From the Department of Medicine, Division of
Endocrinology and Metabolism, University of California, San Diego,
La Jolla, California 92093-0673 and ¶ San Diego Veterans
Administration Medical Research Service and the Whittier Diabetes
Institute, La Jolla, California 92037
Received for publication, August 30, 2002, and in revised form, January 29, 2003
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ABSTRACT |
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We investigated the role of cdc42, a Rho GTPase
family member, in insulin-induced glucose transport in 3T3-L1
adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to
decreased insulin-induced and constitutively active
Gq (CA-Gq; Q209L)-induced GLUT4
translocation. Adenovirus-mediated expression of constitutively active
cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the
maximal insulin response, and this was blocked by treatment with the
phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or
LY294002. Both insulin and CA-Gq expression caused an
increase in cdc42 activity, showing that cdc42 is activated by insulin
and is downstream of G Insulin stimulates glucose transport in skeletal muscle
and adipose tissue by inducing the translocation of the GLUT4 glucose transporter from an intracellular pool to the plasma membrane (1, 2).
Although the signaling pathways and the dynamics of GLUT4 movement have
been intensively studied, the precise mechanisms of GLUT4 translocation
remain incompletely understood. Insulin initiates its signal
transduction cascade by activating the insulin receptor tyrosine
kinase, leading to phosphorylation of phosphoprotein substrates,
activation of PI3-kinase,1
and stimulation of glucose transport (3-6). Phosphorylation of IRS-1,
which binds to and activates PI3-kinase, is one mechanism by which
insulin stimulates PI3-kinase to mediate glucose transport. However,
several reports (7-12) using various approaches indicate that IRS-1 is
not necessarily essential for transport stimulation and that other
pathways exist. Thus, we have shown (6) that the activated insulin
receptor can also phosphorylate the heterotrimeric protein component
G More recently, it has been recognized that a separate
PI3-kinase-independent pathway is initiated by insulin stimulation that must complement the PI3-kinase-dependent pathway to achieve
full GLUT4 translocation (16, 17). This PI3-kinase-independent pathway
involves insulin-mediated localization of CAP-Cbl complexes to membrane
rafts with subsequent recruitment of CrkII-C3G to these
structures, leading to activation of the small Rho family GTPase, TC10.
Insulin-stimulated cortical actin remodeling and polymerization are
necessary for the final steps of GLUT4 movement to the plasma membrane,
and it has been proposed that TC10 interacts with neural
Wiskott-Aldrich syndrome protein in an insulin-dependent manner to mediate cortical actin polymerization (18).
Cdc42, another member of the Rho GTPase family, has 69% homology and
86% similarity to TC10 (16). Insulin can stimulate cdc42 activity, and
cdc42 can facilitate actin rearrangement (19). Furthermore, bradykinin,
a GPCR agonist that couples into G Materials--
Mouse monoclonal anti-cdc42 and anti-p85
antibodies (N-SH2), cdc42 assay kit, and protein A-agarose were
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse
monoclonal anti-phosphotyrosine (PY20) antibody was from Transduction
Laboratories (Lexington, KY). Rabbit polyclonal anti-GLUT4 antibody was
purchased from Chemicon International Inc. (Temecula, CA). Rabbit
polyclonal anti-G Cell Culture and Cell Treatment--
3T3-L1 cells were cultured
and differentiated as described previously (6). Differentiated 3T3-L1
adipocytes were incubated with 100 µM LY294002, 100 or
300 nM wortmannin, 50 µM PD98059, or 0.1%
dimethyl sulfoxide vehicle for 1 or 4 h before each assay. For
adenovirus infection, 3T3-L1 adipocytes were transduced for 16 h
in Dulbecco's modified Eagle's high glucose medium with 5% heat-inactivated serum with the following multiplicity of infection (m.o.i.) and with either the recombinant adenovirus of wild
type-G 2-Deoxyglucose Uptake--
The procedure for glucose uptake was
described previously (22) with some modifications. After 60 h of
adenovirus infection, 3T3-L1 adipocytes were serum-starved for 3 h, and the cells were stimulated with 0.5 or 17 nM insulin
in KRP-Hepes buffer (10 mM Hepes, pH 7.4, 131.2 mM NaCl, 4.7 mM KCl, 1.2 mM
MgSO4, 2.5 mM CaCl2, 2.5 mM NaH2PO4) for 30 min at 37 °C.
The procedure for stimulation by osmotic shock was the same as
described previously (23). Glucose uptake was determined in triplicate
at each point after the addition of 2-[3H]deoxyglucose
(0.1 µCi, final concentration 0.1 mM) in KRP-Hepes buffer
for 5 min at 37 °C.
Microinjection of Antibodies and siRNAs--
Microinjection was
carried out using a semiautomatic Eppendorf microinjection system.
Antibodies for microinjection were concentrated and dissolved at 5 mg/ml in microinjection buffer containing 5 mM sodium
phosphate, pH 7.2, 100 mM KCl and were injected into the
cytoplasm. Five mg/ml sheep IgG was injected into the control cells.
siRNA for cdc42 (guuauccacagacagaugutt), silencing mediator for
retinoid and thyroid hormone receptor (cgagaguucucgcuggacutt), and
insulin receptor (tataccatgaattccagcaactt) were purchased from
Dharmacon. siRNAs were dissolved at 5 µM in
microinjection buffer.
Immunostaining and Immunofluorescence
Microscopy--
Immunostaining of GLUT4 was performed essentially as
described (6). 3T3-L1 adipocytes were stimulated with insulin for 20 min at 37 °C and were fixed in 3.7% formaldehyde in
phosphate-buffered saline (PBS) for 10 min at room temperature.
