1 Department of Medicine, Washington University School of Medicine, St. Louis 63110; and 2 Departments of Physiology and Internal Medicine and Diabetes and Cardiovascular Biology Program, School of Medicine, University of Missouri, Columbia, Missouri 65212
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ABSTRACT |
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There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca2+ in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca2+ induces an increase in GLUT4. We found that raising cytosolic Ca2+ by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca2+ release, and completely blocked by KN93, an inhibitor of Ca2+-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca2+ and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca2+ is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.
5'-adenosine monophosphate-activated protein kinase; gene expression; skeletal muscle; tissue culture; myocyte enhancer factor 2; Ca2+-calmodulin-dependent protein kinase
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INTRODUCTION |
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INSULIN, EXERCISE, AND HYPOXIA all stimulate glucose transport in skeletal muscle by causing movement of the GLUT4 isoform of the glucose transporter from intracellular sites to the cell surface (17). Under normal conditions, i.e., in the absence of insulin resistance, the increase in glucose transport induced by the maximal effects of these stimuli is determined by the quantity of GLUT4 protein present in the muscle (12, 19). Exercise training induces an increase in GLUT4 protein in skeletal muscle (9, 17, 34). This adaptive increase in GLUT4 is associated with a proportional increase in maximally stimulated muscle glucose transport activity (35, 37). The increase in muscle GLUT4 protein induced by exercise occurs very rapidly, with most of the increase occurring within the first 24 h after an exercise bout (35).
There is considerable evidence that activation of AMP-activated protein kinase (AMPK), by the increase in AMP and the decreases in phosphocreatine and ATP that occur in contracting muscle, is involved in the acute stimulation of glucose transport by exercise (10, 21, 28, 40). Recent studies suggest that, in addition to the acute stimulation of glucose transport, activation of AMPK plays a role in increasing the capacity for glucose transport by inducing an increase in GLUT4 in skeletal muscle. This is evidenced by the findings that administration of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) to rats, or exposing muscles to AICAR in vitro, results in a rapid increase in GLUT4 protein (18, 30, 41, 43). AICAR is taken up by muscle cells and converted to the AMP analog ZMP, which activates AMPK (40).
Endurance exercise also induces an increase in muscle mitochondria (15, 16). Activation of AMPK at least partially mimics this effect of exercise (41). In addition to the evidence for involvement of the perturbation in high-energy phosphate concentrations that leads to activation of AMPK, studies on myocytes in culture have suggested that increases in cytosolic Ca2+ can induce increases in mitochondrial enzymes (8, 22). Some of the same adaptive stimuli that induce an increase in mitochondria also result in an increase in GLUT4 in skeletal muscle (4, 9, 17, 36, 41), suggesting the possibility that the pathways involved in mediating adaptive increases in GLUT4 and mitochondria may overlap. This study was conducted to test the hypothesis that raising cytosolic Ca2+ induces an increase in GLUT4 expression in myocytes, to compare the adaptive responses to raising cytosolic Ca2+ and treatment with AICAR, and to obtain information regarding the mechanisms responsible for mediating increased GLUT4 expression.
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MATERIALS AND METHODS |
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Materials. L6 myocytes were purchased from American Type Culture Collection (Manassas, VA), the Ca2+-calmodulin-dependent protein kinase (CAMK) inhibitor KN93 from Calbiochem (La Jolla, CA), MEF2A and GLUT3 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and MEF2D antibody from Transduction Laboratories (Lexington, KY). 2-Deoxy-D-[1,2-3H]glucose was purchased from American Radiolabeled Chemicals (St. Louis, MO). The rest of the reagents were purchased from Sigma Chemical.
Cell culture.
L6 myocytes were maintained at 37°C in 5% CO2 on 100 mM
collagen-coated plastic dishes, or 12-well plates, containing
low-glucose (5 mM) DMEM supplemented with 0.5 mM oleic acid, 1% BSA, 1 mM L-carnitine, 10 mM creatine, 100 µU/ml penicillin, 100 µU/ml streptomycin, 0.25 µg/ml fungizone, and 10% fetal bovine
serum (FBS). Cells were maintained in continuous passage by
trypsinization of subconfluent cultures with 0.25% trypsin. Myoblast
differentiation was induced by switching to medium containing 2%
heat-inactivated horse serum when myoblasts were ~80% confluent.
