Originally published In Press as doi:10.1074/jbc.M301511200 on June 2, 2003
J. Biol. Chem., Vol. 278, Issue 32, 30157-30169, August 8, 2003
Insulin Recruits GLUT4 from Distinct Compartments via Distinct Traffic Pathways with Differential Microtubule Dependence in Rat Adipocytes*
Li-Bin Liu,
Waka Omata,
Itaru Kojima and
Hiroshi Shibata 
From the
Department of Cell Biology, Institute for Molecular and Cellular
Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512,
Japan
Received for publication, February 12, 2003
, and in revised form, May 29, 2003.
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ABSTRACT
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In the present study, we investigated the physiological significance of the
microtubules in the subcellular localization and trafficking of GLUT4 in rat
primary adipocytes. Morphological and biochemical analyses revealed a dose-
and time-dependent disruption of the microtubules by treatment with
nocodazole. With nearly complete disruption of the microtubules, the
insulin-stimulated glucose transport activity was inhibited by 55%. This
inhibition was concomitant with a comparable inhibition of GLUT4 translocation
measured by the subcellular fractionation and the cell-surface GLUT4 labeling
by trypsin cleavage. In addition, the time-course of insulin stimulation of
the glucose transport activity was significantly delayed by microtubule
disruption (t
were 7 and 2.3 min in nocodazole-treated and
control cells, respectively), while the rate of GLUT4 endocytosis was little
affected. The impaired insulin-stimulated glucose transport activity was not
fully restored to the level in control cells by blocking GLUT4 endocytosis,
suggesting that the inhibition was due to the existence of a
microtubule-dependent subpopulation in the insulin-responsive GLUT4 pool. On
the other hand, nocodazole partially inhibited insulin-induced translocation
of the insulin-regulated aminopeptidase and the vesicle-associated membrane
protein (VAMP)-2 without affecting GLUT1 and VAMP-3. In electrically
permeabilized adipocytes, the insulin-stimulated glucose transport was
inhibited by 40% by disruption of the microtubules whereas that stimulated
with GTP
S was not affected. Intriguingly, the two reagents stimulated
glucose transport to the comparable level by disruption of the microtubules.
These data suggest that insulin recruits GLUT4 to the plasma membrane from at
least two distinct intracellular compartments via distinct traffic routes with
differential microtubule dependence in rat primary adipocytes.
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INTRODUCTION
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Insulin stimulates glucose uptake mainly by promoting subcellular
redistribution of a facilitative glucose transporter isoform GLUT4 from
intracellular compartments to the plasma membrane in adipocyte and
skeletal/cardiac muscles
(1–3).
While the subcellular trafficking pathways and the molecular mechanisms by
which insulin recruits GLUT4 to the plasma membrane still remain obscure,
several lines of evidence have suggested that GLUT4 is associated with more
than one intracellular compartment. Morphologically, studies with
immunoelectron microscopy have shown that most of GLUT4 localizes
intracellularly to tubulovesicular structures clustered near the stacks of
Golgi and the endosomes, or scattered throughout the cytoplasm in unstimulated
adipocytes and that insulin decreases GLUT4 from all the intracellular
compartments (4,
5). On the other hand, many
biochemical studies have indicated that insulin-responsive GLUT4 is associated
with the endosomal recycling pathway and with a more specialized postendosomal
compartment (for review, see Ref.
1). Additionally, more recent
works have suggested that insulin recruits GLUT4 to the plasma membrane from
the postendosomal vesicle-associated membrane protein
(VAMP)1-2-positive
compartment as well as from the cation-dependent mannose-6-phosphate receptor
(CD-M6PR)-positive compartment that is dynamically shuttling between endosomes
and the trans-Golgi network (TGN)
(6,
7). While such a diversity of
the subcellular localization of GLUT4 would hamper our understanding of the
insulin-regulated trafficking of GLUT4, it implies that the transit of GLUT4
to and from such distinct subcellular compartments would be regulated by
different signals and trafficking machinery (e.g. Refs.
8 and
9).
The actin and microtubule cytoskeleton networks have been implicated in the
subcellular movements of the GLUT4-containing membranes. While the role of the
actin filaments in insulin-induced GLUT4 translocation has been reported with
relatively consistent results
(10–13),
the relevance of the microtubules to the insulin action has been
controversial. Thus, depolymerization of the microtubules caused a complete
cessation of the basal linear tracking movements of green fluorescent protein
(GFP)-tagged GLUT4, and inhibited insulin-induced GLUT4 translocation and
glucose uptake by 40–70% in 3T3-L1 adipocytes
(14,
15). Likewise, Olson et
al. (16) also showed that
nocodazole treatment of 3T3-L1 adipocytes resulted in about 80% inhibition of
insulin-stimulated translocation of GLUT4. In addition, the GLUT4-containing
vesicles have been shown to be associated with 
-tubulin and polymerized
microtubules under the in vivo and in vitro conditions,
respectively (16,
17). Furthermore, the two
molecular motor proteins, dynein and kinesin, that direct the vesicles along
the microtubules toward the minus and plus ends, respectively, have been
implicated in the subcellular localization and insulin-responsive movements of
GLUT4 (17,
18). These observations have
strongly supported a physiological significance for the microtubules in the
subcellular trafficking of GLUT4. On the other hand, more recent studies
demonstrated that depolymerization of the microtubules with lower
concentrations (2–3 µM) of nocodazole did not affect
insulin-stimulated GLUT4 translocation and glucose uptake
(19,
20). They also argued that the
inhibition of glucose transport with higher concentrations (30
µM or more) of nocodazole derived from a direct inhibition with
the drug of the glucose transporter activity itself.
Thus there remain important issues to be addressed on the role of the
microtubules in the GLUT4 trafficking and the action of nocodazole as a
microtubule-depolymerizing tool. In addition, all of the studies were carried
out using 3T3-L1 adipocytes, while the relevance of the microtubules to the
GLUT4 trafficking has never been precisely studied in primary adipocytes.
Structurally, isolated adipocyte has a spherical shape with a large diameter
(usually 50–100 µm), a huge lipid droplet inside the cell, and the
thin cytoplasm confined beneath the plasma membrane. Because of these
morphological features, the relationship between the microtubules and GLUT4
has not been visualized in primary adipocytes. Functionally, although both
GLUT1 and GLUT4 isoforms are expressed in primary adipocytes, the latter is
far more abundant and virtually accounts for the major portion of the cellular
glucose uptake (21). This
feature makes it easier to study the effect of nocodazole on the glucose
transport activity of GLUT4. Additionally, some of the observations described
in 3T3-L1 adipocytes may not be applicable to primary adipocytes; for example,
endothelin-1 stimulates glucose transport and GLUT4 translocation via a
Gq/11-dependent stimulation of the endosomal recycling in 3T3-L1
adipocytes
(22–24)
whereas it inhibits the insulin effect without affecting the basal glucose
transport in primary adipocytes (Refs.
25 and
26).2
In the present study, we reconstructed the three-dimensional images to
investigate the spatial relationship between the microtubules and GLUT4 in
isolated rat adipocytes. In addition, we investigated the role of the
microtubules in the insulin-stimulated translocation of GLUT4. The results of
our study suggested that insulin recruits GLUT4 to the cell surface from at
least two distinct intracellular compartments via distinct traffic routes with
differential microtubule dependence in rat primary adipocytes.
