From the Programme in Cell Biology, The Hospital for
Sick Children, Toronto, Ontario M5G 1X8 and the ¶ Department of
Biochemistry, University of Toronto, Toronto, Ontario M5G 1A8,
Canada
Received for publication, November 7, 2000, and in revised form, March 16, 2001
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ABSTRACT |
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The intracellular traffic of the glucose
transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here
we make use of L6 myoblasts stably expressing GLUT4 with an exofacially
directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic
rates of the transporter. Insulin caused a rapid
(t1/2 = 4 min) gain, whereas hyperosmolarity (0.45 M sucrose) caused a slow (t1/2 = 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation
followed by addition of hypertonic sucrose, the increase in surface
GLUT4myc was partly additive. Unlike the effect of insulin, the
GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin
or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t1/2 = 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in
response to hyperosmolarity was only partially blocked by inhibition of
tyrosine kinases with erbstatin analog (erbstatin A) and
genistein. However, neither inhibitor interfered with the
ability of hyperosmolarity to block GLUT4myc internalization. We
propose that hyperosmolarity increases surface GLUT4myc by preventing
GLUT4 endocytosis and stimulating its exocytosis via a pathway
independent of phosphatidylinositol 3-kinase activity and of VAMP2 or
VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in
these cells, might mediate membrane fusion of the
hyperosmolarity-sensitive pool.
The glucose transporter 4 (GLUT4)1 is the predominant
glucose transporter of muscle and adipose cells. In untreated
adipocytes, GLUT4 recycles constitutively between the plasma membrane
and intracellular loci (1, 2), with the steady-state distribution favoring the latter. Morphological and biochemical studies have detected GLUT4 in distinct but inter-related intracellular pools, including sorting endosomes, TGN, recycling endosomes, and specialized GLUT4 exocytic vesicles (3-6). GLUT4 endocytosis occurs via
clathrin-coated vesicles, assisted by the GTPase dynamin. Thus,
inhibition of clathrin-coated vesicle formation via K+
depletion (7), interference with dynamin-amphiphysin pairing (8), or
expression of GTPase-deficient dynamin (9, 10), all prevent GLUT4
internalization in adipocytes. Little is known about the traffic of
this transporter in muscle cells, despite the fact that muscle
represents the largest in vivo site of glucose utilization.
Insulin shifts the subcellular distribution of GLUT4 resulting in a new
steady state where a large fraction of GLUT4 resides at the plasma
membrane of skeletal muscle (11-13), primary adipose cells (14, 15),
L6 muscle cells in culture (16), and 3T3-L1 adipocytes (1). Studies in
adipocytes indicate that this shift occurs primarily through the
stimulation of GLUT4 exocytosis (1, 2), but whether or not insulin
inhibits GLUT4 endocytosis is still debatable (1, 2, 17, 18). The
contribution of exocytic and endocytic pathways to insulin action in
muscle cells has not been explored.
Exposing 3T3-L1 adipocytes to hyperosmolar solutions also leads to the
cell surface accumulation of GLUT4. It was postulated that this
accumulation results from the stimulation of GLUT4 exocytosis by
signals different from those elicited by insulin (19-21) through a
so-called "alternative pathway." However, the steady-state analysis used in those studies precluded the distinction of exocytic from endocytic traffic of GLUT4. The well-known effect of hyperosmolarity to
disrupt clathrin function (22, 23) has prompted us to hypothesize that
hyperosmolarity accumulates GLUT4 at the cell surface by inhibiting
GLUT4 endocytosis. We have previously characterized a clone of L6
myoblasts stably expressing GLUT4 tagged with an exofacial myc epitope
(GLUT4myc) (24), which could be used to more accurately measure GLUT4
endocytosis in response to different stimuli.
The objective of this study was to investigate how insulin and
hyperosmolarity affect the recycling of GLUT4 in L6 muscle cells in
culture, to examine how each stimulus modulates the endocytic and
exocytic arms of GLUT4 traffic, and to compare the characteristics of
the donor pools of GLUT4 in each instance. We show that both insulin
and hyperosmolarity induce the subcellular redistribution of GLUT4 from
intracellular loci to the plasma membrane in L6 myoblasts. Whereas
insulin primarily stimulates GLUT4 exocytosis, hyperosmolarity largely
prevents its endocytosis. Although inhibition of tyrosine kinases
prevents significantly insulin-stimulated GLUT4 exocytosis, it does not
affect the hyperosmolarity-induced block of GLUT4 internalization and
only partially blocks hyperosmolarity-triggered GLUT4 accumulation at
the cell surface. The results suggest that insulin draws GLUT4 from a
specific pool that is affected by tetanus toxin and wortmannin. In
contrast, the accumulation of GLUT4 at the cell surface caused by
hyperosmolarity results from both reducing GLUT4 endocytosis and
stimulating its exocytosis. Hyperosmolarity is likely to draw GLUT4
from the recycling endosomal pool and/or an "alternative
pool" that is insensitive to inhibition by tetanus toxin and wortmannin.
Materials--
Human insulin (Humulin R) was purchased from Eli
Lilly Canada Inc. (Toronto); o-phenylenediamine
dihydrochloride (OPD) reagent was from Sigma-Aldrich (Oakville,
Ontario). Mouse monoclonal anti-Myc antibody (9E10) in ascites
fluid was a kind gift from Carmen de Hoog and Dr. Mike Moran
(University of Toronto). Rabbit polyclonal anti-tetanus
toxin-insensitive VAMP (TI-VAMP) antibody (49) was a generous
gift from Dr. Thierry Galli (Institut Curie, Paris, France).
Horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG and
indocarbocyanine (Cy3)-conjugated goat anti-mouse IgG were obtained
from Jackson ImmunoResearch (West Grove, PA). The mammalian expression
vector for enhanced green fluorescence protein, pEGFP-N1, was purchased
from CLONTECH (Palo Alto, CA); pcDNA3 was from
Invitrogen (Carlsbad, CA). The light chain of tetanus toxin (TeTx)
cDNA in pcDNA3 was obtained from Dr. Heiner Niemann (Medizinische Hoshschule, Hanover, Germany), and the cDNAs for GFP-tagged VAMP2 and VAMP3 from Dr. William Trimble (University of
Toronto). Maxi-prep tip DNA purification columns and Effectene transfection kits were from Qiagen (Mississauga, Ontario). All DNA
constructs used in transfections were prepared using Qiagen maxi-prep
columns according to the manufacturer's recommendations.
