Download p38 MAPK regulation of glucose transporter expression

Am J Physiol Regul Integr Comp Physiol 286: R342–R349, 2004.
First published October 30, 2003; 10.1152/ajpregu.00563.2003.
p38␥ MAPK regulation of glucose transporter expression and glucose uptake
in L6 myotubes and mouse skeletal muscle
Richard C. Ho, Oscar Alcazar, Nobuharu Fujii, Michael F. Hirshman, and Laurie J. Goodyear
The Research Division, Joslin Diabetes Center and Department of Medicine,
Harvard Medical School, Boson, Massachusetts 02215
Submitted 26 September 2003; accepted in final form 27 October 2003
Ho, Richard C., Oscar Alcazar, Nobuharu Fujii, Michael F.
Hirshman, and Laurie J. Goodyear. p38␥ MAPK regulation of
glucose transporter expression and glucose uptake in L6 myotubes
and mouse skeletal muscle. Am J Physiol Regul Integr Comp Physiol
286: R342–R349, 2004. First published October 30, 2003; 10.1152/
ajpregu.00563.2003.—Skeletal muscle expresses at least three p38
MAPKs (␣, ␤, ␥). However, no studies have examined the potential
regulation of glucose uptake by p38␥, the isoform predominantly
expressed in skeletal muscle and highly regulated by exercise. L6
myotubes were transfected with empty vector (pCAGGS), activating
MKK6 (MKK6CA), or p38␥-specific siRNA. MKK6CA-transfected
cells had higher rates of basal 2-deoxy-D-[3H]glucose (2-DG) uptake
(P ⬍ 0.05) but lower rates of 2,4-dinitrophenol (DNP)-stimulated
glucose uptake, an uncoupler of oxidative phosphorylation that operates through an insulin-independent mechanism (P ⬍ 0.05). These
effects were reversed when MKK6CA cells were cotransfected with
p38␥-specific siRNA. To determine whether the p38␥ isoform is
involved in the regulation of contraction-stimulated glucose uptake in
adult skeletal muscle, the tibialis anterior muscles of mice were
injected with pCAGGS or wild-type p38␥ (p38␥WT) followed by
intramuscular electroporation. Basal and contraction-stimulated 2-DG
uptake in vivo was determined 14 days later. Overexpression of
p38␥WT resulted in higher basal rates of glucose uptake compared
with pCAGGS (P ⬍ 0.05). Muscles overexpressing p38␥WT showed
a trend for lower in situ contraction-mediated glucose uptake (P ⫽
0.08) and significantly lower total GLUT4 levels (P ⬍ 0.05). These
data suggest that p38␥ increases basal glucose uptake and decreases
DNP- and contraction-stimulated glucose uptake, partially by affecting levels of glucose transporter expression in skeletal muscle. These
findings are consistent with the hypothesis that activation of stress
kinases such as p38 are negative regulators of stimulated glucose
uptake in peripheral tissues.
represents a significant site for whole body
insulin-stimulated glucose disposal (13) and insulin resistance
in this tissue is a characteristic feature in the development of
type 2 diabetes mellitus (12, 20, 43). In addition to diabetes,
skeletal muscle insulin resistance is common to other clinical
and subclinical conditions of obesity, stress, cachexia, pregnancy, starvation, sepsis, and trauma. These conditions are
often associated with systemic factors that activate cellular
signals, many of which are transmitted via mitogen-activated
protein kinases (MAPK). Of the four main MAPK subgroups,
the extracellular-signal regulated kinases (ERK) appear to be
primarily involved in cellular growth and transformation (40),
and the c-Jun NH2-terminal kinases (JNK) and p38 MAPK are
regulated by cytotoxic and environmental stress. JNK has
recently been implicated in obesity- and TNF-␣-mediated insulin resistance by negative regulation of insulin-stimulated
phosphoinositide-3 kinase (PI3K) activity (2, 24). For p38
MAPK, numerous activators of these enzymes are associated
with insulin resistance; however, data regarding the potential
role of p38 MAPK in the regulation of glucose transport in
skeletal muscle have been controversial (17, 49).
