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Centre for Physical Activity and Nutrition, School of Exercise & Nutrition Sciences, Deakin University, Burwood, 3125, Australia
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Abstract |
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O2peak= 3.7 ± 0.8 l min–1) exercised for 60 min at 75 ± 1%O2peak on 7 consecutive days. Muscle samples were obtained before the start of cycle exercise training and 24 h after the first and seventh exercise sessions and analysed for citrate synthase activity, glycogen and glucose transporter 4 (GLUT-4) mRNA and protein expression. Exercise training increased (P < 0.05) citrate synthase by 
20% and muscle glycogen concentration by 40%. GLUT-4 mRNA levels 24 h after the first and seventh exercise sessions were similar to those measured before the start of exercise training. In contrast, GLUT-4 protein expression was increased after 7 days of exercise training (12.4 ± 1.5 versus 3.4 ± 1.0 arbitray units (a.u.), P < 0.05) and although it tended to be higher 24 h after the first exercise session (6.0 ± 3.0 versus 3.4 ± 1.0 a.u.), this was not significantly different (P= 0.09). These results support the suggestion that the adaptive increase in skeletal muscle GLUT-4 protein expression with short-term exercise training arises from the repeated, transient increases in GLUT-gene transcription following each exercise bout leading to a gradual accumulation of GLUT-4 protein, despite GLUT-4 mRNA returning to basal levels between exercise stimuli.
(Received 8 February 2004;
accepted after revision 2 June 2004; first published online 7 June 2004)
Corresponding author M. Hargreaves: Centre for Physical Activity and Nutrition, School of Exercise & Nutrition Sciences, Deakin University, Burwood, 3125, Australia. Email: mharg{at}deakin.edu.au
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Introduction |
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O2peak (Kraniou et al. 2000), suggesting increased GLUT-4 transcription and/or enhanced mRNA stability. It has been proposed that the adaptive increase in skeletal muscle GLUT-4 protein expression arises from the repeated, transient increases in GLUT-gene transcription following each exercise bout (MacLean et al. 2000). Furthermore, these authors argued that since protein has a longer half-life than RNA, there is a gradual accumulation of GLUT-4 protein with repeated exercise bouts, despite GLUT-4 mRNA returning to basal levels between exercise stimuli. The present study was undertaken to test this hypothesis in human skeletal muscle by measuring GLUT-4 mRNA and protein in response to a 7 day exercise training intervention. |
Methods |
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Six healthy, physically active but untrained male subjects (23 ± 1 years old, 84 ± 5 kg body weight) gave their written consent to participate in this study. The experimental procedures and possible risks associated with participation were explained to each subject verbally and in writing before commencment of the study, according to the Declaration ofHelsinki. The study was approved by the Deakin University Human Research Ethics Committee.
Experimental protocol
At least 1 week before starting the exercise training programme, subjects performed an incremental, cycle ergometer (Lode Instruments, Groningen, the Netherlands) test to volitional fatigue for the determination of peak pulmonary oxygen uptake O2peak, which averaged 3.7 ± 0.8 l min–1. Expired gases were analysed using electronic analysers (Applied Electrochemistry, Pittsburgh, PA, USA) and volume measured by a turbine ventilometer. Subjects reported to the laboratory at 8.00 am, after an overnight fast, and an initial muscle sample was obtained under local anaesthesia (1% Xylocaine) from vastus lateralis using the percutaneous needle biopsy technique, with suction. After a brief period of supine rest, subjects then performed their first 60 min exercise training session at 75%O2peak. This exercise session was repeated each day for the next 6 days. Twenty-four hours after completing the first and seventh exercise sessions, subjects again reported to the laboratory in the morning after an overnight fast and muscle samples were obtained under local anaesthesia (1% Xylocaine) from vastus lateralis through separate incisions. All muscle samples were immediately frozen in liquid N2 and stored for later analysis of glycogen, citrate synthase activity, and GLUT-4 mRNA and protein expression. All subjects were provided with a food package (3700 kcal, 74% CHO, 15% fat, 10% protein) to consume for the 24 h prior to the mornings on which muscle samples were obtained.
