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The Stimulation of Glycolysis by Hypoxia in Activated Monocytes Is Mediated by AMP-activated Protein Kinase and Inducible 6-Phosphofructo-2-kinase

The Stimulation of Glycolysis by Hypoxia in Activated Monocytes Is Mediated by AMP-activated... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 34, Issue of August 23, pp. 30778 –30783, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Stimulation of Glycolysis by Hypoxia in Activated Monocytes Is Mediated by AMP-activated Protein Kinase and Inducible 6-Phosphofructo-2-kinase* Received for publication, May 28, 2002 Published, JBC Papers in Press, June 13, 2002, DOI 10.1074/jbc.M205213200 Anne-Sophie Marsin‡, Caroline Bouzin§, Luc Bertrand¶, and Louis Hue From the Hormone and Metabolic Research Unit, University of Louvain Medical School and Christian de Duve International Institute of Cellular and Molecular Pathology, B-1200 Brussels, Belgium The activation of monocytes involves a stimulation of heterotrimeric protein composed of a catalytic () and two glycolysis, release of potent inflammatory mediators, regulatory (, ) subunits (3, 4). It is considered as a “metabolic and alterations in gene expression. All of these pro- master switch” (5), which inactivates key targets that control cesses are known to be further increased under hypoxic anabolic pathways, thereby conserving ATP (1, 2). AMPK is conditions. The activated monocytes express inducible also implicated in the stimulation of glucose uptake that occurs 6-phosphofructo-2-kinase (iPFK-2), which synthesizes in contracting muscle (6, 7). fructose 2,6-bisphosphate, a stimulator of glycolysis. We showed previously that AMPK phosphorylates Ser-466 of During ischemia, AMP-activated protein kinase (AMPK) heart 6-phosphofructo-2-kinase (PFK-2), leading to its activa- activates the homologous heart 6-phosphofructo-2-ki- tion (8). This phenomenon participates in the well known stim- nase isoform by phosphorylating its Ser-466. Here, we ulation of glycolysis by ischemia in the heart. PFK-2/fructose- studied the involvement of AMPK and iPFK-2 in the 2,6-bisphosphatase is a bifunctional enzyme catalyzing the stimulation of glycolysis in activated monocytes under synthesis and degradation of fructose 2,6-bisphosphate (Fru- hypoxia. iPFK-2 was phosphorylated on the homologous 2,6-P ), the most potent stimulator of 6-phosphofructo-1-kinase serine (Ser-461) and activated by AMPK in vitro. The and hence glycolysis. Tissue-specific isozymes of PFK-2/fruc- activation of human monocytes by lipopolysaccharide tose-2,6-bisphosphatase have been identified in mammals. induced iPFK-2 expression and increased fructose 2,6- They possess a conserved catalytic core flanked by variable N- bisphosphate content and glycolysis. The incubation of and C-terminal regulatory domains, and they differ in tissue activated monocytes with oligomycin, an inhibitor of distribution and response to phosphorylation by protein ki- oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, nases (for review see Refs. 9 and 10). Chesney et al. (11) fructose 2,6-bisphosphate content, and glycolysis. In cul- characterized a PFK-2 isozyme, which was induced by proin- tured human embryonic kidney 293 cells, the expression flammatory stimuli and was therefore termed inducible PFK-2 of a dominant-negative AMPK prevented both the acti- (iPFK-2). iPFK-2 is identical to the previously described pla- vation and phosphorylation of co-transfected iPFK-2 by cental isoform (12) and is homologous to heart PFK-2. These oligomycin. It is concluded that the stimulation of gly- isozymes are characterized by their relative PFK-2/fructose- colysis by hypoxia in activated monocytes requires the 2,6-bisphosphatase activities. Under physiological conditions, phosphorylation and activation of iPFK-2 by AMPK. their PFK-2 activity is 100-fold that of their fructose-2,6- bisphosphatase activity (10), indicating that they mainly act as a kinase. The C-terminal regulatory domain of iPFK-2 contains Energy deprivation (e.g. hypoxia and inhibitors of oxidative Ser-461, which lies within a favorable consensus for phospho- phosphorylation such as oligomycin) leads to the activation of 449 467 rylation by AMPK ( KGPNPLMRRNSVTPLAS ), similar the AMP-activated protein kinase (AMPK) via an increase in to that surrounding Ser-466 of heart PFK-2 ( KSQTPVRM- the AMP:ATP ratio (for review see Refs. 1 and 2). AMPK is a RRNSFTPLSS ). A synthetic peptide corresponding to the sequence surrounding Ser-461 in iPFK-2 was indeed shown to * This work was supported by the Belgian Federal Program Interuni- be phosphorylated by AMPK in vitro (8). versity Poles of attraction (P4/23), the Directorate General Higher iPFK-2 is constitutively expressed in several human cancer Education and Scientific Program, French Community of Belgium, The cell lines. This isozyme has also been shown to be induced in Fund for Medical Scientific Research (Belgium), and by European monocytes activated by lipopolysaccharide (LPS) (11), a com- Union contract QLG1-CT-2001-01488 (AMPDIAMET). The costs of pub- lication of this article were defrayed in part by the payment of page ponent of the outer membrane of Gram-negative bacteria, charges. This article must therefore be hereby marked “advertisement” which triggers and mimics an inflammatory response. The in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. response of monocytes to LPS includes the production of cyto- ‡ Research fellow of the National Fund for Scientific Research kines and chemokines, the release of arachidonic acid metabo- (Belgium). § Supported by the Fund for Scientific Research in Industry and lites, and the generation of reactive oxygen species and nitro- Agriculture (Belgium). gen monoxide (13–15). Monocyte activation consumes energy, ¶ Supported by the French Community of Belgium. is glucose-dependent, and involves a stimulation of glycolysis To whom correspondence should be addressed: HORM Unit, ICP- (16 –18). Moreover, in diseased tissues, monocytes are known to UCL 7529, Avenue Hippocrate, 75, B-1200 Brussels, Belgium. Tel.: 32-2-764-74-85; Fax: 32-2-764-75-07; E-mail: [email protected]. accumulate in poorly vascularized hypoxic sites (19, 20). Mono- The abbreviations used are: AMPK, AMP-activated protein kinase; cytes remain functional under such adverse conditions by al- PFK-2, 6-phosphofructo-2-kinase; Fru-2,6-P , fructose 2,6-bisphos- tering gene expression and by switching to anaerobic glycolysis phate; iPFK-2, Inducible 6-phosphofructo-2-kinase; DN, dominant-neg- for ATP production. ative; LPS, lipopolysaccharide; HEK, human embryonic