From the Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received for publication, November 29, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Insulin regulates glucose uptake in adipocytes
and muscle by stimulating the movement of sequestered glucose
transporter 4 (GLUT4) proteins from intracellular membranes to the cell
surface. Here we report that optimal insulin-mediated GLUT4
translocation is dependent upon both microtubule and actin-based
cytoskeletal structures in cultured adipocytes. Depolymerization of
microtubules and F-actin in 3T3-L1 adipocytes causes the
dispersion of perinuclear GLUT4-containing membranes and abolishes
insulin action on GLUT4 movements to the plasma membrane. Furthermore,
heterologous expression in 3T3-L1 adipocytes of the microtubule-binding
protein hTau40, which impairs kinesin motors that move toward the plus
ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no
detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with
the hypothesis that both the actin and microtubule-based cytoskeleton,
as well as a kinesin motor, direct the translocation of GLUT4 to the
plasma membrane in response to insulin.
Physiological glucose homeostasis in humans is largely dependent
on the actions of the hormone insulin, particularly its ability to
inhibit glucose output from the liver and enhance glucose transport into fat and muscle cells. Insulin exerts this latter effect primarily through a process whereby sequestered intracellular GLUT4 glucose transporter proteins are rapidly redistributed to cell surface membranes in which they can catalyze glucose uptake into cells (1-4).
In both the basal and insulin-stimulated states, GLUT4 proteins appear
to cycle between intracellular membrane and plasma membrane locations
(5). However, in the basal state most of the GLUT4 is diverted to
intracellular perinuclear membranes, and the exocytosis rate is slow.
Insulin stimulates exocytosis of GLUT4 by a mechanism that requires the
p85/p110 type phosphatidylinositol 3-kinase, which is recruited
to protein phosphotyrosines in response to activation of the insulin
receptor tyrosine kinase (6-8). Insulin also appears to significantly
inhibit GLUT4 endocytosis (5, 9, 10). However, the precise mechanism
whereby insulin signals to the GLUT4 membrane trafficking machinery
remains obscure.
It is known that other membrane systems, such as lysosomes (11),
mitochondria (12), Golgi membranes (13, 14), and pigment granules (15),
are localized within cells by molecular motors. For example, membrane
vesicles containing melanin appear to be driven along microtubules over
relatively long distances from their perinuclear location in
unstimulated melanocytes to the cell periphery upon elevation of cAMP
levels (16). The complex motor dynein drives movements along
microtubules in the minus direction toward the perinuclear microtubule
organizing center, whereas kinesin motors drive movements toward the
plus growing ends of microtubules (17). Movements of these membranes
over shorter distances seem to require actin filaments (18, 19). Based
on these observations, it has been suggested that the microtubule and
actin filament networks are highly integrated in discharging their
organelle localization functions (20). Recent findings in our
laboratory have implicated a role of the cytoskeleton in the mechanism
of insulin-stimulated GLUT4 translocation in cultured 3T3-L1 adipocytes
(21). These studies revealed that the microtubule protein The major aim of the present studies was to test the hypothesis that
optimal insulin-mediated GLUT4 translocation to the cell periphery
requires the microtubule motor kinesin in the context of an intact
microtubule and F-actin cytoskeleton. Consistent with this concept,
both nocodazole and colchicine were found to inhibit GLUT4
translocation markedly as well as disrupt microtubules in 3T3-L1
adipocytes. Insulin action on GLUT4 was also dependent on intact
F-actin. We took further advantage of the ability of the neuronal
microtubule-associated protein hTau40 to partially inhibit the function
of kinesins when it is expressed heterologously in cultured cells as
described by Mandelkow and co-workers (22, 23). The hTau40 protein
localized to microtubules in 3T3-L1 adipocytes and delayed the initial
appearance of GLUT4 at the cell surface membrane in response to
insulin. The data are consistent with the hypothesis that
insulin-stimulated GLUT4 movements to the cell periphery involve one or
more kinesins.