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EXPERIMENTAL PROCEDURES
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Materials—Nocodazole was purchased from ICN (Costa Mesa, CA)
and dissolved in dimethyl sulfoxide at 20 mM (stock solution). The
maximal final concentrations of nocodazole and Me2SO were 100
µM and 0.5%, respectively. The same concentration of
Me2SO (0.5%) was added to the controls. The
Dk-(62–85) peptide (RETQIAKGNEQSFRVDLRTLLRYY) was synthesized
as described previously (27),
dissolved to a concentration of 1.0 mM in 0.1 M NaCl,
and activated by incubation at 37 °C overnight prior to the addition to
cells. Rabbit polyclonal antibodies against GLUT4 were raised as described
previously (27). Rabbit
polyclonal anti-GLUT1 antibody was a generous gift from Dr. Kuniaki Takata
(Gunma University). Mouse monoclonal anti-
-tubulin (clone TUB 2.1) and
anti-vimentin (clone V9) antibodies were from ICN Biomedicals (Aurora, OH) and
Lab Vision (Fremont, CA), respectively. Rabbit anti-VAMP-2 and sheep
anti-VAMP-3 antibodies were kindly provided by Masami Takahashi (Mitsubishi
Kasei Institute of Life Sciences) and Jeffrey E. Pessin (University of Iowa),
respectively. Rabbit polyclonal anti-insulin responsive aminopeptidase (IRAP)
antibody was kindly donated by Mitsuru Hashiramoto (Ehime University). Alexa
Fluor-labeled anti-mouse IgG and anti-rabbit IgG antibodies were obtained from
Molecular Probes (Eugene, OR). Mouse monoclonal anti-phosphotyrosine antibody
(clone 4G10) was purchased from Upstate (Charlottesville, VA) and Rabbit
anti-phospho-c-Cbl (Tyr-774) and rabbit anti-phospho-Akt (Ser-473) antibodies
were from Cell Signaling Technology (Beverly, MA). GTPS tetralithium
salt was purchased from Roche Applied Science. Endothelin-1 was from Peptide
Institute, Inc. (Osaka, Japan).
Preparation of Isolated Rat Adipocytes and
Permeabilization—Isolated adipocytes were prepared by the
collagenase method from epididymal adipose tissues of Sprague-Dawley rats
(from Charles-River, 
170–220 g)
(28). Unless otherwise
specified, isolated cells were suspended in Buffer A (25 mM
Krebs-Henseleit Hepes buffer supplemented with 40 mg/ml bovine serum albumin
(Fraction V) and 3 mM pyruvate, pH 7.4). The cells to be
permeabilized by electroporation were suspended in high K+/low
Ca2+ buffer designated as Buffer X (118.0 mM
KCl, 4.74 mM NaCl, 0.38 mM CaCl2, 1.0
mM EGTA, 1.18 mM MgSO4, 1.18 mM
KH2PO4, 23.4 mM Hepes/KOH, 40 mg/ml bovine
serum albumin, 3 mM pyruvate, pH 7.4). The electroporation was
carried out four times in a Gene-Pulser (from Bio-Rad) set at 25 microfarads
and 2 kV/cm as described previously
(29).
Visualization of the Microtubules and GLUT4 in Isolated
Adipocytes—For visualization of the microtubules and GLUT4 by laser
confocal microscopy, cells were washed three times with Buffer A and fixed
with 3% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at
room temperature. The cells were permeabilized and nonspecific binding sites
were blocked in PBS containing 0.1% saponin, 1% bovine serum albumin, and 3%
normal goat serum for 45 min at room temperature. The cells were then
incubated with rabbit anti-GLUT4 serum (1:1000 dilution) and mouse
anti--tubulin antibody (1:500) for 2 h at room temperature, and washed
three times with PBS containing 0.1% saponin. Next, the cells were incubated
with Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 568-conjugated
anti-mouse IgG (1:200 dilution) for 1 h at room temperature. Finally, the
cells were washed with PBS containing 0.1% saponin, mounted in 50% glycerol
saturated with n-propyl gallate as an anti-bleaching reagent, and observed
with an epifluorescence microscope (BX-50; Olympus, Tokyo) equipped with a
laser confocal system (MRC-1024; Bio-Rad, Hemel Hempstead, UK). Captured
images were processed with Bio-Rad LaserSharp software. The three-dimensional
images were reconstructed from serial confocal images taken at 0.5 or 1-µm
intervals along the z-axis.
Analysis of GLUT4 Translocation by Confocal Microscopy—After
incubation without or with nocodazole for 30 min at 37 °C, cells were
treated without or with 10 nM insulin for 30 min, and then
immunostained with anti-GLUT4 antibody as described above. Confocal images
from 100 cells were obtained for each condition, and the individual cells were
graded according to the continuity of the GLUT4 fluorescence signals at the
peripheral rim of the cell. Thus, cell scored 0 without any continuous GLUT4
signal, 1 with continuity less than the third of the circumference, 2 with
continuity between one- and two-thirds, and 3 with more than two-thirds.
Extraction and Measurement of Polymerized and Unpolymerized
Tubulin—Polymerized and unpolymerized tubulin were separated as
described by Breitfeld et al.
(30) with a slight
modification. After incubation without or with nocodazole for 30 min at 37
°C, the cells were washed with PBS and incubated with extraction buffer (2
M glycerol, 100 mM MgSO4, 1 mM
EGTA, 0.1% Triton X-100, 0.1 M PIPES/Na, pH 6.9, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml pepstatin A,
10 µg/ml aprotinin) for 30 min at 37 °C. At the end of incubation,
cells were centrifuged at 500 x g for 30 s. The resulting
infranatant below the cell cake, which contains monomeric tubulin, was
incubated with SDS lysis buffer (0.4 M NaCl, 0.5% SDS, and 25
mM Tris/Cl, pH 7.4) for 5 min at room temperature, and boiled for 3
min before centrifugation at 15,000 x g for 2 min. The
resulting supernatant was subjected to SDS-polyacrylamide gel electrophoresis
and immunoblotting with anti--tubulin antibody. Unextracted polymerized
tubulin associated with the cells was recovered by incubation with SDS lysis
buffer for 5 min at 37 °C, followed by centrifugation for 30 s at 500
x g. The infranatant was boiled for 3 min and centrifuged at
15,000 x g for 2 min. The supernatant was subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotting. The immunoblots
were semi-quantified by using NIH image software.
Measurement of 3-O-Methyl-D-glucose Uptake—The cellular
glucose transport activity was estimated by measuring the rate of 0.1
mM 3-O-methyl-D-glucose uptake by the
oil-flotation method as described previously
(29).
Subcellular Membrane Fractionation and Immunoblotting—The
plasma membrane and low density microsomal (LDM) fractions were prepared by
differential and sucrose density gradient centrifugation as described
previously (27). Proteins in
the plasma membrane and LDM fractions were separated on SDS-polyacrylamide
gels and transferred to a polyvinylidene difluoride membrane (from Millipore).
