Akt promotes increased mammalian cell size by stimulating protein synthesis and inhibiting protein degradation

Jesika Faridi,1 Janet Fawcett,2,3 Lihong Wang,1 and Richard A. Roth1

1Department of Molecular Pharmacology, Stanford University, Stanford, California 94305; 2Section of Endocrinology, Veterans Affairs Medical Center, Phoenix 85012; and 3Department of Molecular and Cellular Biology, Arizona State University, Tempe, Arizona 85287

Submitted 23 May 2003 ; accepted in final form 14 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Expression of constitutively active Akt3 was found to increase the size of MCF-7 cells approximately twofold both in vitro and in vivo. A regulatable version of Akt1 (MER-Akt) was also found capable of inducing a twofold increase in the size of H4IIE rat hepatoma cells. Rapamycin, a specific inhibitor of mTOR function, was found to inhibit the Akt-induced increase in cell size by 70%, presumably via inhibition of the Akt-induced increase in protein synthesis. To determine whether Akt could be inhibiting protein degradation, thereby contributing to its ability to induce an increase in cell size, we conducted protein degradation experiments in the H4IIE cell line. Activation of MER-Akt was found to inhibit protein degradation to a degree comparable to insulin treatment. The effects of these two agents on protein degradation were not additive, thereby suggesting that they were acting on a similar pathway. An inhibitor of the phosphatidylinositol 3-kinase pathway, LY-294002, blocked both insulin- and Akt-induced inhibition of protein degradation, again consistent with the hypothesis that both agents were acting on the same pathway. In contrast, rapamycin did not block the ability of either agent to inhibit protein degradation. These results indicate that Akt increases cell size through both mTOR-dependent and -independent pathways and that the latter involves inhibition of protein degradation. These studies are also consistent with the hypothesis that insulin's ability to regulate protein degradation is to a large extent mediated via Akt.

cell growth; protein kinase B; mammalian target of rapamycin; rapamycin


IN THE PAST FEW YEARS, the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway has been shown to play a major role in the control of cell size. The importance of this pathway in the control of cell size initially came from work in Drosophila melanogaster, in which it was shown that overexpression of PI3K Dp110 promotes cell growth in the wing and eye via increasing cell size (16). Further studies in the fruit fly as well as in mammals have demonstrated that additional manipulations of the PI3K/Akt pathway result in alterations in cell size in vivo as well as in vitro (2, 3, 8, 11, 19, 20, 24, 28, 29, 32). The control of cell size by the PI3K/Akt pathway has been in part explained by the ability of this pathway to regulate protein synthesis via the downstream targets the mammalian target of rapamycin (mTOR), ribosomal protein p70 S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1)/PHAS-I (4, 6, 26).

Additionally, for many years, it has been known from work in Saccharomyces cerevisiae that repression of the cell cycle may result in an increase in cell size (7, 10, 23). Later, work in Drosophila also suggested that alterations in cell cycle and ploidy were related to cell size changes (22, 31).

However, cell growth (an increase in cell size) may occur not only by cell cycle dysregulation and increased protein synthesis but also by decreased protein degradation. Whether the PI3K/Akt pathway also stimulates an increase in cell size by regulating protein degradation has not yet been investigated. This would represent a third mechanism by which a cell's size could be increased by this pathway.

In the present study, we have investigated the effect of Akt on cell size. We show that expression of either constitutively active Akt1 or -3 equally increases the size of MCF-7 cells. In addition, activation of a regulatable Akt1 induced a time-dependent increase of cell size in the rat hepatoma H4IIE cells. We found that rapamycin, an inhibitor of mTOR function and subsequent translational control, only partly reversed the effects of Akt on cell size. In contrast, rapamycin did not block the ability of insulin to inhibit protein degradation. Moreover, activation of the regulatable Akt was found to inhibit protein degradation to a comparable degree as insulin, and this was not blocked by rapamycin. These results indicate that Akt can stimulate an increase in cell size by both mTOR-dependent and mTOR-independent pathways and that the latter includes the ability of Akt to inhibit protein catabolism. These results are also consistent with the hypothesis that insulin's ability to regulate protein degradation is to a large extent mediated via its ability to activate Akt.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Dulbecco's modified Eagle's medium (DMEM) was from either GIBCO-Life Technologies (Grand Island, NY) or Sigma (St. Louis, MO), fetal calf serum (FCS) and newborn calf serum (NCS) were from either GIBCO or Gemini (Calabasas, CA), and penicillin and streptomycin were from GIBCO. Biosynthetic human insulin was a gift of Dr. R. Chance (Lilly Research Laboratories), and 4-hydroxytamoxifen (Tam) was from Sigma. [3H]leucine was from ICN (Costa Mesa, CA). LY-294002 and rapamycin were from Calbiochem (San Diego, CA). All other chemicals were of at least reagent grade.

