 |
INTRODUCTION |
One of the many physiologic actions of insulin is to promote
glucose uptake in skeletal muscle, heart, and adipose tissue. Several
lines of evidence have shown that the effect of insulin on glucose
uptake in these tissues is a direct result of the recruitment of the
GLUT41 facilitative glucose
transporter from an intracellular vesicle pool to the plasma membrane
(for recent reviews see Refs. 1 and 2). GLUT4 was first implicated as
the major insulin-responsive glucose transporter when it was shown to
be the predominant isoform expressed in tissues exhibiting
insulin-stimulated glucose uptake (3-6). Prior to the cloning and
identification of GLUT4, it was known that insulin stimulated the
redistribution of a cytochalasin D-inhibitable glucose transport
activity from an intracellular vesicle pool to the plasma membrane (7,
8). It is generally accepted that the translocatable glucose transport
activity is attributable to GLUT4 recruited from an intracellular
location to the plasma membrane.
GLUT4 recycles between the plasma membrane and intracellular
compartments in both the basal and insulin-stimulated states (9-11).
Insulin increases the amount of GLUT4 on the plasma membrane by
stimulating exocytosis of the intracellular GLUT4 pool and by
decreasing the endocytosis of the plasma membrane-associated GLUT4
protein (10, 12-17). Increased exocytosis appears to be the most
important event since complete inhibition of the GLUT4 endocytic
pathway in the basal state does not result in a significant accumulation of GLUT4 at the plasma membrane (18). These data support a
model in which a small portion of the total pool of GLUT4 recycles in
the basal state, with the majority of GLUT4 sequestered in an
insulin-responsive retention pool shown to be associated with
tubulovesicular structures (19).
The mechanism by which GLUT4 vesicles move from their interior location
to the plasma membrane in response to insulin is poorly understood. To
date, research in this area has focused largely on docking and fusion
of the vesicles at the plasma membrane. SNARE (for
soluble NSF attachment protein
receptor) proteins have been shown to play an important
role in the fusion of GLUT4 vesicles with the plasma membrane. It has
been clearly demonstrated that syntaxin 4, VAMP2, and
Syndet/SNAP23 are the major SNARE proteins regulating GLUT4
membrane fusion (20-26). SNARE protein assembly may be mediated by
other syntaxin 4-binding proteins such as Munc18c and the newly cloned
protein, Synip (27-31), but it is not clear that assembly of SNARE
proteins is a rate-limiting step in insulin-mediated GLUT4
translocation. Measurement of GLUT4 translocation in the presence of a
temperature-sensitive Munc18c mutant indicates that membrane fusion is
not rate-limiting in GLUT4 translocation (32). These data suggest that
mechanisms regulating the movement of vesicles from the retention pool
to the plasma membrane need to be explored to determine the specific
steps in GLUT4 translocation that are regulated by insulin.
Regulated insulin-dependent trafficking of GLUT4 vesicles
from the retention pool to the plasma membrane may occur by several mechanisms. GLUT4 vesicles may pass through an endosomal intermediate en route to the plasma membrane. This is unlikely, based on data demonstrating that depletion of the endosomal compartment does not
interfere with insulin-mediated GLUT4 translocation (33, 34).
Alternatively, GLUT4 vesicle trafficking may be regulated by
cytoskeletal elements. GLUT4 secretory vesicles may be tethered to
cytoskeletal structures in a manner similar to endoplasmic reticulum and Golgi membrane structures (35). Insulin stimulation could
induce a release of tethered vesicles, allowing movement to the plasma
membrane by simple diffusion, or insulin may trigger the movement of
vesicles along cytoskeletal tracks as has been observed for regulated
exocytosis of secretory vesicles in other cell systems (36).
Studies examining the role of the actin cytoskeleton in GLUT4
translocation in L6 myotubes, 3T3-L1 adipocytes, and rat adipocytes show that disruption of the actin cytoskeleton by either cytochalasin D
or latrunculin A inhibited insulin-mediated GLUT4 translocation (37-39). In L6 myotubes, disassembly of the actin network did not prevent GLUT4 translocation by stimuli other than insulin, suggesting that the actin cytoskeleton is required for transduction of the insulin
signal to GLUT4 vesicles (40). Disassembly of the actin cytoskeleton in
3T3-L1 cells and L6 myotubes did not interfere with proximal insulin
signaling events but did prevent the relocalization of PI 3-kinase to
GLUT4 vesicles (38, 41).
In this work, we provide evidence that the microtubule cytoskeleton is
required for insulin-mediated GLUT4 translocation. We show that
insulin-mediated GLUT4 translocation requires an intact microtubule
network and that under basal conditions, GLUT4 vesicles associate with
polymerized microtubules.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
3T3-L1 fibroblasts were obtained from the
American Type Tissue Culture repository and were cultured at 37 °C
in 5% CO2 and maintained in Dulbecco's modified Eagle's
medium (DMEM) containing 25 mM glucose and 10% calf serum.
Confluent cultures were induced to differentiate by incubation of the
cells with DMEM plus 25 mM glucose, 10% fetal bovine
serum, 175 nM insulin, 1 µM dexamethasone, and 0.5 mM isobutyl-1-methylxanthine. After 4 days, the
medium was changed to DMEM containing 25 mM glucose, 10%
fetal bovine serum, and 175 nM insulin, with the incubation
period continuing an additional 3 days. Under these conditions, greater
than 95% of the cell population morphologically differentiated into adipocytes.