Following washing, the cells were permeabilized with 0.1% Triton X-100
in PBS for 10 min and blocked with 2% fetal calf serum in PBS for 10 min. The cells were then incubated with anti-GLUT4 antibody in PBS with
2% fetal calf serum overnight at 4 °C. After washing, GLUT4 and
injected IgG were detected by incubation with TRITC-conjugated donkey
anti-rabbit IgG antibody and fluorescein isothiocyanate-conjugated donkey anti-mouse or anti-sheep antibody, respectively, followed by
observation under an immunofluorescence microscope. In all counting
experiments, the observer was blinded to the experimental condition of
each coverslip.
Western Blotting--
Serum-starved 3T3-L1 cells were stimulated
with 17 nM insulin at 37 °C for various times as
indicated in each experiment. The cells were lysed in solubilizing
buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 mM NaF, pH 7.5, for 15 min at 4 °C. The
cell lysates were centrifuged to remove insoluble materials. For
Western blot analysis, whole cell lysates (20-50 µg of protein) were
denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and were resolved by SDS-PAGE. Gels were
transferred to polyvinylidene difluoride membrane (Immobilon-P,
Millipore, Bedford, MA) using Transblot apparatus (Bio-Rad). For
immunoblotting, membranes were blocked and probed with specific
antibodies. Blots were then incubated with horseradish
peroxidase-linked secondary antibodies followed by chemiluminescence
detection, according to the manufacturer's instructions (Pierce).
PI3-kinase Assay--
After 48 h of adenovirus infection,
3T3-L1 adipocytes were starved for 16 h and stimulated with
insulin (17 nM) for 10 min, washed once with ice-cold PBS,
lysed, and subjected to immunoprecipitation (300-500 µg of total
protein) with anti-cdc42 or anti-p110 Cdc42 Assay--
Cdc42 activity was measured according to the
manufacturer's instructions (Upstate Biotechnology, Inc.). After
48 h of adenovirus infections, 3T3-L1 adipocytes were starved for
16 h and stimulated with 17 nM insulin for the
indicated times, washed once with ice-cold PBS, and lysed with lysis
buffer containing 25 mM Hepes, pH 7.5, 150 mM
NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM
Na3VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 25 mM NaF for 15 min at 4 °C. Insoluble
materials were removed by centrifugation. For a negative control, cell
lysate was incubated with 1 mM GDP for 15 min at 30 °C.
Five µg of PAK1-agarose beads, which specifically bound to active
cdc42 (24), were added to the cell lysates and incubated for 1 h
at 4 °C. Agarose beads were washed with lysis buffer three times and
boiled in 2× Laemmli sample buffer. Samples were resolved by SDS-PAGE
and immunoblotted with anti-cdc42 antibody.
Cdc42 Plays a Role in Insulin-induced GLUT4 Translocation and 2-DOG
Uptake--
To evaluate the role of cdc42 in insulin-stimulated GLUT4
translocation, we conducted single cell microinjection studies
using mouse monoclonal (B8) or goat polyclonal (C20) anti-cdc42
antibody, followed by immunofluorescence staining with GLUT4 antibody
in 3T3-L1 adipocytes (Fig.
1A). In the basal state, most
of the cells display GLUT4 staining in the perinuclear region, and
after insulin stimulation there is a marked increase in the proportion
of cells demonstrating GLUT4 localization at the plasma membrane as a
circumferential ring, as described previously (6). Microinjection of
either mouse monoclonal or goat polyclonal anti-cdc42 antibody
decreased 1.7 nM insulin-stimulated GLUT4 translocation by
60-75%. To demonstrate further the importance of cdc42 for
insulin-stimulated GLUT4 translocation, we utilized siRNA to knock down
cdc42 expression followed by measurement of GLUT4 translocation (Fig.
1B). siRNA directed against cdc42 was microinjected into the
cytoplasm of 3T3-L1 adipocytes, and 72 h later, GLUT4
translocation was measured. As seen in Fig. 1B, cdc42 siRNA
led to a 65% decrease in insulin-induced GLUT4 translocation. As a
positive control, siRNA against the insulin receptor was injected,
which completely abolished insulin stimulation. As a negative control,
siRNA against silencing mediator for retinoid and thyroid
hormone receptor, a transcription co-regulating molecule, was also
injected and had no effect.
To examine further the role of cdc42 in insulin-induced glucose
transport, we next measured 2-DOG uptake in 3T3-L1 adipocytes infected
with adenovirus vectors containing either constitutively active cdc42
(CA-cdc42; V12) or dominant negative cdc42 (DN-cdc42; N17). As shown in
Fig. 1C, infection of CA-cdc42 at 80 m.o.i. increased
2-DOG uptake to 61% of the maximal insulin response in the basal state
and further enhanced 2-DOG uptake stimulated by submaximal insulin (0.5 nM). Interestingly, adenovirus-mediated expression of
DN-cdc42 was without effect on insulin stimulation of glucose
transport. Taken together, these results suggest that cdc42 plays an
important role in insulin signaling leading to GLUT4 translocation.
