An Inhibitor of p38 Mitogen-activated Protein Kinase Prevents
Insulin-stimulated Glucose Transport but Not Glucose Transporter
Translocation in 3T3-L1 Adipocytes and L6 Myotubes*
Gary
Sweeney
,
Romel
Somwar
§,
Toolsie
Ramlal
,
Allen
Volchuk
§,
Atsunori
Ueyama
, and
Amira
Klip
§¶
From the
Programme in Cell Biology, Hospital for Sick
Children and § Department of Biochemistry, University of
Toronto, Toronto, Ontario, M5G 1X8 Canada
 |
ABSTRACT |
The precise mechanisms underlying
insulin-stimulated glucose transport still require investigation. Here
we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase
family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes
and L6 myotubes. We found that SB203580, but not its inactive analogue
(SB202474), prevented insulin-stimulated glucose transport in both cell
types with an IC50 similar to that for inhibition of
p38 MAP kinase (0.6 µM). Basal glucose uptake was not
affected. Moreover, SB203580 added only during the transport assay did
not inhibit basal or insulin-stimulated transport. SB203580 did not
inhibit insulin-stimulated translocation of the glucose transporters
GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of
subcellular fractions or by immunofluorescence of membrane lawns. L6
muscle cells expressing GLUT4 tagged on an extracellular domain with a
Myc epitope (GLUT4myc) were used to assess the functional insertion of
GLUT4 into the plasma membrane. SB203580 did not affect the
insulin-induced gain in GLUT4myc exposure at the cell surface but
largely reduced the stimulation of glucose uptake. SB203580 had no
effect on insulin-dependent insulin receptor substrate-1
phosphorylation, association of the p85 subunit of phosphatidylinositol
3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol
3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In
conclusion, in the presence of SB203580, insulin caused normal
translocation and cell surface membrane insertion of glucose
transporters without stimulating glucose transport. We propose that
insulin stimulates two independent signals contributing to stimulation
of glucose transport: phosphatidylinositol 3-kinase leads to glucose
transporter translocation and a pathway involving p38 MAP kinase leads
to activation of the recruited glucose transporter at the membrane.
 |
INTRODUCTION |
The phenomenon of insulin-stimulated glucose transporter (GLUT)
translocation from an intracellular location to the plasma membrane has
been demonstrated in several fat and muscle cell systems since its
initial report in 1980 (1-3). Whether increased plasma membrane
glucose transporter content can fully account for insulin-stimulated
increases in glucose uptake is still being debated (4). It has been
proposed that translocation of GLUTs might only account for as little
as 30% of insulin-stimulated glucose transport (4), with the majority
of insulin-stimulated glucose transport being due to changes in the
intrinsic activity of GLUTs (5-7).
The intracellular signaling machinery employed by insulin in
eliciting glucose transport has also been extensively investigated yet
a precise understanding remains to be achieved (8). To date, only one
signaling molecule involved in GLUT translocation has been
unequivocally identified, i.e. the p85/p110
phosphatidylinositol (PI)
3-kinase1 (9). Debate
currently exists on which insulin receptor substrates activate the PI
3-kinase pathway leading to GLUT translocation (10-12), as well as on
whether the PI 3-kinase effector Akt/PKB is also required (13-15).
There is no evidence for or against the participation of any
insulin-dependent signaling pathway in regulation of the
intrinsic activity of glucose transporters. The mitogen-activated protein kinase (MAPK) cascades represent one of the main intracellular signaling pathways stimulated by mitogenic and stress-inducing stimuli
(16, 17) and their involvement in glucose transport depends on the
tissue and the agonist analyzed (18). Three families of these
proline-directed serine threonine protein kinases, which are activated
by dual phosphorylation on threonine and tyrosine residues, have been
identified (19). One of these is the p38 MAPK (also known as
reactivating kinase, stress-activated protein kinase, and CSAID-binding
protein) family, activation of which has been best characterized in
response to stressors (such as UV light and hyperosmolarity) and
cytokines (such as interleukin-1 or tumor necrosis factor-
) (20). We
have shown that insulin can phosphorylate p38 in L6 myotubes (21, 22)
and insulin has been shown to stimulate p38 activity in hepatoma cells
(23), yet not in skeletal muscle (24).
A group of pyridinyl imidazole compounds were recently reported to bind
to and potently inhibit p38 MAP kinase. One of these, SB203580,
specifically inhibited the
and
isoforms of p38 MAP kinase with
an in vitro IC50 of ~0.6 µM,
while having no effect on the activity of 12 other closely related or
prominent intracellular kinases (25). In KB cells, SB203580 prevented
the stimulation of glucose transport elicited by the potent activator
of p38 MAP kinase anisomycin and by IL-1, but had no effect on
insulin-like growth factor 1-stimulated glucose transport (26).
SB203580 also inhibited anisomycin-stimulated glucose transport in
3T3-L1 adipocytes (27), with an IC50 of <1
µM.