The polyvinylidene difluoride membrane was blocked with solution containing 5%
bovine serum albumin, 10 mM Tris/HCl (pH 7.4) and 154 mM
NaCl for 1 h at room temperature. The blocked membrane was incubated with
rabbit anti-GLUT4 (1:1000 dilution), rabbit anti-GLUT1 (1:1000 dilution),
rabbit anti-VAMP-2 (1:1000 dilution), sheep anti-VAMP-3 (1:1000 dilution), or
rabbit anti-IRAP (1:1000 dilution) antibodies overnight at 4 °C. The
membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated
secondary antibodies for 1 h at room temperature. Following an extensive wash,
the blots were visualized by using ECL Western blotting System (Amersham
Biosciences).
Cell Surface GLUT4 Labeling and Measurement of GLUT4
Endocytosis—GLUT4 in the plasma membrane was labeled by limited
proteolysis with trypsin as described by Czech and Buxton
(31) with a slight
modification. Briefly, adipocytes in Buffer A were stimulated with 10
nM insulin for 20 min at 37 °C, and then incubated for 15 min
in the presence of 3 mM potassium cyanide in order to inhibit GLUT4
recycling by deprivation of metabolic energy. Then the cells were treated
without or with 100 µM nocodazole for 30 min. TPCK-treated
trypsin (a final concentration of 1 mg/ml) was added to the cells for the last
20 min of nocodazole treatment. For measurement of the amount of GLUT4 in the
plasma membrane, soybean trypsin inhibitor was added to a final concentration
of 2 mg/ml at the end of the incubation, and the cells were washed three times
with STE buffer (250 mM sucrose, 10 mM Tris/HCl and 1
mM EDTA/Na, pH 7.4), homogenized with a Dounce glass homogenizer,
and subjected to subcellular membrane fractionation as described above. For
measurement of GLUT4 endocytosis, the trypsin-treated cells were washed three
times with Buffer A after the addition of soybean trypsin inhibitor (2 mg/ml),
resuspended in fresh Buffer A and incubated for 0 (immediately washed), 5, 10,
or 20 min at 37 °C. At the end of the incubation, the cells were washed
three times with STE buffer, homogenized, and subjected to subcellular
fractionation and immunoblotting with anti-GLUT4 antibody.
Measurement of Tubulin Associated with GLUT4-containing
Membranes—The LDM fractions were prepared as described above and
incubated with 5 µl of anti-GLUT4 antiserum and 20 µl (bed volume) of
protein A-Sepharose (Amersham Biosciences) in STE buffer for 2 h at 4 °C.
The beads were then washed three times with STE buffer. The proteins
associated with the immunoadsorbed membranes were solubilized with 1% (v/v)
Nonidet P-40, and subjected to immunoblotting with anti--tubulin
antibody.
Detection of Phosphorylated Proteins—Adipocytes in Buffer A
were washed with STE buffer and homogenized with a Dounce glass homogenizer in
SDS sample buffer (62.5 mM Tris/Cl, pH 6.8, 2% SDS, 10% glycerol, 1
mM Na3VO4) (for phosphotyrosine and
phospho-c-Cbl) or homogenizing buffer (50 mM HEPES/Na, pH7.5, 100
mM KCl, 10% glycerol, 0.2 mM EDTA, 2 mM EGTA,
1 mM dithiothreitol, 1 µM microcystin-LR, 1 µg/ml
pepstatin A, 20 KIU/ml aprotinin, 1 µg/ml leupeptin, 0.2 mM
phenylmethylsulfonyl fluoride, 10 mM NaF, 50 mM
-glycerophosphate) (for phospho-Akt). After centrifugation for 2 min at
3,000 x g, the infranatant fraction below the fat was subjected
to SDS-polyacrylamide gel electrophoresis and immunoblotting with
anti-phosphotyrosine, anti-phospho-c-Cbl (Tyr-774) or anti-phospho-Akt
(Ser-473) antibodies.
Phosphodiesterase Assay—Phoshodiesterase was assayed as
described previously (32) by
incubation of the enzyme with 0.1 µM [3H]cAMP
(PerkinElmer Life Sciences) for 5 min at 30 °C in the presence of 4
mM MgSO4 and 40 mM TES buffer, pH 7.5. After
the incubation, the fat cell phosphodiesterase was heat-denatured, the
[3H]AMP formed was quantitatively degraded with snake venom into
[3H]adenosine, and the latter was separated from unreacted
[3H]cAMP by column chromatography on AG1-X2 (from Bio-Rad).
Phosphodiesterase activity is shown in the unit of pmol of cAMP
decomposed/min/mg of protein. Protein was assayed by the method of Bradford
(33); the standard used was
crystalline bovine serum albumin.
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RESULTS
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To investigate the physiological role of the microtubules in the
subcellular trafficking of GLUT4, we first examined the effect of nocodazole,
a microtubule-depolymerizing reagent, on the integrity of the microtubules in
isolated rat adipocytes by morphological and biochemical methods. Because the
cytoplasm of isolated adipocyte is generally confined to a thin rim beneath
the plasma membrane due to the presence of a lipid droplet, we obtained
confocal images near the cell surface for observation of the microtubules. As
shown in Fig. 1, the
microtubules were observed as filamentous networks developed in the cytoplasm.
Treatment with nocodazole for 30 min at 37 °C resulted in a dose-dependent
disappearance of these filamentous signals
(Fig. 1A). A
significant portion of the microtubules was disrupted with nocodazole at 1
µM, and the disruption seemed nearly complete at 100
µM. With this concentration of nocodazole, we examined the time
course of the disruption of the microtubules. As depicted in
Fig. 1B, nocodazole
caused a time-dependent disruption of the microtubules, with a nearly complete
loss of the filamentous signals at 30 min.
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FIG. 1.
Effects of nocodazole on the microtubule integrity. A,
dose-dependent disruption of the microtubules by nocodazole. Isolated rat
adipocytes in Buffer A were incubated for 30 min without (a and
e) or with 1 (b and f), 10 (c and
g), or 100 (d and h) µM of
nocodazole. The cells were fixed and the microtubules were visualized with
anti--tubulin antibody as described under "Experimental
Procedures." a–d, images obtained by confocal microscopy
at x400 magnification. e–h, enlarged images of
representative cells in a through d, respectively.
Bars, 10 µm. B, time course of disruption of the
microtubules by nocodazole. Adipocytes in Buffer A were incubated without
(a) or with 100 µM nocodazole for 5 (b), 15
(c), or 30 (d) minutes at 37 °C. The cells were then
fixed and immunostained for -tubulin. Bars, 10 µm.
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These morphological data were corroborated by biochemical measurement of
the polymerized tubulin. As illustrated in
Fig. 2, the amount of
polymerized tubulin decreased in a dose-dependent manner by nocodazole
treatment for 30 min at 37 °C, with a concomitant increase in the amount
of tubulin monomer. Approximately 50% of the polymerized tubulin was lost with
nocodazole at 1 µM, and the effect of nocodazole was maximal at
100 µM.
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FIG. 2.
Effect of nocodazole on polymerized and unpolymerized tubulin.
