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Metabolism Clinical and Experimental 56 (2007) 1719 – 1728
www.elsevier.com/locate/metabol
High-fat diet impairs the effects of a single bout of endurance exercise on
glucose transport and insulin sensitivity in rat skeletal muscle
Satsuki Tanaka a , Tatsuya Hayashi a,b,⁎, Taro Toyoda c , Taku Hamada b ,
Yohei Shimizu b , Masakazu Hirata a , Ken Ebihara a , Hiroaki Masuzaki a , Kiminori Hosoda a ,
Tohru Fushiki c , Kazuwa Nakao a
a
Department of Medicine and Clinical Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan
c
Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received 4 May 2007; accepted 10 July 2007
b
Abstract
A single bout of exercise increases the rate of muscle glucose transport (GT) by both insulin-independent and insulin-dependent
mechanisms. The purpose of this study was to determine whether high-fat diet (HFD) feeding interferes with the metabolic activation induced
by moderate-intensity endurance exercise. Rats were fed an HFD or control diet (CD) for 4 weeks and then exercised on a treadmill for
1 hour (19 m/min, 15% incline). Insulin-independent GT was markedly higher in soleus muscle dissected immediately after exercise than in
muscle dissected from sedentary rats in both dietary groups, but insulin-independent GT was 25% lower in HFD-fed than in CD-fed rats.
Insulin-dependent GT in the presence of submaximally effective concentration of insulin (0.9 nmol/L) was also higher in both dietary groups
in muscle dissected 2 hours after exercise, but was 25% lower in HFD-fed than in CD-fed rats. Exercise-induced activation of 5′adenosine
monophosphate–activated protein kinase, a signaling intermediary leading to insulin-independent GT and regulating insulin sensitivity, was
correspondingly blunted in the HFD group. High-fat diet did not affect glucose transporter 4 content or insulin-stimulated Akt
phosphorylation. Our findings provide evidence that an HFD impairs the effects of short-term endurance exercise on glucose metabolism and
that exercise does not fully compensate for HFD-induced insulin resistance in skeletal muscle. Although the underlying mechanism is
unclear, reduced 5′adenosine monophosphate–activated protein kinase activation during exercise may play a role.
© 2007 Elsevier Inc. All rights reserved.
1. Introduction
Physical exercise has profound effects on glucose
metabolism in contracting skeletal muscle. Exercise activates
glucose transport (GT) in skeletal muscle by inducing
translocation of glucose transporter 4 (GLUT4) to the cell
surface by insulin-independent and insulin-dependent
mechanisms (reviewed in Hayashi et al [1]). The activity
of insulin-independent GT is markedly enhanced during
exercise; and this effect wears off within several hours after
exercise, when the postexercise increase in insulin sensitivity
⁎ Corresponding author. Laboratory of Sports and Exercise Medicine,
Graduate School of Human and Environmental Studies, Kyoto University,
Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto, 606-8501, Japan. Tel.: +81 75
753 6640; fax: +81 75 753 6640.
E-mail address: tatsuya@kuhp.kyoto-u.ac.jp (T. Hayashi).
0026-0495/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.metabol.2007.07.017
that leads to insulin-dependent GT becomes prominent.
Wallberg-Henriksson et al [2] showed in isolated rat skeletal
muscle that the rate of insulin-independent GT is maximal
immediately after exercise, whereas the postexercise
increase in insulin sensitivity becomes detectable 3 hours
after exercise. Correspondingly, Price et al [3] showed in
human muscle that postexercise glycogen repletion occurs in
an insulin-independent manner for about 1 hour after
exercise, after which insulin-dependent glycogen repletion
becomes significant. These exercise-stimulated mechanisms
form the basis of practices to prevent individuals from
developing glucose intolerance and to improve glycemic
control in patients with type 2 diabetes mellitus.
It is of interest to know whether exercise-stimulated
GT, including both the insulin-independent and insulindependent components, is normal in the state of insulin
resistance. Although numerous studies have shown that a
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S. Tanaka et al. / Metabolism Clinical and Experimental 56 (2007) 1719–1728
high-fat diet (HFD) causes insulin resistance in muscles at
rest, it is unknown whether an HFD interferes with the
short-term stimulatory effect of exercise on insulin sensitivity. Only one study has addressed this topic and
demonstrated that the postexercise increase in muscle
insulin sensitivity is abolished completely in HFD-fed rats
[4]. In that study, however, insulin-dependent GT was
measured before the insulin-independent glucose uptake
wore off (its activity was still 160% higher than the basal
uptake), indicating that the net effect of exercise on insulin
sensitivity was substantially underestimated because of
residual glucose uptake activity. There is considerable
controversy over whether an HFD alters the effects of
short-term exercise on insulin-independent GT. Most
investigators have reported about 50% reduction in the
rate of muscle GT stimulated by exercise [5-7] and electrical
stimulation [6,8,9] in HFD-fed rodents, although others did
not find these effects [4,10]. Moreover, some studies have
shown that the reduction in insulin-independent GT was not
associated with decreased muscle GLUT4 content [5,6]; but
a conflicting result was also reported [8]. Although Hansen
et al [9] showed that impairment of the exercise-stimulated
GT is associated with decreased GLUT4 translocation to the
cell surface, the responsible signaling mechanism remains to
be elucidated.
