The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase.

The activation of monocytes involves a stimulation of glycolysis, release of potent inflammatory mediators, and alterations in gene expression. All of these processes are known to be further increased under hypoxic conditions. The activated monocytes express inducible 6-phosphofructo-2-kinase (iPFK-2), which synthesizes fructose 2,6-bisphosphate, a stimulator of glycolysis. During ischemia, AMP-activated protein kinase (AMPK) activates the homologous heart 6-phosphofructo-2-kinase isoform by phosphorylating its Ser-466. Here, we studied the involvement of AMPK and iPFK-2 in the stimulation of glycolysis in activated monocytes under hypoxia. iPFK-2 was phosphorylated on the homologous serine (Ser-461) and activated by AMPK in vitro. The activation of human monocytes by lipopolysaccharide induced iPFK-2 expression and increased fructose 2,6-bisphosphate content and glycolysis. The incubation of activated monocytes with oligomycin, an inhibitor of oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, fructose 2,6-bisphosphate content, and glycolysis. In cultured human embryonic kidney 293 cells, the expression of a dominant-negative AMPK prevented both the activation and phosphorylation of co-transfected iPFK-2 by oligomycin. It is concluded that the stimulation of glycolysis by hypoxia in activated monocytes requires the phosphorylation and activation of iPFK-2 by AMPK.

iPFK-2 is constitutively expressed in several human cancer cell lines. This isozyme has also been shown to be induced in monocytes activated by lipopolysaccharide (LPS) (11), a component of the outer membrane of Gram-negative bacteria, which triggers and mimics an inflammatory response. The response of monocytes to LPS includes the production of cytokines and chemokines, the release of arachidonic acid metabolites, and the generation of reactive oxygen species and nitrogen monoxide (13)(14)(15). Monocyte activation consumes energy, is glucose-dependent, and involves a stimulation of glycolysis (16 -18). Moreover, in diseased tissues, monocytes are known to accumulate in poorly vascularized hypoxic sites (19,20). Monocytes remain functional under such adverse conditions by altering gene expression and by switching to anaerobic glycolysis for ATP production.
The mechanisms by which glycolysis is stimulated synergis-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.
In Vitro Studies-For the measurement of kinetic properties, purified 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 Mg⅐[␥-32 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 ϫ 10 6 monocytes/dish) in RPMI 1640 medium with 10% (v/v) fetal calf serum (11). After 2 h of culture, the medium and nonadherent cells were removed by aspiration, and the remaining adherent monocytes were incubated without (resting) or with (activated) 1 g/ml LPS (Escherichia coli 0111:B4, Sigma). The percentage of monocytes in the cultures was Ͼ85% as determined by fluorescenceactivated cell sorter analysis for CD14 expression. The cells were incubated under the conditions and the periods of time indicated in the figure legends. Following incubation, the medium was aspirated, and the cells were immediately lysed in 0.8 ml of ice-cold lysis buffer (8) for enzyme assays or in 0.5 ml of 50 mM NaOH for Fru-2,6-P 2 determination. Total RNA was isolated with the High Pure RNA isolation kit (Roche Molecular Biochemicals). HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum. The transfection protocol was a modified calcium phosphate procedure (22). The cells were incubated under the conditions indicated in the figure legends and lysed in 0.8 ml of ice-cold lysis buffer (8). Unless otherwise stated, the cells were cultured in normoxic conditions (95% O 2 , 5% CO 2 ).
Reverse Transcription-PCR Analysis-RNA was reverse transcribed for 1 h at 37°C with random primers, and cDNA fragments that correspond to interleukin (IL)-1␤ (271 bp) and iPFK-2 (140 bp) were amplified with the primers described previously (11). The cycling program used was 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s in 22 cycles. As a control, ␤-actin cDNA fragment (612 bp) was amplified with the following primers: 5Ј-GGCATCGTGATGGACTCCG-3Ј and 5Ј-GCT-GGAAGGTGGACAGCGA-3Ј (95°C for 30 s, 58°C for 30 s, and 72°C for 45 s in 22 cycles). The amplification cycle number was varied initially to establish unsaturating amplification response. The displayed cycle number allows the illustration of representative differences in the amount of cDNA present.