Four p38 MAPK isoforms have been reported: p38␣ (34),
p38␤ (27, 48), p38␥ (33, 35), and p38␦ (18). Activation of p38
MAPK occurs via dual phosphorylation of conserved TGY
motifs by the MAPK kinases, MKK6 and MKK3 (41). Functional differences between the isoforms are related in part to
their differential expression, activation, and substrate specificity. p38␣ and p38␤ are ubiquitously expressed, while p38␦ is
predominantly expressed in glandular tissues, lung, and kidney
(28, 51). Interestingly, p38␥ (ERK6, SAPK3) and MKK6 are
primarily expressed in skeletal muscle (10, 21, 33).
In studies using the pyridinyl imidazole class of inhibitors,
p38␣ and p38␤ activity has been shown to be required for
insulin-stimulated glucose uptake through a mechanism that
involves increases in the intrinsic activity of GLUT4 (45, 46,
49). In contrast to these studies, activation of p38 by overexpression of a constitutively active MKK6 decreased insulin
signaling, GLUT4 expression, and insulin-stimulated glucose
uptake in 3T3-L1 adipocytes and L6 cells (16, 17). In addition,
overexpression of dominant negative p38␣ or MKK6 leads to
increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes (17). Thus there are data suggesting that p38 is necessary
for insulin effects on glucose transport and other studies
suggesting that p38 is a negative regulator of insulin-stimulated
glucose uptake (16, 17).
Like insulin, exercise is known to increase skeletal muscle
glucose transport. In adult skeletal muscle, exercise and contractile activity increase the phosphorylation and activity of
multiple p38 isoforms via a mechanism that likely involves
activation of MKK6/3 (4, 19, 23). One study has suggested that
the well-known effects of contractile activity to stimulate
glucose transport in skeletal muscle are impaired when muscles
are treated with a p38 inhibitor (47). Interestingly, unlike p38␣
and p38␤, p38␥ is resistant to the effects by known p38
inhibitors (11). Compared with the ␣- and ␤-isoforms of p38,
we have evidence that p38␥ is very highly regulated by muscle
contraction (6). Thus the ␥-isoform is almost exclusively
expressed in skeletal muscle and is highly regulated by skeletal
muscle contraction, yet nothing is known about the role of
Address for reprint requests and other correspondence: L. J. Goodyear,
Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 (E-mail:
[email protected]).
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.
mitogen-activated protein kinase; exercise; GLUT1; GLUT4
SKELETAL MUSCLE
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p38␥ in the regulation of basal and contraction-mediated glucose uptake.
To elucidate the role of p38␥ in the regulation of basal and
contraction-stimulated glucose uptake in L6 myotubes and
skeletal muscle, we transfected and overexpressed wild-type
p38␥ or MKK6 into L6 myotubes and/or adult mouse skeletal
muscle in vivo. We also utilized vector-based small interfering
RNA (siRNA) technology to knock down p38␥ in L6 myotubes. Our findings suggest that p38␥ is involved in the
regulation of GLUT1 expression and basal glucose uptake in
L6 myotubes. Our findings also suggest that p38␥ negatively
regulates GLUT4 expression and contraction-mediated glucose
uptake in adult skeletal muscle in vivo.
EXPERIMENTAL PROCEDURES
Materials. Antibodies used were purchased from the following:
phospho-p38, MKK6, phospho-ATF-2 (Cell Signaling Technology),
p38␥ (Upstate Biotechnology), FLAG (Sigma), GLUT1 (Chemicon),
and GLUT4 (gift from Robert Smith). Anti-mouse and anti-sheep
IgG-horseradish peroxidase (HRP) were from Upstate Biotechnology.
The anti-rabbit IgG-HRP and ECL-Plus Western blotting detection kit
were purchased from Amersham Pharmacia Biotech. 2-Deoxy-D[3H]glucose was from Perkin Elmer.
Plasmid construction. Currently, there are no p38␥ inhibitors
available, so to study p38␥ function in L6 cells we expressed p38␥WT
and constitutively active MKK6 (MKK6CA). The human FLAGp38␥WT and MKK6CA mutants (donated by Dr. Jiahuai Han) were
subcloned from a pcDNA3 vector into a pCAGGS vector (gift from
Dr. J. Miyazaki of Osaka University) (38). The MKK6 mutant was
created by introducing a constitutive negative charge by replacing
serine-207 and threonine-211 with glutamine and has been found to
efficiently activate both endogenous and recombinant p38 (42).