cdc42 Is Downstream of G Constitutively Active Cdc42 Stimulates 2-DOG Uptake in a
PI3-kinase-dependent Manner--
We next examined the
effects of the PI3-kinase inhibitors, wortmannin and LY294002, on
CA-cdc42-induced 2-DOG uptake (Fig. 3A). In the absence of
insulin, expression of CA-cdc42 increased 2-DOG uptake in a
dose-responsive manner with 40 and 80 m.o.i. stimulating to 39 and
56% of the maximal insulin response, respectively. Incubation of the
cells with 100 nM wortmannin or 100 µM
LY294002 for 1 h did not alter 2-DOG uptake stimulated by CA-cdc42
expression, whereas it completely inhibited insulin-stimulated glucose
uptake. However, when the cells were incubated with these inhibitors
for 4 h, not only insulin-induced but also CA-cdc42-induced 2-DOG uptake was inhibited to basal levels. As reported previously (6), overexpression of CA-Gq enhanced 2-DOG uptake, and this was
also inhibited by incubation with 100 nM wortmannin or 100 µM LY294002 for 4 h but not for 1 h. As
expected, incubation with a MEK1 inhibitor, PD98059, for 4 h did
not affect 2-DOG uptake stimulated by insulin, CA-cdc42, or
CA-Gq expression (Fig. 3A). These results
suggest that activated cdc42 and G
Because the lower doses of wortmannin and LY294002 took 4 h to
reach their maximal effects, we further examined the time course and
dose dependence of PI3-kinase inhibition on 2-DOG uptake in cells
expressing CA-cdc42 as well as constitutively active PI3-kinase (p110-CAAX). As seen in Fig. 3B, 48 h after expression
of CA-cdc42, cells were treated with 100 or 300 nM
wortmannin for 1-4 h, and 2-DOG uptake was measured. The
CA-cdc42-induced increase in glucose transport was maximally inhibited
at 300 nM at the first time point, and at the lower dose
(100 nM) transport was inhibited in a gradual,
time-dependent manner reaching maximal inhibition by 4 h. Constitutively active PI3-kinase (p110-CAAX) expression is known to
stimulate 2-DOG uptake in 3T3-L1 cells, and the same inhibitory
experiments were performed in p110-CAAX-expressing cells
with essentially identical results (Fig. 3C). These data indicate that in cells manifesting chronic PI3-kinase activation, there
is a time- and dose-dependent effect of these inhibitors to
suppress glucose transport. This is in contrast to acute effects of
insulin to stimulate transport, which can be inhibited rapidly at a low
or high dose of PI3-kinase inhibitors.
To exclude the possibility that the decrease in CA-cdc42- or
CA-Gq-induced 2-DOG uptake after 4 h of treatment with
the PI3-kinase inhibitors might be because of the toxicity or the
nonspecific effects on general trafficking systems, 2-DOG uptake
stimulated by osmotic shock was measured after 4 h of treatment
with the PI3-kinase inhibitors. Osmotic shock stimulates glucose
transport predominantly through a PI3-kinase-independent mechanism, and as seen in Fig. 3D, pretreatment with 100 nM
wortmannin or 100 µM LY294002 for 4 h did not
inhibit osmotic shock-induced glucose transport.
Insulin and Constitutively Active G Cdc42 Stimulates PI3-kinase Activity--
Since it has been shown
in other systems that cdc42 can bind to the p85 regulatory subunit of
PI3-kinase and stimulate PI3-kinase activity (25, 26), we examined the
relationship between cdc42 and PI3-kinase in 3T3-L1 adipocytes. Fig.
5A shows the co-precipitation of p85 in anti-cdc42 immunoprecipitates after insulin stimulation. Association of p85 with cdc42 was barely detected in the basal state
and was increased by insulin stimulation, with a maximal association at
10 min. Next, we directly measured PI3-kinase activity associated with
cdc42 (Fig. 5, B and C). Insulin increased the PI3-kinase activity in anti-cdc42 immunoprecipitates by 2.8-fold with a
maximal response by 10 min. Expression of CA-cdc42 and CA-Gq (40 m.o.i.) stimulated PI3-kinase activity in the
absence of insulin by 4.4- and 4.2-fold, respectively. Incubation of
the cells with 100 nM wortmannin for 4 h inhibited
PI3-kinase activity stimulated by either CA-Gq, CA-cdc42,
or insulin (Fig. 5B), while 1 h of treatment with
wortmannin inhibited only insulin stimulation (data not shown).
Comparable results were obtained when 100 µM LY294002 was
used (Fig. 5C).
As with the glucose transport data presented in Fig. 3, inhibition of
cdc42-associated PI3-kinase activity was not complete until 4 h
after treatment with 100 nM wortmannin or 100 µM LY294002. To study the dose response and time course
of this inhibition, CA-cdc42 and p110-CAAX-expressing cells were
treated with wortmannin at 100 or 300 nM for up to 4 h, followed by measurement of cdc42-associated PI3-kinase activity. As
seen in Fig. 5, D and E, 300 nM
wortmannin led to maximal inhibition at the first time point studied,
while the inhibition was more gradual, reaching the maximal effect at 4 h at the low dose (100 nM) of wortmannin. These
results are completely consistent with the dose response and time
course of 2-DOG inhibition shown in Fig. 3.
