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Department of Medicine, University of California-San Diego, La Jolla, California 92093; the Department of Cell Biology and Physiology, Washington University School of Medicine (M.M.), St. Louis, Missouri 63110; and the Veterans Administration Research Service (J.M.O.), San Diego, California 92161
Address all correspondence and requests for reprints to: Jerrold M. Olefsky, M.D., Department of Medicine (0673), University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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S), into 3T3-L1
adipocytes. Thirty minutes after the injection of 5 mM
GTPS, 40% of injected cells displayed surface GLUT4 staining
indicative of GLUT4 translocation compared with 55% for
insulin-treated cells and 10% in control IgG-injected cells. Treatment
of the cells with the phosphatidylinositol 3-kinase inhibitor
wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect
on GTPS-mediated GLUT4 translocation. On the other hand, coinjection
of antiphosphotyrosine antibodies (PY20) blocked GTPS-induced GLUT4
translocation by 65%. Furthermore, microinjection of GTPS led to
the appearance of tyrosine-phosphorylated proteins around the periphery
of the plasma membrane, as observed by immunostaining with PY20.
Treatment of the cells with insulin caused a similar
phosphotyrosine-staining pattern. Electroporation of GTPS stimulated
2-deoxy-D-glucose transport to 70% of the extent of
insulin stimulation. In addition, immunoblotting with phosphotyrosine
antibodies after electroporation of GTPS revealed increased tyrosine
phosphorylation of several proteins, including 70- to 80-kDa and 120-
to 130-kDa species. These results suggest that GTPS acts upon a
signaling pathway either downstream of or parallel to activation of
phosphatidylinositol 3-kinase and that this pathway involves
tyrosine-phosphorylated protein(s). |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further clarify the mechanisms of GLUT4 translocation, we examined
the effect of the nonhydrolyzable GTP analog, guanosine
5'-O-(3-thiotriphosphate) (GTPS), on GLUT4 translocation.
GTPS has been reported to stimulate glucose transport and GLUT4
translocation in insulin-responsive cells such as rat adipocytes (2, 3), 3T3-L1 adipocytes (4), and cardiac myocytes (5). It has previously
been shown that treatment of permeabilized 3T3-L1 adipocytes with a
PI-3 kinase inhibitor, wortmannin, only partially inhibits
GTPS-induced GLUT4 translocation (6). Therefore, the effect of
GTPS may converge on the insulin signaling pathway downstream of
PI-3 kinase, which couples to GLUT4 translocation. In the present
study, using immunofluorescence detection of GLUT4 proteins, we show
that microinjection of GTPS into living intact 3T3-L1 adipocytes
stimulates GLUT4 translocation. GTPS-induced GLUT4 translocation is
not affected by treatment of the cells with wortmannin or coinjection
of a GST fusion protein comprised of the SH2 domain of the p85 subunit
of PI-3 kinase. To clarify the possible role of tyrosine
phosphorylation in this PI 3-kinase-independent GLUT4 translocation, we
examined the relationship between GTPS-induced GLUT4 translocation
and phosphotyrosine proteins using microinjection and electroporation
techniques.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S, Lucifer yellow, and all other reagents were
purchased from Sigma Chemical Co. (St. Louis, MO). Electroporation
equipment for adherent cells (Epizap System) was obtained from Ask
Science Products (Ontario, Canada).
Cell culture and microinjection
3T3-L1 cells were cultured and differentiated as previously
described (1). Microinjection of various reagents was carried out using
a semiautomated Eppendorf microinjection system. Cells on glass
coverslips were serum starved for 2 h, microinjected within 30
min, and fixed 30 min after microinjection unless otherwise indicated.
All reagents except PY20 were coinjected with 10 mg/ml sheep IgG for
identification of injected cells.