Cell Culture--
GLUT4myc cDNA was constructed by inserting
the human c-Myc epitope (AEEQKLISEEDLLK, 14 amino acids
recognized by the monoclonal antibody 9E10) into the first ectodomain
of GLUT4 and subcloned into the pCXN2 expression vector (25). A clone
of L6 skeletal muscle cells isolated for high fusion potential (26) was
transfected with pCXN2-GLUT4myc to create a stable cell line,
L6-GLUT4myc (25). L6-GLUT4myc myoblasts were maintained in Assay of Cell Surface GLUT4myc--
Cell surface detection of
GLUT4myc was carried out as previously described (24, 27) with slight
modifications. Briefly, after treatment of quiescent L6-GLUT4myc
myoblasts with or without 100 nM insulin or hypertonic
sucrose solution (0.45 M sucrose in GLUT4myc Endocytosis Assay--
Cells in 24-well tissue culture
plates were stimulated with 100 nM insulin at 37 °C for
30 min. Cells were rinsed 3 times with ice-cold PBS and reacted with
the anti-Myc antibody 9E10 in HPMI to label cell surface GLUT4myc at
4 °C for 1 h. After washing 4 times with ice-cold PBS, the
surface labeled GLUT4myc was allowed to internalize by re-warming the
cells to 37 °C in pre-warmed K+ Depletion--
For the GLUT4myc exocytosis assay,
cells were incubated in K+-free (KCl was replaced with
NaCl) or K+-plus Hepes buffer (containing 140 mM NaCl, 5 mM KCl, 20 mM Hepes (pH
7.4), 2.5 mM MgSO4, and 1 mM
CaCl2) for 2 h prior to detection of cell surface
GLUT4myc. For the GLUT4myc endocytosis assay, following stimulation
with 100 nM insulin for 30 min, cells were washed three
times with ice-cold K+-free or K+-plus (5 mM) Hepes buffer. Cell surface GLUT4myc proteins were labeled with anti-Myc antibody 9E10 in K+-free or
K+-plus (5 mM) Hepes buffer at 4 °C for
1 h. K+ depletion continued during re-warming to
37 °C in K+-free Hepes buffer containing 0.45 M sucrose or K+-plus (5 mM) Hepes
buffer for another hour. The cells were then washed twice with
pre-warmed K+-free or K+-plus (5 mM) Hepes buffer and incubated with the same buffer at 37 °C for 30 min. In the sucrose removal group, 0.45 M
sucrose was present for the first hour of re-warming for endocytosis
and removed for the last 30 min, and K+ (5 mM)
was always added.
Transfection of L6-GLUT4myc Cells--
L6-GLUT4myc myoblasts
were seeded at a density of ~1 × 105 cells/well
onto 18-mm diameter glass coverslips in 12-well tissue culture plates.
The following day, transfection was performed according to the
Effectene product manual, using 3 µl of the Effectene reagent per
transfection condition (as indicated in the figure legends). After
introducing DNA to cells for 5 h, the cultures were washed twice
with PBS and maintained in culture medium for another 43 h until
experimentation. These cells were deprived of serum in culture medium
for 3 h at 37 °C prior to processing for immunofluorescence.
Immunofluorescence--
After serum deprivation, cells were left
untreated or treated with 100 nM insulin or 0.45 M sucrose at 37 °C for 30 min. Indirect immunofluorescence for GLUT4myc translocation was carried out on intact
cells as described (28). All steps were performed at 4 °C unless
otherwise indicated. Cells on coverslips were rinsed twice with
ice-cold PBS prior to incubation with 3% formaldehyde in PBS for 2 min, followed by 0.1 M glycine in PBS for 10 min. The cells
were blocked with 5% goat serum in PBS for 10 min. To detect GLUT4myc,
coverslips were incubated with anti-Myc antibody 9E10 in HPMI for
1 h, washed four times with PBS and incubated in the dark with
secondary antibody (Cy3-conjugated goat anti-mouse IgG, 1:1000 in PBS
containing 3% goat serum) for 1 h. All subsequent steps were
shielded from light. The cells were washed six times with PBS, fixed
with 4% formaldehyde in PBS for 30 min, initiated at 4 °C, but
immediately shifted to room temperature, followed by quenching with 0.1 M glycine for 10 min and three PBS washes of 5 min each at
room temperature. The coverslips were mounted with 10 µl of Antifade
solution. Mounted coverslips were stored at 4 °C prior to analysis
with a Leica TCS 4D fluorescence microscope. To detect TI-VAMP, cells
on coverslips were permeabilized with 0.1% Triton X-100 for 20 min at
room temperature before immunolabeling with anti-TI-VAMP primary
antibody (1:100), followed by Cy3-conjugated goat anti-rabbit IgG
secondary antibody (1:1000) in PBS containing 3% goat serum.
Phosphotyrosine Immunoblotting--
Following experimental
treatment, L6-GLUT4myc myoblasts were rinsed three times with PBS
containing 1 mM sodium vanadate, and the whole cell lysates
were prepared by solubilizing the cells in 125 mM Tris-HCl,
pH 6.8, 4% SDS, 20% glycerol, 7.5% The majority of GLUT4myc Is Located Intracellularly in Untreated L6
Myoblasts--
The exofacial myc epitope of GLUT4myc allowed us to
estimate the proportion of this protein exposed at the cell surface and its total cellular content by analyzing intact and permeabilized cells,
respectively. The amount of myc epitope exposed at the surface of
non-permeabilized cells was determined by a quantitative assay based on
the detection of anti-myc antibody bound to a monolayer of L6
myoblasts. The total myc epitope present in L6 myoblasts was determined
by permeabilization with 0.1% Triton X-100 before immunolabeling with
anti-myc antibody, followed by HRP-conjugated secondary antibody
coupled to an OPD optical densitometric assay. The intracellular
GLUT4myc content was then estimated by subtracting the amount present
on the cell surface from the total cellular content. Under basal
conditions, 90.0 ± 0.6% of the GLUT4myc resides intracellularly
(Fig. 1). Stimulation for 30 min with 100 nM insulin or 0.45 M sucrose elevates the cell
surface content of GLUT4myc to 31.0 ± 1.7% or 30 ± 0.3%,
respectively, with a commensurate reduction in intracellular
GLUT4myc.
