-P₂ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.     

Studies on the entry of fructose-2,6-bisphosphate into chloroplasts

The regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P₂) has an important function in controlling the intermediary carbon metabolism of leaves. Fru-2,6-P₂ controls two cytosolic enzymes involved in the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate (fructose-1,6-bisphosphatase and pyrophosphate, fructose-6-phosphate 1-phosphotransferase) and thereby controls the partitioning of photosynthate between sucrose and starch. It has been demonstrated that Fru-2,6-P₂ is present mainly in the cytosol. Here we present evidence that Fru-2,6-P₂ can be taken up by isolated intact chloroplasts but at a very slow rate (about 0.01 micromoles per milligram of chlorophyll per hour). This uptake is time and concentration dependent and is inhibited by PPi. When provided a physiological concentration of Fru-2,6-P₂ (10 micromolar), chloroplasts accumulated up to 0.6 micromolar Fru-2,6-P₂ in the stroma. Elevated plastid Fru-2,6-P₂ levels had no effect on overall photosynthetic rates of isolated chloroplasts. The results indicate that, while Fru-2,6-P₂ enters isolated chloroplasts at a sluggish rate, caution should be exercised in ascribing physiological importance to effects of Fru-2,6-P₂ on chloroplast enzymes.     

Photosynthetic carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing decreased amounts of fructose 2,6-bisphosphate

The aim of this work was to examine the role of fructose 2,6-bisphosphate (Fru-2,6-P₂) in photosynthetic carbon partitioning. The amount of Fru-2,6-P₂ in leaves of tobacco (Nicotiana tabacum L. cv. Samsun) was reduced by introduction of a modified mammalian gene encoding a functional fructose-2,6-bisphosphatase (EC 3.1.3.46). Expression of this gene in transgenic plants reduced the Fru-2,6-P₂ content of darkened leaves to between 54% and 80% of that in untransformed plants. During the first 30 min of photosynthesis sucrose accumulated more rapidly in the transgenic lines than in the untransformed plants, whereas starch production was slower in the transgenic plants. On illumination, the proportion of ¹⁴CO₂ converted to sucrose was greater in leaf disks of transgenic lines possessing reduced amounts of Fru-2,6-P₂ than in those of the control plants, and there was a corresponding decrease in the proportion of carbon assimilated to starch in the transgenic lines. Furthermore, plants with smaller amounts of Fru-2,6-P₂ had lower rates of net CO₂ assimilation. In illuminated leaves, decreasing the amount of Fru-2,6-P₂ resulted in greater amounts of hexose phosphates, but smaller amounts of 3-phosphoglycerate and dihydroxyacetone phosphate. These differences are interpreted in terms of decreased inhibition of cytosolic fructose-1,6-bisphosphatase resulting from the lowered Fru-2,6-P₂ content. The data provide direct evidence for the importance of Fru-2,6-P₂ in co-ordinating chloroplastic and cytosolic carbohydrate metabolism in leaves in the light. Received: 8 February 2000 / Accepted: 25 April 2000     

Regulatory properties of 6-phosphofructo-1-kinase of the mid-gut of the grasshopper,Poekilocerus bufonius

The fine structure of the mid-gut of Poekilocerus bufonius has been examined and three types of epithelial cells were identified; normal epithelial cells with their apical part possessing well developed microvilli, goblet-like cells containing myelin-like figures and the small basal cells with small and round nuclei, nidi. The regulation of 6-phosphofructo-1-kinase (PFK-1) prepared from the mid-gut of the grasshopper, Poekilocerus bufonius, was studied. Mid-gut PFK-1 displayed cooperativity with respect to fructose-6-phosphate at pH 7.0, and the enzyme was inhibited by high concentrations of ATP. The affinity of the enzyme for fructose-6-phosphate was increased by fru-2,6-P₂ whereas the inhibition of the enzyme by high concentrations of ATP was relieved by fru-2,6-P₂. The activity of mid-gut PFK-1 was highly stimulated in a simultaneous presence of low concentrations of fru-2,6-P₂ and AMP. ADP, AMP and c-AMP were all shown to be activators of the mid-gut PFK-1 with AMP being the greatest effector. The enzyme was not inhibited by citrate either in the presence of low or high concentrations of ATP. These results suggest that the PFK-1 of the mid-gut of the grasshopper is highly regulated with positive stimulators, specially fru-2,6-P₂, whereas the enzyme is not regulated by citrate or glucose-1,6-bisphosphate.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000     

Fructose-2,6-bisphosphate in rat mesenteric lymph nodes

1. The fructose-2,6-bisphosphate (Fru-2,6-P2) content of mesenteric lymph nodes was measured in rats. 2. The effects of Fru-2,6-P2 on the activity of 6-phosphofructo-1-kinase (PFK-1) from rat mesenteric lymph nodes were also studied. 3. The affinity of the enzyme for fructose-6-phosphate was increased by Fru-2,6-P2 whereas the inhibition of the enzyme with high concentrations of ATP was released by Fru-2,6-P2. 4. The activity of lymphocyte PFK-1 was highly stimulated in a simultaneous presence of low concentrations of AMP and Fru-2,6-P2. 5. These results show that rat lymphocyte PFK-1 is highly regulated with Fru-2,6-P2 which means that glycolysis in rat lymphocytes is controlled by Fru-2,6-P2.     