kidney; IL, interleukin. The mechanisms by which glycolysis is stimulated synergis- 30778 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. iPFK-2 Activation by AMPK in Activated Monocytes 30779 tically by LPS and hypoxia are still unknown. We tested whether this synergism results from the phosphorylation and activation of iPFK-2 by AMPK in hypoxia. EXPERIMENTAL PROCEDURES Materials—The construct encoding recombinant polyhistidine- tagged iPFK-2 (21) was a generous gift of R. Bartrons (Barcelona, Spain). Recombinant iPFK-2 was purified (22) from human embryonic kidney (HEK)-293 cells transfected with this construct. Liver AMPK was purified as described previously (23). Wild-type and dominant- negative 1 AMPK constructs were described previously (24). Rabbit polyclonal anti-phospho-S466 (8) and anti-iPFK-2 (11) antibodies were raised against synthetic peptides. These peptides and the SAMS pep- tide (25) were synthesized by V. Stroobant (Ludwig Institute for Cancer Research, Brussels, Belgium). In Vitro Studies—For the measurement of kinetic properties, puri- fied iPFK-2 and heart PFK-2 were incubated with 1 mM MgATP and AMPK at 30 °C (26), and aliquots were taken for PFK-2 assay (27). For determination of phosphorylation, iPFK-2 was incubated with 0.1 mM Mg[- P]ATP (1000 cpm/pmol) and AMPK. Aliquots were taken and analyzed as described previously (26). The amount of purified enzymes used in each experiment is given in the figure legends. Cell Culture—Peripheral blood mononuclear cells were isolated by centrifugation of human whole blood through a density gradient of Ficoll-Paque (Amersham Biosciences) and cultured in Petri dishes (10-cm diameter, 10  10 monocytes/dish) in RPMI 1640 medium with 10% (v/v) fetal calf serum (11). After2hof culture, the medium and FIG.1. Time-dependent changes in phosphorylation and activ- nonadherent cells were removed by aspiration, and the remaining ad- ity of iPFK-2 incubated with AMPK. A, purified iPFK-2 (0.15 mg/ herent monocytes were incubated without (resting) or with (activated) ml) was incubated with 0.1 mM Mg[- P]ATP and purified AMPK (0.6 1 g/ml LPS (Escherichia coli 0111:B4, Sigma). The percentage of unit/ml) with (Œ) or without (‚) AMP (0.2 mM) in a final volume of 50 l. monocytes in the cultures was 85% as determined by fluorescence- Controls (E) were incubated without AMPK. At the indicated times, activated cell sorter analysis for CD14 expression. The cells were incu- aliquots (5 l) were removed for SDS-PAGE and screened using Phos- bated under the conditions and the periods of time indicated in the 32 phorImager for measurement of P incorporation. B, same protocol as figure legends. Following incubation, the medium was aspirated, and in A with 1 mM nonradioactive MgATP in a final volume of 0.1 ml. At the cells were immediately lysed in 0.8 ml of ice-cold lysis buffer (8) for the indicated times, aliquots (10 l) were removed for PFK-2 assay. The enzyme assays or in 0.5 ml of 50 mM NaOH for Fru-2,6-P determina- 2 results are the means  S.E. for three separate experiments. tion. Total RNA was isolated with the High Pure RNA isolation kit (Roche Molecular Biochemicals). HEK-293 cells were cultured in Dul- with bovine serum albumin as a standard. Kinetic constants were becco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf calculated by computer fitting of the data to a hyperbola describing the serum. The transfection protocol was a modified calcium phosphate Michaelis-Menten equation by nonlinear least square regression. One procedure (22). The cells were incubated under the conditions indicated unit of enzyme activity corresponds to the formation of 1 mol (PFK-2) in the figure legends and lysed in 0.8 ml of ice-cold lysis buffer (8). or 1 nmol (AMPK) of product/min under the assay conditions. Unless otherwise stated, the cells were cultured in normoxic conditions (95% O ,5%CO ). RESULTS 2 2 Reverse Transcription-PCR Analysis—RNA was reverse transcribed Phosphorylation and Activation of iPFK-2 by AMPK in for1hat37 °C with random primers, and cDNA fragments that Vitro—Purified iPFK-2 was phosphorylated by AMPK with a correspond to interleukin (IL)-1 (271 bp) and iPFK-2 (140 bp) were stoichiometry close to 0.7 mol of phosphate incorporated/mol of amplified with the primers described previously (11). The cycling pro- gram used was 95 °C for 30 s, 55 °C for 30 s, and 72 °Cfor45sin22 enzyme subunit, indicating phosphorylation at one site. The cycles. As a control, -actin cDNA fragment (612 bp) was amplified with rate and the extent of phosphorylation were stimulated by the following primers: 5-GGCATCGTGATGGACTCCG-3 and 5-GCT- AMP (Fig. 1A), and phosphorylation correlated with PFK-2 GGAAGGTGGACAGCGA-3 (95 °C for 30 s, 58 °C for 30 s, and 72 °C for activation (Fig. 1B). The treatment with AMPK led to a 2.5-fold 45 s in 22 cycles). The amplification cycle number was varied initially to increase in V of PFK-2 with no significant change in K for max m establish unsaturating amplification response. The displayed cycle fructose 6-phosphate or MgATP (Table I). These changes in number allows the illustration of representative differences in the amount of cDNA present. kinetic properties resemble those seen after the phosphoryla- Enzyme and Metabolite Measurements—AMPK (23) and PFK-2 (27) tion of heart PFK-2 by AMPK (Table I) (8). The similarity activities were assayed in a 10 and 20% (w/v) polyethylene glycol 6000 among the sequences surrounding Ser-461 of iPFK-2 and Ser- precipitate, respectively. Fru-2,6-P was measured as described previ- 466 of heart PFK-2 led us to use the antibody raised against the ously (28). 3 phosphorylated Ser-466 of heart PFK-2 (anti-pS466) to study Measurement of [3- H]Glucose Detritiation—The glycolytic flux the phosphorylation of Ser-461 in iPFK2. Immunoblotting with through PFK-1 was estimated by the rate of detritiation of [3- H]glu- cose (29). Monocytes were cultured in 5 ml of RPMI 1640 medium this antibody showed that AMPK phosphorylated Ser-461 of containing 10 mM glucose and activated by LPS for the indicated times. iPFK-2 (Fig. 2). Cells were incubated with oligomycin for 5 min prior to the addition of AMPK Is Activated by Oligomycin and Hypoxia in Resting tracer amounts (0.3 Ci/ml) of radioactive glucose. Samples were re- Monocytes—To activate AMPK, resting monocytes were incu- moved periodically (0 –15 min after the addition of glucose) from the 3 bated under hypoxic conditions or with two known activators of medium to measure the formation of H O. These samples were depro- AMPK, namely 5-aminoimidazole-4-carboxamide riboside (0.5 teinized in 1 M ice-cold perchloric acid. After neutralization and