Physiological Role of Akt in Insulin-Stimulated Translocation of GLUT4 in Transfected Rat Adipose Cells
Li-Na Cong,
Hui Chen,
Yunhua Li,
Lixin Zhou,
Margaret A. McGibbon,
Simeon I. Taylor and
Michael J. Quon
Hypertension-Endocrine Branch National Heart, Lung,
and Blood Institute (L.-N.C., H.C., Y.L., M.J.Q.) and Diabetes
Branch National Institute of Diabetes and Digestive and Kidney
Diseases (L.Z., M.A.M., S.I.T.) National Institutes of
Health Bethesda, Maryland 20892
 |
ABSTRACT
|
---|
Stimulation of glucose transport is among
the most important metabolic actions of insulin. Studies in adipose
cells have demonstrated that insulin stimulates its receptor to
phosphorylate tyrosine residues in IRS-1, leading to activation of
phosphatidylinositol 3-kinase, which plays a necessary role in
mediating the translocation of the insulin-responsive glucose
transporter GLUT4 to the cell surface. Akt is a serine-threonine kinase
recently identified as a direct downstream target of
phosphatidylinositol 3-kinase. A previous study in 3T3-L1 cells showed
that overexpression of a constitutively active mutant of Akt is
sufficient to recruit GLUT4 to the cell surface. Since effects of
overexpression of signaling molecules in tissue culture models do not
always reflect physiological function, we have overexpressed a dominant
inhibitory mutant of Akt in rat adipose cells to investigate the
effects of inhibiting endogenous Akt in a physiologically relevant
insulin target cell. Cells were transfected with either wild type
(Akt-WT), constitutively active (Akt-myr), or dominant inhibitory
(Akt-K179A) forms of Akt, and effects of overexpression of these
constructs on insulin-stimulated translocation of a cotransfected
epitope-tagged GLUT4 were studied. Overexpression of Akt-WT resulted in
significant translocation of GLUT4 to the cell surface even in the
absence of insulin. Interestingly, overexpression of Akt-myr resulted
in an even larger effect that was independent of insulin. More
importantly, overexpression of Akt-K179A (kinase-inactive mutant)
significantly inhibited insulin-stimulated translocation of GLUT4.
Taken together, our data suggest that Akt is not only capable of
stimulating the translocation of GLUT4 but that endogenous Akt is
likely to play a significant physiological role in insulin-stimulated
glucose uptake in insulin targets such as muscle and adipose tissue.
 |
INTRODUCTION
|
---|
Insulin is an important regulator of growth, differentiation, and
metabolism. The ability of insulin to recruit the insulin-responsive
glucose transporter GLUT4 from an intracellular compartment to the cell
surface in muscle and adipose tissue accounts for the majority of
insulins effect to stimulate glucose uptake in the body. We, and
others, have previously shown that the insulin receptor tyrosine
kinase, IRS-1, and phosphatidylinositol 3-kinase (PI3K) are key
elements of the signal transduction pathway responsible for
insulin-stimulated translocation of GLUT4 (Refs. 19 and references
contained therein).
Akt is a protooncogene encoding a serine-threonine kinase (also known
as PKB or RAC-PK). Recently, Akt has been identified as a downstream
target of PI3K that mediates mitogenic actions and antiapoptotic
effects of growth factors such as platelet-derived growth factor
(PDGF), epidermal growth factor, insulin-like growth factor I, and
insulin that are initiated by their cognate receptor tyrosine kinases
(for review see Ref.10). Interestingly, overexpression of
constitutively active mutants of Akt in 3T3-L1 cells results in
spontaneous differentiation of these preadipose cells into an
adipocyte-like phenotype and also results in increased glucose uptake
and elevated levels of cell surface GLUT4 in the absence of insulin in
the differentiated cells (11, 12). Furthermore, glycogen synthase
kinase-3 (GSK3, an enzyme involved with the regulation of glycogen
synthesis by insulin) has been identified as a physiological substrate
for Akt (13). These studies suggest that Akt may participate in
metabolic signaling pathways for insulin (in addition to its mitogenic
functions). However, overexpression of constitutively active mutants in
tissue culture cells may result in effects that do not reflect what
occurs under physiological conditions. In this study, we have
overexpressed wild-type, constitutively active, or dominant inhibitory
forms of Akt in primary cultures of rat adipose cells. Overexpression
of wild type and constitutively active Akt resulted in increased levels
of cell surface GLUT4 in the absence of insulin. More importantly,
overexpression of a dominant inhibitory mutant of Akt resulted in
inhibition of insulin-stimulated translocation of GLUT4. The dominant
inhibitory mutant of Akt that we use in this study is a
kinase-deficient Akt that results from the substitution of alanine for
lysine at position 179 in the canonical ATP-binding domain. This mutant
is not only catalytically inactive but has been shown to inhibit the
activity and actions of endogenous Akt (presumably by competing with
endogenous Akt for other upstream or downstream molecules) (14, 15, 16).
Our results, in a bona fide insulin target cell, suggest a
physiological role for Akt in insulin-stimulated glucose transport that
may also apply to other metabolic actions of insulin.
 |
RESULTS
|
---|
Overexpression of Akt Constructs
To directly evaluate the role of Akt in insulin-stimulated
translocation of GLUT4, we transfected wild type Akt (Akt-WT), Akt-Myr,
Akt-K179A, or Myr-K179A into primary cultures of rat adipose cells. We
confirmed overexpression of all four Akt constructs by immunoblotting
cell extracts isolated from transfected cells with an anti-Akt antibody
(Fig. 1
). In the lane containing cell
extracts from control cells transfected with the empty expression
vector pCIS2, there is a faint band representing endogenous rat Akt.