Materials--
Human Tau40 cDNA construct was kindly
provided by Dr. Mandelkow (22, 23). Rabbit polyclonal anti-GLUT4
antibody was raised against the C-terminal 12 amino acids. Rabbit
polyclonal anti-Tau antibody and anti-tubulin were from Sigma. Goat
polyclonal anti-GLUT4 was from Santa Cruz Biotechnology (Santa Cruz,
CA). The rhodamine-conjugated or fluorescein isothiocyanate
(FITC)1-conjugated goat
anti-mouse rabbit antibodies were from BioSource International (Camarillo, CA). Cy3- and Cy5-conjugated donkey anti-mouse and rabbit IgG were from Jackson Immuno Research (West Grove, PA). Rhodamine-conjugated phalloidin was purchased from Molecular Probes, Inc. (Eugene, OR). Nocodazole and colchicine were
purchased from Sigma, and latrunculin B was obtained from Calbiochem
and dissolved in Me2SO.
Tau Adenovirus Construct and Infection--
Recombinant
adenovirus encoding the hTau40 was constructed after the method
described in He et al. (24). hTau40 cDNA was cloned into
the BglII-XbaI site of pAdTrack-CMV shuttle
vector. This vector was linearized with PmeI and was
electroporated into electrocompetent BJ5183 cells having the pAdEasy-1
plasmid to generate recombinant adenovirus. After the recombinant
plasmid was amplified, DNA was digested with PacI. This
adenovirus was transfected into 293 cells and monitored with
green fluorescent protein expression. After the virus amplification,
cell lysate was stored at Cell Culture and Treatment--
3T3-L1 fibroblasts were grown in
DMEM supplemented with 25 mM glucose, 10% fetal bovine
serum, 50 mg/ml streptomycin, 50 units/ml penicillin. 3T3-L1
fibroblasts (3-4 days postconfluent) were differentiated into
adipocytes by incubating with the same DMEM containing 0.5 mM isobutylmethylxanthine, 0.25 µM
dexamethasone, and 4 µg/ml insulin for 3 days, grown in DMEM with
10% fetal bovine serum and 4 µg/ml insulin for 2 days and then grown
in DMEM with 10% fetal bovine serum for an additional 3-6 days.
For the immunofluorescence experiments, differentiated adipocytes were
replated on glass coverslips using trypsin and collagenase and then
incubated in DMEM containing 25 mM glucose and 10% fetal bovine serum. Before the experiments, adipocytes were incubated overnight in serum-free medium containing 0.5% bovine serum albumin, except when rhodamine-conjugated phalloidin staining was
performed. Concentrations of inhibitors used and incubation times are
noted in Figs. 1-7.
Immunofluorescence Microscopy--
3T3-L1 adipocytes were either
treated or not treated with 30 µM nocodazole, 10 µM latrunculin B for 1 h, or 10 µM
colchicine for 1-3 h at 37 °C. Cells were fixed in 3.7%
formaldehyde in phosphate-buffered saline (PBS) (171 mM
NaCl, 10.1 mM Na2HPO4, 3.35 mM KCl, 1.84 mM KH2PO4,
pH 7.2) or methanol. Expressed hTau40 constructs can be visualized by
extraction with 0.5% Triton X-100 in PBS for 5-10 s before methanol
fixation, which allows unbound protein to diffuse out (25).
Formaldehyde fixation was not applicable because the cellular
localization of Tau is altered (26). Cells were incubated in PBS
containing buffer A (0.5% Triton X-100 in PBS and 1% fetal bovine
serum) for 15 min and then incubated for 2 h with polyclonal
anti-GLUT4, anti-Tau, or monoclonal anti-tubulin antibodies. The
coverslips were washed extensively with buffer A and incubated in a
1:200-1:1000 dilution of the appropriate secondary antibody conjugated
to FITC, rhodamine, or Cy5. For F-actin staining, cells were treated
with 1 unit/ml rhodamine-conjugated phalloidin for 30 min. The
coverslips were washed again extensively with buffer A, rinsed once
with PBS, and mounted on slides with 90% glycerol in PBS and 2.5%
DABCO. The stained cells were observed with confocal microscopy using a
Nikon Diaphot 200 inverted microscope and a MRC1024 processing unit
(Bio-Rad). Zoom factors of 1.0-3.0 were used, and the images were
analyzed by laser-sharp processing software.