Immunostaining and fluorescence microscopy
GLUT4 staining. Immunostaining of GLUT4 was performed
essentially as previously described (1). The cells were fixed in 3.7%
formaldehyde in PBS for 10 min at room temperature. After washing, the
cells were permeabilized and blocked with 0.1% Triton X-100 and 2%
FCS in PBS for 10 min. The cells were then incubated with F349 (1
µg/ml) in PBS with 2% FCS overnight at 4 C. After washing, GLUT4 and
injected IgG were detected by incubation with FITC-conjugated donkey
antirabbit IgG antibody and AMCA-conjugated donkey antisheep or
antimouse IgG antibody, respectively, followed by observation under
immunofluorescence microscope. In all counting experiments, each
coverslip was blindly examined, and the AMCA-positive microinjected
cells on each coverslip were evaluated for the presence of plasma
membrane-associated GLUT4 staining.
Phosphotyrosine staining. Cells were fixed and permeabilized as described above and incubated with mouse monoclonal antiphosphotyrosine antibody (PY20; 20 µg/ml) in PBS with 5% FCS overnight at 4 C. After washing, phosphotyrosine and microinjected cells were identified by incubation with FITC-conjugated donkey antimouse IgG antibody and AMCA-conjugated donkey antisheep IgG antibody, respectively.
Electroporation
Electroporation of adherent 3T3-L1 adipocytes plated on
indium-tin oxide-coated conductive glass (9, 10) was performed using
Epizap System (Ask Science Products) essentially according to the
manufacturer’s instructions. Briefly, on days 7–8 of differentiation,
3T3-L1 adipocytes were plated on a conductive surface glass slide with
a 10 x 32-mm insulating frame and cultured until the cells were
fully differentiated. After washing the cells with PBS, an electrode
was placed on top of the cells, and 5 mM GTPS dissolved
in PBS was added between the electrode and the cells. An electrical
pulse was applied at 50 V with a capacitor setting of 47 µF. One
minute after the pulse, cells were incubated in 
MEM containing 0.1%
BSA at 37 C for the indicated times. With these settings, more than
90% of the cells incorporated the fluorescent dye, Lucifer yellow.
Electroporation with PBS only had no effect on cell viability, GLUT4
staining, or 2-deoxy-D-glucose uptake (data not shown).
2-Deoxy-D-glucose uptake
Glucose transport was measured by a modification of the methods
described by Klip et al. (11). After electroporation, 3T3-L1
adipocytes were incubated in MEM containing 0.1% BSA in the absence
or presence of 100 ng/ml insulin for 1 h at 37 C. Glucose uptake
was determined after the addition of 60 µl substrate [0.1 µCi
2-deoxy-D-[3H]glucose; final concentration,
0.01 mM] for 5 min at 25 C.
Immunoblotting
At the indicated times after electroporation, 3T3-L1 adipocytes
were lysed in RIPA buffer containing 20 mM Tris-HCl (pH
7.5), 140 mM NaCl, 1% Nonidet P-40, 1 mM EDTA,
1 mM sodium orthovanadate, 50 U/ml aprotinin, 1
mM phenylmethylsulfonylfluoride, and 50 mM
sodium fluoride. Laemmli buffer containing 5% 2-mercaptoethanol was
added and boiled for 5 min. Each sample containing 60 µg protein was
subjected to SDS-7.5% PAGE and transferred onto nitrocellulose
filters. The filters were incubated with horseradish
peroxidase-conjugated phosphotyrosine antibody (RC20) or PY20, and
subsequently with horseradish peroxidase-conjugated antimouse IgG
antibody. Chemiluminescence was detected with the ECL system (Amersham,
Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S results in GLUT4 translocation in 3T3-L1
adipocytesS, followed by immunostaining with anti-GLUT4
antibody. Untreated cells display GLUT4 staining mostly localized
around the nucleus, with some staining distributed throughout the
cytoplasm (Fig. 1A
). Insulin treatment
led to an increase in plasma membrane GLUT4 localization, giving the
cells a surface staining pattern that looked like a ring around the
cell (Fig. 1B). As previously described, the percentage of cells that
are positive for GLUT4 translocation increased in a dose- and
time-dependent manner in response to insulin (1). Microinjection of
GTPS caused redistribution of GLUT4 with a staining pattern
indistinguishable from that caused by insulin (Fig. 1, C and D). After
injection of 5 mM GTPS, the percentage of cells
displaying surface GLUT4, indicative of translocation, was 40% at 15
min and declined after 45 min (Fig. 2).
Therefore, we examined GLUT4 translocation 30 min after the injection
of GTPS in the subsequent experiments.