Cell lines and culture conditions. H4IIE rat hepatoma cells were maintained in DMEM supplemented with 5% FCS, 5% NCS, 100 µg/ml streptomycin, and 100 U/ml penicillin. H4IIE cells expressing myristoylated Akt fused to a mutant form of the hormone-binding domain of the estrogen receptor (MER-Akt1) are referred to as H4IIE/MER-Akt1 cells and are as previously described (17). MCF-7 cells were maintained in DMEM-F12 (GIBCO) medium and supplemented with 10% FCS, streptomycin, and penicillin. MCF-7 cells were previously transfected with myristoylated Akt1, Akt3, or vector only and used either as a pool population (A3uncl or Puncl) or cloned, screened for expression, and used as isolated clones (A1–5 or A3B5) (5).

For protein degradation studies, H4IIE cells were grown in 24-well plates (starting density 3.4 x 104 cells/cm2). The growth medium consisted of DMEM with 5% FCS, 5% NCS, and penicillin-streptomycin. Cells were incubated at 37°C in an atmosphere of 5% CO2-95% air. The medium was changed every 2–3 days, and the cells were used when confluent (~5 days).

Cell volume analyses. MCF-7 and H4IIE cells were pelleted, washed once with Hanks' balanced salt solution (GIBCO), and resuspended in PBS containing 0.1% serum and 5 mM EDTA. Cell volume was then determined in a Coulter counter (model Z2).

Cell size and cell cycle analyses. For measurement of cell size using forward scatter units (FSC) with unfixed cells (experiments shown in Fig. 1B), MCF-7 cells were pelleted, washed once with Hanks' balanced salt solution (GIBCO), and resuspended in PBS containing 0.1% serum, 5 mM EDTA, 5 ng/µl propidium iodide (PI; Sigma). Samples were analyzed by fluorescence-activated cell sorters (FACS) analysis (FACSCalibur; Becton Dickinson) for cell size (FSC). PI-positive cells were excluded from the analyses, and the mean of FSC was determined.



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Fig. 1. Constitutively active Akt1 and Akt3 increase the size of MCF-7 cells. Levels of active, phosphorylated Akt (p-Akt) in the parental MCF-7 cells (with or without stimulation by insulin), the plasmid-transfected uncloned pool population (Puncl), the myristoylated (myr)Akt3-transfected uncloned pool population (A3uncl), the clone expressing myrAkt3 (A3B5), and the clone expressing myrAkt1 (A1–5) were assessed by Western blotting with an antibody to p-Akt (Ser473) (A). Sizes of the indicated cells were assessed either by measuring forward light scatter (FSC) via fluorescence-activated cell sorter (FACS; B) or by Coulter counter analysis (C).

 

For measurement of cell size using FSC with fixed cells (experiments shown in Figs. 3B, 4A, and 5, A and B), MCF-7 or H4IIE cells were pelleted, washed once with Hanks' balanced salt solution (GIBCO), and resuspended in 1 ml of PBS containing 0.1% serum and 5 mM EDTA. While vortexing was being performed, 1 ml of 100% ethanol was added to the cell suspensions and incubated at room temperature for 30 min. The cells were then pelleted, resuspended in PBS containing 0.1% serum, 5 mM EDTA, and 32 ng/µl RNase A (Sigma), and incubated at room temperature for 30 min. Next, PI was added at a concentration of 40 ng/µl, and cells were incubated for 10 min at room temperature. Samples were analyzed by FACS analysis for cell size (FSC) and cell cycle (FL-3H).



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Fig. 3. Activation of MER-Akt1 in H4IIE cells induces a time-dependent increase in cell size. Either parental H4IIE cells or cells expressing the regulatable Akt (H4IIE/MER-Akt1) were cultured in serum containing DMEM and treated with either 4-hydroxytamoxifen (Tam; 1 µM) or vehicle (ethanol) as indicated. Cells were either lysed and tested for active Akt by Western blotting with an anti-p-Akt (Ser473) antibody (A) or analyzed for cell size by FACS (B) or by Coulter counter (C). Coulter counter and Western blot analyses were performed after 5 days of Tam treatment, whereas FACS analyses were performed after 1, 3, or 5 days (only the latter 2 are indicated).