Adipocytes in Buffer A were incubated for 30 min with the indicated
concentrations of nocodazole. Then polymerized and unpolymerized tubulin were
extracted as described under "Experimental Procedures" and
subjected to immunoblotting. A, representative immunoblot data for
polymerized and unpolymerized tubulin. B, relative amounts of
polymerized tubulin. The relative intensities of tubulin bands in immunoblots
were measured as described under "Experimental Procedures."
Results are the means ± S.E. (n = 3).
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Previous studies have shown that disruption of the microtubules resulted in
dispersion of the perinuclear GLUT4 compartments in 3T3-L1 adipocytes
(14,
17,
20), while the consequence of
microtubule depolymerization on the subcellular localization of GLUT4 has
never been studied in primary adipocytes. To investigate the spatial
relationship between the microtubules and GLUT4, we reconstructed
three-dimensional images from serial confocal sections of double-immunostained
cells. Fig. 3 illustrates the
two-dimensional projections of the reconstructed three-dimensional images. The
three-dimensional images revealed the microtubules as reticular filamentous
network developed throughout the cytoplasm in the basal cells
(Fig. 3b). GLUT4, on
the other hand, were shown as punctate signals distributed throughout the
cytoplasm with intense accumulation around the nuclei
(Fig. 3a). Some, but
not all of the GLUT4 signals were colocalized with the microtubules
(Fig. 3, c and
d, arrowheads), suggesting association of the
GLUT4-containing membranes with the microtubules. Insulin stimulation did not
cause any significant alterations in the distribution of microtubules
(Fig. 3j), while the
cell surface became diffusely labeled with the GLUT4 signals, with concomitant
decrease in the punctate and perinucelar signals in the cytoplasm, suggesting
translocation of GLUT4 to the cell surface
(Fig. 3i). In
addition, the association of GLUT4 with the microtubules apparently decreased
in insulin-stimulated cells (Fig. 3,
k and l). Nocodazole treatment caused nearly
completely disruption of the microtubules throughout the cytoplasm, resulting
in more random distribution of GLUT4 in the cytoplasm
(Fig. 3e). In
addition, the perinuclear GLUT4 signals became moderately dispersed although
they were still in the vicinity of the nuclei. In such microtubule-disrupted
cells, insulin stimulation apparently caused subcellular redistribution of
GLUT4, but the insulin-induced surface labeling with GLUT4 and decrease in the
cytoplasmic punctate GLUT4 signals were not so consistent among the cells as
in control (nocodazole-untreated) cells
(Fig. 3m).
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FIG. 3.
Reconstructed three-dimensional images of the subcellular localization
of GLUT4 and the microtubules. After incubation in the absence
(a–d and i–l) or presence (e–h
and m–p)of 100 µM nocodazole for 30 min at 37
°C, adipocytes in Buffer A were incubated for an additional 30 min without
(a–d and e–h) or with (i–l and
m–p) 10 nM insulin. The cells were then fixed and
subjected to immunostaining for GLUT4 (a, e, i, and m) and
-tubulin (b, f, j, and n). The three-dimensional
images were reconstructed from serial confocal images taken along the
z-axis as described under "Experimental Procedures."
c, g, k, and o, merged images; d, h, l, and
p, enlarged images of representative cells in c, g, k, and
o, respectively.
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Such inconsistency in the extent of the insulin efffect among
nocodazole-treated cells led us to further analyze the GLUT4 distribution in
the microtubule-deficient state. As we and others previously reported,
insulin-stimulated rat adipocytes showed characteristic continuous GLUT4
signals at the cell surface on confocal planes due to GLUT4 translocation to
the plasma membrane, whereas they were found in the perinuclear region and in
punctate spots distributed throughout the cytoplasm in the basal cells
(12,
34). Hence, we obtained
confocal images from a total of 100 cells for each condition, and individual
cells were graded according to the length of the continuous GLUT4 signals at
the cell surface. As depicted in Fig.
4, most of the basal cells were with only limited continuous GLUT4
signals at the cell surface. Insulin markedly increased the number of cells
with longer continuous GLUT4 signals at the cell surface
(Fig. 4B, left
panel). Disruption of the microtubules with nocodazole had a minor effect
on the surface localization of GLUT4 in the basal cells. In
microtubule-disrupted cells, insulin increased the continuity of cell-surface
GLUT4 signals, but the effect was less consistent than in intact cells
(Fig. 4B, right
panel), suggesting that the insulin action was impaired with disruption
of the microtubules.
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FIG. 4.
Analysis of the cell surface localization of GLUT4. After incubation
in the absence or presence of 100 µM nocodazole for 30 min at 37
°C, adipocytes were incubated for an additional 30 min without or with 10
nM insulin. The cells were then fixed and immunostained for GLUT4.
Confocal images from a total of 100 cells were taken and graded according to
the continuity of the GLUT4 signals at the cell surface as described under
"Experimental Procedures." A, representative images of
the cells treated with none (a), insulin (b), nocodazole
(c), or nocodazole plus insulin (d). Bars, 20
µm. B, effects of insulin on the cell surface localization of
GLUT4 in control (left panel) and nocodazoletreated (right
panel) cells. Shaded columns, without insulin; closed
columns, with insulin.
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We also investigated the subcellular localization of the
vimentin-containing intermediate filaments in microtubule-deficient cells.
Vimentin is associated with insulin-sensitive GLUT4-conataining membranes and
the vimentin-containing intermediate filaments have been implicated in the
perinuclear localization of GLUT4 in 3T3-L1 adipocytes
(17). It has also been shown
that disruption of the microtubules caused reorganization of the
vimentin-containng intermediate filaments into thick bundles localized beneath
the plasma membrane and encapsulating the lipid droplet in 3T3-L1 cells
(19). As depicted in
Fig. 5, the reconstructed
three-dimensional images revealed that rat adipocytes have well developed
networks of the vimentin-containing intermediate filaments throughout the
cytoplasm. Disruption of the microtubules with nocodazole, however, had little
effect on the distribution of the vimentincontaining intermediate
filaments.
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FIG. 5.
Subcellular localization of the vimentin-containing intermediate
filaments in reconstructed three-dimensional images. After incubation in
the absence or presence of 100 µM nocodazole for 30 min at 37
°C, adipocytes were incubated without or with 10 nM insulin for
an additional 30 min. The cells were then fixed and immunostained with
anti-vimentin and anti--tubulin antibodies. The three-dimensional images
were reconstructed from sequential confocal images taken at 1-µm intervals
along the z-axis as described under "Experimental
Procedures." Bars, 20 µm.
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To evaluate the impaired insulin effect in microtubule-disrupted cells, we
next examined the effects of nocodazole on the insulin-stimulated glucose
transport and GLUT4 translocation. As shown in
Fig. 6, nocodazole inhibited
the insulin-stimulated glucose transport activity in a dose-dependent manner
without affecting the basal transport activity. However, there were
discernible differences between the effects of nocodazole on the integrity of
the microtubules (Figs.
