INTRODUCTION

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INSULIN INCREASES THE PERMEABILITY of skeletal muscle to glucose. Furthermore, it is well known that low- or moderate-intensity swimming or running has an insulinlike effect on glucose transport in skeletal muscle (14, 15). In the absence of insulin, in vitro twitch or tetanic contractions induced by electrical stimulation produce an increase in muscle glucose transport (12, 22, 23). Therefore, it is suggested that there are two separate pathways for stimulation of glucose transport in skeletal muscle: one pathway is activated by insulin, the other by muscle contraction.

Several previous studies reported enhanced insulin-stimulated glucose uptake after exercise when the muscle glycogen content was low (5, 7, 28). These results suggest that muscle glycogen content affects insulin-stimulated glucose transport in skeletal muscle. Furthermore, we found recently that in vitro tetani-stimulated glucose transport in rat epitrochlearis (Epi) muscle was negatively correlated with postcontraction muscle glycogen concentration (17). This result also suggests that the lower the concentration of muscle glycogen, the higher the contraction-stimulated glucose transport rate.

In rat Epi muscle, ten 10-s-long in vitro tetani had a maximal effect on glucose transport, and more tetanic contractions did not induce a further increase in glucose transport (11, 17). Furthermore, glucose transport after this protocol was elevated to a level as high as that observed after 120 min of swimming with a weight equal to 2% of body mass (3). Therefore, activation of glucose transport after ten 10-s-long in vitro tetani has been considered as the exercise-induced maximal activation of glucose transport in Epi. However, our previous study showed that mean muscle glycogen concentration was ~15 µmol glucose/g muscle after ten 10-s-long in vitro tetani, and more tetani did not further decrease muscle glycogen content (17). Therefore, it is not known whether a significant negative relationship between postcontraction muscle glycogen and glucose transport exists in the lower range of muscle glycogen concentration (i.e., much less than 15 µmol glucose/g muscle).

Therefore, we examined glucose transport in Epi after high-intensity intermittent exercise that has been used as a tool to deplete muscle glycogen (4). For this purpose, we adopted intermittent swimming with a weight equivalent to 18% of body mass. After this protocol, muscle glycogen concentration decreased to much less than 15 µmol glucose/g muscle, and glucose transport was further increased to a level that was much higher than that observed after ten 10-s-long in vitro tetani.

	METHODS

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Male Sprague-Dawley rats (Crea Japan, Tokyo) with body weights of 90-110 g were used for this study. Ethical approval for this work was obtained from the Committees on Animal Care at the National Institute of Health and Nutrition, Japan.

Treatment of animals and swimming program. All animals were housed in rooms lighted from 7 AM to 7 PM and were maintained with ad libitum feeding on standard chow and water. Room temperature was maintained at 20-22°C. Food was restricted to 8 g on the evening of the last day before the experiment.

Muscle preparation. Rested and exercised rats were anesthetized with an intraperitoneal injection of pentobarbital sodium, 5 mg/100 g body wt, and the Epi muscles were dissected out to measure glucose transport in vitro. Some muscles from high-intensity intermittently swimming rats were incubated at 35°C for 2 h in 3 ml of oxygenated Krebs-Henseleit bicarbonate buffer (KHB) containing 8 mM Na pyruvate and 24 mM mannitol. The gas phase in the flasks was 95% O₂-5% CO₂. The Epi muscle is a small, thin muscle weighing ~25 mg in rats weighing 90-110 g. It is made up predominantly (~85%) of fast-twitch fibers (21).

Electrical stimulation. 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 that connected to an isometric force transducer (TB-653t, Nihon Kohden, Tokyo, Japan), and resting tension was adjusted to 0.5 g. The mounted muscle was immersed in 100 ml KHB containing 32 mM mannitol and 8 mM glucose. The muscles were continuously oxygenated with 95% O₂-5% CO₂ at 35°C. For activation of glucose transport by tetanic 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. Tetanic contractions were repeated 1, 5, 10, 15, and 20 times at a rate of 1 contraction/min.

Insulin treatment. The muscles to be used for the measurement of insulin-stimulated glucose transport activity were placed in 3 ml of oxygenated KHB containing 8 mM Na pyruvate, 24 mM mannitol, 0.1% radioimmunoassay-grade bovine serum albumin, with 2 mU/ml insulin (this concentration of insulin stimulates glucose transport to a maximal level). The gas phase in the flasks was 95% O₂-5% CO₂ at 35°C.

