Glucose: a more powerful modulator of fructose 2,6-bisphosphate levels than insulin in human hepatocytes

This study provides the first experimental evidence of the short-term control of fructose 2,6-bisphosphate (Fru(2,6)P2) levels in adult human hepatocytes. (1) In hepatocytes whose metabolic status resembles the fed state (glycogen-rich), exposure to glucagon (10(-8) M) caused a drastic decrease in the levels of this effector and a significant fall in lactate production rate. Adrenaline, isoprenaline (a beta-adrenergic agonist) and lactate exerted a similar action decreasing Fru(2,6)P2 concentration. (2) In glucagon pre-treated, glycogen- and Fru(2,6)P2-depleted cells (a situation that mimics the fasted state), Fru(2,6)P2 re-synthesis was strictly dependent on glucose availability. (3) Insulin did not seem to exert a direct action on the control of Fru(2,6)P2 in human hepatocytes. The hormone--which failed to enhance Fru(2,6)P2 in glucose-starved cells--did not further increase Fru(2,6)P2 content nor its time-course evolution as compared to hepatocytes incubated with glucose alone. (4) Lactate caused a significant delay in the glucose-induced increase in Fru(2,6)P2 content that could not be prevented by insulin. (5) Data indicate that in human hepatocytes glucose is a more powerful modulator of Fru(2,6)P2 than insulin, and that variations in blood lactate concentration may also play a role in the control of hepatic Fru(2,6)P2 levels during the fasted-to-fed transition in humans.     

Effect of recombinant cytokines on glycolysis and fructose 2,6-bisphosphate in rheumatoid synovial cells in vitro.

Recombinant-derived human interleukin 1 (IL1) alpha and beta and interferon gamma (IFN-gamma) each produced similar increases in rheumatoid synovial cell (RSC) glycolysis, as judged by increased values for glucose uptake, lactate production and cellular fructose 2,6-bisphosphate [Fru(2,6)P2]. Measurement of Fru(2,6)P2 proved to be the most sensitive parameter for an assessment of glycolysis: IL1 alpha, IL1 beta and IFN-gamma all produced a 3-6-fold increase in this metabolite whereas tumour necrosis factor (TNF alpha) was far less effective. Prostaglandin E production was stimulated predominantly by IL1 alpha and IL1 beta rather than by IFN-gamma or TNF alpha. When combinations of cytokines were examined the addition of IFN-gamma with either IL1 alpha, IL1 beta or murine IL1 produced a synergistic increase in cellular Fru(2,6)P2. The three forms of IL1 increased Fru(2,6)P2 via the same pathway, whereas IFN-gamma acted via a different mechanism. The increase in Fru(2,6)P2 in subcultured RSC produced by addition of medium from a primary culture exceeded the maximal effects of any of the single cytokines studied, suggesting the presence of a mixture of cytokines in the primary RSC culture medium.     

Stimulation by insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine-dependent inhibition of phosphofructokinase 2 activation

Activation of glycolysis by insulin in cultured rat hepatocytes is preceded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructose 2,6-bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated. 1. 50 microM ATP completely prevented the 3-5-fold insulin-dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content. 50 microM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin cells with initial high Fru(2,6)P2 content. 2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 microM ATP or 15 microM adenosine 5'[beta, gamma-methylene]triphosphate. ADP and adenosine-5-[gamma-thio]triphosphate were as effective as ATP, whereas 100 microM adenosine 5'[alpha, beta-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis. 3. Extracellular ATP (100 microM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3-phosphate levels stayed constant. Puromycin did not inhibit PFK 2. 4. Both puromycin and ATP prevented the insulin-dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase. 5. 100 microM ATP elevated the cAMP-dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16-fold within 5 min, whereas puromycin was without effect on either level. It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.     