Treatment of 3T3-L1 adipocytes with drugs to modify the microtubule
cytoskeleton were carried out for 3 h in serum-free F-12 Ham's
media. Nocodazole was used at a final concentration of 33 µM, and taxol was used at a final concentration of 12 µM.
Plasma Membrane Sheet Assay--
Preparation of plasma membrane
sheets was carried out as described previously (23). Briefly, cultured
adipocytes grown in a 6-well cluster dish were treated as described in
the figure legends. Following experimental treatment, cells were washed
with ice-cold PBS and attached to the plate with 0.5%
poly-D-lysine. The cells were swollen in hypotonic buffer
and then sonicated to release intracellular contents. Pure plasma
membrane fragments remaining attached to the plastic dish were scraped
into 120 µl of solubilization buffer (1% SDS, 20 mM
Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA). Protein
content of the plasma membrane sheets was measured in a 10-µl aliquot
using a spectrofluorometric assay (42). For this assay, the solubilized
protein sample was diluted to 60 µl in solubilization buffer. The
diluted sample was mixed with 0.25 ml of 0.2 M sodium
borate buffer, pH 9.0, at room temperature for 5 min. A 20-µl aliquot
of fluorescamine solution (0.2 mg/ml in acetonitrile) was added with
vigorous vortexing. Following a 20-min incubation at room temperature,
fluorescence was measured using 395 nm excitation and 460 nm emission
at high sensitivity using a Shimadzu RF5000U spectrofluorophotometer. A
standard curve was generated using bovine serum albumin ranging from
0.1 to 15 µg/ml.
Extraction of Monomeric and Polymeric Tubulin--
Monomeric and
polymeric tubulin were differentially extracted from cells plated on
35-mm dishes according to previous methods (43). Following treatments
as indicated, cell monolayers were washed 2 times with PBS and then
incubated with 0.3 ml of extraction buffer (2 M glycerol,
0.1 M Pipes, pH 7.1, 1 mM MgSO4, 1 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mM benzamidine, 5 µg/ml pepstatin A, 0.5 mM
PMSF, 0.1% Triton X-100) for 30 min at 37 °C. The Triton
X-100-soluble extract contains monomeric tubulin. Polymeric tubulin was
then extracted from the remaining Triton X-100-insoluble material by
extracting in SDS lysis buffer (25 mM Tris, pH 7.4, 0.4 M NaCl, 0.5% SDS) for 5 min at 37 °C. Aliquots from
monomeric and polymeric fractions were fractionated by 10% SDS-PAGE
and Western blotted for
-tubulin.
Whole Cell Detergent Lysates--
100-mm plates of treated
3T3-L1 adipocytes were washed twice with ice-cold TBS followed by
freezing in liquid nitrogen. The plates were thawed on ice and scraped
into 1 ml of 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Hepes, pH 7.4, 2 mM EDTA) containing
phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate,
and 1 mM molybdate) and protease inhibitors (10 µM leupeptin, 10 µg/ml aprotinin, 1.5 mM
pepstatin A, and 1 mM PMSF). The cells were lysed on ice for 20 min, and insoluble material was removed by microcentrifugation for 10 min at 4 °C. Protein concentrations of the detergent lysates were determined by a Bradford protein assay (Pierce) using
manufacturer's specifications.
Electrophoresis and Immunoblotting--
Samples were
fractionated using SDS-PAGE and transferred to PVDF membranes
(Millipore) in transfer buffer (25 mM Tris, 193 mM glycine, pH 8.5) for 3-4 A-h at 4 °C. Membranes
blotted for insulin receptor
-subunit, IRS-1, GLUT4, IRAP, tubulin,
and dynein were blocked with 7-10% dried milk and 0.3% Tween 20 in
TBS. Anti-phosphotyrosine blots were blocked with 2% bovine serum
albumin and 0.1% Tween 20 in TBS. Anti-phosphotyrosine blots were
carried out using PY99 monoclonal antibody (Santa Cruz Biotechnology).
GLUT4 antiserum was provided by Dr. Gwyn Gould, and IRAP antiserum was
provided by Dr. Kostantin Kandror. GLUT1 antibody was provided by Dr.
Samuel W. Cushman. Tubulin antibody was obtained from Sigma (TUB 2.1). Phospho-PKB/Akt-1 antibody specific for Ser-473 was obtained
from New England Biolabs, and antibody against total PKB/Akt-1
was purchased from Transduction Laboratories. Immunoblots were
visualized using an enhanced chemiluminescence system (Pierce) and
quantified using scanning laser densitometry.
2-Deoxyglucose Uptake--
3T3-L1 adipocytes were placed in
Ham's F-12 media containing 0.5% bovine serum albumin for 2 h at
37 °C. Nocodazole was added to a final concentration of 33 µM at designated times. Cell were then washed with KRPH
buffer (5 mM Na2HPO4, 20 mM Hepes, pH 7.4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, and 1% bovine serum albumin) and treated with or
without additional nocodazole to prevent repolymerization of
microtubules and with or without 100 nM insulin added for
20 min. Glucose transport was determined at 37 °C by incubation with
50 µM 2-deoxyglucose uptake containing 0.5 µCi of
2-[3H]deoxyglucose in the absence or presence of 10 µM cytochalasin B. The reaction was stopped after 4 min
by washing the cells 3 times with ice-cold PBS. The cells were
solubilized in 1% Triton X-100 at 37 °C for 30 min, and aliquots
were subjected to scintillation counting or protein assay to calculate
uptake as pmol/min/mg protein.