In the present study we assessed the effect of SB203580 and its
inactive structural analogue, SB202474, on insulin-stimulated glucose
transport. We report that SB203580 inhibits uptake of 2-deoxyglucose or
3-O-methylglucose in muscle and fat cells without interfering with translocation of GLUTs. This effect was not due to
direct binding of SB203580 to GLUTs nor to a nonspecific effect on
other signaling molecules. The results suggest that SB203580 attenuates
the ability of insulin to stimulate the intrinsic activity of glucose
transporter molecules recruited to the plasma membrane.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All cell culture solutions and supplements were
obtained from Life Technologies, Inc. (Burlington, ON, Canada). 3T3-L1
cells were a kind gift from Dr. G. Holman (University of Bath, United Kingdom). L6 cells transfected with Myc-tagged GLUT4 were kindly provided by Dr. Y. Ebina (University of Tokushima, Japan). Human insulin (Humulin) was obtained from Eli Lilly Canada Inc. (Toronto, ON,
Canada). Protein A- and protein G-Sepharose were from Pharmacia (Uppsala, Sweden). Polyclonal anti-GLUT1 and anti-GLUT4 glucose transporter antiserum was from East Acres Laboratories (South Bridge,
MA). Polyclonal antibodies to Akt1 (C-20) and p38 MAP kinase and
monoclonal antibody to Myc were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Akt1 substrate peptide (Crosstide), monoclonal
anti-phosphotyrosine, polyclonal anti-IRS-1, and Ak+2 and Ak+3
antibodies were purchased from Upstate Biotechnology (Lake Placid, NY).
[
-32P]ATP (6000 Ci/mmol) and enhanced
chemiluminescence (ECL) reagents were purchased from Amersham
(Oakville, ON, Canada). 2-D-Deoxy-[3H]glucose
and 3-O-[3H]methylglucose were purchased from
NEN (Mississauga, ON, Canada). ATF-2 peptide was from New England
Biolabs (Mississauga, ON, Canada). Fluorescein
isothiocyanate-conjugated donkey anti-rabbit and horseradish peroxidase-conjugated sheep anti-rabbit and anti-mouse antiserum were
from Jackson Immunoresearch (Baltimore Pike, PA). ProLong antifade
mounting solution was from Molecular Probes (Eugene, OR). Purified
L-
-phosphatidylinositol (PI) was purchased from Avanti
Polar Lipids Inc. (Alabaster, AL). Oxalate-treated TLC Silica Gel H
plates (250 µm) were from Analtech (Newark, DE). o-Phenylenediamine dihydrochloride (OPD reagent) was from
Sigma. Okadaic acid was from Biomol (Plymouth Meeting, PA). SB203580 and SB202474 were from Calbiochem (La Jolla, CA). All electrophoresis and immunoblotting reagents were purchased from Bio-Rad (Mississauga, ON, Canada). All other reagents were of the highest analytical grade.
Cell Culture--
3T3-L1 cells were grown in monolayer culture
in Dulbecco's modified Eagle's medium supplemented with 10% (v/v)
calf serum and 1% (v/v) antibiotic solution (10,000 units/ml
penicillin and 10 mg/ml streptomycin) in an atmosphere of 5%
CO2 at 37 °C. 3T3-L1 fibroblasts were differentiated
into adipocytes as described previously (28). L6 cells from a
spontaneously fusing subclone of the original L6 muscle cells and L6
cells stably overexpressing GLUT4 tagged with a Myc epitope were grown
and differentiated into myotubes as described previously (29). For
transport studies cells were treated with trypsin and seeded in 12-well
plates (2.5-cm diameter well) and maintained in growth medium as
described above except supplemented with 2% fetal bovine serum. Cells
were maintained under the same conditions in 6-well plates for
preparation of whole cell lysates and in 10-cm diameter dishes for immunoprecipitations.
Analysis of p38 MAP Kinase Phosphorylation--
Cells treated
with insulin or SB203580 as indicated were lysed in 1 ml of lysis
buffer containing 137 mM NaCl, 20 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2,
100 µM Na3VO4, 2 mM
PMSF, 10% glycerol (v/v), and 1% Nonidet P-40 (v/v) (pH 7.5). Cell
lysates were then centrifuged for 5 min at 12,000 rpm to remove cell
debris and nuclei. Antiphosphotyrosine antibody (2 µg/condition) was
then added to supernatants for overnight incubation under rotation at
4 °C, followed by addition of 30 µl (10% w/v) of protein
A-Sepharose for 1 h. The immunoprecipitation pellets were washed
three times with PBS containing 0.1% Nonidet P-40 and 100 µM Na3VO4, solubilized in 30 µl
of 2 × Laemmli sample buffer, boiled for 5 min, and separated by
10% SDS-PAGE. Anti-p38 MAP kinase polyclonal antibody (1:1000 dilution) was added to the polyvinylidene difluoride membrane, followed
by goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase
(1:5000 dilution) and protein was visualized by the enhanced
chemiluminescence method.
Assay of p38 MAP Kinase and Akt (PKB)
Activity--
Immunoprecipitation of p38 MAP kinase and Akt isoforms
and analysis of their kinase activity was performed in a similar
fashion to that described previously for Akt1 (30). For both assays, cells were lysed with lysis buffer containing 50 mM HEPES,
pH 7.6, 150 mM NaCl, 10% glycerol (v/v), 1% Triton X-100
(v/v), 30 mM Na4P2O7,
10 mM sodium fluoride, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 1 mM
Na3VO4, 1 mM dithiothreitol, and
100 nM okadaic acid. Polyclonal anti-p38 MAP kinase
antibody (2 µg/condition) precoupled to a mixture of protein A- and
protein G-Sepharose (20 µl (100 mg/ml) each per condition) beads was
added to 200 µg of total protein from cell lysates. Anti-Akt1, -Akt2,
and -Akt3 antibodies were also precoupled to a mixture of protein A-
and protein G-Sepharose (2 µg/condition) and added to 200 µg of
total protein. Antibody coupled beads were washed twice with ice-cold PBS and once with ice-cold lysis buffer before use. Proteins were immunoprecipitated by incubating with the antibody bead complex for
2-3 h under constant rotation (4 °C). Immunocomplexes were isolated
and washed 4 times with 1 ml of wash buffer (25 mM HEPES, pH 7.8, 10% glycerol (v/v), 1% Triton X-100 (v/v), 0.1% bovine serum
albumin, 1 M NaCl, 1 mM dithiothreitol, 1 mM PMSF, 1 µM microcystin, and 100 nM okadaic acid) and twice with 1 ml of kinase buffer (50 mM Tris/HCl, pH 7.5, 10 mM MgCl2,
and 1 mM dithiothreitol). The complexes were then incubated
under constant agitation for 30 min at 30 °C with 30 µl of
reaction mixture (kinase buffer containing 5 µM ATP, 2 µCi of [
-32P]ATP plus 2 µg of ATF-2 as substrate
in p38 MAP kinase assays and 100 µM Crosstide as
substrate in Akt assays). Following the reaction, 30 µl of the
supernatant was transferred onto Whatman p81 filter paper and washed 4 times for 10 min with 3 ml of 175 mM phosphoric acid and
once with distilled water for 5 min. Filters were air-dried and then
subjected to liquid scintillation counting.