Constitutively Active Cdc42 Stimulates GLUT4 Translocation and
2-DOG Uptake in a PKC One of the major actions of insulin is to stimulate GLUT4
translocation in order to increase glucose uptake into target cells. This is accomplished by a complicated, multistep signaling pathway, which remains incompletely understood. In the current study, we have
shown an important role for cdc42, a Rho GTPase family member, in this
process. We show that insulin stimulates cdc42 activity and that
interfering with cdc42 function by microinjection of anti-cdc42
antibody or siRNA into 3T3-L1 adipocytes inhibits insulin-stimulated GLUT4 translocation. Furthermore, we show that cdc42 is downstream of
another insulin-stimulated activator of glucose transport, G TC10 is a small Rho family GTPase that participates in insulin
stimulation of glucose transport and GLUT4 translocation (17). It has
been suggested that TC10 helps mediate cortical actin polymerization, possibly through interaction with neural Wiskott-Aldrich
syndrome protein (18). Cdc42 is also a member of the Rho GTPase family, with a high degree of homology (69%) to TC10 (17), and a role for
cdc42 in mediating actin rearrangement has been suggested (19). With
respect to bradykinin-stimulated actin remodeling, data exist placing
cdc42 downstream of G It should be noted that some recent studies have shown that
transfection of dominant negative cdc42 (DN-cdc42) does not affect insulin-induced glucose transport into 3T3-L1 adipocytes (17), and, at
first approximation, this is not consistent with our above described
results. However, in our own study, we also find that expression of
DN-cdc42 did not inhibit insulin-stimulated glucose transport, and this
result may have to do with the specific nature of this dominant
negative construct. The DN-cdc42 used in the current studies, as well
as in the previous reports (17), contains a point mutation impairing
its ability to phosphorylate PAK1, one of the target molecules of
cdc42. Indeed, we demonstrate that DN-cdc42 inhibits insulin-stimulated
cdc42 activity toward PAK1. However, cdc42 may have multiple target
molecules (19), and it is unknown whether this mutant has a dominant
negative effect on actions of cdc42 other than PAK1 phosphorylation.
Thus, DN-cdc42 inhibits the ability of endogenous cdc42 to bind to
GST-PAK1 beads but does not inhibit insulin-stimulated glucose
transport or insulin-stimulated cdc42-associated PI3-kinase activity
(data not shown). These results raise the possibility that cdc42 may
stimulate PI3-kinase and glucose uptake through a mechanism independent
from its ability to interact with PAK1, and this could provide an
explanation for why DN-cdc42 did not inhibit insulin-induced glucose
transport in the studies by Chiang et al. (17) or in our own experiments.
Based on numerous studies (29-32), it is quite clear that activation
of PI3-kinase is a necessary step for stimulation of GLUT4 translocation and glucose transport. However, the connection between the activated insulin receptor and PI3-kinase stimulation is somewhat more involved. IRS-1 is a major substrate of the insulin receptor, and
tyrosine-phosphorylated IRS-1 can bind to the Src homology 2 domain of
the p85 regulatory subunit of PI3-kinase, resulting in PI3-kinase
enzymatic activation (3). However, a number of studies, using different
approaches, have indicated that IRS-1 is not strictly essential for
glucose transport stimulation and that other signaling pathways may be
involved (7-12). For example, inhibition of IRS-1 activity in 3T3-L1
adipocytes does not impair insulin-stimulated glucose transport (9),
and IRS-1 knockout animals are only mildly insulin-resistant, and
adipocytes from these animals show only a partial defect in
insulin-stimulated glucose transport (10, 11). Additionally, when IRS-1
is activated independently of insulin, glucose transport is not
stimulated (12). Furthermore, we have shown previously (6) that insulin can cause tyrosine phosphorylation of G Supporting this line of reasoning, and consistent with our current
results, it has already been reported that some of the biologic actions
of cdc42 are mediated through activation of PI3-kinase. For example,
cdc42, as well as Rac1, modifies actin organization leading to
increased motility and invasiveness in epithelial cells (35). The
activation of PAK1 and c-Jun NH2-terminal kinase, both of
which are known target molecules of cdc42, is not required for these
functions, but PI3-kinase activation is necessary (35). It has also
been suggested that the Bcr homology domain of p85 directly interacts
with cdc42 and Rac1 (26, 36), and it is quite possible that the
biological actions of cdc42, which require PI3-kinase activation, are
mediated through the interaction of cdc42 with the Bcr homology domain
of p85.
In summary, we have demonstrated an important role for
cdc42 as a novel signaling molecule in the insulin action pathway
leading to GLUT4 translocation and stimulation of glucose transport. We find that cdc42 is downstream of Gq/11 in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct
association of cdc42 and p85, and both insulin treatment and CA-cdc42
expression stimulated PI3-kinase activity in immunoprecipitates with
anti-cdc42 antibody. Furthermore, the effects of insulin, CA-Gq, and CA-cdc42 on GLUT4 translocation or
2-deoxyglucose uptake were inhibited by microinjection of anti-protein
kinase C
(PKC
) antibody or overexpression of a kinase-deficient
PKC
construct. In summary, activated cdc42 can mediate 1)
insulin-stimulated GLUT4 translocation and 2) glucose transport in a
PI3-kinase-dependent manner. 3) Insulin treatment and
constitutively active Gq expression can enhance the cdc42
activity state as well as the association of cdc42 with activated
PI3-kinase. 4) PKC
inhibition blocks CA-cdc42, CA-Gq,
and insulin-stimulated GLUT4 translocation. Taken together, these data
indicate that cdc42 can mediate insulin signaling to GLUT4
translocation and lies downstream of G
q/11 and upstream of PI3-kinase and PKC
in this stimulatory pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11, leading to activation of PI3-kinase and glucose
transport stimulation. Others (13-15) have also found that
G
q/11 can stimulate glucose transport but have failed to observe PI3-kinase dependence of this effect.
