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Cytokine stimulation of aerobic glycolysis in hematopoietic cells exceeds proliferative demand

Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

²Correspondence: Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: craig{at}mail.med.upenn.edu

SPECIFIC AIMS

Extracellular signals stimulate multiple basic processes in mammalian cells including cell growth, proliferation, and glucose metabolism. To determine whether up-regulation of glycolysis is a secondary response to bioenergetic demand or a primary response regulated by signaling events, the relationship between cell growth and glucose metabolism was studied.

PRINCIPAL FINDINGS

1. IL-3 stimulation results in a dose-dependent increase in glycolytic activity
FL5.12 cells are an immortalized hematopoietic cell line dependent on IL-3 for growth, proliferation, and survival. Cells were cultured in 0.01, 0.05, or 0.35 ng/mL recombinant IL-3, concentrations above the threshold necessary to inhibit apoptosis. With higher levels of IL-3, the rate of cell accumulation in culture increased. The size of cells in G₁ and G₂/M phases of the cell cycle was found to be larger with each increment of IL-3.

Glycolytic rates increased dramatically when cells were cultured in higher concentrations of IL-3. Staining with an anti-Glut1 antibody followed by FACS analysis indicated that expression of the Glut1 glucose transporter increased with increasing amounts of growth factor. Enzymatic measurement of hexokinase and PFK-1 activities demonstrated higher activity with higher growth factor availability.

2. IL-3 stimulation results in increased cellular NADH, elevated mitochondrial membrane potential, and a shift from glucose oxidation to lactate production
Cells cultured in increasing concentrations of IL-3 displayed increased levels of NADH derived from glycolysis, as measured by NAD(P)H fluorescence sensitive to iodoacetic acid, an inhibitor of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase. Cells cultured with high levels of IL-3 demonstrated increased mitochondrial membrane potential, independently suggesting an excess production of substrates for electron transport. The oxygen:glucose metabolism ratio, an indicator of the relative extent of glucose oxidation calculated by dividing the oxygen consumption rate by the glycolytic rate, declined with increasing levels of IL-3. In contrast, the ratio of lactate produced to glucose consumed increased as cells were grown in higher concentrations of IL-3.

3. IL-3 stimulation of glycolysis is independent of cell growth
To directly test the role of demand by cell growth on the stimulation of glycolysis, cells were stimulated by IL-3 under conditions in which growth was prevented. Cells cultured in the lowest concentration of IL-3 were switched to the highest concentration of IL-3 for 4 h in the presence or absence of the protein synthesis inhibitor cycloheximide. Cells switched to high IL-3 displayed a statistically significant increase in cell size whereas cells switched to high IL-3 in the presence of cycloheximide remained the same size as cells grown in low IL-3 (Fig. 1 A). PFK-1 activity and the glycolytic rate of cells switched to high IL-3 increased significantly after 4 h and increased to a similar extent in the presence or absence of cycloheximide (Fig. 1B ).

View larger version (11K): [in this window] [in a new window]   Figure 1. IL-3 increases glycolysis independent of changes in cell size. Cells cultured in 0.01 ng/mL IL-3 were switched to media containing 0.35 ng/mL IL-3 for 4 h with or without 5 µg/mL cycloheximide (CHX) or kept in 0.01 ng/mL IL-3. A) Cell growth stimulation by IL-3 is prevented by CHX. After 4 h, cell size was measured by Coulter analysis. Average of 4 independent experiments ± SEM normalized to cell size in cells maintained in 0.01 ng/mL IL-3. Mean cell size was significantly greater for cells stimulated with 0.35 than for 0.01 ng/mL IL-3 (paired t test, 2-tailed P value, P<0.01). B) Glycolytic rate stimulation by IL-3 is not prevented by CHX.Glycolytic rate was measured after 4 h by conversion of glucose to water. Average of 4 independent experiments ± SEM normalized to glycolytic rate of cells maintained in 0.01 ng/mL IL-3.