FLAG-p38 and MKK6 cDNA constructs were excised with HindIII
and XbaI and transferred into the XhoI site between the CAG promoter and a 3⬘-flanking region of a rabbit ␤-globin gene of pCAGGS
expression vector after blunt-end treatment (37). This expression
vector drives a target gene under the CAG (cytomegalovirus immediate-early enhancer-chicken ␤-actin hybrid) promoter. The CAG
promoter has extremely high activity in muscle, as demonstrated in
both transgenic mice (39) and intramuscular DNA injection (50).
Plasmid DNA was grown in E. coli TOP10 cells, extracted using the
Endo-free Plasmid Mega Kit (Qiagen), and the DNA was suspended
in saline.
RNA interference. pSilencer vectors harboring the U6 promoter
(Ambion, TX) were used to drive the expression of four different pairs
of p38␥-specific oligonucleotides. The siRNA constructs contain a U6
promoter followed by a p38␥-specific, 21-mer sense oligonucleotide,
a spacer, the 21-mer antisense oligonucleotide, and a U6 termination
sequence consisting of five thymidines (Fig. 1A). Four pairs of
oligonucleotides were generated and cotransfected with the FLAGp38␥WT plasmid in L6 cells. All four siRNA oligos were successful
in blocking FLAG-p38␥WT expression by ⬎85%, and one set, which
had ⬎94% knockdown efficiency, was used for subsequent experiments. While FLAG-p38␥WT expression was dramatically decreased,
p38␥-specific siRNA did not affect expression of FLAG-p38␤ or
endogenous expression of p38␣ and p38␤ (Fig. 1B). Levels of
endogenous p38␥ were below the limits of detection by immunoblotting; however, it is known that p38␥ is expressed in differentiating
muscle cells as determined by Northern blotting (33).
Cells and transfections. L6 cells were seeded into 12-well plates
and maintained in growth medium consisting of ␣MEM supplemented
with 10% fetal bovine serum in a humidified atmosphere containing
5% CO2-95% air at 37°C. Myoblasts were grown in monolayers and
allowed to reach confluence. Myoblasts were induced to differentiate
by exposure to 2% fetal bovine serum for 6 days. Transient transfections were performed using a total of 2 ␮g of DNA per well, using
lipofection (Lipofectamine 2000, Invitrogen). Recombinant protein
expression was allowed for 48 h, at which time cells were used for the
determination of glucose transport and immunoblotting.
Glucose uptake in L6 myotubes. L6 myotubes were serum-starved
for 5 h in ␣MEM before any treatment. Cells were washed twice in
PBS and incubated in 2,4-dinitrophenol (DNP, 500 ␮M; 10 min).
After stimulation, 2-deoxy-D-[3H]glucose uptake was measured by
incubating cells at room temperature for 5 min in transport solution
(140 mM NaCl, 20 mM HEPES-Na, pH 7.4, 5 mM KCl, 0.5 ␮Ci/ml
2-deoxy-D-[3H]glucose, 2.5 mM MgSO4, 1.0 mM CaCl2). Nonfacilitated glucose uptake was determined in the presence of 10 ␮M
cytochalasin B. Net accumulation of 2-deoxy-D-[3H]glucose was
determined, and rates of uptake were calculated.
Sensitivity to stimulated glucose uptake of L6 cells increases, but
transfection efficiency decreases with differentiation. Therefore, we
determined the optimal stage at which myotubes could be both
effectively transfected and stimulated by DNP. By the eighth day of
Fig. 1. Overexpression of p38␥ in L6 myotubes. L6 myoblasts were seeded and differentiated in 2% FBS. Myotubes were
transfected on day 6 of differentiation, and subsequent experiments were performed 48 h later. A: four pairs of p38␥-specific
oligonucleotides were cloned into the pSilencer vector. FLAG-p38␥ was reduced by 89.6, 97.2, 94.5, and 94.7% response to the
4 different p38␥-siRNAs. Pair 2 exhibited the maximum silencing effect and was used for all subsequent experiments. B:
representative immunoblot with a total p38 antibody showing the specificity of p38␥-specific siRNA. Expression of wild-type p38␥
(p38␥WT) was reduced over 90% in response to p38␥-specific siRNA, while expression of endogenous p38␣/␤ and overexpression
of FLAG-p38␤WT was unaffected.
