Original Article

Skeletal Muscle Adaptation to Exercise Training

AMP-Activated Protein Kinase Mediates Muscle Fiber Type
Shift
Katja S.C. Rockl,1 Michael F. Hirshman,1 Josef Brandauer,1 Nobuharu Fujii,1 Lee A. Witters,2 and
Laurie J. Goodyear1

Regular endurance exercise has profound benefits on overall health, including the prevention of obesity, cardiovascular disease, and diabetes. The objective of this study was
to determine whether AMP-activated protein kinase
(AMPK) mediates commonly observed adaptive responses
to exercise training in skeletal muscle. Six weeks of voluntary wheel running induced a significant (P < 0.05) fiber
type IIb to IIa/x shift in triceps muscle of wild-type mice.
Despite similar wheel running capacities, this traininginduced shift was reduced by 40% in transgenic mice
expressing a muscle-specific AMPK2 inactive subunit.
Sedentary mice carrying an AMPK-activating mutation
(1TG) showed a 2.6-fold increase in type IIa/x fibers but
no further increase with training. To determine whether
AMPK is involved in concomitant metabolic adaptations to
training, we measured markers of mitochondria (citrate
synthase and succinate dehydrogenase) and glucose uptake
capacity (GLUT4 and hexokinase II). Mitochondrial markers increased similarly in wild-type and AMPK2-inactive
mice. Sedentary 1TG mice showed a 25% increase in
citrate synthase activity but no further increase with training. GLUT4 protein expression was not different in either
line of transgenic mice compared with wild-type mice and
tended to increase with training, although this increase
was not statistically significant. Training induced a 65%
increase in hexokinase II protein in wild-type mice but not
in AMPK2-inactive mice. Hexokinase II was significantly
elevated in sedentary 1TG mice, without an additional
increase with training. AMPK is not necessary for exercise
training-induced increases in mitochondrial markers, but it
is essential for fiber type IIb to IIa/x transformation and
increases in hexokinase II protein. Diabetes 56:20622069,
2007

From the 1Research Division, Joslin Diabetes Center and Department of

Medicine, Brigham and Womens Hospital, and Harvard Medical School,
Boston, Massachusetts; and 2Dartmouth Medical School, Hanover, New
Hampshire.
Address correspondence and reprint requests to Laurie J. Goodyear, PhD,
Senior Investigator and Head, Section on Metabolism, Joslin Diabetes Center,
One Joslin Place, Boston, MA 02215. E-mail: laurie.goodyear@joslin.
harvard.edu.
Received for publication 21 February 2007 and accepted in revised form 1
May 2007.
Published ahead of print at http://diabetes.diabetesjournals.org on 18 May
2007. DOI: 10.2337/db07-0255.
AICAR, 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; AMPK,
AMP-activated protein kinase; MHC, myosin heavy chain; PGC, peroxisome
proliferatoractivated receptor- coactivator.
2007 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.

2062

he beneficial effects of regular physical activity

on a variety of pathological conditions such as
obesity, cardiovascular disease, or diabetes are
undisputed (1). These positive effects are most
likely related to a number of adaptations that occur in
skeletal muscle as a response to exercise training. They
allow the muscle to more efficiently use substrates for ATP
production and thus become more resistant to fatigue.
Adaptations of skeletal muscle fibers to exercise training
occur, for example, by the expression of specific contractile proteins (myosin heavy chain [MHC] isoforms) and by
an increase in the activity and content of mitochondria,
also referred to as oxidative capacity (2 4). The exact
molecular mechanisms that underlie these adaptations
remain elusive. A better understanding of the signaling
pathways mediating skeletal muscle adaptations to exercise training may help reveal new approaches for treating
metabolic or cardiovascular diseases, which are rising
rapidly in our increasingly sedentary society.
Muscle fibers have traditionally been classified according to their expression of MHC isoforms as fast twitch
fibers (types IIb, which are not expressed in humans [5],
IIx, and IIa), and slow twitch fibers (type I) (5,6). Type IIb
and type IIx fibers depend mainly on glycolytic pathways
for ATP production, whereas type IIa and type I fibers rely
predominantly on oxidative pathways (5). Endurance exercise training has been shown to increase mitochondria
(2 4). Exercise training also has well-established effects to
change MHC isoform expression, thus provoking a fiber
type switch from type IIb to IIx and IIa and in rare cases
also to type I muscle fibers (7,8). Importantly, mitochondrial biogenesis and fiber type changes can occur independently of each other, suggesting distinct signaling
pathways for both types of adaptive responses (3).
Known as the fuel gauge of the cell, AMP-activated
protein kinase (AMPK) plays a key metabolic role during
exercise. To restore ATP levels, AMPK has been proposed
to increase muscle glucose uptake and to be involved in
fatty acid and carbohydrate metabolism (9 12). AMPK has
previously been suggested as a possible mediator of
muscle fiber plasticity (13); however, a role of AMPK in
training-induced fiber type transformation has not yet
been investigated. AMPK activation by 5-aminoimidazole4-carboxamide-1--D-ribofuranoside (AICAR) has also
been shown to activate transcription factors (12) as well
as to initiate mitochondrial biogenesis (14). Yet, reports on
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K.S.C. ROCKL AND ASSOCIATES

