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1 Medical Research Laboratory and Medical Department M (Endocrinology and Diabetes), Aarhus University Hospital, Aarhus Kommunehospital, and 2 Department of Clinical Pharmacology, University of Aarhus, DK-8000 Aarhus C, Denmark
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Physical activity is known
to increase insulin action in skeletal muscle, and data have indicated
that 5'-AMP-activated protein kinase (AMPK) is involved in the
molecular mechanisms behind this beneficial effect.
5-Aminoimidazole-4-carboxamide-1-
-D-ribofuranoside (AICAR) can be used as a pharmacological tool to repetitively activate
AMPK, and the objective of this study was to explore whether the
increase in insulin-stimulated glucose uptake after either long-term
exercise or chronic AICAR administration was followed by
fiber-type-specific changes in insulin signaling and/or changes in
GLUT-4 expression. Wistar rats were allocated into three groups: an
exercise group trained on treadmill for 5 days, an AICAR group exposed
to daily subcutaneous injections of AICAR, and a sedentary control
group. AMPK activity, insulin-stimulated glucose transport, insulin
signaling, and GLUT-4 expression were determined in muscles
characterized by different fiber type compositions. Both exercised and
AICAR-injected animals displayed a fiber-type-specific increase in
glucose transport with the most marked increase in muscles with a high
content of type IIb fibers. This increase was accompanied by a
concomitant increase in GLUT-4 expression. Insulin signaling as
assessed by phosphatidylinositol 3-kinase and PKB/Akt activity was
enhanced only after AICAR administration and in a
non-fiber-type-specific manner. In conclusion, chronic AICAR
administration and long-term exercise both improve insulin-stimulated glucose transport in skeletal muscle in a fiber-type-specific way, and
this is associated with an increase in GLUT-4 content.
glucose transport; AMP-activated protein kinase; skeletal muscle; insulin signaling; muscle fiber type
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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LONG-TERM EXERCISE IS well known to improve the glucose homeostasis and insulin action in insulin-resistant subjects (11, 12) and is one of the cornerstones in the treatment of Type 2 diabetes mellitus. Furthermore, it has been shown that regular exercise can partly prevent or delay the onset of the disease (18, 27). The ameliorated insulin sensitivity after exercise is mainly due to enhanced insulin action on skeletal muscle, the predominant tissue for insulin-stimulated glucose disposal (10). In skeletal muscle, insulin stimulation leads to activation of a specific intracellular signaling cascade involving the insulin receptor substrate (IRS) family, the lipid kinase phosphatidylinositol (PI) 3-kinase, and also the serine/threonine kinase PKB/Akt (1). Activation of the insulin-signaling cascade leads to a recruitment of the insulin-sensitive glucose transporter (GLUT-4) from an intracellular pool to the surface membrane, thus allowing glucose to enter the cell (24). This transport of glucose across the sarcolemma through GLUT-4 is thought to be the rate-limiting step for glucose utilization (8).
The molecular mechanism of the increased insulin action after long-term exercise in skeletal muscles might partly be explained by an increased GLUT-4 expression (9) and to some extent also by enhanced activity in the insulin-signaling cascade (5, 17). Although several studies have shown that GLUT-4 expression is enhanced only in muscles activated by the chosen exercise program (9), little is known about the muscle fiber specificity of the increased insulin-signaling activity.
The 5'-AMP-activated protein kinase (AMPK) has recently been suggested
as a potential candidate in the signaling process in response to
exercise (19, 28). Interestingly, both long-term treatment
of rats with
5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR), which is an AMPK activator (3, 13, 29), and
exposure for 18 h of in vitro incubated rat epitrochlearis muscle
to AICAR (23) result in enhanced expression of GLUT-4 in
skeletal muscles. Furthermore, the increased GLUT-4 expression is
associated with a concomitant fiber type-related increase in the level
of maximally insulin-stimulated glucose uptake (3),
suggesting that at least parts of this beneficial adaptation to
long-term exercise can be mimicked through chronic AMPK
activation. The possible effects of repetitive AMPK stimulations
with AICAR on activity in the insulin-signaling pathway are,
however, still to be defined.