q/11, can stimulate
glucose transport and actin remodeling in 3T3-L1 adipocytes (20). Since
it has been shown that cdc42 can mediate the effects of
bradykinin-stimulated G
q/11 on actin remodeling (21), we
hypothesized that cdc42 might also mediate signals to GLUT4
translocation. In the current study, we show a novel role for cdc42 as
a downstream activator of G
q/11, which can mediate GLUT4
translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11, anti-p110
, and anti-cdc42 (P1)
antibodies, goat polyclonal anti-cdc42 (C20) antibody, mouse monoclonal
anti-cdc42 antibody (B8), and horseradish peroxidase-linked anti-rabbit
and anti-mouse antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep IgG and fluorescein isothiocyanate-conjugated and
TRITC-conjugated anti-rabbit and anti-mouse IgG antibodies were from
Jackson ImmunoResearch (West Grove, PA). Dulbecco's modified
Eagle's medium and fetal bovine serum were purchased from
Invitrogen. All radioisotopes were from ICN (Costa Mesa, CA). All other
reagents were purchased from Sigma. The GTPase-deficient (constitutively active) Q209L mutant G
q expression
vector and recombinant adenoviruses were described elsewhere (6).
Adenoviruses encoding constitutively active and kinase-deficient PKC
were kindly provided by Dr. Wataru Ogawa (Kobe University,
Japan), and adenoviruses encoding constitutively active cdc42 and
dominant negative cdc42 were kindly provided by Dr. James R. Bamburg
(Colorado State University).
q (40 m.o.i.), constitutively active
mutant-G
q (Q209L) (40 m.o.i.), constitutively active
cdc42 (V12) (40 or 80 m.o.i.), dominant negative cdc42 (N17) (80 m.o.i.), constitutively active PI3-kinase (P110-CAAX) (40 m.o.i.),
constitutively active PKC
(80 m.o.i.) or kinase-deficient PKC
(10-100 m.o.i.), or a control recombinant adenovirus of GFP. The total
amount of adenovirus was adjusted to the same m.o.i. with control
adenovirus in each experiment. Transduced cells were incubated for 48 or 60 h at 37 °C in 10% CO2 and Dulbecco's
modified Eagle's high glucose medium with 10% heat-inactivated serum,
followed by incubation in the starvation media required for the assays.
The efficiency of adenovirus-mediated gene transfer was above 90% as
measured by histocytochemical staining of LacZ-infected cells with
-galactosidase, as we reported previously (6).
antibody for 4 h at
4 °C. Immunocomplexes were precipitated with protein A-plus agarose
(Upstate Biotechnology Inc.). The immunoprecipitates were washed with
the following buffers: (i) PBS, containing 1% Nonidet P-40, 100 µM sodium orthovanadate, pH 7.4; (ii) 100 mM Tris, 0.5 M LiCl, 100 µM sodium
orthovanadate, pH 7.4; and (iii) 10 mM Tris, 100 mM NaCl, 100 µM sodium orthovanadate, pH 7.4. Immunoprecipitates were washed mildly (once with each washing buffer) only in the experiment using LY294002, whereas they were washed
more strictly (twice with each buffer) in the other experiments, because LY294002 was a reversible inhibitor. The washed immunocomplexes were incubated with phosphatidylinositol for 5 min and then with [
-32P]ATP (3000 Ci/mmol) for 5 min at room
temperature. Reactions were stopped with 20 µl of 8 N
HCl, mixed with 160 µl of CHCl3:methanol (1:1). Samples
were centrifuged, and the lower organic phase was applied to a silica
gel TLC plate that had been coated with 1% potassium oxalate. TLC
plates were developed in
CHCl3:CH3OH:H2O:NH4OH (60:47:11.3:2), dried, and exposed to an x-ray film. PI3-kinase activity was quantitated by scanning the film using NIH Image.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Involvement of cdc42 in insulin-induced
glucose transport in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes
on coverslips were serum-starved for 4 h, and mouse monoclonal
(B8) or goat polyclonal (C20) anti-cdc42 antibody
(Ab) or sheep IgG was microinjected. Cells were stimulated
with or without 1.7 nM insulin for 20 min. GLUT4 was
stained as described under "Experimental Procedures." The
percentage of cells positive for GLUT4 translocation was calculated by
counting at least 100 cells at each point. The data are the mean ± S.E. from three independent experiments. B, after 72 h of microinjection of cdc42, insulin receptor (IR), or
silencing mediator for retinoid and thyroid hormone receptor
siRNA, 3T3-L1 adipocytes on coverslips were serum-starved for 4 h
and were stimulated with or without 1.7 nM insulin for 20 min. Cells were then stained for GLUT4 localization. Data represent the
mean ± S.E. of three independent experiments. C,
3T3-L1 adipocytes were infected with adenoviruses expressing
constitutively active (CA-cdc42) or dominant negative cdc42
(DN-cdc42) or control GFP (control). After
48 h of infection, these cells were serum-starved for 3 h and
stimulated with 0.5 or 17 nM insulin for 30 min, and
2-[3H]deoxyglucose uptake was measured as described under
"Experimental Procedures." The data are the mean ± S.E. from
four independent experiments.
q/11 in the
Insulin-GLUT4 Translocation Signaling Pathway--
As we reported
recently (6), an adenovirus encoding constitutively active
Gq (CA-Gq; Q209L) stimulated GLUT4
translocation to 60% of the maximal insulin response, showing a
role for G
q/11 in this insulin action. In order to
examine whether cdc42 is downstream of G
q/11, we
microinjected mouse monoclonal anti-cdc42 antibody (B8) into cells
infected with CA-Gq or CA-cdc42, and similar to the results
with insulin- or CA-cdc42 stimulation, anti-cdc42 antibody injection
inhibited CA-Gq-induced GLUT4 translocation (Fig.