 


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Fig. 4. Rapamycin (Rap) partially reverses the Akt-induced increase in cell size. MCF-7 parental cells or cells expressing myrAkt3 (A3B5) were cultured in serum containing DMEM-F12 and treated as indicated with 100 nM rapamycin or vehicle (DMSO) for 5 days (medium including rapamycin was changed every other day), and then their cell size was measured by FSC (A) or Coulter counter (B).

 


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Fig. 5. Active Akt increases cell size throughout the cell cycle. To measure the size of these cells at different periods in the cell cycle, MCF-7 cells expressing myrAkt3 (A3B5) or control cells (Puncl) were cultured in phenol red-free, serum-free DMEM-F12 to synchronize them. FACS cell cycle (FL-3H) analysis was performed after 9 days of synchronization. Cells were gated as being in either G1 or post-G1 phase. Cell size was measured by FSC for Puncl and A3B5 cells in G1 (A) and post-G1 phase (B).

 

Protein degradation. Protein degradation was measured as described by Gunn et al. (9) with modifications. The growth medium was removed from the cells and replaced with leucine-free DMEM containing 5% FCS, 5% NCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 µCi/ml [3H]leucine. Cells were incubated for 18 h to allow labeling of cellular proteins with [3H]leucine. Labeling medium was replaced with incubation medium (DMEM containing 2 mM unlabeled leucine, 20 mM TES, pH 7.5, and 0.1% BSA) containing insulin (10–12 to 10–6 M) or Tam (0.1–1,000 nM) and incubated for3hat37°C. The incubation was stopped by placing the cells on ice and adding an equal volume of 6 M acetic acid containing 2% Triton X-100 to solubilize the cells. Aliquots of the cell-medium mix were analyzed for protein degradation by precipitation in 10% (final concentration) trichloroacetic acid (TCA). Protein degradation was taken as percent TCA-soluble radioactivity. Experiments in which inhibitors (LY-294002 or rapamycin) were present were carried out in a similar manner except for the addition of a 30-min preincubation time before the addition of insulin or Tam. Statistical comparisons between the different conditions were by ANOVA with Dunnett's multiple comparison posttest. P < 0.05 were considered significant.

Western blot analyses. Cells were disrupted in lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 nM okadaic acid, 30 mM NaF, 2 mM NaPPi, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation for 15 min at 15,000 g before Western blotting. The protein concentrations in each sample were measured, and an equal amount of protein from each cell type was used for Western blotting. Akt phosphorylation was determined by probing Western blots with p-Akt-Ser473 antibody (Cell Signaling). Detection of bound antibody was carried out after a subsequent incubation with secondary antibody and utilizing West Pico Chemiluminescence (Pierce) reagents.

Cell proliferation. H4IIE cells were plated in 60-mm plates in DMEM with 5% FCS and 5% NCS and treated as indicated with vehicle (ethanol) or 1 µM Tam. The media and treatments were changed every 3rd day. After 7 days, triplicate plates were trypsinized, stained with Trypan blue, and counted, excluding Trypan blue-positive cells.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Expression of either active Akt1 or -3 induced an increase in cell size of MCF-7 cells. MCF-7 cells transfected with either empty plasmid (Puncl), a plasmid encoding a constitutively active Akt3 [myristoylated (myr)Akt3], or a constitutively active Akt1 (myrAkt1) (5, 14) were analyzed. Immunoblotting with an anti-p-Akt-Ser473 antibody (to detect the activated, phosphorylated form of the enzyme) demonstrated that the uncloned population of myrAkt3-transfected cells (A3uncl) as well as the clones isolated of myrAkt3 (A3B5)- and myrAkt1 (A1–5)-expressing cells contained active, p-Akt at levels much higher than the total p-Akt in insulin-treated MCF-7 cells (Fig. 1A). During the culturing of these cells, we observed an increased size of the cells expressing the constitutively active Akt. To quantitate this, we measured FSC with a FACS of the different cell lines (this measurement is proportional to the diameter of the cells). An ~30% increase in mean FSC units was observed for the Akt1 and Akt3 high-expressing clones (A1–5 and A3B5; Fig. 1B). To confirm the increase in cell size of these cells, we also measured their size via the use of a Coulter counter (this measurement is proportional to the volume of the cells). By this technique, the A1–5 and A3B5 cell lines were found to have an 80–90% increase in mean cell volume over that observed in the control parental MCF-7 cells (MCF-7; Fig. 1C). The A3uncl pool, which expresses a lower level of active Akt (Fig. 1A) had a lower increase in cell volume (Fig. 1C). These results indicate that both the Akt1 and Akt3 isoforms can induce an increase in cell size and that this increase in cell size is proportional to the amount of active Akt present in the cell.