Consequently, the aim of the present study was to determine whether the observed increase in insulin-stimulated glucose uptake in muscles from long-term exercised rats and rats exposed to repetitive pharmacological AMPK activations by chronic AICAR administration are followed by fiber-type-specific changes in insulin signaling and/or fiber-type-specific changes in GLUT-4 expression.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Male Wistar rats (weight ~70 g) were housed under controlled temperature (22-23°C) and lighting (12:12-h light/dark cycle) and were given free access to water and a standard rat diet. Animals were supplied from M&B A/S (Ry, Denmark) and randomly divided into either an AICAR, exercise, or control group. All experimental procedures were approved by the Danish Animal Experiments Inspectorate and complied with the European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes.
Animal protocol for AMPK-studies.
To determine the level of isoform-specific activation of the AMPK
system, rats were killed immediately either after a 60-min treadmill
run or 60 min after a single AICAR injection (1 mg/g body wt). Soleus
[muscle fiber composition (2): ~84% type I, 16% type
IIa, and 0% type IIb], extensor digitorum longus (EDL) (muscle fiber
composition: ~3% type I, 59% type IIa, and 38% type IIb), and
epitrochlearis [muscle fiber composition (22): ~15% type I, 20% type IIa, and 65% type IIb] were rapidly dissected out,
snap-frozen in liquid nitrogen, and stored at 
80°C until analyzed.
Individual muscles were homogenized as described by Musi et al.
(21) with minor modifications. Muscles were homogenized in
ice-cold lysis buffer (1:25 wt/vol) [20 mM Tris, 50 mM NaCl, 5 mM
Na4P2O7, 50 mM NaF, 250 mM sucrose,
2 mM DTT, 1% Triton X-100, 2 µg/ml aprotinin, 5 µg/ml leupeptin,
0.5 µg/ml pepstatin, 10 µg/ml antipain, 1.5 mg/ml benzamidine, and
100 µmol/l 4-(2-aminoethyl)-benzenesulfonyl fluoride, hydrochloride
(AEBSF); pH 7.4] with a homogenizer operating at maximum speed
twice for 20 s. Insoluble materials were removed by centrifugation
at 14,000 g for 20 min at 4°C, and protein content on the
supernatant was determined with a bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL).
Isoform-specific AMPK activity assay.
Isoform-specific AMPK activity was assessed as previously described
(21) with minor modifications. Antibodies against the catalytic 
1 and 2 subunits of AMPK (Upstate Biotechnology, Lake Placid, NY) coupled to protein A-agarose (Sigma Chemical, St. Louis,
MO) were used for immunoprecipitating from aliquots of protein (200 µg). The immune complexes were washed twice in ice-cold lysis buffer
and twice in wash buffer (240 mM HEPES and 480 mM NaCl; pH 7.0). AMPK
activity was assessed in a buffer containing 40 mM HEPES, 80 mM NaCl,
0.2 mM AMP, 0.2 mM MgATP, 6 µCi [
-32P]ATP 5 mM
MgCl2, and 0.2 SAMS peptide (Upstate Biotechnology) for 20 min at 30°C. The reaction was stopped by spotting 20 µl of reaction
aliquot on Whatman P81 paper and dropping it into 1% phosphoric acid.
The papers were washed six times in 1% phosphoric acid and once with
acetone, and radioactivity was quantified by scintillation
counting (Wallac 1409, Wallac, Turku, Finland).
Immunoblotting for phospho-AMPK (Thr172).
Aliquots of protein were resolved by SDS-PAGE by use of the Bio-Rad
Mini Protean II system (10% polyacrylamide gels), transferred to
nitrocellulose, blocked with 5% nonfat milk in TBST (10 mM Tris, 150 mM NaCl, pH 8.0, and 0.1% Tween 20), and incubated with anti-phospho-AMPK (Thr172) (Cell Signalling, Beverly, MA) for protein
expression of both phosphorylated AMPK-1 and 2. The membranes
were then washed and incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Pierce Chemical) as secondary antibody, and proteins were visualized by BioWest enhanced
chemiluminescence (UVP, Upland, CA) and quantified by UVP BioImaging System.
