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INTRODUCTION |
Virtually all mammalian cells utilize glucose as a major energy
source and thus possess facilitative glucose transporters on their cell
surfaces. The facilitative glucose transporter gene family encodes at
least six isoforms (GLUT1-6) with varying tissue distributions,
subcellular localizations, and kinetics for glucose uptake (1, 2).
Among them, GLUT4, in which expression is strictly limited to muscle
and fat cells, resides in an intracellular compartment under basal
conditions and moves to the cell surface in response to stimulation by
insulin and other factors. In contrast, the ubiquitously expressed
GLUT1 is mainly located on the cell surface, irrespective of
stimulation, and is thus considered to be mainly involved in the
maintenance of basal glucose uptake into the cells.
The mechanisms whereby signal-transducing molecules regulate the
activity of glucose transporters have been extensively studied. At
present, it is clear that activation of phosphatidylinositol 3-kinase
is essential for insulin-induced GLUT4 translocation to the plasma
membrane and increased glucose uptake (3-6), although the mediators
downstream of phosphatidylinositol 3-kinase remain controversial
(7-9). In addition, activation of classical mitogen-activated protein
kinase (MAPK)1 (also termed
extracellular signal-regulated kinase (ERK)), which plays a central
role in cellular transformation, reportedly up-regulates GLUT1
expression, thereby augmenting glucose transport (10, 11).
Recent studies have revealed the existence of at least three
independent MAPK pathways (12-14). Besides ERK, two novel MAPKs have
been identified and designated p38 MAPK (15, 16) and stress-activated
protein kinase (also known as c-Jun N-terminal kinase (JNK)) (8, 17).
Among the mitogenic factors and stress-inducing stimuli that mediate
activation of these enzymes, insulin is reported to activate p38 MAPK
(18, 19). Interestingly, SB203580, a specific inhibitor of p38 MAPK,
inhibits insulin-stimulated increases in glucose transport in both
3T3-L1 adipocytes and L6 myotubes without affecting insulin-stimulated
GLUT4 translocation to the cell surface (20). It has therefore been
hypothesized that p38 MAPK activation is involved in an insulin-induced
enhancement of intrinsic GLUT4 activity on the cell surface. In
addition, p38 MAPK is also activated in response to tumor necrosis
factor (TNF)
, interleukin 1 (IL-1), and hyperosmotic shock
(21-23), reportedly leading to insulin resistance (24-27).
In that context, our study was undertaken to clarify the role of the
p38 MAPK pathway in the regulation of glucose transport. To modulate
p38 MAPK activity, dominant negative p38 MAPK and MAPK kinase (MKK)
mutants as well as constitutively active MKK mutants were overexpressed
in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated
transfection system. In addition, the effects induced by TNF
,
IL-1
, and 200 mM sorbitol were examined in 3T3-L1
adipocytes. We show that MKK6- and MMK3-mediated activation of p38 MAPK
significantly alters expression of GLUT1 and GLUT4, increasing basal
glucose transport and reducing insulin responsiveness.
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EXPERIMENTAL PROCEDURES |
Materials--
3-Isobutyl-1-methylxanthine and
2-deoxy-D-glucose were purchased from Wako Bioproducts.
Enhanced chemiluminescence (ECL) detection system was from Amersham
Pharmacia Biotech. TNF
and IL-1
were from Genzyme Corp.
Inhibitors wortmannin and SB203580 were from Sigma and Calbiochem,
respectively. [
-32P]UTP was from ICN. All other
reagents from commercial sources were of analytical grade.
Antibodies--
Anti-MAPK kinase 1 (MEK1), anti-MKK3, anti-MKK6,
and anti-MKK7 antibodies were purchased from Santa Cruz Biotechnology.
Anti-p38 MAPK and anti-phospho-p38 MAPK
(Thr180/Tyr182) were from New England Biolabs.
Anti-GLUT1 and anti-GLUT4 antisera were raised against the C-terminal
region of GLUT1 (28) and GLUT4 (29), respectively.