Analytical techniques
Muscle for glycogen analysis was freeze dried for approximately 24 h, crushed and then dissected free of visible connective tissue. Dried extracts were incubated in 1 M HCl at 100°C for 2 h, neutralized with 0.67 M NaOH and analysed for glucosyl units using an enzymatic fluorometric technique (Passonneau & Lauderdale, 1974). Citrate synthase activity was measured using the method of Srere (1969) and expressed as micromoles substrate per gram dry weight muscle tissue. Total RNA from 8–10 mg of muscle was isolated using the FastRNA Kit-Green (BIO 101, Vista, CA, USA) protocol and reagents. Total RNA concentration was determined spectrophotometrically at 260 nm. First strand cDNA was generated from 0.5 µg RNA using AMV RT (Promega, Madison, WI, USA) as previously described (Wadley et al. 2001). The cDNA was stored at –20°C until further analysis. Primers were designed using Primer Express software package version 1.0 (Applied Biosystems, Foster City, CA, USA) from gene sequences obtained from GenBank. A BLAST (Altschul et al. 1990) search for each primer confirmed homologous binding to the desired mRNA of human skeletal muscle. Primer sequences for GLUT-4 (forward primer 5'-GCCGGACGTTTGACCAGAT-3' and reverse primer 5'-GGTGTTTCACCTCCTGCTCTA-3') and ß-actin (forward primer 5'-GACAGGATGCAGAAGGAGATTACT-3' and reverse primer 5'-TGATCCACATCTGCTGGAAGGT-3'). Quantification of mRNA expression was performed (in triplicate) by real-time RT-PCR using the ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster City, CA, USA). Fluorescence emission data were captured and mRNA levels were quantified using the critical threshold (CT) value. To compensate for variations in input RNA amounts and efficiency of reverse transcription, ß-actin mRNA was quantified and results were normalized to these values. ß-Actin met the criteria of a housekeeping gene in that there was no evidence of a training effect when ß-actin mRNA was analysed following conversion to a linear numerical value using the expression 2–CT, where CT is the threshold cycle value (Murphy et al. 2003). To ensure that the primers were detecting a single product, samples were subjected to a heat dissociation protocol following the final cycle of PCR (Ririe et al. 1997). Approximately 20 mg of wet muscle tissue were used to measure total crude membrane GLUT-4 protein by immunoblotting. Muscle samples were homogenized (Polytron PT 1200, Kinematica AG, Luzernerstrasse Switzerland) in 0.5 ml homogenization buffer (10 mM Tris-HCl, 1 mM EDTA, 250 mM sucrose and 1 M PMSF, pH 7.4) per 20 mg muscle. Homogenates were spun at 1000g for 5 min at 4°C, the supernatant removed and spun at 150 000g for 1 h at 4°C. The crude membrane pellet was resuspended in approximately 100 µl of homogenization buffer and stored at –80°C. Total protein content of the crude membrane suspension was assessed using a commercially available protein assay kit (BCA protein assay, Pierce, Rockford, IL, USA). Crude membrane suspensions containing 10 µg of total protein were then solubilized with an equal volume of Laemmli sample buffer and incubated for 30 min at 37°C. Fifteen micrograms of protein from each sample were separated using SDS-PAGE on a 10% acrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat skimmed milk solution for 1 h at room temperature and then washed (50 mM Tris, 750 mM NaCl and 0.25% Tween 20) before being incubated overnight at 4°C with the primary, monoclonal GLUT-4 antibody (R & D Systems, Minneapolis, MN, USA), diluted 1:500 in 5% skimmed milk. After the overnight incubation, the membrane was rewashed with 5% non-fat skimmed milk solution and incubated for 1 h at room temperature in 5% blocking buffer containing antimouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, membranes were incubated in an ECL solution for 1 min and placed in an autoradiography cassette (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Immunoreactive bands were highlighted by ECL (Supersignal, Pierce, Roachford, IL, USA), exposed to light-sensitive film for 15–60 s and quantified by densitometry using image analysis software (Kodak Digital Science, New York, NY, USA). An internal control of previously extracted human muscle was used in each gel to normalize for variations in signals across different membranes.
