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. 2001 Sep;21(17):5899-912.
doi: 10.1128/MCB.21.17.5899-5912.2001.

Growth factors can influence cell growth and survival through effects on glucose metabolism

M G Vander Heiden  1 , D R Plas, J C Rathmell, C J Fox, M H Harris, C B Thompson
Affiliations

Growth factors can influence cell growth and survival through effects on glucose metabolism

M G Vander Heiden et al. Mol Cell Biol. 2001 Sep.

Abstract

Cells from multicellular organisms are dependent upon exogenous signals for survival, growth, and proliferation. The relationship among these three processes was examined using an interleukin-3 (IL-3)-dependent cell line. No fixed dose of IL-3 determined the threshold below which cells underwent apoptosis. Instead, increasing growth factor concentrations resulted in progressive shortening of the G(1) phase of the cell cycle and more rapid proliferative expansion. Increased growth factor concentrations also resulted in proportional increases in glycolytic rates. Paradoxically, cells growing in high concentrations of growth factor had an increased susceptibility to cell death upon growth factor withdrawal. This susceptibility correlated with the magnitude of the change in the glycolytic rate following growth factor withdrawal. To investigate whether changes in the availability of glycolytic products influence mitochondrion-initiated apoptosis, we artificially limited glycolysis by manipulating the glucose levels in the medium. Like growth factor withdrawal, glucose limitation resulted in Bax translocation, a decrease in mitochondrial membrane potential, and cytochrome c redistribution to the cytosol. In contrast, increasing cell autonomous glucose uptake by overexpression of Glut1 significantly delayed apoptosis following growth factor withdrawal. These data suggest that a primary function of growth factors is to regulate glucose uptake and metabolism and thus maintain mitochondrial homeostasis and enable anabolic pathways required for cell growth. Consistent with this hypothesis, expression of the three genes involved in glucose uptake and glycolytic commitment, those for Glut1, hexokinase 2, and phosphofructokinase 1, was found to rapidly decline to nearly undetectable levels following growth factor withdrawal.