The purposes of our present study were to determine how
HFD affects insulin-independent and insulin-dependent GT
activated by a single bout of endurance exercise and to
explore the underlying mechanism that leads to the change in
exercise-stimulated glucose utilization. We found that both
components of exercise-induced GT were impaired by an
HFD and that these changes were accompanied by a decrease
in 5′adenosine monophosphate (AMP)–activated protein
kinase (AMPK) activation in skeletal muscle of rats fed an
HFD for 4 weeks.
2. Materials and methods
were killed by cervical dislocation immediately after the
cessation of running; and the soleus muscles were isolated.
Muscles were incubated in 7 mL Krebs-Ringer bicarbonate
buffer containing 2 mmol/L pyruvate (KRBP) at 37°C for 20
minutes, and then 3-O-methyl-D-glucose (3MG) uptake
activity was determined as described previously [11-14].
Muscles dissected from sedentary rats were treated similarly.
Some of the muscles were frozen in liquid nitrogen
immediately after dissection for analysis of isoform-specific
AMPK activity and Western blotting of phosphorylated
AMPKα and phosphorylated acetyl–coenzyme A carboxylase (ACC). Muscles from sedentary animals were also
analyzed by Western blotting of total AMPKα and GLUT4.
For histochemical analysis, soleus muscles were isolated
from sedentary animals and frozen in dry ice-cooled 2methylbutane. Abdominal fat (epididymal, retroperitoneal,
and mesenteric fat pads) was collected from sedentary
animals and weighed. To study the postexercise effect on
insulin-dependent GT, exercised and sedentary rats were
placed in separate cages with free access to drinking water
but without food for 2 hours, after which the rats were killed
by cervical dislocation and the soleus muscles were
dissected. Isolated muscles were incubated for 30 minutes
in KRBP in the absence or presence of half-maximally
effective insulin (0.9 nmol/L) at 37°C, and then 3MG uptake
was determined. Some muscles were frozen in liquid
nitrogen immediately after incubation for Western blotting
of phosphorylated Akt, a signaling intermediary leading to
insulin-stimulated GT. To measure glycogen and triglyceride content, muscles were isolated from sedentary rats and
from exercised rats immediately and 2 hours after exercise,
and frozen in liquid nitrogen. We chose soleus muscle
because our preliminary studies showed that soleus muscle
provided the most prominent activation of insulin-independent and insulin-dependent GT in response to exercise
compared with other muscles including extensor digitorum
longus and epitrochlearis.
2.1. Animals and diets
2.3. 3MG uptake
Male Wistar rats at the time of weaning were purchased
from Clea Japan (Tokyo, Japan). Animals were fed either
control diet (CD) (MF; 3.6 kcal/g, 12% kcal fat, source:
soybean; Oriental Yeast, Tokyo, Japan) or HFD (D12493;
5.2 kcal/g, 60% kcal fat, source: soybean/lard; Research
Diets, New Brunswick, NJ) for 4 weeks. All animal
experiments were approved by the Animal Research
Committee, Graduate School of Medicine, Kyoto University.
To assay GT, incubated muscles were transferred to 2 mL
Krebs-Ringer bicarbonate buffer containing 1 mmol/L 3-O[methyl-3H]-D-glucose (1.5 μCi/mL) (American Radiolabeled Chemicals, St Louis, MO) and 7 mmol/L D-[14C]
mannitol (0.3 μCi/mL) (PerkinElmer Life Science, Boston,
MA) at 30°C and incubated for 10 minutes [11-14]. The
muscles were weighed and processed by incubating them in
450 μL of 1 mol/L NaOH at 80°C for 10 minutes. Digestates
were neutralized with 1 mol/L HCl, and particulates were
precipitated by centrifugation at 20 000g for 2 minutes.
Radioactivity in aliquots of the digested protein was
determined by liquid scintillation counting for dual labels.