Experimental treatments were begun 4 days later, when myotubes were
evident. At this stage, we switched back to a high-serum medium
containing 10% horse serum and 5% FBS. Treatment of myotubes with
caffeine or AICAR, with or without other agents, i.e., dantrolene,
adenosine-9--D-arabino-furanoside (AraA), and KN93,
was for 3 h/day for 5 days. To remove these agents, myotubes were
washed twice with PBS.
Determination of cytosolic Ca2+ levels. Cytosolic Ca2+ was determined using fura 2 epifluorescence digital microscopy, as described earlier (14). Briefly, cells on coverslips were loaded with 5 µM fura 2-AM for 30 min at 37°C. The coverslip was mounted onto a superfusion chamber and superfused with a physiological saline solution containing (in mM) 2 CaCl2, 143 NaCl, 1 MgCl2, 5 KCl, 10 HEPES, and 10 glucose, pH 7.4, and either 5 mM caffeine and/or 10 µM dantrolene. Fura 2 was excited by 340- and 380-nm light and the emitted fluorescence (510 nm) collected by a monochrome charge-coupled device camera that was attached to a computer for data acquisition by the InCa Ratiometric Fluorescence program version 1.2 (Intracellular Imaging). Data are expressed as a ratio (and indicated as ratio units) of the emitted light intensity at 340 and 380 nm excitation, with corresponding levels of Ca expressed in nanomoles.
Glucose transport determinations. Measurement of [2-3H]deoxyglucose uptake was carried out as described by Klip et al. (20). Briefly, 18 h after the last of the 5-day treatments, differentiated myotubes in 12-well plates were rinsed twice with HEPES-buffered saline (in mM: 140 NaCl, 20 Na-HEPES, 2.5 MgSO4, 1 CaCl2, and 5 KCl, pH 7.4). Myotubes were then incubated in serum-free medium for 5 h before treatment with 50 mU/ml insulin for 60 min. Glucose uptake was quantitated by exposing the cells to 10 µM [2-3H]deoxyglucose (1 µCi/ml) for 12 min. Nonspecific uptake was determined by quantitating cell-associated radioactivity in the presence of 10 µM cytochalasin B, which blocks transporter-mediated uptake. At the end of the 12-min period, the uptake buffer was aspirated rapidly, and the cells were washed three times with ice-cold isotonic saline (0.9% NaCl, wt/vol). The cells were lysed in 0.05 N NaOH and associated radioactivity was determined by liquid scintillation counting. Each condition was assayed in duplicate.
Western analysis. Myotubes or epitrochlearis muscles were homogenized in buffer of 10 mM HEPES, 1 mM EDTA, 250 mM sucrose, pH 7.4. Aliquots of homogenate were solubilized in Laemmli sample buffer, loaded onto a 10% SDS-PAGE minigel, and subjected to electrophoresis at 4 W for ~1 h. Proteins were transferred from gel to nitrocellulose membrane at 200 mA for 1 h. Membranes were blocked overnight at 4°C with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20. GLUT4 protein content was determined using a rabbit polyclonal antibody directed against the COOH terminus, as described previously (36). The GLUT4 and GLUT1 antibodies were kindly given to us by Mike Mueckler (Washington University, St. Louis, MO). For determination of MEF2A, the blots were probed with an affinity-purified rabbit antibody directed against the COOH terminus (Santa Cruz Biotechnology). For MEF2D, the blots were probed with a monoclonal antibody (Transduction Laboratories). This was followed by incubation with appropriate horseradish peroxidase-conjugated anti-IgG antibody. Antibody-bound protein was detected using enhanced chemiluminescence.
Animals. This study was approved by the Animal Studies Committee of Washington University. Male Wistar rats weighing ~50 g were obtained from Charles River Laboratories and maintained on a diet of Purina rat chow and water ad libitum. On the day of the experiment, rats were anesthetized with pentobarbital sodium (5 mg/100 g body wt) given intraperitoneally, and the epitrochlearis muscles were removed.
Muscle incubations.