exercise-induced increases in mitochondria through

AMPK signaling have been inconsistent (15,16).
Peroxisome proliferatoractivated receptor- coactivator (PGC)-1 is a major regulator of mitochondrial biogenesis (17). In addition, overexpression of PGC-1 in
transgenic mice promotes muscle fiber type II to I transformation (13). Interestingly, studies in which rats were
treated with AICAR have shown an induction of PGC-1
mRNA expression in skeletal muscle (12), which creates a
possible link between AMPK and PGC-1 signaling.
The interest in exploring the role of AMPK in muscle
adaptations to exercise training lies particularly in its
clinical relevance. Individuals with insulin resistance or
type 2 diabetes tend to have more glycolytic type IIx
(formerly misclassified as type IIb) skeletal muscle fibers
than healthy individuals, and a number of studies have
shown that fiber type distribution correlates with insulin
resistance, as reviewed in Zierath and Hawley in 2004 (18).
This correlation implies that muscle fiber plasticity may
play an essential role in the pathogenesis of diabetes.
AMPK activation, which occurs during exercise (10,19),
may be involved in metabolic changes such as increased
skeletal muscle glucose uptake (10,20) and increased fatty
acid oxidation (9,12). Moreover, experiments with animal
models of type 2 diabetes have provided evidence that
AICAR activation of AMPK can reverse many of the
metabolic defects of these animals in vivo (11,21). Finally,
the AMPK system is the probable target of the antidiabetes
drugs metformin and thiazolidinediones (11,22). Given the
significance of AMPK in exercise-induced metabolic signal
transduction, it is likely that its repeated activation plays
an important role in provoking skeletal muscle adaptations to exercise training.
The overall purpose of our study was to investigate
whether AMPK is necessary for adaptations of skeletal
muscle to endurance training. In particular, we studied
transformations of muscle fiber types, increases in mitochondrial markers, and increases in proteins involved in
skeletal muscle glucose uptake. Using transgenic mouse
models of attenuated AMPK2 or chronically increased
total AMPK activity, we found that AMPK is an important
mediator of muscle fiber type changes and increases in
hexokinase II protein after exercise training, whereas
increases in mitochondrial markers are not dependent on
AMPK activity.
RESEARCH DESIGN AND METHODS
Muscle-specific transgenic mice expressing the inactivating D157A mutation
in their 2 subunit of AMPK (2iTG) were generated on an FVB background
as described previously (23). Basal and contraction-stimulated AMPK2
activity of 2iTG mice is almost abolished, whereas basal AMPK1 activity is
normal, and contraction-stimulated AMPK1 activity is 50% decreased
compared with that of their wild-type littermates (23,24). The 2iTG mice
used for this study were heterozygotes for the inactivating mutation. Musclespecific R70Q1 transgenic mice (1TG) expressing the activating R70Q
mutation in their 1 subunit of AMPK were generated on an FVB background
as described previously (25). Basal total AMPK activity in 1TG mice is
approximately threefold higher than that in wild-type littermates (25). The
1TG mice used for this study were heterozygotes for the activating mutation.
Physiological and behavioral characterization using the Comprehensive Lab
Animal Monitoring System in both transgenic animal models showed that the
respective transgene did not significantly alter locomotive activity, oxygen
consumption, carbon dioxide generation, heat generation, or food and water
consumption (23; M.F.H., L.A.W., L.J.G., unpublished data). Mice were housed
at a constant temperature (20 22C) with a 12 h:12 h light-dark cycle and
received LabDiet rodent chow (Purina Mills, St. Louis, MO) and water ad
libitum.
DIABETES, VOL. 56, AUGUST 2007