蛇肌果糖1，6－二磷酸酯酶果糖2，6－二磷酸和底物抑制的结合部位

在果糖１,６—二磷酸酯酶中果糖２,６—二磷酸可能与底物抑制的作用方式不同,因为蛇肌果糖１,６－二磷酸酯酶ｐＨ９．２的活性受到果糖２,６－二磷酸的抑制,而不受高浓度底物的影响。Ｋ＋能增强果糖２,６—二磷酸对酶活性抑制,并能较大程度地解除过量底物的抑制。快反应流基修饰酶不再受较低浓度果糖２,６—二磷酸的抑制,但高浓度果糖２,６—二磷酸仍能抑制酶活性,其ＩＣ５０增大４０倍。修饰酶受底物抑制的阈值不变。为胰蛋白酶或枯草杆菌蛋白酶限制性酶解的果糖１,６—二磷酸酯酶受过量底物和果糖２,６—二磷酸抑制的行为也不相同。以上结果可能提示在蛇肌果糖１,６—二磷酸酯酸中存在既有别于ＡＭＰ,又有别于过量底物的结合部位。     

Activation of phosphofructokinase 2 by insulin in cultured hepatocytes without accompanying changes of effector levels or cAMP-stimulated protein kinase activity ratios

Activation of glycolysis by insulin in cultured adult rat hepatocytes is accompanied by an activation of phosphofructokinase 2 (PFK 2). PFK 2 activation might be caused by insulin-dependent changes of (a) metabolite levels, (b) basal and (c) Br8cAMP-stimulated cAMP-dependent protein kinase activity; this problem was investigated. 1. Cells cultured with 0.1 nM insulin for 48 h exhibited a low glycolytic rate and low fructose 2,6-bisphosphate [Fru(2,6)P2] levels. Addition of insulin increased Fru(2,6)P2 and Fru(1,6)P2 levels sequentially which points to PFK 2 as first target enzyme of insulin action. 2. Concentrations of Glc6P, Fru6P, phosphoenolpyruvate, glycerol 3-phosphate and citrate, which modulate PFK 2/fructose 2,6-bisphosphatase 2 activity, were not altered by insulin. 3. Activation of PFK 2 by insulin occurred without changes in the levels of total and protein-bound cAMP. Bound cAMP amounted to about 14% of total cAMP. 4. Insulin neither decreased the basal dissociation state of the cAMP-dependent protein kinase nor lowered the sensitivity of the kinase towards cAMP in cell extracts. 5. Addition of the phosphodiesterase-resistant Br8cAMP to the cultures increased cAMP levels 3-4-fold, elevated the protein kinase activity ratio from 0.14 to 0.6 and decreased the Fru(2,6)P2 level and the rate of glycolysis. When Br8cAMP and insulin were given together, insulin was capable of counteracting Br8cAMP in that it activated glycolysis and PFK 2 and elevated the Fru(2,6)P2 level; however, it did not decrease the elevated protein kinase activity ratio. It is concluded that insulin presumably does not activate PFK 2 through changes in cAMP and effector levels or through inhibition of cAMP-dependent protein kinase dissociation. The data support the hypothesis that insulin may act via activation of PFK 2 phosphatase.     

蛇肌果糖1，6－二磷酸酯酶、果糖2，6－二磷酸、AMP的结合部位关系

腺苷－磷酸（ＡＭＰ）对４个快反应巯基被修饰的蛇肌果糖１,６－二磷酸酯酶活性的抑制作用增强,而该修饰的酶受果糖２,６－二磷酸的抑制脱敏。ＡＭＰ对酶抑制为半部位反应,酶受果糖２,６－二磷酸抑制的脱敏则表现为全部位反应。经枯草杆菌蛋白酶限制性酶解的果糖１,６－二磷酸酯酶的Ｋｉ（ＡＭＰ）增大１０倍,但受果糖２,６－二磷酸抑制的性质不变。经胰蛋白酶限制性酶解的果糖１,６－二磷酸酯酶的活性不再为ＡＭＰ抑制,但果糖２,６－二磷酸对该形式酶的抑制作用则明显增强,由于该酶失去受ＡＭＰ的抑制作用,因此ＡＭＰ促进果糖２,６－二磷酸抑制的性质亦随之丧失。据此提出在蛇肌果糖１,６－二磷酸酯酶中果糖２,６－二磷酸不是结合在ＡＭＰ结合部位上的看法。     

Activation of glycolysis by insulin with a sequential increase of the 6-phosphofructo-2-kinase activity, fructose-2,6-bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes

The involvement of 6-phosphofructo-2-kinase, fructose 2,6-bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin-dependent short-term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short-term influence of insulin on these parameters was dependent on the insulin concentration used for the long-term culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as 'insulin cells' and 'control cells', respectively. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two-fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6-phosphofructo-2-kinase remained constant. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6-phosphofructo-2-kinase directly after the medium change. However, both parameters increased during a 1-2-h incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value. Addition of insulin to control cells led to a 5-8-fold stimulation of glycolysis but only after a 30-90-min lag phase. During this lag period insulin strongly increased sequentially the 6-phosphofructo-2-kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin-stimulated glycolysis. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin-independently, led to an immediate increase in glycolysis without a lag phase. It is concluded that in this insulin-sensitive cell system: the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin-dependent stimulation of glycolysis in insulin cells; activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level. The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.     