Determination of 2-Deoxyglucose and 3-O-Methylglucose
Uptake--
L6 myotubes were deprived of serum for 5 h with
-minimal essential medium, 0.1% fetal bovine serum (v/v), and 25 mM glucose (serum-deprivation medium) while 3T3-L1
adipocytes were deprived of serum by incubation in Dulbecco's modified
Eagle's medium for 2 h before experimental manipulations.
2-Deoxyglucose uptake measurements were carried out as described
previously (31). Briefly, following all stimulations and incubations
with inhibitors cell monolayers were washed twice with HEPES-buffered
saline (140 mM NaCl, 20 mM Na-HEPES, 2.5 mM MgSO4, 1 mM CaCl2, 5 mM KCl, pH 7.4) and any remaining liquid was aspirated.
Cells were then incubated for 5 min in HEPES-buffered saline containing
10 µM unlabeled 2-deoxyglucose and 10 µM
D-2-deoxy-[3H]glucose (1 µCi/ml for L6 and
0.5 µCi/ml for 3T3-L1) in the absence of insulin. The reaction was
terminated by washing three times with ice-cold 0.9% NaCl (w/v).
Nonspecific uptake was determined in the presence of 10 µM cytochalasin B. Cell associated radioactivity was
determined by lysing the cells with 0.05 N NaOH, followed by liquid scintillation counting. Total cellular protein was determined by the Bradford method (32). 3-O-Methylglucose uptake was
measured in a similar fashion with the following differences: 50 µM 3-O-methylglucose (4 µCi/ml) was added to
HEPES-buffered saline and uptake allowed to occur for 30 s, a
period over which 3-O-methylglucose uptake is known to be
linear. After this time, cell monolayers were washed three times with 1 mM HgCl2 in saline solution before lysis with 0.05 N NaOH.
Plasma membrane Lawn Formation and Immunofluorescence
Labeling--
Differentiated 3T3-L1 adipocytes, grown on glass
coverslips in 6-well dishes, were treated with SB203580 plus or minus
insulin as described in the figure legends. Plasma membrane lawns
(sheets) were prepared as described previously (33) with slight
modifications. Following the various treatments, the cells were placed
on ice and washed twice in ice-cold PBS. Hypotonic swelling buffer (23 mM KCl, 10 mM Na-HEPES, 2 mM
MgCl2, 1 mM EGTA, pH 7.5) was added in three
quick rinses. Five ml of breaking buffer (70 mM KCl, 30 mM Na-HEPES, 5 mM MgCl2, 3 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, 1 µM pepstatin A, pH 7.5) were added
to each well, and the solution was aspirated up and down using a 1.0-ml
pipette to promote cell breakage. The coverslips were washed three
times in breaking buffer and incubated with cold 3% paraformaldehyde in breaking buffer for 10 min on ice, followed by three washes in PBS.
Excess fixative was quenched with 50 mM
NH4Cl/PBS for 5 min, followed by three washes with PBS at
room temperature. The lawns were subsequently blocked by a 1-h
incubation in 5% goat serum in PBS at room temperature, then incubated
with rabbit anti-GLUT4 antiserum (1:150) for 30 min at room temperature
and washed three times in PBS. Fluorescein isothiocyanate-conjugated donkey anti-rabbit antiserum (1:50) was added for 30 min then rinsed
out with four washes with PBS and the coverslips mounted with ProLong
Antifade mounting solution. Confocal images were obtained using a Leica
TCS 4D laser confocal fluorescence microscope with a 63X objective. All
images shown were collected under identical gain settings.
Quantification was made using NIH Image software.
Fractionation of 3T3-L1 Adipocytes and Immunoblotting for
GLUTs--
Subcellular fractionation was carried out essentially as
described previously (34). Cells were homogenized in ice-cold 255 mM sucrose, 0.5 mM PMSF, 1 µM
pepstatin A, 10 µM E-64, 1 mM EDTA, and 20 mM Na-HEPES (pH 7.4) and the homogenate centrifuged at 19,000 × g for 20 min. The resulting supernatant was
centrifuged at 41,000 × g to pellet the crude plasma
membranes; the supernatant from this step was centrifuged at
180,000 × g for 75 min to yield the low density
microsomes. Crude plasma membranes were further purified by layering on
a sucrose cushion (1.12 M sucrose, 1 mM EDTA,
and 20 mM Na-HEPES, pH 7.4) and centrifuged at 100,000 × g in a Beckman SW-55 rotor for 60 min. The band at the
interface was resuspended in homogenization buffer and pelleted at
40,000 × g for 20 min to yield purified plasma
membranes. All fractions were resuspended in homogenization buffer to a
final concentration of 2-10 mg/ml and stored at
80 °C.
