1 Research Division, Joslin Diabetes Center and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02215; and 2 Endocrine-Metabolism Division, Department of Medicine and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 02755
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ABSTRACT |
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The AMP-activated protein kinase (AMPK) has
been hypothesized to mediate contraction and
5-aminoimidazole-4-carboxamide 1--D-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The
purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPK
1 and AMPK
2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative
AMPK inhibitors adenine 9-
-D-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and
3-O-methyl-D-glucose (3-MG) uptake. There were
dose-dependent increases in AMPK
2 activity and 3-MG uptake in rat
epitrochlearis muscles with treadmill running exercise but no effect of
exercise on AMPK
1 activity. Tetanic contractions of isolated
epitrochlearis muscles in vitro significantly increased the activity of
both AMPK isoforms in a dose-dependent manner and at a similar rate
compared with increases in 3-MG uptake. In isolated muscles, the
putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully
inhibited AICAR-stimulated AMPK
2 activity and 3-MG uptake but had
little effect on AMPK
1 activity. In contrast, these compounds had
absent or minimal effects on contraction-stimulated AMPK
1 and -
2
activity and 3-MG uptake. Although the AMPK
1 and -
2 isoforms are
activated during tetanic muscle contractions in vitro, in
fast-glycolytic fibers, the activation of AMPK
2-containing complexes
may be more important in regulating exercise-mediated skeletal muscle
metabolism in vivo. Development of new compounds will be required to
study contraction regulation of AMPK by pharmacological inhibition.
adenosine 5'-monophosphate-activated protein kinase; contraction
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INTRODUCTION |
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PHYSICAL EXERCISE DECREASES blood glucose concentrations in people with diabetes, and this is due in part to an increase in the rate of glucose uptake into the contracting muscles (4, 20, 33). It is well established that the mechanism by which insulin and contraction stimulate glucose uptake into skeletal muscle involves the translocation of the GLUT-4 glucose transporter to the cell surface (17). However, there is considerable evidence that the underlying mechanisms responsible for insulin and contraction-stimulated GLUT-4 translocation and glucose uptake are different. For example, the combination of contraction and insulin has additive or partially additive effects on glucose uptake in skeletal muscle (17, 21). In the insulin-resistant (fa/fa) rat (24, 25) and people with type 2 diabetes mellitus (23, 44), insulin-stimulated GLUT-4 translocation is impaired (25, 44) but exercise-stimulated GLUT-4 translocation is normal (23, 24). Studies investigating the signaling mechanisms regulating glucose uptake in muscle have shown that phosphatidylinositol 3-kinase (PI 3-kinase) is necessary for insulin-stimulated, but not contraction-stimulated, glucose uptake (10, 27, 28, 43).
There is increasing evidence that the AMP-activated protein kinase
(AMPK) is a key mediator of contraction-stimulated glucose uptake in
skeletal muscle (3, 15, 16, 26, 30). AMPK is active as a
heterotrimer consisting of one catalytic subunit () and two
noncatalytic subunits (
,
) (14, 22). AMPK is activated allosterically in muscle by increases in the
creatine-to-phosphocreatine and AMP-to-ATP ratios and is also activated
by phosphorylation by an upstream kinase (AMPKK) and is inhibited by
the activity of protein phosphatases (13, 14, 32).
Treadmill-running exercise (35, 40) and muscle
contractions induced by electrical stimulation (16, 34,
39) in rats significantly increase AMPK activity. Most studies
suggesting a role for AMPK in the regulation of muscle glucose uptake
are based on experiments using 5-aminoimidazole-4-carboxamide
1-
-D-ribonucleoside (AICAR), a compound that is
converted to 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) within muscle (30). ZMP can then mimic the
effect of AMP to increase AMPK activity (19). Acute AICAR
treatment increases glucose uptake in skeletal muscle (3, 16,
30) and promotes GLUT-4 translocation to the plasma membrane
(26). The combination of insulin and AICAR treatments have
additive effects on glucose uptake, whereas there is no additivity with
the combination of AICAR plus contraction (3, 16).