PI 3-Kinase Assay--
Phosphatidylinositol 3-OH kinase activity
was measured in IRS-1 immune complexes obtained from whole cell
detergent lysates as described above. The detergent-soluble cell
extract was incubated overnight with anti-IRS-1 pre-loaded on protein
A-conjugated Sepharose. Immune complexes were washed and subjected to
the in vitro PI 3-kinase assay as described previously using
bovine phosphatidylinositol (Sigma) and [
-32P]ATP as
substrates (44). Phosphorylated lipids were separated by thin layer
chromatography, visualized by autoradiography, and quantified by
collecting and counting radiolabeled phosphatidylinositol 3-phosphate.
Adipsin Secretion--
Adipsin secretion into cell culture media
was measured as described previously (45). Thirty five-mm dishes of
3T3-L1 adipocytes were treated without or with 33 mM
nocodazole for 3 h in serum-free Ham's F-12 media. The cells were
washed 2 times with fresh media, followed by replacement with 1 ml of
Ham's F-12 media without or with 33 µM nocodazole and/or
100 nM insulin for 30 min. The media were removed, and
proteins were precipitated in the presence of 10% trichloroacetic acid
and deoxycholate as carrier (46). Pelleted proteins were captured by
microcentrifugation and resuspended in 40 µl of 0.25 M
Tris base. Protein concentration was determined using a Bradford assay.
Equivalent protein aliquots were fractionated on 10% SDS-PAGE and
transferred to PVDF. Membranes were immunoblotted using an adipsin
antibody (provided by Dr. Jess Miner, University of Nebraska).
Microtubule Binding Assay--
Microtubules and associated
proteins were polymerized in vitro as described previously
(47). Briefly, 3T3-L1 adipocytes (day 8-10
post-differentiation) were serum-starved in Ham's F-12 media for
3 h and then treated without or with 100 nM insulin for specified times. Cells (from two 100-mm plates per treatment) were
scraped into 500 µl of microtubule stabilizing buffer (5 mM MgSO4, 5 mM EGTA, 35 mM PIPES, pH 7.1, and 142 mM sucrose) containing 1 mM dithiothreitol, protease inhibitors (10 µg/ml leupeptin, 10 mg/ml aprotinin, 0.5 mM benzamidine,
5 µg/ml pepstatin A, 0.5 mM PMSF), and phosphatase
inhibitors (100 mM NaF, 1 mM sodium vanadate,
and 1 mM molybdate). Cells were homogenized at 4 °C by
passage through a 26-guage needle. Homogenates were centrifuged at
100 × g for 10 min to obtain the post-nuclear
supernatant. The post-nuclear supernatant was subjected to a
40,000 × g centrifugation using an SW60 rotor
(Beckman) for 1 h. The supernatant was recovered, and protein
concentration was adjusted to 4 mg/ml with homogenization buffer. Taxol
(1 mM stock solution in Me2SO) was added
to a final concentration of 20 µM, and samples were
incubated at 4 °C for 1 h. Polymerized microtubules and
associated proteins were pelleted by centrifugation at 16,000 × g for 30 min using an SW60 rotor (Beckman). In some
experiments 1% Triton X-100 was added to the 40,000 × g supernatant prior to addition of taxol to solubilize membranes. The 16,000 × g taxol-stabilized pellets
were resuspended in homogenization buffer and protein concentration
measured by Bradford assay. The 16,000 × g
taxol-stabilized pellets were analyzed by SDS-PAGE and transferred to
PVDF membrane and Western blotting.
 |
RESULTS |
Nocodazole Treatment Inhibits Insulin-mediated GLUT4
Translocation--
To determine whether an intact microtubule
cytoskeleton is necessary for insulin-mediated GLUT4 translocation, we
measured GLUT4 translocation in cells treated with 33 µM
nocodazole. This concentration depolymerizes greater than 95% of
microtubules in other cell types (43). To confirm that depolymerization
of the microtubule network occurred in 3T3-L1 adipocytes under this
treatment, we compared monomeric and polymeric tubulin levels in cells
treated without or with 33 µM nocodazole for 3 h by
Western blot analysis. Monomeric tubulin was extracted in 0.1% Triton
X-100 extraction buffer plus protease inhibitors at 37 °C for 15 min. After the Triton X-100-soluble fraction was removed, polymeric
tubulin was extracted with buffer containing 0.4 M NaCl and
0.5% SDS. An equivalent portion of each extract was fractionated by
SDS-PAGE, and samples were probed with an anti-
-tubulin antibody
(Fig. 1A). Nocodazole treatment shifted greater than 95% polymeric tubulin to the monomeric form (Fig. 1A, compare lanes 2 and 4).