Responses of primary human T cells to IL-2 and immortalized murine B cell progenitors to IL-7 were assayed. In primary T cells, acute stimulation with IL-2 caused significant increases in cell size and glycolytic rate. In the presence of cycloheximide, IL-2 stimulated glycolysis but not cell growth. Similarly, in response to IL-7, the B cell progenitor line B23 increased in size and glycolytic rate but displayed cycloheximide-insensitive glycolytic activation.

4. Growth factor-stimulated glycolysis suppresses oxidative metabolism and exceeds proliferative demand
To determine the metabolic and cellular consequences of limiting glycolysis, cells were cultured in reduced concentrations of extracellular glucose. As expected, when cells were switched from 10 mM to 0.4 mM glucose, the glycolytic rate significantly decreased (Fig. 2 A). Cells responded to decreased glucose availability by increasing their rate of oxygen consumption (Fig. 2B ). The effect of the metabolic switch caused by glucose limitation on cell growth and proliferation was investigated. Surprisingly, cell number after 3 days in culture was unaffected by a reduction in glucose availability to 0.4 mM (Fig. 2C ). Glucose limitation had no effect on cell size (Fig. 2D ).

View larger version (19K): [in this window] [in a new window]   Figure 2. IL-3 directed glycolysis suppresses cellular oxygen consumption and exceeds proliferative demand. Cells were cultured in 0.35 ng/mL IL-3 and 10 mM or 0.4 mM glucose. (A) Glycolytic rate is suppressed when cells are shifted from 10 mM to 0.4 mM glucose. Glycolytic rate was measured after two days in culture by measuring the specific conversion of tritriated glucose to water. Average of three independent experiments ± SEM is shown. (B) Oxygen consumption rate is derepressed when cells are shifted from 10 mM to 0.4 mM glucose. Oxygen consumption was measured after two days in culture using an oxygen electrode in a heated, airtight chamber. Average of three independent experiments ± SEM is shown. (C) Cell accumulation in culture is maintained when cells are shifted from 10 mM to 0.4 mM glucose. The concentration of cells was measured after three days in culture. Average of three independent experiments ± SEM is shown. (D) Cell size is maintained when cells are shifted from 10 mM to 0.4 mM glucose. Cell size was measured after two days in culture by Coulter analysis. Average of three independent experiments ± SEMis shown.

CONCLUSIONS AND SIGNIFICANCE

IL-3 and other hematopoietic cytokines can stimulate rates of cellular glycolysis. Two models may explain this effect. The first suggests that cells respond to the energetic demands of IL-3-stimulated growth and proliferation by compensatorily up-regulating glycolysis. Presumably, this homeostatic response would be through sensing and responding to the depletion of glycolytic end products such as NADH, pyruvate, and ATP. The alternate model states that IL-3 directly regulates glycolytic metabolism independent of other IL-3 effects.

Several lines of evidence presented here support the direct regulation model. Glycolysis results in the net consumption of 1 molecule of glucose and 2 molecules each of NAD⁺ and ADP and production of 2 molecules each of pyruvate, NADH, and ATP. In cells with high ATP demand, cytosolic pyruvate and NADH can be oxidized in mitochondria to produce additional ATP. If the bioenergetic requirements of cell growth and proliferation were driving the observed increase in glycolysis, an increased percentage of the pyruvate and NADH produced by glycolysis would be expected to be used by mitochondria. Alternatively, if the glycolytic rate of pyruvate and NADH production exceeded the rate of consumption by mitochondria, these glycolytic end products would accumulate in the cytosol. The finding of increased glycolytic NADH in cells stimulated by IL-3 was inconsistent with a homeostatic response to cytosolic NADH depletion.

Another bioenergetic indicator is the level of mitochondrial membrane potential, reflecting production by electron transport and consumption by oxidative phosphorylation. Elevated bioenergetic demand could decrease mitochondrial potential by decreasing availability of substrates for electron transport or by increasing consumption of the electrochemical gradient. However, cells cultured with higher levels of IL-3 demonstrated increased mitochondrial potential, consistent with the excess production of substrates for electron transport.