Furthermore, similar to contraction-simulated uptake, AICAR-stimulated
uptake is not inhibited by wortmannin, a pharmacological inhibitor of
PI 3-kinase (3, 16). This is evidence that both muscle
contraction and AICAR stimulate glucose uptake through a common
insulin-independent mechanism. A recent study from our group
(15) also showed a close correlation between increases in
AMPK activity and glucose uptake in rat skeletal muscle under numerous
conditions of metabolic stress, suggesting that activation of AMPK may
be a common mechanism mediating insulin-independent glucose uptake
aimed at restoring cellular energy stores.
The AMP analog adenine 9--D-arabinofuranoside (ara-A)
and the adenosine kinase inhibitor iodotubercidin decrease the activity of AMPK in isolated hepatocytes in vitro (19).
Subsequently, it was demonstrated that these two compounds decrease
AICAR- and cyanide-stimulated glucose uptake in isolated rat papillary
muscles (36). Although this proved that ara-A and
iodotubercidin inhibit AICAR- and cyanide-stimulated glucose uptake in
an isolated papillary muscle preparation, AMPK activity was not
measured in this study. The effects of these compounds on
contraction-stimulated AMPK activity and glucose uptake in skeletal
muscle have not been evaluated.
In the present study, we investigated the relationship between isoform-specific AMPK activation and glucose uptake. We examined whether increasing the number of contractions in an isolated muscle preparation in vitro and different exercise intensities in vivo activate AMPK in a dose-dependent manner, and we compared changes in AMPK activity to changes in glucose uptake. We also evaluated the effects of ara-A, iodotubercidin, and 8-bromo-AMP (another AMP analog) on AICAR- and contraction-stimulated glucose uptake and isoform-specific AMPK activity.
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METHODS |
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Experimental animals. Male Sprague-Dawley rats weighing 120-140 g were purchased from Taconic Farms (Germantown, NY). Animals were housed in an animal room maintained at 23°C with a 12:12-h light-dark cycle and fed standard laboratory chow and water ad libitum. Food was removed from rats at 2300, allowing for 4 h of feeding during the dark cycle, and experiments were performed between 0900 and 1100. Protocols for animal use and euthanasia were reviewed and approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and were in accordance with National Institutes of Health guidelines.
Materials.
[-32P]ATP,
3-O-methyl-D-[3H]glucose (3-MG)
and D-[14C]mannitol were obtained from New
England Nuclear (Boston, MA), and protein A/G agarose beads were from
Santa Cruz Biotechnology (Santa Cruz, CA). AICAR, ara-A,
8-bromoadenosine 5'-monophosphate (8-bromo-AMP), 4- amino-5-iodo-7-(
-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (iodotubercidin),
and all other standard reagents were purchased from Sigma Chemical (St.
Louis, MO).
Treadmill exercise studies. Rats were accustomed to a rodent treadmill (Quinton Instruments, Seattle, WA) for 5 min/day for 2 days before the experiment. Rats ran on the treadmill for 1 h at 18 or 32 m/min up a 10% incline. Animals were decapitated immediately after exercise, and epitrochlearis muscles were rapidly dissected, frozen in liquid nitrogen, and used for AMPK activity determination (see Assays for muscle enzymes and metabolites). The time elapsed between cessation of exercise and the freezing of muscles was 1 min, and there was no significant muscle activity of the forelimbs before dissection of the muscles. To measure 3-MG uptake, muscles were mounted on an incubation apparatus as previously described (16), and the elapsed time between cessation of exercise and mounting of the muscles on the apparatus was 2 min. Muscles were then preincubated for 20 min in Krebs-Ringer bicarbonate (KRB) buffer containing 2 mM pyruvate at 37°C and transferred to transport buffer for assessment of glucose uptake (see 3-MG uptake).
Contraction and AICAR treatment in isolated muscles. Animals were killed by decapitation, and the epitrochlearis muscles were rapidly dissected. Both ends of each muscle were tied with suture (silk 4-0) and mounted on the incubation apparatus. The buffers were continuously gassed with 95% O2-5% CO2. Muscles were preincubated in KRB containing 2 mM pyruvate for 30 min at 37°C. For the isolated muscle contraction dose-response studies, muscles were stimulated for 1, 3, 10, or 15 min (one 10-s contraction/min, train rate = 1/min, train duration = 10 s, pulse rate = 100 pulse/s, duration = 0.1 ms, volts = 100 V). The postcontraction experiments had the same preincubation and 10-min contraction protocol as described above; muscles were then incubated at rest for 10, 30, or 60 min in KRB containing 2 mM pyruvate at 37°C. Muscles were then frozen in liquid nitrogen and were used for AMPK activity measurements or to measure glucose uptake (see 3-MG uptake).