Lanes containing extracts from groups of cells transfected with
recombinant Akt constructs show overexpression of recombinant Akt at
levels that are much higher than the endogenous Akt levels. Since only
5% of the adipose cells that have undergone electroporation are
actually transfected (1), we estimate that there is at least 100-fold
overexpression of the recombinant Akt constructs relative to endogenous
Akt in the transfected cells. The constructs with a mutation in the
ATP-binding site (Akt-K179A and Myr-K179A) are predicted to act in a
dominant inhibitory manner that depends on the level of overexpression.
Therefore, we tested the effect of increasing the concentration of
plasmid DNA used for transfection on expression levels for these
constructs. As expected, increasing the concentration of DNA from 4
µg/cuvette to 7 µg/cuvette significantly increased the levels of
overexpression for Akt-K179A (Fig. 1
, compare lanes 4 and 5).

View larger version (21K):
[in this window]
[in a new window]
|
Figure 1. Overexpression of Recombinant Mouse Akt Constructs
in Transfected Rat Adipose Cells
Whole cell homogenates derived from cells transfected with either
pCIS2, Akt-WT, Akt-K179A, Akt-myr, or Myr-K179A were subjected to
immunoblotting with an anti-Akt antibody. In lane 1, containing extract
from cells transfected with the empty expression vector pCIS2, a band
representing endogenous rat Akt can be seen. Overexpression of
recombinant Akt constructs can be seen in lanes 2, 3, and 4 containing
extracts from cells transfected with Akt-WT (4 µg/cuvette), Akt-myr
(4 µg/cuvette), or Akt-K179A (4 µg/cuvette). Lanes 5 and 6 contain
extracts from cells transfected with Akt-K179A or Myr-K179A at higher
DNA concentrations (7 µg/cuvette). By scanning densitometry, the
relative intensities of the bands representing Akt in lanes 16 were
420, 4510, 3790, 2920, 4220, and 4150, respectively. It is possible
that the Akt antibody we used does not detect rat and mouse Akt with
equal efficiency. A representative blot is shown from an experiment
that was repeated independently three times.
|
|
To confirm that the overexpressed Akt constructs were functional,
we assessed Akt kinase activity in adipose cells overexpressing either
Akt-WT or Akt-myr (Fig. 2
). Whole cell
homogenates derived from groups of transfected cells treated without or
with insulin were subjected to immunoprecipitation with an Akt
antibody, and the ability of the immunoprecipitates to stimulate
incorporation of [32P]ATP into the substrate histone 2B
was measured. In control cells transfected with the empty expression
vector pCIS2, there was a small increase in endogenous Akt activity
caused by insulin stimulation. Cells overexpressing Akt-WT showed a
marked increase in detectable Akt activity upon insulin stimulation
(compared with control cells) while cells overexpressing the
constitutively active Akt-myr had high levels of Akt activity in both
the basal and insulin-stimulated states as expected. We did not measure
Akt activity in cells transfected with the kinase-inactive mutants
because complete inhibition of Akt activity in the 5% of cells
that are transiently transfected would be difficult to detect against
the background of 95% untransfected cells.

View larger version (21K):
[in this window]
[in a new window]
|
Figure 2. Akt Kinase Activity in Adipose Cells Overexpressing
Akt-WT or Akt-myr
Whole cell homogenates derived from cells transfected with pCIS2,
Akt-WT, or Akt-myr and treated without or with insulin (60
nM) were subjected to immunoprecipitation with an antibody
against Akt, and the kinase activity in the immunoprecipitates was
assessed by incorporation of [32P]ATP into histone 2B.
Relative intensities of bands representing phosphorylated histone 2B in
lanes 16 were 149, 178, 109, 308, 438, and 537, respectively, as
assessed by PhosphorImager.
|
|
Effects of Overexpression of Akt-WT and Akt-K179A on Translocation
of GLUT4
After confirming overexpression and activity of the
recombinant Akt constructs in transfected adipose cells, we next
determined their effects on the ability of insulin to recruit a
cotransfected epitope-tagged GLUT4 to the cell surface. The insulin
dose-response curve for control cells cotransfected with the empty
expression vector pCIS2 and GLUT4-HA showed a 3-fold increase in cell
surface GLUT4-HA upon maximal insulin stimulation (60 nM)
with an ED50 of 0.06 nM (Fig. 3A
). Interestingly, in the absence of
insulin, overexpression of Akt-WT resulted in significant translocation
of the cotransfected GLUT4-HA to the cell surface to levels that were
approximately 80% of that seen in the control cells treated with a
maximally stimulating dose of insulin. Treatment of cells
overexpressing Akt-WT with insulin increased the amount of GLUT4-HA at
the cell surface to levels that were comparable to levels observed in
the control cells treated with 60 nM insulin. These results
demonstrate that overexpression of wild type Akt is sufficient to cause
translocation of GLUT4 in adipose cells.
To determine whether Akt plays a necessary physiological role in
insulin-stimulated translocation of GLUT4, we overexpressed Akt-K179A,
a point mutant of Akt that does not bind ATP and thus has no kinase
activity. This mutant is predicted to behave in a dominant inhibitory
fashion. When we transfected cells with Akt-K179A at the same DNA
concentration as we used for Akt-WT (4 µg/cuvette), the insulin
dose-response curve for translocation of GLUT4-HA was similar to
that of the control cells (data not shown). These results suggest that
kinase activity is important for the effect of overexpressed Akt-WT on
translocation of GLUT4. More importantly, when we transfected higher
concentrations of Akt-K179A (7 µg/cuvette), we observed a
statistically significant 20% decrease in insulin responsiveness and
2.5-fold decrease in insulin sensitivity with respect to translocation
of GLUT4 (Fig. 3B
). These results are consistent with the idea that
endogenous Akt contributes significantly to insulin-stimulated glucose
transport in a bona fide insulin target tissue under
physiological conditions.