centrif- ugation (10,000  g, 5 min, 4 °C), H O was separated from radioactive mM), a precursor of the AMP analog ZMP or oligomycin (1 M), glucose (30). The release of H O was linear over the 15-min experi- an inhibitor of oxidative phosphorylation. Basal AMPK activity mental period, and the rate was calculated from the average detritia- was low and similar to that measured in normoxic perfused tion rate over 15 min and expressed as nanomoles of glucose detritiated hearts or cells in culture (8) and remained unchanged over the per minute per milligram of protein. This rate may give an underesti- incubation period (Fig. 3). By contrast, AMPK activity progres- mation of the net glycolytic flux because of an incomplete detritiation of sively increased during incubation with oligomycin or hypoxia the tracer (29). Other Methods—Proteins was estimated by the method of Bradford to reach maximal values between 10 and 20 min before decreas- 30780 iPFK-2 Activation by AMPK in Activated Monocytes TABLE I. Effects of AMPK on the kinetic properties of iPFK-2 and heart PFK-2 Purified iPFK-2 or heart PFK-2 (0.1 mg/ml) was incubated with (w/) or without (w/o) AMPK (0.6 unit/ml) with 0.2 mM AMP and 1 mM MgATP in a final volume of 100 lat30°C for 30 min. Aliquots (10 l) were taken for the measurement of PFK-2 activity. PFK-2 was measured at pH 7.1 in the presence of 5 mM MgATP and concentrations of fructose 6-phosphate up to 10 times the K or in the presence of 1 mM fructose 6-phosphate and concentrations of MgATP up to 10 times the K The results are the means  S.E. for three different experiments. iPFK-2 heart PFK-2 w/o w/o w/ AMPK w/ AMPK AMPK AMPK a a V (milliunits/mg protein) 10 125  2 50  5 125  10 max K for fructose 6- phosphate (M) 53 250 457 346  1 K for MgATP (M) 650  32 582  37 930  12 745  5 p  0.01 in comparison with sample incubated without AMPK. FIG.2. Immunoblot of inducible and heart PFK-2 phosphoryl- ated by AMPK with the anti-pS466 antibody. Purified PFK-2 (0.15 mg/ml) was incubated with AMPK (0.6 unit/ml), AMP (0.2 mM), and 1 mM MgATP in a final volume of 20 lat30 °C. After 30 min, samples were removed for SDS-PAGE and immunoblotted with the anti-pS466 antibody. FIG.4. Time-dependent induction of IL-1 and iPFK-2 by LPS in monocytes. A, reverse transcription-PCR analysis of IL-1, iPFK-2, and -actin mRNAs obtained from resting or LPS-activated monocytes. The effects of oligomycin (1 M, 15 min) on iPFK-2 and IL-1 mRNA was analyzed in monocytes cultured for 6 h. B, immunoblot analysis with anti-iPFK-2 antibody on 10 g of protein from extracts of resting or LPS-activated monocytes. The effect of oligomycin (1 M, 15 min) was also verified. effect on AMPK activity (Fig. 5C). Hypoxia and Oligomycin Activate iPFK-2 in LPS-stimulated Monocytes—The effects of oligomycin were tested in cells incu- bated for 15 min. This incubation period was too short to affect iPFK-2 content (mRNA and protein) (Fig. 4). The incubation of FIG.3. Time-dependent activation of AMPK by hypoxia or oli- gomycin in resting monocytes. Resting monocytes were submitted resting and LPS-activated monocytes with oligomycin acti- for the indicated periods of time to normoxia (), hypoxia (95% N ,5% 2 vated AMPK (Fig. 5C). It also activated PFK-2 (Fig. 5A) and CO )(E), 1 M oligomycin (f), or 0.5 mM 5-aminoimidazole-4-carbox- increased Fru-2,6-P concentration (Fig. 5B), these changes amide riboside (Œ). The values are the means  S.E. for at least three only occurring in cells expressing iPFK-2. different preparations. The effect of hypoxia was also investigated and compared ing toward basal levels. The maximal effect of oligomycin on with that of oligomycin. Resting monocytes or monocytes acti- AMPK activity was 2–3-fold greater than that observed un- vated by LPS for 6 h, an incubation period sufficient to induce der hypoxia. The same difference was already observed in iPFK-2, were submitted to hypoxia or oligomycin. In resting perfused rat hearts where oligomycin induced a greater in- and LPS-activated monocytes, this hypoxic episode resulted in crease in the AMP:ATP ratio (8). 5-Aminoimidazole-4-carbox- AMPK activation, which was less pronounced as seen with amide riboside had no effect on AMPK activity in monocytes oligomycin (Fig. 6A). Hypoxia also activated PFK-2 but only in (Fig. 3) as previously reported for rat hearts and human em- LPS-activated cells (Fig. 6B). The hypoxia-induced activation bryonic kidney cells in which ZMP does not accumulate (8). of PFK-2 was less than that observed with oligomycin and iPFK-2 Is Induced by LPS in Monocytes—iPFK-2 expression paralleled AMPK activation (Fig. 6). was measured by reverse transcription-PCR in monocytes Oligomycin Stimulates PFK-1 Flux in Activated Mono- stimulated for up to 12 h with LPS and compared with the cytes—To evaluate the effect of oligomycin on glycolysis, the expression of the early response gene IL-1 taken as a control rate of detritiation of [3- H]glucose, an estimation of the flux of the proinflammatory activation of monocytes. IL-1 and through PFK-1 (29), was measured. The detritiation of iPFK-2 mRNA increased within 0.5 and 1 h (Fig. 4A). Both [3- H]glucose was measured in monocytes activated by LPS for levels of expression were maintained for 12 h as already re- up to 12h, incubated with or without oligomycin (Fig. 5D). A ported by Chesney et al. (11). The increase in iPFK-2 mRNA stimulation of glucose detritiation was observed in LPS-acti- corresponded to an increase in iPFK-2 protein detected by vated monocytes compared with resting monocytes. Moreover, immunoblotting with an anti-iPFK-2 antibody (Fig. 4B) and in oligomycin further increased the flux through PFK-1 in LPS- iPFK-2 activity (Fig. 5A). As expected, Fru-2,6-P concentra- activated monocytes but not in resting monocytes. A compari- tion increased in parallel (Fig. 5B). By contrast, LPS had no son of Fig. 5, A, B, and D, indicates that the increase in iPFK-2 Activation by AMPK in Activated Monocytes 30781 FIG.5. Effect of oligomycin on AMPK and PFK-2 activity, Fru-2,6-P content, and glycolysis in resting and LPS-activated monocytes. Resting monocytes (squares) or LPS-activated monocytes (circles) were incubated for the indicated periods of time. At the indicated time, monocytes were incubated without (open symbols) or with (filled symbols)1 M oligomycin. After 15-min incubation with oligomycin, the cells were lysed for measurement of PFK-2 activity (A), Fru-2,6-P content (B), and AMPK activity (C). D, after 5-min incubation with oligomycin, radioactive glucose was added, and the cells were further incubated for 15 min for measurement of glucose detritiation. The values are the