Insulin and Hyperosmolarity Increase GLUT4myc at the Cell Surface
with Different Time Courses and Sensitivity to Wortmannin--
We next
examined the time course of GLUT4myc appearance at the cell surface in
the presence of insulin or hypertonic sucrose. L6 myoblasts were
treated with insulin or hypertonic sucrose in culture medium at
37 °C for increasing times, and thereafter the cell surface-exposed
myc epitope was reacted with anti-myc antibody at 4 °C followed by
the OPD optical densitometric detection assay. Insulin triggered a
rapid redistribution of GLUT4myc to the cell surface with a
t1/2 of ~4 min. This rapid insulin response peaked
at 15 min (Fig. 2). On the other hand, accumulation of GLUT4myc to the cell surface by hypertonic sucrose was a much slower process with a t1/2 of
~20 min (Fig. 2). Hypertonic sucrose progressively augmented GLUT4myc
presence at the cell surface, peaking after 1 h (Fig. 2).
Because phosphatidylinositol 3-kinase (PI3K) activation by insulin is a
prerequisite for the insulin-dependent translocation of
GLUT4 to the cell surface in muscle and fat cells (29, 30), we
investigated the involvement of PI3K in the
hyperosmolarity-dependent GLUT4myc accumulation at the cell
surface. In line with previous reports, the PI3K inhibitor
wortmannin (100 nM) completely prevented the GLUT4myc gain
at the cell surface produced by insulin. However, it did not inhibit
the hypertonic sucrose-induced cell surface gain of GLUT4myc (Fig.
3). These results suggest that
hyperosmolarity engages signaling pathways different from those
involved in insulin action.
Tetanus Toxin Does Not Block the
Hyperosmolarity-dependent GLUT4myc Accumulation at the Cell
Surface--
SNARE proteins are required for membrane fusion in both
neuronal and non-neuronal cells (31-33). In the final step of GLUT4 translocation, fusion of GLUT4-containing vesicles with the plasma membrane involves fusion complexes consisting of the t-SNARE proteins syntaxin 4 and SNAP23 on the target membrane, and v-SNARES of the
synaptobrevin family on the incoming vesicles (34-38). Tetanus toxin cleaves the synaptobrevins VAMP2 and VAMP3, rendering them inactive as fusogens (39). In L6-GLUT4myc myoblasts, insulin stimulates
GLUT4myc translocation by 2-fold (24, 28, 40). We have recently
reported that transient transfection of tetanus toxin light chain can
inhibit the insulin-stimulated GLUT4myc translocation by 70% in
L6-GLUT4myc myoblasts (40). In the present study, we compared the
effects of tetanus toxin on insulin- and hypertonic sucrose-induced
cell surface accumulation of GLUT4myc. L6-GLUT4myc myoblasts were
transiently transfected with cDNA encoding tetanus toxin light
chain. Experiments were carried out 48 h after transfection.
Following 30 min of treatment with insulin or hypertonic sucrose at
37 °C, cell surface GLUT4myc was labeled with anti-myc antibody at
4 °C and then detected by fluorescence microscopy on intact cells.
Tetanus toxin markedly reduced the insulin-dependent gain
in GLUT4myc at the cell surface (Fig. 4),
confirming our previous observation (40). However, the toxin did not
block the hyperosmolarity-dependent gain in GLUT4myc at the
cell surface (Fig. 4). Similarly, the level of surface GLUT4myc in
unstimulated cells was not affected by tetanus toxin (Fig. 4): the
basal-state levels of surface GLUT4myc were 0.98 ± 0.003 and
0.98 ± 0.005 in pcDNA3 and tetanus toxin-transfected cells,
respectively, compared with a value of 1.00 assigned to basal,
untransfected cells. These results suggest that insulin and
hyperosmolarity draw GLUT4 from distinct intracellular pools that can
be differentiated by their sensitivity to tetanus toxin. Moreover, the
insulin-responsive pool appears to be static, because there is no
contribution of tetanus toxin-sensitive GLUT4 to the basal state.
Because hyperosmolarity-induced GLUT4myc accumulation still occurs when
VAMP2 and VAMP3 are completely cleaved by tetanus toxin (40), we
examined whether tetanus toxin-insensitive VAMP (TI-VAMP) is expressed
in L6 muscle cells. TI-VAMP was first described in epithelial cells and
found to form apical SNARE complexes with syntaxin 3 and SNAP23,
suggesting its involvement in apical exocytosis in epithelial cells
(49). By indirect immunofluorescence, TI-VAMP was detected in L6 muscle
myoblasts and found to be distributed in perinuclear and punctate
cytoplasmic locations (Fig. 5).
Interestingly, the majority of TI-VAMP was found outside of the region
of VAMP2 or VAMP3 judged from the fluorescence of transfected VAMP2-GFP or VAMP3-GFP (Fig. 5). The possible role of TI-VAMP in mediating fusion
of the basal state recycling GLUT4 compartment and/or the hyperosmolarity-drawn compartment will have to be rigorously tested in
the future.
Additive Effect of Insulin and Hyperosmolarity on Surface
GLUT4myc--
If insulin and hyperosmolarity draw GLUT4 from different
intracellular pools, their effects should be additive. A 40-min
stimulation by insulin increased the presence of GLUT4myc at the cell
surface by 2.3- ± 0.1-fold, and 30 min of exposure to hyperosmolarity increased cell surface GLUT4myc by 2.1- ± 0.1-fold (Fig.
6). When insulin was given for the first
10 min, followed by the combined stimuli of insulin and sucrose for 30 min, cell surface GLUT4myc increased 3.2- ± 0.1-fold (Fig. 6). This
higher level of GLUT4myc present at the cell surface is significantly
different from the effect of either insulin or hyperosmolarity alone.
In contrast, when hyperosmolarity preceded insulin addition, the
maximum effect equaled that achieved by hyperosmolarity alone (Fig. 6).
This result is consistent with the notion that hyperosmolarity has the
additional effect of causing insulin resistance (see
"Discussion").
Hyperosmolarity Inhibits GLUT4myc Retrieval from the Cell
Surface--
The cell surface GLUT4myc accumulation brought about by
insulin or hyperosmolarity can result from either the stimulation of
GLUT4myc exocytosis or the inhibition of its endocytosis. To record
GLUT4myc internalization, cells were stimulated with insulin at
37 °C, reacted with anti-myc antibody at 4 °C to label cell surface GLUT4myc and then re-warmed to allow endocytosis in the absence
or presence of insulin or hypertonic sucrose. At the indicated times,
cells were chilled again and the myc antibody-labeled GLUT4myc remaining on the surface was detected by the densitometric assay. The
amount of GLUT4myc remaining at the cell surface at defined time points
was expressed as a percentage of cell surface GLUT4myc level at 0 min
of endocytosis. GLUT4myc was found to be rapidly internalized following
insulin removal with a t1/2 of ~3 min (Fig.