2). To assess further the relative loci
of G
q/11 and cdc42 in this pathway, we microinjected
anti-G
q/11 antibody into cells stimulated by CA-cdc42,
CA-Gq, or insulin. Anti-G
q/11 antibody injection inhibited insulin- or CA-Gq-induced GLUT4
translocation but did not inhibit the effects of CA-cdc42 (Fig. 2).
These data provide further evidence that cdc42 is downstream of
G
q/11 in mediating insulin-stimulated GLUT4
translocation.
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Fig. 2.
Effects of microinjection of anti-cdc42 or
anti-G q/11 antibody on GLUT4
translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were
infected with adenoviruses expressing constitutively active cdc42
(CA-cdc42), constitutively active Gq
(CA-Gq), or control GFP (control). After 48 h of infection, anti-G
q/11 antibody (Ab),
anti-cdc42 antibody, or control sheep IgG was microinjected into the
cells on coverslips. Cells were serum-starved for 4 h and
stimulated with 1.7 nM insulin for 20 min. GLUT4 in the
cells was stained as described under "Experimental Procedures." The
percentage of cells positive for GLUT4 translocation was calculated by
counting at least 100 cells at each point. The data are the mean ± S.E. from three independent experiments.
q/11 can stimulate
glucose uptake in a PI3-kinase-dependent manner in 3T3-L1
adipocytes.
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Fig. 3.
Effects of PI3-kinase inhibitors on
constitutively active cdc42-, constitutively active Gq-,
insulin-, or osmotic shock-induced 2-DOG uptake into 3T3-L1 adipocytes.
A, 3T3-L1 adipocytes were infected with adenoviruses
expressing constitutively active cdc42 (CA-cdc42) or
constitutively active Gq (CA-Gq) or control GFP
(control) with indicated m.o.i. After 48 h of
infection, cells were serum-starved and incubated with 100 µM LY294002 (L), 100 nM wortmannin
(W), 50 µM PD98059 (P), or 0.1%
DMSO (D) for 1 or 4 h. Some cells were stimulated by 17 nM insulin for 30 min (insulin).
2-[3H]Deoxyglucose uptake was measured as described under
"Experimental Procedures." B and C, 3T3-L1
adipocytes were infected with adenoviruses expressing CA-cdc42
(B) or constitutively active PI3-kinase
(p110-CAAX) (C). After 48 h of infection,
cells were serum-starved and incubated with 100 (open
circle) or 300 nM wortmannin (WM)
(closed circle) for the indicated times.
2-[3H]Deoxyglucose uptake was measured as described under
"Experimental Procedures." Open square indicates
2-[3H]deoxyglucose uptake in basal without virus
infection. D, 3T3-L1 adipocytes were serum-starved and
incubated with 100 µM LY294002 (L), 100 nM wortmannin (W), or 0.1% DMSO (D)
for 4 h. They were incubated with 600 mM sorbitol or
17 nM insulin for 30 min, and
2-[3H]deoxyglucose uptake was measured as described under
"Experimental Procedures." The data are the mean ± S.E. from
four (A) or three (B-D) independent
experiments.
q Stimulate Cdc42
Activity--
Because our data indicate that glucose uptake induced by
CA-cdc42 and CA-Gq involves common mechanisms, we further
examined the relationship between cdc42 and G
q/11 before
and after insulin stimulation. We measured the time course of
insulin-induced cdc42 activation using GST-PAK1 as a substrate, which
specifically binds to active cdc42 (24). As shown in Fig.
4A, insulin rapidly stimulated cdc42 activity with a maximal response at 1 min, returning to basal
levels thereafter. We showed recently (6) that insulin treatment led to
tyrosine phosphorylation of G
q/11, and this could
activate G
q/11 as a positive signaling molecule.
Interestingly, the time course of insulin-stimulated tyrosine
phosphorylation of G
q/11 was comparable with the time
course of cdc42 activation (Fig. 4A). Cdc42 activity was
also measured in 3T3-L1 adipocytes after adenovirus expression of wild
type or constitutively active Gq, before and after insulin
stimulation for 1 min (Fig. 4B). Expression of
CA-Gq stimulated cdc42 activity in the basal state and
enhanced the effect of insulin compared with control cells. These data
show that cdc42 is activated by insulin and G
q/11, further suggesting the concept that cdc42 is downstream of
G
q/11 in the insulin signaling cascade. We also assessed
the activity of DN-cdc42 by using this method. As can be seen in Fig.
4C, expression of CA-cdc42 resulted in increased cdc42
activity in the absence of insulin, whereas expression of DN-cdc42 at
80 m.o.i. inhibited the effect of insulin to activate cdc42.
View larger version (20K):
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Fig. 4.
Insulin- or constitutively active
Gq-induced cdc42 activity. A, 3T3-L1 adipocytes
were serum-starved for 16 h and stimulated with 17 nM
insulin for the indicated times. Cdc42 activities were measured by
mixing the cell lysates with GST-PAK1 beads that specifically
recognize active cdc42. Samples were analyzed by Western blotting using
anti-cdc42 antibody. The activity was quantitated by scanning the film
using NIH Image. Tyrosine-phosphorylated G q/11 was
detected by immunoprecipitating (IP) with or without
(negative control (NC)) anti-phosphotyrosine antibody (PY20)
and Western blotting using anti-G
q/11 antibody as
described under "Experimental Procedures." Representative blots are
shown from five (cdc42) or three (G
q/11) independent
experiments. B, 3T3-L1 adipocytes were infected with
adenoviruses expressing wild type Gq
(WT-Gq), constitutively active Gq
(CA-Gq), or control GFP (C). After 48 h of
infection, 3T3-L1 adipocytes were serum-starved for 16 h,
stimulated with or without insulin for 1 min, and lysed. Cdc42 activity
was analyzed as described above. These experiments were repeated twice.