means S.E. for 3–5 different preparations. *, significant effect (p  0.01) of LPS; #, significant effect (p  0.05) of oligomycin in LPS-activated cells. glycolytic flux is remarkably correlated with the increase in Fru-2,6-P content and PFK-2 activity. iPFK-2 Activation by Oligomycin Is Prevented by a Domi- nant-negative Mutant of AMPK—To test the involvement of AMPK in the activation of iPFK-2 by oligomycin, the effect of a dominant-negative mutant of AMPK (1DN AMPK) was inves- tigated in HEK-293 cells. These cells are known to be trans- fected with high efficiency and have been used previously to study the effect of AMPK on the heart PFK-2 activation (8). The transfection of HEK-293 cells with the iPFK-2 construct re- sulted in a 5–10-fold increase in total PFK-2 content (7  1 microunits/mg protein in untransfected cells to 67  15 mi- crounits/mg protein in cells transfected with 5 g of iPFK-2 DNA, n  6). Incubation with oligomycin for 15 min activated both endogenous AMPK (4-fold) and transfected iPFK-2 (2-fold) in a time-dependent manner (Fig. 7, A and B) but had no effect on endogenous PFK-2 (Fig. 7B). In addition, immunoblotting with the anti-pS466 antibody revealed a time-dependent phos- phorylation of iPFK-2 (Fig. 7C). We previously demonstrated the dominant-negative character of the 1DN AMPK construct by verifying that its transfection abolished the oligomycin- induced activation of both endogenous (Fig. 7A) (8) and trans- fected wild-type AMPK in HEK-293 cells (8). We investigated the effect of this dominant-negative AMPK on the activation of iPFK-2 by oligomycin. The co-expression of 1DN AMPK abol- ished both the phosphorylation (Fig. 7D) and activation of FIG.6. Activation of AMPK and PFK-2 by hypoxia in mono- iPFK-2 (Fig. 7B), demonstrating that AMPK mediates the oli- cytes. Resting monocytes or monocytes activated by LPS for 6 h were gomycin-induced activation of iPFK-2 in intact cells. incubated under normoxic conditions (open bars) or submitted to a 15-min incubation under hypoxic condition (95% N ,5%CO ) or with 1 2 2 DISCUSSION M oligomycin (filled bars) as indicated. After this incubation, the cells were lysed for measurement of AMPK (A) and PFK-2 (B) activity. The The results presented here suggest that AMPK and iPFK-2 values are the means  S.E. for four different preparations. *, signifi- are implicated in the stimulation of glycolysis by hypoxia in cant effect (p  0.01) of hypoxia or oligomycin compared with normoxic LPS-activated monocytes. The incubation of resting monocytes control; #, significant effect (p  0.05) of oligomycin compared with with LPS induced the expression of iPFK-2, a PFK-2 isoform hypoxia. 30782 iPFK-2 Activation by AMPK in Activated Monocytes activation and the subsequent phosphorylation and activation of iPFK-2 mediate the stimulation of glycolysis in LPS-acti- vated monocytes. While this work was in progress, a study of the control of glycolysis in macrophages during anoxia was published (31), and the results obtained are at variance with our results. Kawaguchi et al. (31) used the H36.12j macrophage immortal cell line as a model. These tumor-derived cells differ in several respects from the human monocytes used in our study. Similar to many other tumor cells (11), H36.12j cells constitutively overexpress iPFK-2, and their basal cyclic AMP concentration was 30 pmol/g cells, an abnormally high value for unstimu- lated cells. This elevated cyclic AMP concentration would be expected to fully activate cyclic AMP-dependent protein kinase in the resting cells. Although no direct in vitro evidence was presented, the authors (31) suggested that iPFK-2 was consti- tutively phosphorylated and activated by cyclic AMP-depend- ent protein kinase, thus explaining the elevated concentration of Fru-2,6-P under normoxic conditions. In these cells submit- ted to hypoxia, glycolysis was increased, whereas cyclic AMP concentration decreased, leading to a fall in Fru-2,6-P content supposedly mediated by a decrease in PFK-2 activity. From this study, it was concluded that Fru-2,6-P was not involved in the stimulation of glycolysis by hypoxia in these cells. By con- trast in human resting monocytes, iPFK-2 was not expressed, and the concentration of Fru-2,6-P was 2 pmol/mg protein, a concentration that was 10 times lower than that measured in H36.12j cells. Furthermore, we found that the concentration of cyclic AMP in resting monocytes (4 pmol/g cells) was 10-fold lower than in H36.12j cells and remained unchanged in LPS- activated monocytes. In these activated monocytes, which ex- press iPFK-2, hypoxia increased Fru-2,6-P concentrations to maximal values similar to those observed in normoxic H36.12j cells. Therefore, the conclusions drawn from the study of the response of H36.12j macrophages to hypoxia are probably not applicable to normal human monocytes. Few studies have shown striking effects of hypoxia on mono- FIG.7. 1DN AMPK prevents the oligomycin-induced phos- cytes in the absence of additional stimuli. Likewise, in our phorylation and activation of iPFK-2 in HEK-293 cells. HEK-293 experiments, hypoxia alone had no significant effect on glycol- cells were co-transfected with 5 g of iPFK-2 DNA and 5 gof 1DN AMPK DNA (f)or 1 wild-type AMPK DNA as control (). Cells were ysis but increased glycolytic flux after LPS activation. The fact incubated with 0.5 M oligomycin. At the indicated times, cells were that monocyte responses to hypoxia are enhanced by stimu- lysed for measurement of AMPK (A) and PFK-2 (B) activity. The trian- lants such as LPS and interferon- (32–34) reflects the impor- gles indicate endogenous PFK-2 activity in nontransfected cells. The tant role of these stimuli in coordinating monocyte activity. We values are the means  S.E. for four different preparations. C, immu- noblot of phosphorylated iPFK-2 (anti-pS466 antibody) on samples postulate that LPS primes monocytes to respond to hypoxia, taken at the indicated times from cells transfected with iPFK-2 and 1 which inevitably occurs in and around diseased tissues. The wild-type AMPK. NT, untransfected cells. D, immunoblot of phospho- subsequently expressed iPFK-2 could thus be activated by rylated iPFK-2 (anti-pS466 antibody) on samples taken from cells AMPK under hypoxic conditions, thereby furnishing ATP to transfected with iPFK-2 and 1DN AMPK as indicated and incubated boost the inflammatory response. with oligomycin for 10 min. Acknowledgments—We thank R. Bartrons who kindly provided resembling heart PFK-2, which was previously shown to be a iPFK-2 construct, D. Vertommen for help, and C. Beauloye for interest. target of AMPK (8). As observed with heart PFK-2, the phos- We also thank M. H. Rider for help in preparing the paper. phorylation of iPFK-2 by AMPK increased the V of PFK-2 max (2-fold) without changing the K for its substrates. This REFERENCES similarity suggests that phosphorylation occurs at the same 1. Hardie, D. G., and Carling, D. (1997) Eur. J. Biochem. 246, 259 –273 2. Hardie, D. G., and Hawley, S. A. (2001) Bioessays 23, 1112–1119 site, namely Ser-461 in iPFK-2. 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The Stimulation of Glycolysis by Hypoxia in Activated Monocytes Is Mediated by AMP-activated Protein Kinase and Inducible 6-Phosphofructo-2-kinase