GLUT4 Translocation Assay--
GLUT4 translocation was assayed
using plasma membrane lawns as described previously (27). 3T3-L1
adipocytes cultured on glass coverslips were treated as described in
the figure legends. At the end of each experiment, cells were rapidly
washed in PBS followed by a 40-s treatment in PBS containing 0.5 mg/ml
poly-L-lysine (Sigma). The cells were swollen by three
rapid washes in hypotonic buffer (one-third of buffer B), transferred
to buffer B (70 mM KCl, 30 mM HEPES, 5 mM MgCl2, 3 mM EGTA, pH 7.4), and
sonicated using a probe sonicator to generate a lawn of plasma membrane fragments attached to the glass. The membranes were then fixed for 15 min with 3.7% formaldehyde, washed three times with PBS, and blocked
with PBS containing 2% bovine serum albumin for 40 min. To quantify
GLUT4 on lawns, coverslips were incubated with anti-GLUT4 antibody
diluted 1:1000 in PBS containing 0.05% Tween 20 for 2 h at room
temperature. Coverslips were washed five times for 3 min each, and they
were incubated with FITC-conjugated goat anti-rabbit IgG (1:1000) mixed
with 10 µg/ml rhodamine-conjugated wheat germ agglutinin (to identify
plasma membranes). Coverslips were then washed as described earlier and
postfixed for 10 min with 3.7% formaldehyde followed by a final wash
with PBS before mounting on slides with DABCO. Stained cells were
observed using the confocal microscopy system as described above. For
each experiment, samples were run in duplicate, and 10 random
representative images were collected from each coverslip. Fluorescence
intensity was quantified using Adobe Photoshop analysis software.
Intracellular Localization of GLUT4 Is Disrupted by Nocodazole or
Latrunculin B--
Initial experiments were conducted using confocal
microscopy to test the effects of the F-actin-disrupting agent
latrunculin B and the microtubule depolymerizing agent nocodazole on
GLUT4 disposition in 3T3-L1 adipocytes. Minimum concentrations of these agents required for disrupting the cytoskeleton were determined (data
not shown). As depicted in Fig.
1A, cultured adipocytes do not
display actin stress fibers, but cortical actin and thin actin
filaments, especially at the lower focal planes of the cells, can be
visualized by phalloidin staining. Incubation of the cells with 10 µM latrunculin B for 60 min causes dispersion of the
actin filaments and much of the cortical actin. Interestingly, the
normally concentrated perinuclear GLUT4 in these treated cells appears to be partially dispersed and more granular. In addition, many large
vesicular structures containing GLUT4 are visualized throughout the
latrunculin B-treated cells (Fig. 1A). Treatment with 30 µM nocodazole causes a virtually complete disassembly of
microtubules in 3T3-L1 adipocytes as well as a marked dispersion of
perinuclear GLUT4 into a fine punctate appearance throughout the
cytoplasm. The addition of both agents to the cultured adipocytes
yields a disposition of GLUT4 that is also dispersed throughout the
cells. These alterations in GLUT4-containing membrane localizations do not appear to originate from nonspecific cell damage because a normal
perinuclear GLUT4 morphology is restored within 3 h after the
removal of the cytoskeleton-disrupting agents. These data indicate that
the integrity of both F-actin and microtubules is required to
concentrate GLUT4 in the perinuclear region of 3T3-L1 adipocytes.
Disruption of Both Microtubules and F-actin Abolishes GLUT4
Translocation in Response to Insulin--
To determine whether
tubulin- and actin-based cytoskeletal elements are necessary for
optimal GLUT4 movements to the cell surface membrane in response to
insulin, the effects of latrunculin B and nocodazole on the appearance
of GLUT4 on plasma membrane sheets were assessed. In this assay,
adipocytes grown on glass coverslips were sonicated after appropriate
treatments, and the plasma membranes remaining attached to the glass
were probed with anti-GLUT4 and FITC-labeled secondary antibody. The
GLUT4 detected was then normalized to the number of membranes remaining
attached, which was determined by staining with
rhodamine-conjugated wheat germ agglutinin. Fig.
2 shows that insulin causes significant increases in cell surface GLUT4 by 3 min and that a maximal effect is
observed by 15 min (see also Fig. 4). Treatment of the cultured adipocytes with either nocodazole or latrunculin B leads to an approximately 50% inhibition of insulin-stimulated GLUT4
translocation. Remarkably, incubation of 3T3-L1 adipocytes with the
combination of these agents for 60 min completely blocked insulin
action on GLUT4 translocation (Fig. 2). Taken together, these results
demonstrate the requirement of either intact microtubules or intact
F-actin in cultured adipocytes for partial responsiveness of GLUT4 to insulin. In the absence of both cytoskeletal systems, GLUT4 movements to the cell surface are abolished.