7A). Hypertonic sucrose
retained GLUT4myc at the cell surface for the entire time tested (2-60
min) (Fig. 7A). A similar observation was made when using
0.6 M mannitol instead of 0.45 M sucrose as
hyperosmolar challenge (data not shown). In the continued presence of
insulin, cell surface-labeled GLUT4myc attained a slightly higher value
by 10 min after initiation of its internalization (Fig. 7A).
To identify whether this difference resulted from insulin-induced GLUT4
re-exocytosis or a delayed inhibition of its endocytosis rate, we
measured the extent of re-exocytosis of the myc antibody-labeled
GLUT4myc. After labeling the insulin-recruited GLUT4myc transporters at
the plasma membrane, GLUT4myc internalization was allowed to proceed at
37 °C for 20 min in the absence of the hormone, so that ~80% of
the myc antibody-labeled GLUT4myc was internalized. Insulin was then
reintroduced for 10 or 20 min. Under these conditions, the amount of
surface GLUT4myc was elevated by ~35-40% of the level attained by
internalization for 30 or 40 min in the absence of insulin (Fig.
7A, inset) and this increase was equivalent to
the difference between the endocytic curves measured in the continuous
presence and absence of insulin (Fig. 7A). Therefore,
anti-myc-labeled GLUT4myc is recycled back to the cell surface in the
presence of insulin.
We then compared the rates of GLUT4myc internalization in basal and
insulin-stimulated cells. Surface GLUT4myc was labeled with anti-myc
antibody in unstimulated cells. GLUT4myc was found to be internalized
rapidly in the basal state with a t1/2 of ~1.5 min
(Fig. 7B). The presence of insulin caused a minor delay in
the rate of GLUT4 internalization at 10-60 min after the initiation of
internalization (Fig. 7B) in a similar pattern as shown in
Fig. 7A. However, the initial rates of internalization for
the first 5 min, when ~50% to 60% of GLUT4myc disappeared from the
cell surface, were identical under all conditions, i.e. in
the basal state, in the presence of insulin during re-warming, and in
insulin-prestimulated cells (Fig. 7C).
K+ Depletion Causes a Gain in GLUT4myc at the Cell
Surface and Prevents GLUT4myc Internalization--
Disassembly of
clathrin lattices by K+ depletion results in an inhibition
of the endocytosis of LDL and transferrin receptors in fibroblasts and
hepatocytes (22, 41, 42). It also causes the accumulation of GLUT4 at
the cell surface in adipocytes (7). In the present study, we explored
whether inhibition of clathrin-dependent endocytosis by
K+ depletion mimics the hyperosmolarity effects on GLUT4myc
traffic. For the GLUT4myc externalization assay, cells were incubated
with K+-free Hepes buffer for 2 h prior to detection
of cell surface GLUT4myc with the anti-myc antibody coupled to the
densitometric assay. K+ depletion was found to increase
cell surface GLUT4myc by 3.6- ± 0.3-fold (Fig.
8A).
Next, the effect of K+ depletion on GLUT4myc
internalization was analyzed. K+ depletion was carried out
during GLUT4myc labeling with anti-myc antibody at 4 °C for 1 h
and continued during re-warming in the presence of hypertonic sucrose
for another hour. Hypertonic sucrose was added during this period to
maintain the transporters at the cell surface while the intracellular
K+ was being depleted. The cells were then incubated in
K+-free, iso-osmotic (sucrose-free) buffer for 30 min, and
under these conditions, GLUT4myc remained at the cell surface (Fig. 8B). A control experiment confirmed that removal of sucrose
in K+-containing medium allowed full internalization of
GLUT4myc (Fig. 8B). These results suggest that
K+ depletion, like hyperosmolarity, retains GLUT4myc at the
cell surface.
Tyrosine Kinases Are Not Involved in Hyperosmolarity-induced
GLUT4myc Retention at the Cell Surface--
It has been reported that
tyrosine kinases participate in hyperosmolarity-induced GLUT4
externalization in 3T3-L1 adipocytes (19), based on steady-state
measurements of GLUT4 accumulation at the cell surface, which do not
differentiate between exocytic and endocytic events. Therefore, we
examined the effect of the tyrosine kinase inhibitor erbstatin analog
(erbstatin A) on GLUT4myc exocytosis and endocytosis in the presence
of hypertonic sucrose in L6 muscle cells. Inhibition of tyrosine
kinases by erbstatin A (40 µg/ml) prevented significantly
insulin-stimulated GLUT4myc translocation (data not shown).
Hyperosmolarity caused an increase in protein tyrosine phosphorylation,
which was prevented by erbstatin A (Fig.
9A). However, the tyrosine
kinase inhibitor only partially blocked the hypertonic sucrose-induced
GLUT4myc externalization (Fig. 9A). The same results were
obtained when another tyrosine kinase inhibitor, genistein, was used
(Fig. 9B). Interestingly, erbstatin A did not affect the
hypertonic sucrose action on GLUT4myc internalization (Fig.
9C), suggesting that tyrosine kinases are not involved in
hyperosmolarity-induced GLUT4myc retention at the cell surface. Because
erbstatin A partially inhibited GLUT4myc externalization, the
accumulation of GLUT4myc at the surface caused by hypertonic sucrose
appears to result partly from erbstatin A-insensitive GLUT4myc
retention and partly from stimulation of erbstatin A-sensitive GLUT4myc
exocytosis.
GLUT4 is a determinant of insulin sensitivity in muscle and fat
cells. In the L6 skeletal muscle cell line, GLUT4 expression occurs
after differentiation from myoblasts into myotubes (26). We have
previously reported that expression of GLUT4myc in L6 myoblasts leads
to the segregation of the protein to a GLUT4-specific pool, conferring
insulin sensitivity to glucose uptake (24). This conclusion is based on
the finding that, in L6-GLUT4myc myoblasts, the intracellular GLUT4myc
compartment contains the majority of the insulin-regulatable amino
peptidase but less than half of the GLUT1, and the sensitivity of
glucose uptake to insulin is markedly improved. Indeed, we confirm in
the present study that 90% of the GLUT4myc resides
intracellularly.2 Upon
insulin or hypertonic sucrose stimulation, 30% of the total cellular
GLUT4myc is redistributed to the cell surface within 30 min. These
results demonstrate that, as with previous observations in 3T3-L1
adipocytes, GLUT4myc is vastly retained in the intracellular pool in
the basal state and is redistributed to the cell surface in response to
insulin and hyperosmolarity in L6-GLUT4myc myoblasts.