Marsin, Anne-Sophie; Bouzin, Caroline; Bertrand, Luc; Hue, Louis
Journal of Biological Chemistry – Aug 1, 2002
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 34, Issue of August 23, pp. 30778 –30783, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Stimulation of Glycolysis by Hypoxia in Activated Monocytes Is Mediated by AMP-activated Protein Kinase and Inducible 6-Phosphofructo-2-kinase* Received for publication, May 28, 2002 Published, JBC Papers in Press, June 13, 2002, DOI 10.1074/jbc.M205213200 Anne-Sophie Marsin‡, Caroline Bouzin§, Luc Bertrand¶, and Louis Hue From the Hormone and Metabolic Research Unit, University of Louvain Medical School and Christian de Duve International Institute of Cellular and Molecular Pathology, B-1200 Brussels, Belgium The activation of monocytes involves a stimulation of heterotrimeric protein composed of a catalytic () and two glycolysis, release of potent inflammatory mediators, regulatory (, ) subunits (3, 4). It is considered as a “metabolic and alterations in gene expression. All of these pro- master switch” (5), which inactivates key targets that control cesses are known to be further increased under hypoxic anabolic pathways, thereby conserving ATP (1, 2). AMPK is conditions. The activated monocytes express inducible also implicated in the stimulation of glucose uptake that occurs 6-phosphofructo-2-kinase (iPFK-2), which synthesizes in contracting muscle (6, 7). fructose 2,6-bisphosphate, a stimulator of glycolysis. We showed previously that AMPK phosphorylates Ser-466 of During ischemia, AMP-activated protein kinase (AMPK) heart 6-phosphofructo-2-kinase (PFK-2), leading to its activa- activates the homologous heart 6-phosphofructo-2-ki- tion (8). This phenomenon participates in the well known stim- nase isoform by phosphorylating its Ser-466. Here, we ulation of glycolysis by ischemia in the heart. PFK-2/fructose- studied the involvement of AMPK and iPFK-2 in the 2,6-bisphosphatase is a bifunctional enzyme catalyzing the stimulation of glycolysis in activated monocytes under synthesis and degradation of fructose 2,6-bisphosphate (Fru- hypoxia. iPFK-2 was phosphorylated on the homologous 2,6-P ), the most potent stimulator of 6-phosphofructo-1-kinase serine (Ser-461) and activated by AMPK in vitro. The and hence glycolysis. Tissue-specific isozymes of PFK-2/fruc- activation of human monocytes by lipopolysaccharide tose-2,6-bisphosphatase have been identified in mammals. induced iPFK-2 expression and increased fructose 2,6- They possess a conserved catalytic core flanked by variable N- bisphosphate content and glycolysis. The incubation of and C-terminal regulatory domains, and they differ in tissue activated monocytes with oligomycin, an inhibitor of distribution and response to phosphorylation by protein ki- oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, nases (for review see Refs. 9 and 10). Chesney et al. (11) fructose 2,6-bisphosphate content, and glycolysis. In cul- characterized a PFK-2 isozyme, which was induced by proin- tured human embryonic kidney 293 cells, the expression flammatory stimuli and was therefore termed inducible PFK-2 of a dominant-negative AMPK prevented both the acti- (iPFK-2). iPFK-2 is identical to the previously described pla- vation and phosphorylation of co-transfected iPFK-2 by cental isoform (12) and is homologous to heart PFK-2. These oligomycin. It is concluded that the stimulation of gly- isozymes are characterized by their relative PFK-2/fructose- colysis by hypoxia in activated monocytes requires the 2,6-bisphosphatase activities. Under physiological conditions, phosphorylation and activation of iPFK-2 by AMPK. their PFK-2 activity is 100-fold that of their fructose-2,6- bisphosphatase activity (10), indicating that they mainly act as a kinase. The C-terminal regulatory domain of iPFK-2 contains Energy deprivation (e.g. hypoxia and inhibitors of oxidative Ser-461, which lies within a favorable consensus for phospho- phosphorylation such as oligomycin) leads to the activation of 449 467 rylation by AMPK ( KGPNPLMRRNSVTPLAS ), similar the AMP-activated protein kinase (AMPK) via an increase in to that surrounding Ser-466 of heart PFK-2 ( KSQTPVRM- the AMP:ATP ratio (for review see Refs. 1 and 2). AMPK is a RRNSFTPLSS ). A synthetic peptide corresponding to the sequence surrounding Ser-461 in iPFK-2 was indeed shown to * This work was supported by the Belgian Federal Program Interuni- be phosphorylated by AMPK in vitro (8). versity Poles of attraction (P4/23), the Directorate General Higher iPFK-2 is constitutively expressed in several human cancer Education and Scientific Program, French Community of Belgium, The cell lines. This isozyme has also been shown to be induced in Fund for Medical Scientific Research (Belgium), and by European monocytes activated by lipopolysaccharide (LPS) (11), a com- Union contract QLG1-CT-2001-01488 (AMPDIAMET). The costs of pub- lication of this article were defrayed in part by the payment of page ponent of the outer membrane of Gram-negative bacteria, charges. This article must therefore be hereby marked “advertisement” which triggers and mimics an inflammatory response. The in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. response of monocytes to LPS includes the production of cyto- ‡ Research fellow of the National Fund for Scientific Research kines and chemokines, the release of arachidonic acid metabo- (Belgium). § Supported by the Fund for Scientific Research in Industry and lites, and the generation of reactive oxygen species and nitro- Agriculture (Belgium). gen monoxide (13–15). Monocyte activation consumes energy, ¶ Supported by the French Community of Belgium. is glucose-dependent, and involves a stimulation of glycolysis To whom correspondence should be addressed: HORM Unit, ICP- (16 –18). Moreover, in diseased tissues, monocytes are known to UCL 7529, Avenue Hippocrate, 75, B-1200 Brussels, Belgium. Tel.: 32-2-764-74-85; Fax: 32-2-764-75-07; E-mail: [email protected]. accumulate in poorly vascularized