To determine whether this increase in cell volume would persist in vivo, we compared the size of the cells in a tumor formed with the myrAkt3-expressing cells (A3B5) with those formed by the plasmid-transfected pool population of MCF-7 cells (Puncl). Hematoxylinstained sections of tumors from animals injected with Puncl (Fig. 2A) or A3B5 (Fig. 2B) cells were analyzed by morphometric analysis. The average number of pixels per cell ± SD was determined after analyzing >=50 cells of each type (Fig. 2C). We detected about a twofold increase in the in vivo cell size of myrAkt3-overexpressing MCF-7 cells compared with the control cells.



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Fig. 2. Increased size of MCF-7 cells expressing myrAkt3 is maintained in vivo in xenografts. MCF-7 cells transfected with either empty plasmid (Puncl) or myrAkt3 (A3B5) were subcutaneously injected into nude mice. After 28 days, tumors were harvested, sectioned, and stained with hematoxylin. Tumor sections from animals injected with Puncl (A) or A3B5 (B) cells were subjected to morphometric analysis. The boundary of each cell was drawn and the number of pixels inside each cell determined. The average number of pixels per cell ± SD was determined after analyzing >=50 cells of each type (C).

 

Regulatable Akt induces an increase in size of a rat hepatoma cell line. Because the aforementioned studies were performed with cells that had been selected for expression of a constitutively active Akt, a long time had elapsed between expression of the Akt and the size determination. We therefore examined the size of a rat hepatoma cell (called H4IIE) that expresses a regulatable version of Akt1 (MER-Akt1) (12). The MER-Akt1 enzyme is rapidly activated (<=30 min) after treatment of the cells with Tam (17), and it remains active as observed by the presence of the p-Akt in these cells during the course of these experiments (Fig. 3A). In contrast, the control H4IIE cells treated with Tam show no activated p-Akt (Fig. 3A). The size of the H4IIE cells was monitored after addition of Tam to the cells. A 3% increase in mean FSC units was observed 3 days after start of the treatment with Tam of the H4IIE/MER-Akt1 cells, and a 22% increase in FSC was observed after 5 days of treatment (Fig. 3B). In contrast, no increase in cell size was observed when these same cells were treated with the vehicle used to dissolve the Tam (ethanol; Fig. 3B), and no increase in size of the parental H4IIE cells was observed after treatment with Tam (data not shown). In addition, 5 days of treatment with Tam caused an ~70–75% increase in mean cell volume in the H4IIE/MER-Akt1 cells compared with similarly treated H4IIE or H4IIE/MER-Akt1 cells treated with vehicle (ethanol; Fig. 3C).

Rapamycin does not completely inhibit the Akt-induced increase in cell size. Because Akt can induce an increase in cell size by stimulating protein synthesis through the mTOR pathway, we utilized an inhibitor of mTOR (rapamycin) to assess the role of this pathway in the subsequent increase in cell size (6). A3B5 and Puncl cells were treated with 100 nM rapamycin for 5 days, and their sizes were measured by FACS analysis. The previously observed 30% increase in mean FSC units of the A3B5 cells over that of the Puncl cells was dramatically reduced, although the A3B5 cells still appeared somewhat larger than the Puncl cells (Fig. 4A). To better assess the sizes of these cells, we also analyzed the effect of this treatment by measuring the sizes of the cells with the Coulter counter (Fig. 4B). When parental and A3B5 cells were treated for 5 days with 100 nM rapamycin, we observed a 9 and 69% decrease in cell volume, respectively (Fig. 4B). Thus the rapamycin-treated A3B5 cells were still ~44% larger than the rapamycin-treated parental cells. Controls verified that the rapamycin concentration used here completely blocked the phosphorylation of the downstream substrates p70S6K and ribosomal S6 protein (data not shown). In addition, in the H4IIE/MERAkt1, rapamycin again decreased the Tam-induced increase in cell size only by ~70% (data not shown).