Measurement of GLUT4myc Translocation in L6
Myotubes--
The movement of Myc-tagged GLUT4 to the cell surface was
measured by an antibody-coupled colorimetric assay (35) as follows. Quiescent L6 GLUT4myc cells treated as indicated in the legend for
Table II were washed once with PBS, fixed with 3% paraformaldehyde in
PBS for 3 min at room temperature, and the fixative was immediately neutralized by incubation with 1% glycine in PBS at 4 °C for 10 min. The cells were blocked with 10% goat serum and 3% bovine serum
albumin in PBS at 4 °C for at least 30 min. Primary antibody (anti-c-Myc, 9E10) was then added into the cultures at a dilution of
1:100 and maintained for 30 min at 4 °C. The cells were extensively washed with PBS before introducing peroxidase-conjugated rabbit anti-mouse IgG (1:1000). After 30 min at 4 °C, the cells were extensively washed and 1 ml of o-phenylenediamine
dihydrochloride reagent (0.4 mg/ml o-phenylenediamine
dihydrochloride and 0.4 mg/ml urea hydrogen peroxide in 0.05 M phosphate/citrate buffer) was added to each well for 10 min at room temperature. The reaction was stopped by addition of 0.25 ml of 3 N HCl. The supernatant was collected and the
optical absorbance was measured at 492 nm.
Detection of IRS-1 Phosphorylation and Association of
p85--
3T3-L1 adipocytes were treated with 100 nM
insulin for 5 min then IRS-1 was immunoprecipitated and tyrosine
phosphorylation of IRS-1 and association of the p85 subunit of PI
3-kinase was determined essentially as described previously (31).
Briefly, immunoprecipitated proteins were resolved by 7.5% SDS-PAGE
and then electrotransferred onto polyvinylidene difluoride membranes. To detect tyrosine-phosphorylated IRS-1 the upper part of the blot was
probed with anti-phosphotyrosine antibody (monoclonal, 1:5000 dilution)
and protein detected by the enhanced chemiluminescence method using
sheep anti-mouse immunoglobulin conjugated to horseradish peroxidase as
secondary antibody. The lower part of the blot was probed with anti-p85
antibody (polyclonal, 1:1000 dilution) and protein detected using
anti-rabbit IgG conjugated to horseradish peroxidase.
Assay of PI 3-Kinase Activity Associated with IRS-1--
To
determine PI 3-kinase activity, cell extracts were prepared exactly the
same way as for IRS-1 immunoprecipitation and PI 3-kinase activity was
measured on IRS-1 immunoprecipitates as described previously (36).
Briefly, the ability of PI 3-kinase associated with IRS-1 to convert
phosphatidylinositol to phosphatidylinositol 3-phosphate was detected
by separation of these lipids by thin layer chromatography (TLC).
Detection and quantitation of [32P]phosphatidylinositol
3-phosphate on the TLC plates were done using a Molecular Dynamics
PhosphorImager System (Sunnyvale, CA).
Statistical Analysis--
Statistical analysis was performed
using either unpaired Student's t test or analysis of
variance test (Fischer, multiple comparisons) as indicated in the
figure legends.
 |
RESULTS |
Stimulation of Phosphorylation and Activity of p38 MAP Kinase by
Insulin--
Activation of p38 MAP kinase involves dual
phosphorylation on tyrosine and threonine. Insulin stimulated
phosphorylation of p38 MAP kinase in 3T3-L1 adipocytes measured by
phosphotyrosine immunoprecipitation followed by Western blotting with
anti-p38 MAP kinase antibody (Fig.
1A). We have previously shown
a similar result in L6 myotubes (22). SB203580, which interacts with
the ATP-binding domain of p38 MAP kinase (37), did not affect the basal
or insulin-stimulated level of p38 phosphorylation. An in vitro kinase assay was then used to measure the ability of p38 MAP
kinase to phosphorylate one of its known natural substrates, ATF-2.
Insulin caused an approximately 5-fold increase in p38-mediated ATF-2
phosphorylation and this effect was inhibited by SB203580 (Fig.
1B).

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Fig. 1.
Stimulation of p38 MAP kinase phosphorylation
and activity by insulin in 3T3-L1 adipocytes and effect of
SB203580. 3T3-L1 adipocytes were treated with or without 10 µM SB203580 for 20 min followed by insulin treatment (100 nM) for 5 min, as indicated. A, tyrosine
phosphorylation of p38 MAP kinase was analyzed as described under
"Experimental Procedures." B, in vitro kinase
activity of p38 MAP kinase was determined as described under
"Experimental Procedures." Results are from one representative
experiment from at least three replicates.
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Effect of SB203580 and SB202474 on 2-Deoxyglucose and
3-O-Methylglucose Uptake--
Fig.
2A shows a dose response of
the effect of SB203580 on the uptake of 3H-labeled
2-deoxyglucose into 3T3-L1 adipocytes. The compound was given to cells
for 20 min prior to addition of insulin. Treatment with the hormone
alone for 30 min caused an increase in glucose uptake of over 4-fold
(control, 7.4 ± 0.7 pmol/min/mg protein: insulin, 26.9 ± 4.1 pmol/min/mg protein (p < 0.05)). Pretreatment with
a range of concentrations of SB203580 from 1 nM to 0.1 mM prevented stimulation by insulin in a
dose-dependent fashion. The IC50 for this
effect was calculated to be 0.75 µM. Fig. 2A also shows that, over the same range of concentrations, SB203580 had no
effect on basal 2-deoxyglucose uptake in these cells. Fig. 2B shows that the dose-dependent inhibitory
effect of SB203580 on insulin-stimulated 2-deoxy-D-glucose
transport was also observed in L6 myotubes. In this case the
IC50 was 0.1 µM and again SB203580 had no
effect on basal glucose uptake.

