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INTRODUCTION |
One of the major problems in diabetes mellitus is the disruption
of whole body glucose homeostasis. Glucose homeostasis is maintained by
a balance of hepatic glucose production and cellular glucose uptake and
metabolism (1). Glucose transport is the rate-limiting step in glucose
metabolism. Among the six known facilitative glucose transporters
(GLUT1 to -5 and GLUT7) (2), GLUT1 and GLUT4 are expressed in
insulin-responsive tissues, such as adipose tissue and cardiac and
skeletal muscle. GLUT4, which translocates from an intracellular
membrane compartment to the plasma membrane after insulin stimulation,
is particularly important in regulating postprandial glucose uptake. It
is now known that, besides the insulin signaling pathway, other
mechanisms also stimulate GLUT4 translocation and glucose uptake. For
example, exercise induces GLUT4 translocation and glucose uptake in
skeletal muscle through an insulin-independent pathway (3, 4). Also,
introduction of GTP analogs, such as
GTP
S,1 into 3T3-L1
adipocytes, and activation of
1-adrenergic receptors, stimulate glucose uptake independent of insulin (5-7).
Endothelin (ET), originally isolated from cultured porcine aortic
endothelial cells, is a peptide with 21 amino acid residues (8). Three
distinct members of the ET family, namely, ET-1, ET-2, and ET-3, have
been identified in humans through cloning (9). Binding of ETs to
G-protein-coupled receptors (GPCRs) in tissues and cells activates
various signaling molecules such as protein kinase C (PKC), PI
3-kinase, and extracellular signal-related kinases (10). Two types of
mammalian ET receptors, ETA and ETB, have been
characterized and purified (11, 12), and their cDNA have been
cloned (13, 14). ETA receptor is selective for ET-1 and
ET-2, while ETB receptor binds ET-1, ET-2, and ET-3 with
equal affinity. ET-1 is thought to play important roles in various
pathophysiological conditions.
Recently, several reports have shown that insulin stimulates ET-1
secretion from endothelial cells and also enhances ET-1 binding to its
receptors (15). It has also been shown that the plasma ET-1 level is
elevated in type II diabetes patients with microvascular complications,
suggesting that ET-1 may be involved in diabetes-related complications
such as microangiopathy (15, 16). Although there is an interest in
investigating the role of ET-1 in the development of diabetic
complications, very little is known about whether or not the ET system
is involved in glucose metabolism. In our studies to examine whether
ET-1 interacts with insulin in regulating glucose transport, we are
surprised to find that ET-1 alone stimulates glucose uptake. This
report shows for the first time in an unequivocal manner that the
ETA receptor is expressed in 3T3-L1 adipocytes and that
ET-1 stimulates GLUT4 translocation and glucose uptake in these cells
via an insulin-independent pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
3H-Labeled
2-deoxy-D-glucose (2-DOG) (26 Ci/mmol) was purchased from
NEN Life Science Products. ET-1 and ET-3 were purchased from American
Peptide Co. (Sunnyvale, CA). A-216546 and A-192621 were synthesized at
Abbott Laboratories. Other reagents were of analytical grade.
Cell Culture--
The 3T3-L1 fibroblasts were cultured in
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
serum (FBS). For experiments, cells were plated at 20,000 cells/well in
48-well plates or 10,000 cells/well in 96-well plates in DMEM
containing 10% FBS. After 3 days, cells were fed with DMEM containing
10% FBS. On day 4, cells at 100% confluency were treated with
induction medium (DMEM with 10% FBS, 400 nM insulin, 250 nM dexamethasone, and 0.5 mM isobutylmethylxanthine) for 3 days and then changed back to DMEM containing 10% FBS on day 7. Cells were fed again on day 10 with DMEM
containing 10% FBS and used for studies between day 12 and day 14 (or
as indicated) after plating. At that time, fat droplets were observed
in ~70% of the cells.