Epitrochlearis muscles were incubated as described previously
(30). Briefly, muscles of sedentary rats were incubated in tissue culture medium in the presence of 1.0 µM ionomycin or 4.0 mM
caffeine or vehicle (control) in glass vials in a shaking incubator maintained at 37°C. The vials, containing 2 ml of medium, were gassed
continuously with 95% O2-5% CO2 throughout
the incubation. The incubation medium consisted of -MEM (GIBCO-BRL
1200-063), 10% fetal bovine serum (GIBCO-BRL), 50 µU/ml
purified pork insulin (Ilectin II, Eli Lilly), 100 µU/ml penicillin,
100 µg/ml streptomycin, and 0.25 µg/ml fungizone. The medium was
sterilized by filtration through 0.2-µm Millipore filters. The medium
was replaced with fresh medium after 6 and 12 h of incubation.
After an 18-h-long incubation period, the muscles were washed in 2 ml
of PBS for 10 min, blotted, clamp frozen, and stored at
80°C until
they were used for measurement of GLUT4 and MEF2A and MEF2D.
Statistics. Results are expressed as means ± SE. Statistically significant differences were determined with Student's t-test or ANOVA, as appropriate.
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RESULTS AND DISCUSSION |
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Caffeine induces a sustained increase in cytosolic
Ca2+.
Caffeine has been shown to release Ca2+ from isolated
sarcoplasmic reticulum (SR) preparations and, at high concentrations, to cause muscle contractions (1, 32). Because we have
found single-cell measures of cytosolic Ca2+ in response to
caffeine and other agonists (14) to be similar to those in
intact tissues (3), we conducted a series of experiments on the more convenient L6 myotube preparation. As shown in Fig. 1, in the presence of 10 µM dantrolene,
a compound that inhibits Ca2+ release from the SR, exposure
of myocytes to 5 mM caffeine resulted in a modest increase in cytosolic
Ca2+. On replacement of the dantrolene-containing medium
with dantrolene-free medium, the cytosolic Ca2+ response
was potentiated and sustained more than fourfold above basal levels. In
light of this evidence that 5 mM caffeine raises cytosolic
Ca2+, we used 5 mM caffeine in our subsequent experiments
on these cells.
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AICAR induces an increase in GLUT4 in L6 myotubes.
To validate L6 myotubes as an appropriate model in which to study the
regulation of GLUT4 induction, we examined the effect of AICAR. This
compound is transported into cells and converted to the AMP analog ZMP,
resulting in the activation of AMPK (40). It has been
shown that administration of AICAR to rats results in an increase in
muscle GLUT4 (18, 41), and we have shown that incubation
of rat epitrochlearis muscles with AICAR in culture medium induces a
significant increase in GLUT4 protein (30). As shown in
Fig. 2, exposure of L6 myotubes to 1.0 mM
AICAR for 3 h/day for 5 days induced an increase in GLUT4 protein.
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An inhibitor of AMPK prevents the AICAR-induced increase in GLUT4. AraA is taken up by muscle cells and converted to AraATP, which is a competitive inhibitor of AMPK (11, 29). As shown in Fig. 2, AraA prevents the increase in GLUT4 induced by AICAR. This finding supports the interpretation that activation of AMPK is the mechanism by which AICAR induces an increase in GLUT4.
Raising cytosolic Ca2+ induces an
increase in GLUT4.
The finding that AICAR induces an increase in GLUT4 in L6
myotubes provided evidence that these cells are suitable for studies of
the regulation of GLUT4 induction. We therefore investigated the effect
of exposing L6 myotubes to 5 mM caffeine for 3 h/day. As shown in Fig.
3, exposure to caffeine induced a highly
significant increase in GLUT4. This effect was partially blocked by 10 µM dantrolene (Fig. 3), which also blocked ~66% of the
caffeine-induced increase in cytosolic Ca2+ (Fig. 1). As
shown in Fig. 4, the increases in GLUT4
were associated with significant increases in insulin-stimulated
glucose transport activity.
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GLUT1 and GLUT3 do not increase in response to caffeine or AICAR.