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Fig. 2.
Dose-dependent inhibition of
insulin-stimulated 2-deoxy-D-glucose transport by SB203580
in 3T3-L1 adipocytes and L6 myotubes. Differentiated 3T3-L1
adipocytes (A) and L6 myotubes (B) were treated
with increasing concentrations of SB203580 for 20 min prior to the
addition of 100 nM insulin (closed circles) for
an additional 30 min in the same medium as indicated (basal = open circles). Cells were rinsed then
2-deoxy-D-glucose uptake measured as described under
"Experimental Procedures." Results shown are the mean ± S.E.
of at least three individual experiments within which each point was
assayed in triplicate.
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Fig. 3 compares the effect of SB203580
with that of the structurally similar compound, SB202474, on
insulin-stimulated 2-deoxyglucose and 3-O-methylglucose
transport in 3T3-L1 adipocytes. SB202474 is unable to inhibit p38 MAP
kinase and in this context is considered to be a functionally inactive
analogue of SB203580 (38). As expected from results presented in Fig.
1A, SB203580 (10 µM) inhibited insulin-stimulated 2-deoxyglucose uptake by approximately 60% (p < 0.05) (Fig. 3A). However, SB202474 (10 µM) had no effect on the ability of insulin to stimulate
2-deoxyglucose uptake. Neither SB203580 nor SB202474 had any effect on
basal 2-deoxyglucose transport level (Fig. 3A).

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Fig. 3.
Comparison of the effect of SB203580 and
SB202474 on insulin-stimulated 2-deoxy-D-glucose and
3-O-methylglucose transport in 3T3-L1 adipocytes.
Cells were treated with 10 µM SB203580 or 10 µM SB202474 for 20 min prior to stimulation with 100 nM insulin for 30 min where indicated.
2-Deoxy-D-glucose (A) or
3-O-methylglucose uptake (B) were then measured
as described under "Experimental Procedures." Results shown are
mean ± S.E. of three individual experiments within which each
point was assayed in triplicate.
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To explore whether the effect of SB203580 is on glucose transport
activity and not on subsequent metabolism (phosphorylation) of the
sugar, we analyzed its action on uptake of the nonmetabolizable glucose
analogue, 3-O-methylglucose. Fig. 3B shows that
SB203580 (10 µM) prevented the stimulation of
3-O-methylglucose uptake by insulin to a similar extent as
it prevented stimulation of 2-deoxyglucose uptake. The inactive
analogue SB202474 had no effect on insulin-stimulated
3-O-methylglucose uptake.
In the experiments described above cells were preincubated for 20 min with SB203580 before insulin stimulation. In order to test whether
SB203580 has direct effects on the hexose transport process itself,
cells were treated with or without insulin and SB203580 was added only
during the 5-min period of the glucose uptake assay. By this protocol,
SB203580 had no effect on either basal or insulin-stimulated
2-deoxyglucose uptake in either 3T3-L1 adipocytes or L6 myotubes (Table
I).
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Table I
Effect of addition of SB203580 to assay buffer during measurement
of 2-deoxy-D-glucose uptake
3T3-L1 adipocytes and L6 myotubes were treated with or without 100 nM insulin for 30 min prior to measurement of
2-deoxy-D-glucose uptake as described under "Experimental
Procedures." 2-Deoxyglucose uptake was measured in the presence or
absence of SB203580 (10 µM) during the 5-min assay
period. Results shown are the mean ± S.E. of three replicates
within one representative of three individual experiments. Each
condition was assayed in triplicate.
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Effect of SB203580 on Translocation of GLUT1 and GLUT 4 to the
Plasma Membrane in Response to Insulin in 3T3-L1 Adipocytes--
Two
methods were used to assess the effect of SB203580 on insulin-induced
glucose transporter translocation in 3T3-L1 adipocytes. First, the
content of plasma membrane-associated glucose transporters was analyzed
by preparation of plasma membrane lawns and subsequent immunofluorescence detection of GLUT1 and GLUT4. Fig.
4A shows an increase in the
plasma membrane lawn content of both GLUT1 and GLUT4 in response to
insulin, which was unaffected by preincubation of cells with SB203580.
Quantification of these results gave the following values for GLUT4
levels on membrane lawns (in relative units): basal, 1.00 ± 0.11;
insulin, 3.75 ± 0.40; insulin plus SB203580, 3.53 ± 0.14. The levels of GLUT1 on membrane lawns were as follows: basal, 1.00 ± 0.12; insulin, 1.75 ± 0.08; insulin plus SB203580, 1.92 ± 0.11. There was also no effect of SB203580 on GLUT1 and GLUT4 levels
under basal conditions (results not shown).

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Fig. 4.
Effect of SB203580 on insulin-stimulated
translocation of GLUT1 and GLUT4 to the plasma membrane in 3T3-L1
adipocytes. 3T3-L1 adipocytes were treated with or without 10 µM SB203580 for 20 min followed by insulin treatment (100 nM) for 30 min where indicated. A, translocation
of GLUT1 and GLUT4 was assessed by plasma membrane lawn formation and
immunofluorescence as described under "Experimental Procedures."
Experiments were performed three times and images shown are from one
representative experiment. B, cell homogenates were
fractionated to produce low density microsome (LDM) and
plasma membrane (PM) fractions which were then analyzed by
SDS-PAGE and immunoblotting as described under "Experimental
Procedures." In each case, experiments were performed three times and
images shown are from one representative experiment.