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. (27). After exercise, electrical stimulation, or the incubation with insulin, the muscles were transfered to flasks containing 3 ml of KHB with 40 mM mannitol and were incubated with shaking for 15 min at 29°C to remove glucose. In the case of insulin stimulation, 2 mU/ml of insulin were added to the 3 ml of KHB. The muscles were then incubated for 20 min at 29°C in 2 ml of KHB containing 1 mM 2-deoxy-(1,2-³H)glucose (1.5 mCi/mmol) and 39 mM (U-¹⁴C)mannitol (8 µCi/mmol) (Moravek Biochemicals). In the case of insulin stimulation, 2 mU/ml of insulin were also added to 2 ml of KHB. The gas phase in the flasks during both the rinse and incubation periods was 95% O₂-5% CO₂. Hansen et al. (10) reported that the accumulation of 2-DG remains linear until the total intracellular 2-DG-6-phosphate 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 (27). 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 methods according to Lowry and Passonneau (18) 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 4 ml of Aquasol-2 (NEN) and were 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 the total muscle water. Glucose transport activity is expressed as micromoles of 2-DG per milliliter intracellular water per 20 minutes.

Statistics. All values are means ± SE. Statistical comparisons of the groups were made by one-way analysis of variance, and each group was compared with Fisher's paired least significant difference analysis. The correlation analysis was done with Pearson's test. Statistical significance was defined as P < 0.05.

	RESULTS

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Effects of high-intensity intermittent swimming on muscle glucose transport and glycogen in rat Epi muscle. When rats swam with a weight equal to 18% of body mass, muscle glycogen concentration in Epi decreased as the number of bouts of swimming was increased (Table 1). Eight bouts of swimming reduced muscle glycogen concentration to a level of 7.6 ± 0.5 µmol glucose/g muscle (Table 1). 2-DG transport also increased as the number of bouts of swimming was increased (Table 1).

Effects of in vitro tetanic contractions on muscle glucose transport and glycogen in rat Epi muscle. Muscle glycogen concentration in Epi decreased with increasing numbers of in vitro tetani (Table 2). After five tetani, muscle glycogen concentration was decreased, compared with the basal level (P < 0.01), but more tetani did not significantly induce any further decrease in muscle glycogen (Table 2). Twenty tetani reduced muscle glycogen concentration to a level of 13.7 ± 1.0 µmol glucose/g muscle (Table 2). 2-DG transport also increased with increasing numbers of in vitro tetani (Table 2). Although 2-DG transport was significantly increased after five tetani compared with the basal level (P < 0.01), more tetani did not result in further increase in 2-DG transport (Table 2).

Effects of low-intensity continuous swimming on muscle glucose transport and glycogen in rat Epi muscle. When rats swam with a weight equal to 3% of body mass, muscle glycogen concentration in Epi decreased as the duration of swimming was increased (Table 3). 2-DG transport also increased as the duration of swimming was prolonged to 120 min (Table 3).

Effects of high-intensity intermittent swimming, in vitro tetani, and combined stimuli on muscle glucose transport and glycogen in rat Epi. Fifteen tetanic contractions, which provide a maximal effect of in vitro tetani on 2-DG transport, resulted in a 3.2-fold increase in 2-DG transport rate above the basal level (Table 4). Eight bouts of high-intensity swimming induced a 7.3-fold increase in the rate of 2-DG transport above the basal level, which was significantly greater than that induced by 15 tetani alone (P < 0.01; Table 4). We also examined the effects of swimming followed by 15 tetani in vitro on 2-DG transport. The combined effects of swimming and subsequent in vitro tetani on 2-DG transport were not significantly greater than the effect of swimming alone (Table 4).

Effects of high-intensity intermittent swimming, insulin, and combined stimuli on muscle glucose transport and glycogen in rat Epi muscle. Incubation of Epi muscle with 2 mU/ml of insulin increased 2-DG transport from a basal value of 0.33 ± 0.04 µmol · ml intracellular water¹ · 20 min¹ (n = 8) to 1.34 ± 0.07 µmol · ml intracellular water¹ · 20 min¹ (n = 5). High-intensity intermittent swimming followed by 15 tetani in vitro increased 2-DG transport to 2.32 ± 0.13 µmol · ml intracellular water¹ · 20 min¹ (n = 5). Furthermore, swimming followed by in vitro tetani and subsequent insulin (2 mU/ml) incubation increased 2-DG transport to 3.41 ± 0.29 µmol · ml intracellular water¹ · 20 min¹ (n = 7). Thus the combined effects of insulin and swimming followed by in vitro tetani were additive.