For the inhibitor studies, isolated rat epitrochlearis muscles were preincubated in KRB containing 2 mM pyruvate at 37°C for 30 min in the presence or absence of ara-A (2.5 mM), 8-bromo-AMP (1 mM), or iodotubercidin (10 µM). These concentrations were determined on the basis of previous reports (36) and our own preliminary dose-response studies. The muscles were then incubated for 20 min in buffer containing 2 mM AICAR or contracted for 10 min as described above. When added, the inhibitors ara-A, 8-bromo-AMP, and iodotubercidin were present throughout the entire incubation. Muscles were then immediately frozen in liquid nitrogen and subsequently analyzed for AMPK activity or used to measure glucose uptake (see 3-MG uptake).3-MG uptake. 3-MG uptake was measured in 2 ml KRB containing 1 mM 3-O-methyl-D-[3H]glucose (1.5 µCi/ml) and 1 mM D-[14C]mannitol (0.45 µCi/ml) at 30°C for 10 min. AICAR (2 mM), ara-A (2.5 mM), 8-bromo-AMP (1 mM), and iodotubercidin (10 µM) were added to the buffer if they had been present during the previous incubation period. Muscles were processed, radioactivity was determined by liquid scintillation counting for dual labels, and the transport rate was determined as previously described (16).
Assays for muscle enzymes and metabolites.
For the measurement of isoform-specific AMPK activity, muscles were
homogenized in ice-cold lysis buffer (1:100, wt/vol) containing 20 mM
Tris · HCl (pH 7.4), 1% Triton X-100, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM sodium pyrophosphate, 2 mM dithiothreitol, 4 mg/l leupeptin, 50 mg/l trypsin inhibitor, 0.1 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride and then centrifuged at 14,000 g for 20 min at 4°C. The supernatants (200 µg protein) were immunoprecipitated with isoform-specific antibodies to the 1 or
2 catalytic subunits of AMPK and protein A/G beads. These are
anti-peptide antibodies made to the amino acid sequences
DFYLATSPPDSFLDDHHLTR (339) of
1 and
MDDSAMHIPPGLKPH (352) of
2 (38).
Immunoprecipitates were washed twice in lysis buffer and twice in wash
buffer (240 mM HEPES and 480 mM NaCl). Kinase reactions were performed
in 40 mM HEPES (pH 7.0), 0.1 mM SAMS peptide (6), 0.2 mM
AMP, 80 mM NaCl, 0.8 mM dithiothreitol, 5 mM MgCl2, and 0.2 mM ATP (2 µCi [
-32P]ATP) in a final volume of 40 µl for 20 min at 30°C. At the end of the reaction, a 20-µl
aliquot was removed and spotted on Whatman P81 paper. The papers were
washed for 20 min six times in 1% phosphoric acid and once with
acetone. Radioactivity was quantitated with a scintillation counter.
Statistical analysis. Data are expressed as means ± SE. Comparison of means was by one-way ANOVA followed by post hoc comparison using the Fisher's protected least significant difference method. For comparison of two means, an unpaired Student's t-test was performed. P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of exercise intensity on AMPK activation and 3-MG uptake.
We measured isoform-specific AMPK activity and 3-MG uptake in rat
epitrochlearis muscles in response to different intensities of
treadmill exercise. Rats performed treadmill running at 18 or 32 m/min
for 1 h, up a 10% grade. Figure
1A shows that, with lower-intensity exercise, there was no increase in 1 activity and
only a tendency for
1 to increase with higher-intensity exercise. On
the other hand, there was a tendency for an increase in
2 activity
with low-intensity exercise and a significant increase in
2 activity
with higher-intensity exercise (2.1-fold above sedentary,
P < 0.05). 3-MG uptake increased twofold above
sedentary levels with low-intensity exercise (P < 0.05) and 4.6-fold with high-intensity exercise (P < 0.01, Fig. 1B). Muscle glycogen concentrations decreased
24% below baseline with low-intensity exercise (P < 0.05) and 52% with high-intensity exercise (P < 0.001), confirming that both exercise intensities of treadmill exercise
caused significant recruitment of epitrochlearis muscles. These
findings show that, under these conditions, AMPK
2 is preferentially
activated during treadmill exercise and that the magnitude of the
increases of AMPK
2 activity and glucose uptake depends on exercise
intensity. Western blotting of immunoprecipitates and supernatants
showed that the efficiency of immunoprecipitation was 75% for AMPK
1 and 85% for AMPK
2 and that there was no difference between basal and stimulated samples.