To help rule out the possibility that differences we observed in the
insulin dose-response curves for cells overexpressing the various Akt
constructs are due to effects of these constructs on expression of
GLUT4-HA, we evaluated total levels of GLUT4-HA in cells cotransfected
with GLUT4-HA and the various Akt constructs. Total membrane fractions
derived from each group of transfected cells were immunoblotted with an
anti-HA antibody (Fig. 4
). The results of
this experiment demonstrate that there is no detectable effect of
overexpressing the various Akt constructs on the total level of
GLUT4-HA in transfected cells. Thus, any differences in the insulin
dose-response curves of cells overexpressing the Akt constructs are
most likely due to effects of these constructs on signal transduction
pathways related to translocation of GLUT4.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 4. Cotransfected Cells Express Comparable Levels of
GLUT4-HA
Total membrane fractions prepared from cells co-transfected with
GLUT4-HA (2 µg/cuvette) and either pCIS2, Akt-WT, or Akt-myr (4
µg/cuvette), or Akt-K179A or Myr-K179A (7 µg/cuvette) were
subjected to immunoblotting with the anti-HA antibody HA-11. Cells
transfected with pCIS2 alone represent a negative control since these
cells do not express GLUT4-HA (lane 1). Comparable levels of GLUT4-HA
are seen for cells cotransfected with GLUT4-HA and either pCIS2,
Akt-WT, Akt-myr, Akt-K179A, or Myr-K179A (lanes 26, density in
arbitrary units = 1310, 1160, 954, 1089, and 1120, respectively).
A representative blot is shown from an experiment that was repeated
independently twice.
|
|
Effects of Overexpression of Akt-myr and Myr-K179A on Translocation
of GLUT4
Since localization to the cell membrane is thought to play an
important role in the activation of Akt, we also investigated effects
of overexpressing Akt constructs that are targeted to the cell membrane
as a result of having a myristoylation sequence fused in-frame with the
N terminus of Akt. In the absence of insulin, overexpression of Akt-myr
in adipose cells resulted in a dramatic translocation of the
cotransfected GLUT4-HA to the cell surface to levels that were
approximately 150% of the levels observed in the control cells treated
with a maximally stimulating dose of insulin (Fig. 5A
). Interestingly, this effect was not
influenced significantly by insulin treatment. These results
demonstrate that targeting wild type Akt to the cell membrane is
sufficient to generate a signal for translocation of GLUT4 that exceeds
that produced by maximal insulin stimulation under physiological
conditions.
To determine whether Akt kinase activity is important for mediating the
effects of the membrane-targeted Akt on translocation of GLUT4, we
overexpressed a point mutant of the Akt-myr construct (Myr-K179A)
that does not bind ATP and therefore has no kinase activity.
Transfection of adipose cells with high concentrations of Myr-K179A (7
µg DNA/cuvette) resulted in an inhibition of insulin-stimulated
translocation of GLUT4 that was similar to that observed with Akt-K179A
(Fig. 5B
). These results demonstrate that targeting of Akt to the cell
membrane is not sufficient to mediate translocation of GLUT4 in the
absence of kinase activity. Furthermore, the inhibitory effect of
overexpression of Myr-K179A is consistent with a physiological role for
endogenous Akt in insulin-stimulated translocation of GLUT4. As with
Akt-WT and Akt-K179A, the fact that overexpression of Akt-myr and
Myr-K179A did not significantly affect expression of GLUT4-HA (Fig. 4
)
suggests that any differences observed in the insulin dose-response
curves for cells overexpressing Akt-myr or Myr-K179A are due to effects
of these Akt constructs on signaling pathways.
 |
DISCUSSION
|
---|
The insulin receptor belongs to a large family of ligand-activated
tyrosine kinases that includes receptors for growth factors such as
insulin-like growth factor I, PDGF, epidermal growth factor, and
fibroblast growth factor. Although insulin and these other growth
factors mediate a diverse array of biological effects, the signal
transduction pathways for their receptors share many common elements.
For example, PI3K is activated when the SH2 domains of the
p85-regulatory subunit of PI3K bind to specific phosphorylated tyrosine
motifs located either on the growth factor receptors themselves or on
substrates of the receptors such as IRS-1 (for review see Refs. 4 and
17). PI3K is involved with both metabolic signaling by insulin (3) and
mitogenic signaling by other growth factors (18). However, activation
of PI3K by growth factors such as PDGF is not sufficient to mimic
metabolic effects of insulin in adipose cells (19, 20). Thus,
specificity in insulin signaling must be generated by other cell
type-specific mechanisms such as subcellular localization of specific
PI3K-signaling complexes (21), the use of multiple isoforms of PI3K
(18), integration of multiple upstream signals (22), or divergent
downstream pathways.
Recently, Akt has been identified as a direct downstream target of PI3K
that plays an important role in mediating the mitogenic and
antiapoptotic effects of several growth factors including insulin (10, 14, 15, 16, 23, 24, 25, 26, 27). Products of PI3K such as
phosphatidylinositol-3,4-diphosphate bind to the N-terminal PH domain
of Akt and may serve to recruit Akt to the cell membrane and activate
its serine-threonine kinase activity (28, 29). The oncogenic form of
Akt (v-Akt) contains a Gag sequence that is myristoylated and results
in targeting of v-Akt to the cell membrane and constitutive activation
(30). Using a myristoylated form of Akt lacking the PH domain, Kohn
et al. (12) recently showed that overexpression of this
constitutively active mutant resulted in recruitment of GLUT4 to the
cell surface in differentiated 3T3-L1 cells. Although these experiments
suggest activated Akt is sufficient to recruit GLUT4, they did not
determine whether Akt is a necessary factor in insulin-stimulated
translocation of GLUT4.
