1 Division of Molecular Physiology, School of Life Sciences, Dundee University, Wellcome Trust Biocentre, Dundee, DD1 5EH Scotland, UK; and 2 Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah 84602
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ABSTRACT |
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The effects of
endurance training on the response of muscle AMP-activated protein
kinase (AMPK) and acetyl-CoA carboxylase (ACC) to moderate treadmill
exercise were examined. In red quadriceps, there was a large activation
of 2-AMPK and inactivation of ACC in response to exercise. This
response was greatly reduced after training, probably because of
reduced metabolic stress. In white quadriceps, there were no effects of
exercise on AMPK or ACC, but
2-activity was higher after training
because of increased phosphorylation of Thr172. In soleus,
there were small increases in
2-activity during exercise that were
not affected by training. The expression of all seven AMPK subunit
isoforms was also examined. The
2- and
2-isoforms were most
highly expressed in white quadriceps, and
3 was expressed in red
quadriceps and soleus. There was a threefold increase in expression of
3 after training in red quadriceps only. Our results suggest that
3 might have a special role in the adaptation to endurance exercise
in muscles utilizing oxidative metabolism.
3-isoform; acetyl-coenzyme A carboxylase; GLUT4; muscle
mitochondrial enzymes; muscle gene expression
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INTRODUCTION |
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ENDURANCE
EXERCISE TRAINING causes adaptations of skeletal muscle that
allow it to adopt a more oxidative, as opposed to glycolytic, mode of
energy metabolism. This is achieved by the initiation of numerous
changes in protein expression (2, 45). For example, the
expression of the glucose transporter GLUT4 and the enzyme hexokinase
II is upregulated, whereas the expression of many glycolytic enzymes is
downregulated. On the other hand, mitochondrial proteins, including
those involved in electron transport, the tricarboxylic acid cycle, and
fatty acid oxidation, are upregulated, as are the size and number of
the mitochondria themselves (22). Although these changes
are well documented, the intracellular signaling pathways that mediate
the changes in gene and protein expression are not understood.
However, some of the changes in protein expression induced by endurance
training are also produced by feeding rats -guanidinoproprionic
acid, a creatine analog that causes a fall in cellular ATP and
phosphocreatine (28, 31). This suggested that cellular
energy charge might be one of the key signals.
The AMPK-activated protein kinase (AMPK) is the downstream component of
a protein kinase cascade that is activated in an ultrasensitive manner
by a drop in cellular energy charge (16, 17, 46, 48). The
key signals that trigger this activation are a rise in AMP coupled with
a fall in ATP (19) and/or a fall in phosphocreatine (36). The system is activated by exercise in rat
(37, 38, 47) or human (12, 51) muscle, as
well as by electrical stimulation of rat muscle (21, 24,
44). Via phosphorylation of acetyl-CoA carboxylase (ACC)
(50) and a consequent decrease in malonyl-CoA (29), this activation appears to underlie the acute
stimulation of fatty acid oxidation during exercise. AMPK
phosphorylates and inactivates the ACC-1/-isoform of ACC (found in
liver and adipose tissue) at three sites, with phosphorylation of
Ser79 being responsible for inactivation
(9, 14). Although the sites phosphorylated by AMPK on the
ACC-2/
-isoform, which is expressed in skeletal muscle, have not been
characterized in detail, AMPK does cause phosphorylation and
inactivation of this isoform (50). An antibody raised
against a phosphopeptide based on the sequence around Ser79
on rat ACC-1/
detected a large increase in phosphorylation of ACC in
response to exercise in human muscle biopsies (6, 40). This suggests that the equivalent site on human ACC-2/
(Ser221) may be phosphorylated by AMPK in vivo.
Activation of AMPK in response to acute exercise also appears to account, at least in part, for the increased translocation of GLUT4 to the plasma membrane and consequent increase in glucose uptake (20, 21, 27, 29). This hypothesis was recently confirmed by using transgenic mice in which the AMPK activity in skeletal muscle was ablated by expression of a dominant negative mutant (32), whereby the effect of contraction on glucose uptake was partly abolished.