2.2. Exercise and muscle sampling
The rats were accustomed to a rodent treadmill (Muromachi Kikai, Kyoto, Japan) by running at 14 to 18 m/min
on a 15% grade for 5 minutes on the day before the
experiment. After an overnight fast, rats performed treadmill
running at 19 m/min on a 15% grade for 1 hour or were kept
sedentary. To study insulin-independent GT, exercised rats
2.4. Isoform-specific AMPK activity assay
Muscles were treated as described [11-14]. Frozen
muscles were homogenized in ice-cold lysis buffer (1:40
S. Tanaka et al. / Metabolism Clinical and Experimental 56 (2007) 1719–1728
wt/vol) containing 20 mmol/L Tris-HCl (pH 7.4), 1% Triton
X-100, 50 mmol/L NaCl, 250 mmol/L sucrose, 50 mmol/L
NaF, 5 mmol/L sodium pyrophosphate, 2 mmol/L dithiothreitol, 4 mg/L leupeptin, 50 mg/L soybean trypsin
inhibitor, 0.1 mmol/L benzamidine, and 0.5 mmol/L
phenylmethylsulfonyl fluoride, and centrifuged at 14 000g
for 30 minutes at 4°C. The supernatants (100 μg of protein)
were immunoprecipitated with antibodies directed against
the α1 or α2 catalytic subunits of AMPK [11] and protein
A–Sepharose CL-4B (Amersham, Buckinghamshire, United
Kingdom). Kinase reactions were performed in the presence
of SAMS peptide [11], and then 32P incorporation was
quantitated with a scintillation counter.
2.5. Western blotting
For analysis of phosphorylated AMPKα, total AMPKα,
phosphorylated ACC, and phosphorylated Akt, muscles
were homogenized in lysis buffer used for isoform-specific
AMPK activity. Lysates were solubilized in Laemmli sample
buffer containing mercaptoethanol and boiled. For analysis
of GLUT4, muscles were homogenized in ice-cold buffer
containing 250 mmol/L sucrose, 20 mmol/L 2-[4-(2-hydroxyethyl)-1-piperadinyl] ethonsulforic acid (HEPES) (pH 7.4),
and 1 mmol/L EDTA, and centrifuged at 1200g for 5 minutes.
The supernatant was centrifuged at 200 000g for 60 minutes
at 4°C. The resulting pellet was solubilized in Laemmli
sample buffer containing dithiothreitol. Samples were
subjected to sodium dodecyl sulfate–polyacrylamide gel
electrophoresis, and proteins were transferred to polyvinylidene difluoride membranes (PolyScreen; PerkinElmer,
Wellesley, MA). Blocked membranes were incubated with
phosphospecific AMPKα Thr172 (Cell Signaling Technology, Beverly, MA), total AMPKα (Cell Signaling Technology), phosphospecific ACC Ser79 (Upstate Biotechnology,
Lake Placid, NY), phosphospecific Akt (Ser473 ) (Cell
Signaling Technology), and GLUT4 (Biogenesis; South
Coast, United Kingdom) antibodies. Proteins were visualized
with enhanced chemiluminescence reagents (Amersham).
The signal was quantified with a Lumino-Image Analyzer
LAS-1000 System (Fuji Photo Film, Tokyo, Japan).
2.6. Histochemical analysis
Serial sections (10 μm thick) were used for muscle fiber
typing and intramyocellular lipid (IMCL) measurement. To
determine muscle fiber type (type I, IIa), myosin adenosine
triphosphatase (ATPase) staining was performed as
described [15,16]. Sections were incubated in acidic (30
mmol/L sodium barbital and 50 mmol/L sodium acetate,
adjusted to pH 4.3 with HCl) or alkaline buffer (50 mmol/L
CaCl2 and 75 mmol/L NaCl, adjusted to pH 10.6 with
NaOH) and then incubated in staining buffer (2.8 mmol/L
adenosine triphosphate, 50 mmol/L CaCl2, 75 mmol/L
NaCl, adjusted to pH 9.4 with NaOH), followed by
immersion in 1% CaCl2, 2% CoCl2, and 1% (NH4)2S. The
sections were treated in ethanol and xylol, dried in the air,
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and then mounted with Aquatex (Merk, Darmstadt,
Germany). Fiber type distribution was determined by
counting the number of each fiber type in 100 contiguous
fibers in a muscle section. To determine IMCL content, the
oil red O (ORO) staining procedure and stained area
measurement were performed as described [17]. Sections
were incubated with formaldehyde-methanol (1:1 vol/vol)
and then incubated with ORO solution followed by
extensive wash with distilled water. The sections were
dried in the air and then mounted with Aquatex. Images
from each section were saved as gray-scale images, and the
digitized data were then analyzed using the freeware ImageJ
software (http://rsb.info.nih.gov/). The amount of IMCL in
each fiber was quantified as the percentage of the area
occupied by ORO-stained droplets (total area occupied by
lipid droplets of a muscle fiber) × 100/total cross-sectional
area of the fiber. Lipid area was calculated for each of 3
different fields within the section, and a mean percentage
was then calculated for each muscle [17].