In control cells, approximately two-thirds of the total tubulin was
polymeric, and the remaining third was monomeric (Fig. 1A,
compare lanes 1 and 2).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Nocodazole depolymerizes tubulin in vivo
(A). Monomeric (M) tubulin was extracted with
0.1% Triton X-100 from control (Con) and nocodazole
(Noc)-treated cells. Polymeric (P) tubulin was
extracted with 0.4 M NaCl and 0.5% SDS from the
Triton-insoluble material. Nocodazole treatment inhibits
insulin-mediated GLUT4 translocation. GLUT4 accumulation in purified
plasma membrane sheets was measured following 20 min of treatment with
100 nM insulin. B shows 1 independent experiment
done in duplicate. C shows the quantitation of 4 independent
experiments (mean and S.D.). Data were analyzed by two-tailed
t test. Asterisk indicates p < 0.005 comparing control and nocodazole treated cells. IB,
immunoblot.
|
|
After establishing the efficacy of nocodazole treatment for
depolymerization of the microtubule network in 3T3-L1 adipocytes, we
examined insulin-mediated GLUT4 translocation under identical treatment
conditions. Insulin-dependent GLUT4 translocation to the
plasma membrane was assessed by measuring GLUT4 levels in purified
plasma membrane fractions prepared by sonication of monolayers of
3T3-L1 adipocytes (23, 48). Quantification of protein levels in this
fraction by Western blot analysis provides an estimate of the
accumulation of GLUT4 in the plasma membrane of an entire plate of
cells. Cells were treated for 3 h with 33 µM
nocodazole followed by treatment without or with 100 nM
insulin for 30 min. Isolated plasma membrane fragments were solubilized
and analyzed by Western blot. Basal levels of GLUT4 in plasma membrane
sheets were not affected by nocodazole treatment (Fig. 1B, lanes
3 and 4). In contrast, nocodazole inhibited recruitment
of GLUT4 to the plasma membrane following insulin treatment
(p < 0.005) (Fig. 1B, lanes 7 and
8). Quantification of four independent experiments by laser
densitometry is shown in Fig. 1C.
In the previous experiment, insulin-mediated GLUT4 translocation was
measured after 3 h of nocodazole treatment; however, a time course
experiment revealed that complete depolymerization of microtubules in
3T3-L1 cells occurred between 15 and 30 min of nocodazole treatment
(data not shown). To confirm that the effects of nocodazole on
insulin-mediated GLUT4 translocation occurred at early time points
paralleling microtubule depolymerization, we measured GLUT4
translocation after 20 min of nocodazole treatment (Fig.
2A). Cells were treated with
nocodazole for 20 or 120 min prior to insulin treatment and plasma
membrane sheets prepared as described above. Results of two independent
time course experiments are shown in Fig. 2A. Densitometric
analysis of the Western blot reveals a 4-fold increase in GLUT4 protein
following insulin treatment in the absence of nocodazole (Fig.
2A, lanes 1-4). In contrast to control conditions, the
insulin-mediated GLUT4 translocation was increased 1.4- and 1.6-fold,
respectively, in cells treated for 20 and 120 min with nocodazole (Fig.
2A, lanes 5-12). Thus, the effects of nocodazole
on insulin-mediated GLUT4 translocation do not likely results from
progressive changes in the cell that develop overtime in the presence
of nocodazole.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2.
Insulin-mediated GLUT4 translocation is
inhibited to the same extent after short and long exposure to
nocodazole. GLUT4 accumulation in purified membrane sheets was
measured following 20 min of treatment with 100 nM insulin
in cells pretreated for 0, 20, or 120 min in the presence of 33 µM nocodazole. A shows results from two
independent experiments. Insulin-mediated 2-deoxyglucose uptake was
measured following 20 min of treatment with 100 nM insulin
in cells pretreated for 0 (control), 20, or 120 min in the presence of
33 µM nocodazole. B shows the mean and
standard error from 4 independent experiments performed in duplicate.
Data were analyzed by analysis of variance to compare insulin-mediated
2-deoxyglucose uptake between treatment group. Asterisk
indicates p < 0.01 comparing control and
nocodazole-treated cells.
|
|
In a complementary experiment, we measured insulin-mediated
2-deoxyglucose uptake in 3T3-L1 adipocytes treated for 0, 20, or 120 min with 33 µM nocodazole. Insulin treatment caused a
5-fold increase in 2-deoxyglucose uptake under control conditions
(Fig. 2B). Nocodazole treatment for both 20 and 120 min
significantly inhibited insulin-mediated 2-deoxyglucose uptake, similar
to the inhibition of GLUT4 translocation (Fig. 2).
Nocodazole Treatment Does Not Affect Proximal Insulin Receptor
Signaling--
Nocodazole treatment of cells significantly reduces
microtubule-based vesicle transport; however, it has also been shown to elicit effects on the cell including inhibition of protein synthesis, disruption of plasma membrane subdomains, and changes in some types of
cellular signaling (49). Any of these perturbations could lead to
changes in early insulin signaling events. To determine whether
nocodazole interferes with GLUT4 translocation by decreasing steady
state levels of relevant proteins, we measured the levels of insulin
receptor, IRS-1, and GLUT4 protein in nocodazole-treated cells. 3T3-L1
adipocytes were incubated with 33 µM nocodazole for
3 h and then treated without or with 100 nM insulin
for 20 min at 37 °C. Nocodazole did not alter steady state protein
levels of IRS-1, insulin receptor
-subunit, or GLUT4 protein (data
not shown).
The first event in insulin signaling is the activation of the intrinsic
tyrosine kinase activity of the insulin receptor upon ligand binding.
This leads to both mitogenic and metabolic effects of the cascade.
Another early event in insulin signaling is the tyrosine
phosphorylation of intracellular insulin receptor substrates of the IRS
family (50). Phosphotyrosine residues on the IRS isoforms act as
binding sites for specific SH2 domain molecules, including
phosphatidylinositol 3-OH kinase (PI 3-kinase) (50, 51). Activation of
PI 3-kinase is required for most, if not all, intracellular actions of
insulin, including GLUT4 translocation (52). In both rat adipocytes and
3T3-L1 adipocytes, PI 3-kinase binds predominantly to IRS-1 (53, 54).