3H-Labeled 2-DOG Uptake--
Cells in 48-well plates
(or 96-well plates as indicated) were washed once with 500 µl/well
(200 µl/well) of serum-free DMEM, and incubated in 500 µl/well (200 µl/well) of serum-free DMEM for 3 h at 37 °C. Cells were then
washed twice with 500 µl/well (200 µl/well) of glucose-free
serum-free DMEM, followed by incubating in 500 µl/well (200 µl/well) of glucose-free DMEM for 30 min at 37 °C. In experiments
using kinase inhibitors, the agents were added at the beginning of this
30-min incubation period except for wortmannin, which was added 10 min
before the addition of insulin or ET-1. Afterward, insulin and/or ET-1
(or others as indicated) were added, and cells were incubated for
another 30 min at 37 °C. 3H-Labeled 2-DOG (final
concentration of 50 µM, 0.33 µCi/ml) was added, and
cells were incubated for 20 min at 37 °C. The incubation was stopped
by washing cells with 500 µl/well (200 µl/well) of ice-cold
phosphate-buffered saline (PBS) twice. Cells were dissolved in 0.1 N NaOH for scintillation counting.
[125I]ET-1 Binding to Cells--
Cells in 48-well
culture plates were incubated with 125I-labeled ET-1 in 0.2 ml/well of buffer 1 (Earle's solution; 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, buffered
with 25 mM Hepes, pH 7.4) containing protease inhibitors (5 µg/ml pepstatin A, 0.01 mM phosphoramidon, 0.1 mM phenylmethylsulfonyl fluoride) for 4 h at 4 °C.
After incubation, cells were washed twice with 0.5 ml/well of PBS,
followed by solubilization in 0.5 ml of 0.1 N NaOH before
counting. Nonspecific binding was determined in the presence of 1 µM ET-1.
Measurement of PI Hydrolysis--
PI hydrolysis was measured as
described previously (17). Briefly, cells in 48-well culture plates
were labeled with 1 µCi/well of myo-[3H]inositol for
24 h. Cells were washed with PBS and then incubated with buffer 1 containing protease inhibitors (as described above) and 10 mM LiCl for 60 min before being challenged with ET-1 for 30 min. ET challenge was terminated by the addition of 50 µl of 1 N NaOH, and the mixture was immediately neutralized by
adding 50 µl of 1 N HCl. The samples were treated with
1.5 ml of chloroform/methanol (1:2, v/v). Total inositol phosphates
were extracted after adding chloroform and water to give final
proportions of chloroform/methanol/water of 1:1:0.9 (v/v/v). The upper
aqueous phase (1 ml) was retained and analyzed by batch chromatography
using the anion exchange resin AG1-X8 (Bio-Rad). Total water-soluble
inositol phosphates were eluted from the resin by 6 ml of 1 M ammonium formate with 0.1 N formic acid after
the resin was washed with 6 ml of 60 mM sodium formate with
5 mM sodium tetraborate.
Immunofluorescence Staining and Confocal Microscopy--
The
3T3-L1 cells on day 12 after the induction medium treatment were
treated with collagenase I (200 units/ml) and plated in two-chamber
slides in DMEM containing 10% FBS 24 h before the experiment.
Cells were put into serum-free medium for 3 h and then stimulated
with or without ET-1 (10 nM) or insulin (100 nM) for 10 min (or as indicated). Afterward, cells were
washed with PBS for 30 s, fixed with methanol at
20 °C for 20 min, rinsed with PBS three times, and incubated with PBS containing
10% rabbit serum for 30 min at 37 °C. The slides were then
incubated with an anti-GLUT4 antibody (1:50 dilution) derived from goat
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in PBS with 10%
rabbit serum for 2 h at 37 °C. After the incubation, slides
were rinsed with PBS three times and then incubated with
fluorescein-conjugated rabbit anti-goat IgG (1:50 dilution, Jackson
ImmunoResearch Laboratories, West Grove, PA) for 30 min at 37 °C,
followed by another three rinses with PBS. The slides were mounted, and
pictures were taken using a Bio-Rad MRC1000 confocal microscope linked
to an image analyzer.
Insulin-responsive Aminopeptidase (IRAP)
Translocation--
Cells in 24-well plates were incubated in
serum-free DMEM (1 ml/well) for 3 h at 37 °C and then incubated
with insulin or ET-1 for 10 min. Cells were quickly chilled to 4 °C,
washed briefly with 1 ml/well of PBS, washed once more with 1 ml/well
of buffer 2 (PBS, 0.1 mM CaCl2, 1 mM MgCl2). Cells were then incubated with 0.4 ml/well of 1.5 mg/ml N-hydroxysuccinimide-SS-biotin (Pierce) in 10 mM HEPES, 2 mM CaCl2, 150 mM NaCl, pH 8.5, on ice with constant agitation for 20 min.