2.7. Muscle glycogen and triglyceride content measurement
Glycogen content was assayed as described [12,14].
Frozen muscles were weighed and digested in 1 mol/L
NaOH (1:9 wt/vol) at 80°C for 10 minutes. The digestates
were neutralized with 1 mol/L HCl, and then 6 mol/L HCl
was added to obtain a final concentration of 2 mol/L HCl.
The digestates were incubated at 85°C for 2 hours and then
neutralized with 5 mol/L NaOH. The concentration of
hydrolyzed glucose residues was measured enzymatically
using the hexokinase glucose assay reagent (Glucose CII
Test; Wako, Osaka, Japan). Triglyceride content was
measured as described [18]. Total lipids were extracted
from muscles with isopropyl alcohol–heptane (1:1 vol/vol)
and saponified in ethanolic KOH (0.5 mol/L). Free glycerol
concentration was then determined using a commercial kit
(Triglyceride E Test; Wako).
2.8. Blood sample analysis
Blood samples were collected from the tail vein using
heparinized glass tube 3 days before the experimental day
after an overnight fast. Plasma levels for glucose (GlutestAce; Sanwa Kagaku Kenkyusyo, Nagoya, Japan), insulin
(rat insulin ELISA kit; Morinaga, Yokohama, Japan), leptin
(rat leptin radioimmunoassay kit; LINCO, St Charles, MO),
triglycerides (Triglyceride E Test; Wako), and lactate
(Lactate Pro; Arkray, Kyoto, Japan) were measured. Lactate
concentration was also measured on the experimental day
immediately after exercise.
2.9. Statistical analysis
Results are presented as means ± SE. The significance
of difference between 2 groups was evaluated using
Student t test. Multiple means were compared by analysis
of variance followed by post hoc analysis using Dunn's
procedure. P b .05 was considered statistically significant.
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3. Results
3.1. Metabolic parameters in rats fed the CD and HFD
Table 1 summarizes the basic characteristics of the CDand HFD-fed rats (Table 1). Rats fed the HFD for 4 weeks
were slightly heavier and had higher plasma concentrations
of glucose, insulin, triglycerides, and leptin than did
CD-fed rats.
3.2. HFD increases IMCL in soleus
We analyzed the influence of HFD on IMCL concentration by ORO staining (Fig. 1A-B). Muscle fiber type was
determined by myofibrillar ATPase histochemical staining
(Fig. 1C-F). The fiber type proportions did not differ
significantly between CD-fed and HFD-fed rats (CD,
80.7% ± 1.2% type I and 19.3% ± 1.2% type IIa fibers;
HFD, 79.2% ± 2.5% type I and 20.8% ± 2.5% type IIa
fibers). In both muscle fiber types, the IMCL content in
HFD-fed rat muscle was twice as high as that in muscles
from CD-fed rats. In CD-fed rats, the IMCL content was
2.3% ± 0.5% in type I fibers and 5.9% ± 1.3% in type IIa
fibers. In HFD-fed rats, the respective values were 4.5% ±
0.9% and 12.8% ± 2.0% (P b .05 vs CD) (Fig. 1G). The
IMCL content in the total soleus muscle was also twice as
high in the HFD group than in the CD group (3.0% ± 0.7% vs
6.2% ± 1.0%, P b .05) (Fig. 1G).
3.3. HFD attenuates activation of insulin-independent GT
induced by one bout of endurance exercise
To analyze insulin-independent GT stimulated by one
bout of exercise, soleus muscles were dissected and 3MG
uptake was determined ex vivo in the absence of insulin
immediately after exercise (Fig. 2). Exercise elicited
significant activation of insulin-independent 3MG uptake
by 3.4 times in muscles from CD-fed rats and by 2.9 times
in muscles from HFD-fed rats. However, the rate of insulinindependent 3MG uptake stimulated by exercise was 25%
lower in muscles from HFD-fed rats (0.15 ± 0.02 μmol/[g h])
than in muscles from CD-fed rats (0.11 ± 0.01 μmol/[g h])
Table 1
Metabolic parameters in rats under HFD and CD feeding
Body weight (g)
Food intake (kcal/d)
Plasma glucose (mg/dL)
Plasma insulin (mg/dL)
Plasma triglycerides (mg/dL)
Plasma leptin (ng/mL)
Abdominal fat (g)
CD
HFD
193 ± 2
54 ± 1
78 ± 2
1.3 ± 0.1
98 ± 4
1.6 ± 0.2
5.3 ± 0.5
203 ± 3 ⁎
58 ± 1 ⁎
88 ± 2 ⁎
2.5 ± 0.2 ⁎
122 ± 8 ⁎
7.6 ± 0.4 ⁎
9.7 ± 0.4 ⁎
Male Wistar rats at the time of weaning were fed CD or HFD for 4 weeks.