As well as these acute effects on muscle metabolism, there is increasing evidence that chronic activation of the AMPK system might underlie some of the long-term effects of endurance exercise training. These studies have mainly been performed by chronic treatment of rats with 5-aminoimidazole-4-carboxamide (AICA) riboside, an agent that is converted inside the cell to AICA ribotide (ZMP), which activates the AMPK system without disturbing AMP or ATP levels (8, 29). This treatment results in increased expression of GLUT4, increased activity of hexokinase and mitochondrial enzymes, and increased glycogen content (23, 49, 54), all of which are also seen in response to endurance training. AICA riboside treatment in vivo also prevented the decrease in GLUT4 content induced by denervation (35) and increased insulin-stimulated glucose transport in isolated muscle (4). In genetically obese mice, AICA riboside treatment also caused increased expression of GLUT4 and hexokinase and improved glucose tolerance (33). These observations, and the finding that the antidiabetic drug metformin activates AMPK in vivo (55), greatly strengthen our previous proposals (48) that activation of the AMPK system is a promising target for treatment of type 2 diabetes.
Although these findings suggest that AMPK is intimately involved in
both the acute and the chronic effects of exercise in skeletal muscle,
little is known about the effects of endurance training on the
expression or response of the AMPK system itself. In this study, we
have addressed this in rat red and white quadriceps and soleus muscles,
and we find that the response is dependent on the muscle type.
Intriguingly, endurance training caused a threefold increase of
expression of the 3-subunit of AMPK in red quadriceps muscle,
although not in soleus or white quadriceps. This is one of the first
reports of regulation of AMPK at the level of protein expression.
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MATERIALS AND METHODS |
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Animal care and training protocol. Male Sprague-Dawley (SAS:VAF) rats (Sasco, Wilmington, MA) were housed in individual cages in a temperature-controlled (21°C) room with a 12:12-h light-dark cycle. Rats assigned to the trained group were run on motor-driven rodent treadmills, 5 days/wk, in two 1-h sessions, morning and afternoon. The initial treadmill speed was 16 m/min (grade 15%). The speed was gradually increased so that after 4 wk, these rats were running at 31 m/min (grade 15%). They were maintained at this training intensity and duration for at least three additional weeks. Rats assigned to the untrained group were run for 5 min/day (same speed as trained group) to accustom them to the treadmill. Rats in the trained group were allowed to eat ad libitum, whereas untrained rats had their food intake restricted so that they gained weight at the same rate as the trained rats. Trained rats weighed 319 ± 9 g, and nontrained rats weighed 320 ± 11 g at the end of the experiment. Preliminary analysis of a group of untrained rats fed ad libitum indicated that their AMPK activities were similar to those of the untrained food-restricted group, so their further analysis was not pursued.
Three days before the final exercise test, trained rats were run for 2 h, and untrained rats were run for 5 min. Jugular catheters were installed with rats under ether anesthesia. The next day, trained rats were run for 1 h in the morning and 1 h in the afternoon. The next day, these rats ran 1 h in the morning only. Untrained rats ran 5 min/day during this period. All rats were given 25 g of rat chow on the evening before being killed. On the third day after catheterization, rats from trained and untrained groups were anesthetized by intravenous injection of pentobarbital sodium via the catheter, either at rest or after running on the treadmill for 5 min at 16 m/min and 5 min at 31 m/min (15% grade). The soleus muscles and the red and white regions of the quadriceps (vastus lateralis) muscle were quickly removed and immediately freeze-clamped between stainless steel blocks at liquid nitrogen temperature. Tissues were kept frozen belowAntibodies.
Sheep anti-1, anti-
2, anti-
1, anti-
2, and anti-PT172
antibodies have been described previously (7, 41, 53).
Sheep anti-
2 and anti-
3 antibodies were raised against the
synthetic peptides CSVFSLPDSKLPGDK and CPGLGEGAQSGPAA [residues
43-56 of rat
2 and rat sequence equivalent to 72-84 of
human
3 (D. Carling, personal communication), respectively, plus
NH2-terminal cysteines for coupling]. Sheep antibodies
were raised and affinity purified as described previously
(53). A rabbit antiserum raised against the bacterially
expressed COOH-terminal region of rat
1 has been described
previously (52).