Measurement of [3-3 H]Glucose
Detritiation-The glycolytic flux through PFK-1 was estimated by the rate of detritiation of [3-3 H]glucose (29). Monocytes were cultured in 5 ml of RPMI 1640 medium containing 10 mM glucose and activated by LPS for the indicated times. Cells were incubated with oligomycin for 5 min prior to the addition of tracer amounts (0.3 Ci/ml) of radioactive glucose. Samples were removed periodically (0 -15 min after the addition of glucose) from the medium to measure the formation of 3 H 2 O. These samples were deproteinized in 1 M ice-cold perchloric acid. After neutralization and centrifugation (10,000 ϫ g, 5 min, 4°C), 3 H 2 O was separated from radioactive glucose (30). The release of 3 H 2 O was linear over the 15-min experimental period, and the rate was calculated from the average detritiation rate over 15 min and expressed as nanomoles of glucose detritiated per minute per milligram of protein. This rate may give an underestimation of the net glycolytic flux because of an incomplete detritiation of the tracer (29).
Other Methods-Proteins was estimated by the method of Bradford with bovine serum albumin as a standard. Kinetic constants were calculated by computer fitting of the data to a hyperbola describing the Michaelis-Menten equation by nonlinear least square regression. One unit of enzyme activity corresponds to the formation of 1 mol (PFK-2) or 1 nmol (AMPK) of product/min under the assay conditions.

RESULTS
Phosphorylation and Activation of iPFK-2 by AMPK in Vitro-Purified iPFK-2 was phosphorylated by AMPK with a stoichiometry close to 0.7 mol of phosphate incorporated/mol of enzyme subunit, indicating phosphorylation at one site. The rate and the extent of phosphorylation were stimulated by AMP (Fig. 1A), and phosphorylation correlated with PFK-2 activation (Fig. 1B). The treatment with AMPK led to a 2.5-fold increase in V max of PFK-2 with no significant change in K m for fructose 6-phosphate or MgATP (Table I). These changes in kinetic properties resemble those seen after the phosphorylation of heart PFK-2 by AMPK (Table I) (8). The similarity among the sequences surrounding Ser-461 of iPFK-2 and Ser-466 of heart PFK-2 led us to use the antibody raised against the phosphorylated Ser-466 of heart PFK-2 (anti-pS466) to study the phosphorylation of Ser-461 in iPFK2. Immunoblotting with this antibody showed that AMPK phosphorylated Ser-461 of iPFK-2 (Fig. 2).
AMPK Is Activated by Oligomycin and Hypoxia in Resting Monocytes-To activate AMPK, resting monocytes were incubated under hypoxic conditions or with two known activators of AMPK, namely 5-aminoimidazole-4-carboxamide riboside (0.5 mM), a precursor of the AMP analog ZMP or oligomycin (1 M), an inhibitor of oxidative phosphorylation. Basal AMPK activity was low and similar to that measured in normoxic perfused hearts or cells in culture (8) and remained unchanged over the incubation period (Fig. 3). By contrast, AMPK activity progressively increased during incubation with oligomycin or hypoxia to reach maximal values between 10 and 20 min before decreas-

iPFK-2 Activation by AMPK in Activated Monocytes
ing toward basal levels. The maximal effect of oligomycin on AMPK activity was ϳ2-3-fold greater than that observed under hypoxia. The same difference was already observed in perfused rat hearts where oligomycin induced a greater increase in the AMP:ATP ratio (8). 5-Aminoimidazole-4-carboxamide riboside had no effect on AMPK activity in monocytes (Fig. 3) as previously reported for rat hearts and human embryonic kidney cells in which ZMP does not accumulate (8).
iPFK-2 Is Induced by LPS in Monocytes-iPFK-2 expression was measured by reverse transcription-PCR in monocytes stimulated for up to 12 h with LPS and compared with the expression of the early response gene IL-1␤ taken as a control of the proinflammatory activation of monocytes. IL-1␤ and iPFK-2 mRNA increased within 0.5 and 1 h (Fig. 4A). Both levels of expression were maintained for 12 h as already reported by Chesney et al. (11). The increase in iPFK-2 mRNA corresponded to an increase in iPFK-2 protein detected by immunoblotting with an anti-iPFK-2 antibody (Fig. 4B) and in iPFK-2 activity (Fig. 5A). As expected, Fru-2,6-P 2 concentration increased in parallel (Fig. 5B). By contrast, LPS had no effect on AMPK activity (Fig. 5C).
The effect of hypoxia was also investigated and compared with that of oligomycin. Resting monocytes or monocytes activated by LPS for 6 h, an incubation period sufficient to induce iPFK-2, were submitted to hypoxia or oligomycin. In resting and LPS-activated monocytes, this hypoxic episode resulted in AMPK activation, which was less pronounced as seen with oligomycin (Fig. 6A). Hypoxia also activated PFK-2 but only in LPS-activated cells (Fig. 6B). The hypoxia-induced activation of PFK-2 was less than that observed with oligomycin and paralleled AMPK activation (Fig. 6).
Oligomycin Stimulates PFK-1 Flux in Activated Monocytes-To evaluate the effect of oligomycin on glycolysis, the rate of detritiation of [3-3 H]glucose, an estimation of the flux through PFK-1 (29), was measured. The detritiation of [3-3 H]glucose was measured in monocytes activated by LPS for up to 12h, incubated with or without oligomycin (Fig. 5D). A stimulation of glucose detritiation was observed in LPS-activated monocytes compared with resting monocytes. Moreover, oligomycin further increased the flux through PFK-1 in LPSactivated monocytes but not in resting monocytes. A comparison of Fig. 5, A, B, and D, indicates that the increase in 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 l at 30°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 m or in the presence of 1 mM fructose 6-phosphate and concentrations of MgATP up to 10