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Effect of contraction number on AMPK activation and 3-MG uptake.
In vitro muscle contractions increase total AMPK activity as measured
in ammonium sulfate precipitates (16). To determine whether there is a dose-dependent increase in isoform-specific AMPK
activity with contraction, isolated epitrochlearis muscles were
contracted tetanically, with one 10-s contraction/min for various
periods of time. Figure 2 shows that a
single contraction was enough to begin to increase the activity of both
the 1 and
2 isoforms. Maximal activity of both AMPK isoforms was
observed with 10 contractions, with no further increase with 15 contractions. Importantly, there was a striking similarity between the
increases in AMPK
1 and -
2 activities and the increases in 3-MG
uptake (Fig. 2). Muscle glycogen concentrations tended to decrease with a single contraction (17% below baseline) and decreased significantly with higher numbers of contractions (28% decrease with 3 and 32% decrease with 10 contractions, P < 0.05). No further
decreases in glycogen concentrations were observed with 15 contractions.
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AMPK activity and glucose uptake in the period after contraction.
The period after contraction is characterized by enhanced glucose
uptake into muscle (12). To determine whether AMPK
activity remains elevated after contractions, rat epitrochlearis
muscles were contracted in vitro for 10 min and then incubated at rest for an additional 10, 30, or 60 min. The activity of both AMPK isoforms
decreased rapidly after the cessation of contraction (t1/2 = 8 min). In contrast, the rate of
decrease in 3-MG uptake was much slower, with only a 48% decrease by
60 min after the contractions (Fig. 3).
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Effect of putative AMPK inhibitors on isoform-specific AMPK
activity and glucose uptake.
We evaluated the effect of preincubating isolated skeletal muscle with
the reported AMPK inhibitors ara-A (2.5 mM) and iodotubercidin (10 µM) and the AMP analog 8-bromo-AMP (1 mM). After preincubation, muscles were treated for 20 min with AICAR (2 mM) or contracted for 10 min in the presence or absence of the different compounds. Contraction
force was measured by a force transducer, and tracings were obtained
using a chart recorder. The compounds did not have any effect on
contraction force. Ara-A did not alter basal levels of AMPK1
activity (Fig. 4A),
2
activity (Fig. 4B), or 3-MG uptake (Fig. 4C).
Ara-A fully inhibited AICAR-simulated AMPK
2 activity and 3-MG uptake
(P < 0.05) but had no effect on AMPK
1 activity. In
contrast, ara-A had no inhibitory effect on contraction-stimulated AMPK
1 or -
2 activity and had a mild effect on 3-MG uptake (30% decrease, P < 0.05). Similar to ara-A, 8-bromo-AMP
inhibited AICAR-stimulated AMPK
2 activity and 3-MG uptake but had
minimal inhibitory effects on contraction-stimulated AMPK activity and
3-MG uptake (data not shown). During the preliminary dose-response
studies, higher concentrations of the compounds did not result in
further decreases in AICAR- or contraction-stimulated AMPK activity .
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DISCUSSION |
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In skeletal muscle, both the 1 and
2 isoforms of the AMPK
catalytic subunit are expressed (38). In INS-1 cells,
there are differences in the subcellular localization of AMPK
1 and -
2, with
1 localized predominantly in the cytosol whereas
2 is
found in both the cytosol and the nucleus (37). A study
done in rat liver also showed that AMPK
1 and AMPK
2 have different substrate specificity (42). These differences in the
subcellular localization (37) and substrate specificity
(42) between AMPK
1 and AMPK
2 suggest that there are
distinct functions of the isoforms in the regulation of metabolic
processes (22). Our current data, showing that in
epitrochlearis muscles the
2 but not the
1 isoform of the
catalytic subunit of AMPK was activated during treadmill exercise along
with recent work showing that moderate-intensity exercise
preferentially activates the
2 isoform in human skeletal muscle
(9, 41), also support the concept of differential regulation of the
1 and
2 isoforms.
