The P2X7 receptor is a key modulator of aerobic glycolysis

Abstract

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.

Figure 1

P2X7R expression stimulates cell metabolism. HEK293-P2X7 (open bars) and HEK293-mock cells (closed bars) were incubated in 17 or 4 mM glucose-supplemented medium, and cell proliferation (a), intracellular ATP content (b) and lactate output (c) were measured as described in Materials and Methods. Intracellular ATP and lactate output were assessed after a 24-h incubation under the different experimental conditions. Data are average±S.D. of multiple determinations (n=9 for ATP measurements; n=12 for lactate measurements). Statistical significance, *P <0.05 and **P <0.03

Figure 2

P2X7R expression is needed for ATP or FCCP-stimulated lactate output. HEK293-P2X7 (open bars) and HEK293-mock cells (closed bars) were incubated in 17 or 4 mM glucose-supplemented medium for 24 h in the absence or presence of ATP, FCCP or the P2X7R blocker A740003. Data are average ±S.D. of multiple determinations (n=3). Statistical significance, *P <0.05 and **P <0.03

Figure 3

Lactate release is dependent upon P2X7R stimulation in the human neuroblastoma ACN cell line. 25 × 10³ ACN cells per well were plated in 11 mM (open bar) or 4 mM (closed bar) glucose medium and lactate release was measured after a 24-h incubation in the absence or presence of either ATP or the selective P2X7R blocker A740003. Data are average±S.D. of multiple determinations (n=3). Statistical significance, *P <0.05 and **P <0.03

Figure 4

P2X7R expression causes overexpression of the key glycolytic enzymes G3PDH and PFK. HEK293-P2X7 (open bars) and HEK293-mock cells (closed bars) were incubated in 17 or 4 mM glucose-supplemented medium, and G3PDH (a) and PFK (b) expression was measured by quantitative PCR and ELISA, respectively. G3PDH mRNA content is expressed as a RQ ratio normalized onto P2Y1 mRNA. G3PDH and PFK determinations are average±S.D. of three separate experiments, each performed in duplicate. Statistical significance, *P <0.05; **P <0.03; and n.s., not significant

Figure 5

P2X7R expression causes overexpression of PKM2 and PDHK1 and inhibition of PDH activity. HEK293-P2X7 (open bars) and HEK293-mock cells (closed bars) were incubated in 17 or 4mM glucose-supplemented medium, and PKM2 (A) and PDHK1 (B) expression was measured by western blot. Protein bands (a) in panels (A and B) were quantified by densitometry (b) and normalized onto endogenous actin band. PKM2 and PDHK1 determinations are from one experiment representative of four similar. PDH (C) activity was measured as described in Materials and methods and expressed in arbitrary units. Data are averages±S.D. of multiple determinations (n=3). Statistical significance, *P <0.05

Figure 6

Glucose depletion causes a decrease in mitochondrial potential. HEK293-P2X7 (A) and HEK293-mock (B) cells were incubated under the different conditions for 24 h, and at the end of this incubation were labeled with TMRM (panels a and d) and Mito Tracker Green (panels b and e). Image acquisition and analysis were performed as described in Materials and Methods. Panels (c and f) show merge of the red and green channels

Figure 7

Glucose depletion causes upregulation of Glut1 in HEK293-P2X7, but not in HEK293-mock, cells. Samples were processed as described in Materials and Methods and protein bands (a) were quantified by densitometry (b) and normalized onto endogenous actin band. HEK293-P2X7, open bars and HEK293-mock, closed bars. One experiment representative of four similar is shown

Figure 8

HEK293-P2X7 cells posses larger glycogen stores than HEK293-mock cells. HEK293-P2X7 (a and b) and HEK293-mock (c and d) were plated in 24 well per plates and incubated under the different experimental conditions as described in Materials and Methods. After 24 h, they were PAS-stained and analyzed by phase contrast microscopy with a × 40 objective. Glycogen deposits are revealed by the pink stain. For each experimental condition, three wells per plate were analyzed

Figure 9

P2X7R expression increases Akt/PKB phosphorylion and HIF-1α expression. HEK293-P2X7 (open bars) and HEK293-mock cells (closed bars) were incubated for 24 h under the different experimental conditions and processed as detailed in Materials and Methods. Protein bands (a) were quantified by densitometry (b and c) and normalized onto endogenous actin band. One experiment representative of six similar is shown. HIF-1α expression (d) was determined by ELISA as described in Materials and Methods. Data are average±S.D. of multiple determinations (n=3). Statistical significance, *P <0.05 and **P <0.03