1A and
2) and on the
insulin-stimulated glucose transport activity. First, the half-maximal
concentration of nocodazole (about 1 µM) to depolymerize the
microtubules had little effect on the insulin-stimulated glucose transport
activity. Second, the maximal concentration of nocodazole (100
µM), which caused nearly complete disruption of the
microtubules, inhibited the insulin-stimulated glucose transport activity by
only 55%. Such discrepancies in the nocodazole-sensitivity led us to examine
whether the inhibition of the glucose transport activity was associated with
inhibition of GLUT4 translocation in nocodazole-treated cells. As illustrated
in Fig. 7, the subcellular
membrane fractionation assay showed that insulin-induced GLUT4 translocation
was partially (by 47%) inhibited by treatment with the maximal concentration
of nocodazole. In another assay, we measured the amounts of the cell surface
GLUT4 by trypsin cleavage, which generates a 35-kDa fragment of the
transporter (27,
31). As shown in
Fig. 8, insulin stimulation
caused a 5-fold increase in the trypsin-cleaved 35-kDa fragment of GLUT4 in
the plasma membrane fraction. This insulin effect was inhibited by 65% in
nocodazole-treated cells, suggesting that insulin-induced insertion of GLUT4
into the plasma membrane was actually inhibited in microtubule-disrupted
cells. Thus, it seemed that the integrity of the microtubules is required for
insulin to fully stimulate GLUT4 translocation and glucose transport although
the insulin action would not be affected unless a considerable portion (50% or
more) of the microtubules is disrupted.
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FIG. 6.
Effects of nocodazole on the basal and insulin-stimulated glucose
transport. Adipocytes in Buffer A were incubated for 30 min with the
indicated concentrations of nocodazole. The cells were then incubated in the
absence ( ) or the presence (•) of 10 nM insulin for an
additional 30 min, and subjected to the glucose transport assay. Results are
the means ± S.E. (n = 3–6).
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FIG. 7.
Effect of nocodazole on insulin-stimulated GLUT4 translocation.
After incubation for 30 min in the absence or the presence of 100
µM nocodazole, cells were incubated for an additional 30 min
without or with 10 nM insulin. At the end of incubation, the cells
were washed and subjected to subcellular fractionation and immunoblotting for
GLUT4. A, representative immunoblot data for GLUT4 in the plasma
membrane (PM) and low density microsomal (LDM) fractions.
B, relative amounts of GLUT4 in immunoblots. The relative intensities
of GLUT4 bands were quantified as described under "Experimental
Procedures." Results are the means ± S.D. of three
determinations.
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FIG. 8.
Cell surface GLUT4 labeling by trypsinization. A,
representative immunoblot data for the trypsin-cleaved 35-kDa fragments of
GLUT4 in the plasma membrane fractions. After incubation for 30 min in the
absence or the presence of 100 µM nocodazole, cells were
stimulated without or with insulin (10 nM) for an additional 30
min, and then incubated with 3 mM potassium cyanide for 15 min.
TPCK-treated trypsin (final concentration, 1 mg/ml) was added to the
incubation buffer, and incubation was continued for an additional 15 min. At
the end of incubation, soybean trypsin inhibitor (final concentration, 2
mg/ml) was added to the buffer, and the cells were washed, homogenized and
subjected to subcellular fractionation and immunoblotting for GLUT4.
B, relative amounts of the 35-kDa fragments of GLUT4 in the plasma
membrane fraction. The relative intensities of the 35-kDa bands were
quantified as described under "Experimental Procedures." Results
are the means ± S.D. of three determinations.
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To exclude the possibility that nocodazole inhibited the insulin action by
any mechanisms unrelated to the microtubules, we investigated the effects of
nocodazole on the insulin signaling as well as on the glucose transporter
activity itself. Immunoblot analyses of tyrosine-phosphorylated proteins in
cell lysates showed that insulin stimulated tyrosine phosphorylation of
proteins corresponding in size to IRS-1/2 (180–190 kDa), the
-subnit of the insulin receptor (95 kDa) and IRS-3 (60 kDa)
(Fig. 9A,
arrows). Insulin-mediated tyrosine phosphorylation of these proteins
was not affected by nocodazole treatment
(Fig. 9A). Likewise,
nocodazole treatment did not affect insulin-induced tyrosine phosphorylation
of c-Cbl, a component of the PI 3-kinase-independent signaling pathway
involved in GLUT4 translocation
(35)
(Fig. 9B,
arrow). In addition, we did not see any inhibitory effects of
nocodazole on the insulin-induced phosphorylation of protein kinase B/Akt
(Fig. 9C) or
activation of the insulin-sensitive cAMP phosphodiesterase, a downstream
target of protein kinase B
(36)
(Fig. 9D).
Furthermore, as shown in Fig.
10, neither nocodazole (100 µM) nor wortmannin (100
nM) inhibited the glucose transport activity in KCN-treated cells
that had been stimulated with insulin, whereas phloretin, a direct inhibitor
of glucose transporters, significantly inhibited the cellular glucose uptake,
indicating that nocodazole was without effect on the glucose transporter
activity itself. Thus, it seemed unlikely that the inhibition of the insulin
effect on glucose transport in nocodazole-treated adipocytes derived from
attenuation of the proximal insulin signals or direct inhibition of the
glucose transporter activity.
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FIG. 9.
Effects of nocodazole on insulin signals. A and B,
effects of nocodazole on insulin-dependent tyrosine phosphorylation.
Adipocytes in Buffer A were treated without or with nocodazole (100
µM) for 30 min at 37 °C. Then the cells were stimulated
without or with insulin (10 nM) for 20 min (A) or 1 min
(B). At the end of incubation, cells were washed and solubilized in
SDS sample buffer. The proteins in the lysates were separated on
SDS-polyacrylamide gel and immunoblotted with anti-phosphotyrosine antibody
(A) or anti-phospho-c-Cbl antibody (B). C, effect
of nocodazole on insulin-stimulated protein kinase B phosphorylation.
Adipocytes in Buffer A were incubated without or with nocodazole (100
µM) for 30 min and then stimulated without or with insulin (10
nM) for 20 min. At the end of incubation, the cells were washed
with STE and homogenized in homogenizing buffer as described under
"Experimental Procedures." The lysates were subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotting with
anti-phospho-Akt (Ser-473) and anti-Akt antibodies. D, effect of
nocodazole on insulin-dependent stimulation of phosphodiesterase. Adipocytes
in Buffer A were incubated for 30 min without or with nocodazole (100
µM) and stimulated without (open columns) or with
insulin (2 nM) (closed columns) for 15 min. The
phosphodiesterase activity was assayed as described under "Experimental
Procedures." Results are the means ± S.E. (n =
3–6). NCZ, nocodazole.
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FIG. 10.
Effect of nocodazole on the glucose transport in KCN-treated cells.
Panel A, adipocytes in Buffer A were treated without or with
nocodazole (100 µM), wortmannin (100 nM), or
phloretin (1 mM) for 30 min at 37 °C. Then the cells were
stimulated without or with insulin (10 nM) for 20 min. At the end
of incubation, the cellular glucose transport activity was assayed. Panel
B, adipocytes in Buffer A were stimulated without or with insulin (10
nM) for 20 min at 37 °C, followed by treatment with 3
mM KCN for an additional 15 min. The cells were then incubated in
the absence or the presence of nocodazole (100 µM), wortmannin
(100 nM), or phloretin (1 mM) for 30 min. At the end of
incubation, the cellular glucose transport activity was assayed. Results are
the means ± S.E. (n = 3–6). *, p < 0.01
(versus insulin alone). Noc, nocodazole; Wort,
wortmannin; Phlo, phloretin.