	DISCUSSION

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In the present study, the high-intensity intermittent swimming increased glucose transport in rat Epi to a much higher level than that induced by repeated 10-s-long in vitro tetani alone (Table 4). A previous study (3) reported that ten 10-s-long in vitro tetanic contractions increased glucose transport to a level as high as that achieved after four bouts of 30-min swimming with a weight equal to 2% of body mass. Therefore, glucose transport after ten 10-s-long in vitro tetani has been considered as the exercise-induced maximal activation of glucose transport in Epi. However, the present study showed that high-intensity intermittent swimming raises glucose transport in rat Epi to a higher level than do ten 10-s-long in vitro tetani.

We do not know whether other in vitro contraction- or exercise-related protocols increase glucose transport to a higher level than that observed after ten 10-s-long in vitro tetani. Therefore, it is not, so far, plausible to conclude that this high-intensity intermittent swimming protocol induces the exercise-related maximal stimulus for glucose transport. However, our finding showed that the high-intensity intermittent swimming protocol increases glucose transport to a higher level than that induced by ten 10-s-long in vitro tetani, which was regarded as the exercise-related maximal stimulus for glucose transport in Epi.

We previously showed that glucose transport in Epi after repeated in vitro tetani was negatively correlated with postcontraction muscle glycogen content (17). In the present study, we also demonstrated a significant negative correlation between glucose transport and muscle glycogen content at the end of high-intensity intermittent swimming (Fig. 1). Furthermore, the data of muscle glycogen and glucose transport after in vitro tetani fell on the same regression line obtained from the data after high-intensity intermittent swimming (Fig. 1). Therefore, these results suggest that glucose transport in Epi is influenced by glycogen content regardless of the mode of contraction (in vitro tetani or swimming). In the present study, high-intensity intermittent swimming reduced muscle glycogen concentration in Epi to a level of 7.6 µmol glucose/g muscle, which has never been observed after repeated 10-s-long in vitro tetani (Table 1). Therefore, the higher glucose transport induced by high-intensity intermittent swimming seems to be related to a lower muscle glycogen content after swimming than that observed after repeated 10-s-long in vitro tetani. However, a causal mechanism explaining the negative correlation between muscle glycogen and glucose transport after exercise is not known. Some novel experimental approach seems necessary to explain the high glucose transport activity after high-intensity intermittent swimming.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Relationship between mean value of postexercise glycogen concentration and mean rate of 2-deoxy-D-glucose (2-DG) transport in rat epitrochlearis muscle after 1 (), 5 (), 10 (), 15 (), and 20 () in vitro tetanic contractions; 10 (), 30 (), and 120 () min of low-intensity continuous swimming; 1 (), 3 (), and 8 () bouts of high-intensity intermittent swimming; and 8 bouts of high-intensity swimming following 15 in vitro tetani (). Data are shown in Tables 1, 2, 3, and 4. Values are means ± SE.

In the present study, 120-min-long, low-intensity swimming reduced muscle glycogen concentration to a lower level and activated glucose transport to a greater extent than did ten 10-s-long in vitro tetani (Tables 2 and 3), whereas a previous study (3) reported that low-intensity swimming with the same duration activated glucose transport to the same extent as did ten 10-s-long in vitro tetani. Apparently, the results obtained from the two studies are in conflict. Because the weight attached to the swimming rat (3% of body weight) in the present study was heavier than that used in the previous study (2% of body weight), the total muscle work (probably substrate consumption) of the Epi during swimming in the present study is thought to be greater than that in the previous study. Therefore, it is possible that muscle glycogen content in the present study was lower than that in the previous study. Consequently, we believe that this apparent difference can also be explained by our hypothesis that the lower the glycogen content in muscle, the higher the glucose transport activity.