Cell Culture--
3T3-L1 fibroblasts were initially maintained
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
donor calf serum (Life Technologies, Inc.) under an atmosphere of 90%
air, 10% CO2 at 37 °C. Differentiation was induced 2 days after the cells reached confluence by replacing their normal
culture medium with DMEM supplemented with 0.5 mM
3-isobutyl-1-methylxanthine, 4 µg/ml dexamethasone (Sigma), and 10%
fetal bovine serum (Life Technologies, Inc.) for 48 h. Thereafter,
the cells were incubated for an additional 4-10 days in DMEM
supplemented with 10% fetal bovine serum; the medium was changed every
other day. With this protocol, more than 90% of the cells expressed
the adipocyte phenotype (3).
L6 myoblasts were initially grown in DMEM supplemented with 10% fetal
calf serum under an atmosphere of 95% air, 5% CO2 at 37 °C, after which they were seeded onto 10-cm plastic culture dishes at a density of 3,000 cells/cm2. To promote their
fusion into myotubes, they were rendered quiescent by incubation for 10 days in DMEM containing 2% serum. Myotube formation was assessed
according to the percentage of nuclei present in multinucleated
myotubes. With this protocol, 80-90% of myoblasts were fused into myotubes.
Construction of MAPK Mutants and Myristoylated Akt--
Plasmids
containing the cDNAs encoding constitutively active MEK1
(LASDSE-MEK1), MKK3 (EE-MKK3), MKK6 (EE-MKK6), and MKK7 (DED-MKK7)
mutants and dominant negative MKK6 (AA-MKK6) and p38 MAPK (p38 MAPK-AF)
mutants were prepared as follows. LASDSE-MEK1 was constructed by
substituting serine 218 with aspartic acid, serine 222 with glutamic
acid, and two critical leucines (Leu11 and
Leu37) in the nuclear export signal sequence of
Xenopus MAPKK with alanines (30). EE-MKK3 was constructed by
replacing serine 189 and threonine 193 with glutamic acids (31).
EE-MKK6 and AA-MKK6 was constructed by substituting serine 207 and
threonine 11 with glutamic acid and alanine, respectively (31). p38
MAPK-AF was constructed by substituting threonine 180 and threonine 182 in the TGY motif with alanine and phenylalanine, respectively (31). DED-MKK7 was constructed by substituting serine 287 with aspartic acid,
threonine 291 with glutamic acid, and serine 293 with aspartic acid.
Myristoylated Akt (Myr-Akt), which contains an src
myristoylation signal sequence, was described previously (32).
Gene Transduction--
To obtain recombinant adenoviruses, the
expression cosmid cassette pAdexCAwt was ligated with the cDNA
encoding LacZ from Escherichia coli or one of the
aforementioned mutants, respectively, after which homologous
recombination between the recombinant cosmid cassette and its parental
virus genome was carried out as described previously (33). Infection of
3T3-L1 (5, 34) and L6 cells (35) with the indicated adenoviruses was
carried out as described previously. Assessing triglyceride content of
the infected cells confirmed that overexpression of the MAPK family
mutants did not affect differentiation of 3T3-L1 cells into adipocytes
(data not shown).
Immunoblotting--
3T3-L1 adipocytes in a 12-well tissue
culture dishes were lysed, boiled in Laemmli buffer containing 10 mM dithiothreitol, and subjected to SDS-polyacrylamide gel
electrophoresis (PAGE). Immunoblotting was performed using an ECL
system according to the manufacturer's instructions.
p38 MAPK Assay--
Adipocytes were serum-deprived for 3 h
before being exposed to 100 nM insulin for 5 min. p38 MAPK
activity was assayed using a specific kit (New England Biolabs)
according to the manufacturer's instructions. Briefly, the cells were
lysed, immunoprecipitated with immobilized anti-phospho-p38 MAPK
(Thr180/Tyr182) antibody, and immunoblotted
with anti-phospho-ATF2 (Thr71) antibody, a natural
substrate of p38 MAPK.