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S-induced GLUT4 translocation is not blocked by inhibition of
PI 3-kinaseS-induced glucose transport is only partially inhibited by the
PI-3 kinase inhibitor wortmannin in permeabilized cells (6). In our
study also, treatment of the cells with 1 µM wortmannin,
which completely inhibited insulin-induced GLUT4 translocation (Fig. 3) did not inhibit GLUT4 translocation
induced by microinjection of GTPS (Fig. 3). We also examined the
effect of a GST-p85 SH2 fusion protein on GTPS-induced GLUT4
translocation. We have previously shown that microinjection of GST-p85
SH2 inhibits both insulin-induced mitogenesis (8) and insulin-induced
GLUT4 translocation (1). Coinjection of this GST-SH2 fusion protein
with GTPS into 3T3-L1 adipocytes had no effect on GLUT4
translocation (Fig. 1
, E and F, and Fig. 3). Taken together, these
results show that inhibition of the enzymatic activity of PI 3-kinase
or its proper intracellular targeting does not affect GTPS-induced
GLUT4 intracellular targeting.
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S-induced GLUT4 translocation), but had no effect on basal cell
surface GLUT4 content, which confirms previous results (1). To examine
the possible role of tyrosine phosphorylation in GTPS-induced GLUT4
translocation, we coinjected PY20 with GTPS into 3T3-L1 adipocytes
and evaluated GLUT4 staining of each injected cell. Each coverslip
injected with the various reagents was blindly evaluated and counted
for GLUT4 translocation-positive cells. The results revealed that
coinjection of PY20 significantly inhibited GTPS-induced GLUT4
translocation by 65% (Fig. 1, G and H, and Fig. 4A). Coinjection of
phosphotyrosine with PY20 neutralized the inhibitory effect of PY20 on
GTPS-induced GLUT4 translocation (Fig. 4A), indicating that the
effect of PY20 was specific for phosphotyrosine residues. To further
confirm the specificity of the PY20 antibody, we examined the effects
of different concentrations of PY20 and unrelated antibodies on
GTPS-induced GLUT4 translocation. Microinjection of PY20 inhibited
the effect of GTPS on GLUT4 translocation in a dose-responsive
manner when injected at concentrations ranging from 2.5–10.0 mg/ml
(Fig. 4B). Neither anti-Ras antibody (1), anti-Shc antibody (1), nor
sheep IgG, each at a concentration of 10 mg/ml, inhibited
GTPS-induced GLUT4 translocation (Fig. 4B).
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SS-induced GLUT4
translocation indicates that one or more tyrosine-phosphorylated
proteins are involved in the GTPS mechanism. Thus, we next
examined the effect of GTPS on phosphotyrosyl staining, as assessed
by indirect immunofluorescence microscopy with the PY20 antibody. In
the unstimulated state, specific staining with PY20 was hardly observed
throughout the cell (Fig. 5A). After
injection of GTPS, the injected cells displayed discrete punctate
staining with PY20 around the periphery of the plasma membrane (Fig. 5, B and C). Stimulation with insulin caused a similar staining pattern,
although the intensity was less than that in GTPS-injected cells
(Fig. 5D). In the insulin-stimulated cells, staining of phosphorylated
IRS proteins was not observed in the cytoplasm, presumably because of
the relatively diffuse nature of the localization of these
proteins.
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S stimulates glucose transport and induces tyrosine
phosphorylation of 70- to 80-kDa and 120- to 130-kDa proteinsS, we performed
electroporation of GTPS into monolayer 3T3-L1 adipocytes, followed
by measurements of glucose transport and immunoblotting with
phosphotyrosine antibodies. Compared with the cells electroporated with
PBS only, introduction of GTPS stimulated
2-deoxy-D-glucose uptake of the cells by 2.8-fold, which
was 69% of the insulin effect (Fig. 6).
This indicated that GTPS was successfully introduced into the cells,
and that the subsequent translocation of GLUT4 to the cell surface was
functionally significant, leading to increased uptake of glucose.