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In the next series of experiments, we investigated the mechanisms of
inhibition of insulin-induced GLUT4 translocation in mictotubule-disrupted
cells. Since the amount of GLUT4 on the plasma membrane depends on the balance
between exocytotic recruitment and endocytosis of the transporter, the
inhibition of insulin-induced GLUT4 translocation in microtubule-deficient
cells may derive from altered recycling rate of GLUT4 in the
insulin-responsive pool; either slowed exocytosis or accelerated endocytosis
of GLUT4. Another possiblity is that the insulin recruits GLUT4 from two
distinct compartments with different microtubule dependence in the traffic to
the cell surface; one is totally dependent on the microtubules in trafficking
to the plasma membrane while another is less or not dependent on the
microtubule integrity. The GLUT4 in the former compartment would not be able
to translocate to the plasma membrane in the absence of the microtubules,
resulting in a decrease in the maximal insulin effect.
To elucidate these points, we measured the time course of insulin
stimulation of the glucose transport activity in nocodazole-treated cells. As
shown in Fig. 11, the
time-course of insulin stimulation of the glucose transport activity was
significantly delayed in microtubule-disrupted cells (t were
7 and 2.3 min in nocodazole-treated and control cells, respectively). This
delay was not brought about simply as a consequence of a decline in the
maximal transport activity because a reduction of the insulin concentration
from 10 to 0.3 nM resulted in an 50% decrease in the maximal
glucose transport activity without a significant alteration in the time course
of activation.
Next, we examined the rate of GLUT4 endocytosis in nocodazole-treated
cells. As depicted in Fig. 12,
the time-course of endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4
was little affected by nocodazole treatment. These data suggested that the
delay in the insulin activation of the glucose transport activity was not
caused by accelerated endocytosis but due to slowed exocytosis of GLUT4.
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FIG. 12.
Effect of nocodazole on the time course of GLUT4 endocytosis.
Adipocytes in Buffer A were stimulated with 10 nM insulin for 20
min at 37 °C. The cells were then incubated with 3 mM potassium
cyanide for 15 min, followed by incubation without or with nocodazole (100
µM) for an additional 30 min. Then TPCK-treated trypsin was
added to the cells at a final concentration of 1 mg/ml, and incubation was
continued for 20 min. At the end of the incubation, soybean trypsin inhibitor
(final concentration, 2 mg/ml) was added to the incubation buffer and the
cells were washed with Buffer A. The cells were resuspended in fresh Buffer A
and incubated for 0 (immediately washed), 5, 10, or 20 min at 37 °C. At
the end of the incubation, the cells were washed with STE buffer, homogenized,
and then subjected to subcellular fractionation. The LDM fractions were
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with
anti-GLUT4 antibody as described under "Experimental Procedures."
A, representative immunoblot data. B, relative amounts of
the 35-kDa fragments of GLUT4 in the LDM fraction. The relative intensities of
the 35-kDa bands were quantified as described under "Experimental
Procedures." Results are the means ± S.D. of three
determinations. Open circles, without nocodazole; closed
circles, with nocodazole.
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Thirdly, we tested if the impaired maximal glucose transport activity would
be restored by inhibition of GLUT4 endocytosis to the same level as in control
cells. To this end, we used the MHC class-I antigen-derived peptide,
Dk-(62–85), which has been shown to nearly completely inhibit
GLUT4 endocytosis (27,
37). As shown in
Fig. 13, inhibition of GLUT4
endocytosis resulted in a marked enhancement of the insulin-stimulated glucose
transport activity in control cells, consistent with previous studies
(37,
27). In nocodazole-treated
cells, the insulin-stimulated glucose transport activity was enhanced by
Dk-(62–85) peptide, but was never restored to the same level
as in control cells. These data suggest that the inhibition of the
insulin-stimulated glucose transport and GLUT4 translocation in
microtubule-deficient cells was not caused solely by altered recycling rate of
GLUT4 in the whole insulin-responsive pool. Instead, these data indicate the
existence of a microtubule-dependent subpopulation in the insulin-responsive
GLUT4 pool.
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FIG. 13.
Inhibition of GLUT4 endocytosis does not restore the glucose transport
activity in nocodazole-treated cells. After incubation without () or
with (•) 100 µM of nocodazole for 30 min at 37 °C,
adipocytes in Buffer A were incubated with 10 nM insulin for 15
min. At the end of incubation, 50 µM Dk-(62–85)
peptide was added, and the cells were incubated for an additional 30 min. The
glucose transport activity was assayed at the indicated time points. Results
are the means ± S.E. (n = 3–6).
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To characterize the microtubule-dependent and independent membrane traffic,
we investigated the effects of nocodazole on insulin-induced translocation of
other membrane-associated proteins. As shown in
Fig. 14, nocodazole had minor
effects on the insulin-stimulated translocations of GLUT1 and VAMP-3/
cellubrevin, both proteins localized to the endosomal recycling system,
whereas those of IRAP and VAMP-2 was significantly but incompletely inhibited
by nocodazole treatment. These results are consistent with previous
observations that microtubule depolymerization did not affect
insulin-stimulated translocation of the transferrin receptor
(14) and GLUT1
(16) and suggest that
insulin-stimulated recruitment of GLUT4 from the endosomal system to the
plasma membrane does not require the microtubules integrity. On the other
hand, it seems that insulin also recruits GLUT4 by a microtubule-dependent
mechanism to the cell surface from non-endosomal compartments that may be
shared with IRAP and VAMP-2.
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FIG. 14.
Effects of nocodazole of insulin-induced translocation of GLUT1, IRAP,
and VAMPs. After incubation for 30 min in the absence or the presence of
100 µM nocodazole, adipocytes in Buffer A were incubated for an
additional 30 min with 10 nM insulin. At the end of incubation, the
cells were washed with STE buffer, homogenized, and subjected to subcellular
fractionation and immunoblotting for GLUT1, IRAP, VAMP-2, and VAMP-3.
PM, plasma membrane fractions; LDM, low density microsomal
fractions.
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To further confirm the notion that the insulin recruits GLUT4 to the plasma
membrane from distinct compartments with differential microtubule dependence,
we compared the sensitivity to nocodazole of the insulin- and
GTPS-stimulated glucose transport in electrically permeabilized
adipocytes. Previous studies have shown that while both insulin and
GTPS stimulate glucose transport and GLUT4 translocation
(38,
39,
27), GTPS selectively
stimulates recycling via the endosomal system whereas, in addition to this
pathway, insulin also stimulates the movement of GLUT4 from a compartment that
is distinct from the endosomal recycling system
(9). Since we previously
observed increased sensitivity to H-7, a protein kinase inhibitor, by
electroporation of rat adipocytes suspended in
high-K+/low-Ca2+ buffer
(29), we first examined the
effect of nocodazole on the microtubules in electrically permeabilized cells.
As shown in Fig. 15,
electrically permeabilized cells showed higher sensitivity to nocodazole
compared with intact cells; the microtubules were completely disrupted with
nocodazole at 10 µM (Fig.
