Am J Physiol Endocrinol Metab 285: E964–E972, 2003.
First published July 22, 2003; 10.1152/ajpendo.00239.2003.
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
1
Department of Molecular Pharmacology, Stanford University, Stanford, California 94305;
Section of Endocrinology, Veterans Affairs Medical Center, Phoenix 85012; and 3Department
of Molecular and Cellular Biology, Arizona State University, Tempe, Arizona 85287
2
Submitted 23 May 2003; accepted in final form 14 July 2003
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
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: [email protected]).
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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.
The costs of publication of this article were defrayed in part by the
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marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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Faridi, Jesika, Janet Fawcett, Lihong Wang, and
Richard A. Roth. Akt promotes increased mammalian cell
size by stimulating protein synthesis and inhibiting protein degradation. Am J Physiol Endocrinol Metab 285:
E964–E972, 2003. First published July 22, 2003; 10.1152/
ajpendo.00239.2003.—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, LY294002, 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.
Akt INHIBITS PROTEIN DEGRADATION
MATERIALS AND METHODS
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[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 for 3 h at 37°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 ␮g/ml aprotinin, 10 ␮g/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 ␮M 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
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-pAkt-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
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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 ␮g/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 ⫻ 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/␮l 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.
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/␮l RNase A
(Sigma), and incubated at room temperature for 30 min.
Next, PI was added at a concentration of 40 ng/␮l, 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).
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 ␮g/ml streptomycin, and 0.5 ␮Ci/ml
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Akt INHIBITS PROTEIN DEGRADATION
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). HematoxylinAJP-Endocrinol Metab • VOL
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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).
stained 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 ⱖ50
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.
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 (ⱕ30 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).
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Akt INHIBITS PROTEIN DEGRADATION
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
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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).
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.
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 insulininduced 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
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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 ⱖ50 cells of each type (C).
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Akt INHIBITS PROTEIN DEGRADATION
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
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 ␮M) or vehicle (ethanol) as indicated. Cells were either
lysed and tested for active Akt by Western blotting with an anti-pAkt (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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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 (2–4, 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
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.
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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).
Akt INHIBITS PROTEIN DEGRADATION
E969
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.
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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).
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
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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 ␮M) or vehicle (ethanol), as
indicated. After 7 days, cells were counted. Results shown are means
of 3 experiments.
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Akt INHIBITS PROTEIN DEGRADATION
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
␮M) 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 ␮M 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 ␮M)
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 ␮M 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.
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 activaAJP-Endocrinol Metab • VOL
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.
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tion 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
Akt INHIBITS PROTEIN DEGRADATION
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.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
DISCLOSURES
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).
18.
19.
20.
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