In contrast to the differential regulation of AMPK1 and AMPK
2
during in vivo exercise, we found that both isoforms are activated in
isolated epitrochlearis muscles contracted in vitro. These muscles are composed mostly of fast-glycolytic (FG) fibers and there is
evidence that treadmill running in rats causes more depletion of
glycogen in fast-oxidative glycolytic (FOG) and slow-oxidative (SO)
fibers than in FG fibers (2), suggesting that FOG and SO
fibers might be recruited more significantly during treadmill running.
In the present study, we observed significant depletion of muscle
glycogen during treadmill exercise, especially at the higher intensity
(52%), confirming the recruitment of epitrochlearis muscles during the
protocol. This fall in glycogen was associated with significant
stimulation of AMPK
2 but not -
1 activity; nevertheless, in future
studies it will be necessary to evaluate the effect of exercise on
isoform-specific AMPK activity in different fiber types.
A previous study using gastrocnemius and soleus muscles found that,
during in situ contractions induced by electrical stimulation of the
sciatic nerve, the 2 but not the
1 AMPK isoform was activated (39). This is in accordance with the effect of exercise on
the activity of the AMPK isoforms found in the present study but
opposite to the response observed with isolated muscle contractions.
The cause of the discrepancy in AMPK
1 activation induced by these models is yet to be determined; however, these findings suggest that
the changes in energy status induced by exercise are more similar to
the effects of sciatic nerve-stimulated contractions in situ than to
the changes induced by contraction of isolated muscles in vitro. A
recent study showed that super-maximal sprint cycle exercise for
30 s leads to activation of both AMPK isoforms in human skeletal
muscle (5). Therefore, it is possible that the
1
isoform is more resistant and is activated in situations of extreme
contraction intensity, such as in vitro tetanic contraction of isolated
muscles and sprint exercise.
It has been postulated that AMPK is a key mediator of exercise-induced
glucose uptake in muscle (3, 15, 16, 26, 30). If the
activity of this enzyme increases in a dose-dependent manner in
parallel with increases in glucose uptake, this would further strengthen the possibility of an important role for AMPK in mediating rates of glucose uptake. As shown in Fig. 1, the degree of activation of AMPK2 showed a similar trend compared with the increases in 3-MG
uptake in response to running exercise. We also found a strong relationship between the activation of the AMPK
1 and -
2 isoforms and the rate of contraction-stimulated 3-MG uptake (Fig. 2). The AMPK
activity assay used in the current study is performed under saturating
concentrations of AMP measuring only AMPKK-induced changes in AMPK
activity. Therefore, because potential allosteric activation of the
AMPK isoforms during exercise is not determined by the assay, it is
possible that there is some degree of AMPK
1 activation in vivo that
is not detected using currently available assay systems.
There is evidence that the soleus muscle (SO fiber) is particularly resistant to the effects of AICAR in increasing glucose uptake and AMPK activity (Ref. 7 and T. Hayashi, M. F. Hirshman, and L. J. Goodyear, unpublished data). Recently, in a model using muscles saturated with glycogen, contraction led to an increase in glucose uptake but no change in total AMPK activity in soleus muscle, whereas in white gastrocnemius muscle, both glucose uptake and AMPK activity increased in response to contraction. This finding suggests that, in soleus under these specific conditions, contraction-mediated glucose uptake is AMPK independent (8). In isolated soleus muscles from fasted rats (not glycogen supercompensated), we found that contraction increases the activity of both AMPK isoforms as well as glucose uptake (T. Hayashi, M. F. Hirshman, and L. J. Goodyear, unpublished data). Thus contraction-induced changes in AMPK activity may vary depending on the preexisting glycogen content. It will be important to measure isoform-specific AMPK activity in the glycogen-supercompensated model used by Derave et al. (8), because the lack of an increase in total AMPK activity in the supercompensated red muscle with contraction was still associated with a significant alteration in acetyl-CoA carboxylase activity, a sensitive intracellular reporter of AMPK activity.