Protocol for long-term exercise and chronic AICAR administration. Animals in the long-term exercise group were accustomed to running for 5 min/day for 2 days on an Exer-3/6 rodent treadmill (Columbus Instruments, Columbus, OH). Subsequently the workload and duration were increased to 20 m/min, at a 10% incline, 60 min/day for 5 successive days. AICAR-exposed animals received daily subcutaneous injections of AICAR (Toronto Research Chemicals, North York, ON, Canada) (1 mg/g body wt) for 5 days as described previously (29). All treadmill running and AICAR injections were carried out in the morning after the rats had free access to food during the night. Sedentary rats served as control animals.
Muscle preparations for long-term study.
Rats were killed by cervical dislocation after an overnight fast and
24 h after the last training session or AICAR injection. Therefore, the results obtained in the long-term study reflect the
chronic adaptations induced by exercise or AICAR administration. Soleus, EDL, and epitrochlearis were rapidly but carefully dissected out and used for measurements of glucose uptake, insulin signaling, and
total GLUT-4 content. Muscles used for determination of total GLUT-4
content were snap-frozen in liquid nitrogen directly after removal and
stored at 80°C until assayed.
Muscle incubation.
All muscles (epitrochlearis, EDL, and soleus) used for measurement of
glucose uptake or insulin signaling were incubated for 20 min at 30°C
in 5 ml of oxygenated Krebs-Henseleit buffer (117 mM NaCl, 4.7 mM KCl,
2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 24.6 mM NaHCO3; pH 7.4) containing 5 mM
HEPES, 20 mM mannitol, and 0.1% BSA in the presence or absence of
insulin (60 nmol/l) by using a shaking water bath allowing continuously
gassing of the buffer (95% O2-5% CO2).
Muscles used for insulin-signaling measurements were trimmed and frozen
in liquid nitrogen and stored at 80°C until analyzed, whereas
muscles used for measurement of glucose uptake were further incubated
for 10 min in Krebs-Henseleit buffer containing 5 mM HEPES, 12 mM
[14C]mannitol (8 µCi/mmol), and 8 mM
3-O-[3H]methylglucose (3-OMG; 437 µCi/mmol) (NEN, Boston, MA) with or without insulin. Glucose
transport activity was assessed as previously described
(20) and presented as micromoles glucose analog
accumulated per milliliter of intracellular water per hour.
Muscle preparations for insulin-signaling assays. Incubated muscles were homogenized as described by Wojtaszewski et al. (30). In short, muscles were homogenized in ice-cold solubilization buffer (50 mM HEPES, 137 mM NaCl, 10 mM Na4P2O7, 10 mM NaF, 1 mM MgCl2, 1 mM CaCl2, 1% NP-40, 10% glycerol, 2 mM Na3VO4, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 µg/ml antipain, 1.5 mg/ml benzamidine, and 100 µmol/l AEBSF; pH 7.4) and rotated for 1 h at 4°C. Insoluble materials were removed by centrifugation at 16,000 g for 60 min at 4°C, and protein content on the supernatant was determined with a bicinchoninic acid protein assay reagent (Pierce Chemical).
PI3-kinase assay.
PI3-kinase activity was assessed as previously described
(30) with minor modifications. Briefly, aliquots of
protein were immunoprecipitated overnight with protein
A-agarose-coupled anti-IRS-1 or anti-IRS-2 antibody (Upstate
Biotechnology). The immune complexes were washed, and PI3-kinase
activity was assessed directly on the protein A-agarose complex in a
buffer containing 10 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 75 µM ATP, 50 mM NaCl, and 6 µCi [-32P]ATP (NEN).
Reaction products were resolved by thin-layer chromatography and were
quantified by using a phosphoimager (Packard BioScience, Meriden, CT).
Protein expression and phosphorylation of PKB/Akt. Aliquots of protein were resolved by SDS-PAGE as described in Immunoblotting for phospho-AMPK (Thr172). Anti-PKB/Akt antibody (New England BioLabs, Beverly, MA) was used for protein expression of PKB/Akt and antiphospho-PKB/Akt (Ser473) antibody (New England BioLabs) for expression of phosphorylated PKB/Akt. Proteins were visualized and quantified as described in Immunoblotting for phospho-AMPK (Thr172).
PKB/Akt activity assay.
