Journal of Applied Physiology
Vol. 83, No. 2, pp. 429-433, August 1997
METABOLISM

More tetanic contractions are required for activating glucose transport maximally in trained muscle

Kentaro Kawanaka, Izumi Tabata, and Mitsuru Higuchi

Laboratories of Health Evaluation and Exercise Physiology, Division of Health Promotion, National Institute of Health and Nutrition, Toyama 1-23-1, Shinjuku-city, Tokyo 162, Japan

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Kawanaka, Kentaro, Izumi Tabata, and Mitsuru Higuchi. More tetanic contractions are required for activating glucose transport maximally in trained muscle. J. Appl. Physiol. 83(2): 429-433, 1997.Exercise training increases contraction-stimulated maximal glucose transport and muscle glycogen level in skeletal muscle. However, there is a possibility that more muscle contractions are required to maximally activate glucose transport in trained than in untrained muscle, because increased glycogen level after training may inhibit glucose transport. Therefore, the purpose of this study was to investigate the relationship between the increase in glucose transport and the number of tetanic contractions in trained and untrained muscle. Male rats swam 2 h/day for 15 days. In untrained epitrochlearis muscle, resting glycogen was 26.6 µmol glucose/g muscle. Ten, 10-s-long tetani at a rate of 1 contraction/min decreased glycogen level to 15.4 µmol glucose/g muscle and maximally increased 2-deoxy-D-glucose (2-DG) transport. Training increased contraction-stimulated maximal 2-DG transport (+71%; P < 0.01), GLUT-4 protein content (+78%; P < 0.01), and resting glycogen level (to 39.3 µmol glucose/g muscle; P < 0.01) on the next day after the training ended, although this training effect might be due, at least in part, to last bout of exercise. In trained muscle, 20 tetani were necessary to maximally activate glucose transport. Twenty tetani decreased muscle glycogen to a lower level than 10 tetani (18.9 vs. 24.0 µmol glucose/g muscle; P < 0.01). Contraction-stimulated 2-DG transport was negatively correlated with postcontraction muscle glycogen level in trained (r = 0.60; P < 0.01) and untrained muscle (r = 0.57; P < 0.01).

muscle glycogen; epitrochlearis; 2-deoxy-D-glucose transport

INTRODUCTION

EXERCISE TRAINING INCREASES both insulin- and contraction-stimulated glucose transport activity in skeletal muscle (1, 16, 17, 20). The increments in insulin- and contraction-stimulated maximal glucose transport induced by training paralleled the increase in glucose transporter protein (GLUT-4) content (1, 16, 17, 20). Recently, Henriksen and Halseth (10) reported that elevation of muscle glycogen level by exercise training interferes with insulin-stimulated glucose tranport and overrides the parallelism between the insulin-stimulated glucose transport and GLUT-4 protein content. However, it is not known whether elevation of muscle glycogen level interferes with contraction-stimulated glucose transport. If the increase in muscle glycogen level by training interferes with contraction-stimulated glucose transport, there is a possibility that more muscle contractions are required for maximally activating glucose transport in trained than in untrained muscle.

The purpose of the present study was to compare the number of muscle contractions required for activating glucose transport maximally in trained and untrained rat epitrochlearis muscles and to investigate the relationship between the number of tetanic contractions, postcontraction muscle glycogen level, and contraction-stimulated glucose transport in trained and untrained muscle. The results indicate that more contractions are required for activating glucose transport maximally in trained than in untrained muscle. Furthermore, this result may be explained by the increased muscle glycogen level after training.

METHODS

Animal care and exercise training program. Male Sprague-Dawley rats (Crea Japan, Tokyo) with initial body weights of 50-60 g were used for this study. All animals were housed in rooms lighted from 7 AM to 7 PM and maintained with ad libitum feeding on standard chow and water. Room temperature was maintained at 20-22°C.