hypoxic sites (19, 20). Mono- The abbreviations used are: AMPK, AMP-activated protein kinase; cytes remain functional under such adverse conditions by al- PFK-2, 6-phosphofructo-2-kinase; Fru-2,6-P , fructose 2,6-bisphos- tering gene expression and by switching to anaerobic glycolysis phate; iPFK-2, Inducible 6-phosphofructo-2-kinase; DN, dominant-neg- for ATP production. ative; LPS, lipopolysaccharide; HEK, human embryonic kidney; IL, interleukin. The mechanisms by which glycolysis is stimulated synergis- 30778 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. iPFK-2 Activation by AMPK in Activated Monocytes 30779 tically by LPS and hypoxia are still unknown. We tested whether this synergism results from the phosphorylation and activation of iPFK-2 by AMPK in hypoxia. EXPERIMENTAL PROCEDURES Materials—The construct encoding recombinant polyhistidine- tagged iPFK-2 (21) was a generous gift of R. Bartrons (Barcelona, Spain). Recombinant iPFK-2 was purified (22) from human embryonic kidney (HEK)-293 cells transfected with this construct. Liver AMPK was purified as described previously (23). Wild-type and dominant- negative 1 AMPK constructs were described previously (24). Rabbit polyclonal anti-phospho-S466 (8) and anti-iPFK-2 (11) antibodies were raised against synthetic peptides. These peptides and the SAMS pep- tide (25) were synthesized by V. Stroobant (Ludwig Institute for Cancer Research, Brussels, Belgium). In Vitro Studies—For the measurement of kinetic properties, puri- fied iPFK-2 and heart PFK-2 were incubated with 1 mM MgATP and AMPK at 30 °C (26), and aliquots were taken for PFK-2 assay (27). For determination of phosphorylation, iPFK-2 was incubated with 0.1 mM Mg[- P]ATP (1000 cpm/pmol) and AMPK. Aliquots were taken and analyzed as described previously (26). The amount of purified enzymes used in each experiment is given in the figure legends. Cell Culture—Peripheral blood mononuclear cells were isolated by centrifugation of human whole blood through a density gradient of Ficoll-Paque (Amersham Biosciences) and cultured in Petri dishes (10-cm diameter, 10  10 monocytes/dish) in RPMI 1640 medium with 10% (v/v) fetal calf serum (11). After2hof culture, the medium and FIG.1. Time-dependent changes in phosphorylation and activ- nonadherent cells were removed by aspiration, and the remaining ad- ity of iPFK-2 incubated with AMPK. A, purified iPFK-2 (0.15 mg/ herent monocytes were incubated without (resting) or with (activated) ml) was incubated with 0.1 mM Mg[- P]ATP and purified AMPK (0.6 1 g/ml LPS (Escherichia coli 0111:B4, Sigma). The percentage of unit/ml) with (Œ) or without (‚) AMP (0.2 mM) in a final volume of 50 l. monocytes in the cultures was 85% as determined by fluorescence- Controls (E) were incubated without AMPK. At the indicated times, activated cell sorter analysis for CD14 expression. The cells were incu- aliquots (5 l) were removed for SDS-PAGE and screened using Phos- bated under the conditions and the periods of time indicated in the 32 phorImager for measurement of P incorporation. B, same protocol as figure legends. Following incubation, the medium was aspirated, and in A with 1 mM nonradioactive MgATP in a final volume of 0.1 ml. At the cells were immediately lysed in 0.8 ml of ice-cold lysis buffer (8) for the indicated times, aliquots (10 l) were removed for PFK-2 assay. The enzyme assays or in 0.5 ml of 50 mM NaOH for Fru-2,6-P determina- 2 results are the means  S.E. for three separate experiments. tion. Total RNA was isolated with the High Pure RNA isolation kit (Roche Molecular Biochemicals). HEK-293 cells were cultured in Dul- with bovine serum albumin as a standard. Kinetic constants were becco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf calculated by computer fitting of the data to a hyperbola describing the serum. The transfection protocol was a modified calcium phosphate Michaelis-Menten equation by nonlinear least square regression. One procedure (22). The cells were incubated under the conditions indicated unit of enzyme activity corresponds to the formation of 1 mol (PFK-2) in the figure legends and lysed in 0.8 ml of ice-cold lysis buffer (8). or 1 nmol (AMPK) of product/min under the assay conditions. Unless otherwise stated, the cells were cultured in normoxic conditions (95% O ,5%CO ). RESULTS 2 2 Reverse Transcription-PCR Analysis—RNA was reverse transcribed Phosphorylation and Activation of iPFK-2 by AMPK in for1hat37 °C with random primers, and cDNA fragments that Vitro—Purified iPFK-2 was phosphorylated by AMPK with a correspond to interleukin (IL)-1 (271 bp) and iPFK-2 (140 bp) were stoichiometry close to 0.7 mol of phosphate incorporated/mol of amplified with the primers described previously (11). The cycling pro- gram used was 95 °C for 30 s, 55 °C for 30 s, and 72 °Cfor45sin22 enzyme subunit, indicating phosphorylation at one site. The cycles. As a control, -actin cDNA fragment (612 bp) was amplified with rate and the extent of phosphorylation were stimulated by the following primers: 5-GGCATCGTGATGGACTCCG-3 and 5-GCT- AMP (Fig. 1A), and phosphorylation correlated with PFK-2 GGAAGGTGGACAGCGA-3 (95 °C for 30 s, 58 °C for 30 s, and 72 °C for activation (Fig. 1B). The treatment with AMPK led to a 2.5-fold 45 s in 22 cycles). The amplification cycle number was varied initially to increase in V of PFK-2 with no significant change in K for max m establish unsaturating amplification response. The displayed cycle fructose 6-phosphate or MgATP (Table I). These changes in number allows the illustration of representative differences in the amount of cDNA present. kinetic properties resemble those seen after the phosphoryla- Enzyme and Metabolite Measurements—AMPK (23) and PFK-2 (27) tion of heart PFK-2 by AMPK (Table I) (8). The similarity activities were assayed in a 10 and 20% (w/v) polyethylene glycol 6000 among the sequences surrounding Ser-461 of iPFK-2 and Ser- precipitate, respectively. Fru-2,6-P was measured as described previ- 466 of heart PFK-2 led us to use the antibody raised against the ously (28). 