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differentiation, we were able to measure a twofold increase in glucose
uptake with DNP stimulation. Transfection efficiency of ⬃45% was
observed on day 8 of differentiation judged by LacZ transfection (on
day 6) and X-gal staining for ␤-galactosidase expression. Therefore,
all subsequent experiments were performed according to this protocol.
DNA injection into skeletal muscle and in vivo electroporation.
Animal protocols were approved by the Joslin Diabetes Center Institutional Animal Care and Use Committee and were in accordance
with National Institutes of Health guidelines. DNA injection and in
vivo electroporation were performed by a modification of the method
of Aihara and Miyazaki (3). Mice (ICR, Taconic) weighing 25–30 g
were anesthetized with an intraperitoneal injection of pentobarbital
sodium (90 mg/kg), and 100 ␮g of FLAG-p38/pCAGGS plasmid in
25 ␮l of saline was injected into the tibialis anterior muscle of one leg
using an insulin syringe with a 29-gauge needle. For the control,
empty pCAGGS vector in 25 ␮l of saline was injected into the
opposite leg. Square-wave electrical pulses (200 V/cm) were applied
eight times at a rate of 1 pulse/s with each pulse being 20 ms in
duration using an electric pulse generator. The electrodes consist of a
pair of stainless needles inserted and fixed 5 mm apart into the tibialis
anterior muscles.
Previous work in our lab has determined the distribution and
efficiency of gene transfer by x-gal staining to detect the activity of
␤-galactosidase (the lacZ gene product). Our results show that ␤-galactosidase activity is detected in almost all areas of the tibialis
anterior and that ␤-galactosidase expression is observed not only on
the surface but also deep within the tissue. The percentage of fibers
expressing ␤-galactosidase was estimated as 85.7 ⫾ 2.3%. Our data
demonstrate that a very high proportion of muscle fibers express the
foreign gene using the parameters in the in vivo electroporation
section above (15).
In situ contraction. Fourteen days after the in vivo electroporation
protocol, 12-h overnight fasted mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (90 mg/kg). The sciatic
nerves were bilaterally isolated, and electrodes were placed around
each nerve and then interfaced with a Grass model S88 electrical
stimulation unit. Hindlimb muscles were either stimulated to induce
contractions for 15 min (1 train/s, 500-ms train duration, 100 Hz,
0.1-ms duration, 1–5 V) or remained unstimulated to serve as sham
controls.
Glucose uptake in skeletal muscle in vivo. Baseline blood samples
were collected from the tail, the jugular vein was catheterized, and an
intravenous bolus of 2-deoxy-D-[3H]glucose (10 ␮Ci/mouse) was
administered. After the injection of the tracer, mice were subjected to
the in situ muscle contraction protocol (15 min). A matched group of
animals was used as controls (no contraction) for the determination of
basal glucose uptake. Blood samples were obtained at 5, 10, 15, 25,
35, and 45 min for the determination of blood glucose and 2-deoxy3
D-[ H]glucose specific activity. After collection of the last blood
sample, animals were killed, and the tibialis anterior muscle was
removed and snap-frozen in liquid nitrogen. Accumulation of 2-deoxy-D-[3H]glucose in the tissue was determined, and rates of uptake
were calculated (26).
Immunoblotting. After the experimental protocols skeletal muscle
samples were homogenized in lysis buffer (20 mM Tris䡠HCl, 5 mM
EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM NaVO4, 1% NP-40, 10
␮g/ml aprotinin, 10 ␮g/ml leupeptin, 3 mM benzamidine, 1 mM
PMSF), and total protein concentrations were determined by the
Bradford method (Bio-Rad). Samples were resolved by 8% SDSPAGE, transferred to nitrocellulose membranes, blocked in 5% milk,
and probed using the respective antibodies. Immunocomplexes were
detected using chemiluminescence.
Statistical analysis. Results are expressed as means ⫾ SE or
transformed and expressed relative as a percentage of the mocktransfected control. ANOVA and Student’s paired and unpaired t-tests
were used (P ⬍ 0.05).