Rats were randomly assigned to either a sedentary control or 15-days' swimming training group. Training group rats swam 2 h/day in four 30-min bouts separated by 5 min of rest. After the first 30-min bout, a weight equal to 2% of body weight was tied to the body of the rat. The rats swam with the weight attached for the remaining three exercise bouts. All rats swam in a barrel filled to a depth of 50 cm and an average surface area of 190 cm²/rat. Training started at 3:30 PM and ended at 6 PM every day. At 11:00 AM to 3:00 PM on the next day (17-21 h after the last bout of exercise), groups of trained and control rats were anesthetized with an intraperitoneal injection of pentobarbital sodium, 5 mg/100 g body wt. Food intake for all rats was restricted to 8 g from 7:00 PM on the last day before the experiment. Afterward, epitrochlearis muscles were dissected out to measure glucose transport in vitro and GLUT-4 protein content. A period of 17-21 h is long enough to permit the acute effect of exercise on insulin responsiveness to wear off (2).

Muscle preparation. Because epitrochlearis muscle is a thin muscle (<2 mm), it can be incubated intact and used to measure glucose transport in vitro (21). Additional epitrochlearis muscles were trimmed free of connective tissue and frozen for subsequent measurements of GLUT-4 protein concentration (6).

Electrical stimulation. After dissection, epitrochlearis muscles were allowed to recover for 30 min in oxygenated Krebs-Henseleit bicarbonate buffer (KHB) containing 8 mM glucose and 32 mM mannitol. For electrical stimulation, the distal end of the muscle was attached to a vertical Lucite rod containing two platinum electrodes. The proximal end was clipped to a jeweler's chain and attached to an isometric force transducer (TB-653t, Nihon Kohden, Tokyo, Japan). The mounted muscle was immersed in 100 ml KHB and continuously oxygenated with 95% O₂-5% CO₂ at 35°C. For activation of glucose transport by contractions, the muscles were stimulated with supramaximal square-wave pulses of 0.2-ms duration with an electrical stimulator (SEN-7203, Nihon Kohden). Tetanic contractions were produced by stimulating the muscle at 100 Hz for 10 s. To investigate the effects of tetanic contraction number, 10, 20, and 25 tetanic contractions were produced at a rate of 1 contraction/min. Also, to investigate the effects of tetanic contraction frequency, 20 tetanic contractions were produced at a rate of 1 contraction/0.5 min or 1 contraction/min.

Measurement of glucose transport activity and glycogen concentration. Glucose transport activity was measured by using the glucose analog 2-deoxy-D-glucose (2-DG) and the procedure of Young et al. (21). After the electrical stimulation or the initial incubation periods, the muscles were transfered to flasks containing 3 ml of KHB with 40 mM mannitol and incubated with shaking for 10 min at 29°C to remove glucose. The muscles were then incubated for 20 min at 29°C in 2 ml of KHB containing 1 mM 2-[1,2-³H]deoxy-D-glucose (1.5 mCi/mmol) and 39 mM [U-¹⁴C]mannitol (8 µCi/mmol) (Moravek Biochemicals). The gas phase in the flasks during both the rinse and incubation periods was 95% O₂-5% CO₂. Hansen et al. (9) reported that accumulation of 2-DG remains linear until the total intracellular 2-DG concentration exceeds 30 mM. Therefore, under our experimental conditions, 2-DG uptake accurately reflects glucose transport activity. After incubation, the muscles were blotted briefly on filter paper dampened with incubation medium, trimmed, and frozen in liquid N₂. The extracellular space and intracellular 2-DG concentrations were determined as described previously (21). The samples were weighed and homogenized in 0.3 M perchloric acid, and an aliquot of the homogenate was stored for later determination of glycogen concentration. Glycogen concentrations were determined by enzymatic method according to Lowry and Passonneau (13) after acid hydrolysis. The remainder of the homogenate was centrifuged at 1,000 g. Aliquots of the muscle extracts and of the incubation media were placed in scintillation vials containing 2 ml of Aquasol-2 (NEN) and counted in a liquid scintillation counter with channels preset for simultaneous ³H and ¹⁴C counting. The amount of each isotope present in the samples was determined, and this information was used to calculate the extracellular space and the intracellular concentration of 2-DG. The intracellular water content of the muscles was calculated by subtracting the measured extracellular space water from total muscle water. Glucose transport activity is expressed as micromoles 2-DG per milliliter intracellular water per 20 minutes.