The PI 3-kinase activity immunoprecipitated with either anti-IRS-1 or
anti-phosphotyrosine antibodies is increased in both intracellular
membranes and cytosol following insulin stimulation (53-56).
Tyrosine-phosphorylated proteins were solubilized in 1% Nonidet P-40
and cell lysates and then analyzed by immunoblotting with
anti-phosphotyrosine antibody. Insulin treatment increased tyrosine
phosphorylation of proteins corresponding in size to the
-subunit of
the insulin receptor and IRS-1. Insulin-mediated tyrosine
phosphorylation of these proteins was not affected by nocodazole
treatment (Fig. 3A).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Nocodazole treatment does not affect
phosphorylation of the IRS-1 or insulin receptor
-subunit. Cells were stimulated with
100 nM insulin for 30 min and extracted with 1% Nonidet
P-40 lysis buffer. Lysates were fractionated on SDS-PAGE, transferred
to PVDF membranes, and probed using an anti-phosphotyrosine antibody
(A). Nocodazole does not inhibit PI 3-kinase activity
associated with IRS-1. PI 3-kinase activity was assayed in IRS-1
immunoprecipitated complexes following 30 min of insulin treatment in
control (Con) and nocodazole (Noc)-treated cells.
PI 3-kinase products were separated by thin layer chromatography. The
band corresponding to PI 3-phosphate was cut and quantitated by
scintillation counting. An autoradiogram experiment is shown, and the
histogram shows quantitation of 4 experiments (mean and S.D.)
(B). Phosphorylation of PKB was measured using a
phosphoserine-specific antibody against phosphorylated Ser-473. Total
PKB protein was blotted in the same lysate. Cells lysates were prepared
as described above. Results of 3 independent cell lysates are shown.
IB, immunoblot (C).
|
|
To determine whether immediate downstream signaling events other than
tyrosine phosphorylation were modified by nocodazole treatment, we
assayed the IRS-1-associated PI 3-kinase activity. Control and
nocodazole-treated cells were treated without or with 100 nM insulin for 30 min. IRS-1 complexes were
immunoprecipitated from 1% Nonidet P-40 cell extracts and assayed for
PI 3-kinase activity under standard conditions (48). IRS-1-associated
PI 3-kinase activity increased 16-fold under insulin treatment (Fig. 3B). Nocodazole treatment did not significantly alter PI
3-kinase activity associated with IRS-1 (p = 0.7) in
either basal or insulin-treated cells. These data indicate that an
intact microtubule system is not required for
insulin-dependent association of PI 3-kinase with
phosphorylated IRS-1.
Insulin treatment activates protein kinase B (PKB) activity through a
PI 3-kinase-dependent mechanism (57). The pleckstrin homology domain on the N terminus of PKB interacts with the
phospholipid product of PI 3-kinase (PI-3,4,5-P3). This
interaction induces a conformational change in PKB that permits
phosphorylation of PKB at two regulatory sites (Thr-308 and Ser-473),
activating the kinase (58). To determine whether nocodazole treatment
interfered with activation of PKB, we measured phosphorylation of PKB
at serine 473 in cell lysates using a phospho-PKB-specific antibody recognizing the phosphoserine. The ratio of phospho-PKB to total PKB
was calculated from the densitometric analysis of the Western blots.
The ratio in control cells for three independent experiments was
112 ± 1.2 (mean ± S.D.) compared with a ratio 92 ± 6.9 in nocodazole-treated cells (Fig. 3C). This 18%
reduction in the ratio of phospho-PKB to total PKB was statistically
significant (p < 0.02); however, the physiologic
significance of this decrease is not certain. Nocodazole treatment did
not prevent insulin-mediated phosphorylation of PKB at Ser-473.
Nocodazole Does Not Effect Insulin-mediated Adipsin Secretion or
GLUT1 Translocation--
To determine whether depolymerization of the
microtubule network interferes specifically with insulin-mediated GLUT4
translocation, we examined other insulin-mediated exocytosis pathways
that are independent of the GLUT4 pathway. First, we examined the
effects of nocodazole treatment on insulin-mediated secretion of
adipsin. Recently, two groups have shown that insulin stimulates a
2-3-fold increase in adipsin secretion through an
ARF6-dependent pathway that passes through an endosomal
intermediate on its way to the plasma membrane (34, 45). We measured
adipsin secretion into culture media over a 30-min incubation period in
control and nocodazole-treated cells without or with 100 nM
insulin (Fig. 4). Quantification of three
independent experiments is shown in Fig. 4B. Insulin treatment caused a 3-fold increase in adipsin secretion in both control
and nocodazole-treated cells, indicating that an intact microtubule
network is not required for this process.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 4.
Insulin-mediated adipsin secretion into media
for 30 min without or with 100 nM insulin treatment in
cells treated without (control) or with 33 µM nocodazole. Cell media
were harvested and precipitated in the presence of 10% trichloroacetic
acid. Equivalent samples (based on total protein) were fractionated
using SDS-PAGE and blotted for adipsin. Autoradiograph of three
independent experiments is shown in A. B shows
quantitation of 3 independent experiments performed in triplicate.