The data depicted in Figs. 1 and 2 are consistent with the recent work
on several other cell types revealing that the functions of
microtubules and F-actin in localizing intracellular membranes are
highly coordinated (for review see Ref. 20). Axoplasmic vesicles in the
giant squid axon were shown to translocate on both microtubules and
actin filaments, and these vesicles apparently can switch from one
system to the other during movements (28). Early endosomes containing
Rab5 appear to be regulated initially by cortical actin and then
by microtubule-based motors as they move within the cytoplasm (29).
Furthermore, regulated exocytosis in embryonic sea urchin cells in
response to wounding was found to require both microtubule- and
actin-dependent stages based on sensitivity to anti-kinesin
antibodies and a myosin ATPase inhibitor (30). Pigment granules within
frog melanocytes display short range movements that are dependent on
F-actin integrity (19), and depolymerization of actin causes
exaggerated microtubule-dependent aggregation of pigment
granules in unstimulated cells (18). When cells are stimulated under
these conditions, granule dispersion is restricted to the plus ends of
microtubules rather than being spread out. These and other observations
have led to the hypothesis that specific molecular mechanisms, possibly
involving nonconventional myosin, CLIP-170, and dynein, operate to
switch vesicles between microtubule and F-actin tracks (20). Further
experiments will be required to test this concept vigorously with
respect to GLUT4 translocation.
The hTau40 Protein Associates with Microtubules and Delays GLUT4
Translocation to the Cell Surface Membrane--
If GLUT4-containing
vesicles translocate on microtubule tracks in response to insulin
before connecting to an actin-based system of movement to the cell
surface, one might expect a more complete inhibition of insulin action
upon the destruction of microtubules by nocodazole (see Fig. 2).
However, nocodazole itself releases GLUT4-containing vesicles from the
juxtanuclear region of adipocytes, presumably due to disruption of
dynein motors that concentrate GLUT4-containing vesicles, and thus may
partially mimic the ability of insulin to engage GLUT4-containing
vesicles with peripheral actin filaments. According to this model,
insulin promotes the movement of GLUT4 toward the plus ends of
microtubules through the actions of kinesin motors on microtubules,
whereas nocodazole promotes outward mobility of GLUT4 through removal of the normally predominant minus end-directed movement.
To test this hypothesis, we performed heterologous expression of the
microtubule-binding human Tau40 protein (Fig.
3A) in cultured adipocytes
through recombinant adenovirus infection. Tau40 is normally a neuronal
protein, but its expression in cultured Chinese hamster ovary cells has
been shown to disrupt kinesin-mediated movements of intracellular
membranes to the cell periphery (22, 23). For example, mitochondria and
intermediate filaments are inappropriately localized to the
juxtanuclear region of these cells stably expressing Tau, and the
expansion of the endoplasmic reticulum throughout the cytoplasm in
neuroblastoma cells expressing high levels of Tau is inhibited.
Importantly, the expression of Tau has also been shown to inhibit the
rate of exocytosis of vesicles containing the transferrin receptor
(22). These effects of Tau are reported to result from the ability of
the protein to affect the frequencies of attachment and detachment of
vesicles to microtubules rather than the speed of microtubule-based
motors. In particular, reversals of vesicle movements from minus end to
plus end are greatly reduced by the expression of Tau, effectively
favoring movements in the minus end direction. By reducing attachment
of kinesins to microtubules, we reasoned that Tau expression may partially inhibit insulin-mediated GLUT4 translocation.
Fig. 3B shows the localization of expressed hTau40 protein
in cultured adipocytes. In the absence of nocodazole, hTau40 was observed to localize with microtubules, whereas in the presence of the
drug, the structural features of both microtubules and hTau40 were
dispersed. Thus, the expression of hTau40 decorates rather than
disrupts microtubule structures, which is consistent with previous
results (22, 23). Similarly, the expression of hTau40 had no detectable
effect on the perinuclear disposition of GLUT4 in 3T3-L1 adipocytes,
whereas under similar conditions nocodazole treatment dispersed
GLUT4-containing vesicles throughout the cytoplasm (Fig.
3B). These results show that heterologously expressed hTau40
protein associates with microtubules in cultured adipocytes, consistent
with its reported role as a modulator of motility on these cytoskeletal
tracks. Importantly, as expected hTau40 protein does not detectably
disrupt microtubules in 3T3-L1 adipocytes (Fig. 3B).