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RESULTS
Overexpression of constitutively active MKK6 increases
basal glucose uptake in L6 myotubes. To study the regulation of
basal glucose uptake by p38␥, we transfected L6 myotubes with
MKK6CA alone or with p38␥-specific siRNA. Expression of the
activating MKK6 mutant resulted in an increase in basal rates of
glucose uptake in the myotubes (Fig. 2A). These changes in basal
glucose uptake were likely due to alterations in GLUT1 protein
expression, as there was a tendency for GLUT1 expression to be
higher in cells overexpressing MKK6CA compared with
pCAGGS transfected cells. (Fig. 2B). Because MKK6 activates
all p38 isoforms, we also designed vector-based siRNA constructs
Fig. 2. Overexpression of constitutively active MKK6 in L6 myotubes. L6
myoblasts were seeded and differentiated in 2% FBS. Myotubes were transfected on day 6 of differentiation, and subsequent experiments were performed
48 h later. A: levels of constitutively active MKK6 (MKK6CA) overexpression
in L6 myotubes were similar to endogenous skeletal muscle MKK6 expression.
Endogenous levels of MKK6 in L6 cells are below the detection limit by
immunoblotting. Overexpression of MKK6CA significantly increased basal
glucose uptake in L6 myotubes. Cotransfection with p38␥-specific siRNA
reversed the effect of MKK6CA on basal glucose uptake in L6 myotubes. B:
changes in basal glucose uptake associated with overexpression of MKK6CA
and p38␥-specific siRNA were paralleled by changes in total GLUT1 protein
expression. Results shown are means ⫾ SE of at least 3 independent experiments within which each point was assayed in duplicate. *Significant difference (P ⬍ 0.05).
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to selectively knockdown p38␥ expression in L6 cells. The increases in basal glucose uptake in response to MKK6CA overexpression were reversed when cells were cotransfected with the
p38␥-specific siRNA (Fig. 2A).
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Overexpression of p38 in injected muscles. In adult skeletal
muscle, the expression of FLAG-p38␥ was 10-fold higher
compared with the endogenous expression of p38␥ (Fig. 3A).
The high degree of overexpression did not affect endogenous
p38␥ expression (Fig. 3A). Using a pan-p38 antibody that
recognizes all p38 isoforms, Fig. 3B shows that overexpression
of FLAG-p38␥ did not affect total expression of endogenous
p38 isoforms. Recombinant FLAG-p38␥ is functional, as evidenced by the finding that it is phosphorylated in response to in
situ muscle contractions (Fig. 3C). Total (endogenous ⫹ recombinant) p38 phosphorylation under both the basal and
contraction-stimulated conditions was significantly increased
in FLAG-p38WT muscles compared with pCAGGS controls
(Fig. 3C). The changes in p38 phosphorylation in muscles
overexpressing p38␥WT were associated with increases in
phosphorylation of the p38 downstream substrate ATF-2
(Fig. 3D).
p38␥ positively regulates basal glucose uptake in adult
skeletal muscle. We next used this gene transfer method to
overexpress p38␥WT in skeletal muscle of free-living animals
to determine whether basal glucose uptake and GLUT1 expression are regulated by p38␥. Skeletal muscles overexpressing
p38␥WT exhibited significantly higher basal rates of glucose
uptake compared with pCAGGS-transfected muscles (Fig. 4A).
This increase was not due to detectable changes in GLUT1
expression in the muscle (Fig. 4B, 85.8 ⫾ 13.5 vs. 100.0 ⫾
17.9%, P ⫽ 0.54), nor was it due to increases in GLUT4
(described below). Collectively, the data from L6 cells and
adult skeletal muscle suggest that p38␥ positively regulates
basal glucose uptake.