The 3T3-L1 cell line is a tissue culture model derived from primitive
mouse mesodermal cells that can differentiate into an adipocyte-like
phenotype under the appropriate conditions (31). These tissue culture
cells have been extremely useful for understanding the regulation and
control of adipocyte differentiation (32, 33). However, these cells do
not always differentiate completely or uniformly. Furthermore, they do
not display the full repertoire of genes expressed in primary adipose
cells. For example, ob gene mRNA levels in differentiated 3T3-L1 cells
are <1% of the levels observed in freshly isolated rat adipose
cells. Finally, these tissue culture cells are much less responsive
than isolated rat adipose cells with respect to the effects of insulin
and other hormones on glucose transport and metabolism.
To address the physiological role of Akt in insulin-stimulated glucose
transport in a bona fide insulin target cell, we have
overexpressed a dominant inhibitory mutant of Akt in primary cultures
of rat adipose cells to determine whether endogenous Akt is necessary
for insulin-stimulated translocation of GLUT4.
Overexpression of Akt Constructs in Adipose Cells
Transfection of rat adipose cells with the various Akt constructs
resulted in high levels of overexpression similar to what we previously
observed with other recombinant genes in our system (1, 3, 34, 35). In
addition, we were able to demonstrate that increasing the concentration
of DNA during transfection for the K179A construct led to a comparable
increase in the expression of these constructs. Interestingly, Akt-WT
had somewhat higher levels of expression than Akt-K179A when the
same concentrations of DNA were used for transfection. This finding is
similar to a previous report in transfected 293 cells overexpressing
wild type and kinase-dead forms of Akt (15). In the case of Akt-WT and
Akt-myr, we were also able to demonstrate that overexpression of these
recombinant proteins resulted in Akt activity that was consistent with
the level of expression and expected kinase activity. That is, the
ability of Akt-WT to phosphorylate histone 2B was increased in response
to insulin while Akt-myr showed high constitutive kinase activity.
Unfortunately, the 5% transfection efficiency of our adipose cell
transfection system (1) limits our ability to directly measure
decreases in Akt activity expected with the kinase-inactive mutants
Akt-K179A and Myr-K179A because of the background activity of 95%
of the untransfected cells.
As discussed previously (35), the use of epitope-tagged GLUT4 allows us
to distinguish and study transfected cells without interference from
nontransfected cells. The fact that levels of GLUT4-HA were comparable
in all groups of transfected cells suggests that changes in cell
surface GLUT4 caused by overexpression of the various Akt constructs
are due to effects on insulin signal transduction pathways rather than
effects on the total level of expression of GLUT4. Although highly
unlikely, it is formally possible that effects of the Akt constructs on
translocation of GLUT4 are due, in part, to effects of Akt on levels of
expression of upstream signaling molecules such as the insulin receptor
or IRS-1. We do not believe this is the case, however, because we have
previously demonstrated that 20-fold overexpression of these upstream
molecules does not have as large an effect on translocation of GLUT4 as
overexpression of the constitutively active Akt (1, 2). Nevertheless,
it is difficult for us to directly rule out an effect of Akt on
expression level of these upstream signaling molecules because it is
not possible for us to assess the amounts of endogenous insulin
receptor and IRS-1 exclusively in the 5% of cells that are transfected
with the Akt constructs. In our cotransfection experiments, we used at
least twice as much DNA for the Akt constructs as we did for GLUT4-HA
to increase the likelihood that cells transfected with GLUT4-HA were
also transfected with the vector of interest. If some fraction of cells
were transfected only with GLUT4-HA, our results would underestimate
the differences between control and experimental groups. We estimate
that at least 95% of cells expressing GLUT4-HA also express the
cotransfected second plasmid under our experimental conditions (3).
Recruitment of GLUT4 by Akt-WT and Akt-myr
Overexpression of Akt-WT resulted in significant translocation of
GLUT4 to the cell surface in the absence of insulin. These results are
similar to what we previously observed with overexpression of either
wild type insulin receptors or IRS-1 in adipose cells (1, 2).
Presumably, there is a small signal present even in the absence of
insulin that can be amplified by an excess of Akt-WT. The existence of
this basal level of signaling is also supported by our recent
demonstration that overexpression of the protein tyrosine phosphatase
PTP1B in adipose cells led to a decrease in the amount of cell surface
GLUT4 present in both the absence and presence of insulin (35). As with
overexpression of the insulin receptor or IRS-1, insulin stimulation of
adipose cells overexpressing Akt-WT resulted in a further increase in
the amount of cell surface GLUT4 to a level that was comparable to that
observed in control cells treated with a maximally stimulating
concentration of insulin. Thus, although overexpression of Akt-WT was
sufficient to recruit GLUT4, this did not result in a larger effect
than insulin alone could elicit. In contrast, overexpression of the
constitutively active mutant Akt-myr resulted in dramatic translocation
of GLUT4 to the cell surface at levels that significantly exceeded
those achievable by insulin stimulation of the control cells.
