Glucose uptake and metabolic stress in rat muscles stimulated electrically with different protocols

J Appl Physiol 91: 1237 1244, 2001. Glucose uptake and metabolic stress in rat muscles stimulated electrically with different protocols RUNE ASLESEN, 1,2 * ELLEN M. L. ENGEBRETSEN, 3 * JESPER FRANCH, 4 AND JØRGEN JENSEN 1,3 1 Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo; 2 The Norwegian University of Sport and Physical Education, N-0806 Oslo; 3 Department of Physiology, National Institute of Occupational Health, N-0033 Oslo, Norway; and 4 Institute of Sport Science and Physical Education, Odense University, DK-5230 Odense, Denmark Received 30 November 2000; accepted in final form 17 May 2001 Aslesen, Rune, Ellen M. L. Engebretsen, Jesper Franch, and Jørgen Jensen. Glucose uptake and metabolic stress in rat muscles stimulated electrically with different protocols. J Appl Physiol 91: 1237 1244, 2001. In the present study, the relationship between the pattern of electrical stimulation and glucose uptake was investigated in slow-twitch muscles (soleus) and fast-twitch muscles (epitrochlearis) from Wistar rats. Muscles were stimulated electrically for 30 min in vitro with either single pulses (frequencies varied between 0.8 and 15 Hz) or with 200-ms trains (0.1 2 Hz). Glucose uptake (measured with tracer amount of 2-[ 3 H]deoxyglucose) increased with increasing number of impulses whether delivered as single pulses or as short trains. The highest glucose uptake achieved with short tetanic contractions was similar in soleus and epitrochlearis (10.9 0.7 and 12.0 0.8 mmol kg dry wt 1 30 min 1, respectively). Single pulses, on the other hand, increased contraction-stimulated glucose uptake less in soleus than in epitrochlearis (7.5 1.1 and 11.7 0.5 mmol kg dry wt 1 30 min 1, respectively; P 0.02). Glucose uptake correlated with glycogen breakdown in soleus (r 0.84, P 0.0001) and (epitrochlearis: r 0.91, P 0.0001). Contraction-stimulated glucose uptake also correlated with breakdown of ATP and PCr and with reduction in force. Our data suggest that metabolic stress mediates contraction-stimulated glucose uptake. glycogen; adenosine 5 -triphosphate; force; fiber type; fatigue *R. Aslesen and E. M. L. Engebretsen contributed equally to this work. Address for reprint requests and other correspondence: J. Jensen, Dept. of Physiology, National Institute of Occupational Health, PO Box 8149 Dep., N-0033 Oslo, Norway (E-mail: jorgen.jensen@ stami.no). CONTRACTILE ACTIVITY AND INSULIN are the two main stimuli of glucose uptake in skeletal muscle (34). Although it has been more than 100 yr since Chauveau and Kaufmann (3) reported that contractile activity increases glucose uptake in skeletal muscle, the signaling pathway for contraction-stimulated glucose uptake is still unknown (34). Contraction and insulin, however, regulate glucose uptake via different signaling pathways. Insulin stimulates glucose uptake via phosphatidylinositol 3-kinase, but this kinase is not involved in contraction stimulated glucose uptake (27, 39). Glucose transport in skeletal muscle is regulated by translocation of the glucose transporter GLUT-4 from intracellular vesicles to the plasma membrane. Both insulin and contractile activity stimulate translocation of GLUT-4, but it is believed that insulin and contraction translocate different pools of GLUT-4 (6, 32). The total amount of GLUT-4 and glucose uptake during insulin stimulation is higher in type I than in type II fibers of rats (2, 11, 15, 21, 34). There is, however, controversy about the ability of contractile activity to stimulate glucose uptake in different fiber types. Most studies using treadmill running report higher glucose uptake in type I fibers (21, 35), whereas most studies using electrical stimulation report higher glucose uptake in type II fibers (11, 23, 31, 36). The low level of glucose uptake in type II fibers during treadmill running may be due to lack of recruitment of these fibers rather than the inability of contractile activity to stimulate glucose uptake in vivo. Only a few detailed studies exist concerning the relationship between the intensity of electrical stimulation and glucose uptake (23, 27, 30). In general, these studies show that the glucose uptake increases with increasing stimulus intensity (23, 27, 30, 