Measurement of immunoreactive GLUT-4 protein. Epitrochlearis muscles were homogenized with a glass homogenizer (Kontes, Vineland, NJ) in 9 ml of buffer containing 0.25 M sucrose, 1 mM EDTA, and 10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.5, at 4°C. The homogenate thus obtained was centrifuged at 175,000 g for 60 min at 4°C. This pellet (the membrane fraction) was suspended in 490 µl of buffer containing 1 mM EDTA and 10 mM Tris · HCl, pH 7.5, at 4°C, and blended vigorously in a Vortex mixer until the visible pellets were completely dispersed. Then, this solution was solubilized by adding 10 µl of 0.35 M (10% wt/vol) sodium dodecyl sulfate (SDS), mixed well in a Vortex mixer, and kept for 10 min at room temperature. This solution was transferred to a microcentrifuge tube and centrifuged at 10,000 g for 15 min to remove unsolubilized materials. These solubilized membranes were used for the assay of protein and GLUT-4 protein concentration. In a previous study (6), it was shown that almost all GLUT-4 protein was recovered in this procedure.

GLUT-4 protein was assayed as described by Ezaki et al. (6). The solubilized membranes were incubated for 10 min at 37°C in a solution containing 2.5% SDS, 75 mM dithiothreitol, 1.7 M (12.5% vol/vol) glycerol, 361 mM (0.025% wt/vol) bromophenol blue, and 12.5 mM Tris · HCl, pH 7.0. SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (12) with a 0.56 M (4% wt/vol) stacking gel and a 1.4 M (10% wt/vol) resolving gel. Immunoblotting of electrophoresis gels was performed as described previously (5). Proteins in gels were electrophoretically transferred to polyvinylidine difluoride sheets (Immobilon, Millipore, Bedford, MA) in a transfer buffer. The sheets were incubated successively with antibodies to glucose transporters for 24 h and ¹²⁵I-labeled protein A for 24 h at 4°C. Autoradiography was performed with Kodak XAR film (Rochester, NY) at 70°C for 4-12 h. To quantify the glucose transporters, we cut out pieces of sheet containing the GLUT-4 proteins and counted radioactivity in a gamma counter. The background was estimated by counting a region with no labeled band and then subtracted. To compare the amount of transporters in different sheets, we applied a fixed amount of transporters to each gel, and radioactivity of the corresponding bands was normalized.

Statistics. All values are expressed as means ± SE. The significance of differences between two groups was assessed by using Student's unpaired t-test. Statistical comparisons of the mutiple groups were made by two-way analysis of variance (ANOVA), and each group was compared with means comparisons test (Super ANOVA, Abacus Concepts, Berkeley, CA). The correlation analysis was done with Pearson's test. Statistical significance was defined as P < 0.05.

RESULTS

Effects of swimming training on body weights, food consumption, and total GLUT-4 protein content in epitrochlearis muscle. Body weights were significantly decreased in trained rats (P < 0.01) compared with untrained rats (Table 1). Mean food consumption during training period was 17.4 g/day in the control group and 17.2 g/day in the training group. Trained epitrochlearis muscle displayed a 78% greater GLUT-4 protein level (P < 0.01) compared with the control group (Table 1).

Table  1.   Effect of swimming training on body weight and GLUT-4 protein concentration in epitrochlearis muscle Untrained Trained P Body weight, g 168.8 ± 3.5 151.0 ± 2.0 <0.01 (16) (16) GLUT-4 protein in Epi, % mean value of untrained 100 ± 9.8 (7) 177.5 ± 8.6 (8) <0.01 Values are means ± SE; nos. in parentheses represent no. of rats. Glucose transporter GLUT-4 protein concentration levels in a crude membrane fraction were determined by immunoblotting. Epi, epitrochlearis muscle.

Basal glucose transport and glycogen level in nonstimulated untrained and trained epitrochlearis muscle. In the nonstimulated state, there was no statistically significant difference in basal 2-DG transport between trained and untrained muscles (Fig. 1). However, in the nonstimulated state, muscle glycogen concentration was significantly greater (+48%; P < 0.01) in trained than in untrained muscles (Fig. 2). There was no significant correlation between basal 2-DG transport and muscle glycogen level in either trained or untrained muscles (Fig. 3).