Furthermore, this effect was independent of insulin. It is likely that
overexpression of Akt-myr results in a higher level of Akt activity at
the cell membrane than is achievable by insulin stimulation of
endogenous Akt in the control cells. However, this is not merely a
function of the amount of Akt present because comparable overexpression
of Akt-WT did not have as large an effect as Akt-myr (even with insulin
stimulation). It is also possible that targeting of overexpressed Akt
to the cell membrane stimulates pathways for signaling recruitment of
GLUT4 that may not be operative under physiological conditions. For
example, even though Ras probably does not contribute to
insulin-stimulated translocation of GLUT4 under physiological
conditions, we previously reported that overexpression of
constitutively active mutants of Ras results in a similarly large
effect to recruit GLUT4 in adipose cells (although in this case,
insulin treatment results in a further increase in cell surface GLUT4)
(3). Our results with Akt-myr are consistent with those of Kohn
et al. (12), who showed that overexpression of a
myristoylated mutant of Akt lacking its PH domain had similar effects
on translocation of GLUT4 in differentiated 3T3-L1 adipocytes.
Akt-K179A and Myr-K179A Inhibit Translocation of GLUT4
Because overexpression of a signaling protein can lead to events
that are not related to the physiological functions of that protein, it
is useful to assess the effects of inhibiting endogenous Akt on
insulin-stimulated translocation of GLUT4. Toward this end we used Akt
constructs that have a mutation in the ATP-binding domain rendering the
kinase catalytically inactive. Importantly, the PH domain and other
regions of the molecule are intact so that overexpressed Akt-K179A
or Myr-K179A can presumably compete with endogenous Akt for upstream or
downstream factors. Indeed, the catalytically inactive mutant Akt-K179A
has been extensively characterized (15, 25, 26, 36) and has been shown
to have dominant inhibitory effects in other contexts (23, 24, 28).
When Akt-K179A was transfected into adipose cells using the same
concentration of DNA as was used for Akt-WT and Akt-Myr, the resulting
insulin dose-response curve was similar to that of the control
cells. This suggests that intact kinase activity is important for
mediating the effect of Akt-WT on translocation of GLUT4. When higher
concentrations of Akt-K179A were used, we observed inhibition of
insulin-stimulated translocation of GLUT4 with significant decreases in
both insulin sensitivity and responsiveness. Thus, endogenous Akt is
likely to contribute importantly to the physiological regulation of
GLUT4 by insulin. The significance of these results is highlighted by
comparison with results from our previous study on the role of Ras in
insulin-stimulated translocation of GLUT4 (3). In that study, we showed
that while overexpression of constitutively active mutants of Ras leads
to recruitment of GLUT4, overexpression of a dominant inhibitory mutant
had no effect on insulin-stimulated translocation of GLUT4, leading us
to conclude that Ras does not play a physiological role in
insulin-stimulated glucose transport. Our observation of an expression
level-dependent effect of Akt-K179A on inhibition of GLUT4 recruitment
is consistent with the putative dominant inhibitory mechanism of
Akt-K179A (i.e. competition with endogenous Akt for
limiting factors). Using a dominant inhibitory mutant of PI3K, we
previously demonstrated nearly complete inhibition of
insulin-stimulated translocation of GLUT4 in adipose cells (3).
Interestingly, even though Akt is a direct downstream target of PI3K,
overexpression of Akt-K179A did not have as large an effect as the
dominant inhibitory mutant of PI3K. It is possible that a further
increase in the level of expression of Akt-K179A would result in
further inhibition of GLUT4 recruitment. Because of limitations on the
total amounts of DNA that we can use in our system (37), we were not
able to test this possibility. It is also likely that there are
multiple downstream effectors of PI3K or other signaling molecules that
contribute to the effect of insulin on recruitment of GLUT4. For
example, using a catalytically inactive mutant of Syp we recently
demonstrated a small role for Syp in insulin-stimulated translocation
of GLUT4 in adipose cells (35). Thus, maximal inhibition of endogenous
Akt may lead to only partial inhibition of translocation of GLUT4
because there are other effectors of PI3K that contribute to this
effect.
Overexpression of Myr-K179A had an inhibitory effect on recruitment of
GLUT4 that was similar to that seen with Akt-K179A. This suggests that
localization of Akt to the membrane is not sufficient to mediate
effects on recruitment of GLUT4 in the absence of kinase activity.
Furthermore, even when localized to the cell membrane, Myr-K179A is
presumably able to compete with endogenous Akt for limiting factors and
results in inhibition of insulin-stimulated translocation of GLUT4. In
addition, the magnitude of inhibition caused by both Akt-K179A and
Myr-K179A was similar and supports the idea that endogenous Akt is not
the only effector of PI3K in this action of insulin. These results
further support a physiological role for Akt in insulin-stimulated
glucose transport.
In summary, overexpression of a constitutively active Akt has a greater
effect on recruitment of GLUT4 than overexpression of wild-type Akt.
More importantly, overexpression of catalytically inactive mutants of
Akt in primary cultures of rat adipose cells inhibits
insulin-stimulated translocation of GLUT4. Our results strongly suggest
a physiological role for Akt in insulin-stimulated glucose transport in
insulin target tissues.
 |
MATERIALS AND METHODS
|
---|
DNA Vector Constructions
pCIS2
An expression vector that generates high expression levels in
transfected rat adipose cells (37) was used as the parent vector for
subsequent constructions.
GLUT4-HA
Complementary DNA coding for human GLUT4 with the influenza
hemagglutinin epitope (HA1) inserted in the first exofacial loop of
GLUT4 was subcloned into the pCIS2 vector (1).
Akt-WT
A 1.4-kb XbaI/BamHI fragment containing the cDNA
for mouse Akt-1 [the generous gift of Drs. P. N. Tsichlis and K.