Training protocol. Seven-week-old female 2iTG and 1TG mice and

corresponding wild-type littermates were randomly assigned to housing in
individual cages with or without running wheels (Nalgene, Rochester, NY) for
a total of 6 weeks. Completed wheel revolutions and time spent running were
continuously monitored. On day 42, all mice were removed from their cages
3 4 h before being killed by cervical dislocation. Preliminary experiments
from our laboratory have shown that wheel cage running has the most
pronounced training effects (as determined by increases in citrate synthase
activity) in triceps brachii. We therefore chose to use this muscle for our
studies. After the mice were killed, triceps muscles from both forelegs were
immediately removed. One triceps of each animal was snap frozen in liquid
nitrogen and stored at 80C. The other triceps was embedded in Tissue-Tek
O.C.T. compound (Sakura Finetek, Torrance, CA), quick frozen in isopentane
cooled by liquid nitrogen, and stored at 80C until cryosections were
prepared for histology. All experiments were in accordance with guidelines of
the Institutional Animal Care and Use Committee of the Joslin Diabetes
Center and the National Institutes of Health.
Blood glucose, body weight, and food consumption. At weeks 0, 2, 4, and
6, blood glucose, body weight, and food consumption were recorded. Blood
glucose measurements were performed between 3 and 4 P.M. by obtaining
blood samples from the tail of fully conscious mice, using a OneTouch Ultra
portable glucometer (Lifescan, Mipitas, CA). Body weight and food consumption were determined using a digital scale (model CS200; Ohaus, Pine Brook,
NJ).
Immunoblot analysis. Frozen muscles were pulverized and homogenized as
described previously (26), and protein concentrations were determined via
the Bradford assay. Skeletal muscle proteins (40 g) were resolved by
SDS-PAGE for Western blot analysis (27). Antibody-bound proteins were
visualized on film using chemiluminescence detection reagents (Perkin-Elmer
Life Sciences, Boston, MA). Protein bands were scanned with an ImageScanner (Amersham Biosciences) and quantitated by densitometry (FluorChem 2.0; Alpha Innotech, San Leandro, CA). Commercially available primary
antibodies were PGC-1 (Calbiochem, San Diego, CA), hexokinase II (Santa
Cruz Biotechnology, Santa Cruz, CA), and GLUT4 (Chemicon International,
Temecula, CA). The PGC-1 antibody was a gift from Dr. Bruce Spiegelman
and was produced as described (28).
Electrophoretic separation of MHC isoforms. A 10-mg portion of pulverized triceps from each animal was homogenized on ice in a lysis buffer (pH
6.5) containing 300 mmol/l KCl, 100 mmol/l KH2PO4, 50 mmol/l K2HPO4, and 1
mmol/l EDTA and then centrifuged at 12,000g for 1 min. Total protein content
was assessed via the Bradford assay. Electrophoretic separation of MHC
isoforms was performed as described previously (29) with slight modifications. Briefly, 1 g of skeletal muscle proteins was resolved using an
SDS-PAGE gel (0.75 mm) consisting of a stacking gel with 4% acrylamide-bis
(50:1) and 30% glycerol and a separating gel with 8% acrylamide-bis (50:1) and
30% glycerol. The upper running buffer consisted of 300 mmol/l Tris (base),
450 mmol/l glycine, 0.3% SDS, and 10 mmol/l mercaptoethanol. The lower
running buffer consisted of 50 mmol/l Tris (base), 75 mmol/l glycine, and 0.05%
SDS. The gels were run on an electrophoresis Minigel system (Bio-Rad
Laboratories, Hercules, CA) at 4C at a constant current of 6 mA/gel for 25 h.
After the electrophoresis, the gels were fixed for 20 min in 7% acetic acid and
50% methanol, stained for 1 h with GelCode Blue Stain Reagent (Pierce
Biotechnology, Rockford, IL), and washed with double-distilled H2O overnight. Gels were scanned with the ImageScanner, and the relative proportion
of each MHC isoform was quantitated by densitometry. Bands were identified
according to their migration characteristics as described previously (29). In
triceps muscle, electrophoresis resulted in two distinct bands. The upper band
corresponded to combined MHC IIa and IIx (referred to as MHC IIa/x) and the
lower band to MHC IIb. In soleus muscle, an additional lower band was
identified as MHC I, and, as expected, this band was not present in triceps,
which is a mainly glycolytic muscle as determined by ATPase staining.
Succinate dehydrogenase staining. Mid-belly cross-sections of triceps
muscle were cut at 8 m in a cryostat (20C). After drying for 5 min at room
temperature, the sections were incubated for 30 min at 37C in the incubation
solution (pH 7.6) containing 6.5 mmol/l sodium phosphate monobasic, 43.5
mmol/l sodium phosphate biphasic, 0.6 mmol/l nitroblue tetrazolium (N6876;
Sigma), and 50 mmol/l sodium succinate. The sections were then rinsed three
times for 30 s each in physiological saline and for 5 min in 15% ethanol and
coverslipped with Faramount aqueous mounting medium (DakoCytomation)
(30).
Citrate synthase activity. Citrate synthase activity was determined in
muscle lysates spectrophotometrically at room temperature (412 nm) to
detect the transfer of sulfhydryl groups to 5,5-dithiobis-2-nitrobenzoate using
saturating concentrations of substrates and cofactors: 80 mmol/l Tris-HCl, 0.1
mmol/l 5,5-dithiobis-2-nitrobenzoate, 0.4 mmol/l acetyl-CoA, and 0.6 mmol/l
oxaloacetate (31). Citrate synthase activity was calculated as micromoles per
minute per gram of protein.
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AMPK MEDIATES MUSCLE FIBER TYPE SHIFT