IB, immunoblot.
|
|
GLUT1 is also recruited to the plasma membrane in response to insulin
(59, 60). Two lines of evidence suggest that the GLUT1 pathway is
independent of the GLUT4 pathway. First, intracellular vesicles
containing GLUT1 are largely distinct from vesicles containing GLUT4
(59, 60). Second, accumulation of GLUT1 in the plasma membrane is not
dependent on the syntaxin 4 SNARE protein, which is required for GLUT4
translocation (23). To determine whether insulin-dependent
GLUT1 translocation was inhibited by nocodazole treatment, the plasma
membrane sheets probed for GLUT4 (see Fig. 1B) were next
probed for GLUT1 (Fig. 5A). In
contrast to its effect on GLUT4, nocodazole treatment had no effect on
insulin-mediated GLUT1 translocation.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
Nocodazole treatment does not inhibit
insulin-mediated GLUT1 translocation. GLUT1 accumulation in
purified plasma membrane sheets was measured following 20 min of
insulin treatment. A shows the results of 1 independent
experiment done in duplicate. B shows the quantitation of 3 independent experiments (mean ± S.D.).
|
|
Taxol Treatment Inhibits Insulin-mediated GLUT4
Translocation--
It is possible that the effect of nocodazole on
GLUT4 translocation was independent of its effects on the microtubule
cytoskeleton, but rather due to some unrelated cytotoxic effect. To
test this, we treated the cells with 12 µM taxol to
stabilize the microtubule network. In vivo polymerization of
microtubules by taxol has been shown to inhibit vesicular exocytosis in
other systems (36, 49). We reasoned that if a dynamic microtubule
network is required for GLUT4 exocytosis, taxol treatment should also
inhibit this process. To show that taxol treatment polymerized the
microtubule network in 3T3-L1 cells, we compared monomeric and
polymeric tubulin in cells treated without or with 12 µM
taxol (Fig. 6A). Taxol treatment resulted in a complete conversion of Triton X-100 extractable tubulin (monomeric) to polymeric tubulin (Fig. 6A, compare
lanes 1 and 2). Plasma membrane sheets were
prepared from control and taxol-treated cells that were stimulated
without or with 100 nM insulin for 20 min. Accumulation of
GLUT4 in response to insulin was inhibited by 40% in plasma membrane
sheets prepared from taxol-treated cells (p < 0.01)
(Fig. 6B, compare lanes 5 and 6 with
lanes 7 and 8). Quantitation of three independent
experiments by laser densitometry is shown in Fig. 6C.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Taxol polymerizes tubulin in
vivo. A shows monomeric and polymeric
tubulin extracted from cells treated without or with 12 µM taxol for 3 h in serum-free media. Monomeric
tubulin was extracted with 0.1% Triton X-100 from control and
taxol-treated cells. Polymeric tubulin was extracted with 0.4 M NaCl and 0.5% SDS from the Triton-insoluble material.
B shows that taxol treatment inhibits
insulin-mediated GLUT4 accumulation in purified plasma membrane sheets
that was measured following 20 min of insulin treatment
(p < 0.003). C shows results of 1 of 3 independent experiments performed in duplicate. IB,
immunoblot.
|
|
GLUT4 Vesicles Associate with Microtubules in Vitro--
Although
our examination of insulin-signaling pathways was not exhaustive, it
was sufficient to conclude that nocodazole treatment did not affect
proximal insulin signaling events at the cell surface or decrease
steady state levels of proteins known to be involved in proximal
insulin signaling. A plausible explanation consistent with our
observations is that destabilization of the microtubule network
interferes with vesicle translocation and/or with later events in
insulin signaling that lead to translocation. To begin to examine these
possibilities, we performed an in vitro microtubule binding
assay (47) to determine whether GLUT4 vesicles associate directly with microtubules.
Tubulin was polymerized in vitro by addition of taxol to a
post-40,000 × g supernatant from 3T3-L1 cells
homogenized in microtubule stabilization buffer without detergent. The
40,000 × g supernatant was obtained by centrifuging
the post-nuclear supernatant at 40,000 × g, and this
fraction consisted of low density vesicles including GLUT4 vesicles and
cytosolic proteins including tubulin dimers (47, 56). Addition of 20 µM taxol at 4 °C polymerized ~60% of tubulin (data
not shown). Polymerized tubulin was pelleted from the taxol-treated
40,000 × g supernatant by slower speed centrifugation
at 16,000 × g. Tubulin appeared in the 16,000 × g pellets only in the presence of taxol (Fig.
7, lane 1). The 16,000 × g supernatant (obtained from the 16,000 × g
spin of the 40,000 × g supernatant) (Fig. 7,
lane 5) depicts a proportional loading of the supernatant
from samples not treated with taxol to indicate the level of each
protein in the 40,000 × g supernatant prior to taxol
treatment. The 16,000 × g pellet was probed for associated proteins by Western blot analysis. IRS-1, GLUT4, and the
insulin-responsive aminopeptidase (IRAP), a protein that exclusively colocalizes with GLUT4 vesicles (61), were associated with the 16,000 × g pellets, and this association was
dependent on taxol (Fig. 7, lane 2). The
-subunit of the
insulin receptor was not detected in this pellet even though insulin
receptor was present in the 40,000 × g supernatant
(Fig. 7, compare lanes 2 and 5). Insulin receptor
was not present the 16,000 × g pellets,
demonstrating that vesicles containing the insulin receptor do not bind
microtubules. Together, these results indicate that our assay for
vesicle binding to polymerized microtubules is specific.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 7.