3 phosphorylated Ser-466 of heart PFK-2 (anti-pS466) to study Measurement of [3- H]Glucose Detritiation—The glycolytic flux the phosphorylation of Ser-461 in iPFK2. Immunoblotting with through PFK-1 was estimated by the rate of detritiation of [3- H]glu- cose (29). Monocytes were cultured in 5 ml of RPMI 1640 medium this antibody showed that AMPK phosphorylated Ser-461 of containing 10 mM glucose and activated by LPS for the indicated times. iPFK-2 (Fig. 2). Cells were incubated with oligomycin for 5 min prior to the addition of AMPK Is Activated by Oligomycin and Hypoxia in Resting tracer amounts (0.3 Ci/ml) of radioactive glucose. Samples were re- Monocytes—To activate AMPK, resting monocytes were incu- moved periodically (0 –15 min after the addition of glucose) from the 3 bated under hypoxic conditions or with two known activators of medium to measure the formation of H O. These samples were depro- AMPK, namely 5-aminoimidazole-4-carboxamide riboside (0.5 teinized in 1 M ice-cold perchloric acid. After neutralization and centrif- ugation (10,000  g, 5 min, 4 °C), H O was separated from radioactive mM), a precursor of the AMP analog ZMP or oligomycin (1 M), glucose (30). The release of H O was linear over the 15-min experi- an inhibitor of oxidative phosphorylation. Basal AMPK activity mental period, and the rate was calculated from the average detritia- was low and similar to that measured in normoxic perfused tion rate over 15 min and expressed as nanomoles of glucose detritiated hearts or cells in culture (8) and remained unchanged over the per minute per milligram of protein. This rate may give an underesti- incubation period (Fig. 3). By contrast, AMPK activity progres- mation of the net glycolytic flux because of an incomplete detritiation of sively increased during incubation with oligomycin or hypoxia the tracer (29). Other Methods—Proteins was estimated by the method of Bradford to reach maximal values between 10 and 20 min before decreas- 30780 iPFK-2 Activation by AMPK in Activated Monocytes TABLE I. Effects of AMPK on the kinetic properties of iPFK-2 and heart PFK-2 Purified iPFK-2 or heart PFK-2 (0.1 mg/ml) was incubated with (w/) or without (w/o) AMPK (0.6 unit/ml) with 0.2 mM AMP and 1 mM MgATP in a final volume of 100 lat30°C for 30 min. Aliquots (10 l) were taken for the measurement of PFK-2 activity. PFK-2 was measured at pH 7.1 in the presence of 5 mM MgATP and concentrations of fructose 6-phosphate up to 10 times the K or in the presence of 1 mM fructose 6-phosphate and concentrations of MgATP up to 10 times the K The results are the means  S.E. for three different experiments. iPFK-2 heart PFK-2 w/o w/o w/ AMPK w/ AMPK AMPK AMPK a a V (milliunits/mg protein) 10 125  2 50  5 125  10 max K for fructose 6- phosphate (M) 53 250 457 346  1 K for MgATP (M) 650  32 582  37 930  12 745  5 p  0.01 in comparison with sample incubated without AMPK. FIG.2. Immunoblot of inducible and heart PFK-2 phosphoryl- ated by AMPK with the anti-pS466 antibody. Purified PFK-2 (0.15 mg/ml) was incubated with AMPK (0.6 unit/ml), AMP (0.2 mM), and 1 mM MgATP in a final volume of 20 lat30 °C. After 30 min, samples were removed for SDS-PAGE and immunoblotted with the anti-pS466 antibody. FIG.4. Time-dependent induction of IL-1 and iPFK-2 by LPS in monocytes. A, reverse transcription-PCR analysis of IL-1, iPFK-2, and -actin mRNAs obtained from resting or LPS-activated monocytes. The effects of oligomycin (1 M, 15 min) on iPFK-2 and IL-1 mRNA was analyzed in monocytes cultured for 6 h. B, immunoblot analysis with anti-iPFK-2 antibody on 10 g of protein from extracts of resting or LPS-activated monocytes. The effect of oligomycin (1 M, 15 min) was also verified. effect on AMPK activity (Fig. 5C). Hypoxia and Oligomycin Activate iPFK-2 in LPS-stimulated Monocytes—The effects of oligomycin were tested in cells incu- bated for 15 min. This incubation period was too short to affect iPFK-2 content (mRNA and protein) (Fig. 4). The incubation of FIG.3. Time-dependent activation of AMPK by hypoxia or oli- gomycin in resting monocytes. Resting monocytes were submitted resting and LPS-activated monocytes with oligomycin acti- for the indicated periods of time to normoxia (), hypoxia (95% N ,5% 2 vated AMPK (Fig. 5C). It also activated PFK-2 (Fig. 5A) and CO )(E), 1 M oligomycin (f), or 0.5 mM 5-aminoimidazole-4-carbox- increased Fru-2,6-P concentration (Fig. 5B), these changes amide riboside (Œ). The values are the means  S.E. for at least three only occurring in cells expressing iPFK-2. different preparations. The effect of hypoxia was also investigated and compared ing toward basal levels. The maximal effect of oligomycin on with that of oligomycin. Resting monocytes or monocytes acti- AMPK activity was 2–3-fold greater than that observed un- vated by LPS for 6 h, an incubation period sufficient to induce der hypoxia. The same difference was already observed in iPFK-2, were submitted to hypoxia or oligomycin. In resting perfused rat hearts where oligomycin induced a greater in- and LPS-activated monocytes, this hypoxic episode resulted in crease in the AMP:ATP ratio (8). 5-Aminoimidazole-4-carbox- AMPK activation, which was less pronounced as seen with amide riboside had no effect on AMPK activity in monocytes oligomycin (Fig. 6A). Hypoxia also activated PFK-2 but only in (Fig. 3) as previously reported for rat hearts and human em- LPS-activated cells (Fig. 6B). The hypoxia-induced activation bryonic kidney cells in which ZMP does not accumulate (8). of PFK-2 was less than that observed with oligomycin and iPFK-2 Is Induced by LPS in Monocytes—iPFK-2 expression paralleled AMPK activation (Fig. 6). was measured by reverse transcription-PCR in monocytes Oligomycin Stimulates PFK-1 Flux in Activated Mono- stimulated for up to 12 h with LPS and compared with the cytes—To evaluate the effect of oligomycin on glycolysis, the expression of the early response gene IL-1 taken as a control rate of detritiation of [3- H]glucose, an estimation of the flux of the proinflammatory activation of monocytes. IL-1 and through PFK-1 (29), was measured. The detritiation of iPFK-2 mRNA increased within 0.5 and 1 h (Fig. 4A). Both [3- H]glucose was measured in monocytes activated by LPS for levels of expression were maintained for 12 h as already re- up to 12h, incubated with or without oligomycin (Fig. 5D). A ported by Chesney et al. (11). The increase in iPFK-2 mRNA stimulation of glucose detritiation was observed in LPS-acti- corresponded to an increase in iPFK-2 protein detected by vated monocytes compared with resting monocytes. Moreover, immunoblotting with an anti-iPFK-2 antibody (Fig. 4B) and in oligomycin further increased the flux through PFK-1 in LPS- iPFK-2 activity (Fig. 5A). As expected, Fru-2,6-P concentra- activated monocytes but not in resting monocytes. A compari- tion increased in parallel (Fig. 5B). By contrast, LPS had no son of Fig. 5, A, B, and D, indicates that the increase in iPFK-2 Activation by AMPK in Activated Monocytes 30781 FIG.5. Effect of oligomycin on AMPK and PFK-2 activity, Fru-2,6-P content, and glycolysis in resting and LPS-activated monocytes. Resting monocytes (squares) or LPS-activated monocytes (circles) were incubated for the indicated periods of time. At