TABLE 1
Baseline parameters of sedentary and trained wild-type and 2iTG mice

n
Body weight (g)
Total food intake (g/g body wt)
Blood glucose (mg/dl)
Distance (km/day)
Speed (m/min)

Wild-type
sedentary

Wild-type
trained

2iTG
sedentary

2iTG
trained

10
20.67 0.58
8.79 0.51
170 5.0
NA
NA

9
19.37 0.72
9.44 0.36
167 4.5
4.56 0.66
24.06 0.79

9
20.22 0.76
7.61 0.20*
162 4.8
NA
NA

10
20.51 0.38
9.01 0.31
169 3.3
4.56 0.58
24.36 0.55

Data are means SE. *P 0.05 (vs. wild type for respective condition); P 0.05 (vs. sedentary). NA, not applicable.
Statistical analysis. Data are means SE. All data were compared using
two-way ANOVA and Tukeys post hoc analysis. Pearsons correlation coefficients were used to describe the linear association between variables. The
differences between groups were considered significant when P 0.05.

RESULTS

Baseline parameters and exercise training performance. Body weight and blood glucose concentrations
were not different between the study groups throughout
the training period (Tables 1 and 2). All training groups
showed a tendency for an increase in food intake compared with the sedentary control groups, which was
statistically significant in the trained 2iTG and 1TG
mice. Running distances and running speeds did not differ
between transgenic mice and their wild-type littermates
and were maintained throughout the training period (Tables 1 and 2 and Figs. 1A and 2A).
Training-induced fiber type shift from IIb to IIa/x is
reduced in 2iTG mice. We hypothesized that one of the
responses of skeletal muscle to chronic intermittent (exercise training) or chronic sustained (1TG mice) AMPK
activation may be the mediation of muscle fiber type
transformation. To study fiber type changes, we performed
electrophoretic separation of MHC isoforms in triceps
muscle, a mainly glycolytic muscle with very few type I
fibers, as determined by ATPase staining. Electrophoretic
separation of MHC isoforms resulted in two distinct bands
that were identified according to their migration characteristics as previously described (29). The lower band
corresponded to MHC IIb, and the upper band corresponded to a combination of MHC IIa and IIx (Fig. 1B).
Further separation of MHC IIa and IIx was not achieved, a
difficulty that has been reported earlier (32). To signify
that the highest migrating band may contain both MHC IIa
and IIx isoforms, we refer to this band as IIa/x.
In wild-type mice, exercise training significantly increased the relative proportion of type IIa/x fibers (Fig.
1B). Exercise training also induced an increase of type
IIa/x fibers in 2iTG mice. However, the proportion of type
IIa/x fibers in trained 2iTG mice was significantly lower