The biotin cross-linking process was repeated once more. To stop the
cross-linking reaction, cells were washed once with 1 ml/well of buffer
2 containing 100 mM glycine and followed by incubation in
this buffer (1 ml/well) on ice for 20 min with constant agitation.
Afterward, cells were washed once with PBS and lysed in 0.6 ml/well of
lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH
7.5, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin). Cells were removed from the well
into a microcentrifuge tube, homogenized by a microultrasonic cell
disruptor (Kontes), and then centrifuged at 10,000 × g
for 20 min. The supernatant (0.4 ml/sample) was incubated with 50 µl
of a 50% slurry of streptavidin-agarose beads (Pierce) for 24 h
at 4 °C with constant agitation. The beads were collected by
centrifugation and washed three times with 1 ml/each of lysis buffer,
followed by one wash with water. The proteins were released from the
beads by incubating beads with 20 µl of 5× SDS-PAGE sample buffer
containing 0.5%
-mercaptoethanol. Samples were analyzed by
SDS-PAGE using a 4-12% gradient gel (Novex, San Diego, CA).
SDS-PAGE and Western Blot Analysis--
Samples were resolved by
SDS-PAGE using a gradient gel (as indicated; Novex, San Diego, CA) and
proteins were electrophoretically transferred to a polyvinylidene
difluoride membrane (Immobilon-P, 0.45-µm pore size, Millipore,
Corp., Burlington, MA) for Western blotting. The membrane was blotted
with 5% nonfat dry milk in PBS-T (10 mM Tris, pH 8.0, 0.15 M NaCl, 0.1% Tween 20) for at least 30 min and then
incubated with primary antibodies in 5% bovine serum albumin
(anti-IRAP, 5000-fold dilution; anti-phospho-Akt, 2000-fold dilution;
both derived from rabbits) for 18 h at 4 °C. The anti-IRAP
antibody was obtained from Metabolex (Hayward, CA), and the
anti-phospho-Akt antibody was from New England Biolabs (Beverly, MA).
For the detection of proteins with tyrosine phosphorylation, the
membrane was blotted with PBS-T containing 1% nonfat dry milk and 1%
bovine serum albumin, and the primary antibody was a mouse monoclonal
antibody specific for phosphorylated tyrosine (from Santa Cruz
Biotechnology; 10,000-fold dilution). The membrane was washed with
PBS-T and incubated with a horseradish peroxidase-labeled anti-rabbit
or anti-mouse antibody (Pierce) for at least 1 h at 25 °C. The
paper was then incubated with detection reagent containing luminol in
an alkaline buffer. The specific bands were visualized by exposing the
paper to blue light-sensitive autoradiography films.
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RESULTS |
Effects of ET-1 and Insulin on Glucose Uptake in 3T3-L1
Adipocytes--
To investigate whether ET-1 interacts with insulin in
regulating cellular functions, we first examined glucose uptake
stimulated by insulin in the presence or absence of ET-1. Fig.
1A shows that insulin-stimulated 2-DOG uptake in a dose-dependent manner,
with a 5-fold stimulation at 100 nM. ET-1 (10 nM) alone stimulated 2-DOG uptake by 2.6-fold in the
absence of insulin. An additive effect on stimulating 2-DOG uptake was
observed when cells were treated with ET-1 and a low concentration (0.1 or 1 nM) of insulin. To further confirm the observation
that ET-1 alone stimulates glucose uptake in the 3T3-L1 adipocytes, we
compared the effects of ET-1 and angiotensin II (ANG II). Fig.
1B shows that, at three different concentrations examined
(0.1, 1, and 10 nM), ET-1 stimulated 2-DOG by 2-2.5-fold.
As a comparison, ANG II at the same concentrations, a GPCR (angiotensin
II receptor) agonist and a potent vasoactive agent, which has been
shown to stimulate lipogenesis in 3T3-L1 and human adipose cells (18),
did not show a significant effect. Fig. 1C shows that the
effect of ET-1 at 1 nM was completely blocked by 0.1 µM A-216546, an antagonist selective for ETA
receptor (19). As a comparison, 0.1 µM A-192621, an
antagonist selective for ETB receptor (20), had no effect
in blocking ET-1-stimulated glucose uptake. These results show that
ET-1 alone stimulates glucose uptake via activation of the
ETA receptor.

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Fig. 1.