15A). At this concentration of nocodazole, the
insulin-stimulated glucose transport activity was inhibited by 40%, whereas
the GTPS-stimulated glucose transport activity was not affected
(Fig. 15B).
Intriguingly, insulin and GTPS stimulated the glucose transport
activity to the comparable level in the absence of the microtubules. Thus, it
seemed that the GTPS recruits GLUT4 to the cell surface presumably from
the endosomal recycling system by a microtubule-independent mechanism whereas
insulin recruits GLUT4 to the cell surface from at least two distinct
compartments with differential microtubule dependence. In addition, such
differential sensitivity to nocodazole between the effects of insulin and
GTPS provides further evidence that nocodazole does not inhibit
insulin-stimulated glucose transport by direct inhibition of the transporter
activity.
We also examined the subcellular localization of GLUT4 and the microtubules
in GTPS-stimulated cells. The reconstructed three-dimensional images
depicted that GTPS like insulin caused diffuse labeling of the cell
surface with the GLUT4 signals both in the control
(Fig. 16A, e and
f) and nocodazole-treated cells
(Fig. 16A, g and
h). However, compared with insulin-stimulated cells (see
Fig. 3), there remained more
punctate GLUT4 signals in the cytoplasm with GTPS stimulation, some of
which were still associated with the microtubules
(Fig. 16A, e and
f). To further assess the differences between the effects
of insulin and GTPS, we measured the amount of tubulin associated with
the GLUT4-containing membranes immunoisolated from insulin- or
GTPS-stimulated cells. As illustrated in
Fig. 16B,
-tubulin was present in the immunoisolated GLUT4-containing membranes in
the basal state. Significantly, insulin but not GTPS markedly reduced
the association of -tubulin with the GLUT4-containing membranes. These
results are consistent with our morhological data and the observation that
insulin causes dissociation of -tubulin from the GLUT4-containing
membranes in the insulin-sensitive fractions in 3T3-L1 adipocytes
(17). Our data also support
the notion that the two reagents recruit GLUT4 by distinct mechanisms with
differential microtubule dependence.
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FIG. 16.
Effects of insulin or GTPS on the association of tubulin with the
GLUT4-containing membranes. A, reconstructed three-dimensional
images of the subcellular localization of GLUT4 and the microtubules in
electrically permeabilzed cells. Electrically permeabilized adipocytes in
Buffer X were incubated for 30 min at 37 °C without (a, b, e, and
f) or with (c, d, g, and h)10 µM
nocodazole, and then stimulated without (a, b, c, and d) or
with (e, f, g, and h) 1 mM GTPS for 20
min. The cells were fixed and immunostained for GLUT4 (green) and
-tubulin (red). The three-dimensional images were reconstruced
as described under "Experimental Procedures." a, c, e,
and g, lower magnification (x400) images; b, d, f, and
h, higher magnification (x1000) images. B, the
association of tubulin with the GLUT4-containing membranes. Electrically
permeabilized adipocytes in Buffer X were incubated without or with insulin
(10 nM) or GTPS (1 mM) for 20 min. Then cells
were washed, homogenized, and subjected to subcellular fractionation. The
GLUT4-containing membranes were immunoisolated from the LDM fractions and
solubilized as described under "Experimental Procedures." Proteins
in the solubilized fraction were immunoblotted for -tubulin and
GLUT4.
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DISCUSSION
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The results of the present study showed that insulin recruits GLUT4 to the
plasma membrane from at least two distinct intracellular compartments via
distinct traffic routes with differential microtubule dependence in rat
primary adipocytes.
First, microtubule disruption with nocodazole resulted in only a partial
(about 50%) inhibition of the insulin-stimulated glucose transport
(Fig. 6) and GLUT4
translocation (Figs. 7 and
8). Importantly, the impaired
insulin effect was not fully restored to the level in control cells by
inhibition of GLUT4 endocytosis (Fig.
13), suggesting that the inhibition did not derive solely from
alterations in the recycling rate of the whole insulin-responsive GLUT4.
Instead, our data clearly indicate the existence of two GLUT4 subpopulations;
one is unable to translocate to the cell surface in response to insulin in the
absence of the microtubules while another is not or less
microtubule-dependent.
Second, although the latter subpopulation of GLUT4 can undergo
insulin-induced translocation to the plasma membrane, the time course of
insulin activation of the glucose transport activity was markedly
(3-fold) delayed by disruption of the microtubules
(Fig. 11). Since the rate of
GLUT4 endocytosis was not significantly affected
(Fig. 12), it is likely that
the delay originated from slowed exocytosis of this subpopulation of GLUT4.
The apparent slowdown of insulin-induced GLUT4 exocytosis could be interpreted
in two ways. One interpretation is that insulin recruits these two
subpopulations of GLUT4 via distinct routes with different traffic kinetics,
and a faster traffic route is more susceptible to and eliminated by
microtubule disruption, unveiling another slower traffic pathway that is
independent of the microtubules. Alternatively, in addition to the
microtubule-dependent route, another routes may also be affected at some
step(s) by disruption of the microtubules, causing a slowdown of GLUT4
exocytosis. In either case, our data show the existence of two distinct GLUT
subpopulations that take distinct traffic routes to the cell surface with
differential microtubule dependence.
Third, in electrically permeabilized adipocytes, the insulin-stimulated
glucose transport was inhibited by 40% by disruption of the microtubules
whereas that stimulated with GTPS was not affected
(Fig. 15). Intriguingly, both
insulin and GTPS stimulated the glucose transport activity to the
comparable level in the absence of the microtubules. In the light of previous
observations that GTPS selectively stimulates recycling via the
endosomal system, these data suggest that insulin recruits GLUT4 from the
endosomal and non-endosomal compartments, and the former may correspond to the
GLUT4 subpopulation that is not or less dependent on the microtubules and the
latter to the microtubule-dependent one. This notion also seems consistent
with the results that insulin-stimulated translocation of GLUT1 and VAMP-3 but
not that of IRAP and VAMP-2 was insensitive to microtubule disruption
(Fig. 14) as well as with the
observation by other investigators that insulin-induced translocation of the
transferrin receptor was not affected by microtubule disruption
(14). It is unclear at present
to what subcellular compartment(s) the microtubule-dependent GLUT4
subpopulation localizes, but since insulin-stimulated translocation of IRAP
and VAMP-2 was partially inhibited by microtubule disruption, GLUT4 seems to
share the compartment(s) with IRAP and VAMP-2.