Subcellular compartmentation of fructose 2,6-bisphosphate in oat mesophyll cells

Evacuolated mesophyll protoplasts from oat (Avena sativa L.) were fractionated by a membrane-filtration technique. This method of rapid quenching of metabolic reactions permitted estimation of the in-vivo pools of fructose 2,6-bisphosphate (Fru2,6bisP) in the cytosol, chloroplasts and mitochondria. Vacuolar Fru2,6bisP was calculated as the difference between control protoplasts and evacuolated ones. The results indicate that Fru2,6bisP is exclusively cytosol-located in oat mesophyll protoplasts. Assuming a cytosolic volume of about 2 pl per evacuolated protoplast, the cytosolic concentration there was 11 M if protoplasts were in darkness. Illumination of either control or evacuolated protoplasts resulted in a significant decrease in the Fru2,6bisP content within 5 min.Abbreviations EPs evacuolated protoplasts - Fru2,6bisP fructose 2,6-bisphosphate - PFP fructose 6-phosphate kinase (pyrophosphate-dependent), EC 2.7.1.90 - PEPCase phosphoenolpyruvate carboxylase, EC 4.1.1.31     

cDNA sequence and kinetic properties of human lung fructose(1, 6)bisphosphatase

A cDNA encoding fructose(1,6)bisphosphatase was isolated from total human lung RNA. The cDNA contained an open reading frame encoding 337 amino acids. The determined nucleotide sequence of the lung cDNA was significantly different from muscle cDNA and slightly differed from human liver cDNA in a single mutation (Gly-336 for Ala-336) and a T for C substitution in position 648. The human lung fructose(1, 6)bisphosphatase [Fru(1,6)Pase] was isolated and its kinetic parameters were compared with liver and muscle isoenzymes. Values of kcat for the lung Fru(1,6)Pase were lower than for the liver and muscle enzyme. Like the liver isoenzyme, lung Fru(1,6)Pase is significantly less inhibited by AMP than the muscle enzyme. The values of I0.5 were 9.5, 9.8, and 0.3 microM for the liver, lung, and muscle enzyme, respectively. The lung enzyme was slightly more sensitive to fructose(2,6)bisphosphate [Fru(2,6)P2] inhibition than the liver enzyme. Ki was 75 microM for the lung and 96 microM for the liver enzyme. The synergistic effect of AMP and Fru(2,6)P2 on the lung and liver Fru(1,6)Pase was also observed. In the presence of AMP the corresponding values of Ki for Fru(2,6)P2 were 16 microM for the lung and 10 microM for the liver enzyme.     

Rapid oscillations in fructose 2,6-bisphosphate levels in plant tissues

The fructose 2,6-bisphosphate (Fru 2,6-P₂) content of pea, Pisum sativum, roots and leaves were measured following flooding with water and found to change in times of minutes and to exhibit oscillatory-type changes. Each organ changes its Fru 2,6-P₂ content in a unique pattern in response to environmental disturbances such as flooding or light. For example, when roots of intact illuminated pea plants are flooded, roots decrease their Fru 2,6-P₂ content while simultaneously leaves increase their Fru 2,6-P₂ content; but both organs exhibit oscillatory-type patterns within flooding time of about 30 minutes. Half-change times can be as rapid as 2 to 3 minutes. The endogenous extractable activity of the root pyrophosphate-dependent phosphofructokinase also exhibits an oscillatory pattern upon root immersion slightly after Fru 2,6-P₂ changes occur. We postulate from these results that Fru 2,6-P₂ is a primary signal molecule which enables plants to regulate their metabolism to cope with changing environments.     