iPFK-2 Activation by AMPK in Activated Monocytes
glycolytic flux is remarkably correlated with the increase in Fru-2,6-P 2 content and PFK-2 activity.
iPFK-2 Activation by Oligomycin Is Prevented by a Dominant-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 investigated in HEK-293 cells. These cells are known to be transfected 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 resulted in a 5-10-fold increase in total PFK-2 content (7 Ϯ 1 microunits/mg protein in untransfected cells to 67 Ϯ 15 microunits/mg protein in cells transfected with 5 g of iPFK-2 DNA, n ϭ 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 phosphorylation of iPFK-2 (Fig. 7C). We previously demonstrated the dominant-negative character of the ␣1DN AMPK construct by verifying that its transfection abolished the oligomycininduced activation of both endogenous (Fig. 7A) (8) and transfected 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 abolished both the phosphorylation (Fig. 7D) and activation of iPFK-2 (Fig. 7B), demonstrating that AMPK mediates the oligomycin-induced activation of iPFK-2 in intact cells. DISCUSSION The results presented here suggest that AMPK and iPFK-2 are implicated in the stimulation of glycolysis by hypoxia in LPS-activated monocytes. The incubation of resting monocytes with LPS induced the expression of iPFK-2, a PFK-2 isoform

iPFK-2 Activation by AMPK in Activated Monocytes
resembling heart PFK-2, which was previously shown to be a target of AMPK (8). As observed with heart PFK-2, the phosphorylation of iPFK-2 by AMPK increased the V max of PFK-2 (ϳ2-fold) without changing the K m for its substrates. This similarity suggests that phosphorylation occurs at the same site, namely Ser-461 in iPFK-2. This finding is further supported by the fact that (i) stoichiometry of phosphorylation was close to 1, (ii) phosphorylation of iPFK-2 could be detected using an antibody raised against the phosphorylated Ser-466 of heart PFK-2, and (iii) a peptide containing the sequence surrounding Ser-461 in iPFK-2 was phosphorylated by AMPK (8). The incubation of LPS-activated monocytes with oligomycin or under hypoxia activated AMPK and PFK-2, increased Fru-2,6-P 2 concentration, and stimulated glycolysis, which all followed the same time course. Finally, the oligomycin-induced phosphorylation and activation of iPFK-2 were completely blocked by dominant-negative AMPK in HEK-293 cells cotransfected with iPFK-2. Therefore, during hypoxia, AMPK activation and the subsequent phosphorylation and activation of iPFK-2 mediate the stimulation of glycolysis in LPS-activated 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 Ͼ30 pmol/g cells, an abnormally high value for unstimulated 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 constitutively phosphorylated and activated by cyclic AMP-dependent protein kinase, thus explaining the elevated concentration of Fru-2,6-P 2 under normoxic conditions. In these cells submitted to hypoxia, glycolysis was increased, whereas cyclic AMP concentration decreased, leading to a fall in Fru-2,6-P 2 content supposedly mediated by a decrease in PFK-2 activity. From this study, it was concluded that Fru-2,6-P 2 was not involved in the stimulation of glycolysis by hypoxia in these cells. By contrast in human resting monocytes, iPFK-2 was not expressed, and the concentration of Fru-2,6-P 2 was ϳ2 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 LPSactivated monocytes. In these activated monocytes, which express iPFK-2, hypoxia increased Fru-2,6-P 2 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 monocytes in the absence of additional stimuli. Likewise, in our experiments, hypoxia alone had no significant effect on glycolysis but increased glycolytic flux after LPS activation. The fact that monocyte responses to hypoxia are enhanced by stimulants such as LPS and interferon-␥ (32-34) reflects the important role of these stimuli in coordinating monocyte activity. We postulate that LPS primes monocytes to respond to hypoxia, which inevitably occurs in and around diseased tissues. The subsequently expressed iPFK-2 could thus be activated by AMPK under hypoxic conditions, thereby furnishing ATP to boost the inflammatory response.