Glucose Transport Assay--
3T3-L1 adipocytes and L6 myotubes
in 24-well culture dishes were serum-starved for 3 h in DMEM
containing 0.2% bovine serum albumin, and then they were incubated for
45 min in glucose-free Krebs-Ringer phosphate buffer (in
mM: 137 NaCl, 4.7 KCl, 10 sodium phosphate (pH 7.4), 0.5 MgCl2, and 1 CaCl2) (3). Basal and stimulated
uptake of 2-deoxy-D-[3H]glucose was then
measured as described previously (29). Experiments were carried out in
the presence and absence of glucose transport inhibitors.
Subcellular Fractionation--
Fractionation of subcellular
membranes from 3T3-L1 adipocytes was carried out essentially as
described previously (36). Aliquots of subcellular membrane fractions
containing equal amounts of protein were subjected to SDS-PAGE,
followed by immunoblotting using anti-GLUT1 and anti-GLUT4 antisera and
an ECL detection system.
RNA Extraction--
Total cell RNA was isolated from 3T3-L1
adipocytes using an Isogen RNA isolation kit (Nippon Gene, Tokyo,
Japan). RNA concentrations were estimated based on absorbance at 260 nm, and 10 µg of RNA from each sample were used for the RNase
protection assay described below.
Preparation of Riboprobes--
Riboprobes were synthesized as
described previously (37). To obtain mouse GLUT1 and GLUT4 cDNAs,
polymerase chain reaction was performed based on the reported sequences
using mouse cDNA libraries. The amplified fragments, which
corresponded to nucleotides 790-939 of mouse GLUT1 cDNA and
790-946 of mouse GLUT4 cDNA, respectively, were subcloned into a
TA vector. These resultant plasmids were then linearized with
BamHI and HindIII, respectively, and used for
in vitro transcription.
RNase Protection Assay--
RNase protection assays were carried
out according to the manufacturer's instructions (RPA III; Ambion,
Austin, TX). Pooled samples of 10 µg of total RNA from adipocytes
were hybridized with the riboprobes for GLUT1 and GLUT4. After
treatment with RNase, the protected fragments were resolved on 5%
polyacrylamide-urea gels and subjected to autoradiography, and the band
intensities were determined using a Molecular Imager GS-525.
Statistical Analysis--
Results were expressed as means ± S.E. Comparisons were made using unpaired Student's t
test. Values of p < 0.05 were considered statistically significant.
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RESULTS |
Overexpression of MAPK Family Mutants in 3T3-L1
Adipocytes--
LASDSE-MEK1, p38 MAPK-AF, AA-MKK6, EE-MKK6, EE-MKK3,
and DED-MKK7 were overexpressed in 3T3-L1 adipocytes using an
adenovirus transfection system (Fig. 1).
Their respective levels of expression were 5-, 11-, 10-, 15-, 8-, and
8-fold greater than that in control cells overexpressing LacZ. Similar
results were obtained with L6 myotubes (data not shown).

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Fig. 1.
Overexpression of MAPK family mutants.
3T3-L1 adipocytes were infected with recombinant adenoviruses
containing LacZ (control) or the indicated mutants as follows.
A, LASDSE-MEK1; B, AA-MKK6, EE-MKK6, EE-MKK3, and
p38 MAPK-AF; and C, DED-MKK7. Equal amounts of protein
isolated from the cells were subjected to SDS-PAGE and then
immunoblotted using specific antibodies as probes.
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Effects of p38 MAPK and MKK6 Mutants on p38 MAPK Activity in 3T3-L1
Adipocytes in the Presence and Absence of Insulin--
p38 MAPK, which
is activated by insulin-mediated dual phosphorylation on threonine 180 and tyrosine 182 in the TGY motif, phosphorylates a number of
downstream targets, including ATF2. We overexpressed p38 MAPK-AF,
AA-MKK6, EE-MKK6, and EE-MKK3 in 3T3-L1 adipocytes, after which p38
MAPK activities were measured by immunoblotting anti-phospho-p38 MAPK
(Thr180/Tyr182) immunoprecipitates with
anti-phospho-ATF2 (Thr71) antibody (Fig.
2). As shown previously, insulin did
indeed activate p38 MAPK in control 3T3-L1 adipocytes overexpressing
LacZ. By contrast, overexpression of EE-MKK6 led to marked activation
of p38 MAPK irrespective of insulin stimulation, whereas overexpression of p38 MAPK-AF or AA-MKK6 strongly inhibited insulin-induced activation of p38 MAPK. EE-MKK3 also activated p38 MAPK; however, the magnitude of
the enhancement was smaller than seen with EE-MKK6 (data not shown).