In a previous study by Gulve et al. (9), when the Epi was allowed to recover for 3 h after prolonged low-intensity swimming in incubation medium without glucose, glucose transport returned to basal levels. In this study, during the recovery period, muscle glycogen in Epi was not resynthesized, because the incubation medium did not contain glucose. Furthermore, when Epi was allowed to recover in incubation medium without glucose for 2 h after high-intensity intermittent swimming, we observed that glucose transport returned to near-basal levels while muscle glycogen content was as low as that observed immediately after the swim (unpublished observations). Therefore, this suggests that a reduction of muscle glycogen itself does not initiate the biochemical reactions that lead to activation of glucose transport. In addition to reduced muscle glycogen content, other factor(s) may be necessary for activating glucose transport. The transient increase in the cytoplasmic Ca²⁺ during muscle contraction may be a possible factor. Previous studies (1, 13, 24, 26) suggest that an increase in cytoplasmic Ca²⁺ that activates phosphorylase kinase and induces a burst of glycogenolysis activates the process that results in increased glucose transport. An increase in cytoplasmic Ca²⁺ may increase glucose transport through activation of a Ca²⁺-activated enzyme, in cooperation with a reduction in muscle glycogen.

Muscle contraction stimulates glucose transport by translocating glucose transporter GLUT-4-containing vesicles from an intracellular pool to the plasma membrane (6, 8, 19). An increase in GLUT-4 at the plasma membrane is considered to be due both to stimulation of translocation from an intracellular pool to the plasma membrane and to a decrease in the rate of endocytosis (16, 25). In terms of explaining the data obtained in the present investigation, we raise the hypothesis that translocation of GLUT-4 vesicles is interfered with by muscle glycogen. Coderre et al. (2) 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 possible that the more muscle glycogen is broken down, the more GLUT-4 is freed from glycogen, resulting in an increased intracellular pool of free GLUT-4 available in response to muscle contraction. Thus less muscle glycogen binding to intracellular GLUT-4 vesicles may facilitate GLUT-4 vesicles to undergo translocation in response to muscle contraction. We speculate that another factor (for example, Ca²⁺), other than a reduction in muscle glycogen, may trigger the translocation of GLUT-4 vesicles, and that the amount of muscle glycogen binding to GLUT-4 vesicles may control the degree of GLUT-4 vesicle translocation.

Previous studies in which rat Epi muscle was used indicated that the maximal effects of insulin and in vitro tetani on glucose transport in Epi were additive (11, 22). Therefore, these previous studies suggest the hypothesis that muscle contractile activity and insulin increase glucose transporter through two different mechanisms. In the present study, although the effect of high-intensity intermittent swimming on glucose transport was much greater than the effect of in vitro tetani, the combined effects of swimming and insulin on glucose transport were also additive (see RESULTS). Therefore, the present study supports the hypothesis that there are two separate mechanisms of glucose transport activation in skeletal muscle.

During exercise, muscle glycogen is catabolized to lactate or CO₂ for the resynthesis of ATP. When rats swam with a weight equal to 18% of body mass, the blood lactate concentrations were significantly elevated from resting levels of 1.3 ± 0.2 mM (n = 7) to 10.4 ± 0.3 mM (n = 7), and the mean exhaustion time was 63 s (see METHODS). Because the increase in blood lactate concentration roughly reflects production of lactate in active skeletal muscle, large amounts of glycogen in active skeletal muscle are considered to be catabolized to lactate during the high-intensity swimming. Furthermore, Medbo and Tabata (20) demonstrated that most of the glycogen broken down during 60 s of exhaustive exercise was catabolized to lactate. On the other hand, rats with a weight equal to 3% of body mass could swim for >120 min. During such a low-intensity exercise, the blood lactate concentration did not increase above resting levels. Therefore, consumed glycogen during low-intensity swimming was assumed to be metabolized not to lactate but to CO₂. However, the data of muscle glycogen and glucose transport after low-intensity continuous swimming fell on the same regression line obtained from the data after high-intensity swimming (Fig. 1). Therefore, we suggest that the effect of exercise on glucose transport is mediated by a process associated with a decrease in muscle glycogen content, regardless of the end product from glycogen breakdown.

In summary, high-intensity intermittent swimming increases glucose transport in rat Epi to a higher level than that induced by ten 10-s-long in vitro tetani, which has been regarded as the maximal exercise-related stimulus for glucose transport. This result may be explained by the lower muscle glycogen level after high-intensity intermittent swimming than after in vitro tetani, because there was a significant negative correlation between glucose transport and muscle glycogen concentration in Epi after the high-intensity swimming and the in vitro tetani.