Aliquots of protein were immunoprecipitated overnight with protein
G-agarose coupled anti-PKB/Akt1 antibody (Upstate Biotechnology) or
protein A-agarose coupled PKB/Akt2 antibody (Aviva Antibody, San
Diego, CA). The complex was washed twice in solubilization buffer
containing additional NaCl (500 mM in total), twice in buffer
containing 50 mM Tris, 0.1 mM EGTA, 5 mM DTT, and 0.03% Brij 35, pH
7.5, and once in kinase buffer (20 mM MOPS, 25 mM -glycerol
phosphate, 5 mM EGTA, 1 mM Na3VO4, and 1 mM
DTT; pH 7.2). PKB activity was assessed in kinase buffer containing 0.1 mM ATP, 1.88 mM MgCl2, 4 µCi [-32P]ATP,
0.01 mM PKA-inhibitor peptide, 0.1 mM PKB-specific peptide (Upstate Biotechnology) at a final volume of 40 µl at 30°C for 10 min. At the end of the reaction, a 20-ml aliquot was removed and
spotted on Whatman P81 paper. The papers were washed six times for 20 min in 1% phosphoric acid and once with acetone, and radioactivity was
quantified by scintillation counting (Wallac 1409, Wallac).
Total muscle GLUT-4 content. Total crude membranes were prepared from ~20 mg of epitrochlearis, EDL, or soleus muscles as previously described (3). Proteins were visualized and quantified as described in Immunoblotting for phospho-AMPK (Thr172).
Statistical analysis. All data are presented as means ± SE. Statistical evaluation of the data was done by one-way ANOVA. When analysis revealed significant differences, a post hoc test was used to correct for multiple comparisons (Student-Newman-Keuls test). Differences between groups were considered statistically significant if P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Immunoblotting for phospho-AMPK (Thr172) and isoform-specific acute
AMPK activity.
A single injection of AICAR or 60 min of treadmill run resulted in a
rise in AMPK-2 activity (Fig. 1) and
phospho-AMPK (Thr172) expression (Table
1) in all three muscles investigated. The
greatest increase was observed in epitrochlearis muscles, in which
AMPK-activity rose 5.6-fold in AICAR-injected animals and 3.7-fold in
exercised animals (P < 0.01, n = 5-6). Protein amount of phosphorylated AMPK rose 2.2-fold after an
AICAR injection and 1.6-fold after exercise (P < 0.01, n = 5-6). In EDL and soleus muscles, AICAR injection and exercise both resulted in ~3-fold rise in AMPK activity (P < 0.01, n = 5-6); likewise,
AICAR injection and exercise resulted in a significant ~40% rise in
phosphorylation in EDL (P < 0.01, n = 5-6) whereas only a nonsignificant tendency to an increase in AMPK
phosphorylation was noted in soleus muscles during both regimes. No
significant changes in AMPK-1 activity were observed (Table 1).
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3-OMG transport.
Maximal insulin-stimulated glucose transport activity was markedly
increased in the predominant type IIb fiber-containing epitrochlearis
muscles from both exercised (9.40 ± 0.49 µmol · ml
1 · h1)
and AICAR-injected (12.07 ± 0.87 µmol · ml1 · h1)
animals compared with the control group (7.15 ± 0.45 µmol · ml1 · h1),
resulting in a 31 and 69% (P < 0.01, n = 12-18) increase in exercised and
AICAR-injected animals, respectively (Fig.
2A). Furthermore, a
significant increase in maximally insulin-stimulated glucose uptake was
found in the mixed type II EDL muscle in both the exercised and
AICAR-injected animals compared with controls [on average 24.5 and
27.2% (P < 0.01, n = 12-18) for
exercise and AICAR, respectively] (Fig. 2B). In contrast,
insulin-stimulated 3-OMG-transport did not differ between the groups in
the red type I soleus muscle. Basal noninsulin-stimulated glucose
transport was significantly (P < 0.05) lower in
AICAR-exposed animals in all muscle types compared with controls and
exercised animals (76.2% in epitrochlearis, 54.3% in EDL, and 82.8%
in soleus compared with control animals).
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IRS-1- and IRS-2-associated PI3-kinase activity.