Statistical analysis
Data were analysed using a one-way analysis of variance and Newman–Kuels post hoc test when significant differences were detected at the P= 0.05 level. All data are presented as means ±S.E.M.
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Results |
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O2peak. Citrate synthase activity increased by 20% after the short-term training programme, providing evidence of the normal training-induced increase in muscle oxidative capacity and verifying the effectiveness of the current protocol. Muscle glycogen concentration increased by 42% (Table 1). GLUT-4 mRNA, measured 24 h after the first and seventh exercise sessions, was similar to that measured before the start of exercise training (Fig. 1). In contrast, GLUT-4 protein expression was increased after 7 days of exercise training (12.4 ± 1.5 versus 3.4 ± 1.0 arbitrary units (a.u.), P < 0.05, Fig. 1) and although it tended to be higher 24 h after the first exercise session (6.0 ± 3.0 versus 3.4 + 1.0 a.u., Fig. 1), this was not statistically significant (P= 0.09).
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Discussion |
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It has been shown previously that a single exercise bout increases skeletal GLUT-4 protein expression in rodents (Ren et al. 1994; Kuo et al. 1999; Chibalin et al. 2000). Studies in humans are less clear. Griewe et al. (2000) reported an increase in GLUT-4 protein expression 8 and 22 h after completion of a single, moderate-intensity exercise bout. In contrast, when measured 4 h after exercise, there was no significant change in skeletal muscle GLUT-4 expression in the study by Wojtaszewski et al. (2000), although GLUT-4 levels were 23% higher after exercise. Similarly, in the present study GLUT-4 protein tended (P= 0.09) to be higher 24 h after the first exercise training session, but the small sample size and variable response prevented the 76% increase from being statistically significant. After 7 days of exercise training, GLUT-4 protein increased 3.6-fold in the present study. This is consistent with previous studies demonstrating rapid up-regulation of GLUT-4 protein in response to short-term exercise training (Gulve & Spina, 1995; Houmard et al. 1995; Phillips et al. 1996). Similarly, there is a rapid decrease in GLUT-4 levels in human skeletal muscle with detraining (McCoy et al. 1994; Vukovich et al. 1996). The molecular signals responsible for the increase in GLUT-4 protein are yet to be fully elucidated (Dohm, 2002). However, recent results implicate increased intracellular calcium and energy status, as sensed by AMP-activated protein kinase (AMPK), in the up-regulation of GLUT-4 in L6 myocytes (Ojuka et al. 2002). The transcription factor myocyte enhancer factor 2 (MEF2) also appears to be important, since the elevated GLUT-4 levels in response to increased calcium and AMPK activation were associated with increased expression of both the MEF2A and MEF2D isoforms (Ojuka et al. 2002). Whatever the cellular mechanisms, their induction, and the subsequent increase in GLUT-4 protein expression, are dependent on a regular exercise stimulus.
In the present study, muscle glycogen levels were increased by 42%. Studies in transgenic mice overexpressing either GLUT-4 or glycogen synthase in skeletal muscle have demonstrated that both are critical for muscle glycogen synthesis, although the enhanced muscle glycogen storage observed in trained athletes appears to be more related to increased skeletal muscle GLUT-4 expression (Hickner et al. 1997; Griewe et al. 1999). Furthermore, the increase in both GLUT-4 gene and protein expression observed in the immediate postexercise period appears to be linked to the restoration of muscle glycogen (Holloszy, 2003). We have previously observed that postexercise muscle glycogen storage following a standardized dietary carbohydrate intake was correlated with GLUT-4 expression (McCoy et al. 1996). Thus, it is likely that the increase in GLUT-4 protein expression in the present study contributed to the enhanced muscle glycogen storage.
In summary, we have shown that steady-state increases in GLUT-4 protein following 7 days of exercise training were not accompanied by similar increases in GLUT-4 mRNA. These results support the suggestion (MacLean et al. 2000) that the adaptive increase in skeletal muscle GLUT-4 protein expression with short-term (7 days) exercise training arises from the repeated, transient increases in GLUT-4 gene transcription following each exercise bout.
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Acknowledgements |
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Author's present address
Giorgos N. Kraniou: Department of Nutrition and Dietetics, Harokopio University, Athens 176-71, Greece.
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