Datta (27)] was blunt-ended and ligated in the sense orientation into
the HpaI site in the multiple cloning region of pCIS2.
Akt-myr
BglII/BamHI fragment containing the cDNA for
mouse Akt-1 with a myristoylation sequence from pp60 c-src
(38) fused in-frame with the N terminus of Akt (a generous gift from
Drs. P.N. Tsichlis and K. Datta) was blunt-ended and ligated in the
sense orientation into the HpaI site in the multiple cloning
region of pCIS2.
Akt-K179A
Point mutant of Akt-WT with a substitution of alanine for lysine at
position 179 was constructed using a mutagenic oligonucleotide 5'-GC
TAC TAT GCC ATG GCG ATC CTC AAG AAG G-3' and the
MORPH site-specific plasmid DNA mutagenesis kit according to the
manufacturers instructions (5 prime 3 prime, Inc., Boulder, CO). The
creation of the mutation introduced a new NcoI site. In
addition, the mutation was confirmed by direct sequencing.
Myr-K179A
Point mutant of Akt-myr with a substitution of alanine for lysine at
position 179 was constructed exactly as described above for
Akt-K179A.
Isolated Rat Adipose Cell Preparation
Isolated adipose cells were prepared from the epididymal fat
pads of male rats (170200 g, CD strain, Charles River Breeding
Laboratories, Wilmington, MA) by collagenase digestion as described
(37, 39).
Electroporation
Isolated adipose cells were transfected by electroporation as
described (35, 37). Cells from multiple cuvettes were pooled to obtain
the necessary volume of cells for each experiment as described (see
Table 1
for number of cuvettes and amount
of DNA used).
Assay for Cell Surface Epitope-Tagged GLUT4
Twenty hours after electroporation, adipose cells were processed
as described (1, 2, 3, 35) and treated with insulin at final
concentrations of 0, 0.024, 0.072, 0.3, or 60 nM at 37 C
for 30 min. Cell surface epitope-tagged GLUT4 was determined by using
the anti-HA1 mouse monoclonal antibody HA-11 (Berkeley Antibody
Company, Richmond, CA) at a final dilution of 1:2000 in conjunction
with [125I]-sheep anti-mouse IgG as described (35). Cells
transfected with the empty expression vector pCIS2 were used to
determine nonspecific binding of the antibodies. Typically, the
nonspecific binding was 20% of the total binding to cells transfected
with GLUT4-HA and maximally stimulated with insulin. The actual
specific counts were comparable from experiment to experiment (see
figure legends). The lipid weight from a 200-µl aliquot of cells was
determined as described (40) and used to normalize the data for each
sample.
Immunoblotting of Akt and GLUT4-HA
Expression of recombinant Akt-WT, Akt-myr, Akt-K179A, Myr-K179A,
and GLUT4-HA was confirmed by immunoblotting extracts of cells that
were prepared at the same time and had undergone transfection in
parallel with the cells used for the translocation assay described
above. Cells from 12 cuvettes were pooled for each group. Whole cell
homogenates were prepared from cells cotransfected with GLUT4-HA
(2 µg/cuvette) and either pCIS2, Akt-WT, Akt-myr, (4
µg/cuvette) or Akt-K179A or Myr-K179A (7 µg/cuvette). To determine
relative levels of GLUT4-HA in each group of transfected cells, total
membrane fractions were prepared from the whole cell homogenate by
centrifuging 30 min at 400,000 x g at 4 C. The pellet
containing the total membrane fraction was resuspended in 300 µl TES
buffer (20 mM Tris, 1 mM EDTA, 8.73% sucrose,
10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin
inhibitor, 10 µg/ml BSA, 1 mM phenylmethylsulfonyl
fluoride, pH 7.4, 4 C). The protein concentration in each sample was
measured and aliquots containing 60 µg of protein were subjected to
SDS-PAGE. The contents of the gel were transferred to nitrocellulose
and immunblotted with the monoclonal anti-HA antibody HA-11 (final
concentration, 5 µg/ml). Immunolabeled bands were visualized using an
antibody against mouse IgG in conjunction with an enhanced
chemiluminescent detection system (ECL, Amersham, Arlington Heights,
IL).
For immunodetection of Akt constructs, whole cell homogenates
containing equal amounts of protein (80 µg) were solubilized in
Laemmli sample buffer and subjected to SDS-PAGE. The contents of the
gel were transferred to nitrocellulose, and the Akt protein was
detected with a polyclonal anti-Akt antibody (Upstate Biotechnology
Inc., Lake Placid, NY) at a final concentration of 1 µg/ml. Bands
were visualized using an antibody against mouse IgG in conjunction with
an ECL detection system (Amersham).
Akt Kinase Assay
To assess the kinase activity of the recombinant Akt-WT and
Akt-myr in transfected adipose cells, cells were transfected with
either the empty expression vector pCIS2, Akt-WT, or Akt-myr (4 µg
DNA/cuvette, 15 cuvettes per group) and treated without or with insulin
(60 nM) for 2 min, and whole cell homogenates of each group
were prepared as described above. Samples containing 200 µg protein
from each group were subjected to immunoprecipitation with an antibody
against Akt, and kinase activity in the immunoprecipitates was assessed
by incorporation of [32P]ATP into the substrate histone
2B as described (27). Samples were separated on a 12% SDS-PAGE, and
the phosphorylation of histone 2B in each sample was quantified using
PhosphorImager analysis of the gel (Molecular Dynamics, Sunnyvale,
CA).