The effect of hTau protein expression on GLUT4 responsiveness to
insulin in 3T3-L1 adipocytes was then tested using the plasma membrane
sheet assay as shown in Fig. 2. After 12 or 15 min of insulin
stimulation, GLUT4 at the plasma membrane was maximally elevated and
was no different in adipocytes infected with control adenovirus
versus hTau40 adenovirus. However, expression of hTau40 in
the cultured adipocytes caused a significant inhibition of approximately 65% in the GLUT4 translocated to the cell surface 3 min
after the addition of insulin. This delay in insulin-stimulated GLUT4
translocation due to hTau40 expression is similar to the delay in
exocytosis of transferrin receptor-containing vesicles in
Tau-expressing Chinese hamster ovary cells (22). These data are
consistent with the hypothesis that hTau40 interference with kinesin-mediated transport along microtubules retards the rate of GLUT4
exocytosis in response to insulin.
Our data show a good correlation between the reported ability of hTau40
to partially inhibit kinesin activity (22, 23) and its partial
disruption of GLUT4 translocation, as reflected in the retardation of
GLUT4 movements to the cell surface (Fig. 4). Furthermore, the data in Fig. 2 show
a requirement of microtubule integrity for optimal GLUT4 translocation.
Taken together, these data strongly support an obligatory role of both
microtubules and kinesin motor activity in directing GLUT4 movements
toward the plasma membrane. These considerations led to the prediction that the full effect of insulin on the increase in cell surface GLUT4
would actually be permanently inhibited rather than be simply delayed
by depolymerization of microtubules in cultured adipocytes. We tested
this prediction by monitoring plasma membrane GLUT4 during prolonged
insulin treatments of cultured adipocytes. For these experiments, we
used either colchicine or nocodazole to disrupt microtubules in these
cells. As shown in Fig. 5, a low concentration (10 µM) of colchicine disrupts microtubules
in 3T3-L1 adipocytes in 1-2 h, whereas by 2 h of treatment, the
perinuclear GLUT4 also is dispersed fully. In contrast to the
transient effect observed for hTau40 (Fig. 4), colchicine or nocodazole
inhibits the effect of insulin by 50-70% even after cells are exposed
to insulin for 60 min (Fig. 6). Similar
experiments were conducted using latrunculin B to disrupt F-actin in
cultured adipocytes (Fig. 7). The results
of such experiments were consistent with the results obtained by
depolymerization of microtubules, stable inhibition of full GLUT4
translocation even after a 60-min exposure of 3T3-L1 adipocytes to
insulin. These data indicate that the full effect of insulin on
translocation of GLUT4 is dependent upon intact microtubules as well as
intact F-actin.
It is noteworthy that a previous report appeared indicating that
colchicine treatment of adipocytes caused a delay in the full effect of
insulin on hexose transport activity, but that the stimulatory effect
of prolonged treatment with insulin was not affected (31). Our data
indicate that GLUT4 movements are stably inhibited by colchicine (Fig.
6) and that insulin-stimulated deoxyglucose uptake likewise is stably
inhibited by colchicine (data not shown). The previous work did not
monitor GLUT4 itself, and it is not clear that glucose transport
activity simply reflects plasma membrane GLUT4 in those studies.
Further work will be required to evaluate whether the effects of other
glucose transporter isoforms might have contributed to the hexose
uptake measured under the experimental conditions used in the previous studies.
Taken together, the data presented here provide a compelling case for
the involvement of the cytoskeleton in both the perinuclear localization of GLUT4 and its movements in response to insulin. The
cytoplasmic dispersion of perinuclear GLUT4 in response to nocodazole
(Fig. 1) or colchicine (Fig. 5) and its partial inhibition of GLUT4
translocation (Fig. 2) indicate that both plus end-directed and minus
end-directed microtubule-based motors are involved in GLUT4
localization and movements. It is unlikely that nonspecific effects of
nocodazole and colchicine account for these effects, because
perinuclear GLUT4 dispersion is observed when microtubules are
disrupted by an alternative approach (21), and in the present studies,
GLUT4 translocation is also modulated by the disruption of kinesin
function (Fig. 4). A recent report also describes the use of colchicine
to implicate a role of microtubules in GLUT4 movements (32). It is
noted that in addition to microtubule depolymerization, the effects of
nocodazole but not colchicine can also partially inhibit insulin
signaling to Akt. The data we present implicating actin filament
involvement also are consistent with recent findings showing an
association of GLUT4 with actin bundles in cultured muscle cells (33)
and with the reported dependence of GLUT4 translocation in primary
adipocytes on intact F-actin (34). An important question for future
investigation is how insulin signaling directly regulates the
interactions between GLUT4-containing membranes and cytoskeletal
elements that direct their movements.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-tubulin
and the intermediate filament protein vimentin are present in the
preparations of GLUT4-containing membranes and confirmed their
association with vesicles containing GLUT4 by electron microscopy.