Citrate inhibition of rat-kidney cortex phosphofructokinase

The regulatory properties of citrate on the activity of phosphofructokinase (PFK) purified from rat-kidney cortex has been studied. Citrate produces increases in the K_0.5 for Fru-6-P and in the Hill coefficient as well as a decrease in the V_max of the reaction without affecting the kinetic parameters for ATP as substrate. ATP potentiates synergistically the effects of citrate as an inhibitor of the enzyme. Fru-2,6-P₂ and AMP at concentrations equal to K_a were not able to completely prevent citrate inhibition of the enzyme. Physiological concentrations of ATP and citrate produce a strong inhibition of renal PFK suggesting that may participate in the control of glycolysisin vivo.Abbreviations PFK 6-Phosphofructo-1-kinase (EC 2.7.1.11) - Fru-6-P Fructose 6-phosphate - Fru-2,6-P₂ Fructose 2,6-bisphosphate     

Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P₂), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P₂. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (K _a) of spinach leaf PFP for Fru-2,6-P₂ in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the K _a of spinach leaf PFP for Fru-2,6-P₂ was up to 2000-fold greater than that measured in the optimised assay and V _max decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P₂). When assayed under optimum conditions in the gluconeogenic direction, the K _a of spinach leaf PFP for Fru-1,6-P₂ was approximately 50 μM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased K _a up to 25-fold, and decreased V _max by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the K _a of PFP for Fru-2,6-P₂ to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P₂ levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the K _a of PFP for Fru-1,6-P₂ to an extent that activation of PFP by this compound is unlikely to be physiologically relevant. Received: 21 July 2000 / Accepted: 15 September 2000     

Enzymatic preparation of high-specific-activity β-d-[6,6′-H]fructose-2,6-bisphosphate: Application to a sensitive assay for fructose-2,6-bisphosphatase

β-d-Fructose-2,6-bisphosphate (Fru-2,6-P₂) is an important regulator of eukaryotic glucose homeostasis, functioning as a potent activator of 6-phosphofructo-1-kinase and inhibitor of fructose-1,6-bisphosphatase. Pharmaceutical manipulation of intracellular Fru-2,6-P₂ levels, therefore, is of interest for the treatment of certain diseases, including diabetes and cancer. [2-³²P]Fru-2,6-P₂ has been the reagent of choice for studying the metabolism of this effector molecule; however, its short half-life necessitates frequent preparation. Here we describe a convenient, economical, one-pot enzymatic preparation of high-specific-activity tritium-labeled Fru-2,6-P₂. The preparation involves conversion of readily available, carrier-free d-[6,6′-³H]glucose to [6,6′-³H]Fru-2,6-P₂ using hexokinase, glucose-6-phosphate isomerase, and 6-phosphofructo-2-kinase. The key reagent in this preparation, bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from human liver, was produced recombinantly in Escherichia coli and purified in a single step using an appendant C-terminal hexa-His affinity tag. Following purification by anion exchange chromatography using triethylammonium bicarbonate as eluant, radiochemically pure [6,6′-³H]Fru-2,6-P₂ having a specific activity of 50 Ci/mmol was obtained in yields averaging 35%. [6,6′-³H]Fru-2,6-P₂ serves as a stable, high-specific-activity substrate in a facile assay capable of detecting fructose-2,6-bisphosphatase in the range of 10⁻¹⁴ to 10⁻¹⁵ mol, and it should prove to be useful in many studies of the metabolism of this important biofactor.     

Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters.

Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms.     

Central Cavity of Fructose-1,6-bisphosphatase and the Evolution of AMP/Fructose 2,6-bisphosphate Synergism in Eukaryotic Organisms

The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P₂) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P₂ synergism in pFBPase is absent in eFBPase. Fru-2,6-P₂ induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P₂ synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser⁴⁵ → His substantially fills the central cavity of pFBPase, and the triple mutation Ser⁴⁵ → His, Thr⁴⁶ → Arg, and Leu¹⁸⁶ → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P₂ on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P₂ exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P₂ complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P₂ synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P₂ synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.     

Reduced Cardiac Fructose 2,6 Bisphosphate Increases Hypertrophy and Decreases Glycolysis following Aortic Constriction

This study was designed to test whether reduced levels of cardiac fructose-2,6-bisphosphate (F-2,6-P₂) exacerbates cardiac damage in response to pressure overload. F-2,6-P₂ is a positive regulator of the glycolytic enzyme phosphofructokinase. Normal and Mb transgenic mice were subject to transverse aortic constriction (TAC) or sham surgery. Mb transgenic mice have reduced F-2,6-P₂ levels, due to cardiac expression of a transgene for a mutant, kinase deficient form of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) which controls the level of F-2,6-P₂. Thirteen weeks following TAC surgery, glycolysis was elevated in FVB, but not in Mb, hearts. Mb hearts were markedly more sensitive to TAC induced damage. Echocardiography revealed lower fractional shortening in Mb-TAC mice as well as larger left ventricular end diastolic and end systolic diameters. Cardiac hypertrophy and pulmonary congestion were more severe in Mb-TAC mice as indicated by the ratios of heart and lung weight to tibia length. Expression of α-MHC RNA was reduced more in Mb-TAC hearts than in FVB-TAC hearts. TAC produced a much greater increase in fibrosis of Mb hearts and this was accompanied by 5-fold more collagen 1 RNA expression in Mb-TAC versus FVB-TAC hearts. Mb-TAC hearts had the lowest phosphocreatine to ATP ratio and the most oxidative stress as indicated by higher cardiac content of 4-hydroxynonenal protein adducts. These results indicate that the heart’s capacity to increase F-2,6-P₂ during pressure overload elevates glycolysis which is beneficial for reducing pressure overload induced cardiac hypertrophy, dysfunction and fibrosis.