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Fig. 2.
The effects of p38 MAPK and MKK6 mutants on
p38 MAPK activity in the presence and absence of insulin. 3T3-L1
adipocytes were infected with recombinant adenoviruses containing LacZ
(control), p38 MAPK-AF, AA-MKK6, or EE-MKK6. After exposing some to 100 nM insulin for 5 min, the cells were lysed,
immunoprecipitated (IP) with anti-phospho-p38 MAPK
(Thr180/Tyr182) antibody, and immunoblotted
(WB) with anti-phospho-ATF2 (Thr71) antibody.
Bars, means ± S.E. from three independent experiments;
**p < 0.01 versus the indicated
control.
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Effect of Dominant Negative p38 MAPK and MKK6 Mutants on Glucose
Transport in 3T3-L1 Adipocytes--
Fig.
3A shows the basal and
insulin-stimulated 2-deoxy[3H]glucose uptake into 3T3-L1
adipocytes overexpressing LacZ or dominant negative p38 MAPK (p38-AF)
and MKK6 (AA-MKK6) mutants. In each case, insulin markedly enhanced
glucose uptake. Moreover, overexpression of either p38-AF or AA-MKK6
significantly enhanced the insulin-induced increases in glucose uptake,
with the latter having the greater degree. Based on these results, it
seems clear that p38 MAPK activation is not essential for
insulin-induced increases in glucose uptake.

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Fig. 3.
A, effects of dominant negative p38 and
MKK6 mutants on insulin-induced increases in glucose transport. 3T3-L1
adipocytes were infected with recombinant adenoviruses containing LacZ
(control), p38 MAPK-AF, or AA-MKK6, after which some were exposed to
100 nM insulin for 15 min, and
2-deoxy-D-[3H]glucose transport was measured
as described under "Experimental Procedures." B, effect
of wortmannin (wort) on constitutively active MKK6
mutant-induced glucose transport. 3T3-L1 adipocytes were infected with
recombinant adenoviruses containing LacZ or EE-MKK6. Some cells were
then preincubated with 100 nM wortmannin for 15 min and/or
100 nM insulin for 15 min, after which
2-deoxy-D-[3H]glucose uptake was assayed.
Bars, means ± S.E. from three independent experiments;
*p < 0.05 and **p < 0.01 versus the indicated control. N.S., not
significant.
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On the other hand, overexpression of a constitutively active form of
MKK6 (EE-MKK6) led to an ~30-fold increase in glucose uptake by
3T3-L1 adipocytes, even in the absence of insulin, and insulin
stimulation further increased glucose uptake by ~50% (Fig. 3B). Pretreatment for 15 min with 100 nM of
wortmannin, a phosphatidylinositol 3-kinase inhibitor, abolished the
insulin-induced increase in glucose uptake by cells overexpressing
either LacZ or EE-MKK6, although wortmannin had no effect on the
increase in glucose uptake induced by EE-MKK6.
Comparison of the Effects of Constitutively Active MKK
Mutants on Glucose Transport in 3T3-L1 Adipocytes and L6
Myotubes--
As mentioned in the Introduction, there exist at least
three MAPK pathways. To examine the roles of the respective MKK
isoforms involved in these pathways, the effects of overexpressing
constitutively active forms of MKK3 (EE-MKK3), MKK6 (EE-MKK6), MEK1
(LASDSE-MEK1), and MKK7 (DED-MKK7) in 3T3-L1 adipocytes and L6 myotubes
were compared. Maximal overexpression of EE-MKK3, EE-MKK6, and
LASDSE-MEK1 induced ~3-, 30-, and 15-fold, respectively, increases in
glucose uptake by 3T3-L1 adipocytes, whereas DED-MKK7 had no effect on glucose transport (Fig.
4A).

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Fig. 4.