Body weight, abdominal fat weight, and plasma parameters were measured
at the end of week 4. Blood samples were obtained after an overnight fast at
9:00 to 11:00 AM. Data are means ± SE; n = 6 to 19 per group.
⁎ P b .05 vs CD-fed group.
(P b .05). Basal glucose uptake was not affected by
dietary manipulation.
3.4. One bout of endurance exercise activates
insulin-dependent GT after exercise, but does not
fully compensate for insulin resistance in muscle from
HFD-fed rats
The effect of the HFD on insulin-dependent GT is shown
in Fig. 3. The rate of insulin-dependent 3MG uptake was
59% lower in muscles from sedentary HFD-fed rats than in
sedentary CD-fed rats (0.12 ± 0.02 vs 0.05 ± 0.01 μmol/[g
h], P b .05), indicating marked insulin resistance in the HFDfed animals (Fig. 3; insulin+, sedentary). Two hours after
exercise, when insulin-independent 3MG uptake stimulated
by exercise had declined significantly (Fig. 3; insulin−, 2
hours postexercise), insulin-dependent 3MG uptake was
markedly higher in muscles from exercised animals than in
muscles from sedentary rats in both dietary groups (Fig. 3;
insulin+, 2 hours postexercise). The net increase in the rate of
insulin-stimulated 3MG uptake was similar in both dietary
groups (CD, 0.33 ± 0.03 μmol/[g h] vs HFD, 0.27 ± 0.02
μmol/[g h]). However, the rate of insulin-stimulated 3MG
uptake was still 25% lower in HFD-fed rats than in CD-fed
rats (0.36 ± 0.03 vs 0.27 ± 0.03 μmol/[g h], P b .05).
3.5. HFD attenuates muscle AMPKα2 activation by one
bout of endurance exercise
We evaluated whether the HFD affects muscle AMPK
activity, a signaling intermediary leading to insulinindependent GT [12,19-21] and regulation of insulin
sensitivity [22-24]. Neither diet nor one bout of exercise
had an effect on AMPKα1 activity (Fig. 4A). In contrast,
exercise increased AMPKα2 activity by 1.6 times in muscle
from CD-fed rats (P b .05), whereas in muscle from HFDfed rats, AMPKα2 activation did not change significantly
(Fig. 4B). Interestingly, the basal AMPKα2 activity was
1.3 times higher in muscle from HFD-fed rats than from
CD-fed rats (P b .05), whereas the AMPKα2 activity
immediately after exercise was similar in both dietary
groups (Fig. 4B). Therefore, the exercise-mediated response
of AMPKα2 activity was significantly lower in HFD-fed
than in CD-fed rats. This is consistent with the findings of
AMPKα2 activity: exercise increased phosphorylation of
the Thr172 residue of AMPKα, an essential site for full
kinase activation (Fig. 4C), and the Ser79 residue of ACC,
a known substrate of muscle AMPK (Fig. 4D), in muscles
from CD-fed animals, but not in HFD-fed animals. The
protein level of AMPKα did not differ between muscles
from both dietary groups (Fig. 4E).
3.6. One bout of endurance exercise dose not affect
nsulin-stimulated phosphorylation of muscle Akt in CD- and
HFD-fed rats
To determine whether HFD impairs the downstream of
phosphatidylinositol 3-kinase (PI-3 kinase), we measured the
S. Tanaka et al. / Metabolism Clinical and Experimental 56 (2007) 1719–1728
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Fig. 1. High-fat diet increases IMCL content in soleus muscle. Representative transverse sections of soleus muscle dissected from CD-fed (A, C, and E) and
HFD-fed (B, D, and F) rats (80× magnification). A and B, Oil red O staining of IMCL. Oil red O stains neutral lipid (mainly triglycerides) with an orange-red tint,
and lipid droplets are seen as distinct spots of stain (A, B). C and D, Myosin ATPase staining (pH 4.3). Light and dark fibers are type IIa and I, respectively (C, D).
E and F, Myosin ATPase staining (pH 10.6). Light and dark fibers are type I and IIa, respectively (E, F). G, Fiber type–specific IMCL content, expressed as a
percentage of the area of lipid stained. Data are means ± SE; n = 7 per group. *P b .05 vs CD-fed group.