C, 3T3-L1 adipocytes were infected with adenoviruses
expressing dominant negative cdc42 (DN-cdc42),
constitutively active cdc42 (CA-cdc42), or control GFP
(control). After 48 h of infection, 3T3-L1 adipocytes
were serum-starved for 16 h, stimulated with or without insulin
for 1 min, and lysed. Cdc42 activity was analyzed as described above. A
sample for a negative control (NC) was prepared as described
under "Experimental Procedures." Representative blot is shown from
three independent experiments.
View larger version (36K):
[in a new window]
Fig. 5.
Association of p85 and PI3-kinase activity in
immunoprecipitates with cdc42 antibody. A, 3T3-L1 adipocytes
were serum-starved for 16 h, stimulated with 17 nM
insulin for the indicated times, and immunoprecipitated (IP)
with or without (negative control (NC)) anti-cdc42 antibody.
Immunoprecipitates were analyzed by Western blotting using anti-p85
antibody as described under "Experimental Procedures." A
representative blot is shown from three independent experiments.
B and C, 3T3-L1 adipocytes were infected with
adenoviruses expressing constitutively active cdc42
(CA-cdc42), constitutively active Gq
(CA-Gq), or control GFP (control). After 48 h of infection, 3T3-L1 adipocytes were serum-starved for 16 h and
incubated with wortmannin (B) or LY294002
(C) for 4 h. PI3-kinase activity in immunoprecipitates
with cdc42 antibody was measured as described under "Experimental
Procedures." A representative film is shown, and the bar
graph shows the mean ± S.E. from four independent
experiments. PI3P, phosphatidylinositol 3-phosphate.
D and E, 3T3-L1 adipocytes were infected with
adenoviruses expressing CA-cdc42 (D) or constitutively
active PI3-kinase (p110-CAAX) (E). After 48 h of
infection, cells were serum-starved for 16 h and incubated with
100 (open circle) or 300 nM wortmannin
(closed circle) for 1, 2, or 4 h. PI3-kinase activity
in immunoprecipitates using cdc42 antibody (D) or p110
antibody (E) was measured as described under "Experimental
Procedures." A representative film is shown, and the graph
represents the mean ± S.E. of four independent experiments.
-dependent Manner--
It has been
reported (27, 28) that PKC
is a component of the insulin signaling
cascade and plays an important role in insulin-induced GLUT4
translocation downstream of PI3-kinase. Since we recently showed that
PKC
was also required for CA-Gq-induced GLUT4
translocation (6), we determined whether PKC
was a participant in
the cdc42 pathway leading to GLUT4 translocation and glucose uptake.
First, we measured the effect of anti-PKC
antibody microinjection on
GLUT4 translocation stimulated by insulin, CA-cdc42, or
CA-Gq. Anti-PKC
antibody injection completely blocked
CA-cdc42-stimulated GLUT4 translocation, similar to the results with
insulin stimulation or CA-Gq expression.
Adenovirus-mediated expression of constitutively active PKC
stimulated GLUT4 translocation to the same extent as insulin, further
arguing that PKC
is a participant in the insulin signaling pathway
leading to GLUT4 translocation (Fig. 6A). Next, we examined the
effect of a kinase-deficient mutant of PKC
(K273E) on insulin- or
CA-cdc42-induced 2-DOG uptake (Fig. 6B). Adenoviral gene
transfer of this mutant PKC
resulted in a dose-dependent
inhibition of 2-DOG uptake stimulated by either 17 nM
insulin or CA-cdc42 expression. Taken together, these results further
argue that both G
q/11 and cdc42 mediate
insulin-stimulated GLUT4 translocation and glucose transport by a
common signaling pathway in which G
q/11 lies upstream of
cdc42 and that both are upstream of PKC
.
View larger version (17K):
[in a new window]
Fig. 6.
Effects of microinjection of
anti-PKC antibody on CA-cdc42-induced GLUT4
translocation and effects of overexpression of kinase-deficient
PKC
on CA-cdc42-induced glucose uptake in
3T3-L1 adipocytes. A, 3T3-L1 adipocytes were infected
with adenoviruses expressing constitutively active cdc42
(CA-cdc42), constitutively active Gq
(CA-Gq), constitutively active PKC
(CA-PKC
), or control GFP (control). After
48 h of infection, anti-cdc42 antibody (Ab) (+) or
sheep IgG (
) was microinjected into the cells on coverslips. Cells
were serum-starved for 4 h and incubated with 1.7 nM
insulin for 20 min. GLUT4 in the cells was stained as described
under "Experimental Procedures." The percentage of cells positive
for GLUT4 translocation was calculated by counting at least 100 cells
at each point. The data are the mean ± S.E. from three
independent experiments. B, 3T3-L1 adipocytes were
infected with adenovirus expressing kinase-deficient PKC
(KD-PKC
) with the indicated m.o.i. Adenoviruses
expressing constitutively active cdc42 (CA-cdc42) with
40 m.o.i. or control adenovirus (control) were also
co-infected to adjust the total amount of adenovirus. After
48 h of infection, these cells were serum-starved for 3 h and
stimulated by 17 nM insulin for 30 min, and
2-[3H]deoxyglucose uptake was measured as described under
"Experimental Procedures." The data are the mean ± S.E. from
three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11, and that the effects of cdc42 to facilitate GLUT4
translocation are mediated through PI3-kinase. Taken together, these
results describe a novel role for cdc42 as a signaling molecule within the insulin action cascade ultimately mediating GLUT4 translocation.