Disruption of the intermediate filaments and microtubules in 3T3-L1
adipocytes by microinjection of a vimentin-derived peptide caused the
dispersion of perinuclear GLUT4. These and other findings (21) are
consistent with the hypothesis that the molecular motor dynein directs
the movements of GLUT4-containing membranes to the minus ends of
microtubules in the juxtanuclear region of cultured adipocytes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C. For the adenovirus infection,
3T3-L1 adipocytes were grown and differentiated on coverslips in 6-well
plates and then infected at a multiplicity of infection of 50 with
either a control virus expressing green fluorescent protein only or the recombinant adenovirus encoding hTau40 adenovirus. On the next day,
cells were serum-starved overnight in Dulbecco's modified Eagle's
medium (DMEM) with 0.5% bovine serum albumin, and the experiments were
started after 46 h of infection. This protocol resulted in an
infection efficiency of at least 80% of adipocytes.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effect of nocodazole and latrunculin B
treatment on microtubules, F-actin, and the localization of GLUT4 in
3T3-L1 adipocytes. A, differentiated 3T3-L1 adipocytes
were treated with 10 µM latrunculin B ((+)
LB) or without 10 µM latrunculin B
(( ) LB) for 60 min at 37 °C, and F-actin and
GLUT4 (
GLUT4) were visualized. Latrunculin B
treatment disrupted the F-actin completely. Perinuclear GLUT4 vesicles
were dispersed and enlarged. B, cells were treated with 30 µM nocodazole ((+) Noc) or without
30 µM nocodazole ((
) Noc) for 60 min, and microtubules (
Tubulin) and GLUT4 were
visualized. Nocodazole treatment disrupted the microtubules completely.
Perinuclear GLUT4-containing vesicles were dispersed. C,
cells were treated with latrunculin B and nocodazole as noted above.
GLUT4 vesicles were dispersed and enlarged.
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Fig. 2.
Treatment of 3T3-L1 adipocytes with
nocodazole and latrunculin B abolishes GLUT4 translocation in response
to insulin. A, after the serum starvation, 3T3-L1
adipocytes were treated without or with 30 µM nocodazole
(Noc) or 10 µM latrunculin B (LB)
for 60 min at 37 °C, and then they were treated with or without 100 nM insulin for 15 min. Lawns of adipocyte plasma membranes
were generated as described under "Experimental Procedures." The
lawns were incubated with rabbit anti-GLUT4 antibody followed by
FITC-conjugated secondary antibody. These images are representative
fields from three independent experiments. B, translocated
GLUT4 protein in the lawns as shown in A were quantified by
measuring the fluorescence intensity using Adobe Photoshop software.
These values were obtained by random counting of >300 cell sheets from
three independent experiments and were normalized to total cell sheet
numbers in each experiment. Results are the means ± S.D.
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Fig. 3.
Effects of nocodazole treatment and
overexpression of hTau40 protein on microtubules and GLUT4 localization
in 3T3-L1 adipocytes. A, bar diagram of the
long human Tau isoform (hTau40) used for expression in 3T3-L1
adipocytes in this study. The hTau40 protein can be subdivided into an
N-terminal projection domain and a C-terminal microtubule binding
domain. hTau40 contains four repeats (R1-R4) that are
flanked by a proline-rich region (P1 and P2) that
extends into the projection domain and harbors most of the Ser-Thr-Pro
motifs. B, differentiated 3T3-L1 adipocytes were infected
with hTau40 recombinant adenovirus (c, d, g, h, k, l,
o, and p) or control virus with no insert (a, b,
e, f, i, j, m, and n). The cells were then treated with
30 µM nocodazole ((+) Noc) (e-h
and m-p) for 60 min at 37 °C. The cells were fixed with
methanol (see "Experimental Procedures") and subjected to confocal
fluorescence microscopy. Shown are representative fields from three to
four independent experiments. Note that nocodazole disrupted the
microtubules and dispersed GLUT4 vesicles, but hTau40 overexpression
had little effect on GLUT4 morphology.