Effects of insulin, ET-1, and angiotensin II
on 2-DOG uptake in the 3T3-L1 adipocytes. Cells in 96-well plates
were incubated with increasing concentrations of insulin in the
presence or absence of 10 nM ET-1 for 30 min (A)
or increasing concentrations of ET-1 or angiotensin II for 30 min
(B) and then assayed for the 2-DOG uptake as described under
"Materials and Methods." C, cells were incubated with 1 nM ET-1 in the presence of A-216546 (0.1 µM)
or A-192621 (0.1 µM). Each value represents the mean ± S.D. of four determinations. Statistical significance was determined
by the unpaired Student's t test. *, p < 0.001; **, p < 0.05.
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Effect of ET-1 Dependent on Adipocyte Differentiation--
Next,
we examined whether the effect of ET-1 on glucose uptake is dependent
on the differentiation state of the 3T3-L1 cells. Fig.
2 shows that, in undifferentiated 3T3-L1
cells, both insulin and ET-1 exhibited little or no effect on
stimulating 2-DOG uptake. In comparison, insulin at 100 nM
stimulated 2-DOG uptake in differentiated adipocytes by 9-fold (Fig.
2A), while ET-1 at 100 nM stimulated 2-DOG
uptake in adipocytes by 3-fold (Fig. 2B). The results show that the effect of ET-1, like that of insulin, is dependent upon the
differentiation state of the 3T3-L1 cells. From four independent experiments, the glucose uptake stimulation in 3T3-L1 adipocytes by 100 nM ET-1 in comparison with no treatment was 2.47 ± 0.23-fold (mean ± S.E.), and the EC50 value for ET-1
was determined to be 0.29 ± 0.13 nM. This effect is
consistent with the EC50 values observed in other
ET-1-mediated biological responses (21).

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Fig. 2.
Effects of insulin and ET-1 on 2-DOG uptake
in differentiated versus undifferentiated 3T3-L1
cells. Cells in 48-well plates were incubated with increasing
concentrations of insulin for 30 min (A) or increasing
concentrations of ET-1 for 30 min (B) and then assayed for
the 2-DOG uptake. The undifferentiated cells were maintained at ~80%
confluency in DMEM containing 10% FBS. The differentiated cells were
prepared as described under "Materials and Methods." Each value
represents the mean ± S.D. of four determinations.
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It is known that the expression of several proteins such as insulin
receptor and GLUT4 increases during 3T3-L1 differentiation. We examined
whether the expression of ET receptors was altered by differentiation.
The number of ET binding sites was determined in saturation binding
studies. As shown in Fig. 3A,
ET-1 binding to undifferentiated cells reached a plateau when free ET-1
concentration in the buffer was 0.6 nM. The Scatchard plot
(Fig. 3A, inset) resulted in a straight line and
yielded a maximum binding (Bmax) value of 670 fmol/106 cells and an equilibrium dissociation constant
(Kd) value of 0.52 nM. ET-1 saturation
binding in differentiated cells yielded a similar result with a
Bmax value of 640 fmol/106 cells and
a Kd value of 1.3 nM (not shown). From
three independent experiments, the Bmax and
Kd values were determined to be 890 ± 124 fmol/106 cells and 0.99 ± 0.67 nM
(mean ± S.E.) for the undifferentiated cells and 963 ± 191 fmol/106 cells and 1.65 ± 0.81 nM for the
differentiated cells. The results show that the 3T3-L1 cells express a
large number of ET receptors (~560,000 sites/cell), and the number of
ET binding sites is not significantly altered after cell
differentiation, although the affinity of ET-1 for the receptor seems
to be lower in the differentiated cells.

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Fig. 3.
ET-1 binding studies and ET-1-evoked PI
hydrolysis in 3T3-L1 cells. A, saturation binding
studies. Undifferentiated 3T3-L1 cells in 48-well plates were incubated
for 4 h at 4 °C with increasing concentrations of
125I-labeled ET-1 in the absence ( ) or presence ( ) of
1 µM unlabeled ET-1. Specific binding ( ) was
determined by subtraction of nonspecific binding ( ) from total
binding ( ). Each value represents the mean ± S.D. of three
determinations. Inset, Scatchard analysis of the data.