Polymerized microtubules bind GLUT4 vesicles
and IRS-1 in vitro. A 40,000 × g supernatant was derived from the post-nuclear supernatant
of a cell homogenate from cells treated with 100 nM insulin
for 0, 5, or 20 min. This cellular fraction, which contained low
density microsomal membranes and cytosolic proteins, was treated
without or with 20 µM taxol to polymerize tubulin. The
polymerized tubulin and associated proteins were pelleted by slow speed
centrifugation (16,000 × g). The pellet (16,000 × g pellets (16 K P)) and a sample from
the supernatant of the 16,000 × g pellet that was not
treated with taxol (16,000 × g supernatant (16 K
SN)) were analyzed by Western blot. The 16,000 × g pellets were probed for GLUT4 and IRAP, insulin
receptor -subunit, IRS-1, and -tubulin. IB,
immunoblot.
|
|
Insulin treatment did not affect the polymerization of tubulin;
however, IRS-1, GLUT4, and IRAP bindings to polymerized tubulin were
decreased in response (Fig. 7, lanes 3 and 4).
The decrease in GLUT4 and IRAP binding to polymerized microtubules in
response to insulin is consistent with data indicating a flux from
intracellular membrane compartments to the plasma membrane. In contrast
to GLUT4, the abundance of IRS-1 in the 40,000 × g
supernatant was the same in basal and insulin-treated cells (data not
shown). Thus, the decreased association of IRS-1 with polymerized
tubulin from the insulin-treated 40,000 × g
supernatant indicates that insulin treatment decreases the affinity of
IRS-1 for microtubules.
To determine whether microtubule-associated IRS-1 was phosphorylated in
response to insulin, the 16,000 × g pellets and
16,000 × g supernatant from cells were probed with
anti-phosphotyrosine antibody. IRS-1 appearing in the
16,000 × g pellets was tyrosine-phosphorylated (Fig.
8, lanes 4 and 5)
following insulin treatment. Phosphorylated IRS-1 levels were similar
in the 16,000 × g pellets and the 16,000 × g supernatant (Fig. 8, compare lanes 4 and
5 with lanes 7 and 8), indicating that
phosphorylation state of IRS-1 does not affect its binding to
polymerized tubulin.

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 8.
Phosphorylated IRS-1 binds microtubules
in vitro. The 16,000 × g
pellets (16 K P) and 16,000 × g
supernatant (16 K SN) from cells treated as described in
Fig. 9 were blotted with anti-phosphotyrosine antibodies.
|
|
Characterization of GLUT4 Vesicle Binding to Microtubules in
Vitro--
In vitro microtubule binding was performed in a
detergent-free cell homogenate; it was therefore possible that the
association of GLUT4 vesicles with microtubules was mediated either
directly through GLUT4, through IRAP, or through an undetermined
vesicle-associated protein. To determine whether GLUT4 or IRAP bind to
microtubules through direct protein interactions, we performed the
microtubule binding assay in the 40,000 × g
supernatant after adding 1% Triton X-100 to solubilized membranes.
Addition of detergent to the 40,000 × g supernatant
had no effect on taxol-induced polymerization of tubulin (Fig.
9, lanes 2 and 4).
Detergent also had no effect on IRS-1 binding to polymerized tubulin,
indicating that IRS-1 binds microtubules through a protein-protein
interaction. Contrasting with IRS-1, detergent treatment of the
40,000 × g supernatant abolished binding of GLUT4 and
IRAP to polymerized tubulin (Fig. 9, lanes 2 and
4) indicating that the association of both GLUT4 and IRAP
with microtubules depends on the continuity of the GLUT4/IRAP vesicle
membrane. This experiment demonstrates that GLUT4 vesicles bind to
microtubules via a vesicle-associated protein, and this intermediate
protein is not GLUT4 or IRAP. Specificity of protein binding to
microtubules is also confirmed by this experiment since IRSl-1, but not
GLUT4 or IRAP, binds microtubules in the presence of detergent.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 9.
Detergent solubilization of the 40,000 × g supernatant prevents GLUT4 and IRAP binding to
polymerized microtubules in vitro. The
microtubule binding assay was performed as described in Fig. 6 except
that the 40,000 × g supernatant was treated with or
without 0.1% Triton X-100 to solubilize membranes prior to addition of
taxol. The 16,000 × g pellets (16 K
P) were blotted for GLUT4, IRAP, IRS-1, and -tubulin.
IB, immunoblot.
|
|
 |
DISCUSSION |
Insulin stimulates the translocation of an intracellular storage
pool of vesicles that contain the GLUT4 facilitative glucose transporter to the cell surface. Subcellular fractionation studies have
biochemically defined the insulin-responsive GLUT4 storage pool (62,
63). Immunocytochemical analysis of the GLUT4 storage vesicles in brown
adipose tissue indicates that GLUT4 is localized to tubulo-vesicular
structures near the trans-Golgi network, as well as discrete vesicles
distributed throughout the cytoplasm (19). The observation that GLUT4
storage vesicles are located at a distance from the plasma membrane,
rather than docked and awaiting fusion, implies that an insulin-induced
signal triggers the movement of storage vesicles to the plasma membrane
after which fusion occurs. Movement of intracellular vesicles to the plasma membrane, as opposed to regulation membrane fusion, has recently
been shown to be a rate-limiting step in insulin-stimulated GLUT4
translocation (32). It is of considerable interest to understand
movement of GLUT4 vesicles in response to insulin at the molecular
level. Our studies indicate that the microtubule cytoskeleton plays a
fundamental role in this process.
We demonstrated that destabilization of the microtubule cytoskeleton
completely inhibits GLUT4 translocation to the plasma membrane. This
observation strongly implies that recruitment of GLUT4 vesicles to the
plasma membrane surface requires an intact microtubule network. A
reasonable assumption, based on known functions of microtubules in
other secretory pathways, is that microtubules act as a filament upon
which the GLUT4 vesicle moves to its cell surface location (36).