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Since the lack of effect of SB203580 on glucose transporter levels
associated with the plasma membrane in the face of inhibition of
glucose transport was not anticipated, we also analyzed the insulin-stimulated glucose transporter translocation by subcellular fractionation. This approach allowed us to test the effect of SB203580
on both the internal and the plasma membranes. Equal amounts of
membrane protein from subcellular fractions isolated from cells treated
with or without SB203580 and with or without insulin were analyzed by
SDS-PAGE and immunoblotting (Fig. 4B). The results confirmed
the observation made in plasma membrane lawns that SB203580 does not
prevent the translocation of either GLUT1 or GLUT4 to the plasma
membrane from intracellular stores.
Effect of SB203580 on Insulin-stimulated 2-Deoxyglucose Uptake and
GLUT4 Translocation in L6-GLUT4myc Myotubes--
It was conceivable
that, in the presence of SB203580, GLUT4 vesicles might be docked but
not fused with the plasma membrane, and that membrane lawns and
subcellular fractionation may not be able to differentiate between
these two states. To address this possibility, we analyzed the effect
of SB203580 on the insulin-dependent incorporation of GLUT4
molecules tagged with a Myc epitope that is exofacial when GLUT4 is
inserted in the plasma membrane. This approach allows one to analyze
not only the GLUT4 translocation to the cell surface but also the
functional insertion of transporter molecules. In L6 myotubes stably
expressing Myc-tagged GLUT4 (L6-GLUT4myc), we confirmed that SB203580
(10 µM) prevented the insulin-dependent stimulation of 2-deoxyglucose uptake by ~50% (Table
II). The exposure of Myc-tagged GLUT4 on
the cell surface, however, was not inhibited by SB203580 (Table II).
Collectively, the results presented so far suggest that SB203580
inhibits insulin-dependent glucose transport but not
glucose transporter arrival at the cell surface.
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Table II
Effect of SB203580 on 2-deoxyglucose transport and GLUT4 translocation
in L6 GLUT4myc myotubes
L6 myotubes transfected with Myc-tagged GLUT4 were treated with or
without 10 µM SB203580 for 20 min followed by 100 nM insulin for 30 min in the same medium, where indicated.
Cells were rinsed then 2-deoxyglucose uptake or Myc epitope exposure at
the cell surface were measured as described under "Experimental
Procedures." Results are expressed relative to basal values and
represent mean ± S.E. of three individual experiments within
which each point was assayed in triplicate.
|
|
Effect of SB203580 on IRS-1 Phosphorylation, Association with
the p85 Subunit of PI 3-Kinase and Stimulation of PI 3-Kinase Activity
in 3T3-L1 Adipocytes--
An early step in the insulin signaling
pathway necessary for stimulation of glucose transport is tyrosine
phosphorylation of IRS molecules to activate PI 3-kinase. This pathway
is required for translocation of GLUTs but it is not known if it can
also regulate the intrinsic activity of GLUTs. We therefore examined the effect of SB203580 on the ability of insulin to phosphorylate IRS-1, in order to gain information on its mechanism of action leading
to prevention of stimulation of glucose uptake by insulin. IRS-1 was
immunoprecipitated from 3T3-L1 adipocyte cell lysates treated with and
without insulin or SB203580, resolved by 7.5% SDS-PAGE, then subjected
to Western blotting with anti-phosphotyrosine antibody. Fig.
5A shows that the
insulin-induced tyrosine phosphorylation of IRS-1 was not affected by
SB203580. The amount of p85 associated with IRS-1 immunoprecipitates
was markedly increased by insulin and this effect was also not
prevented by SB203580 (Fig. 5A). We next determined the
effect of SB203580 on the stimulation of PI 3-kinase activity by
insulin using an in vitro kinase assay to determine the
ability of PI 3-kinase associated with IRS-1 in cell lysates to
incorporate 32P into phosphatidylinositol. Fig.
5B shows that insulin activated PI 3-kinase activity
approximately 10-fold and that SB203580 had no effect on
insulin-stimulated or basal PI 3-kinase activity in IRS-1
immunoprecipitates (Fig. 5B).

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|
Fig. 5.
Effect of SB203580 on insulin-stimulated
signaling pathways implicated in the regulation of glucose
transport. 3T3-L1 adipocytes were treated with or without 10 µM SB203580 for 20 min followed by insulin treatment (100 nM) for 5 min where indicated. All assays were conducted as
described under "Experimental Procedures." A,
phosphorylation of IRS-1 and association of the p85 subunit of PI
3-kinase with IRS-1. B, IRS-1-associated PI 3-kinase
activity. C, activity of Akt1, Akt2, and Akt3. For
A and C results are from one representative
experiment from at least three replicates. The mean ± S.E. of
three individual experiments is presented for B.
|
|
Effect of SB203580 on Insulin-stimulated Akt Activity--
Akt is
activated by products of PI 3-kinase and several studies have suggested
a role for Akt in insulin-stimulated glucose transport in 3T3-L1
adipocytes and L6 myotubes (14, 39). To examine the effect of SB203580
on stimulation of Akt1 (PKB
), Akt2 (PKB
), and Akt3 (PKB
) by
insulin these kinases were immunoprecipitated with isoform-specific
antibodies and in vitro activity determined using Crosstide
as substrate. We found that insulin stimulated all three isoforms in
3T3-L1 adipocytes and this stimulation was not affected by SB203580
(Fig. 5C). SB203580 also had no effect on basal Akt1, Akt2,
or Akt3 activities.
 |
DISCUSSION |
A variety of stimuli initiate the sequence of hierarchical protein
phosphorylation leading to activation of p38 MAP kinase, the most
potent being stresses such as osmotic shock and ultraviolet radiation,
as well as inflammatory cytokines (20). p38 MAP kinase is activated by
phosphorylation on threonine and tyrosine residues. At least two MAP
kinase kinases, MKK3 and MKK6, have been identified as the dual
specificity kinases that phosphorylate and activate p38 MAP kinase
(40). Previously identified MKK kinases (MEKK1-3) were found to be
unable to activate MKK3/6, however, recently the MEKK homologue TAK-1
has been confirmed to activate MKK3/6 while both Rac- and p21-activated
kinases have been implicated as functioning further upstream of MKK3/6
(20). Several cellular substrates have been identified to date for p38
MAP kinase, including MAP kinase-activated protein 2/3 and ATF2 (20).