B, competition binding studies. Differentiated 3T3-L1 cells
in 48-well plates were incubated with 0.1 nM
125I-labeled ET-1 in the presence of increasing
concentrations of unlabeled ligands for 4 h at 4 °C. Results
are expressed as % of control (specific binding in the absence of
unlabeled ligand). Nonspecific binding, determined in the presence of 1 µM of ET-1, was subtracted from total binding to give
specific binding. Each value represents the mean ± S.D. of three
determinations. C, PI hydrolysis. Cells in 48-well plates
were prelabeled with myo-[3H]inositol (1 µCi/well) for 16 h.
Cells were challenged with various concentrations of ET-1 in buffer 1 with 10 mM LiCl for 30 min at 37 °C. Results were
calculated by normalizing AG1-X8-bound radioactivity at each point to
that of control (no addition of ET). Each value represents the mean of
two determinations.
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To find out what subtypes of ET receptors are expressed in the
differentiated 3T3-L1 cells, a competition binding study comparing ET-1
and ET-3 was conducted. Fig. 3B shows that unlabeled ET-1 effectively inhibited specific 125I-labeled ET-1 binding
(IC50 = 0.03 nM). ETB-selective
ligand ET-3 was much less effective in inhibiting specific
125I-labeled ET-1 binding with an IC50 value of
449 nM. These results show that ET receptor in 3T3-L1
adipocytes is predominantly the ETA subtype, which is
consistent with the observation in Fig. 1C.
To investigate whether other biological responses stimulated by ET-1
are also dependent on cell differentiation, we examined ET-1-evoked PI
hydrolysis in these cells. Fig. 3C shows that ET-1 stimulated PI hydrolysis in a dose-dependent manner. The
EC50 values were 0.17 nM for undifferentiated
cells versus 0.28 nM for differentiated cells.
The maximal stimulation was reached at ~1 nM ET-1 in both
cases, although the maximal stimulation was higher in the
undifferentiated cells. The results suggest that ET-1 stimulated PI
hydrolysis in both undifferentiated and differentiated cells in a
similar manner, possibly mediated by a Gq protein known
to be linked to PI hydrolysis.
Taken together, these results show that the differentiation of 3T3-L1
cells into adipocytes does not have a significant effect on the
expression of the ET receptor or on ET-1-stimulated PI hydrolysis.
However, differentiation specifically affects ET-1-stimulated glucose
uptake, suggesting that ET-1-stimulated glucose uptake may be linked to
GLUT4, which is expressed at a low level in the undifferentiated cells
but is greatly up-regulated during differentiation.
Effect of ET-1 on the Translocation of IRAP and GLUT4--
To
investigate whether ET-1 stimulated GLUT4 translocation,
immunofluorescence staining and confocal microscopy were employed. In
the control slides without ET-1 or insulin treatment, cells were found
to have GLUT4 located in punctate structures distributed throughout the
cytoplasm with intense staining concentrated on some spots and a low
level of immunofluorescence in the plasma membrane; a typical example
was shown in Fig. 4A,
part a. As expected, cells treated with insulin
(100 nM) exhibited a higher level of immunofluorescence in
the plasma membrane (Fig. 4A, part f),
indicating GLUT4 translocation to the plasma membrane. Cells treated
with ET-1 (10 nM) also exhibited an increase in the level
of immunofluorescence in the plasma membrane (Fig. 4A,
parts b-d). The effect of ET-1 on GLUT4
translocation was time-dependent with the optimal effect observed at 20 min (Fig. 4A, part c),
which was blocked by 10 µM A-216546 (Fig. 4A,
part e). The results suggest that both insulin and ET-1 stimulate GLUT4 translocation, although the effect of ET-1 may
be less than that of insulin. The results are consistent with the
observation in the 2-DOG uptake studies.

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Fig. 4.
Effects of insulin and ET-1 on the
translocation of GLUT4 and IRAP in 3T3-L1 adipocytes.
A, immunofluorescence staining of GLUT4. Differentiated
3T3-L1 cells were treated with collagenase I and plated on chamber
slides in DMEM containing 10% FBS for 24 h. Cells were put in
serum-free DMEM for 3 h before treated with medium (a,
control), or 10 nM ET-1 (b, 10 min;
c, 20 min; d, 30 min), or 10 nM ET-1
in the presence of 10 µM A-216546 for 20 min
(e), or 100 nM insulin for 10 min
(f). Cells were then fixed and stained with an anti-GLUT4
antibody as described under "Materials and Methods." B,
Western blot of membrane-associated IRAP. Differentiated 3T3-L1 cells
in 24-well plates were treated with insulin or ET-1 (concentrations as
indicated) for 10 min. Plasma membrane-associated proteins were
cross-linked to biotin and isolated by streptavidin-agarose beads. The
lysates were analyzed by SDS-PAGE and Western blotting as described
under "Materials and Methods." The SDS-PAGE was also detected by
silver staining to make sure that equal amounts of proteins were used
for the samples. The molecular mass of IRAP is 165 kDa. The results are
representative of three different experiments.