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Figures

FIG. 1
FIG. 1
Cell proliferation is responsive to the availability of growth factor. (A) Cells were cultured in medium containing the indicated amounts of IL-3. After gradual adaptation of at least 1 week to defined levels of IL-3, cells were plated at equivalent densities; the numbers of cells in the culture were measured at the indicated times. A graph of the mean cell number (and one standard error of the mean [SEM]) over time is shown. (B) Cells cultured as described for panel A were switched to medium containing 0.35 ng of IL-3/ml for 1 day, and the numbers of cells were determined at the indicated times. The mean cell number (and SEM) over time is shown. (C) Cells were cultured with different amounts of IL-3 as described for panel A but with the addition of the caspase inhibitor zVAD-fmk. The mean cell numbers (and SEM) at the indicated times are shown. (D) Cells expressing increased levels of Bcl-xL were cultured in different amounts of IL-3 as described for panel A. The mean cell numbers (and SEM) at the indicated times are shown.
FIG. 2
FIG. 2
Cell growth and cell cycle progression are decreased as growth factor becomes limiting. (A) The volumes of cells cultured in medium containing the indicated amounts of IL-3 were measured using a Coulter Z2 particle analyzer. The mean cell volume (and standard error of the mean [SEM]) is shown. (B) The cell cycle characteristics of cells growing in medium containing the indicated amounts of IL-3 were determined by propidium iodide staining and flow cytometry. The number of cells in each phase of the cell cycle is shown. (C) Cells growing with the indicated amounts of IL-3 were cultured in the presence of BrdU. Cells were fixed at the indicated times, and the percentages of cells incorporating BrdU in immunostained samples were determined by flow cytometry. The percentages of BrdU-positive (BrdU+) cells in the culture over time are shown. (D) Cells cultured in medium containing different amounts of IL-3 were switched to medium containing 0.35 ng of IL-3/ml. The mean cell volume (and SEM) after 1 day in culture with the increased IL-3 concentration is shown.
FIG. 3
FIG. 3
The rate of glycolysis correlates with both the amount of growth factor present and cell size. (A) The rates of glycolysis in cells growing with the indicated amounts of IL-3 were determined by measuring the conversion of 5-3H-glucose to 3H-H2O. The mean glycolytic rate (and standard error of the mean) is shown. (B) The glycolytic rates and cell volumes from multiple independent determinations are shown. A positive correlation between cell size and the rate of glycolysis was identified (P < 0.01).
FIG. 4
FIG. 4
Cells cultured with larger amounts of growth factor are more susceptible to cell death following growth factor withdrawal. (A) Cells cultured with the indicated amounts of IL-3 were withdrawn from growth factor, and cell viability was measured by propidium iodide exclusion using flow cytometry. The mean viability (and standard error of the mean [SEM]) following growth factor withdrawal over time is shown. (B) Cells growing in medium supplemented with the indicated amounts of IL-3 (withdrawn from:) were washed and switched directly to medium containing the indicated amounts of IL-3 (to:) (−IL-3 indicates no IL-3). The mean cell viability (and SEM) after 48 h of growth factor limitation is shown.
FIG. 5
FIG. 5
Mitochondria experience a limitation in substrate availability following growth factor withdrawal. (A) Oxygen consumption in cells cultured for 12 h in the presence (+IL-3) or absence (−IL-3) of IL-3 was measured, and the measurement of oxygen consumption was performed before and after (+FCCP) the addition of the protonophore FCCP. FCCP collapses the proton gradient present across the mitochondrial inner membrane, enabling the consumption of oxygen to be limited only by substrate availability. The mean rate of oxygen consumption (and standard error of the mean [SEM]) under each of the above conditions is shown. dPO2/dt, change in oxygen tension divided by change in time. (B) Cells cultured in the presence of absence of IL-3 for 12 h were fractionated into mitochondrial (M) and cytosolic (S) fractions. Fractions were probed for cytochrome c or cytochrome oxidase IV as a control for cellular fractionation. (C) Fluorometric measurement of NADH was performed using intact cells before and after the addition of FCCP. The difference between the mean steady-state fluorescence values (and SEM) before and after FCCP addition is plotted.
FIG. 6
FIG. 6
Magnitude of change in rate of glycolysis correlates with rate of cell death. Cells adapted to growth in medium containing the indicated amounts of IL-3 were cultured for 12 h in the presence (+IL-3) or absence (−IL-3) of IL-3. The mean glycolytic rate (and standard error of the mean) for each condition is shown. The change in glycolysis (Δ) upon IL-3 withdrawal is also shown.
FIG. 7
FIG. 7