Insulin Stimulates GLUT4 Exocytosis and Hyperosmolarity Inhibits
Its Internalization--
GLUT4myc undergoes rapid internalization upon
insulin removal. Half of the surface-labeled GLUT4myc is internalized
within 3 min. Even in the presence of insulin, the rate of GLUT4myc
internalization is not appreciably slowed down, having approximately
the same t1/2 of 3 min. These results suggest that
insulin does not regulate GLUT4 internalization in L6-GLUT4myc myoblasts. This contrasts with observations made in fat cells where a
small proportion of insulin-induced gain in surface GLUT4 appears to be
due to inhibition of GLUT4 endocytosis (1, 18). The 3-min half-time
measured for GLUT4myc internalization in L6 myoblasts is very similar
to the 4.2- or 3-min half-time for GLUT4 or insulin-regulatable amino
peptidase endocytosis, respectively, reported for 3T3-L1 adipocytes in
the presence of insulin (1, 43).
GLUT4 internalizes via clathrin-coated pits (7, 44). K+
depletion and hypertonic shock are two strategies known to perturb the
formation of clathrin coats (22, 23, 41) by preventing the interaction
between clathrin and adaptors proteins (22). We demonstrate here that
K+ depletion mimics hyperosmolarity by causing a gain in
GLUT4myc at the cell surface and preventing GLUT4myc endocytosis. These results strongly support the concept that hyperosmolarity accumulates GLUT4 at the cell surface, at least in part, through inhibition of
GLUT4 endocytosis.
Hyperosmolarity is a stress stimulus that activates a tyrosine kinase
pathway (19, 45), and tyrosine kinase activity is required for the
surface gain in GLUT4 in 3T3-L1 adipocytes (19). However, it is
unlikely that this effect is related to the retention of GLUT4 at the
cell surface, because an inhibitor of the tyrosine kinases, erbstatin
A, was unable to prevent the inhibition of GLUT4myc endocytosis by
hyperosmolarity in muscle cells (Fig. 9C). Erbstatin A and
genistein prevented the hyperosmolarity-induced GLUT4 externalization
by only ~50% (Fig. 9, A and B). We propose that inhibition of GLUT4 endocytosis accounts for 50% of the GLUT4 surface accumulation, and the remaining 50% arises from the
stimulation of GLUT4 exocytosis in response to hyperosmolarity. Chen
et al. (19) reported complete prevention of
hyperosmolarity-induced GLUT4 translocation by inhibition of tyrosine
kinases. Therefore, it is conceivable that GLUT4 endocytosis is not
blocked by hyperosmolarity in 3T3-L1 cells.
Insulin and Hyperosmolarity Draw GLUT4 from Different Intracellular
Pools--
GLUT4 accumulated at the cell surface with very different
time courses in response to insulin and hypertonic sucrose. We
speculate that there may be an insulin-regulated exocytic GLUT4 pool in L6 muscle cells, which can be rapidly mobilized by insulin and that
hyperosmolarity draws GLUT4 from an alternative pool of GLUT4 and/or
the recycling endosomes. Supporting this concept that insulin and
hyperosmolarity draw GLUT4 from different intracellular pools in L6
muscle cells is their differential sensitivity to tetanus toxin.
VAMP2 and VAMP3 are expressed in muscle and fat cells (46-48) and
reside in different GLUT4 pools in both muscle and fat cells (5, 40,
48). As shown earlier (40) and confirmed here, tetanus toxin reduced
the insulin-dependent GLUT4myc translocation in L6
myoblasts. Our previous study also demonstrated that the reduction can
be rescued by the tetanus toxin-resistant mutant VAMP2 but not by the
toxin-resistant mutant VAMP3, suggesting that VAMP2 but not VAMP3 is
required for GLUT4 vesicle fusion with the plasma membrane in response
to insulin (40). We now show that expression of tetanus toxin does not
alter GLUT4myc externalization caused by hyperosmolarity. These results
support the concept that insulin and hyperosmolarity recruit GLUT4 from different intracellular pools, one requiring VAMP2 and another one that
is tetanus toxin-insensitive. Thus, neither VAMP2 nor VAMP3 are the
fusogenic v-SNARE for the incorporation of GLUT4 vesicles from
recycling endosomes into the plasma membrane. It is conceivable that a
tetanus toxin-insensitive VAMP such as TI-VAMP (49) could mediate
fusion of the recycling endosome with the plasma membrane in muscle
cells. Indeed, TI-VAMP was detected in L6-GLUT4myc myoblasts, and its
localization was partially distinct from that of VAMP2 or VAMP3. The
possible role of TI-VAMP in fusion of the hyperosmolarity-drawn pool is
under investigation.
The action of insulin and hyperosmolarity on GLUT4myc externalization
was partly additive. A previous study failed to observe an additive
effect in 3T3-L1 adipocytes where the cells were pretreated with
hyperosmolar solution prior to exposure to insulin (20). We also failed
to observe any additive effect when we incubated L6-GLUT4myc myoblasts
in the same manner. The lack of additivity under these latter
conditions may be due to the inhibition of insulin signaling by
hyperosmolarity at the level of Akt, as reported previously (20). In
contrast, treating L6 muscle cells with insulin, followed by the
addition of hyperosmolar solution, caused a further increase in the
surface GLUT4myc compared with the effect of either stimulus alone.
Hypertonic sucrose must draw GLUT4myc from an alternative GLUT4 pool
and/or recycling endosomal pool to account for the appearance of
additional GLUT4myc at the cell surface, and as shown in Fig. 7, also
retains GLUT4myc at the cell surface. Hence, hyperosmolarity causes a
compounded gain of GLUT4 molecules at the cell surface.