than that in their trained wild-type littermates. Some of the

variability in fiber type adaptations in trained wild-type
mice can be explained by the differences in total running
distance. Figure 1C illustrates the fact that running distance correlated strongly with the relative proportion of
type IIa/x fibers in wild-type mice. In 2iTG mice, this
correlation was absent.
Increase of type IIa/x muscle fibers in 1TG mice. In
a second approach to investigate the role of AMPK activity
in fiber type transformation, we performed muscle fiber
type analysis in triceps of sedentary and trained 1TG
mice, a genetic model of chronic AMPK activation (25)
(Fig. 2B). Training significantly increased type IIa/x fibers
in wild-type mice. Sedentary 1TG mice had a 2.6-fold
higher proportion of type IIa/x fibers than their sedentary
wild-type littermates, and exercise training did not further
increase type IIa/x fibers in the transgenic animals. Figure
2C illustrates total running distances and corresponding
type IIa/x fibers in wild-type and 1TG mice. There was no
significant correlation between running distance and type
IIa/x fibers in these training groups.
Decreased mitochondrial markers in 2iTG mice but
normal adaptive response to exercise training. Exercise training induces a variety of metabolic adaptations
that lead to improvements in fatty acid oxidation (33) and
glucose metabolism (34). To study whether AMPK mediates exercise traininginduced increases in oxidative capacity, we assessed mitochondrial markers. Citrate
synthase activity was 14% lower (P 0.05, main effect)
in triceps of 2iTG mice compared with their wild-type
littermates (Fig. 3A). Exercise training, however, induced
a similar increase in citrate synthase activity in 2iTG and
wild-type mice. Succinate dehydrogenase staining of triceps mid-belly cross-sections supports these results (Fig.
3B). The stain was more intense in muscle sections from
all trained animals, suggesting that ablation of AMPK2
activity has little effect on the increase in mitochondria in
response to exercise training.

TABLE 2
Baseline parameters of sedentary and trained wild-type and 1TG mice

n
Body weight (g)
Total food intake (g/g body wt)
Blood glucose (mg/dl)
Distance (km/day)
Speed (m/min)

Wild-type
sedentary

Wild-type
trained

1TG
sedentary

1TG
trained

8
20.71 0.43
8.00 0.31
148 3.8
NA
NA

7
20.60 0.34
8.63 0.28
157 2.6
2.07 0.24
22.45 0.50

7
20.59 0.45
7.50 0.25
154 5.6
NA
NA

8
21.14 0.78
8.92 0.39*
156 3.1
2.26 0.31
22.34 0.59

Data are means SE. *P 0.05 (vs. sedentary). NA, not applicable.
2064

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K.S.C. ROCKL AND ASSOCIATES

FIG. 1. MHC profile in sedentary and trained wild-type and 2iTG

muscle. 2iTG mice and their wild-type littermates performed 6 weeks
of voluntary wheel cage running or were kept as sedentary controls. A:
Average daily running distances were calculated from continuous
recordings. Triceps muscles were harvested and processed for electrophoresis. B: MHC isoforms were resolved by SDS-PAGE electrophoresis and visualized by Coomassie blue staining, resulting in two distinct
bands for MHC IIa/x (upper band) and MHC IIb (lower band). The
relative proportion of MHC IIa/x isoform in sedentary and trained
wild-type and 2iTG mice was compared by two-way ANOVA. The data
are means SE. *P < 0.05 (versus sedentary); **P < 0.01 (versus
sedentary); #P < 0.05 (versus wild type for respective condition). C:
The correlation between total running distance and percentage of type
IIa/x fibers of each study animal is shown for wild-type (left graph) and
2iTG mice (right graph). sed, sedentary.