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To further demonstrate that indeed ET-1 induces the translocation of
GLUT4-containing vesicles in the 3T3-L1 adipocytes, we compared the
effects of ET-1 and insulin on IRAP translocation using a sensitive
cell surface biotinylation method (for details, see "Materials and
Methods"). IRAP is an aminopeptidase that is one of the major
polypeptides enriched in GLUT4-containing vesicles and is known to
co-translocate with GLUT4 (22). Fig. 4B shows that both
insulin and ET-1 caused an increase in membrane-associated IRAP in a
dose-dependent manner, suggesting that ET-1, like insulin, indeed stimulates the translocation of GLUT4-containing vesicles from
cytosol to the plasma membrane.
Effect of ET-1 on Proteins Involved in the Insulin Signaling
Pathway--
To investigate whether ET-1 treatment affects insulin
signaling molecules, we examined the effect of ET-1 on the
phosphorylation of Akt, IRS-1, and the
-subunit of the insulin
receptor (IR
). Fig. 5A
(left) shows that insulin at 100 nM stimulated
the tyrosyl phosphorylation of IRS-1 and IR
after 10 min of
incubation, while ET-1 had no effect. The identities of IRS-1 and IR
were confirmed using anti-IRS-1 and anti-IR
antibodies in Western
blot analysis (not shown). Interestingly, ET-1 stimulated the tyrosyl
phosphorylation of a 75-kDa protein in a time-dependent
manner (Fig. 5A). Fig. 5B shows that insulin
stimulated Akt phosphorylation in a dose-dependent manner,
while ET-1 had no effect. These results suggest that ET-1 treatment
does not stimulate phosphorylation of IR
, IRS-1, or Akt. Probably,
ET-1 stimulates glucose uptake via an insulin-independent pathway.

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Fig. 5.
. Effects of insulin and ET-1 on the
phosphorylation of IRS-1, ER , and Akt in 3T3-L1 adipocytes.
A, differentiated 3T3-L1 cells in 48-well plates were
treated with 100 nM insulin for 10 min or ET-1 for 40 min
(left) or 100 nM ET-1 for 10 or 30 min
(right). Cells were lysed in a buffer containing 50 mMTris-HC1, 1% Nonidet P40, 1 mM sodium
orthovanadate and then centrifuged at 10,000 × g to remove nuclei and
debris. The supernatants were retained to be analyzed by SDS-PAGE and
Western blotting. The samples were blotted using an
anti-phosphotyrosine antibody as described. B,
differentiated 3T3-L1 cells were treated with increasing concentrations
of insulin or ET-1 FOR 10 min, and cells were lysed in the SDS-PAGE
sample buffer. The samples were blotted using an anti-phospho-Akt
antibody. The results ate representative of three different
experiments.
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Effects of Kinase Inhibitors on ET-1-stimulated Glucose Uptake in
3T3-L1 Adipocytes--
To investigate the mechanism of ET-1-stimulated
glucose uptake, we examined the roles of PKC, PI 3-kinase, and MAPK,
the three kinases known to be activated by ET-1 (10). Fig.
6, A-C, shows that
bisindolylmaleimide (bisindo; an inhibitor of PKC), PD98059 (PD; an inhibitor of MEK1/2), and wortmannin (an inhibitor
of PI 3-kinase) seemed to partially inhibit the effect of ET-1.
However, these agents also inhibited the basal glucose uptake. The
insets in Fig. 6, A-C, show that, after
normalizing ET-1-stimulated glucose uptake by the basal glucose uptake
in the presence of the inhibitor, none of the inhibitors had a
significant effect. In control experiments, 1 µM
wortmannin blocks insulin-stimulated glucose uptake in 3T3-L1 adipocytes by >80%, while 80 µM PD98059 inhibits
ET-1-stimulated extracellular signal-related kinases 1/2 in human
smooth muscle cells by >50%
(23).2 We then tested the
effect of genistein, a general tyrosine kinase inhibitor. Fig.