Both upstream regulation of p38 MAP kinase and downstream effectors
seem to be determined in a cell-specific fashion. Four isoforms of p38 MAP kinase have been identified (p38
,
,
, and
) (41). Of these, p38
and p38
are ubiquitously distributed and inhibited by
SB203580 (42). In addition to these isoforms, skeletal muscle also
expresses the p38
isoform (43). The specific isoforms found in
3T3-L1 cells have not been clearly defined although use of specific
primers and the reverse transcription-polymerase chain reaction
approach suggested predominantly p38
and some p38
expression.2
Regulation of p38 MAP kinase by insulin is an often overlooked
signaling phenomenon and again control of p38 MAP kinase by this
hormone appears to occur in a tissue-specific manner. It was
established in Chinese hamster ovary cells that insulin-induced MKK3/6
phosphorylation and activation of p38 MAP kinase (44) and insulin
stimulation of p38 MAP kinase was also demonstrated in hepatoma cells
(23). Conversely, insulin decreased tyrosine phosphorylation of p38 and
inhibited p38 activity in postmitotic fetal neurons where the hormone
is a potent survival factor (45). Two studies have established
activation of p38 MAP kinase by insulin in skeletal muscle (46, 47),
yet in a third study no stimulation of p38 MAP kinase was detected
(24). Less information is available on regulation of p38 MAP kinase
activity by insulin in adipose cells. A preliminary communication
reported that insulin stimulated p38 MAP kinase activity in 3T3-L1
fibroblasts and adipocytes (48). In the present study we further
substantiate this finding and furthermore, show that p38 activation by
insulin is inhibited by SB203580.
We have shown that SB203580, a cell permeant inhibitor of p38 MAP
kinase, attenuates the stimulation of glucose transport by insulin.
Translocation of GLUT1 and GLUT4 to the cell surface is undoubtedly an
important component of the stimulation of glucose transport by insulin
in 3T3-L1 adipocytes. However, some uncertainty still remains over
whether enrichment of the plasma membrane with GLUTs can completely
account for insulin-stimulated glucose uptake (4), and one estimate
suggests that it contributes to only 30% of the total gain in glucose
transport. We utilized two different approaches with 3T3-L1 adipocytes
(preparation of plasma membrane lawns followed by immunofluorescence or
immunoblotting of subcellular fractions) to determine if the inhibition
of insulin-stimulated glucose transport by SB203580 could be explained
by a decreased amount of glucose transporters at the plasma membrane.
Surprisingly, we discovered that in the presence of SB203580, insulin
was able to elicit normal increases in the amount of GLUT1 and GLUT4 at the plasma membrane without a corresponding stimulation of glucose transport. Therefore, the results reveal a strategy to dissociate increased glucose transporter number at the cell surface from increased
glucose transport activity.
One drawback with both of these approaches is that complete
confidence that glucose transporters are inserted into the plasma membrane bilayers is not assured. Indeed, some studies have postulated that in some instances glucose transporter-containing vesicles can be
docked but occluded from the membrane (49, 50). Therefore, to determine
if the newly arrived glucose transporters were properly inserted into
the plasma membrane we utilized the recently characterized L6 muscle
cells stably overexpressing GLUT4 that is tagged with a Myc epitope on
the exofacial surface (35). These cells can be used to measure the
actual incorporation of Myc-tagged GLUT4 into the plasma membrane by
detecting exposure of the Myc epitope on the surface of intact cells.
Importantly, insulin-induced translocation and incorporation of
GLUT4myc into the plasma membrane was not affected by SB203580.
Collectively, the results with 3T3-L1 adipocytes or L6-GLUT4myc cells
suggest that insertion of glucose transporters into the plasma membrane
in response to insulin is not sufficient to account for the observed
increased flux of glucose across the membrane. Instead, another
unidentified event or signal which is prevented by SB203580 is required
to convert the newly arrived cell surface transporters into functional
proteins after insertion into the plasma membrane. Given that the
maximal effect of SB203580 (10 µM) was to inhibit
insulin-stimulated glucose transport by around 50-70%, we suggest
that full insulin-stimulated glucose transport may require a
combination of increased GLUT translocation and increased intrinsic
activity of GLUTs.