Comparison of the effects of constitutively
active MKK family mutants on glucose transport. 3T3-L1 adipocytes
(A) and L6 myotubes (B) were infected with
recombinant adenoviruses containing LacZ (control), EE-MKK3, EE-MKK6,
LASDSE-MEK1, DED-MKK7 or myr-Akt, after which
2-deoxy-D-[3H]glucose transport was measured.
Bars, means ± S.E. from three independent experiments;
**p < 0.01 versus LacZ.
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As reported previously, a membrane-targeted, constitutively active form
of Akt (myr-Akt) also increased glucose transport in both 3T3-L1
adipocytes and L6 myotubes (Figs. 4, A and
B).
Effect of Overexpressing EE-MKK6 or LASDSE-MEK1 on the Subcellular
Distribution of Glucose Transporter in 3T3-L1 Adipocytes--
To
understand better the mechanism underlying the increase in glucose
transport induced by MKK6 and MEK1, subcellular fractionation of 3T3-L1
adipocytes was performed, and the GLUT1 and GLUT4 contents of the
plasma membrane (PM) and low density microsome (LDM) fractions were
analyzed. We found that EE-MKK6 and LASDSE-MEK1 markedly increased
levels of GLUT1 protein in both the PM and LDM fractions (Fig.
5, A and B),
whereas levels of GLUT4 protein were markedly diminished (Fig. 5,
C and D).

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Fig. 5.
Effect of EE-MKK6 or LASDSE-MEK1
overexpression on the subcellular distribution of glucose transporter
isoforms. LDM (A and C) and PM (B
and D) fractions were prepared from 3T3-L1 adipocytes
overexpressing LacZ (control), EE-MKK6, or LASDSE-MEK1, after which it
was subjected to SDS-PAGE and immunoblotted using anti-GLUT1
(A and B) or anti-GLUT4 (C and
D) antiserum as a probe. Arrows indicate the
respective protein bands. Bars, means ± S.E. of the
fold increase above basal for three independent experiments.
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Effect of Overexpressing EE-MKK6 or LASDSE-MEK1 on Expression of
Glucose Transporter mRNA in 3T3-L1 Adipocytes--
By using an
RNase protection assay, we found that, as compared with control (LacZ),
expression of GLUT1 mRNA was significantly increased, whereas that
of GLUT4 mRNA was significantly diminished in 3T3-L1 adipocytes
overexpressing either EE-MKK6 or LASDSE-MEK1 (Fig.
6).

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Fig. 6.
The effect of EE-MKK6 or LASDSE-MEK1
overexpression on transcription of glucose transporter isoforms.
Samples (10 µg) of total RNA from 3T3-L1 adipocytes overexpressing
LacZ (control), EE-MKK6, or LASDSE-MEK1 were subjected to an RNase
protection assay using radiolabeled antisense riboprobes for GLUT1
(A) and GLUT4 (B) mRNA. Protected fragments
were resolved on 5% polyacrylamide-urea gels and subjected to
autoradiography. The intensities of RNase-protected bands were analyzed
with a molecular imager. Bars, means ± S.E. from three
independent experiments; **p < 0.01 versus
LacZ.
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Time-dependent Effect of SB203580 on Stimulated
Glucose Transport in 3T3-L1 Adipocytes Overexpressing EE-MKK6--
To
confirm whether or not MKK6-induced changes in glucose transport and
GLUT1/4 expression were mediated via p38 MAPK, the effects of SB203580,
a specific p38 MAPK inhibitor, were examined. As shown in Fig.
7, SB203580 time-dependently
inhibited the increase in glucose uptake induced by overexpression of
EE-MKK6 in 3T3-L1 adipocytes; an almost complete blockade was achieved
when cells were preincubated with SB203580 for 48 h.

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Fig. 7.
Effect of SB203580 on stimulated glucose
transport. 3T3-L1 adipocytes overexpressing LacZ (control),
EE-MKK6, or myr-Akt were preincubated with 10 µM SB203580
for the indicated times, after which uptake of
2-deoxy-D-[3H]glucose was assayed.
Bars, means ± S.E. from three independent experiments;
**p < 0.01 versus no SB203580.
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By contrast, preincubation with SB203580 had no significant effect on
the increase in glucose uptake induced by overexpression of
myr-Akt.