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Fig. 4.
Expression of microtubule-binding protein
hTau40 delays GLUT4 translocation in response to insulin in 3T3-L1
adipocytes. A, 3T3-L1 adipocytes were infected with
hTau40 adenovirus or control virus as described under "Experimental
Procedures". After the overnight serum starvation, cells were treated
without or with 100 nM insulin for 3-15 min. Lawns of
plasma membrane were then generated as described under "Experimental
Procedures". The lawns were incubated with rabbit anti-GLUT4 antibody
followed by FITC-conjugated secondary antibody. Shown are
representative fields from five different experiments. B,
translocated GLUT4 protein in the lawns as shown in A were
quantified by measuring the fluorescence intensity using Adobe
Photoshop software. Fluorescence intensity was normalized to total cell
numbers in each experiment. These data represent averages of five
independent experiments (500-1000 cell sheets/condition). A
significant decrease of translocation can be seen at 3 min after
insulin stimulation in Tau-expressing cells (open circles)
and control virus-infected cells (closed circles).
Results are the means ± S.D. Asterisk,
p < 0.01 versus control, n = 5.
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Fig. 5.
Effect of colchicine treatment on the
cytoskeleton and GLUT4 localization in 3T3-L1 adipocytes. After
the serum starvation, differentiated 3T3-L1 adipocytes were treated
with or without 10 µM colchicine for 1 or 2 h at
37 °C, and microtubules ( Tubulin) and GLUT4
were visualized. A 1- or 2-h treatment with colchicine disrupted
microtubules almost completely, but in a 1-h treated cell, perinuclear
GLUT4 vesicles still existed.
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Fig. 6.
Both nocodazole and colchicine inhibit GLUT4
translocation in response to prolonged exposure of cultured adipocytes
to insulin. A, after the serum starvation, 3T3-L1
adipocytes were treated with 30 µM nocodazole
(Noc) for 1 h or with 10 µM colchicine
for 2 h at 37 °C and then were treated with 100 nM
insulin for 0-60 min. Lawns of adipocyte plasma membrane were
generated as described under "Experimental Procedures." The lawns
were incubated with rabbit anti-GLUT4 antibody followed by
FITC-conjugated secondary antibody. These images are representative
fields from three or five independent experiments. B,
translocated GLUT4 protein in the lawns as shown in A were
quantified by measuring the fluorescence intensity using Adobe
Photoshop software. These values were obtained by random counting of
>300 cell sheets from three or five independent experiments and were
normalized to total cell sheet numbers in each experiment. Results are
the means ± S.D.
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Fig. 7.
Latrunculin B inhibits GLUT4 translocation in
response to prolonged exposure of cultured adipocytes to insulin.
A, after the serum starvation, 3T3-L1 adipocytes were
treated with 10 µM latrunculin B (LB) for
1 h at 37 °C and then were treated with 100 nM
insulin for 0-60 min. Lawns of adipocyte plasma membrane were
generated as described under "Experimental Procedures." The lawns
were incubated with rabbit anti-GLUT4 antibody followed by
FITC-conjugated secondary antibody. These images are representative
fields from five independent experiments. B, translocated
GLUT4 protein in the lawns as shown in A were quantified by
measuring the fluorescence intensity using Adobe Photoshop software.
These values were obtained by random counting of >500 cell sheets from
five independent experiments and were normalized to total cell sheet
numbers in each experiment. Results are the means ± S.D.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mandelkow for kindly providing the human Tau40 cDNA construct and Jane Erickson for excellent assistance with preparation of this manuscript.
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FOOTNOTES |
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* This work is supported by National Institutes of Health Grant DK30898 (to M. P. C.).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.
To whom correspondence should be addressed: Program in Molecular
Medicine, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-2254;
Fax: 508-856-1617; E-mail: Michael.Czech@umassmed.edu.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M010785200
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ABBREVIATIONS |
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The abbreviations used are: GLUT4, glucose transporter 4; FITC fluorescein isothiocyanate, DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Dabco, 1,4-diazabicyclo[2.2.2]octane; CLIP, cytoplasmic linker protein.
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