Statistical Analysis
Insulin dose-response curves were compared using multivariate
ANOVA. Paired t tests were used to compare individual points
where appropriate. P values of less than 0.05 were
considered statistically significant. The insulin dose-response
curves were fit to the equation y = a + b [x/(x + k)] using a
Marquardt-Levenberg nonlinear least squares algorithm. When plotted on
linear-log axes, this equation gives a sigmoidal curve where the
parameters are associated with the following properties: a = basal
response; a + b = maximal response; k = half-maximal dose
(ED50); and x = concentration of insulin.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. G.I. Bell for supplying the human GLUT4 cDNA, Dr.
C. Gorman for the pCIS2 expression vector, and Drs. T. N. Tsichlis
and K. Datta for the mouse Akt cDNA constructs. We thank Dr. L. Kohn
for helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael J. Quon, M.D., Ph.D., Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-103, 10 Center Drive MSC 1754, Bethesda, Maryland 20892-1754.
This work was supported in part by a Research Award from the American
Diabetes Association (to M.J.Q.).
Received for publication June 3, 1997.
Revision received August 20, 1997.
Accepted for publication September 5, 1997.
 |
REFERENCES
|
---|
-
Quon MJ, Guerre-Millo M, Zarnowski MJ, Butte AJ, Em M,
Cushman SW, Taylor SI 1994 Tyrosine kinase-deficient mutant human
insulin receptors (Met1153>Ile) overexpressed in transfected rat
adipose cells fail to mediate translocation of epitope-tagged GLUT4.
Proc Natl Acad Sci USA 91:55875591[Abstract]
-
Quon MJ, Butte AJ, Zarnowski MJ, Sesti G, Cushman SW, Taylor
SI 1994 Insulin receptor substrate 1 medi-ates the stimulatory
effect of insulin on GLUT4 translocation in transfected rat adipose
cells. J Biol Chem 269:2792027924[Abstract/Free Full Text]
-
Quon MJ, Chen H, Ing BL, Liu ML, Zarnowski MJ, Yonezawa
K, Kasuga M, Cushman SW, Taylor SI 1995 Roles of 1-phosphatidylinositol
3-kinase and ras in regulating translocation of GLUT4 in transfected
rat adipose cells. Mol Cell Biol 15:54035411[Abstract]
-
Quon MJ, Butte AJ, Taylor SI 1994 Insulin signal transduction
pathways. Trends Endocrinol Metab 5:369376
-
Tamemoto H, Kadowaki T, Tobe K, Yagi T, Sakura H, Hayakawa T,
Terauchi Y, Ueki K, Kaburagi Y, Satoh S, et al 1994 Insulin resistance
and growth retardation in mice lacking insulin receptor substrate-1.
Nature 372:182186[CrossRef][Medline]
-
Araki E, Lipes MA, Patti ME, Bruning JC, Haag BR, Johnson
RS, Kahn CR 1994 Alternative pathway of insulin signalling in mice with
targeted disruption of the IRS-1 gene. Nature 372:186190[CrossRef][Medline]
-
Tanti JF, Gremeaux T, Grillo S, Calleja V, Klippel A,
Williams LT, Van Obberghen E, Le Marchand-Brustel Y 1996 Overexpression of a constitutively active form of phosphatidylinositol
3-kinase is sufficient to promote Glut 4 translocation in adipocytes.
J Biol Chem 271:2522725232[Abstract/Free Full Text]
-
Frevert EU, Kahn BB 1997 Differential effects of
constitutively active phosphatidylinositol 3- kinase on glucose
transport, glycogen synthase activity, and DNA synthesis in 3T3-L1
adipocytes. Mol Cell Biol 17:190198[Abstract]
-
Czech MP 1995 Molecular actions of insulin on glucose
transport. Annu Rev Nutr 15:441471[CrossRef][Medline]
-
Franke TF, Kaplan DR, Cantley LC 1997 PI3K: downstream AKTion
blocks apoptosis. Cell 88:435437[Medline]
-
Magun R, Burgering BM, Coffer PJ, Pardasani D, Lin Y, Chabot
J, Sorisky A 1996 Expression of a constitutively activated form of
protein kinase B (c-Akt) in 3T3L1 preadipose cells causes spontaneous
differentiation. Endocrinology 137:35903593[Abstract]
-
Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a
constitutively active Akt Ser/Thr kinase in 3T3L1 adipocytes
stimulates glucose uptake and glucose transporter 4 translocation.