Overexpression of constitutively active MKK6 decreases
DNP-stimulated glucose uptake in L6 myotubes. We next
investigated the role of p38␥ in stimulated glucose uptake. L6
myotubes were transfected with MKK6CA and stimulated with
DNP, an uncoupler of oxidative phosphorylation, to determine
whether p38␥ regulates increases in glucose uptake caused by
cellular energy demand. DNP-stimulated glucose uptake was
significantly lower in cells overexpressing MKK6CA compared with pCAGGS-transfected cells (Fig. 5). Interestingly,
the effect of MKK6CA on DNP-stimulated glucose uptake was
reversed when cells were cotransfected with p38␥-specific
siRNA (Fig. 5). Thus knockdown of p38␥ completely restores
the impairment in glucose uptake caused by overexpression of
MKK6CA. While the effects of MKK6 and p38␥-siRNA on
basal glucose uptake are paralleled by changes in GLUT1
Fig. 3. Overexpression of p38␥ in adult mouse skeletal muscle. Empty vector
or FLAG-p38␥ constructs were injected into the tibialis anterior muscles of
anesthetized mice (n ⫽ 6). Animals were allowed to recover and recombinant
proteins were overexpressed for 14 days. A: in vivo transfection or overexpression of FLAG-p38␥ does not affect endogenous expression of p38␥
(bottom band). Lanes: 1, nontransfected control; 2, empty vector transfection
(pCAGGS); 3, FLAG-p38␥ transfection. Expression of FLAG-p38␥WT in
lane 3 was 10-fold higher compared with endogenous p38␥ (lanes 1 and 2). B:
overexpression of FLAG-p38␥WT did not affect endogenous expression of
other p38 isoforms. C: using an antibody that recognizes the activating
phosphorylation sites of all p38 isoforms, recombinant FLAG-p38␥WT was
phosphorylated and regulated by 15 min of in situ contraction. Overexpression
of FLAG-p38␥ results in significant increases in both basal and in situ
contraction-stimulated p38 phosphorylation. D: ATF-2 phosphorylation is
increased by in situ contraction. Increased p38␥ from muscles overexpressing
FLAG-p38␥WT is shown by elevations in ATF-2 phosphorylation compared
with pCAGGS controls. *Significant difference (P ⬍ 0.05).
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p38␥ negatively regulates contraction-mediated glucose uptake. Contractile activity also increases glucose uptake through
a mechanism that may also involve cellular energy demand.
Because p38␥ is highly activated by exercise, we determined
whether p38␥ regulates glucose uptake in contracting skeletal
muscle. Mice injected with empty vector or p38␥WT into the
tibialis anterior muscle were anesthetized, and in situ contraction-stimulated glucose uptake was determined. The mean
contraction-mediated glucose uptake was 16% lower in skeletal muscle overexpressing p38␥WT compared with pCAGGStransfected muscles, and this effect was close to reaching
statistical significance (P ⫽ 0.08, Fig. 6A). Total GLUT4
expression was significantly lower in skeletal muscle overexpressing FLAG-p38␥WT (Fig. 6B). Thus these data suggest
that p38␥ is a negative regulator of GLUT4 expression and,
subsequently, attenuates the increases in glucose uptake in
response to changes in cellular energy demand (DNP stimulation and contractile activity in L6 cells and adult skeletal
muscle, respectively).
DISCUSSION
Fig. 4. Overexpression of p38␥WT increases basal glucose uptake in skeletal
muscle. Empty vector or FLAG-p38␥ constructs were injected into the tibialis
anterior muscles of anesthetized mice. Animals were allowed to recover, and
recombinant proteins were overexpressed for 14 days. A: anesthetized mice
(n ⫽ 6) were injected with 2-deoxy-D-[3H]glucose, and tibialis anterior
muscles were removed 45 min later. Accumulation of 2-deoxy-D-[3H]glucose
was determined, and basal rates of glucose uptake were calculated. Muscles
overexpressing FLAG-p38␥WT exhibited significantly higher rates of basal
glucose uptake compared with pCAGGS transfected muscles. B: levels of total
GLUT1 protein were not different between the groups. *Significant difference
(P ⬍ 0.05).
Mitogen-activated protein kinases, particularly JNK and p38
MAPK, are activated under conditions of environmental and
cellular stress. These physiological and cellular perturbations
are commonly associated with skeletal muscle insulin resistance, and recent studies suggest that JNK is a negative
regulator of insulin signaling in 3T3-L1 adipocytes (24). How-
expression, the effects on DNP-stimulated glucose uptake were
not associated with detectable changes in GLUT4 protein
levels (data not shown).
Fig. 5. Overexpression of constitutively active MKK6 decreases DNP-stimulated glucose uptake in L6 myotubes. Overexpression of MKK6CA significantly decreased DNP-stimulated glucose uptake. Cotransfection of MKK6CA
with p38␥-specific siRNA reversed the negative effect of MKK6CA alone on
DNP-stimulated glucose uptake. Results shown are means ⫾ SE of at least 3
independent experiments within which each point was assayed in duplicate.
