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Fructose 2,6-Bisphosphate and the Control of Glycolysis by Glucocorticoids and by Other Agents in Rat Hepatoma Cells1

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Fructose 2,6-Bisphosphate and the Control of Glycolysis by Glucocorticoids and by Other Agents in Rat Hepatoma Cells1 [CANCER RESEARCH 45, 4263-4269, September 1985] Fructose 2,6-Bisphosphate and the Control of Glycolysis by Glucocorticoids and by Other Agents in Rat Hepatoma Cells1 Anne M. Loiseau,2 Guy G. Rousseau, and Louis Hue3 Hormone ana Metabolic Research Unit, International Institute of Cellular and Molecular Pathology, and Louvain University Medical School, 75 Avenue Hippocrate, B-1200, Brussels, Belgium ABSTRACT hepatocytes and HTC cells as models, we have compared sev eral aspects of glycolysis in normal and cancer cells. HTC cells The rate, key enzymes, and several metabolites of glycolysis were chosen because of their high degree of dedifferentiation in rat hepatoma (HTC) cells have been compared to those in rat and high rate of glycolysis (7). Moreover, when studying the hepatocytes. At 5 to 10 mw glucose, lactate release was greater glycolysis of HTC cells, we had found that this process is in HTC cells. This could be explained in part by the absence of stimulated by glucocorticoid hormones, presumably via a recep key gluconeogenic enzymes, by the substitution of glucokinase tor-mediated mechanism (8). We have now examined whether by hexokinase, and by an increase in phosphofructokinase 1 and fructose 2,6-bisphosphate is present in HTC cells and whether pyruvate kinase activity. In addition, fructose 2,6-bisphosphate, their PFK1 is sensitive to this stimulator. We show that this is the most potent stimulator of phosphofructokinase 1, was iden the case and that fructose 2,6-bisphosphate could be involved tified in HTC cells and shown to stimulate phosphofructokinase in the control of glycolysis by glucocorticoids in HTC cells. We 1 partially purified from these cells. have also examined whether agents known to decrease fructose Dexamethasone increased the release of lactate in HTC cells. 2,6-bisphosphate concentration in liver would do so in HTC cells This glucocorticoid increased the concentration of fructose 2,6- and whether this would correlate with an inhibition of glycolysis. bisphosphate and the Vmaxof the enzyme that catalyzes its synthesis, phosphofructokinase 2. The data were consistent with MATERIALS AND METHODS an indirect effect at the gene level, mediated by glucocorticoid receptors. Dexamethasone had no effect on the other rate- Cell Culture. HTC cell lines 4 and 99 are derived from the "wild" type limiting glycolytic enzymes. Several agents (adenosine, dibutyryl originally received from Dr. G. M. Tomkins. The variant cell line 268EC, cyclic adenosine 3':5'-monophosphate, ethanol, antimycin) partially resistant to glucocorticoids, was a gift of Dr. E. B. Thompson. known to decrease fructose 2,6-bisphosphate in hepatocytes All experiments, except those in Table 2, were performed with cell line 4. The cells were grown (9) as suspension cultures in Swim's Medium were without effect on this stimulator in HTC cells. DL-Glyceral- S-77 containing 10% (v/v) newborn calf serum. Where indicated (serum- dehyde inhibited glycolysis in HTC cells and eventually killed them. Although this substance decreased fructose 2,6-bisphos free medium), the latter was replaced by a solution of bovine serum albumin (10 g/liter) in phosphate-buffered saline (0.15 M NaCI:2.5 rriM phate, inhibition of glycolysis through an action at another level KCI:8 rnw Na2HPO4:1.5 rriM KH2PO4, pH 7.45). Glucose concentration in could not be ruled out. the culture medium was 17.5 mM and 18.3 mw in absence and presence of serum, respectively. Incubation of Cells. Two types of incubations were performed. In the INTRODUCTION first one, 2 ml of a suspension containing about 5 x 106 HTC cells/ml of Krebs-Henseleit bicarbonate medium (10) were incubated like hepato Unlike normal cells, many cancer cells maintain a high glycolytic cytes (11 ) at 37°Cin a rotatory incubator under an O2:CO2 atmosphere rate under aerobic conditions. The biochemical mechanism of (19:1) for no longer than 1 h, in the presence of glucose at the concen this phenomenon is not fully understood (1-3). The discovery (4) trations indicated. For the measurement of fructose 2,6-bisphosphate, of fructose 2,6-bisphosphate led us to reassess the issue. Fruc 0.5-ml aliquots were taken and immediately frozen in tubes kept in liquid tose 2,6-bisphosphate, a naturally occurring molecule, is the N2; the frozen cells were then extracted in 50 mM NaOH as described most potent stimulator of PFK1," one of the rate-limiting enzymes earlier (12). For the measurement of glucose, lactate, and metabolites, 1 of glycolysis. It is synthetized from fructose 6-phosphate and ml of incubation mixture was deproteinized by 1 ml of 10% (w/v) HCIO4; ATP by PFK2 and hydrolyzed