Molecular cloning, sequence analysis, and expression of a human liver cDNA coding for fructose-6-P,2-kinase:fructose-2,6-bisphosphatase

A cDNA coding for 378 amino acids from the C-terminus of the human liver bifunctional enzyme, Fructose-6-phosphate,2-kinase:Fructose-2,6-bisphosphatase was isolated, sequenced, and expressed in E. coli K38. The expressed protein, identified by specific immunoassay, showed Fru 2,6-bisphosphatase activity but no Fru 6-P,2-kinase activity, demonstrating directly that the Fru 2,6-bisphosphatase activity resides in the C-terminal region. The Km for Fru 2,6-P2 was 4.3 microM. Fru 6-P was a noncompetitive inhibitor (Ki = 2.9 microM), and formed a phosphorylated intermediate when incubated with Fru 2,6[2-32P]P2. The subunit Mr of the enzyme was 36,600, and the active enzyme showed Mr = 37,000 by gel filtration.     

Control of fructose 2,6-bisphosphate levels in rat macrophages by glucose and phorbol ester

The presence of fructose 2,6-bisphosphate (Fru 2,6-P2) in elicited peritoneal macrophages of rat was examined. These cells possess an active phosphofructokinase-2 which is diminished by citrate and only slightly inhibited by glycerol 3-phosphate. Phosphofructokinase-1 submaximal activity was increased 26-fold by the addition of 1 microM Fru 2,6-P2. Incubation of cells without glucose decreased the amount of Fru 2,6-P2 to zero, but further addition of 5 mM glucose increased the levels of the sugar ester 20-fold. In addition, the presence of phorbol ester potentiated the synthesis of Fru 2,6-P2. By contrast phenylisopropyladenosine or prostaglandin F2 alpha inhibited the production of Fru 2,6-P2.     

Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6-bisphosphate levels

The aim of this work was to investigate the role of fructose 2,6-bisphosphate (Fru 2,6-P₂) during photosynthesis. The level of Fru 2,6-P₂ in tobacco plants was elevated by the introduction of a modified mammalian gene encoding 6-phosphofructo-2-kinase (6-PF-2-K). Estimates of the metabolite control coefficient (C) for Fru 2,6-P₂ levels in response to increased 6-PF-2-K activity, suggest that small increases in 6-PF-2-K activity have little effect upon steady-state Fru 2,6-P₂ levels (_C = +0.08 for a 0–58% increase in 6-PF-2-K activity). However, larger changes resulted in dramatic rises in Fru 2,6-P₂ levels (C = +3.35 for 206–268% increase in 6-PF-2-K activity). Transgenic plants contained Fru 2,6-P₂ levels in the dark that ranged from 104 to 230% of the level in wild-type tobacco. Plants with altered levels of Fru 2,6-P₂ were used to determine the effects of this signal metabolite upon carbohydrate metabolism during the initial phase of the light period. Here we provide direct evidence that Fru 2,6-P₂ contributes to the regulation of carbon partitioning in tobacco leaves by inhibiting sucrose synthesis.     

Mutations in the charged residues of the amino terminus of rat liver fructose 6-phosphate,2-kinase:Fructose 2,6-bisphosphatase: effects on regulation.

Amino and carboxyl termini of the bifunctional enzyme Fru 6-P, 2-kinase:Fru 2,6-bisphosphatase regulate the relative activities of the kinase/phosphatase. The N-terminus of the rat liver bifunctional enzyme is highly basic, containing a protein kinase A phosphorylation site that regulates these enzyme activities in a reciprocal manner. To determine the role of charged residues in the N-terminal peptide, mutant enzymes were constructed in which these residues were altered to residues carrying opposite charges, and the effect on the catalytic properties, thermal lability, and susceptibility to trypsin digestion and phosphorylation by protein kinase A was determined. Most of these mutations decreased k(cat)/K(ATP) and/or k(cat)/K(Fru) (6-P) of the kinase and increased k(cat)/K(Fru 2,6-P2) of the phosphatase. These mutant enzymes were more susceptible to trypsin digestion, phosphorylation by protein kinase A, and thermal inactivation. In general, the effect was greater with amino acid residues located more distant from the N-terminus. The resulting changes were not as large as observed with the phosphorylated enzyme. Mutation of Ser22 to Pro produced large changes in the kinetic properties comparable to those of phosphorylation, suggesting that the flexible region of the N-terminus containing five serines (Ser20 to S24) is essential for the enzyme activities. These results indicated that the charged residues as well as Ser20-Ser24 in the N-terminus of the liver Fru 6-P,2-kinase:Fru 2,6-Pase are essential in the allosteric regulation and probably involved in interactions with the catalytic domains that induce a conformation that has high Fru 6-P,2-kinase and low Fru 2,6-Pase activities. Any disruption of this N-terminal interaction results in inhibition of the kinase and activation of the phosphatase.     