Lastly, insulin and hyperosmolarity engage different
signals in their action. PI3K activation is required for
insulin-dependent GLUT4 translocation in fat and muscle
cells (29, 30, 50-53). In contrast, the hyperosmolarity-induced
accumulation of GLUT4 at the cell surface is not prevented by the PI3K
inhibitor wortmannin in 3T3-L1 adipocytes
(19).3 Here we demonstrate
that PI3K activity is not required for GLUT4myc externalization caused
by hyperosmolarity in L6 myoblasts.
Current models suggest that in 3T3-L1 adipocytes the increased amount
of GLUT4 at the surface in response to insulin is due to recruitment of
GLUT4 from a putatively static exocytic pool and from a continuously
recycling pool (54-56). Our results suggest that in muscle cells a
major effect of insulin is to cause GLUT4myc exocytosis from a specific
pool (likely specialized vesicles), which is distinct from the pool
mobilized by hyperosmolarity and from the basal-state recycling pool.
Fig. 10 illustrates the pathways of
intracellular GLUT4 traffic highlighted by this study.
In summary, the majority (90%) of GLUT4myc is located intracellularly
in L6 myoblasts in the basal state. In the presence of insulin, 70% of
GLUT4myc resides intracellularly and 30% recycles back to the cell
surface with a t1/2 of ~4 min. Under hyperosmolar
stress, 65% of the GLUT4myc remains intracellularly and 35% recycles
with a t1/2 of ~20 min. Insulin and
hyperosmolarity draw GLUT4 from different pools. Insulin recruits GLUT4
primarily from a specialized pool that requires the participation of
PI3K and VAMP2. Hyperosmolarity accelerates GLUT4 exocytosis from an
alternative pool and/or the recycling pool that are PI3K-independent
and tetanus toxin-insensitive, and it also retains the GLUT4 molecules
at the cell surface by blocking their endocytosis. This latter action
significantly contributes to the accumulation of GLUT4 at the cell
surface by hyperosmolarity. The possible interconnections among the
specialized, alternative, and recycling pools require further investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MEM
culture medium supplemented with 10% (v/v) fetal bovine serum in a
humidified atmosphere containing 5% CO2 and 95% air at
37 °C. Before use, cell monolayers were rinsed with serum-free
-MEM and then serum depleted for 3 h in the medium.
-MEM) at 37 °C as
indicated, cells in 24-well tissue culture plates were placed on ice
and rinsed twice with ice-cold phosphate-buffered saline (PBS), blocked
with 5% (v/v) goat serum in PBS for 15 min, and then reacted with the
anti-Myc antibody 9E10 (8 µg/ml in HEPES-modified RPMI (HPMI)
containing 3% goat serum) at 4 °C for 1 h. After four washes
with ice-cold PBS, cells were fixed with 3% (v/v) formaldehyde in PBS
at 4 °C for 10 min, followed by quenching with 0.1 M
glycine in PBS for 10 min and incubation with secondary antibody
(HRP-conjugated donkey anti-mouse IgG, 1:1000 in PBS containing 3%
goat serum) at 4 °C for 1 h. The cell plates were washed six
times with PBS, then 1 ml/well 0.4 mg/ml OPD reagent (the HRP
substrate) was added at room temperature for 20-30 min. The reaction
was stopped by addition of 0.25 ml of 3 N HCl and the
optical absorbance was measured at 492 nm. To measure total cellular
GLUT4myc, cells were permeabilized with 0.1% (v/v) Triton X-100 in PBS
at 4 °C for 30 min prior to incubation with the anti-myc antibody 9E10.
-MEM. Insulin 100 nM or
sucrose 0.45 M was present in the pre-warmed media. The
cell plates were placed on a 37 °C water bath before transferring to
a 37 °C incubator. At the indicated times, cell plates were placed
on ice, washed once with ice-cold PBS and then fixed with 3%
formaldehyde in PBS for 10 min, quenched with 0.1 M glycine
in PBS for 10 min, blocked with 5% goat serum in PBS for 15 min,
incubated with HRP-conjugated secondary antibody in PBS for 1 h,
reacted with OPD reagent, and the optical absorbance was measured at
492 nm as for the GLUT4myc exocytosis assay. The amount of GLUT4myc
remaining on the cell surface at any time point after re-warming was
expressed as a percentage of the cell surface GLUT4myc level at 0 min
of endocytosis. When measuring GLUT4myc endocytosis in the basal state,
cells were kept in
-MEM without any stimulus before being reacted
with anti-myc antibody.
-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µM E64, 1 µM leupeptin, 25 µM pepstatin, and 1 mM sodium vanadate. The lysates were then homogenized by
passing through syringe with the needle of 25G
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of insulin and hyperosmolarity on the
subcellular distribution of GLUT4myc in L6-GLUT4myc myoblasts.
Confluent, quiescent L6-GLUT4myc myoblasts were incubated with or
without 100 nM insulin or 0.45 M sucrose at
37 °C for 30 min. GLUT4myc exposed at the cell surface was reacted
with anti-Myc antibody and coupled to the OPD optical densitometric
detection assay outlined under "Experimental Procedures." The total
GLUT4myc present in whole cells was determined by permeabilization of
the cells with 0.1% Triton X-100 before immunolabeling with anti-Myc
antibody. Intracellular content = total cellular content cell surface content. Shown are the means ± S.E. of three
separate experiments each performed in triplicate; *, p < 0.05 versus basal cell surface GLUT4myc levels by ANOVA
with post-testing. Error bars missing were smaller than the
line.
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Fig. 2.
Insulin and hyperosmolarity increase the
amount of GLUT4myc at the cell surface with different time
courses. Confluent, quiescent L6-GLUT4myc myoblasts were incubated
with or without 100 nM insulin or 0.45 M
sucrose at 37 °C for 5 to 120 min. GLUT4myc exposed at the cell
surface was reacted with anti-Myc antibody and coupled to the OPD
optical densitometric detection assay as described under
"Experimental Procedures." Shown are the means ± S.E. of
three to four separate experiments each performed in triplicate;
p < 0.05 by ANOVA.
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Fig. 3.
Wortmannin does not prevent the GLUT4myc gain
at the cell surface induced by hyperosmolarity. Confluent,
quiescent L6-GLUT4myc myoblasts were treated with 100 nM
wortmannin for 20 min prior to and during subsequent 30 min of
incubation with 100 nM insulin or 0.45 M
sucrose. GLUT4myc exposed at the cell surface was reacted with anti-Myc
antibody and coupled to the OPD optical densitometric detection assay
as described under "Experimental Procedures." Shown are the
means ± S.E. of three separate experiments each performed in
triplicate; *, p < 0.05 versus
wortmannin-untreated basal cells by ANOVA with post-testing.