The period after contraction is characterized by increased glucose disposal into skeletal muscle (12). In the present study, we found that the activity of both isoforms decreased rapidly in the postcontraction period, whereas 3-MG uptake remained elevated (Fig. 3). This suggests that, although AMPK may be involved in initiating increases in glucose uptake during contraction, sustained enzyme activity is not necessary to maintain uptake after contraction. Studies of GLUT-4 vesicle kinetics suggest that plasma membrane GLUT-4 is elevated immediately and 30 min after treadmill exercise in rats, and that by 2 h after exercise, plasma membrane GLUT-4 returns to baseline values (11). Therefore, AMPK may be involved in triggering GLUT-4 to translocate to the plasma membrane, but another mechanism may be responsible for maintaining transporters at the membrane in the period after exercise. This mechanism could be AMPK independent, or it could be due to the prolonged activation of one or more putative "downstream" substrates of AMPK.
Although most data now show that AMPK is activated during contraction
concurrently with an increase in glucose uptake (3, 15, 16, 26,
30), studies evaluating the effects of AMPK inhibitors on
stimulated enzyme activity could provide more direct evidence of the
enzyme's involvement in exercise-mediated glucose uptake. In isolated
rat hepatocytes, the AMP analog ara-A, a precursor of ara-ATP,
significantly inhibited AMPK activity in the absence of AMP
(19). The competitive inhibitor of adenosine kinase
iodotubercidin (18, 31), which is also a potent
serine-threonine kinase inhibitor (29), had similar
inhibitory effects on AMPK (19). Subsequently, in isolated
heart papillary muscles, these two compounds were shown to inhibit
AICAR- and cyanide-stimulated glucose uptake, but the effect of the
compound on AMPK activity was not determined (36). We
found that iodotubercidin, ara-A, and 8-bromo-AMP are not effective
inhibitors of the AMPK1 isoform, suggesting that the
1 isoform is
resistant to the effects of these compounds. These compounds
did, however, significantly inhibit AICAR-stimulated AMPK
2 activity
and glucose uptake. Ara-A had a mild AMPK-independent inhibitory effect
on contraction-stimulated glucose uptake, suggesting that, similar to
the glycogen-supercompensated model, in certain situations AMPK may not
be indispensable for contraction-regulated uptake. The ability of the
inhibitors to blunt AICAR-stimulated AMPK
2 activity and not
contraction-stimulated activity suggests that AICAR and contraction do
not necessarily share a common pathway to increase AMPK activity and
glucose uptake. However, it is also possible that these compounds are
acting upstream of AMPK and not directly inhibiting the kinase. In
fact, iodotubercidin has recently been shown to alter AICAR-stimulated
AMPK activity by decreasing the concentrations of ZMP (1).
Ara-A and 8-bromo-AMP could also be competing with AICAR at the level
of the nucleoside transporter and/or adenosine kinase, reducing
intracellular availability of ZMP. Overall, the lack of effect of the
compounds on contraction-stimulated AMPK activity makes them
unsatisfactory agents for the study of AMPK as a possible mediator of
contraction-induced glucose uptake. The use of more specific and potent
AMPK inhibitors or the development of AMPK knockout animal models will
be necessary to fully clarify this issue and to definitively determine
the role of AMPK in regulating skeletal muscle metabolism.
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ACKNOWLEDGEMENTS |
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This work was supported by Grants AR-42338 and AR-45670 (to L. J. Goodyear) and DK-35712 (to L. A. Witters) from the National Institutes of Health and by grants from the Foundation for Total Health Promotion, the Meiji Life Foundation of Health and Welfare, the Nakatomi Foundation, and the Descente and Ishimoto Memorial Foundation for the Promotion for Sports Science (to T. Hayashi). N. Fujii is supported by a postdoctoral fellowship for research abroad from the Japan Society for the Promotion of Science. T. Hayashi was a Mary K. Iacocca Fellow at the Joslin Diabetes Center.
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FOOTNOTES |
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* N. Musi and T. Hayashi contributed equally to this study.
Address for reprint requests and other correspondence: L. J. Goodyear, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 (Email: Laurie.Goodyear{at}joslin.harvard.edu).
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.
Received 14 August 2000; accepted in final form 15 January 2001.
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