Electroporation with other nucleotides, i.e. GTP or ATP, had
no effect on glucose transport, and the effects of GTPS plus insulin
were equal to those of insulin alone (data not shown). Immunoblotting
of the cell lysates revealed that electroporation with GTPS
stimulated protein tyrosine phosphorylation (Fig. 7A). In particular, tyrosine
phosphorylation of 70- to 80-kDa and 120- to 130-kDa species was
significantly increased, with maximum phosphorylation 20 min after
electroporation (Fig. 7A). This effect of GTPS was inhibited in a
dose-dependent manner by the tyrosine kinase inhibitor genestin, but
not by wortmannin (not shown). In contrast, electroporation with PBS,
GTP, or ATP was without significant effect on these proteins (Fig. 7, A
and B). Treatment of the cells with insulin stimulated tyrosine
phosphorylation of 95- and 180-kDa proteins 5 min after insulin
stimulation, and these presumably represent the insulin receptor
ß-subunit and IRS-1, respectively (Fig. 7C). In addition, at later
time points, insulin treatment caused tyrosine phosphorylation of the
70- to 80-kDa protein, which appeared to be the same protein as that
tyrosine phosphorylated by GTPS (Fig. 7C). As a control, stimulation
of cells with LPA (a ligand for G protein-coupled receptors) did not
stimulate glucose transport of tyrosine phosphorylation of 120- to
130-kDa or 70- to 80-kDa proteins (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S into 3T3-L1 adipocytes stimulates GLUT4 translocation and
that this effect was not blocked by inhibition of PI 3-kinase enzymatic
activity or targeting of PI 3-kinase to its intracellular sites of
action. However, the effect of GTPS was blocked by coinjection of
antiphosphotyrosine antibodies. We also used an electroporation system
to show that GTPS can stimulate both glucose transport and protein
tyrosine phosphorylation. Although it has been known that GTPS can
stimulate glucose transport, the novel findings in this paper are that
GTPS can do this by mediating GLUT4 translocation in 3T3-L1
adipocytes, that GTPS acts in the signaling pathway either
downstream of or parallel to PI 3-kinase, and that its effects are
dependent on the induction of one or more downstream
phosphotyrosine-containing proteins.
GTPS is a poorly hydrolyzed GTP analog that can bind to various
classes of GTP-binding proteins, converting them to the active
GTP-bound form. Alternatively, binding of GTPS can inactivate other
classes of guanosine triphosphatases by inhibiting their
GTP-hydrolyzing function. Thus, it is possible that GTP-binding
proteins activated or inactivated by GTPS could stimulate exocytosis
or inhibit the endocytosis of GLUT4 proteins. Indeed, evidence for both
stimulation of exocytosis and inhibition of endocytosis has been
reported (3). Recently, a role of heterotrimeric G proteins in insulin
signaling has been suggested, as transgenic mice expressing antisense
RNA to Gi2 display insulin resistance (23). In these
mice, deficiency of Gi2 leads to an increase in protein
tyrosine phosphatase activity and attenuation of insulin-stimulated
tyrosine phosphorylation of IRS-1 (23). Therefore, the effect of
Gi2 appears to lie upstream of that of PI 3-kinase. As
our results show that the effect of GTPS is independent or
downstream of PI 3-kinase, it seems unlikely that GTPS-induced GLUT4
translocation is mediated through stimulation of Gi2.
GTPS could potentially activate Ras protein, but we have previously
demonstrated that p21ras is not necessary
for GLUT4 translocation (1), making this also an unlikely target of
GTPS in mediating GLUT4 translocation. Other classes of GTP-binding
proteins include members of the Rho and Rab families. Rab family
members could be potentially stimulated by binding GTPS, and they
have been implicated in various steps of vesicle trafficking (24). In
fact, evidence exists implicating Rab4 in insulin-induced GLUT4
translocation, consistent with the possibility that Rab4 is a target of
GTPS in this system (25, 26, 27). Another GTP-binding protein, dynamin,
which functions by hydrolyzing GTP to GDP, has been implicated in
clathrin-mediated endocytosis (28). Thus, inactivation of dynamin-like
guanosine triphosphatase by binding to GTPS may block endocytosis of
GLUT4, leading to an increased amount of GLUT4 on the plasma
membrane.