Effects of SB203580 on Glucose Transport and Expression of GLUT1/4
in 3T3-L1 Adipocytes Overexpressing Constitutively Active MKK
Mutants--
Preincubation for 24 h with 10 µM
SB203580 significantly inhibited the increase in glucose transport
normally induced by overexpression of EE-MKK6 and EE-MKK3 in 3T3-L1
adipocytes but not that induced by LASDSE-MEK1 (Fig.
8). Moreover, preincubation with SB203580 restored expression of both GLUT1 and GLUT4 protein to control levels
in 3T3-L1 adipocytes overexpressing EE-MKK3 or EE-MKK6 (Fig.
9). By contrast, the effects of
overexpressing LASDSE-MEK1 on expression of GLUT1 and GLUT4 protein
were unaffected by SB203580.

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Fig. 8.
Effects of SB203580 on MKK family
mutant-stimulated glucose transport. 3T3-L1 adipocytes
overexpressing LacZ (control), EE-MKK3, EE-MKK6, LASDSE-MEK1, or
DED-MKK7 were incubated for 24 h with or without 10 µM SB203580, after which uptake of
2-deoxy-D-[3H]glucose was assayed.
Bars, means ± S.E. from three independent experiments;
**p < 0.01 versus the indicated
control.
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Fig. 9.
Effect of SB203580 on altered expression of
glucose transporter isoforms. Total cellular protein prepared from
3T3-L1 adipocytes overexpressing LacZ (control), EE-MKK3, EE-MKK6,
LASDSE-MEK1, or EE-MKK7 were subjected to SDS-PAGE and immunoblotted
using anti-GLUT1 (A) or anti-GLUT4 (B) antiserum
as a probe. Arrows indicate the respective protein bands.
Bars, means ± S.E. from three independent
experiments.
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Effects of TNF
, IL-1
, and Hyperosmolarity on p38 MAPK
Phosphorylation, Glucose Transport, and GLUT1 Expression in 3T3-L1
Adipocytes--
As shown in Fig. 10,
briefly (30 min) exposing 3T3-L1 adipocytes to TNF
, IL-1
, or 200 mM sorbitol up-regulated phosphorylation of p38 MAPK.
Consistent with these findings, more prolonged (24 h) exposures to
these stimuli significantly elevated glucose transport in 3T3-L1
adipocytes (Fig. 11A, striped
bars). Similar results were obtained with cells overexpressing
EE-MKK6 and EE-MKK3 (Figs. 10 and 11A, solid bars). In
addition, incubating cells for 24 h with TNF
, IL-1
, or 200 mM sorbitol up-regulated GLUT1 expression (Fig. 11B,
striped bars) and down-regulated GLUT4 expression (data not shown)
in a manner similar to that seen with overexpression of EE-MKK6 and
EE-MKK3 (Fig. 11B, solid bars).

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Fig. 10.
Effects of various stressful stimuli and
MKK3/6 on p38 phosphorylation. Total cellular proteins prepared
from 3T3-L1 adipocytes were incubated with 10 ng/ml TNF , 10 ng/ml
IL-1 , or 200 mM sorbitol for 30 min or were infected
with recombinant adenoviruses containing LacZ (control), EE-MKK3, or
EE-MKK6, after which they were subjected to SDS-PAGE and immunoblotted
using anti-phospho-p38 MAPK (Thr180/Tyr182)
antibody as a probe. In this experiment, the level of EE-MKK6
overexpression was attenuated so that the degrees of p38 MAPK
activation were similar under all conditions. Bars,
means ± S.E. from three independent experiments.
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Fig. 11.
Effects of various stressful stimuli and
MKK3/6 on glucose transport and GLUT1 expression. 3T3-L1
adipocytes were incubated for 24 h with 10 ng/ml TNF , 10 ng/ml
IL-1, or 200 mM sorbitol or were infected with recombinant
adenoviruses containing LacZ (control), EE-MKK3, or EE-MKK6 as
indicated. A,
2-deoxy-D-[3H]glucose uptake. B,
total cellular proteins prepared from the indicated 3T3-L1 adipocytes
were subjected to SDS-PAGE and immunoblotted using anti-GLUT1 antiserum
as a probe. The arrow indicates the GLUT1 protein band.