into fructose 6-phosphate and P> the deproteinized supernatants were neutralized by a 3 M KOH:1 M KHCC-3 mixture. by fructose 2,6-bisphosphatase. In liver, fructose 2,6-bisphos In the second type of incubation, performed to study the effect of phate allows glycolysis to proceed even if ATP, an inhibitor of glucocorticoids, the cells in culture medium were agitated in a rotatory PFK1, is present (for a review, see Refs. 5 and 6). Using rat incubator under atmospheric air. Except when mentioned otherwise, the 1This research was supported in part by a grant from the Caisse Generate cells were taken in their exponential phase of growth and resuspended d'Epargne et de Retraite (Belgium) and from the Fonds de la Recherche Scientifique in serum-free medium. Steroids in ethanol or ethanol alone (final concen Medicate (3.4539.81). tration, < 0.01%) was added to the cultures. For the measurement of 2 Fellow of Institut pour l'Encouragement de la Recherche Scientifique dans fructose 2,6-bisphosphate, 10 ml of cells were centrifuged (5000 x g for l'Industrie et l'Agriculture (Belgium). 1 min, 0°C),and the cell pellet was kept frozen at -80°C until further 3 Maîtrede Recherches of the Fonds National de la Recherche Scientifique (Belgium). To whom requests should be addressed, at UCL-ICP 7529, 75 Avenue processed. For the measurement of glucose and lactate, aliquots of the Hippocrate, B-1200 Brussels, Belgium. culture medium were deproteinized as described above. •Theabbreviations used are: PFK1, phosphofructokinase 1 (ATP:o-fructose-6- Enzyme Assays. Frozen pellets of HTC cells (corresponding to about phosphate 1-phosphotransferase, EC 2.7.1.11); PFK2, phosphofructokinase 2 (ATP:o-fructose-6-phosphate 2-phosphotransferase, EC 2.7.1.-); HTC, hepatoma 200 mg of cells) or livers from rats fasted for 24 h were homogenized at 0-4°C with an UltraTurrax in 4 volumes of 0.1 M KCI:0.1 mM EDTA:2 tissue culture. Received 10/2/84; revised 1/18/85; accepted 3/25/85. m«MgCbiSO mM 4-(2-hydroxyethyl)-1-piperazineethanolsulfonic acid at CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4263 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1985 American Association for Cancer Research. CONTROL OF GLYCOLYSIS IN RAT HEPATOMA CELLS pH 7.2. After centrifugation (10,000 x g for 10 min), appropriate dilutions in HTC cells as compared with those in the liver of normal rats of the supernatant were used for enzyme assays. The activities of fasted for 24 h. In agreement with earlier work (7, 24, 25), the hexokinase (EC 2.7.1.1) (13), glucokinase (EC 2.7.1.2) (13), glucose-6- enzyme profile of cancer cells differed quantitatively and quali phosphatase (EC 3.1.3.9) (14), fructose-1,6-bisphosphatase (EC tatively from that of normal cells. The activity of gluconeogenic 3.1.3.11 ) (15), pyruvate kinase (EC 2.7.1.40) (16), lactate dehydrogenase (EC 1.1.1.28) (17), PFK1 (EC 2.7.1.11 ) (18), and PFK2 (EC 2.7.1.-) (19) enzymes was decreased or absent, whereas that of glycolytic were measured under Vâ„¢,conditions. Measurement of tyrosine amino- enzymes was increased. One qualitative change was the replace transferase (EC 2.6.1.5) and alkaline phosphodiesterase I (EC 3.1.4.1) ment of glucokinase by hexokinase. Table 1 also shows the concentration of several metabolites. Hexoses 6-phosphate were was made as described previously (9). For the partial purification of PFK1, frozen pellets of HTC cells and rat more concentrated, and phosphoenolpyruvate was less concen livers were homogenized in 5 volumes of 50 HIM NaF:1 mw dithioerythri- trated in HTC cells than in hepatocytes; no significant difference tol:1 rriM ATP-Mg:50 mw Tris-HCI at pH 8. After centrifugation (10,000 in ATP and fructose 1,6-bisphosphate was observed. Glycogen x g for 10 min), the enzyme present in the supernatant was precipitated was not detectable. by 33% (w/v) ammonium sulfate, collected by centrifugation (15,000 x The rate of lactate release and the concentration of fructose g for 10 min), resuspended in 0.1 M KF:1 HIM dithioerythritol:50 mw 2,6-bisphosphate of HTC cells were measured after incubation potassium phosphate:0.5 mw MgCI2:10% (w/v) glycerol:50 mw Tris-HCI with various concentrations of glucose under aerobic and anaer at pH 7.6, and applied on a column (0.7 x 5 cm) of ATP:agarose obic conditions. The relationship between glucose concentration equilibrated in the latter medium. PFK1 was eluted from the column in the presence of 1 mw ATP-Mg. All the steps were performed at 0-4°C. and lactate release was hyperbolic, with an apparent Km for glucose of about 1 rriM (Chart ~\A).In contrast, this relationship The enzyme was purified 100- to 150-fold. The kinetic measurement of PFK1 activity was performed as described previously (20). Analytical Methods. Glucose and glycogen (21), glucose 6-phos- m phate, fructose 6-phosphate, lactate, ATP, and phosphoenolpyruvate ^ 5 were measured enzymically in neutralized HCIO4 extracts (22).
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