Active Akt increases cell size throughout the cell cycle. Because we have observed that Akt increases the percentage of cells in G2/M (data not shown), which might result in an increase in mean FSC units, we wanted to distinguish whether the Akt-induced increase in cell size was due to changes in cell cycling or whether the increase in cell size occurs throughout the cell cycle. After synchronization of the cells, we determined the relative size of the A3B5 cells and Puncl cells in either the G1 or post-G1 phase. An ~20% increase in mean FSC units was observed for the cells expressing active Akt3 over the control cells in both the G1 and post-G1 phases (Fig. 5). Thereby, active Akt increases cell size throughout the cell cycle and is not a result of cell cycle dysregulation.

Akt activation decreases protein degradation but not cell number. Because an inhibitor of the insulin-induced increase in protein synthesis, rapamycin, did not completely block the increase in cell size induced by Akt (Fig. 4), it suggested that Akt could also be acting on another pathway to stimulate an increase in cell size. Because cell size is also dependent on the rate of protein turnover, we assessed whether Akt would also inhibit protein degradation, a well-known effect of insulin (18). To do these studies, we utilized the rat hepatoma cells expressing the regulatable version of Akt (the H4IIE/MER-Akt1 cells). A control experiment verified that, in the parental H4IIE cells and the H4IIE/MER-Akt1 cells, insulin (at concentrations 10–12 to 10–6 M) inhibited protein degradation similarly in these two cell lines (Fig. 6A). In contrast, Tam, which activates the MER-Akt1 (12), inhibited the protein degradation in the H4IIE/MER-Akt1 cells but not in the parental cells (Fig. 6B). The extent of maximal inhibition of protein degradation in these cells by Tam (15%) was comparable to the extent of maximal inhibition by insulin. The dose of Tam required to inhibit degradation also corresponds to the dose required to activate Akt in these cells (17).



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Fig. 6. Akt activation inhibits protein degradation but does not increase cell numbers. A: effect of insulin on protein degradation in H4IIE cells with or without a Tam-regulatable MER-Akt1 construct. Cells were incubated with indicated concentrations of insulin. Control H4IIE and H4IIE/MER-Akt1 cells degraded 11.0 ± 1.4%/3 h and 13.0 ± 1.2%/3 h, respectively, of their protein in the absence of insulin. B: activation of Akt inhibits protein degradation in H4IIE/MER-Akt1 cells. Cells were incubated with indicated concentrations of Tam. Control H4IIE and H4IIE/MER-Akt1 cells degraded 12.1 ± 1.6%/3 h and 12.9 ± 0.9%/3 h, respectively, in the absence of Tam. Data are means ± SE for 3–6 individual experiments carried out in triplicate, and statistical significance was as shown. ***P < 0.001 for H4IIE/Mer-Akt1 vs. control cells. C: activation of Akt does not effect proliferation. Equal nos. of H4IIE and H4IIE/MER-Akt1 cells were grown in the presence of either Tam (1 µM) or vehicle (ethanol), as indicated. After 7 days, cells were counted. Results shown are means of 3 experiments.

 

To test whether Akt activation also affects cell cycling, we measured cell proliferation over the course of Tam treatment. We treated both parental and H4IIE/MER-Akt1 cells with Tam or vehicle for 7 days and then measured total cell numbers. We did not observe any significant effect of Akt activation on total cell numbers (Fig. 6C).

On the mechanism whereby insulin inhibits protein degradation. To further examine the mechanism whereby insulin inhibits protein degradation, H4IIE/MER-Akt1 cells were treated with or without insulin in the presence or absence of Tam (Fig. 7A). As observed above, a similar decrease in protein degradation was found with Akt activation as was seen with insulin addition. Moreover, we did not observe an additive decrease in protein degradation in the presence of both insulin and Tam (Fig. 7A), consistent with the hypothesis that insulin utilizes the Akt pathway to inhibit protein degradation.



