Expression of mouse 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 mRNA alternative splice variants in hypoxia

Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) mRNA alternative splice variants was studied in different mouse tissues in hypoxic conditions in vivo. Significant increase of the expression of PFKFB-3 mRNA was observed in the mouse lungs, testes and brain in hypoxia. Several new alternative splice variants of PFKFB-3 mRNA were identified in the lung, testis, brain and skeletal muscle. They have different length and amino acid sequence of C-terminal regulatory part. However, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase catalytic domains were identical. Moreover, the expression of different alternative splice variants of PFKFB-3 mRNA has shown tissue specificity and different levels of induction in hypoxic conditions in vivo. Results of this investigation indicate a possible role of PFKFB-3 splice isoform in cell adaptation to hypoxic conditions.     

Insulin induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells

Fructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 microM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide.     

Effect of silver nanoparticles on the expression of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-2 mRNA and its alternative splice variants in different rat organs

Bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) is represented by several alternative splice variants and plays a significant role in the glycolysis regulation in the brain, lung, testis and heart cells. The expression of PFKFB-2 mRNA and its alternative splice variants in these rat vital organs after single intratracheal injection of silver nanoparticles was studied. It was shown that the expression of PFKFB-2 mRNA is significantly changed in different rat tissues under silver nanoparticles action. The effect of silver nanoparticles on the expression of PFKFB-2 mRNA was observed one day after its injection to animals. In 3 and 14 days the effect of silver nanoparticles was increased (in testes) or kept on the approximately same level (in other investigated tissues). The expression of PFKFB-2 mRNA in most tissues is returned to its control levels one year after the injection of silver nanoparticles to the rats. It was also shown that the expression of alternative splice variants of PFKFB-2 mRNA without functional activity of 6-phosphofructo-2-kinase is significantly increased in different tissues 1, 3 and 14 days after single injection of silver nanoparticles. The results of this investigation demonstrate clearly that silver nanoparticles significantly affect the expression of PFKFB-2 mRNA on the alternative splicing level in different vital organs and show their effect on the important mechanisms of metabolism regulation in the cells on the level of key enzyme gene expression.     

Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1-4) expression in vivo

When oxygen becomes limiting, cells shift primarily to a glycolytic mode for generation of energy. A key regulator of glycolytic flux is fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric regulator of 6-phosphofructo-1-kinase (PFK-1). The levels of F-2,6-BP are maintained by a family of bifunctional enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB or PFK-2), which have both kinase and phosphatase activities. Each member of the enzyme family is characterized by their phosphatase:kinase activity ratio (K:B) and their tissue-specific expression. Previous work demonstrated that one of the PFK-2 isozyme genes, PFKFB-3, was induced by hypoxia through the hypoxia-inducible factor-1 (HIF-1) pathway. In this study we examined the basal and hypoxic expression of three members of this family in different organs of mice. Our findings indicate that all four isozymes (PFKFB-1-4) are responsive to hypoxia in vivo. However, their basal level of expression and hypoxia responsiveness varies in the different organs studied. Particularly, PFKFB-1 is highly expressed in liver, heart and skeletal muscle, with the highest response to hypoxia found in the testis. PFKFB-2 is mainly expressed in the lungs, brain and heart. However, the highest hypoxia responses are found only in liver and testis. PFKFB-3 has a variable low basal level of expression in all organs, except skeletal muscle, where it is highly expressed. Most importantly, its hypoxia responsiveness is the most ample of all three genes, being strongly induced in the lungs, liver, kidney, brain, heart and testis. Further studies showed that PFKFB-1 and PFKFB-2 were highly responsive to hypoxia mimics such as transition metals, iron chelators and inhibitors of HIF hydroxylases, suggesting that the hypoxia responsiveness of these genes is also regulated by HIF proteins. In summary, our data demonstrate that PFK-2 genes are responsive to hypoxia in vivo, indicating a physiological role in the adaptation of the organism to environmental or localized hypoxia/ischemia.     

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression is regulated by diet composition and ration size in liver of gilthead sea bream, Sparus aurata

Modulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P(2)ase) gene expression by diet composition and ration size was studied in the liver of gilthead sea bream, Sparus aurata. From five different types of diet supplied to fish, those with either high carbohydrate/low protein or high carbohydrate/low lipid content stimulated 6PF-2-K/Fru-2,6-P(2)ase expression at the levels of mRNA, immunodetectable protein and kinase activity as well as promoting higher fructose-2,6-bisphosphate (Fru-2,6-P(2)) values. The expression of the bifunctional enzyme and Fru-2,6-P(2) levels showed also direct dependence on the quantity of diet supplied. These findings demonstrate for the first time nutritional regulation of 6PF-2-K/Fru-2,6-P(2)ase at mRNA level by diet composition and ration size and suggest that the carnivorous fish S. aurata can adapt its metabolism, by stimulation of liver glycolysis, to partial substitution of protein by carbohydrate in the diet. In addition, the expression of 6PF-2-K/Fru-2,6-P(2)ase can be used as an indicator of nutritional condition.     

Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver.

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.     

Fructose 2,6-bisphosphate and fructose-6-P 2-kinase in Saccharomyces cerevisiae in relation to metabolic state in wild type and fructose-6-P 1-kinase mutant strains

In wild type Saccharomyces cerevisiae, fructose-6-P is known to be in much lower amounts than needed to saturate fructose-6-P 1-kinase in vitro, and the same is true for a mutant with reduced affinity for fructose-6-P, even though its in vivo fructose-6-P concentration is much higher than normal. Both the wild type and mutant fructose-6-P 1-kinases were activated in vitro by fructose-2,6-P2 in the 0.1 microM concentration range, and the effector was present in more than adequate amounts. Hence, it is likely to be necessary for sufficient flux through the fructose-6-P 1-kinase reaction in vivo, and the data also fit with fructose-2,6-P2 acting at different sites on the enzyme from fructose-6-P. In growth on glucose, a variety of wild type strains contained 5-10 microM fructose-2,6-P2, and various fructose-6-P 1-kinase mutant strains had levels of up to 150 microM in the presence of glucose. Fructose-2,6-P2 was also found (0.5-10 microM) in derepressed cultures after glucose exhaustion and in growth on pyruvate. Activities of fructose-6-P 2-kinase in the various strains and situations are also presented. The data generally indicate a correlation between levels of fructose-2,6-P2 and fructose-6-P and suggest that fructose-2,6-P2 is not rapidly degraded after glucose exhaustion.     

Effect of tolbutamide on fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase in rat liver

The effects of tolbutamide on the activities of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were examined using rat hepatocytes. Tolbutamide stimulated fructose-6-phosphate,2-kinase activity and inhibited fructose-2,6-bisphosphatase activity, resulting in an increase of fructose-2,6-bisphosphate level. Changes in the activities of the enzyme by tolbutamide were due to variation in the Km value, but not dependent on alteration of Vmax. Glucagon inhibition of fructose-2,6-bisphosphate formation resulting from an inactivation of fructose-6-phosphate,2-kinase and an activation of fructose-2,6-bisphosphatase was released by tolbutamide. Tolbutamide stimulation of fructose-2,6-bisphosphate formation through regulation of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase may produce enhancement of glycolysis and inhibition of gluconeogenesis in the liver.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by o-phthalaldehyde.

The two activities of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by o-phthalaldehyde. Absorbance and fluorescence spectra of the modified enzyme were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme subunit). The inactivation of 6-phosphofructo-2-kinase by o-phthalaldehyde was faster than the inactivation of fructose-2,6-bisphosphatase, which was concomitant with the increase in fluorescence. The substrates of 6-phosphofructo-2-kinase did not protect the kinase against inactivation, whereas fructose-2,6-bisphosphate fully protected against o-phthalaldehyde-induced inactivation of the bisphosphatase. Addition of dithiothreitol prevented both the increase in fluorescence and the inactivation of fructose-2,6-bisphosphatase, but not that of 6-phosphofructo-2-kinase. It is proposed that o-phthalaldehyde forms two different inhibitory adducts: a non-fluorescent adduct in the kinase domain and a fluorescent isoindole derivative in the bisphosphatase domain. A lysine and a cysteine residue could be involved in fructose-2,6-bisphosphate binding in the bisphosphatase domain of the protein.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Interaction with N-bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate

N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.     

Increases in hepatic fructose-2,6-bisphosphate level and fructose-6-phosphate,2-kinase activity in rats with ventromedial lesions of the hypothalamus

To investigate altered fructose-2,6-bisphosphate (fructose-2,6-P2) metabolism, we measured fructose-2,6-P2 levels and fructose-6-phosphate,2-kinase (fructose-6-P,2-kinase) activities in various tissues, including liver, kidney, heart, and skeletal muscle, of ventromedial hypothalamus (VMH)-lesioned rats during feeding and starvation. The plasma insulin level was 6 times or more higher in these rats than in the controls. The fructose-2,6-P2 level in liver was much greater in VMH-lesioned rats than in the controls: 15.1 +/- 2.2 nmol/g tissue versus 7.7 +/- 0.7 in the fed state, 5.3 +/- 1.1 versus 1.6 +/- 0.4 in the starved state. In kidney, heart, and skeletal muscle, fructose-2,6-P2 levels were not different between the two animal groups. The activity of hepatic fructose-6-P,2-kinase remained high after 20 h of starvation in VMH-lesioned rats, whereas it was decreased markedly in the controls. The hepatic concentration of fructose-6-phosphate was also high in VMH-lesioned rats. Both fructose-6-P,2-kinase activity and fructose-6-phosphate concentration in the liver of starved VMH-lesioned rats were comparable to those of control rats in fed conditions. These results indicate that the alteration of fructose-2,6-P2 metabolism is characteristic of liver in VMH-lesioned rats, and that the increase in hepatic fructose-2,6-P2 may activate hepatic glycolysis not only during feeding but also during starvation, leading to the enhanced lipogenesis in these obese rats.