The ability of exogenous growth factor receptors to maintain cell viability correlates with their ability to sustain glycolysis. (A) FL5.12 cells transfected with EpoR (closed squares), PDGF-R (closed diamonds), or nothing (control) (open squares) were withdrawn from IL-3 and placed in medium containing Epo (−IL-3/+Epo), PDGF (−IL-3/+PDGF), or no exogenous growth factors (−IL-3). The mean cell viability (and standard error of the mean [SEM]) for each condition over time is shown. (B) Cells transfected with EpoR, PDGF-R, or nothing (Control) were cultured in the presence of IL-3 or withdrawn from IL-3 and placed in medium containing Epo (−IL-3/+Epo), PDGF (−IL-3/+PDGF), or no exogenous growth factors (−IL-3) for 12 h. The mean rate of glycolysis (and SEM) measured in each population of cells is shown.
FIG. 8
FIG. 8
Cells growing with more growth factor are more susceptible to death following nutrient limitation. (A) Equal numbers of cells growing with 0.35 ng of IL-3/ml and 11 mM glucose were washed and resuspended in buffer containing different amounts of glucose. The mean glycolytic rate (and standard error of the mean [SEM]) at the indicated concentrations of glucose was determined and is shown. (B) Cells adapted to culturing with the indicated concentrations of IL-3 were washed and resuspended in medium containing the same amounts of IL-3 but containing only 0.05 mM glucose. Cell viability was determined at the indicated times following glucose withdrawal by propidium iodide exclusion and flow cytometry. The mean cell viability (and SEM) for each condition over time is shown.
FIG. 9
FIG. 9
Cell death following glucose limitation mimics cell death induced by growth factor withdrawal. (A) Control (Neo) and Bcl-xL-expressing cells cultured with 0.35 ng of IL-3/ml were washed and resuspended in medium containing the same amount of IL-3 but only 0.02 mM glucose. The mean cell viability (and standard error of the mean [SEM]) following glucose limitation over time is shown. (B) Mitochondrial potential was assessed using the potentiometric dye TMRE with cells growing for 6 h in the presence or absence of IL-3 (upper panel) or with high or low concentrations of glucose (10 or 0.02 mM, respectively) (lower panel). Note that the same histogram is used for the 10 mM glucose plus IL-3 condition in both panels. (C) Bax conformation was detected in mitochondrial fractions from cells growing for 15 h in the presence (+IL-3) or absence (−IL-3) of IL-3 or with 0.02 mM glucose. Mitochondrial lysates were prepared, immunoprecipitated (IP) using a conformation-specific anti-Bax antibody, and immunoblotted (IB) for Bax. (D) Control (Neo) and Bcl-xL-expressing cells were cultured with 0.35 ng of IL-3/ml in the presence of 11 mM glucose (Control) or 0.02 mM glucose (glucose withdrawal) for 18 h prior to subcellular fractionation. The amounts of cytochrome c (Cyt. c) and cytochrome oxidase subunit IV (Cox IV) present in the mitochondrial (M) and cytosolic (S) fractions were determined by Western blotting. Cox IV, an integral membrane protein located in the inner mitochondrial membrane, served as a control to demonstrate the absence of mitochondria in the cytosolic fraction.
FIG. 10
FIG. 10
Expression of Glut1 improves viability following IL-3 withdrawal. (A) Cells were transfected with control vector or with Glut1 expression vector. Transfected cells were permeabilized and stained in a two-step reaction with a Glut1-specific antibody and analyzed by flow cytometry. Cells stained in the absence of the primary antibody (Ab) are included as a control. The data are representative of the results for three independent clones. (B) Cells transfected with control vector (Neo) or with Glut1 expression vector were withdrawn from IL-3 for 1 day, and viability was determined by propidium iodide staining with a flow cytometer. Error bars show the standard deviation of triplicate samples. (C) RNA was prepared from control vector (Neo)- and Bcl-xL-expressing cells withdrawn from IL-3 at the indicated times. A Northern blot was serially hybridized with probes specific for actin, tubulin, Glut1, hexokinase 2 (Hk-2), and PFK-1 (Pfk-1).
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References

    1. Ashcroft S J, Weerasinghe L C, Bassett J M, Randle P J. The pentose cycle and insulin release in mouse pancreatic islets. Biochem J. 1972;126:525–532. - PMC - PubMed
    1. Auger K R, Serunian L A, Soltoff S P, Libby P, Cantley L C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989;57:167–175. - PubMed
    1. Boise L H, Gonzalez-Garcia M, Postema C E, Ding L, Lindsten T, Turka L A, Mao X, Nunez G, Thompson C B. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993;74:597–608. - PubMed
    1. Chakrabarti R, Jung C Y, Lee T P, Liu H, Mookerjee B K. Changes in glucose transport and transporter isoforms during the activation of human peripheral blood lymphocytes by phytohemagglutinin. J Immunol. 1994;152:2660–2668. - PubMed
    1. Conlon I, Raff M. Size control in animal development. Cell. 1999;96:235–244. - PubMed

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