Measurement of glucose, lactate, and glycogen. Glucose (1) and lactate (13) were measured by spectrophotometric methods on neutralized perchloric acid extracts of blood. Muscle glycogen was determined as described in Ref. 34.
Analysis of metabolic enzymes and GLUT4.
Citrate synthase (39) and hexokinase (43)
were determined as described previously. GLUT4 was determined by
Western blotting (23) with polyclonal
anti-GLUT4-4670-1704 (Biogenesis, Sandown, NH) and horseradish
peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech,
Arlington Heights, IL). Muscles for ACC determination were ground to
powder under liquid nitrogen. The powder was weighed and then
homogenized in a buffer containing 100 mM mannitol, 50 mM NaF, 10 mM
Tris, 1 mM EDTA, 10 mM -mercaptoethanol, pH 7.5, and proteolytic
enzyme inhibitors (10 ml/l aprotonin, 10 mg/l leupeptin, and 10 mg/l
antitrypsin). The homogenate was immediately centrifuged (48,000 g; 30 min). The ACC was precipitated from the supernatant by
addition of 144 mg ammonium sulfate/ml and by stirring for 30 min on
ice. The precipitate was collected by centrifugation (48,000 g; 20 min). The pellet was dissolved in 10% of the original
volume of the homogenate buffer and was centrifuged again to remove
insoluble protein. ACC activity was determined on the supernatant at
citrate-magnesium acetate concentrations ranging from 0 to 20 mM, as
described previously (47). The ACC activity data were
fitted to the Hill equation [v = (Vmax
V0) · Cn/(Ka + Cn), where Vmax is the
maximum velocity, V0 is activity in the absence of citrate, Ka is the activation constant for
citrate, C is citrate concentration, and n is the Hill
coefficient] by use of the Grafit program (Sigma, St. Louis, MO).
Expression of ACC was determined by analyzing 15 µg of extract
protein per lane by SDS-PAGE in 3-8% Tris-acetate gels (NuPAGE,
Invitrogen), with detection by ExtrAvidin (Sigma) and enhanced
chemiluminescence (Amersham Pharmacia Biotech). The results were
recorded by digital photography by use of a Kodak EDAS120 system, with
comparison of net intensities by use of Kodak 1D software.
Analysis of AMPK.
Muscles were rapidly dissected out and frozen in liquid nitrogen. Small
segments of muscle (20-50 mg, longitudinal sections in the case of
soleus) were ground to a powder under liquid nitrogen and homogenized
in 100 µl of ice-cold 50 mM Tris · HCl (pH 7.5 at 4°C), 50 mM NaF, 5 mM Na pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 1 mM benzamidine, 1 mM phenylmethane sulfonyl fluoride,
1% (vol/vol) Triton X-100, and 10% (vol/vol) glycerol by use of a
motor-driven pestle in a 1.5-ml microcentrifuge tube. The homogenate
was kept on ice for 30 min and then centrifuged (4,000 g, 30 min, 4°C). The supernatants were removed and their protein
concentrations determined (3). Immunoprecipitation was
performed using 20 µg of sheep anti-1, anti-
2, anti-
1, anti-
2, or anti-
3 antibodies, or 10 µg of anti-
1 plus 10 µg anti-
2, coupled to protein G-Sepharose. AMPK assays on the
resuspended precipitates were performed as described previously
(18). Western blotting was carried out by running SDS-PAGE
in 4-12% gradient gels (NuPAGE, Invitrogen), transferring to
nitrocellulose (Bio-Rad, 0.45 µm), and probing with affinity-purified
antibodies as follows: anti-
1 (0.2 µg/ml), anti-
2 (0.2 µg/ml), anti-
2 (0.32 µg/ml), anti-
1 (1.2 µg/ml), anti-
2
(1.2 µg/ml), anti-
3 (0.9 µg/ml), anti-PT172 (1.6 µg/ml). Serum
containing the anti-
1 antibody was used at a dilution of 1:10,000.