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4. Discussion
Fig. 2. High-fat diet attenuates activation of insulin-independent GT induced
by one bout of moderate-intensity endurance exercise. Rats ran on a
treadmill for 1 hour at 19 m/min at a 15% incline. Soleus muscles were
isolated from exercised rats immediately after the cessation of running and
from rats kept sedentary. Isolated muscles were incubated for 20 minutes in
the absence of insulin, and 3MG uptake was determined. Data are means ±
SE; n = 5 to 9 per group. *P b .05 vs CD-fed group.
phosphorylation of the Ser473 residue of Akt in muscles
dissected 2 hours after exercise and stimulated with insulin in
vitro (Fig. 5). Despite the significant increase in insulinstimulated 3MG uptake in muscles from exercised rats (Fig.
3), Akt phosphorylation in muscle was not affected by
exercise in CD- or in HFD-fed rats. The level of basal and
insulin-stimulated Akt phosphorylation was not affected by
the dietary manipulation.
3.7. HFD dose not change muscle GLUT4 protein level
To test the possibility that the decrease in insulinindependent and insulin-dependent GT activity caused by
HFD is mediated by a reduction in the GLUT4 content of
skeletal muscle, we measured soleus muscle GLUT4 protein
content (Fig. 6). The GLUT4 protein content did not differ
between soleus muscles from CD- and HFD-fed rats.
Endurance exercise has long been advocated as beneficial
for patients with insulin resistance associated with type 2
diabetes mellitus and obesity. This is based partly on the
observation that, even in people with insulin resistance,
endurance exercise stimulates muscle glucose uptake in
skeletal muscle by 2 distinct mechanisms: one insulin
independent and one insulin dependent (reviewed in Hayashi
et al [1]). Reversal of the short-term increase in GT after
cessation of contractile activity is followed by a marked
increase in the sensitivity of muscle to insulin. We found a
significant increase in insulin-independent GT followed by
insulin-dependent GT in rat soleus muscle after a 1-hour bout
of treadmill running in both the CD-fed and HFD-fed rats. In
both dietary groups, the mild increase in blood lactate
concentration (b4 mmol/L) (Results) and significant reduction in muscle glycogen content (Table 2) after exercise
suggest that rats performed moderate-intensity endurance
exercise and that muscle glucose utilization was substantially
activated by exercise. Similar lactate concentration and
glycogen content also indicate that the exercise intensity did
not differ between the dietary groups.
Although we found that exercise activated GT in skeletal
muscle from both CD- and HFD-fed rats, the absolute rates
of insulin-independent and insulin-dependent GT were lower
in muscles from HFD-fed rats than in muscles from CD-fed
rats; in contrast, muscle GLUT4 levels were similar between
the groups (Figs. 2 and 3). Our results are consistent with
previous studies demonstrating that exercise-stimulated
insulin-independent GT is impaired in muscles from HFDfed rats in the absence of a reduction in muscle GLUT4
content [5,6]. Liu et al [4] measured insulin-dependent GT in
3.8. HFD does not change muscle glycogen concentration,
but increases muscle triglyceride content
Basal and postexercise glycogen concentrations were
similar in soleus muscle in CD- and HFD-fed rats (Table 2).
In contrast, muscle triglyceride content was higher in
muscle from HFD-fed rats than from CD-fed rats (Table 2)
(P b .05).
3.9. Effect of one bout of endurance exercise on blood
lactate concentration
To evaluate the intensity of exercise, we measured blood
lactate concentration at rest and immediately after exercise.
Compared with basal values, exercise increased blood
lactate by 1.8 times in CD-fed rats (from 1.7 ± 0.2 to
3.1 ± 0.1 mmol/L, P b .05) and by 2.1 times in HFD-fed rats
(from 1.6 ± 0.1 to 3.4 ± 0.2 mmol/L, P b .05). Blood lactate
concentration did not differ between dietary groups.
Fig. 3. One bout of endurance exercise activates insulin-dependent GT after
exercise, but does not fully compensate for reduced insulin sensitivity in
muscle from HFD-fed rats. Soleus muscles were isolated from exercised rats
2 hours after exercise and from sedentary rats. Isolated muscles were
incubated for 30 minutes in the absence (insulin−) or presence of 0.9 nmol/L
insulin (insulin+), and 3MG uptake was determined. Baseline (insulinindependent) activity was subtracted in each diet group. Data are means ±
SE; n = 10 per group. *P b .05 vs CD-fed group.
S. Tanaka et al. / Metabolism Clinical and Experimental 56 (2007) 1719–1728
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Fig. 4. High-fat diet attenuates muscle AMPKα2 activation induced by one bout of endurance exercise. Soleus muscles were isolated from exercised rats
immediately after exercise and from sedentary rats. Isoform-specific AMPK activity was determined in anti-AMPKα1 (A) and α2 (B) immunoprecipitates.