J Biol Chem 271:3137231378[Abstract/Free Full Text]
-
Cross DAE, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated by
protein kinase B. Nature 378:785789[CrossRef][Medline]
-
Burgering BM, Coffer PJ 1995 Protein kinase B (c-Akt) in
phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599602[CrossRef][Medline]
-
Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen
P, Hemmings BA 1996 Mechanism of activation of protein kinase B by
insulin and IGF-1. EMBO J 15:65416551[Abstract]
-
Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C,
Coffer P, Downward J, Evan G 1997 Suppression of c-Myc-induced
apoptosis by Ras signalling through PI(3)K and PKB. Nature 385:544548[CrossRef][Medline]
-
van der Geer P, Hunter T, Lindberg RA 1994 Receptor
protein-tyrosine kinases and their signal transduction pathways. Annu
Rev Cell Biol 10:251337[CrossRef]
-
Carpenter CL, Cantley LC 1996 Phosphoinositide kinases. Curr
Opin Cell Biol 8:153158[CrossRef][Medline]
-
Quon MJ, Chen H, Lin CH, Zhou L, Zarnowski MJ, Klinghoffer
R, Kazlauskas A, Cushman SW, Taylor SI 1996 Effects of overexpressing
wild-type and mutant PDGF receptors on translocation of GLUT4 in
transfected rat adipose cells. Biochem Biophys Res Commun 226:587594[CrossRef][Medline]
-
Isakoff SJ, Taha C, Rose E, Marcusohn J, Klip A, Skolnik EY 1995 The inability of phosphatidylinositol 3-kinase activation to
stimulate GLUT4 translocation indicates additional signaling pathways
are required for insulin-stimulated glucose uptake. Proc Natl Acad
Sci USA 92:1024710251[Abstract]
-
Heller-Harrison RA, Morin M, Guilherme A, Czech MP 1996 Insulin-mediated targeting of phosphatidylinositol 3-kinase to
GLUT4-containing vesicles. J Biol Chem 271:1020010204[Abstract/Free Full Text]
-
Weiss FU, Daub H, Ullrich A 1997 Novel mechanisms of RTK
signal generation. Curr Opin Genet Dev 7:8086[CrossRef][Medline]
-
Kulik G, Klippel A, Weber MJ 1997 Antiapoptotic signalling by
the insulin-like growth factor I receptor, phosphatidylinositol
3-kinase, and Akt. Mol Cell Biol 17:15951606[Abstract]
-
Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM,
Segal RA, Kaplan DR, Greenberg ME 1997 Regulation of neuronal survival
by the serine-threonine protein kinase Akt. Science 275:661665[Abstract/Free Full Text]
-
Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison
DK, Kaplan DR, Tsichlis PN 1995 The protein kinase encoded by the Akt
proto-oncogene is a target of the PDGF-activated phosphatidylinositol
3-kinase. Cell 81:727736[Medline]
-
Kohn AD, Kovacina KS, Roth RA 1995 Insulin stimulates the
kinase activity of RAC-PK, a pleckstrin homology domain containing
ser/thr kinase. EMBO J 14:42884295[Abstract]
-
Datta K, Bellacosa A, Chan TO, Tsichlis PN 1996 Akt is a
direct target of the phosphatidylinositol 3-kinase. Activation by
growth factors, v-src and v-Ha-ras, in Sf9 and mammalian cells. J
Biol Chem 271:3083530839[Abstract/Free Full Text]
-
Klippel A, Kavanaugh WM, Pot D, Williams LT 1997 A specific
product of phosphatidylinositol 3-kinase directly activates the protein
kinase Akt through its pleckstrin homology domain. Mol Cell Biol 17:338344[Abstract]
-
Franke TF, Kaplan DR, Cantley LC, Toker A 1997 Direct
regulation of the Akt proto-oncogene product by
phosphatidylinositol-3,4-bisphosphate. Science 275:665668[Abstract/Free Full Text]
-
Ahmed NN, Franke TF, Bellacosa A, Datta K, Gonzalez-Portal ME,
Taguchi T, Testa JR, Tsichlis PN 1993 The proteins encoded by c-akt and
v-akt differ in post-translational modification, subcellular
localization and oncogenic potential. Oncogene 8:19571963[Medline]
-
Spiegelman BM, Choy L, Hotamisligil GS, Graves RA, Tontonoz P 1993 Regulation of adipocyte gene expression in differentiation and
syndromes of obesity/diabetes. J Biol Chem 268:68236826[Free Full Text]
-
MacDougald OA, Lane MD 1995 Transcriptional regulation of gene
expression during adipocyte differentiation. Annu Rev Biochem 64:345373[CrossRef][Medline]
-
Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of
adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated
transcription factor. Cell 79:11471156[Medline]
-
Ing BL, Chen H, Robinson KA, Buse MG, Quon MJ 1996 Characterization of a mutant GLUT4 lacking the N-glycosylation site:
studies in transfected rat adipose cells. Biochem Biophys Res Commun 218:7682[CrossRef][Medline]
-
Chen H, Wertheimer SJ, Lin CH, Katz SL, Amrein KE, Burn P,
Quon MJ 1997 Protein-tyrosine phosphatases PTP1B and Syp are modulators
of insulin-stimulated translocation of GLUT4 in transfected rat adipose
cells. J Biol Chem 272:80268031[Abstract/Free Full Text]
-
Andjelkovic M, Jakubowicz T, Cron P, Ming XF, Han JW, Hemmings
BA 1996 Activation and phosphorylation of a pleckstrin
homology domain containing protein kinase (RAC-PK/PKB) promoted by
serum and protein phosphatase inhibitors. Proc Natl Acad Sci USA 93:56995704[Abstract/Free Full Text]
-
Quon MJ, Zarnowski MJ, Guerre-Millo M, de la Luz Sierra M,
Taylor SI, Cushman SW 1993 Transfection of DNA into isolated rat
adipose cells by electroporation: evaluation of promoter activity in
transfected adipose cells which are highly responsive to insulin after
one day in culture. Biochem Biophys Res Commun 194:338346[CrossRef][Medline]
-
Klippel A, Reinhard C, Kavanaugh WM, Apell G, Escobedo MA,
Williams LT 1996 Membrane localization of phosphatidylinositol 3-kinase
is sufficient to activate multiple signal-transducing kinase pathways.
Mol Cell Biol 16:41174127[Abstract]
-
Karnieli E, Zarnowski MJ, Hissin PJ, Simpson IA, Salans LB,
Cushman SW 1981 Insulin- stimulated translocation of glucose transport
systems in the isolated rat adipose cell. Time course, reversal,
insulin concentration dependency, and relationship to glucose transport
activity. J Biol Chem 256:47724777[Medline]
-
Cushman SW, Salans LB 1978 Determinations of adipose cell size
and number in suspensions of isolated rat and human adipose cells. J
Lipid Res 19:269273[Abstract]