As shown in Table 2, maximal insulin
stimulation (60 nmol/l) led to a 9.0-, 8.5-, and 8.0-fold increase in
IRS-1-associated PI3-kinase activity over basal activity in
epitrochlearis, EDL, and soleus, respectively. In response to AICAR
exposure, insulin-stimulated IRS-1-associated PI3-kinase activity was
further enhanced in all three muscles characterized by different fiber-
type composition (60.6% in epitrochlearis, 36.1% in EDL, and 80.1%
in soleus compared with control animals; P < 0.05, n = 12-14). In contrast, muscles from exercised
rats did not differ in insulin-stimulated IRS-1-associated PI3-kinase
activity from control rats, even in the epitrochlearis muscles, in
which exercise resulted in a 31% increase in insulin-stimulated glucose transport compared with control rats. In all three muscles, basal IRS-1- and IRS-2-associated PI3-kinase activity as well as
insulin-stimulated IRS-2-associated PI3-kinase (Table 2) showed no
significant difference between the three groups.
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Protein expression, phosphorylation, and activity of PKB.
Neither exercise nor AICAR administration altered PKB protein
expression (data not shown). Insulin-stimulated PKB and activity and phosphorylated PKB were increased by ~50% in all of the three muscles in the AICAR-injected group compared with controls as shown in
Table 3. However, in the exercised
animals, insulin did not increase PKB activity further compared with
the sedentary controls in either epitrochlearis, EDL, or soleus
muscles, consistent with our results for IRS-1-associated PI3-kinase
activity.
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Muscle GLUT-4 protein content.
Measurements of total crude membrane content of GLUT-4 protein in the
three investigated muscles are displayed in Fig.
3. Muscles from AICAR-exposed and
exercised animals showed an increase in GLUT-4 content that was most
prominent in epitrochlearis with a 40.8% (P < 0.05)
increase in the exercised-trained and a 97.8% (P < 0.01) increase in the AICAR-injected group compared with the sedentary
controls (n = 8-12). In the EDL muscle, AICAR
administration and long-term exercise resulted in an almost equal
~45% increase in GLUT-4 content (P < 0.01, n = 6-12). In contrast, GLUT-4 content in the
soleus muscles the did not differ significantly from control animals
(n = 6-12).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study demonstrates that 5 days of AICAR exposure or
long-term treadmill exercise increased insulin-stimulated 3-OMG
transport in rat skeletal muscles in a fiber-type-specific manner and
that this was almost paralleled by an enhanced GLUT-4 expression in the
same muscles investigated. Our data from the isoform-specific AMPK
activity in these muscles indicate that the adaptations could be
mediated through this system, as the acute effect of either one
injection of AICAR or a single bout of exercise increases AMPK-2
activity with the same fiber-type specificity as the increased GLUT-4
expression. AMPK-1 activity does not seem to be involved in the
adaptations because neither AICAR injections nor treadmill exercise
increased AMPK-1 activity.
The effect of long-term AMPK-activation on the activity in the insulin-signaling cascade is another potential cause for increased insulin-stimulated glucose uptake. The molecular mechanisms by which AMPK activity augments insulin signaling have not yet been revealed, but a recent study has shown that AMPK can phosphorylate IRS-1 on Ser-789 in mouse C2C12 myotubes when these are exposed to AICAR, thereby establishing a link to the insulin-signaling cascade (14). This phosphorylation does not alone affect the activity of the IRS-1-associated PI3-kinase, but simultaneous AICAR exposure enhances the activity induced by insulin.
Interestingly, our study showed that long-term AICAR administration increased the activity in the proximal insulin-signaling cascade 24 h after the last injection of AICAR. The increase was noticed on the IRS-1-associated PI3-kinase level, a key mediator of insulin-signaling activity. At the same level, IRS-2 has been shown to be able to mediate the insulin signaling in mice lacking IRS-1 through association with the PI3-kinase (15), but chronic AICAR administration did not affect the activity of the insulin-stimulated IRS-2-associated PI3-kinase. The exercised animals showed no increase in the activity of either insulin-stimulated IRS-1 or IRS-2-associated PI3-kinase compared with sedentary control animals.