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Fig. 4.
Tetanus toxin reduces the
insulin-dependent gain, but not the
hyperosmolarity-dependent accumulation, of GLUT4myc at the
cell surface. L6-GLUT4myc myoblasts were left untransfected or
co-transfected with 0.45 µg of EGFP cDNA and 0.45 µg of
pcDNA3 or tetanus toxin light chain (TeTx) cDNA as indicated.
After 48 h, cells were serum-starved for 3 h and left
untreated or exposed to 100 nM insulin or 0.45 M sucrose for 30 min as shown. Surface GLUT4myc in intact
cells was detected with anti-Myc antibody coupled to the indirect
immunofluorescence assay as described under "Experimental
Procedures." Shown are cell images from one typical experiment.
A, anti-Myc surface staining is shown in untransfected cells
at 100× magnification. B, shown also is the anti-Myc
surface staining at 40× magnification in both untransfected ( TeTx)
and tetanus toxin-transfected (+TeTx) cells with the corresponding EGFP
expression in transfected cells in the same field of view.
Arrowheads point to the toxin-transfected cells.
Bar, 5 µm. C, cell-surface GLUT4myc labeling in
untransfected and transfected cells was quantitated using National
Institutes of Health IMAGE software. Shown are the means ± S.E.
of three separate experiments each from nine fields of view. The
results were expressed relative to control basal, which was assigned a
value of 1; *, p < 0.05 versus
untransfected basal cells by ANOVA with post-testing. Error
bars missing were smaller than the line.
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Fig. 5.
Tetanus toxin-insensitive VAMP is present in
L6 muscle cells. L6-GLUT4myc myoblasts were left
untransfected or transfected with 0.6 µg of GFP-tagged VAMP2
(V2-GFP) or VAMP3 (V3-GFP) cDNAs as
indicated. After 48 h, cells were serum-starved for 3 h and
TI-VAMP expression in permeabilized cells was detected with
anti-TI-VAMP antibody coupled to the indirect immunofluorescence assay
as described under "Experimental Procedures." Shown are cell images
from one typical experiment. Bar, 5 µm.
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Fig. 6.
Insulin and hyperosmolarity have partly
additive effects on the accumulation of GLUT4myc at the cell
surface. Confluent, quiescent L6-GLUT4myc myoblasts were incubated
with 100 nM insulin for 20-40 min, 0.45 M
sucrose for 25-30 min, or when both stimuli were applied, insulin was
given for the first 10 min (Ins + Suc) or sucrose for the
first 5 min (Suc + Ins), followed by the combined stimuli
for 20-30 min. GLUT4myc exposed at the cell surface was reacted with
anti-Myc antibody and coupled to the OPD optical densitometric
detection assay as described under "Experimental Procedures." Shown
are the means ± S.E. of three separate experiments each performed
in triplicate; *, p < 0.05 versus basal;
**, p < 0.05 versus insulin or sucrose;
***, p < 0.05 insulin versus sucrose + insulin by ANOVA with post-testing.
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Fig. 7.
Hyperosmolarity inhibits endocytosis of
GLUT4myc, whereas insulin has little effect on the endocytosis.
Confluent, quiescent L6-GLUT4myc myoblasts were incubated in the
presence or absence of 100 nM insulin at 37 °C for 30 min, reacted with anti-Myc antibody at 4 °C for 1 h to label
cell surface GLUT4myc and then re-warmed to allow endocytosis in the
absence or presence of 100 nM insulin or 0.45 M
sucrose for 2-60 min. At the indicated times, cells were chilled again
and the Myc antibody-labeled GLUT4myc remaining on the surface was
measured by the OPD optical densitometric detection assay as described
under "Experimental Procedures." The amount of GLUT4myc
remaining on the cell surface at any time point after re-warming
was expressed as a percentage of the cell surface GLUT4myc level at 0 min of endocytosis. A, time course of GLUT4myc internalization. Here surface GLUT4myc was labeled with anti-Myc
antibody in cells pre-stimulated with insulin. Inset,
confluent, quiescent L6-GLUT4myc myoblasts were stimulated with 100 nM insulin at 37 °C for 30 min, reacted with anti-Myc
antibody at 4 °C for 1 h to label cell surface GLUT4myc and
then re-warmed to allow endocytosis in the absence of insulin for 20 min. Insulin was then added for 10 or 20 min. Cells were chilled again
and the Myc antibody-labeled GLUT4myc remaining on the surface was
analyzed by the OPD optical densitometric detection assay as described
under "Experimental Procedures." Shown are the means ± S.E.
of two to five separate experiments each performed in triplicate.
B, time course of GLUT4myc internalization. Here surface
GLUT4myc was labeled with anti-Myc antibody in insulin-unstimulated
cells. Shown are the means ± S.E. of two to five separate
experiments each performed in triplicate. C, after being
reacted with anti-Myc antibody at 4 °C for 1 h to label cell
surface GLUT4myc, cells were re-warmed to allow endocytosis in the
absence or presence of insulin for 5 min. Shown are the means ± S.E. of two to three separate experiments each performed in triplicate.
p > 0.05 by ANOVA with post-testing. Surface GLUT4myc
was labeled with anti-Myc antibody in cells pre-stimulated with insulin
(open bars) or unstimulated cells (filled
bars).
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Fig. 8.