Antibody binding to the blots was detected by enhanced
chemiluminescence as for ACC.
Statistical analysis. Unless stated otherwise, results are presented as means ± SE, and statistical significance was determined by one-way ANOVA using Bonferroni's comparison of selected data sets for post hoc analysis.
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RESULTS |
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Metabolic effects of endurance training.
Rats were subjected to a 7-wk program of endurance training involving
two 1-h bouts of treadmill exercise per day. Untrained controls were
run for 5 min per day to accustom them to the treadmill. Trained or
untrained rats were either killed at rest or immediately after 10 min
of treadmill exercise. Table 1 shows
blood glucose and lactate levels for the four groups. The glucose
levels were rather constant except that there was a moderate increase
in trained rats (but not in untrained rats) after 10 min of exercise
(P < 0.001). There was a marked increase in blood
lactate (P < 0.001) in untrained animals after 10 min
of exercise that was not observed in trained animals. Table
2 shows glycogen contents of the three muscle types with and without training and acute exercise. The glycogen
content of muscle from trained animals was significantly higher than
that from untrained animals in all three muscle types (before acute
exercise, 1.6-fold, 1.4-fold, and 1.3-fold higher in red quadriceps,
white quadriceps, and soleus, respectively). In all muscle types,
treadmill exercise caused decreases in glycogen content, although these
were small in magnitude in white quadriceps, and in this case the
decrease was significant only in trained animals. Table
3 shows data for the activities of
citrate synthase and hexokinase and for the expression of GLUT4.
Training caused significant increases in citrate synthase in all three
muscle types, in hexokinase in both red quadriceps and soleus, and in GLUT4 in red quadriceps only. Similar effects have been demonstrated previously, and they confirm that the training regimen was effective.
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Immunoprecipitation of AMPK isoforms from skeletal muscle.
To measure AMPK activity, muscles were dissected rapidly and
immediately frozen in liquid nitrogen. The frozen tissue was homogenized in the presence of protein phosphatase inhibitors, and a
low-speed supernatant fraction was prepared. Western blotting (not
shown) revealed that only a small proportion (<15%) of AMPK-1 and
-
2 subunits remained in the pellet fraction. AMPK complexes were
immunoprecipitated from the supernatant using anti-
1 antibodies, anti-
2 antibodies, or a mixture of the two. Control experiments (not
shown) demonstrated that the AMPK activity depleted from the
supernatants was quantitatively recovered in the immunoprecipitates and
that a mixture of anti-
1 and anti-
2 antibodies would completely remove all AMPK activity from the supernatant.
Effects of endurance training on activity of AMPK isoforms in red
quadriceps muscle.
Figure 1A shows that, in red
quadriceps muscle, the activity of AMPK complexes containing the
2-isoform was ~10-fold higher than the activity of complexes
containing the
1-isoform. There may have been small increases in
1-activity after acute exercise, but they were not statistically
significant. In untrained animals there was a large (2.6-fold,
P < 0.001) activation of
2-complexes in response to
an acute bout of treadmill exercise. However, in trained animals, the
effect of acute exercise on
2-activity appeared to be greatly
reduced, and any increases were no longer statistically significant.
The
2-activities measured after acute exercise were significantly
lower (54%, P < 0.001) in trained animals compared with untrained animals. Because of the low
1-activity in this tissue, the results for the total activity essentially mirrored those
for
2-activity.
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Effects of endurance training on activity of AMPK isoforms in white
quadriceps muscle.
In white quadriceps muscle, a second pattern was seen. Figure
1B shows that, as in red quadriceps, the activity of AMPK
complexes containing the 2-isoform were almost 10-fold higher than
those containing the
1-isoform, and there were no effects of either acute exercise or endurance training on
1-activity. In both
untrained and trained animals, any activation of
2-complexes in
response to acute exercise was also small and not statistically
significant. A difference from the results in red quadriceps was that
the
2-activity was elevated after training whether it was measured
in animals that had (2.0-fold, P < 0.01) or had not
(2.2-fold, P < 0.05) been subjected to a bout of acute
exercise. Because of the low
1-activity in this tissue, the results
for the total activity essentially mirrored those for
2-activity.