the indicated time, monocytes were incubated without (open symbols) or with (filled symbols)1 M oligomycin. After 15-min incubation with oligomycin, the cells were lysed for measurement of PFK-2 activity (A), Fru-2,6-P content (B), and AMPK activity (C). D, after 5-min incubation with oligomycin, radioactive glucose was added, and the cells were further incubated for 15 min for measurement of glucose detritiation. The values are the means S.E. for 3–5 different preparations. *, significant effect (p  0.01) of LPS; #, significant effect (p  0.05) of oligomycin in LPS-activated cells. glycolytic flux is remarkably correlated with the increase in Fru-2,6-P content and PFK-2 activity. iPFK-2 Activation by Oligomycin Is Prevented by a Domi- nant-negative Mutant of AMPK—To test the involvement of AMPK in the activation of iPFK-2 by oligomycin, the effect of a dominant-negative mutant of AMPK (1DN AMPK) was inves- tigated in HEK-293 cells. These cells are known to be trans- fected with high efficiency and have been used previously to study the effect of AMPK on the heart PFK-2 activation (8). The transfection of HEK-293 cells with the iPFK-2 construct re- sulted in a 5–10-fold increase in total PFK-2 content (7  1 microunits/mg protein in untransfected cells to 67  15 mi- crounits/mg protein in cells transfected with 5 g of iPFK-2 DNA, n  6). Incubation with oligomycin for 15 min activated both endogenous AMPK (4-fold) and transfected iPFK-2 (2-fold) in a time-dependent manner (Fig. 7, A and B) but had no effect on endogenous PFK-2 (Fig. 7B). In addition, immunoblotting with the anti-pS466 antibody revealed a time-dependent phos- phorylation of iPFK-2 (Fig. 7C). We previously demonstrated the dominant-negative character of the 1DN AMPK construct by verifying that its transfection abolished the oligomycin- induced activation of both endogenous (Fig. 7A) (8) and trans- fected wild-type AMPK in HEK-293 cells (8). We investigated the effect of this dominant-negative AMPK on the activation of iPFK-2 by oligomycin. The co-expression of 1DN AMPK abol- ished both the phosphorylation (Fig. 7D) and activation of FIG.6. Activation of AMPK and PFK-2 by hypoxia in mono- iPFK-2 (Fig. 7B), demonstrating that AMPK mediates the oli- cytes. Resting monocytes or monocytes activated by LPS for 6 h were gomycin-induced activation of iPFK-2 in intact cells. incubated under normoxic conditions (open bars) or submitted to a 15-min incubation under hypoxic condition (95% N ,5%CO ) or with 1 2 2 DISCUSSION M oligomycin (filled bars) as indicated. After this incubation, the cells were lysed for measurement of AMPK (A) and PFK-2 (B) activity. The The results presented here suggest that AMPK and iPFK-2 values are the means  S.E. for four different preparations. *, signifi- are implicated in the stimulation of glycolysis by hypoxia in cant effect (p  0.01) of hypoxia or oligomycin compared with normoxic LPS-activated monocytes. The incubation of resting monocytes control; #, significant effect (p  0.05) of oligomycin compared with with LPS induced the expression of iPFK-2, a PFK-2 isoform hypoxia. 30782 iPFK-2 Activation by AMPK in Activated Monocytes activation and the subsequent phosphorylation and activation of iPFK-2 mediate the stimulation of glycolysis in LPS-acti- vated monocytes. While this work was in progress, a study of the control of glycolysis in macrophages during anoxia was published (31), and the results obtained are at variance with our results. Kawaguchi et al. (31) used the H36.12j macrophage immortal cell line as a model. These tumor-derived cells differ in several respects from the human monocytes used in our study. Similar to many other tumor cells (11), H36.12j cells constitutively overexpress iPFK-2, and their basal cyclic AMP concentration was 30 pmol/g cells, an abnormally high value for unstimu- lated cells. This elevated cyclic AMP concentration would be expected to fully activate cyclic AMP-dependent protein kinase in the resting cells. Although no direct in vitro evidence was presented, the authors (31) suggested that iPFK-2 was consti- tutively phosphorylated and activated by cyclic AMP-depend- ent protein kinase, thus explaining the elevated concentration of Fru-2,6-P under normoxic conditions. In these cells submit- ted to hypoxia, glycolysis was increased, whereas cyclic AMP concentration decreased, leading to a fall in Fru-2,6-P content supposedly mediated by a decrease in PFK-2 activity. From this study, it was concluded that Fru-2,6-P was not involved in the stimulation of glycolysis by hypoxia in these cells. By con- trast in human resting monocytes, iPFK-2 was not expressed, and the concentration of Fru-2,6-P was 2 pmol/mg protein, a concentration that was 10 times lower than that measured in H36.12j cells. Furthermore, we found that the concentration of cyclic AMP in resting monocytes (4 pmol/g cells) was 10-fold lower than in H36.12j cells and remained unchanged in LPS- activated monocytes. In these activated monocytes, which ex- press iPFK-2, hypoxia increased Fru-2,6-P concentrations to maximal values similar to those observed in normoxic H36.12j cells. Therefore, the conclusions drawn from the study of the response of H36.12j macrophages to hypoxia are probably not applicable to normal human monocytes. Few studies have shown striking effects of hypoxia on mono- FIG.7. 1DN AMPK prevents the oligomycin-induced phos- cytes in the absence of additional stimuli. Likewise, in our phorylation and activation of iPFK-2 in HEK-293 cells. HEK-293 experiments, hypoxia alone had no significant effect on glycol- cells were co-transfected with 5 g of iPFK-2 DNA and 5 gof 1DN AMPK DNA (f)or 1 wild-type AMPK DNA as control (). Cells were ysis but increased glycolytic flux after LPS activation. The fact incubated with 0.5 M oligomycin. At the indicated times, cells were that monocyte responses to hypoxia are enhanced by stimu- lysed for measurement of AMPK (A) and PFK-2 (B) activity. The trian- lants such as LPS and interferon- (32–34) reflects the impor- gles indicate endogenous PFK-2 activity in nontransfected cells. The tant role of these stimuli in coordinating monocyte activity. We values are the means  S.E. for four different preparations. C, immu- noblot of phosphorylated iPFK-2 (anti-pS466 antibody) on samples postulate that LPS primes monocytes to respond to hypoxia, taken at the indicated times from cells transfected with iPFK-2 and 1 which inevitably occurs in and around diseased tissues. The wild-type AMPK. NT, untransfected cells. 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Published: Aug 1, 2002

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