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Fig. 7. Studies on mechanism(s) whereby insulin and Akt inhibit protein degradation. A: lack of additivity of Akt activation and insulin on inhibition of protein degradation in H4IIE/MER-Akt1 cells. Cells were incubated with 0, 2, or 10 nM Tam in the presence or absence of 100 nM insulin, as indicated. Data are means ± SE of 2–4 individual experiments carried out in triplicate, and statistical significance was as shown. **P < 0.01. B: effect of LY-294002 (40 µM) and rapamycin (200 nM) on inhibition of protein degradation by insulin in H4IIE cells. Inhibitors were added to cells 30 min before addition of 1 µM insulin. Data are means ± SE for 6–8 individual experiments carried out in triplicate, and statistical significance was as shown. *P < 0.05 and **P < 0.01. C: effect of LY-294002 (40 µM) and rapamycin (200 nM) on inhibition of protein degradation by insulin or Tam in H4IIE/MER-Akt 1 cells. Inhibitors were added to cells 30 min before addition of either 1 µM insulin or 10 nM Tam. Data are means ± SE for 3–8 individual experiments carried out in triplicate, and statistical significance was as shown. *P < 0.05 and ***P < 0.001.

 

To further explore this hypothesis, the PI3K inhibitor LY-294002 was utilized (30). This inhibitor blocks the insulin-stimulated activation of Akt as well as the ability of Tam to activate the regulatable version of Akt (13, 17). In the presence of LY-294002, the insulin-induced inhibition of protein degradation was completely blocked in the parental H4IIE cells (Fig. 7B). In the H4IIE/MER-Akt1 cells, the effect of either insulin or Tam on protein degradation was blocked (Fig. 7C), consistent with the hypothesis that insulin utilizes the Akt pathway to inhibit protein degradation.

To determine whether mTOR was involved in the inhibition of protein degradation, we utilized the mTOR inhibitor rapamycin. Rapamycin did not affect the ability of insulin to inhibit protein degradation in the parental H4IIE cells (Fig. 7B), nor did it affect the ability of insulin and Akt to inhibit protein degradation in the H4IIE/MER-Akt1 cells (Fig. 7C).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
A number of studies of both mammals and lower organisms have documented a role for the PI3K/Akt pathway in the regulation of the size of cells (24, 6, 8, 11, 16, 19, 20, 24, 29, 32). In agreement with this previous work, overexpression of either constitutively active Akt1 or Akt3 was observed in the present work to induce an increase in the size of MCF-7 cells. This increase in cell size was observed by two different assays, both by FSC as detected in a FACS and by measurements of cell size in a Coulter counter. These results indicate that both of these isoforms of Akt can induce an equivalent increase in cell size. Moreover, an increase in cell size was observed even in the case of the Akt3-expressing cells after they were implanted in a mouse. This increase in cell size in vivo was demonstrated by a distinct technique, morphometric analysis of the cells.1 Finally, an increase in cell size could be documented in a rat hepatoma cell line expressing a regulatable Akt after this enzyme was activated. We could utilize this latter system to demonstrate that the increase in cell size was a slow process, requiring 5 days to reach the maximum approximately twofold increase in cell volume.

In previous studies, much of the increase in cell size was attributed to the ability of the PI3K/Akt pathway to regulate protein synthesis via its known ability to regulate mTOR (12, 27). Two of the main downstream targets of the PI3K/Akt/mTOR pathway, p70S6K and PHAS-I/4E-BP1, contribute to the effect of Akt on protein synthesis (6). However, our data show that Akt increases cell size through both mTOR-dependent and mTOR-independent pathways. In the present studies, rapamycin, an inhibitor of the mTOR pathway, was observed to block ~70% of the Akt-mediated increase in cell size. The inability of rapamycin to completely reverse the phenotype of increased cell size occurred despite its ability to completely block the phosphorylation (activation) of the downstream substrates of mTOR, p70S6K and S6.

To confirm in both of the Akt-overexpressing cell lines that cell cycle dysregulation was not involved in the Akt-induced increase in cell size, we measured either cell size differences throughout the cell cycle (for the MCF-7 cells expressing constitutively active Akt3) or cell number differences after Akt activation (for the H4IIE/MER-Akt1 cells). In the case of the MCF-7 cells, the increase in cell size was independent of the cell cycle, indicating that changes in the cell cycle induced by Akt do not contribute to the observed changes in cell size (Fig. 4). In the case of the H4IIE/MER-Akt1 cells, there were no differences in cell numbers after Akt activation even though the cells were increasing in size during the course of the experiment. Thus both of these studies suggest that the Akt-induced increase in cell size was independent of cell cycle dysregulation.