Yeast fructose-2,6-bisphosphate 6-phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase.

Yeast fructose-2,6-bisphosphate 6-phosphatase has been purified 7000-fold by heat treatment, poly(ethylene glycol) precipitation, ion-exchange chromatography with Q-Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin-A-Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6-bisphosphate [Fru(2,6)P2] as well as with p-nitrophenyl phosphate (NpP) showing a pH optimum at pH 6-6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69-72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000-fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 degrees C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6-phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6-phosphatase.     

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes

The role of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) in developing leaves was studied using wild-type tobacco (Nicotiana tabacum L.) and transformants with decreased expression of PFP. (i) The leaf base, which is the youngest and most actively growing area of the leaf, had 2.5-fold higher PFP activity than the leaf tip. T3 transformants, with a 56-95% decrease in PFP activity in the leaf base and an 87-97% decrease in PFP activity in the leaf tip, were obtained by selfing and re-selfing individuals from two independent transformant lines. (ii) Other enzyme activities also showed a gradient from the leaf base to the leaf tip. There was a decrease in PFK and an increase in fructose-6-phosphate,2-kinase and plastidic fructose-1, 6-bisphosphatase, whereas cytosolic fructose-1,6-bisphosphatase activity was constant. None of these gradients was altered in the transformants. (iii) Fructose-2,6-bisphosphate (Fru2,6bisP) levels were similar at the base and tip of wild-type leaves in the dark. Illumination lead to a decrease in Fru2,6bisP at the leaf tip and an increase in Fru2,6bisP at the leaf base. Compared to wild-type plants, transformants with decreased expression of PFP had up to 2-fold higher Fru2,6bisP at the leaf tip in the dark, similar levels at the leaf tip in the light, 15-fold higher levels at the leaf base in the dark, and up to 4-fold higher levels at the leaf base in the light. (iv) To investigate metabolic fluxes, leaf discs were supplied with 14CO2 in the light or [14C]glucose in the light or the dark. Discs from the leaf tip had higher rates of photosynthesis than discs from the leaf base, whereas the rate of glucose uptake and metabolism was similar in both tissues. Significantly less label was incorporated into neutral sugars, and more into anionic compounds, cell wall and protein, and amino acids in discs from the leaf base. Metabolism of 14CO2 and [14C]glucose in transformants with low PFP was similar to that in wild-type plants, except that synthesis of neutral sugars from 14CO2 was slightly reduced in discs from the base of the leaf. (v) These results reveal that the role of PFP in the growing cells in the base of the leaf differs from that in mature leaf tissue. The increase in Fru2,6bisP in the light and the high activity of PFP relative to cytosolic fructose-1,6-bisphosphatase in the base of the leaf implicate PFP in the synthesis of sucrose in the light, as well as in glycolysis. The large increase in Fru2,6bisP at the base of the leaf of transformants implies that PFP plays a more important role in metabolism at the leaf base than in mature leaf tissue. Nevertheless, there were no major changes in carbon fluxes, or leaf or plant growth in transformants with below 10% of the wild-type PFP activity at the leaf base, implying that large changes in expression can be compensated by changes in Fru2,6-bisP, even in growing tissues.     

Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells

Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.     

Purification of the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus uberis and an investigation of its existence in different forms.

The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo.     

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.     

Fructose 2,6-bisphosphate in isolated foetal hepatocytes

Fru 2,6-P2 was present in isolated foetal hepatocytes at a concentration of 1.6 nmol per g cells. When foetal hepatocytes were exposed to glucagon no changes were observed either in the concentration of Fru 2,6-P2 and lactate release or in the activities of 6-phosphofructo-2-kinase and pyruvate kinase. Incubation of purified 6-phosphofructo-2-kinase with the catalytic subunit of protein kinase did not change the enzyme activity. The inhibition by sn-glycerol 3-phosphate was much lower for the foetal than for adult enzyme. These results suggest that an isoenzyme of 6-phosphofructo-2-kinase in foetal hepatocytes different from that of adult hepatocytes may be present.