Contraction-stimulated glucose transport and postcontraction glycogen level in untrained epitrochlearis muscle. As shown in Fig. 1, in untrained muscle, 10 tetani at a rate of 1 contraction/min resulted in a 3.4-fold (P < 0.01) increase in 2-DG transport compared with basal level. There was no statistically significant difference between the 10 and 20 tetani-stimulated 2-DG transport rates.

In untrained muscle stimulated by 10 tetani, muscle glycogen concentration was decreased by 42% (P < 0.01) from basal level (from 26.6 to 15.4 µmol glucose/g muscle; P < 0.01, Fig. 2).

There was a significant negative correlation between contraction-stimulated 2-DG transport and postcontraction muscle glycogen level in untrained muscle (P < 0.01, Fig. 3). Furthermore, there was a significant positive correlation between contraction-induced increase in 2-DG transport and decrease in muscle glycogen in untrained muscle (r = 0.57, P < 0.01).

Contraction-stimulated glucose transport and postcontraction glycogen level in trained epitrochlearis muscle. As shown in Fig. 1, in trained muscle, 10 tetani at a rate of 1 contraction/min resulted in a 3.7-fold increase (P < 0.01) in 2-DG transport compared with basal level. However, 20 tetani at a rate of 1 contraction/min increased 2-DG transport to higher level (5.9-fold increase compared with basal level, P < 0.01) than 10 tetani. When the muscle was stimulated by 10 tetani at a rate of 1 contraction/min, significant training effect on the rate of 2-DG transport was not seen. However, when the muscle was stimulated by 20 tetani at a rate of 1 contraction/min, the rate of 2-DG transport was significantly higher in trained compared with untrained muscle (+71%; P < 0.01). There was no statistically significant difference between the effects of 20 and 25 tetani on 2-DG transport. When the muscle was stimulated by 20 tetani at a rate of 1 contraction/0.5 min, 2-DG transport was 30% lower than with stimulation at 1 contraction/min (P < 0.01).

In trained muscle, glycogen concentration was decreased by 39% from basal level (from 39.3 to 24.0 µmol glucose/g muscle; P < 0.01) in response to 10 tetani at a rate of 1 contraction/min (Fig. 2). Furthermore, 20 tetani at a rate of 1 contraction/min decreased muscle glycogen level to lower level (18.9 µmol glucose/g muscle, 52% from basal level; P < 0.01) than 10 tetani (Fig. 2). There was no statistically significant difference in the effects of 20 and 25 tetani on muscle glycogen level (Fig. 2). Postcontraction glycogen level in muscles stimulated by 20 tetani at a rate of 1 contraction/0.5 min was 65% higher than when stimulated at a rate of 1 contraction/min (31.3 vs. 18.9 µmol glucose/g muscle; P < 0.01).

There was a significant negative correlation between contraction- stimulated 2-DG transport and postcontraction muscle glycogen level in trained muscle (P < 0.01, Fig. 3). Furthermore, there was a significant positive correlation between contraction-induced increase in 2-DG transport and decrease in muscle glycogen in trained muscle (r = 0.60, P < 0.01).

DISCUSSION

In this study, 15-days of swimming training increased the maximal value of contraction-stimulated glucose transport on the next day after training (Fig. 1), although there is a possibility that this training effects is due, at least in part, to the last bout of exercise or to weight loss. The increase in contraction-stimulated maximal glucose transport activity is thought to be due to the increase in total GLUT-4 protein content (Table 1). However, more tetanic contractions were required for activating glucose transport maximally in trained than in untrained muscles (Fig. 1). From this result, it is suggested that some factor(s) derived from exercise training interferes with contraction-stimulated glucose transport and blunts the responsiveness of muscle glucose transport to contraction stimulus.

The increases in contraction-stimulated glucose transport paralleled the decreases in muscle glycogen level (Figs. 1 and 2). Also, there was a significant negative correlation between contraction-stimulated glucose transport and postcontraction glycogen level in trained and untrained rat epitrochlearis muscle (Fig. 3). Because exercise training increased the resting level of muscle glycogen, it seems possible that elevation of muscle glycogen level by exercise training interferes with contraction-stimulated glucose transport and that a greater number of tetanic contractions is, therefore, needed to lower muscle glycogen to a level that is compatible with maximal activation of glucose transport.