Increased mitochondrial markers in 1TG mice. In

sedentary 1TG mice, citrate synthase activity was 13%
higher than that in their wild-type littermates (Fig. 4A).
Similarly, succinate dehydrogenase staining suggested an
DIABETES, VOL. 56, AUGUST 2007

FIG. 2. MHC profile in wild-type and 1TG muscle. The same experimental procedures as described for Fig. 1 were carried out using 1TG
mice and their wild-type littermates. The data are means SE. *P <
0.05 (versus sedentary); ##P < 0.01 (versus wild type for respective
condition). sed, sedentary.

increase of mitochondria in sedentary 1TG mice compared with their wild-type littermates as shown in representative micrographs (Fig. 4B). We did not observe any
further increase in mitochondrial markers attributable to
exercise training in 1TG mice.
PGC-1 and PGC-1 protein expression. The transcriptional coactivator PGC-1 has previously been linked
to fiber type II to I transformation and mitochondrial
biogenesis (13,17). The PGC-1 homolog, PGC-1, has
recently been proposed to drive muscle fiber type IIx
formation (35). In our study, PGC-1 protein was not
altered in either line of transgenic mice compared with
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AMPK MEDIATES MUSCLE FIBER TYPE SHIFT

FIG. 3. Mitochondrial markers in sedentary and trained wild-type and

2iTG triceps muscle. Citrate synthase activity assays as well as
succinate dehydrogenase staining were performed to assess differences in mitochondrial content between sedentary and trained wildtype and 2iTG muscle. A: Citrate synthase activity is expressed
relative to wild-type sedentary (1.0 activity unit equals 487 23 mol
min1 g protein1). Data are means SE. **P < 0.01 (versus
sedentary); #P < 0.05 (versus wild type). B: Mid-belly cross-sections of
triceps muscle were obtained and stained for succinate dehydrogenase
activity. Note the marked difference in stain intensity between sedentary and trained muscle in both wild-type and 2iTG mice. sed,
sedentary.

wild-type animals or after exercise training (data not

shown). In contrast, protein expression of PGC-1 was
influenced by genotype as well as training. Ablation of
AMPK2 activity in 2iTG mice was associated with a 32%
decrease in PGC-1 protein expression (P 0.05, main
effect) (Fig. 5A). Exercise training, however, increased
PGC-1 in 2iTG mice to an extent similar to that in
wild-type mice (54% vs. 55%). Sedentary 1TG mice expressed elevated levels of PGC-1 protein compared with
their corresponding wild-type littermates (Fig. 5B), and
there was a tendency for training to increase PGC-1 in
1TG mice (28%, P 0.06).
GLUT4 and hexokinase II protein expression. Muscle
glucose uptake is controlled by glucose transport across
the cell membrane, mainly by GLUT4, as well as intracellular phosphorylation by hexokinase II (36,37). Both
2066

FIG. 4. Mitochondrial markers in sedentary and trained wild-type and

1TG muscle. A: Citrate synthase activity is expressed relative to
wild-type sedentary (1.0 activity unit equals 373 22 mol min1 g
protein1). The data are means SE. **P < 0.01 (versus sedentary);
#P < 0.05 (versus wild type); ##P < 0.01 (versus wild type for
respective condition). B: Representative micrographs of mid-belly
cross-sections of triceps muscle stained for succinate dehydrogenase
activity. sed, sedentary.

GLUT4 and hexokinase II have previously been reported

to be upregulated by exercise training (38 40). In the
current study, training-induced increases in GLUT4 protein expression did not reach statistical significance (main
effect: P 0.06 [Fig. 6A] and 0.12 [Fig. 6B]), and no
differences were observed between genotypes. Hexokinase II protein expression was not different in sedentary
2iTG and wild-type mice (Fig. 6C). However, whereas
exercise training induced a 65% increase in hexokinase II
in wild-type mice, there was no significant change in
2iTG mice. 1TG mice showed an overall higher expression of hexokinase II than their wild-type littermates with
no significant additional increase after exercise training
(Fig. 6D).
DISCUSSION

We investigated the role of AMPK in skeletal muscle

adaptations to exercise training. For this purpose, we used
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K.S.C. ROCKL AND ASSOCIATES

FIG. 5. PGC-1 protein expression. After 6 weeks of voluntary wheel

cage running, triceps muscles of 2iTG (A) and 1TG mice (B) were
studied for PGC-1 protein expression by immunoblotting with antiPGC-1 antibody. Data are means SE. *P < 0.05; **P < 0.01 (versus
sedentary); #P < 0.05 (versus wild type for respective condition). sed,
sedentary; tr, trained; a.u., arbitrary units.