The results presented also show that insulin phosphorylated and
activated p38 MAP kinase in 3T3-L1 adipocytes. As expected, SB203580
inhibited insulin-stimulated kinase activity but had no effect on
insulin-stimulated phosphorylation, thus did not interfere with the
upstream signaling pathway involved in activation of p38 MAP kinase by
insulin. Based on its sensitivity to SB203580, a role for p38 MAPK has
been suggested in the stimulation of glucose transport by anisomycin in
3T3-L1 adipocytes and interleukin-2 in KB cells (26, 27). Anisomycin
did not induce a redistribution of glucose transporters to the cell
surface and hence it was proposed to stimulate the intrinsic activity
of GLUT1 glucose transporters that are already present at the cell
surface (27). Similarly, noradrenaline stimulates glucose transport
into rat adipocytes by enhancing the functional activity of GLUT1, in
this case via a cAMP-dependent mechanism (51). There is
also precedence for activation of glucose transporters as part of the
mechanism by which insulin stimulates glucose transport in fat cells
(7, 52). A detailed study of this activation mechanism has been complicated by the fact that unlike anisomycin and noradrenaline, insulin causes a redistribution of glucose transporters to the cell
surface. Hence, it has been difficult to clearly demonstrate the
requirement for activation of transporters in the face of increased
amounts of transporters on the cell surface. However, due to
discrepancies in the degree of insulin-stimulated PI 3-kinase activity
and glucose uptake in 3T3-L1 adipocytes it has recently been suggested
that an additional pathway is necessary for full insulin-stimulated
glucose transport (52). Moreover, we have recently shown that whereas
membrane-permeant lipid products of PI 3-kinase cannot reproduce the
insulin stimulation of glucose uptake, they effectively restore the
inhibition of insulin action caused by wortmannin (53). These results
also suggest that signals parallel to PI 3-kinase are necessary for the
stimulation of glucose transport in 3T3-L1 adipocytes. In the present
study we provide evidence to support this hypothesis and suggest that
insulin stimulates both translocation and intrinsic activity of GLUTs.
Our results are consistent with the hypothesis that the IRS
PI
3-kinase
Akt pathway is involved in GLUT translocation. We also
suggest another parallel signaling pathway involving p38 MAP kinase,
which culminates in increased intrinsic activity of GLUTs, required for
full stimulation of glucose transport.
An alternative possibility is that SB203580 could bind directly to the
newly recruited glucose transporters, thereby inhibiting their
activity. Several years ago such a scenario was contemplated for the
diterpine forskolin which inhibited insulin-stimulated glucose
transport in a manner unrelated to its stimulation of adenylate cyclase
(54). Indeed, forskolin was found to affect glucose transport by a
direct interaction with GLUT4 without affecting GLUT1 (55). In
contrast, the effect of SB203580 could not be shown to occur from
direct inhibition of glucose transporters since insulin-stimulated
glucose transport was not altered by the presence of SB203580 during
the 5-min transport assay. This result strongly suggests that SB203580
does not interact directly with glucose transporters. Instead, our
results favor the interpretation that SB203580 inhibits
insulin-dependent glucose transport by interfering with an
intracellular signal. This conclusion is supported by the observations
that: (a) the IC50 of SB203580 for the
inhibition of glucose transport in 3T3-L1 adipocytes (0.75 µM) was almost identical to that reported for specific
inhibition of p38 MAP kinase (0.6 µM), and (b)
an inactive structural analogue of SB203580 (SB202474) was without
effect on insulin-stimulated glucose transport in 3T3-L1 adipocytes.
These considerations place p38 MAP kinase in the insulin signal
transduction pathway leading to the stimulation of glucose transport.
Transport of glucose into cells is followed almost immediately by its
phosphorylation to produce glucose 6-phosphate. One possible
explanation for our observations obtained measuring 2-deoxyglucose uptake could have been that SB203580 affects metabolism of this glucose
analogue. To examine this possibility we assessed the effects of
SB203580 on insulin-stimulated transport of the nonmetabolizable analog
3-O-methylglucose but obtained similar results to those seen
with 2-deoxyglucose. This suggests that inhibition of glucose transport
by SB203580 is manifest at the level of transport and not at the
subsequent metabolism of glucose.
The lack of effect of SB203580 on insulin-stimulated translocation of
GLUT1 and GLUT4 to the cell surface suggested that this inhibitor does
not affect the previously characterized upstream signaling events
required for this process. Indeed, the state of insulin-induced
phosphorylation or activation of several elements of the insulin
signaling pathway that are thought be essential for
insulin-dependent GLUT translocation were not affected by SB203580. Insulin-stimulated phosphorylation of IRS-1 was normal, as
was association of the regulatory (p85) subunit of PI 3-kinase with
IRS-1. Activation of PI 3-kinase and Akt1, Akt2, and Akt3 by insulin
were also unaffected by SB203580. The inability of SB203580 to inhibit
insulin-stimulated activation of all three isoforms of Akt also
suggests that, contrary to previous suggestion (56), p38 MAPK is not in
the signaling pathway leading to the activation of Akt in response to insulin.
In conclusion, we have shown that the inhibitor of p38 MAP kinase,
SB203580, can prevent insulin-stimulated glucose uptake in 3T3-L1
adipocytes and L6 muscle cells. This inhibitory effect is not due to a
nonspecific effect on insulin signaling pathways nor directly on
glucose transporters. In addition, the action of SB203580 does not lie
at the level of glucose metabolism. Interference with the translocation
of glucose transporters is not the basis for the inhibitory effect of
SB203580. We propose that glucose transporters are activated following
their translocation to the plasma membrane and that this activation is
prevented by SB203580, likely by its ability to inhibit
insulin-dependent activation of p38 MAP kinase.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Y. Ebina (University of
Tokushima, Japan) for introducing GLUT4myc into L6 muscle
cells and Dr. G. Hollman (University of Bath, United Kingdom) for
providing the 3T3-L1 cells. Thanks are also extended to Dr. P. Bilan
for helpful comments on this manuscript.
 |
FOOTNOTES |
*
This work was suppoted by a grant from the Canadian Diabetes
Association (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.
¶
To whom correspondence should be addressed: Programme 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{at}sickkids.on.ca.
2
P. Scherer, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
PI 3-kinase, phosphatidylinositol 3-kinase;
IRS-1, insulin receptor substrate-1;
PKB, protein kinase B;
MAPK, mitogen-activated protein kinase;
MKK, MAP
kinase kinase;
ATF-2, activating transcription factor-2;
IL-1, interleukin-1;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel
electrophoresis;
PMSF, phenylmethylsulfonyl fluoride.
 |
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