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. 2003 Oct;23(20):7315-28.
doi: 10.1128/MCB.23.20.7315-7328.2003.

Akt-directed glucose metabolism can prevent Bax conformation change and promote growth factor-independent survival

Jeffrey C Rathmell  1 , Casey J Fox, David R Plas, Peter S Hammerman, Ryan M Cinalli, Craig B Thompson
Affiliations

Akt-directed glucose metabolism can prevent Bax conformation change and promote growth factor-independent survival

Jeffrey C Rathmell et al. Mol Cell Biol. 2003 Oct.

Abstract

The serine/threonine kinase Akt is a component of many receptor signal transduction pathways and can prevent cell death following growth factor withdrawal. Here, we show that Akt inhibition of cell death is not dependent on new protein translation. Instead, Akt inhibition of cell death requires glucose hydrolysis through glycolysis. Akt was found to regulate multiple steps in glycolysis via posttranscriptional mechanisms that included localization of the glucose transporter, Glut1, to the cell surface and maintenance of hexokinase function in the absence of extrinsic factors. To test the role of glucose uptake and phosphorylation in growth factor-independent survival, cells were transfected with Glut1 and hexokinase 1 (Glut1/HK1) cells. Glut1/HK1 cells accumulated Glut1 on the cell surface and had high glucose uptake capacity similar to that of cells with constitutively active Akt (mAkt). Unlike mAkt-expressing cells, however, they did not consume more glucose, did not maintain prolonged phosphofructokinase-1 protein levels and activity, and did not maintain pentose phosphate shuttle activity in the absence of growth factor. Nevertheless, expression of Glut1 and HK1 promoted increased cytosolic NADH and NADPH levels relative to those of the control cells upon growth factor withdrawal, prevented activation of Bax, and promoted growth factor-independent survival. These data indicate that Bax conformation is sensitive to glucose metabolism and that maintaining glucose uptake and phosphorylation can promote cell survival in the absence of growth factor. Furthermore, Akt required glucose and the ability to perform glycolysis to prevent Bax activation. The prevention of Bax activation by posttranscriptional regulation of glucose metabolism may, therefore, be a required aspect of the ability of Akt to maintain long-term cell survival in the absence of growth factors.