L6 myotubes also contain the GLUT1 and GLUT3 isoforms of the
glucose transporter; this likely accounts for the high basal levels of
glucose transport (Fig. 4). In contrast to the response of GLUT4,
neither GLUT1 nor GLUT3 protein increased in L6 myotubes treated for 3 h/day for 5 days with either AICAR or caffeine (Fig. 5). Thus the adaptive responses to
raising cytosolic Ca2+ or activating AMPK are specific for
the GLUT4 isoform. This finding is in keeping with the observation that
exercise induces an increase in GLUT4 but not in GLUT1 in rat skeletal
muscle (37).
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Responses of MEF2 to caffeine and AICAR.
Recent studies have shown that there is a MEF2-binding site in the
GLUT4 promoter that is essential for GLUT4 expression in skeletal
muscle (24, 27, 38). Insulin deficiency results in a
decrease in MEF2A protein in striated muscle, and this finding likely
accounts for the decrease in muscle GLUT4 associated with insulin
deficiency (27). The expression of GLUT4 in striated muscle is dependent on binding of a MEF2A-MEF2D heterodimer to the
GLUT4 promoter (27). These findings motivated us to
examine the effect of incubating L6 myotubes with caffeine or AICAR on MEF2A and MEF2D protein concentrations. As shown in Fig.
6, caffeine induced approximately twofold
increases in MEF2A (Fig. 6A) and MEF2D (Fig. 6B)
protein levels. These increases in MEF2A and MEF2D were partially
prevented by 10 µM dantrolene. AICAR also induced increases in MEF2A
and MEF2D (Fig. 7).
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KN93 blocks the caffeine-induced increases in GLUT4, MEF2A, and MEF2D. CAMK I, II, and III are closely related enzymes that belong to the same protein kinase subfamily as AMPK (5). AMPK and the CAMKs recognize the same amino acid consensus sequence, and it has been shown that AMPK and CAMK I have very similar substrate recognition requirements (5, 6, 23, 39). Because activation of AMPK induces an increase in GLUT4 in muscle (18, 30, 41), it seemed possible that activation of a CAMK by Ca2+ might mediate the increases in MEF2A, MEF2D, and GLUT4 by phosphorylation by CAMK of the same substrates that are phosphorylated by AMPK. As a preliminary step in evaluating this possibility, we determined whether the CAMK inhibitor KN93, which is equally effective in inhibiting all three CAMKs (5, 13), prevents the caffeine-induced increases in MEF2A, MEF2D, and GLUT4. As shown in Figs. 3 and 6, KN93 completely inhibited the increases in MEF2A, MEF2D, and GLUT4 proteins in myocytes exposed to caffeine. Evidence obtained using an inhibitor can obviously not be used as proof for the correctness of a hypothesis; however, this finding is consistent with the hypothesis that activation of a CAMK is involved in mediating the increases in MEF2A, MEF2D, and GLUT4 induced by an increase in cytosolic Ca2+ and points the direction for future, more sophisticated studies. These could, for example, involve transfection of myocytes with a constitutively active form of CAMK.
Responses of rat epitrochlearis muscles to caffeine or ionomycin in
vitro.
L6 myocytes differ considerably from adult skeletal muscle in their
pattern of glucose transporter expression and regulation (17). Therefore, to obtain information regarding whether
our findings are applicable to adult skeletal muscle, we incubated rat
epitrochlearis muscles with caffeine or ionomycin in culture medium for
18 h. As shown in Fig. 8, both
caffeine and ionomycin induced ~25% increases in GLUT4, MEF2A, and
MEF2D proteins in rat epitrochlearis muscles.
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ACKNOWLEDGEMENTS |
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We are grateful to Victoria Reckamp for expert assistance with preparation of the manuscript.
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FOOTNOTES |
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This research was supported by National Institutes of Health Grants AG-00425 and DK-18986 to J. O. Holloszy and HL-62552 to M. Sturek. E. O. Ojuka and T. E. Jones were supported by National Institute on Aging Institutional National Research Service Award AG-00078, and B. R. Wamhoff was supported by an American Heart Association doctoral fellowship.
Address for reprint requests and other correspondence: J. O. Holloszy, Dept. of Internal Medicine, Washington Univ. School of Medicine, Campus Box 8113, 4566 Scott Ave., St. Louis, MO 63110 (E-Mail: jhollosz{at}im.wustl.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.
10.1152/ajpendo.00512.2001
Received 12 November 2001; accepted in final form 3 January 2002.
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