Bioenergetic demand was also assessed by measuring cellular oxygen consumption in response to growth factor stimulation. The oxygen:glucose metabolism ratio was calculated by dividing the rate of oxygen consumption by the rate of glycolysis. Maximal oxidation of glucose would yield an oxygen:glucose metabolism ratio of 6:1 whereas incomplete oxidation would result in lower values. Cells cultured in 0.01 ng/mL IL-3 consumed 3.3 mol of oxygen/glucose whereas those cultured in 0.35 ng/mL IL-3 consumed only 0.7 mol of oxygen/glucose. This suggested that growth factor receptor engagement shifts cells away from an oxidative toward a glycolytic form of metabolism.

The finding of increased production of lactate on a per glucose basis was inconsistent with a model based on the depletion of pyruvate by mitochondrial oxidation or macromolecular biosynthesis causing compensatory pyruvate production. That is, increasing concentrations of growth factor induced cells to use a progressively smaller fraction of their metabolized glucose for either macromolecular synthesis or mitochondrial oxidative phosphorylation.

Experiments in which IL-3 stimulation occurred while cell growth was prevented also support the direct activation model. Cells did not grow in response to IL-3 in the presence of cycloheximide, but demonstrated a significant increase in the activity of the enzyme rate-limiting for glycolysis, PFK-1, and in the total glycolytic rate. Similar effects were noted in primary T cells in response to IL-2 and B cell progenitors in response to IL-7. Thus, the up-regulation of glycolysis seen was not dependent on new protein synthesis. These results demonstrate that the increase in glycolytic activity when cells are grown in high concentrations of growth factor cannot simply be a secondary response to cell growth. Furthermore, primary regulation of glycolysis independent of cell growth is not limited to FL5.12 cells in response to IL-3 but may be observed in other hematopoietic cell types, including primary cells, in response to a number of cytokines.

After IL-3 stimulation, glycolysis increased dramatically, but when glycolytic rates were reduced by limiting extracellular glucose (and thus production of mitochondrial substrates such as NADH and pyruvate decreased), cellular oxygen consumption did not decline. Rather, oxygen consumption increased significantly when extracellular glucose was reduced 25-fold. Although glycolysis is directly controlled by extrinsic signals, mitochondrial oxygen consumption appears to be regulated homeostatically by intracellular bioenergetic conditions: under glucose-replete conditions, elevated rates of glycolysis are sufficient to suppress oxidative phosphorylation below maximal. Despite decreased rates of glycolysis, cells maintained growth factor-directed rates of growth and proliferation. Therefore, IL-3 directed glycolysis is not a compensatory response to increased growth and proliferation, and actually is in excess of what is needed to support cell growth and proliferation.

Recently it has been recognized that cell growth and proliferation are independently but coordinately regulated by extrinsic signals in metazoan cells. We suggest that regulation of glycolysis should be considered a parallel pathway downstream of growth factor signaling (Fig. 3) . Moreover, we reason that the shift of cancer cells to a glycolytic form of metabolism may merely be due to growth factor-independent activation of growth factor signaling pathways. Enforced glycolytic metabolism may constitute a therapeutic target for cancer.

View larger version (18K): [in this window] [in a new window]   Figure 3. IL-3 directs glycolysis in parallel to growth and proliferation. Growth factors independently but coordinately regulate multiple processes in metazoan cells, including growth and proliferation. Data presented here suggest that glycolysis is another extrinsically directed process. Glycolysis is regulated at numerous steps including glucose transport (GLUT1), hexokinase (HK), phosphofructokinase-1 (PFK-1), and lactate dehydrogenase. Furthermore, IL-3 stimulates a shift away from oxidative towards glycolytic metabolism. Elevation of glycolytic rates inhibits mitochondrial oxygen consumption below maximal levels, implying that mitochondrial metabolism is a homeostatic sensor of intracellular bioenergetics.

FOOTNOTES

¹ These authors contributed equally.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1001fje; doi: 10.1096/fj.03-1001fje

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