Movement of vesicles between cellular compartments can occur solely by
membrane fusion and fission events, but an acute response to insulin
utilizing regulated fusion would require that vesicles be predocked at
the target membrane. This does not appear to be the case with GLUT4
vesicles, since insulin stimulates the recruitment of GLUT4 vesicles
from varied locations within the cell.
Disruption of the microtubule network is a profound alteration of a
major cellular structure that may have global effects on cellular
processes and vesicular trafficking. To control for this possibility,
we studied other insulin-mediated vesicular trafficking pathways
independent of GLUT4 exocytosis. Neither the insulin-mediated secretion
of adipsin nor translocation of GLUT1 were found to be affected by
disruption of the microtubule network. Thus, the microtubule network is
required for a subset of membrane trafficking events in these cells,
and this subset includes insulin-mediated GLUT4 exocytosis.
We demonstrated that GLUT4 vesicles specifically bind polymerized
tubulin in vitro, implying that this interaction occurs in vivo. In the in vitro microtubule binding
assay, we demonstrated that GLUT4 vesicle binding to microtubules was
specific, since insulin receptor-containing vesicles did not associate
with polymerized tubulin under conditions in which GLUT4 vesicles
associate with polymerized tubulin (Fig. 9). Thus the microtubule
network may act as a scaffold upon which GLUT4 storage vesicles are
tethered awaiting insulin stimulation and/or a track upon which they
travel after a signal has been transduced to the vesicles.
Our data suggest that the microtubule network may provide a link
between the insulin-signaling pathway and GLUT4 vesicle translocation. Although we demonstrated that proximal signaling events through activation of PKB were not affected by destabilization of microtubules in vivo (with nocodazole treatment), it is possible that the
pathway(s) of the insulin-signaling cascade leading specifically to
GLUT4 vesicle recruitment is localized to microtubules. In support of this hypothesis, we observed that IRS-1 specifically bound to polymerized tubulin. Interestingly, recent work indicates that IRS-1 is
bound to cytoskeletal elements, but the specific cytoskeletal components have not yet been identified (56). Our data suggest that
microtubules may be an in vivo binding site for IRS-1.
Other studies have investigated the role of the actin cytoskeleton in
insulin-mediated GLUT4 translocation. Disruption of the actin
cytoskeleton using either cytochalasin D or latrunculin A
partially inhibited GLUT4 translocation in 3T3-L1 cells (38) and
completely disrupted this process in rat adipocytes (39). Although a
direct association between GLUT4 vesicles has not yet been
demonstrated, experimental evidence suggests that an intact actin
cytoskeleton is required for recruitment of PI 3-kinase activity to the
GLUT4 vesicle (38). It has not yet been established that recruitment of
PI 3-kinase to GLUT4 vesicles is required for insulin-mediated GLUT4
translocation. In a recent study, it was shown that targeting of
constitutively active PI 3-kinase to GLUT4 vesicles is not sufficient
on its own to cause redistribution of GLUT4 vesicles to the plasma
membrane (64); however, that does not prove that it is not necessary
for translocation. It is of considerable interest to determine whether
the microtubule cytoskeleton in addition to the actin cytoskeleton is
involved in the insulin-mediated recruitment of PI 3-kinase to GLUT4 vesicles.
There are numerous potential roles for the microtubule network in
insulin-mediated GLUT4 translocation. One possibility is that the
microtubule network may provide a supporting structure that tethers
intracellular GLUT4 vesicles in the basal state. Insulin signaling
would release the vesicles allowing them to move to the plasma membrane
by simple diffusion. If this were correct, complete depolymerization of
the microtubule network would be expected to result in accumulation of
GLUT4 at the plasma membrane in the basal state. We did not observe a
change in plasma membrane GLUT4 levels in basal cells treated with
nocodazole; however, we cannot preclude the possibility that GLUT4
vesicles were docked at the plasma membrane, but not fused, in the
nocodazole-treated cells. Alternatively, the microtubule network may
provide a supporting structure upon which GLUT4 vesicles travel to the
plasma membrane. It is possible that GLUT4 vesicles are tethered to
microtubules in vivo in the basal state and that insulin
promotes association of the GLUT4 vesicle with a microtubule motor
protein. Support of this model requires the identification of a
microtubule motor protein that is regulated by insulin and associates
with GLUT4 vesicles. There are numerous examples of vesicular
trafficking along microtubule filaments. For instance, the regulation
of bile secretion is a regulated exocytotic process in liver cells that has been shown to require an intact microtubule system (65). This
system resembles GLUT4 translocation in that the bile-containing vesicles are recruited to the plasma membrane upon stimulation of cells
with an appropriate ligand.
In the present study, we provide functional and biochemical data
demonstrating that insulin-mediated GLUT4 translocation is dependent on
the microtubule cytoskeleton in 3T3-L1 adipocytes. The role of the
microtubule network in this process is under investigation. We
demonstrated the GLUT4 vesicles bind polymerized tubulin in vitro, and this suggests that the microtubule network may act to
scaffold and compartmentalize GLUT4 vesicles. Our data also suggest
that the microtubule network may provide a structure for the
insulin-signaling cascade to intersect directly with GLUT4 vesicles and
stimulate their exocytosis. Further studies focusing on the role(s) of
the microtubule cytoskeleton in insulin-mediated GLUT4 translocation
are currently in progress.