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Effects of endurance training on activity of AMPK isoforms in
soleus muscle.
Figure 1C shows that, in marked contrast to red or white
quadriceps, the activities of AMPK complexes containing the 1- and
2-isoform were almost equal, due to a slightly higher
1-activity and to a much lower
2-activity. There appeared to be small increases in both
1- and
2-activity in response to acute exercise, although by analysis of variance these were significant only for
2 after training (3.6-fold, P < 0.001) and for total activity
(
1 plus
2) both with (2.9-fold, P < 0.001) and
without (1.8-fold, P < 0.01) training. In soleus
muscle, prior training appeared to have no significant effects.
Effect of endurance training on the activity of ACC.
As a marker of AMPK activity in vivo, we also measured the activity of
ACC. Table 4 shows the effect of an acute
bout of exercise on the citrate dependence (Ka)
and Vmax of ACC in the three muscle types with
animals that either had or had not been subjected to prior endurance
training. We have shown previously that phosphorylation of rat muscle
ACC by AMPK causes a marked increase in Ka and a
more modest decrease in Vmax (50).
In red quadriceps of untrained animals, there was a robust inactivation of ACC in response to acute exercise due to both a decrease in Vmax and an increase in
Ka (Table 4). By contrast, in trained animals,
there was no significant effect on Ka, and the
effect on Vmax was much reduced. In white
quadriceps, there were no significant effects of exercise on ACC
activity in either the trained or untrained state. In soleus muscle,
there appeared to be a small inactivation of ACC in response to acute
exercise in untrained animals, as evidenced by a significant increase
in Ka (Table 4). In trained animals, acute
exercise had no effect on ACC activity in soleus muscle.
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Effect of endurance training on the expression of AMPK subunit
isoforms in the different muscles.
Figure 3 shows Western blot analysis of
equal amounts of protein from the 14,000-g supernatant
fraction of the three muscle types before and after the training
protocol. All of the extracts shown in Fig. 3 were from animals killed
at rest, although similar results were obtained when muscles were
obtained after a bout of acute exercise (not shown). Blots were probed
with affinity-purified antipeptide antibodies against 1,
2,
2,
1,
2, and
3, and with an anti-
1 antiserum raised against
bacterially expressed
1 that also cross-reacts with
2. All
polypeptides migrated with the approximate molecular mass expected from
the amino acid sequence (
1, 63 kDa;
2, 63 kDa;
1, 32 kDa;
2, 30 kDa;
1, 35 kDa;
2, 63 kDa; and
3, 55 kDa). The
results revealed several interesting features regarding the expression
of AMPK subunit isoforms in these muscle types, which will be discussed
further. Quantification of the results (Fig.
4) showed that any minor changes in
expression of
1,
2,
1,
2,
1, or
2 in response to
endurance training were not significant in any of the muscle types.
However, there was a threefold increase in
3-expression in red
quadriceps that was highly significant (P < 0.001). No
data are shown for
3 in white quadriceps, because the 55-kDa
3-polypeptide was not detectable in extracts of this muscle type.
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DISCUSSION |
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The effects of endurance training observed in this study are clearly dependent on the muscle type, and we will discuss the results for each type in turn.
Red quadriceps.
The red quadriceps is a muscle that would be utilized particularly
during running exercise of the moderate intensity utilized in this
study. In untrained animals, 2-complexes were activated 2.6-fold by
acute exercise, and this correlated with a marked inactivation of ACC.