On the other hand, while there is a controversy on the mechanism of action
of nocodazole especially in 3T3-L1 adipocytes
(14,
15,
16,
19,
20), our results show that
nocodazole has no direct inhibitory effect on the glucose transporter activity
(Fig. 10). Under our
experimental conditions, the maximal concentration (100 µM) of
nocodazole to depolymerize the microtubules did not inhibit the glucose
transport activity in ATP-deprived cells that had been stimulated with
insulin. This was further supported by the observation that insulin and
GTPS showed different sensitivity to nocodazole in stimulation of
glucose transport in electrically permeabilized cells
(Fig. 15B). Our
results are seemingly inconsistent with recent reports that lower
concentrations (2–3 µM) of nocodazole disrupt the
microtubules without affecting insulin-induced GLUT4 translocation in 3T3-L1
adipocytes (19,
20). The authors argued that
nocodazole directly inhibits the glucose transporter activity rather than
GLUT4 translocation. We do not rule out the possibility that nocodazole is a
more potent inhibitor of GLUT1 than GLUT4 becasue 3T3-L1 adipocytes express a
considerable amount of GLUT1 in addition to GLUT4 while rat adipocytes mainly
express the latter isoform. It has been reported that the sensitivity to
indinavir is different among the GLUT isoforms
(40). It is also possible that
the difference may derive from the experimental methods for measuring the
effect of nocodazole on the glucose transporter activity. While we measured
the initial uptake rate of 3-O-methylglucose at 37 °C in the
absence of ATP (Fig. 9), both
groups measured the time-dependent accumulation of 2-deoxyglucose at 4 °C.
The latter reflects and does not discriminate two metabolic steps of
2-deoxyglucose; transport and phosphorylation by glucokinase with the
consumption of ATP. In addition, it was previously shown in rat adipocytes
that lowering of the temperature activates the glucose transport activity and
GLUT4 translocation in an ATP-dependent manner, suggesting incomplete arrest
of the subcellular membrane movements at low temperature
(41). Further study will be
needed to elucidate these points. Our data are consistent with the notion that
nocodazole inhibits the insulin action by interfering with the GLUT4
trafficking rather than the activity of glucose transporter. It is unlikely
that nocodazole inhibited insulin-induced GLUT4 translocation by affecting the
insulin signaling since it had little effects on the insulin stimulation of
tyrosine phosphorylation of the proximal signaling proteins
(Fig. 9, A and
B), protein kinase B/Akt
(Fig. 9C), the low
Km cAMP phosphodiesterase
(Fig. 9D) and
translocation of GLUT1 and VAMP-3 (Fig.
14).
While our data thus demonstrated that the microtubules play a significant
role in insulin-induced GLUT4 translocation, one issue remains to be addressed
that there was an apparent discrepancy between the effects of nocodazole on
the microtubules and the insulin-stimulated glucose transport (Figs.
2 and
6). The insulin-stimulated
glucose transport was not affected until a considerable portion (50% or more)
of the microtubules was disrupted. A similar discrepancy was observed by other
investigators in 3T3-L1 adipocytes
(19). This makes a remarkable
contrast to the close correlation between the actin filament integrity and the
insulin action (12). Since
GTPS-stimulated glucose transport was little affected with nocodazole
(Fig. 15B), it seems
that insulin-induced recruitment of the non-endosomal microtubule-dependent
GLUT4 subpopulation is not obstructed until a considerable portion (50% or
more) of the microtubules is disrupted. One possible explanation is that there
may occur a subcellular shift of GLUT4 from the microtubule-dependent to the
independent (or less dependent) compartments by treatment with lower
concentrations of nocodazole. In this respect, the observation by Shigematsu
et al. (20) seems
intriguing. Their study showed a time-dependent accumulation of GLUT4 beneath
the plasma membrane after the addition of a low concentration (3
µM) of nocodazole without alteration in the perinuclear
localization of the transporter. They also showed that the GLUT4 compartments
confined just beneath the plasma membrane were responsive to insulin in the
absence of the microtubules, but unable to be sorted back to the perinuclear
compartments. Thus, such a subcellular shift of GLUT4 may obscure the
inhibitory effect of nocodazole on insulin-induced GLUT4 translocation and
cause an apparent insensitivity to nocodazole. Alternatively, disruption of
the microtubules with lower concentrations of nocodazole may potentiate the
insulin action on GLUT4 translocation by unknown mechanisms. While it has long
been known that disruption of the microtubules promotes actin stress fibers
formation and changes in cell morphology (e.g. Ref.
42), it was recently reported
that GEF-H1, a microtubule-associated Rho guanine nucleotide exchange factor
is activated by microtubule disruption
(43). Additionally,
microtubule breakdown may stimulate other cellular signaling pathways involved
in GLUT4 translocation (for review see Ref.
44). Since nocodazole did not
stimulate the basal glucose transport activity
(Fig. 6), such mechanisms do
not seem to directly activate GLUT4 translocation, but may facilitate the
insulin-stimulated trafficking of GLUT4 to the plasma membrane. Although the
microtubules have been implicated in endocytic pathways
(45,
46), it is unlikely that
microtubule disruption augmented the insulin effect by inhibition of GLUT4
endocytosis since nocodazole had little effect on the rate of GLUT4
endocytosis (Fig. 12).
Finally, there still remains a possibility that nocodazole affected the GLUT4
trafficking by microtubule-unrelated mechanisms. Other methods to manipulate
the microtubules may help to clarify this point.
Since our original manuscript was submitted, Semiz et al.
(47) reported that
conventional kinesin KIF5B is required for insulin-stimulated GLUT4
translocation. Their data are consistent with our conclusions that the
microtubules play an indispensable role in insulin-induced movement of GLUT4
to the plasma membrane.
In summary, the present study provides important insights into the
physiological significance of the microtubules in the subcellular localization
and trafficking of GLUT4 in primary adipocytes. Our results showed that there
are two distinct GLUT4 subpopulations with differential microtubule dependence
in trafficking to the plasma membrane. The microtubule-independent (or less
dependent) GLUT4 subpopulation seems to localize to the endosomal system,
while the compartment(s) to which the microtubule-dependent subpopulation
localizes remains to be elucidated. The GLUT4 distribution among the
compartments may affect the amplitude and kinetics of the cellular response to
insulin when the microtubules are disrupted. With the unique morphological and
functional features, the distribution and transit of GLUT4 among the
intracellular compartments may be more dependent on the microtubules in
primary adipocytes than in other types of cells including 3T3-L1 adipocytes.
Further dissection of the trafficking pathways of GLUT4 is apparently
necessary to elucidate the physiological role of the microtobules in the
insulin action.
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FOOTNOTES
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* This work was supported by a grant-in-aid for scientific research from the
Ministry of Education, Culture, Sports, Science, and Technology of Japan. The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 81-27-220-8836; Fax:
81-27-220-8893; E-mail:
hshibata{at}showa.gunma-u.ac.jp.
1 The abbreviations used are: VAMP, vesicle-associated membrane protein;
IRAP, insulin-regulated aminopeptidase; TGN, trans-Golgi network;
CD-M6PR, cation-dependent mannose 6-phoshate receptor; Me2SO,
dimethyl sulfoxide; HRP, horseradish peroxidase; PI 3-kinase, phosphoinositide
3-kinase; GTPS, guanosine 5'-O-(3-thiotriphosphate);
H-7, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; PBS, phosphate-buffered
saline; PIPES, 1,4-piperazinediethanesulfonic acid.
2 H. Shibata and L. B. Liu, unpublished observations.
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ACKNOWLEDGMENTS
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</
TOP
ABSTRACT
INTRODUCTION
) or with (
) 100 µM of nocodazole. The cells
were then incubated in the presence of 10 nM (
) for 20 min. At the end of incubation, the cellular
glucose transport activity was assayed. Results are the means ± S.E.
(n = 3–6).