Bars, means ± S.E. from three independent experiments;
**p < 0.01 versus LacZ.
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 |
DISCUSSION |
Glucose transport is known to be affected by cell differentiation
and oncogenic transformation, as well as by various hormones and growth
factors. Such modulation has often been attributed to altered
expression of one or more glucose transporter isoforms (i.e.
GLUT1-6) (1, 2) or to modulation of glucose transporters' (particularly GLUT4) capacity to translocate to the plasma membrane (38-40). The current study was undertaken to understand better the
role of MKK6/3-p38 MAPK activation on the regulation of glucose transport and glucose transporter expression.
Activation of p38 MAPK Is Not Essential for Insulin-induced
Increases in Glucose Uptake--
It was recently reported that
treatment with SB203580, a specific p38 MAPK antagonist, inhibited
insulin-induced glucose uptake by both 3T3-L1 adipocytes and L6
myotubes, without inhibiting translocation of GLUT1 or GLUT4 (20).
Consequently, it was hypothesized that activation of p38 MAPK plays a
key role in the insulin-induced enhancement of intrinsic GLUT4 activity
on the cell surface. On the other hand, p38 MAPK is also known to be
activated by stressful stimuli, such as TNF
, IL-1, and hyperosmotic
shock (21-23), which appear to induce insulin resistance (41-43). To
clarify whether or not activation of p38 MAPK is indeed essential for
insulin-induced increases in glucose transport, we overexpressed
dominant negative p38 MAPK and MKK6 mutants, which markedly suppressed
endogenous p38 MAPK activity but significantly enhanced insulin-induced
glucose uptake by 3T3-L1 adipocytes. We also observed that 10 µM SB203580 almost completely blocked insulin-induced
activation of p38 MAPK but that the insulin-induced increase in glucose
uptake was reduced by only 30% (data not shown). A higher
concentration of SB203580 (100 µM) inhibited the
insulin-induced increase in glucose uptake by more than 90%; however,
at this concentration, SB203580 also significantly suppressed the
activity of Akt kinase (data not shown). It therefore appears that the
inhibition of insulin-induced increases in glucose uptake by
SB203580 was not mediated by an effect on p38 MAPK activity but
by an effect on some other molecule(s), perhaps Akt or GLUT4 itself.
Furthermore, it appears unlikely that activation of p38 MAPK plays a
significant role in the regulation of the insulin-induced increases in
glucose transport mediated by GLUT4.
Activation of p38 MAPK Leads to Up-regulation of GLUT1 and
Down-regulation of GLUT4--
We found that the overexpression of
constitutively active MKK6 or MKK3 mutants markedly increased
expression of GLUT1 protein in both 3T3-L1 adipocytes and L6 myotubes,
whereas expression of GLUT4 was diminished. These effects were
reflected by a significant increase in basal glucose transport and
diminished insulin-induced glucose transport, respectively. That
SB203580 inhibited MMK6/3-induced alterations in glucose uptake and
expression of GLUT1/4 makes it highly likely that p38 MAPK plays a
central role in the regulation of glucose metabolism.
Most interesting, overexpression of constitutively active MEK1, which
induces chronic activation of ERK, yielded the same result as did
overexpression of constitutively active MKK6 and MKK3. Previously,
up-regulation of GLUT1 by oncogenes fps, src, and
ras or by 12-O-tetradecanoylphorbol-13-acetate
was shown to underlie the increased glucose transport related to
cellular transformation and/or proliferation (10, 11). The
GLUT1 gene contains two enhancer elements as follows:
one in the 5' region contains one serum-response element, two
12-O-tetradecanoylphorbol-13-acetate-response elements
(TREs), one cyclic AMP-response element, and three GC boxes; and
the other, located in the second intron, contains one cyclic
AMP-response element and two TREs (44). Both of these enhancers are
involved in the augmented GLUT1 gene
transcription induced by ras and v-src, by
serum and by platelet-derived growth factor (11, 44, 45). Since
transcription activator protein-1 is a target of ERK1/2 (46-48), the
association of activator protein-1 with TRE may contribute to the
activation of the MEK1-ERK pathway by acting at the 5' region
enhancer(s) of the GLUT1 gene to up-regulate its
expression and thus increase glucose transport. Indeed, this phenomenon
may be central to the cellular transformation and/or proliferation
mediated by activation MEK1-ERK pathway.