Several reports have indicated that PI 3-kinase is a critical component
of insulin-induced GLUT4 translocation (1, 6, 18, 19, 20, 21, 22). Thus, using the
single cell microinjection system, we have shown that introducing a GST
fusion protein containing an SH2 domain of the p85 subunit of PI
3-kinase into 3T3-L1 adipocytes inhibits insulin-stimulated GLUT4
translocation (1), presumably by behaving as an intracellular
competitive inhibitor of the binding of endogenous PI 3-kinase to its
proper intracellular targets. In addition, wortmannin, which inhibits
the enzymatic activity of the p110 catalytic subunit of PI 3-kinase,
also inhibits insulin-stimulated glucose transport and GLUT4
translocation (6, 20), whereas expression of a constitutively active
form of the p110 stimulates GLUT4 translocation (22). However, the
current studies clearly demonstrate that the GTPS effect on GLUT4
redistribution was not inhibited by treating cells with wortmannin or
coinjecting cells with GST-p85 SH2. Therefore, neither the enzymatic
activity of PI 3-kinase nor its proper intracellular targeting are
necessary for the GTPS effect, indicating that GTPS acts
downstream of PI 3-kinase or in a pathway completely parallel to that
of PI 3-kinase.
It has previously been established that microinjection of
antiphosphotyrosine antibodies can block insulin-induced DNA synthesis
in fibroblasts as well as GLUT4 translocation in 3T3-L1 adipocytes (1),
and this is consistent with the general idea that activation of the
insulin receptor tyrosine kinase with subsequent phosphorylation of
receptor substrates such as IRS-1, IRS-2, Shc, and Gab1 leads to
activation of a signaling cascade that includes the Ras/MAP kinase and
PI 3-kinase pathways (12, 13, 14, 15). However, our observation that
coinjection of antiphosphotyrosine antibodies effectively blocks
GTPS-induced GLUT4 translocation indicates that additional tyrosine
phosphorylation events, independent of the insulin receptor kinase, are
necessary for the effects of GTPS and might also be involved in
insulin-induced GLUT4 translocation.
To further pursue the role of phosphotyrosine proteins in the GTPS
effect, we assessed tyrosine phosphorylation events by immunostaining
cells with antiphosphotyrosine antibodies using immunofluorescence
detection. In basal cells, no phosphotyrosine staining was observed.
Upon microinjection of GTPS, clustering of phosphotyrosine staining
appeared in a discrete, punctate distribution at the periphery of the
plasma membrane. Insulin stimulation of cells induced a similar
phosphotyrosine-staining pattern, although the intensity was somewhat
less than that induced by GTPS. We extended this observation by
performing antiphosphotyrosine immunoblotting of cell lysates prepared
from GTPS-electroporated cells. We found that GTPS induced the
tyrosine phosphorylation of several proteins, including one at 70–80
kDa and another at 120–130 kDa. It is reasonable to suggest that the
phosphotyrosine proteins that appear after GTPS might be involved in
GLUT4 translocation, and this certainly is consistent with the fact
that microinjection of antiphosphotyrosine antibodies inhibits the
effect of GTPS on GLUT4 distribution. In this regard, GTPS did
not cause tyrosine phosphorylation of the insulin receptor ß-subunit
or IRS-I/II, and this coupled with the finding that the effects of
GTPS on glucose transport were not inhibited by wortmannin or
microinjection of the p85 SH2 domain indicate that GTPS stimulates
glucose transport through a tyrosine phosphorylation event downstream
of (or parallel to) the insulin receptor and IRS-I/II, and independent
of PI3 kinase.
In summary, we have demonstrated that GTPS can directly lead to
GLUT4 translocation in 3T3-L1 adipocytes, and that this effect is
mediated by a signaling pathway downstream of or parallel with the
activation and targeting of PI 3-kinase. Furthermore, we have shown
that the GTPS effect is dependent on tyrosine-phosphorylated
proteins. The specific GTP-binding protein(s) and the downstream
tyrosine-phosphorylated proteins involved in this process remain to be
defined.
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Footnotes |
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Received June 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-toxin-permeabilized rat adipose cells.
J Biol Chem 266:4037–4040S and localization of GLUT4 to clathrin lattices. J Cell Biol 117:1181–11962. Nature 379:840–844[CrossRef][Medline]
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