Hespel and Richter (11) demonstrated that precontraction muscle glycogen level was negatively correlated to contraction-stimulated muscle glucose uptake. High levels of precontraction muscle glycogen enhance glycogen breakdown (19). Therefore, from their studies, one possibility that was suggested is that a large increase of intracellular metabolites produced by extensive glycogen breakdown inhibits glucose transport. When precontraction muscle glycogen level is high, lactate production is higher than when glycogen level is low (19). Therefore, Richter (18) suggested the possibility that a larger decrease in pH following higher lactate production during contractions may be responsible for the lower rate of glucose transport observed in the muscles with high glycogen concentrations as compared with the low-glycogen muscles. However, in our study, in which the amount of glycogen breakdown was varied by differences in the number of tetanic contractions, the amount of glycogen breakdown was positively related to contraction-stimulated glucose transport (data in RESULTS). Therefore, it is unlikely that a large increase of intracellular metabolites produced by extensive glycogen breakdown interferes with contraction-stimulated glucose transport.

From our results, it is not clear whether the extent of muscle glycogen breakdown or the final level to which glycogen decreased is important to activate glucose transport. However, Hespel and Richter (11) demonstrated that contraction-stimulated glucose transport was greater in glycogen-depleted muscle than in glycogen-supercompensated muscle. In their study (11), the amount of glycogen breakdown was smaller in glycogen-depleted muscle than in glycogen-supercompensated muscle. Therefore, it is unlikely that larger amount of muscle glycogen breakdown activates glucose transport to higher level.

Muscle contraction stimulates glucose transport by translocating GLUT-4 from an intracellular pool to the plasma membrane (4, 8, 14). The mechanism by which muscle contraction induces GLUT-4 translocation is not known. In our study, a significant correlation between basal glucose transport and muscle glycogen level was not observed (Fig. 3). Nolte et al. (15) demonstrated that depletion of skeletal muscle glycogen by prior in vivo exposure to epinephrine induced only a little increase in basal glucose transport. Therefore, it appears that muscle glycogen breakdown itself does not induce, but only modifies, GLUT-4 translocation.

Coderre et al. (3) reported that ~30% of the GLUT-4 in rat skeletal muscle coprecipitated with glycogen and that transporters could be released from the glycogen particles by amylase digestion. Therefore, it may be speculated that, as muscle glycogen level increases, more GLUT-4 is bound to glycogen, resulting in reduced GLUT-4 translocation in response to muscle contraction. Thus, when more glycogen is broken down, glucose transport may be increased to a higher level by freeing up more GLUT-4 from glycogen.

In humans, Gollnick et al. (7) demonstrated that glucose uptake in an exercising leg was higher when muscle glycogen level was low than when it was normal. Our finding of a significant negative correlation between contraction-stimulated glucose transport and postcontraction glycogen level (Fig. 3) is consistent with their study. From these results, the possibility is raised that glycogen content negatively influences glucose uptake in human muscle during exercise by interference in the process of contraction-stimulated glucose transport.

In summary, more tetanic contractions are required to maximally activate glucose transport in trained than in untrained muscle. This result might be explained by the increased muscle glycogen level after exercise training, because there was a significant negative correlation between contraction-stimulated glucose transport and postcontraction glycogen level.

ACKNOWLEDGEMENTS

The authors appreciate the generous gift from Dr. Osamu Ezaki (National Institute of Health and Nutrition, Tokyo, Japan) of the antibody against GLUT-4 protein. They also thank Dr. John O. Holloszy (Washington University School of Medicine, St. Louis, MO) for generous suggestions and help in editing the English version of this paper.

FOOTNOTES

Address for reprint requests: K. Kawanaka, Division of Health Promotion, National Institute of Health and Nutrition, Toyama 1-23-1, Shinjuku-city, Tokyo 162, Japan (E-mail: mhiguchi{at}nih.go.jp).

Received 16 December 1996; accepted in final form 14 April 1997.

REFERENCES