Muscles were also subjected to Western blot analysis using antiphosphorylated AMPK (C), antiphosphorylated ACC (D), and anti-AMPKα (E) antibodies. Fold
increases are expressed relative to the activity of muscles from the sedentary CD-fed group. Data are means ± SE; n = 13 to 16 per group. *P b .05 vs CD-fed
group, †P b .05 vs corresponding sedentary group.
epitrochlearis muscle in the presence of half-maximally
effective concentration of insulin (0.8 nmol/L) after rats
swam for 2 hours. They found no increase after exercise in
insulin-dependent GT in muscles from HFD-fed rats because
of the maintenance of a high level of insulin-independent GT
in skeletal muscle even at 3.5 hours after exercise. In
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Table 2
Muscle glycogen and triglyceride concentrations in rats under HFD and CD
feeding
Sedentary
Immediate postexercise
Muscle glycogen (μmol/g wet weight)
CD
23.6 ± 0.8
7.6 ± 0.6 †
HFD
22.4 ± 0.6
8.9 ± 0.4 †
Muscle triglycerides (μmol/g wet weight)
CD
6.1 ± 0.5
4.9 ± 0.1 †
⁎
HFD
7.8 ± 0.2
7.1 ± 0.5 ⁎
2 h postexercise
18.1 ± 1.0 ‡
15.9 ± 0.5 ‡
5.0 ± 0.3 †
6.5 ± 0.5 † ⁎
Soleus muscles were isolated from sedentary and exercised rats immediately
and 2 hours after exercise, and glycogen and triglycerides concentrations
were determined. Data are means ± SE; n = 6 to 14 per group.
⁎ P b .05 vs CD-fed group.
†
P b .05 vs sedentary group.
‡
P b .05 vs immediate postexercise group.
Fig. 5. One bout of endurance exercise does not affect insulin-stimulated
phosphorylation of Akt in CD- and HFD-fed rats. Soleus muscles were
isolated from exercised rats 2 hours after exercise and from sedentary rats,
and then incubated for 30 minutes in the presence of insulin (0.9 nmol/L).
Muscles were subjected to Western blot analysis using antiphosphorylated
Akt antibody. Fold increases are expressed relative to the level in muscles
from the sedentary CD-fed group. Data are means ± SE; n = 9 per group.
contrast, insulin-independent GT was substantially lower 2
hours after exercise in our study; and insulin-dependent
GT was lower in muscles from HFD-fed animals (Fig. 3).
Thus, it seems reasonable to speculate that prolonged
HFD feeding evokes “exercise resistance” as well as
insulin resistance; that is, HFD does not allow physiological exercise to activate GT or insulin sensitivity to the
level achieved by similar exercise in skeletal muscle of
CD-fed animals.
The blunted AMPK activation during exercise may be
part of the mechanism leading to impaired exercise-
Fig. 6. High-fat diet feeding does not change muscle GLUT4 protein level.
The GLUT4 protein level was determined in soleus muscle using Western
blot analysis. Fold increases are expressed relative to the level in the CD-fed
group. Data are means ± SE; n = 6 to 7 per group.
stimulated glucose metabolism in skeletal muscle. In the
present study, short-term exercise increased the activity of
the α2 isoform of muscle AMPK from baseline in CD-fed
rats, but not in HFD-fed rats. Correspondingly, exercise
stimulated the phosphorylation of AMPKα Thr172 and ACC
Ser79 only in muscles from CD-fed animals. AMPK is a
heterotrimeric serine-threonine protein kinase comprising a
catalytic α subunit and regulatory β and γ subunits. Two
distinct α isoforms, α1 and α2, are expressed in skeletal
muscle; and both isoforms can be activated in response to
muscle contraction by AMP-independent or AMP-dependent mechanisms [13]. AMPK has been implicated in a
number of exercise-stimulated metabolic events in skeletal
muscle, including insulin-independent GT and GLUT4
translocation [12,19-21], insulin sensitivity [22-24], fatty
acid oxidation by the inactivation of ACC [12,25,26],
GLUT4 expression [12,27-31], and glycogen utilization
[14,32-34]. The mechanism underlying the significantly
higher basal α2 activity in muscle from HFD-fed rats than in
CD-fed rats (Fig. 4B) is unknown; but increased serum leptin
concentration might play a role because, in vivo, both short[35] and long-term [36] administrations of leptin activate
AMPKα2 activity.