transgenic mouse models with either attenuated (2iTG

mice) or increased (1TG mice) AMPK activity and studied long-term effects of endurance training. Our main
finding was that AMPK2 is an important mediator of
training-induced muscle fiber type changes as shown by
decreased fiber type transformation in trained 2iTG mice.
The fiber type profile of 1TG mice with increases in type
IIa/x muscle fibers further emphasizes the significance of
AMPK activity in the determination of muscle fiber types.
Previous studies showed that AMPK activity in 1TG mice
is not further increased by acute bouts of exercise (25).
Hence, exercise training does not result in repeated periods of (additional) AMPK activation in these mice, which
may explain the absence of a training-induced increase in
type IIa/x muscle fibers in 1TG mice.
Although inactive AMPK2 in 2iTG mice was associated with a significant reduction in training-induced fiber
type transformation, the fiber type IIb to IIa/x shift was not
completely inhibited. These data suggest that AMPK2 is
not solely responsible for this skeletal muscle adaptation.
Preserved AMPK1 activity may account for the residual
response. However, data from a variety of studies suggest
that AMPK activity in skeletal muscle is almost exclusively
a function of the 2 isoform (20,41). Hence, it is more
likely that alternative fiber typeregulating pathways remain responsive in 2iTG mice and account for the
residual fiber type adaptation. In particular, the Ca2/
calmodulin-dependent protein phosphatase calcineurin
has been proposed as an important regulator of fiber type
II to I transformation (42).
It seems possible that AMPK2 and calcineurin signaling are parallel pathways in muscle fiber type adaptations,
possibly being differentially regulated by certain stimuli,
such as innervation patterns or exercise intensity. Our
transgenic mouse model with chronically active AMPK
(1TG) has a marked increase in type IIa/x fibers but no
increase in type I fibers in triceps muscle. This response
closely imitates the physiological response to endurance
exercise training, which typically leads to muscle fiber
type IIb to IIa transformations (7,8). Conversely, expresDIABETES, VOL. 56, AUGUST 2007

FIG. 6. GLUT4 and hexokinase II protein expression. After 6 weeks of

voluntary wheel cage running, triceps muscle of 2iTG (A and C) and
1TG mice (B and D) was studied for GLUT4 (A and B) hexokinase (C
and D) protein expression by immunoblotting with anti hexokinase II
and anti-GLUT4 antibody. Data are means SEM. **P < 0.01 (versus
sedentary); #P < 0.05; ##P < 0.01 (versus wild type). HXKII, hexokinase II; sed, sedentary; tr, trained; a.u., arbitrary units.

sion of constitutively active calcineurin mainly induces

slow-twitch type I fibers (42). This kind of fiber type twitch
has been seen after powerful stimuli such as very rigorous
exercise training programs in humans (8) and electrical
stimulation or cross-innervation in animal studies (43).
Thus, calcineurin may be the predominant mediator of
fiber type II to I transformations, whereas AMPK2 appears particularly important for mediating type IIb to IIa/x
transformations as seen in response to moderate intensity
exercise training. It will be intriguing to further investigate
Ca2-dependent regulators of fiber type changes in combination with the AMPK pathway and their distinct responses to various stimuli.
AMPK activation has previously been reported to induce
mitochondrial biogenesis (14,16). We did not directly
assess mitochondrial content in the present study. However, the reduction in citrate synthase activity in 2iTG
mice and its increase in 1TG mice support the role of
AMPK for baseline mitochondrial content. Thus, we were
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AMPK MEDIATES MUSCLE FIBER TYPE SHIFT