*Significant difference (P ⬍ 0.05). Basal glucose uptake values from Fig. 2
included as reference.
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Fig. 6. Overexpression of p38␥WT results in decreases in in situ contractionmediated glucose uptake in adult mouse skeletal muscle. Empty vector or
p38␥WT constructs were injected into the tibialis anterior muscles of anesthetized mice. Animals were allowed to recover and recombinant proteins were
overexpressed for 14 days. Anesthetized mice (n ⫽ 6) were injected with
2-deoxy-D-[3H]glucose, and in situ contraction by peroneal nerve stimulation
was conducted for 15 min. The tibialis anterior muscles were removed 30 min
after contraction. A: accumulation of 2-deoxy-D-[3H]glucose was determined,
and rates of contraction-stimulated glucose uptake were calculated. B: muscles
overexpressing wild-type p38␥ exhibited significantly lower total GLUT4
levels. *Significant difference (P ⬍ 0.05).
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ever, previous data regarding the role of p38 in the regulation
of glucose uptake in skeletal muscle are controversial. The
majority of these studies have focused exclusively on the
ubiquitously expressed p38␣ and p38␤ isoforms using p38
inhibitors. The workhorse among p38 inhibitors has been the
pyridinyl imidazole derivatives (SB compounds), which are
only effective against p38␣ and p38␤ activity. Unfortunately,
these compounds have also been shown to inhibit Akt (32),
JNK (9), ERK (36, 44), and nucleoside transporters (25) in a
variety of cell lines. Skeletal muscle p38␥ is unique in that it is
insensitive to inhibition by known p38 inhibitors (18).
In the current study, we suggest that p38␥ positively regulates basal glucose uptake in L6 myotubes. Overexpression of
constitutively active MKK6, which activates all p38 isoforms,
resulted in increases in rates of basal glucose uptake. This
increase was associated with a trend for higher cellular GLUT1
content. Our data support previous findings that overexpression
of a constitutively active MKK6 mutant upregulated GLUT1
expression and increased basal glucose uptake in 3T3-L1
adipocytes and L6 myotubes (17). Increases in GLUT1 in
3T3-L1 adipocytes and L6 cells have also been reported using
a constitutively active MKK3 mutant, which, unlike MKK6,
does not activate p38␤ (14, 27). Therefore, it appears that p38␤
is not required for the regulation of GLUT1. Interestingly, the
effects of overexpression of the activating MKK6 mutant on
basal glucose uptake and GLUT1 expression observed in the
present study were completely reversed by cotransfection with
p38␥-specific siRNA oligonucleotides. These results suggest
that the effects of MKK6 activity on basal glucose uptake and
GLUT1 expression in L6 myotubes are primarily mediated by
the p38␥ isoform.
In agreement with our data from L6 cells, we also found that
p38␥ increases basal glucose uptake in adult skeletal muscle.
Overexpression of p38␥WT resulted in significantly elevated
rates of basal glucose uptake; however, this was not accompanied by detectable changes in levels of total GLUT1 expression
compared with pCAGGS-transfected muscles. Our data are
consistent with previous reports suggesting that p38 is involved
in the regulation of basal glucose uptake and demonstrate that
the ␥ isoform is pivotal in mediating this effect in skeletal
muscle (14, 17, 25).
We also show that overexpression of constitutively active
MKK6 significantly attenuated DNP-stimulated glucose uptake in L6 myotubes. These data suggest that the p38␥ isoform
is primarily responsible for this effect since full recovery of
DNP-stimulated glucose uptake resulted when L6 cells were
cotransfected with p38␥-specific siRNA oligos. We did not
observe significant changes in GLUT4 expression in response
to overexpression of MKK6CA or p38␥-siRNA; however, L6
cells are known to exhibit relatively low levels of GLUT4, and
changes in protein expression may be below the limits of
detection by immunoblotting.