Both of these effects of acute exercise were greatly reduced in the
trained animals. The attenuation of the exercise-induced activation of
AMPK is likely to be the mechanism for the blunting of the decline in
ACC activity and malonyl-CoA content of red quadriceps reported
previously to occur in response to endurance training in rats
(25). During the training period, this muscle would have
experienced changes in gene expression that would bring about
adaptation to aerobic exercise. We believe, therefore, that a bout of
treadmill exercise of the same intensity would cause less of a
metabolic stress in the trained animals, as indicated by the lack of an
increase in blood lactate in response to the exercise bout in these
animals. This lower degree of metabolic stress would result in more
modest effects on AMP, ATP, and phosphocreatine levels that would
account for the lower degree of activation of AMPK and inactivation of
ACC. Pilot measurements of ATP and phosphocreatine on red quadriceps
samples from each treatment group failed to detect significant
differences (not shown). However, it takes a minimum of 2-3 min to
dissect out the muscles, and changes in ATP and phosphocreatine may not
be stable for that long. Effects on AMPK activity are due to
phosphorylation, and the differences appear to persist, although we
cannot completely rule out the possibility that some changes occurred
during dissection of the muscles. Another problem with detection of
changes in nucleotides is our finding that the AMPK system is
ultrasensitive (19), so that a very small change in the
AMP-to-ATP ratio can produce a large change in AMPK activity. However,
in agreement with our interpretation, endurance training has been
reported to attenuate the falls in phosphocreatine and ATP and the rise
in AMP in rat red gastrocnemius in response to electrical stimulation,
where more rapid freezing of the tissue is possible (11).
A second explanation of the reduced response to the same acute bout of exercise in trained animals compared with untrained animals is that the
glycogen content of the muscle was higher (Table 2). AMPK activation by
contraction has been reported to be markedly attenuated by a high
muscle glycogen content in two different studies (10, 26),
although the mechanism remains unclear.
White quadriceps.
The fibers of the white quadriceps muscle are probably not recruited to
a large extent during the moderate exercise intensity used in this
study. It is therefore not surprising that there were no significant
effects of acute exercise on either AMPK or ACC in this muscle, either
with or without prior training. Citrate synthase was the only one of
the three markers of training to show a small increase in activity. In
white quadriceps, glycogenolysis and glycolysis are major pathways that
provide ATP for contraction. These pathways can be controlled by direct
allosteric effects of AMP and ATP on phosphorylase and
phosphofructokinase (as well as by phosphorylation of the former in
response to elevated Ca2+ and/or cAMP), and there is
currently no evidence that AMPK has any role in controlling these
enzymes. The only effect observed on AMPK activity in white quadriceps
was that the activity of 2-complexes, and the total activity, were
significantly increased after training. This correlated with increased
phosphorylation of the activating site (Thr172) on the
-subunits, as judged by Western blotting with a phosphospecific antibody that does not distinguish between
1 or
2 (Fig. 2). No
changes in expression of any AMPK subunits were evident. The mechanism
behind the increased phosphorylation of AMPK in white quadriceps
remains unclear, but our results show that the basal activity of AMPK
is increased after training. Because long-term activation of AMPK has
been found to increase glycogen content in gastrocnemius/plantaris
muscles (23), the higher basal activity of AMPK might
contribute to the higher glycogen content observed in white quadriceps
after training (Table 2).
Soleus.
In rats, this muscle contains predominantly type I, slow, oxidative
fibers. The activity of 1-containing AMPK complexes was slightly
higher, and that of
2-complexes markedly lower, than in red or white
quadriceps. There appeared to be small increases in both
1 and
2
in response to acute exercise, although these were significant only for
2 after training and for the total activity with or without
training. Acute exercise also had a small effect on ACC activity in
soleus muscle of untrained but not trained animals. There were no
significant effects of training on the expression of any AMPK subunit isoforms.
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ACKNOWLEDGEMENTS |
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We thank Dustin S. Rubink and Jared Bernotski for training the rats.
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FOOTNOTES |
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* These authors contributed equally to this study.
This study was supported by Project Grant RD99/0001901 from Diabetes UK (to D. G. Hardie) and by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-41438 (to W. W. Winder). K. J. Mustard was supported by a studentship from the Biotechnology and Biological Sciences Research Council (UK) and by a grant from Novo-Nordisk.
Address for reprint requests and other correspondence: D. G. Hardie, Division of Molecular Physiology, School of Life Sciences, Dundee Univ., Wellcome Trust Biocentre, Dow St., Dundee, DD1 5EH Scotland, UK (E-mail: d.g.hardie{at}dundee.ac.uk).
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.
First published March 12, 2002;10.1152/ajpendo.00404.2001
Received 12 September 2001; accepted in final form 12 March 2002.
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