MKK6/3 and MKK7 activate p38 MAPK and JNK, respectively (31, 49-52).
In 3T3-L1 adipocytes, activation of JNK appears to induce increased
synthesis of glycogen (18), and in the present study, we found that
MKK6/3, but not MKK7, up-regulated expression of GLUT1 mRNA. Thus,
the regulation of GLUT1 gene transcription may not be
mediated entirely by activator protein-1 but by a more complex
mechanism involving other factors.
On the other hand, the GLUT4 gene, which
contains 11 exons and 10 introns, possesses a promoter region
containing a weak "TATA" box sequence homology, a CCAAT box to
which CCAAT/enhancer binding protein likely binds (53), four potential
binding sites for nuclear transcription factor Sp1 (54), a skeletal
muscle-specific activation domain (55). Together, these domains play a
crucial role in the up-regulation of GLUT4 expression during adipose
and muscle cell differentiation. In that respect, we found no
significant alteration in cellular triglyceride content in 3T3-L1
adipocytes overexpressing the various MAPK family mutants studied; thus
the down-regulation of GLUT4 expression in 3T3-L1 adipocytes
overexpressing constitutively active forms of MKK6/3 and MEK1 cannot be
attributed to the dedifferentiation from adipocytes to fibroblasts.
In many cases, expression of GLUT1 and GLUT4 are regulated oppositely,
but whether changes in the expression of one affect the expression of
the other remains unknown. If such interdependence exists,
down-regulation of GLUT4 might be induced by the elevated intracellular
glucose concentration mediated by up-regulation of GLUT1. However,
there is reportedly no significant alteration in the GLUT4 expression
in the skeletal muscle of transgenic mice overexpressing GLUT1 (56). We
therefore suggest that expression of GLUT1 and GLUT4 is regulated
independently, although further study will be necessary to resolve this
issue definitively.
Involvement of p38 MAPK Activation in the Insulin Resistance
Induced by TNF
, IL-1
, and Hyperosmolarity--
Several studies
have shown that chronically exposing adipocytes to TNF
or oxidant
stress markedly decreases their GLUT4 content as a result of decreased
GLUT4 gene transcription and a reduced half-life of
its mRNA (26, 42, 57, 58). Conversely, chronic exposure to TNF
increases expression of GLUT1 (27, 58, 59), although a contradictory
result was obtained in one study (60). In the present study, we found
that prolonged (24 h) exposure to TNF
, IL-1
, or hyperosmolarity
increased glucose transport, increased GLUT1 expression, and decreased
GLUT4 expression in a manner similar to that seen in cells
overexpressing constitutively active MKK6. These phenomena were all
attenuated by SB203580, implicating p38 MAPK in their occurrence.
Exposure to TNF
, IL-1
, and hyperosmolarity is also known to lead
to insulin resistance (24-27). Consistent with the aforementioned findings, down-regulation of GLUT4 and up-regulation of GLUT1 are
considered to be components of the mechanism underlying such insulin
resistance, although reduced expression of insulin receptor substrate-1 (61) and inhibition of insulin-induced tyrosine phosphorylation of insulin receptor may also be involved in
TNF
-induced insulin resistance (41, 62).
In summary, activation of the MKK6/3-p38 MAPK pathway markedly enhances
glucose transport by up-regulating GLUT1 expression, irrespective of
the stimulus. This effect may help cells meet the increased energetic
demands incurred when they are under stress, perhaps determining
whether cells survive or succumb to apoptosis, a process in which the
MKK6/3-p38 MAPK pathway also plays a role. In addition, although
further study will be required to clarify whether the MKK6/3-p38 MAPK
pathway is indeed activated under conditions associated with insulin
resistance (e.g. obesity), we anticipate that p38 MAPK
activation will prove to be a key component of stress-induced insulin
resistance in fat and muscle cells.