Although the predominant activation of AMPKα2 by
exercise is consistent with most previous studies [12,37,38],
we have recently reported that AMPKα1 activity is more
sensitive to physical or physiological stress than AMPKα2 is
and that AMPKα1 activity increases markedly during
dissection, whereas AMPKα2 activity does not change
[13]. Thus, it may be difficult to measure the α1 activity
because it is disturbed by additional activation during
dissection; only after high-intensity exercise, when the
activation by muscle contraction exceeds that of the isolating
stimuli, would AMPKα1 activity be detectable. In our 2006
study [13], we stabilized isolated muscle in KRBP for 60
minutes, which decreased α1 activity to a constant level and
allowed us to observe the activation of AMPKα1 by
electrical stimulation. This α1 activation was associated
with corresponding increases in AMPKα phosphorylation,
insulin-independent GT, and ACC phosphorylation.
S. Tanaka et al. / Metabolism Clinical and Experimental 56 (2007) 1719–1728
Moreover, α1 was activated even by low-intensity contraction, which was characterized by the absence of an increase
in AMP concentration or in the ratio of AMP to adenosine
triphosphate. These observations suggest that the α1 isoform
is the predominant form activated by low-intensity contractions and lead us to believe that activities of both α2 and α1
isoforms increase in response to moderate-intensity treadmill
exercise used in our current study.
The mechanisms underlying the postexercise increase in
insulin sensitivity, which probably relates to GLUT4
translocation in exercised muscle [39], are presumed to be
mediated by multiple factors, including AMPK [22-24],
muscle glycogen concentration, humoral factors, and
autocrine-paracrine mechanisms (reviewed in Hayashi et
al [1]). In the present study, Akt phosphorylation in
response to insulin stimulus in muscle of HFD-fed rats
did not decrease at rest or after exercise despite the blunted
insulin-dependent GT. This result is consistent with a
previous study showing that muscle insulin resistance
induced by an HFD is not accompanied by impairment of
Akt activation [40] and with studies demonstrating that the
postexercise increase in insulin sensitivity does not
accompany an enhancement of the insulin signal
[22,39,41,42]. We conclude that some functional changes
in the GLUT4 translocation system or signal transduction
mechanism distal to Akt phosphorylation play a role in
HFD-induced insulin resistance and impaired postexercise
increase in insulin sensitivity.
People with type 2 diabetes mellitus exhibit remarkable
muscle insulin resistance and abnormal lipid metabolism,
which are also common among HFD-fed individuals. In
contrast, previous studies have shown that the insulinindependent GT system activated by exercise is intact in
diabetic people, unlike in the case of HFD-fed experimental
animals. Kennedy et al [43] showed that muscles from
people with type 2 diabetes mellitus retain the capacity to
translocate GLUT4 to the sarcolemma in response to shortterm exercise. Correspondingly, Musi et al [38] demonstrated that exercise normally activates muscle AMPK in
people with type 2 diabetes mellitus. We note that
individuals with type 2 diabetes mellitus generally have
normal muscle GLUT4 protein levels [44,45]. Similarly, the
genetically insulin-resistant obese Zucker rat has severe
defects in insulin-stimulated glucose uptake [46] and
GLUT4 translocation [47] despite normal levels of total
muscle GLUT4 protein [46]. In contrast, these animals have
normal increases in contraction-stimulated glucose uptake
[48] and GLUT4 translocation [49,50]. To our knowledge,
no study has addressed whether insulin sensitivity increases
normally after a single bout of endurance exercise in people
with type 2 diabetes mellitus or obese Zucker rats. Our
finding that the HFD attenuated contraction-induced GT in
muscle raises the possibility that the pathophysiological
condition induced by the HFD differs from the condition
exhibited by humans with type 2 diabetes mellitus and
genetically insulin-resistant animals.
1727
In summary, our study provides new evidence to suggest
that moderate-intensity endurance exercise activates both
insulin-independent and insulin-dependent components of
muscle GT even when combined with HFD-induced insulin
resistance. However, these metabolic effects are significantly
reduced by an HFD; and consequently, exercise cannot
compensate totally for muscle insulin resistance in HFD-fed
rats. Although the precise mechanism is not clear, an HFD
may evoke these metabolic impairments by reducing AMPK
activation in contracting skeletal muscle. Appropriate fat
intake might be important for efficient activation of glucose
metabolism by exercise.
Acknowledgments
We thank Takao Shirai, Kyoto University, for technical
suggestions and Yoko Koyama and Kaoru Ijiri for secretarial
assistance. We also thank the Radioisotope Research Center
of Kyoto University for instrumental support in the radioisotope experiments. This work was supported by a research
grant from the Japan Society for the Promotion of Science (to
Tatsuya Hayashi). Taro Toyoda was supported by the
Research Fellowship of the Japan Society for the Promotion
of Science for Young Scientists.
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