K+ depletion mimics
hyperosmolarity in causing a gain in GLUT4myc at the cell surface and
preventing GLUT4myc endocytosis. A, assay of cell
surface GLUT4myc: confluent, quiescent L6-GLUT4myc myoblasts were
incubated with K+-free or K+-plus (5 mM) Hepes buffer at 37 °C for 2 h. For the group of
K+ depletion with sucrose, cells were incubated with
K+-free Hepes buffer at 37 °C for 2 h and 0.45 M sucrose was added for the last 30 min. GLUT4myc exposed
at the cell surface was reacted with anti-Myc antibody and coupled to
the OPD optical densitometric detection assay as described under
"Experimental Procedures." Shown are the means ± S.E. of
three separate experiments each performed in triplicate; *,
p < 0.05 versus basal by ANOVA with
post-testing. B, GLUT4myc endocytosis assay: confluent,
quiescent L6-GLUT4myc myoblasts were treated in three different manners
(Sucrose, Sucrose-removal, and
K+ depletion) as shown schematically. All
conditions were stimulated with 100 nM insulin at 37 °C
for 30 min, reacted with anti-Myc antibody in K+-free or
K+ plus (5 mM) Hepes buffer at 4 °C for
1 h. Cells were re-warmed to 37 °C in K+-free Hepes
buffer containing 0.45 M sucrose or K+-plus (5 mM) Hepes buffer for another hour. Cells were then
incubated with K+-free, iso-osmolar Hepes buffer or
K+-plus (5 mM) Hepes buffer at 37 °C for 30 min. In the sucrose removal group, 0.45 M sucrose was
present for the first hour of re-warming for endocytosis and removed
for the last 30 min, and K+ (5 mM) was always
added. Cells from all conditions were chilled again and the Myc
antibody-labeled GLUT4myc remaining on the surface was analyzed by the
OPD optical densitometric detection assay as described under
"Experimental Procedures." The amount of GLUT4myc remaining on the
cell surface at defined time points was expressed as a percentage of
the cell surface GLUT4myc level at 0 min of endocytosis. Shown are the
means ± S.E. of three to five separate experiments each performed
in triplicate; *, p < 0.05 versus sucrose
removal by ANOVA with post-testing.
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Fig. 9.
Inhibition of tyrosine kinase blocks the
GLUT4myc gain at the cell surface but does not prevent the inhibition
of GLUT4myc internalization induced by hyperosmolarity.
A, confluent, quiescent L6-GLUT4myc myoblasts were treated
with 40 µg/ml erbstatin A for 20 min prior to and during subsequent
30 min of incubation with 0.45 M sucrose. C,
cells were left untreated with sucrose; S, cells were
treated with sucrose; EA, erbstatin A. a, assay
of cell surface GLUT4myc: GLUT4myc exposed at the cell surface was
reacted with anti-Myc antibody and coupled to the OPD optical
densitometric detection assay as described under "Experimental
Procedures." Shown are the means ± S.E. of 8 to 11 separate
experiments each performed in triplicate; *, p < 0.05 versus erbstatin A-untreated basal cells by ANOVA with
post-testing; **, p < 0.05 sucrose versus
erbstatin A + sucrose by ANOVA with post-testing. b,
hypertonic sucrose stimulates tyrosine phosphorylation. After
stimulation described as above, whole cell lysates were prepared and
subjected to immunoblotting with PY99 phosphotyrosine antibody as
described under "Experimental Procedures." The molecular weight of
the phosphotyrosine protein shown is ~190 kDa. B,
confluent, quiescent L6-GLUT4myc myoblasts were treated with 300 µM genistein for 20 min prior to and during subsequent 30 min of incubation with 0.45 M sucrose. C, cells
were left untreated with sucrose; S, cells were treated with
sucrose; Gen, genistein. a, assay of cell surface
GLUT4myc: GLUT4myc exposed at the cell surface was reacted with
anti-Myc antibody and coupled to the OPD optical densitometric
detection assay as described under "Experimental Procedures."
b, hypertonic sucrose stimulates tyrosine phosphorylation.
After stimulation described as above, whole cell lysates were prepared
and subjected to immunoblotting with PY99 phosphotyrosine antibody as
described under "Experimental Procedures." The molecular weight of
the phosphotyrosine protein shown is ~190 kDa. C, GLUT4myc
endocytosis assay: confluent, quiescent L6-GLUT4myc myoblasts were
stimulated with 100 nM insulin at 37 °C for 30 min, and
reacted with anti-Myc antibody at 4 °C for 1 h in the absence
or presence of 40 µg/ml erbstatin A. Cells were re-warmed to 37 °C
to allow endocytosis for 1 h in the absence or presence of 0.45 M sucrose and 40 µg/ml erbstatin A. Cells were chilled
again and the Myc antibody-labeled GLUT4myc remaining on the surface
was analyzed by the OPD optical densitometric detection assay as
described under "Experimental Procedures." The amount of GLUT4myc
remaining on the cell surface at defined time points was expressed as a
percentage of the cell surface GLUT4myc level at 0 min of endocytosis.
Shown are the means ± S.E. of three separate experiments each
performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Model of intracellular GLUT4 traffic
pathways. In untreated L6-GLUT4myc myoblasts, 10% of GLUT4myc
resides at the cell surface (1). In response to
hyperosmolarity, 25% of GLUT4myc recycles to plasma membrane through
translocation from an alternative pool and/or the recycling pool
(2) and remains at the cell surface due to inhibition of
endocytosis. The exocytic event stimulated by hyperosmolarity is
PI3K-independent and tetanus toxin-insensitive. Approximately 20% of
the GLUT4myc translocates from a GLUT4-specific exocytic pool to the
plasma membrane in response to insulin that requires the participation
of PI3K and VAMP2 (3). Approximately 45% of the GLUT4myc
remains intracellularly in stimulated cells (4).
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ACKNOWLEDGEMENTS |
---|
We thank Zayna A. Khayat and Leonard Foster for advice, Dr. Zhi Liu for technical assistance, and Dr. Philip J. Bilan for discussions and comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by the Medical Research Council of Canada/Canadian Institutes for Health Research Grant MT 7307 (to A. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by postdoctoral fellowships from the Banting and Best Diabetes Center, the Hospital for Sick Children, and the Canadian Diabetes Association.
To whom correspondence should be addressed: Program in Cell
Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6392; Fax: 416-813-5028; E-mail:
amira@sickkids.on.ca.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M010143200
2 In a previous report, a shorter incubation time with 0.1% Triton X-100 resulted in incomplete cell permeabilization and underestimation of the intracellular content of GLUT4myc (24).
3 A small inhibition was seen in one study using wortmannin concentrations of 100 nM or higher, which might have inhibited other pathways (21).
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ABBREVIATIONS |
---|
The abbreviations used are: GLUT4, glucose transporter 4; OPD, o-phenylenediamine dihydrochloride; HPMI, HEPES-modified RPMI; TeTx, tetanus toxin; PI3K, phosphatidylinositol 3-kinase; erbstatin A, erbstatin analog; VAMP, vesicle-associated membrane protein; TI-VAMP, tetanus toxin-insensitive VAMP; GFP, green fluorescence protein; EGFP, enhanced GFP; MEM, minimal essential medium; PBS, phosphate-buffered saline; E64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; SNAP, soluble NSF accessory protein; SNARE, SNAP receptor.
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