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Figures

FIG. 1.
FIG. 1.
Akt does not require protein synthesis to promote survival of growth factor-deprived cells. Control (Neo) and myristoylated Akt (mAkt)-expressing cells were withdrawn from IL-3 in medium without inhibitors or in medium containing the PI3-K inhibitor LY or the protein translation inhibitor CHX. The inhibitors were replenished on day 2. Treatment of cells with LY resulted in the dephosphorylation and inactivation of mAkt by day 2, while mAkt remained phosphorylated in the absence of IL-3 (data not shown). The cells were analyzed in culture by flow cytometry at various time points to determine viability by PI exclusion. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations.
FIG. 2.
FIG. 2.
Akt requires a hydrolyzable glucose substrate to prevent death upon growth factor withdrawal. Control (Neo) (A) and mAkt-transfected IL-3-dependent FL5.12 (B) cells were withdrawn from IL-3 in the absence of glucose or in the presence of 10 mM glucose or 10 mM 2-DOG, a glucose analog that cannot be metabolized through glycolysis. At various time points, cells were analyzed by flow cytometry to determine viability by propidium iodide exclusion. (C) The glycolytic rates of Neo and mAkt cells were measured after 10 h in the absence of IL-3. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations.
FIG. 3.
FIG. 3.
Constitutively active Akt promotes glucose uptake not by regulating Glut1 protein levels but by promoting Glut1 cell surface localization. (A) Control (Neo) and mAkt-expressing IL-3-dependent cells were cultured in the presence of IL-3 or in the absence of IL-3 for 5 or 10 h. The cells were then harvested, and their ability to uptake the nonhydrolyzable glucose analog 2-DOG was measured. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations. (B) Control and mAkt cells were analyzed by Western blot for expression of the glucose transporter Glut1, either in the presence of IL-3 or after 10 h of withdrawal from IL-3. Each lane was loaded with 10 μg of protein. (C) Control (Neo) and mAkt cells were stained by immunofluorescence for Glut1 localization in the presence of IL-3 and after 10 h in the absence of IL-3. Each micrograph is individually contrasted to allow the best visualization of Glut1 localization. The bars represent 5 μm. (D) Plasma membranes were isolated from control and mAkt-expressing cells in the presence of IL-3 or after 12 h in the absence of IL-3, and 10 μg of protein was analyzed by Western blot for cell surface expression of Glut1.
FIG. 4.
FIG. 4.
Glut1 expression promotes accumulation of Glut1 surface localization. (A and B) Control and mAkt-expressing FL5.12 cells were compared to cells transfected with Glut1, HK1, or Glut1 and HK1 together (Glut1/HK1) for Glut1 expression (A) and HK1 expression (B). (C) Glut1 localization was determined in control, mAkt, and Glut1 cells by immunofluorescence staining of permeabilized cells for Glut1 protein. Each micrograph is individually contrasted to allow the best visualization of Glut1 localization. Surface Glut1 immunofluorescence of Glut1-overexpressing cells was sufficiently bright that intracellular Glut1 became obscured upon contrasting the images (data not shown). The bars represent 5 μm.
FIG. 5.
FIG. 5.
Constitutively active mAkt cells have enhanced hexokinase activity that they maintain upon withdrawal from IL-3. (A) Control (Neo) and mAkt cells were grown in IL-3 or withdrawn from IL-3 for 12 h. Whole-cell lysates were analyzed for total hexokinase activity. (B) Mitochondria were isolated from control and mAkt cells in the presence of IL-3 or after 12 h in the absence of IL-3, and 10 μg of protein was analyzed for hexokinase activity. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations.
FIG. 6.
FIG. 6.
Constitutively active Akt increases total cellular glucose consumption. Control (Neo), Glut1, HK1, Glut1/HK1, and mAkt cells were cultured in the presence of IL-3 and observed each day for 5 days. (A) Cell proliferation was determined by measuring cell concentration. Glucose consumption (B) and lactate production (C) were determined by measuring their concentrations in the media. The values shown are the means of the results for triplicate cultures; error bars indicate standard deviations.
FIG. 7.
FIG. 7.
Akt prevents loss of PFK1 protein levels and activity upon growth factor withdrawal. Control (Neo), Glut1/HK1, and mAkt cells were grown in the presence of IL-3 or for 12 h in the absence of IL-3. (A) RNA was isolated and PFK1 expression was probed by northern hybridization. (B) Whole-cell lysates were analyzed by Western blot for expression of PFK1 protein. Purified rabbit muscle PFK1 is shown as a size standard. (C) Cell lysates were tested for PFK1 activity. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations.
FIG. 8.
FIG. 8.
Glut1/HK1 cells partially and mAkt cells fully maintain NAD(P)H levels upon removal from IL-3. Control (Neo), Glut1/HK1, and mAkt cells were grown in the presence of IL-3 (A) or withdrawn from IL-3 (B) for 12 h. The cells were then subjected to spectrofluorimetric analysis for NAD(P)H fluorescence to determine the total, mitochondrial-accessible, and cytosolic-accessible NAD(P)H content of each cell type. After establishing a baseline for fluorescence, the cells were treated with the protonophore FCCP to stimulate oxidation of mitochondrial NADH (added at the vertical line). Cytosolic NADH content was then estimated for each sample by observing the decrease in NADH fluorescence over time after the addition of mPyruvate (added at the vertical line). Through the action of the cytosolic enzyme lactate dehydrogenase, mPyruvate causes the depletion of cytosolic NADH. (C) Pentose phosphate shuttle activity was determined in control, Glut1/HK1, and mAkt cells in the presence of IL-3 or after 12 h in the absence of IL-3. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations.
FIG. 9.
FIG. 9.
Coexpression of Glut1 and HK1 increases survival in the absence of IL-3 and prevents change in Bax conformation. (A) Cells were withdrawn from IL-3, and viabilities at various time points were measured by flow cytometry by propidium iodide exclusion. The values shown are the means of the results for cell survival from triplicate samples; error bars indicate standard deviations. (B) Bax activation was measured by flow cytometry in cells that were maintained in IL-3 or withdrawn from IL-3 for 12 h. Percentages for cells staining positive for change in Bax conformation are given. (C) Control (Neo) and mAkt-expressing cells were cultured in the presence or absence of glucose or IL-3, as indicated, for 12 h, and the percentage of cells with activated Bax was determined. (D) Control (a), Glut1/HK1 (b), mAkt (c), and Bcl-xL (d)-overexpressing FL5.12 cells were cultured in the absence of IL-3 either with (solid lines) or without (dashed lines) the glycolytic inhibitor IAA. Cell viabilities at various time points after the start of culture were measured by flow cytometry by propidium iodide exclusion. The values shown are the means for the results of cell survival from triplicate samples; error bars indicate standard deviations.
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