6D shows that genistein at 1 and 10 µM had no effect. However, genistein at 100 µM inhibited
ET-1-stimulated glucose uptake by ~70%. These results suggest that
ET-1-stimulated glucose uptake in the 3T3-L1 adipocytes is independent
of PKC, PI 3-kinase, or the mitogen-activated protein kinase pathway
but may be mediated by a genistein-sensitive tyrosine kinase.

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Fig. 6.
Effects of kinase inhibitors on insulin or
ET-1 stimulated 2-DOG uptake in 3T3-L1 adipocytes. Cells in
48-well plates were treated with different concentrations of
bisindolylmaleimide (bisindo) (A), wortmannin
(B), PD98059 (C), or genistein (D) for
30 min before being treated with 10 nM ET-1 for another 30 min. The uptake of 2-DOG was then assayed. Each value represents the
mean of four determinations. Statistical significance was determined by
the unpaired Student's t test. *, p < 0.001.
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DISCUSSION |
It is known that signal transduction mediated by certain GPCRs
interacts with the insulin signaling pathway to regulate cellular functions in a complicated manner. In some cases, the interaction leads
to insulin resistance and a decrease in insulin-stimulated intracellular signaling. In others, the GPCRs and insulin work in
an additive manner to stimulate a cellular function. For example, it
has been shown that angiotensin II causes an acute inhibition of both
basal and insulin-stimulated PI 3-kinase activity in the rat heart and
in rat aortic smooth muscle cells (24, 25). In cardiomyocytes
isolated from adult rat hearts, the effect of insulin on glucose uptake
can be partially blocked by modifying G-proteins with cholera toxin,
yet isoprenaline alone, like insulin, increases glucose transport (26).
Also, in both cardiomyocytes and brown adipocytes, adrenergic
stimulation induces GLUT4 translocation and glucose uptake (27, 28).
Furthermore, the introduction of GTP analogs such as GTP
S into
3T3-L1 adipocytes stimulates GLUT4 translocation and glucose uptake
independent of insulin (5-7). Possibly, GPCRs play a role in the
pathogenesis of insulin resistance and cardiovascular diseases by
either modulating glucose uptake or directly interacting with
insulin signaling.
In this report, we show for the first time, in an unequivocal manner,
that ETA receptor is expressed in 3T3-L1 adipocytes and
that ET-1 stimulates GLUT4 translocation and glucose uptake in these
cells. In comparison with insulin, which stimulates glucose transport
by a magnitude of 6-10-fold in 3T3-L1 adipocytes, the effect of ET-1
may seem modest (2-3-fold stimulation). However, an additive effect is
observed when adipocytes are treated with low concentrations of insulin
(
1 nM) and ET-1 simultaneously, suggesting that they may
be acting through independent pathways. Our studies examining the
phosphorylation of IR
, IRS-1, and Akt confirm that ET-1 has no
effect on the early signaling molecules activated by insulin. Is the
effect of ET-1 mediated by GLUT4 in the 3T3-L1 adipocytes? Results from
studies comparing the differentiated versus undifferentiated
3T3-L1 cells show that the numbers of ET-1 binding sites are not
significantly different in undifferentiated versus
differentiated cells, but ET-1-stimulated glucose uptake is
significantly higher in the differentiated cells. These results suggest
that the effect of ET-1 on glucose uptake is probably linked to the
presence of GLUT4 in one state versus the other. Indeed, we
show that ET-1 directly stimulates the translocation of GLUT4 and IRAP.
Our results demonstrate that immunofluorescence staining using the
confocal microscopy is a sensitive method to examine GLUT4
translocation stimulated by ET-1 or insulin, although the effect of
ET-1 on glucose uptake is ~25% of that of insulin. Furthermore, the
cell surface biotinylation method, which has been used to detect IRAP
translocation, is an extremely sensitive way to examine the increase of
a protein in the plasma membrane (22). Fig. 4B shows that
both insulin and ET-1 stimulate IRAP translocation in a
dose-dependent manner. We do not have an explanation of why
ET-1 stimulates the increase in membrane-associated IRAP to the same
degree as insulin, because the effect of ET-1 on glucose uptake is
clearly less than that of insulin.