q/11 in this process (21). Because
bradykinin can also stimulate glucose transport under certain
conditions (20), and because we have previously demonstrated an
important role for G
q/11 as a mediator of
insulin-stimulated GLUT4 translocation, we postulated a role for cdc42
in insulin-stimulated glucose transport. The current studies fully
support this idea and also argue strongly that cdc42 lies downstream of
G
q/11 in this pathway. Thus, insulin stimulates
G
q/11 as well as cdc42, and both of these events are
necessary for full stimulation of GLUT4 translocation.
Adenovirus-mediated expression of CA-cdc42 increased GLUT4
translocation and glucose transport independent of insulin, and these
effects of CA-cdc42 were attenuated by inhibitors of PI3-kinase.
Moreover, microinjection of cdc42 antibody inhibits insulin- and
CA-Gq-stimulated GLUT4 translocation, and CA-Gq
expression stimulates cdc42 activity. Furthermore, microinjection of
G
q/11 antibody inhibits insulin- but not
CA-cdc42-stimulated GLUT4 translocation. These results place cdc42
downstream of G
q/11 in this insulin stimulatory cascade.
Finally, insulin stimulates PKC
activity in a
PI3-kinase-dependent manner (6, 27, 28), and microinjection of anti-PKC
antibody blocks insulin, CA-Gq-, and
CA-cdc42-stimulated GLUT4 translocation, indicating that all of these
molecules participate in a common signaling pathway. These results do
not argue in any way against a role for TC10 as a mediator of GLUT4
translocation, and it is quite possible that both of these small Rho
family GTPase proteins participate and may comprise parallel or
redundant steps to mediate this important biologic effect of insulin.
q/11 and that
microinjection of G
q/11 inhibitory reagents into 3T3-L1
adipocytes blocks insulin-stimulated glucose transport. We also found
that constitutively active G
q stimulates glucose
transport by itself in a PI3-kinase-dependent manner (6).
Additionally, other ligands such as bradykinin and ET-1, which
receptors couple into G
q/11, can also stimulate GLUT4
translocation (33). Some of these studies have shown that the effects
of G
q/11 are PI3-kinase-dependent (6, 33),
whereas others (13-15) have been unable to demonstrate this linkage.
In the current study, we find that adenovirus-mediated expression of
CA-Gq leads to activation of cdc42 and that
CA-Gq as well as CA-cdc42 can stimulate glucose transport.
These stimulatory effects are inhibited by 1 h of treatment with
300 nM wortmannin, whereas 100 nM wortmannin
took 4 h to reach full inhibitory effect. Because these
constitutively active proteins were expressed in cells through adenovirus-mediated gene transfer, and the assays were performed 48 h after infection, CA-Gq and CA-cdc42 had
considerable time to exert their effects. Because both wortmannin and
LY294002 inhibit ATP binding to PI3-kinase (34), in the case of
constitutively active preactivation, the ATP-binding site of PI3-kinase
is already occupied by endogenous ATP before the treatment with the
inhibitors. It is possible that higher concentrations of inhibitors or
longer term treatments are necessary to replace ATP under these
conditions. The dose response and time course studies for inhibition of
2-DOG and PI3-kinase activity are fully consistent with this idea (Fig. 3, B and C, and Fig. 5, D and
E). This may explain why some of the earlier studies were
unable to show PI3-kinase dependence of G
q/11 effects.
Another possibility is that insulin and cdc42 could utilize somewhat
different PI3-kinase isoforms to mediate their effects and that
different isoforms of PI3-kinase could have different sensitivity to
inhibition by wortmannin. In any event, under the current experimental
conditions, the effects of CA-cdc42 as well as CA-Gq are
clearly inhibited by wortmannin and LY294002.
q/11 in this signaling
system and lies upstream of PI3-kinase and PKC
. Our experiments show that the various components of this pathway are necessary for full and
efficient insulin stimulation of glucose transport in 3T3-L1
adipocytes. Because stimulation of glucose transport is a major action
of insulin, and because insulin resistance to glucose transport
stimulation is a central component of a number of disease states such
as Type II diabetes, obesity, etc., it is possible that functional
abnormalities in this pathway may participate in the mechanisms of
insulin resistance in the pathophysiologic conditions in man.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. James R. Bamburg (Colorado State
University) for providing adenoviruses encoding constitutively active
cdc42 and dominant negative cdc42, Dr. Wataru Ogawa (Kobe University, Japan) for providing adenoviruses encoding constitutively active PKC
and dominant negative PKC
, and Elizabeth Hansen for editorial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Research Grant DK 33651, the Veterans Administration San Diego Health Care System, Research Service, and the Whittier Institute for Diabetes.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.
§ Supported by an American Diabetes Association Mentor-based Fellowship Award.
To whom correspondence should be addressed: Dept. of Medicine
(0673), University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0673. Tel.: 858-534-6651; Fax: 858-534-6653; E-mail:
jolefsky@ucsd.edu.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M208904200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PI3-kinase, phosphatidylinositol 3-kinase; 2-DOG, 2-deoxyglucose; CA-Gq, constitutively active Gq; PKC, protein kinase C; m.o.i., multiplicity of infection; GFP, green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; DN-cdc42, dominant negative cdc42.
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