We have previously reported that compared with the p38␣
and p38␤ isoforms, p38␥ was highly activated after prolonged
exercise in humans (6); however, the physiological role of
exercise-stimulated p38␥ signaling in skeletal muscle has remained obscure. Here, we demonstrate that total GLUT4 expression is significantly lower in skeletal muscle overexpressing p38␥WT in vivo. This was associated with decreases in in
situ contraction-mediated glucose uptake, suggesting that p38␥
negatively influences contraction-mediated glucose uptake in
AJP-Regul Integr Comp Physiol • VOL
R347
skeletal muscle in vivo. The effect of p38␥ activation on
contraction-stimulated glucose uptake is at least partially due
to the negative regulation of GLUT4 protein expression in
skeletal muscle. Our data agree with a previous report that
showed that constitutively active MKK6/3 mutants downregulated GLUT4 expression in 3T3-L1 adipocytes (17). Recent
studies have shown an upregulation of MAPK (including p38)
in adipose tissue from individuals with type 2 diabetes and
have suggested that the p38 pathway, in particular, might
contribute to the loss of GLUT4 expression observed in adipose tissue from type 2 diabetic patients (8, 31). However,
these studies did not determine isoform-specific effects of p38
signaling in the regulation of GLUT4 expression. Our data
indicate that p38␥ is involved in the regulation of GLUT4
expression in skeletal muscle in vivo and, therefore, may
represent a novel target for the regulation of glucose homeostasis in this tissue.
While the present data are the first to suggest that p38␥ is a
negative regulator of GLUT4 expression in adult skeletal
muscle in vivo, the effect of p38␣ and p38␤ on stimulated
glucose transport has yet to be resolved. Klip and colleagues
(49) have reported that insulin-activated p38, and the stimulation of glucose uptake, was reduced by preincubation of
3T3-L1 adipocytes and L6 myotubes with p38 inhibitors.
These authors concluded that insulin activates GLUT4 intrinsic
activity, most likely by utilizing a p38-dependent signal in L6
cells. The same group has shown that contraction-stimulated
2-deoxyglucose glucose transport was reduced by up to 50%
when isolated muscles were pretreated with SB-203580 (47).
On the other hand, studies have indicated that increases in
glucose uptake resulting from hyperosmolarity, insulin, or
osmotic shock are not impaired by treatment with p38 inhibitors (5, 30). Another study reported that activation of p38 by
treatment of cells with anisomycin did not stimulate glucose
transport (30). Interestingly, both ERK and JNK are activated
by exercise (19), and, like p38␥ described here, ERK and JNK
have been shown to positively regulate GLUT1 expression and
negatively regulate insulin-stimulated glucose uptake in cell
systems (2, 7, 16, 17). While our data indicate that p38␥
negatively regulates DNP-stimulated glucose uptake in L6
cells and contraction-mediated glucose uptake in skeletal muscle, the possibility remains that p38␣ and p38␤ may have the
opposite effect. This is plausible considering different isoformspecific tissue expression, cellular localization, activation, and
downstream signaling. For example, p38␥ is the only known
MAPK to exhibit a PSD-95 discs-large ZO-1 (PDZ) binding
domain (22), which confers the ability for specific downstream
signaling to proteins such as syntrophin (22), aquaporin-4 (1),
and potentially nitric oxide synthase (29).
In conclusion, our results demonstrate that p38␥ MAPK
signaling positively regulates GLUT1 expression and basal
glucose uptake in L6 myotubes and adult skeletal muscle. Our
results also identify p38␥ as a negative regulator of GLUT4
expression and DNP- and contraction-stimulated glucose uptake in L6 myotubes and adult skeletal muscle, respectively.
MAPKs are involved in the regulation of numerous cytokine
signaling networks, which have commonly been associated
with peripheral insulin resistance. Here, we demonstrate the
isoform-specific involvement of p38␥ in the regulation of
glucose uptake in L6 myotubes and skeletal muscle in vivo.
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P38␥
REGULATION OF GLUCOSE UPTAKE IN SKELETAL MUSCLE
ACKNOWLEDGMENTS
We express gratitude to Dr. Amira Klip for providing the L6 cells, Dr. J.
Miyazaki (Osaka University, Japan) for providing the pCAGGS vector, and
Dr. Jiahuai Han (The Scripps Research Institute) for providing the p38 and
MKK6 constructs.
GRANTS
This work was supported by National Institutes of Health Grants R01-AR45670, R01-AR-42238 (L. J. Goodyear) and by F32-AR-049662 Individual
Kirschstein National Research Service Award (R. C. Ho).
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