A well-known effect of insulin is to inhibit protein catabolism (18). Thus, in insulin-deficient states like diabetes, there is an increase in protein catabolism. The ability of insulin to inhibit protein catabolism has been attributed to its ability to regulate lysosomal function (21), the ubiquination process (15), the Ca2+-dependent degradation pathway (25), or a direct effect of insulin on proteasome function (1).

To determine whether the increase in cell size could be due in part to an effect of Akt on protein degradation, we directly measured whether activation of Akt in the H4IIE/MER-Akt1 cells would affect protein degradation. The extent of activation of Akt in these cells is comparable to that induced by insulin (17). The activation of Akt was found capable of inhibiting protein degradation to a level that was comparable to that induced by insulin under the conditions we have utilized to study protein degradation. Although the effect of both insulin and Akt on protein degradation is relatively small (2–3% less protein being degraded during the 3-h assay), such an effect would be expected to accumulate with time. Thus, in 24, 48, and 72 h, one would predict (assuming that the rates of protein degradation remain constant and that these differences are maintained over time) increases of 20, 40, and 60% in cellular protein, respectively. This slow rate of increase in cell proteins is consistent with the observed time course of increase in cell volume that was observed with Akt activation. The inhibition of degradation by both Akt activation and insulin treatment was also found to be unaffected by rapamycin. In contrast, the ability of both of these agents to inhibit degradation was blocked by an inhibitor of PI3K, LY-294002. These results are therefore consistent with the hypothesis that the ability of the PI3K/Akt pathway to induce an increase in cell size is, in part, mediated via an mTOR-independent pathway of inhibition of protein degradation. Moreover, these results are consistent with the hypothesis that the mechanism whereby insulin regulates protein degradation is via its ability to activate Akt. However, it should be noted that the present studies have utilized only cell lines that are partially transformed. It will therefore be important to verify these findings in normal cells and in vivo.

In summary, the present studies indicate that there are three primary mechanisms by which a cell may increase its size (Fig. 8). Two of these mechanisms, cell cycle dysregulation and a stimulation of protein synthesis, have received much attention. In contrast, the third mechanism, inhibition of protein degradation, remains less well understood. From our studies, we do not have any data suggesting that cycle dysregulation plays a role in the Akt-induced increase in cell size. Our data do suggest that Akt increases cell size through both mTOR-dependent and mTOR-independent pathways. The mTOR-dependent pathway presumably involves translational upregulation of protein synthesis via cap-dependent translation (eIF4E) or 5'-TOP translation involving ribosomal proteins or elongation factors (ribosomal S6 and others). From the present studies, we cannot exclude the possibility that a component of the mTOR-independent pathway for increasing cell size may also involve translational upregulation of protein synthesis. However, it is clear from our data that an mTOR-dependent pathway is not involved in the insulin- and Akt-induced inhibition of protein degradation. Future work should be focused on investigating the mechanism by which insulin/Akt inhibits protein degradation.



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Fig. 8. Schema for mammalian target of rapamycin (mTOR)-dependent and -independent pathways of Akt-mediated increase in cell size. Akt-mediated increase in cell size can result either from an increase in protein synthesis, which is mTOR dependent, or via an mTOR-independent pathway, like the inhibition of protein degradation. PI3K, phosphatidylinositol 3-kinase; 4E-BP1, eukaryotic initiation factor 4E-binding protein-1.

 


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported in part by Department of Defense grant DAMD 17–00–1-04445, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-34926 (to R. A. Roth), a Veterans Affairs Merit Review (to W. C. Duckworth/J. Fawcett) and sequential fellowships from an NIH training grant in Diabetes, Endocrinology and Metabolism (NIDDK DK-07217) and National Cancer Institute (PHS CA-09302) training grant (to J. Faridi).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. David Botstein for the use of the Coulter counter, Dr. Garry Nolan for the use of a FACS, and Dr. William Duckworth for support.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Roth, Dept. of Molecular Pharmacology, Stanford University, CCSR, 269 Campus Dr., Stanford, CA 94305–5174 (E-mail: rroth{at}stanford.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The fold increase in volume calculated for the morphometric analysis, 2.8, was larger than the approximately twofold increase in volume determined by the use of the Coulter counter. It is therefore possible that, in vivo, the expression of active Akt may have an even larger effect on a cell's volume. Back


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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