To further elucidate the effects on the insulin signaling, we looked at
two isoforms of the serine/threonine kinase PKB/Akt, a target for
PI3-kinase activity that has been suggested as an intermediate in the
insulin-signaling cascade downstream of the PI3-kinase level
(4). PKB/Akt1 is widely expressed and is the
predominant isoform in most tissues, whereas PKB/Akt2 is mostly
expressed in insulin-sensitive tissues (16), and recent studies of mice lacking PKB/Akt2 have shown insulin resistance and a
diabetes mellitus-like syndrome, indicating an important role for this
isoform in insulin signaling (6). Long-term AICAR administration also increased the activity of both the and isoforms of PKB in all muscles examined; in contrast, no enhanced activity was noticed in the exercised animals compared with controls.
The increase in insulin signaling in the AICAR-treated rats was roughly equal in the three muscles investigated and did not follow the fiber-type specificity of the increased insulin-stimulated glucose transport. This finding shows that it is possible to increase the activity in the proximal part of the insulin-signaling pathway without increasing glucose uptake. Interestingly, in rats undergoing surgical stress, PI3-kinase and PKB activity have also been found to be increased even though glucose uptake in skeletal muscles was decreased (25).
The fact that the insulin-signaling activity was found to be enhanced after chronic AICAR treatment but not after exercise may implicate differences in the response of muscles to chronic AICAR exposure and long-term exercise. However, Chibalin et al. (5) found that long-term exercised rats exhibited elevated insulin-signaling activity in epitrochlearis muscles 24 h after the last bout of exercise. This is in agreement with our data from the AICAR-injected group. The discrepancy between the unchanged insulin-signaling activity in our exercised animals and the study by Chibalin et al. may be due to the training protocol used. In the latter, rats were exercised by 6 h of swimming every day in contrast to the 1 h of treadmill running used in our study. The long 6-h training program might have increased the AMPK activity for a much longer period than the 1-h treadmill running we were using, and this might have resulted in the increase in insulin-signaling activity. In this context, it would be important in future studies to clarify the duration of the elevated AMPK activity in skeletal muscles after in vivo AICAR administration and to define the muscle fiber-type specificity of very long daily exercise like 6 h of swimming.
The marked effect on insulin-stimulated glucose uptake in our exercise group without any changes in insulin-signaling activity demonstrates that increased signaling is not mandatory for increased glucose uptake. This finding is in agreement with two very recent studies, one on healthy middle-aged men completing a 7-day exercise program that resulted in an increased insulin-stimulated glucose transport but no observed changes on the PI3-kinase or PKB activity (26), and another study on the obese Zucker rat in which a 7-wk exercise program also enhanced insulin-stimulated glucose transport without any effect on insulin signaling (7).
In summary, our results show that increased insulin-stimulated glucose transport after exercise and AICAR exposure correlates with the increase in GLUT-4 protein expression. Enhancement of the insulin signal transduction after chronic AICAR administration as shown in this study or by different forms of long-term exercise might also be involved, although we found the activity of the insulin-signaling pathway to be increased in type I muscle fibers after AICAR exposure without a concomitant increase in glucose uptake. This finding could be important for the assessment of future initiatives in the treatment of Type 2 diabetes mellitus because measures focusing solely on increasing the activity in the insulin-signaling cascade may not always result in a beneficial effect on the deranged glucose transport.
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ACKNOWLEDGEMENTS |
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E. Hornemann, E. Carstensen, and H. Petersen are thanked for excellent technical assistance. N. Musi and L. Goodyear, Joslin Diabetes Center, Harvard Medical School, Boston, MA, are thanked for stimulating discussions and technical advice.
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FOOTNOTES |
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The study was supported by grants from The Novo Nordic Foundation; Institute of Experimental Clinical Research, University of Aarhus; the Danish Medical Research Council; the Danish Diabetes Association; Carl J. Beckers Fond; Enid Ingemanns Fond; Kirsten Anthonius Mindelegat; Fonden til Lægevidenskabens Fremme; Agnes and Poul Friis' Fond, and the Torben Frimodt and Alice Frimodt Foundation.
Address for reprint requests and other correspondence: S. Lund, Medical Dept. M (Endocrinology and Diabetes), Aarhus Univ. Hospital, Aarhus Kommunehospital, DK-8000 Aarhus C, Denmark (E-mail: sl{at}dadlnet.dk).
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 December 20, 2002;10.1152/japplphysiol.00250.2002
Received 25 March 2002; accepted in final form 18 December 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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P < 0.01 vs. control.