intrigued to find that training-induced increases in mitochondrial markers (citrate synthase activity and succinate
dehydrogenase) occurred normally in 2iTG mice. These
data suggest that AMPK2 activity is not necessary for
training-induced increases in mitochondria. Alternative
pathways, such as the aforementioned calcineurin signaling pathway (44), may play a critical role in mediating
exercise traininginduced increases in mitochondria.
However, recent reports have questioned the involvement
of calcineurin in these training adaptations (45,46).
Clearly, more studies are needed to comprehensively
understand the mechanisms leading to exercise traininginduced mitochondrial biogenesis.
The transcriptional coactivator PGC-1 is known as a
principal factor in muscle fiber plasticity by inducing
mitochondrial biogenesis and fast-to-slow fiber type transformation (13). PGC-1 has been described to be downstream of AMPK signaling (12). In the present study,
consistent with the proposed AMPK/PGC-1 signaling
pathway, 2iTG mice showed decreased levels of PGC-1
protein, whereas 1TG mice had increased PGC-1 expression. Interestingly, exercise training increased PGC-1
protein even when AMPK2 activity was inhibited (2iTG
mice). Hence, in addition to AMPK, there must be other
pathways that regulate PGC-1 protein expression during
exercise training. Ca2/calcineurin signaling as well as the
p38/mitogen-activated protein kinase pathway are possible
candidates that have been reported upstream of PGC-1
(13,47,48). Thus, PGC-1 may represent a point of convergence of various signaling pathways in skeletal muscle
plasticity. The PGC-1 homolog, PGC-1, has recently
been shown to mediate the formation of type IIx muscle
fibers (35). In contrast to PGC-1, however, PGC-1
protein expression was unchanged in either line of transgenic mice and after exercise training. Hence, the present
study provides no evidence for a role of PGC-1 in
training-induced muscle fiber type transformations.
In states of impaired glucose metabolism, endurance
training leads to improvements in glucose tolerance (49).
Two major regulators of glucose uptake into the muscle
cell are the glucose transporter GLUT4 and hexokinase II.
Whereas training-induced increases in GLUT4 expression
have been described in rats and humans (50), only a few
studies have examined GLUT4 in trained mice. In our
study, GLUT4 protein expression resulted in a nonsignificant increase in wild-type triceps muscle by 24 and 28%.
These mild changes are comparable to previously published training effects (1726% increases) in gastrocnemius
muscle of wild-type and AMPK2 knockout mice (16). The
minimal changes in GLUT4 expression in trained mouse
muscle are consistent with previous reports that adaptations of skeletal muscle to exercise training are more
subtle in mice than in rats or humans, which may be due
to the higher basal metabolism in mice (51).
Hexokinase II phosphorylates glucose upon entering the
muscle cell and may be the major rate-limiting factor for
glucose uptake in the insulin- or exercise-stimulated state
(37). AMPK and exercise training have been suggested to
play a role in the regulation of hexokinase (16,40). In our
study, hexokinase II protein expression was substantially
increased in association with exercise training and chronically activated AMPK. Interestingly, in triceps muscle of
2iTG mice, exercise training did not upregulate hexokinase II. This observation conflicts with data from Jorgensen et al. (16) showing normal increases in hexokinase
II protein expression in trained gastrocnemius muscle of
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AMPK2 whole-body knockout mice. The reason for this

discrepancy is not entirely clear, as both AMPK2 knockout and 2iTG mice lack AMPK2 activity. Although the
different mouse lines might yield conflicting results, it is
more likely that there are muscle-specific differences
between triceps and gastrocnemius in responsiveness to
AMPK activity. The exercise-induced increase of hexokinase II suggests increased capacities for glucose phosphorylation as an adaptation to endurance training. Our
results imply that this effect requires AMPK2 activity,
thus extending the importance of this enzyme for exercise
training adaptations in skeletal muscle.
There is strong epidemiological evidence that regular
physical activity improves insulin resistance (1). Furthermore, individuals with diabetes appear to have a distinct
skeletal muscle phenotype with a prevalence of type IIx
fibers and impaired oxidative capacity (18,52). These
observations suggest that skeletal muscle adaptations to
exercise training may play an important role in the prevention of diabetes. The underlying molecular mechanisms, however, are only partly understood. We
demonstrate here that AMPK plays a critical role in
training adaptations of skeletal muscle. Although our
results suggest that the increase in oxidative capacity is
mediated through AMPK-independent pathways, we show
the significance of AMPK signaling for transforming skeletal muscle fiber type IIb to IIa/x as well as for increasing
hexokinase II protein expression. In the long run, a better
understanding of the molecular mechanisms that link
exercise training to improvement or prevention of type 2
diabetes should reveal new targets for treatment and
provide fundamental knowledge of the complex physiological processes in this disease.
ACKNOWLEDGMENTS

This work was supported by the National Institutes of

Health (Grants R01DK068626 and R01AR45670 to L.J.G.,
Grant DK35712 to L.A.W., Training Grant T32DK07260-29
to J.B., and Diabetes Endocrinology Research Grant
DK36836) and by a fellowship within the Postdoc Program
of the German Academic Exchange Service DAAD (to
K.S.C.R.).
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