The receptor binding studies show that 3T3-L1 cells express
predominantly the ETA receptor. Consistent with the binding
studies, A-216546, an antagonist selective for the ETA
receptor, completely blocks the effect of ET-1 on glucose uptake, while
A-192621, an antagonist selective for the ETB receptor,
does not have an effect. Although in this report we did not address the
issue of which G-protein is involved in ET-1-stimulated glucose uptake,
ET-1 probably activates Gq and PLC-
in these cells due
to the observation that ET-1 stimulates PI hydrolysis in both
undifferentiated and differentiated 3T3-L1 cells. Gq is
coupled to phosphoinositide-specific phospholipase C-
, which
hydrolyzes phosphatidylinositol 4,5-bisphosphate to form inositol
1,4,5-triphosphate and 1,2-diacylglycerol. Interestingly, although PKC
is the downstream target of phospholipase C-
and PI hydrolysis, our
results show that the PKC inhibitor does not have a significant effect
on ET-1-stimulated glucose uptake, suggesting that PKC is not involved,
consistent with the observation by Kishi et al. (6) on the
PAF receptor and the
1a-adrenergic receptor. Furthermore, wortmannin and PD98059 do not affect the 2.5-fold of
stimulation in glucose uptake induced by ET-1, suggesting that PI
3-kinase and the mitogen-activated protein kinase pathway are not
involved. Genistein, which has been shown to inhibit GTP
S-stimulated GLUT4 translocation (7), seems to inhibit ET-1-stimulated glucose uptake at a high concentration (100 µM). It is
interesting to note that, in the Western blot analysis using the
anti-phosphotyrosine antibody, we observe an increase in the tyrosyl
phosphorylation of a 75-kDa protein after ET-1 treatment. Possibly, a
genistein-sensitive tyrosine kinase plays a role in mediating
ET-1-stimulated glucose uptake. We are in the process of investigating
whether the 75-kDa protein is involved in ET-1-stimulated glucose uptake.
It is known that the skeletal muscle plays a central role in glucose
metabolism, and impairment in glucose metabolism in the skeletal muscle
often results in diabetes. Although this report mainly focuses on the
3T3-L1 adipocytes, we have found from both reverse transcription-PCR
and receptor binding studies that human skeletal muscle cells express
predominantly ETA receptor with Bmax
and Kd values of 81.6 fmol/106 cells (or
49,000 sites/cell) and 0.14 nM for ET-1 binding. In membranes prepared from rat skeletal muscle (soleus), ET-1 binding is
of high affinity with Bmax and
Kd values of 58.4 fmol/mg of protein (or 3.5 × 1010 sites/mg of protein) and 0.15 nM. In
addition, we have observed that ET-1 stimulates glucose uptake in
neonatal rat cardiomyocytes (29). These results imply that the ET-1
system may play a role in glucose metabolism in both adipose and muscle
tissues and is potentially a useful model to study the link between
GPCRs and insulin signaling.
Does the finding that ET-1 stimulates GLUT4 translocation and glucose
uptake have any physiological significance? We propose two possible
scenarios. The first is that ET-1 is involved in exercise/hypoxia-induced glucose uptake, which occurs in an
insulin-independent manner. It is now well accepted that the plasma
ET-1 level is significantly elevated under hypoxic conditions (30). It
has also been reported that exercise tends to elevate ET-1 in
plasma and in major organs such as heart and kidney (31-34), although these results are not as conclusive as the hypoxia studies (35). So
far, there is no report on whether hypoxia/exercise-induced increase in
ET-1 is linked to glucose metabolism. A second completely different
scenario is that chronic elevation of ET-1 localized in the skeletal
muscle may cause a constant, albeit modest, increase in glucose influx
into the muscle, which may result in insulin resistance from glucose
toxicity. Infusion of ET-1 into rats has been shown to induce insulin
resistance in one study (36) but to reduce the blood glucose level in
another (37, 38). More studies will be needed to further examine
whether ET-1 plays a physiological or pathophysiological role in
glucose metabolism.
In conclusion, we have shown that 3T3-L1 adipocytes express
ETA receptor. ET-1 alone stimulates glucose uptake in these
cells. The effect of ET-1 on glucose uptake is dependent on the
differentiation of the adipocytes, suggesting a link to the expression
of GLUT4, and consistent with the observation that ET-1 activates IRAP
and GLUT4 translocation in adipocytes.