31). Besides, more intense electrical stimulation is required in the slow-twitch soleus muscles than in fast-twitch muscles for activation of glucose uptake (8, 23, 31). The signaling pathway for contraction-stimulated glucose uptake has received much interest because contractile activity stimulates glucose uptake in insulin-resistant muscles (11). Knowledge about the relationship between the intensity of the electrical stimulation and glucose uptake is important to explore the signaling pathway for contraction-stimulated glucose uptake. The present study was designed to investigate thoroughly the relationship between stimulation pattern and glucose uptake in the fast-twitch epitrochlearis and the slowtwitch soleus muscle. 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. http://www.jap.org 8750-7587/01 $5.00 Copyright 2001 the American Physiological Society 1237

1238 METABOLIC STRESS AND GLUCOSE UPTAKE IN SKELETAL MUSCLES Several lines of evidence have suggested that metabolic stress may play a role in contraction-stimulation glucose uptake. Recently, AMP-activated protein kinase has been suggested to mediate the effect of contraction (12, 13, 19). Glycogen, however, is also important for regulation of glucose uptake (16, 17, 21, 22), but so far the relationship between glycogen breakdown and glucose uptake has not been studied when muscles with normal glycogen concentration are stimulated with different intensities for the same period of time. The second purpose of this study was, therefore, to investigate the relationship between contractionstimulated glucose uptake and breakdown of glycogen. Furthermore, relationships between contraction-stimulated glucose uptake and reduction in concentration of ATP and phosphocreatine (PCr) and reduction in force were investigated. MATERIALS AND METHODS Animals. Male Wistar rats were obtained from Møllegård Breeding Center (Lille Skensved, Denmark) and housed for at least 1 wk at 21 C and a 12:12-h light-dark cycle (light from 7:00 AM to 7:00 PM). The rats had free access to rat chow and water. The experiments were performed during the light cycle (10:00 AM to 2:00 PM), and the weight of the rats on the day of experiment was 120 150 g. The experiments were conducted in conformity with the laws and regulations controlling experiments on live animals in Norway and the European Convention for the Protection of Vertebrate Animals used in Experimental and Other Scientific Purposes. Muscle preparation and incubation. The rats were anaesthetized with an intraperitoneal injection of 7.5 mg pentobarbital (50 mg/ml), and the epitrochlearis and soleus muscles were dissected out. The epitrochlearis muscles were studied intact, whereas the soleus muscles were split in two strips. Some muscles were used for measurements of force development, and other muscles were used for measurements of glucose uptake. All muscles were preincubated for 30 40 min in 3.5 ml Krebs Henseleit buffer containing 5.5 mm glucose, 2 mm sodium acetate, 5 mm HEPES, and 0.1% bovine serum albumin (Fraction V, Sigma Chemical) before stimulation and measurements of force or glucose uptake as described previously (2). The preincubations and all other incubations were performed at 30 C while 95% O 2 and 5% CO 2 were continuously gassed through the buffer. Force measurements. The apparatus for measurements of force development was built at our institute (The Institute of Basic Medical Sciences, University of Oslo) according to the description by Nesher et al. (29). The force transducer (FORT 100, Precision Instruments, Sarasota, FL) was connected to an amplifier (built at The Institute of Basic Medical Sciences, University of Oslo), and the force was printed directly (Omni scribe recorder, Houston Instrument, Austin, TX). The equipment was calibrated by hanging a 20-g weight ( 0.2 N) on the force transducer. Muscles were mounted at the resting tension where the highest twitch force was obtained ( 0.5 g) and rested for at least 30 min. Force development was measured during 30 min when muscles were stimulated with trains 200 ms long at a frequency of 100 Hz (square-wave pulses of 0.2-ms duration and 10-V amplitude) delivered every 0.5, 1, 2, 5, or 10 s (2, 1, 0.5, 0.2, and 0.1 Hz). After the stimulation, the muscles were removed from the contraction apparatus and frozen in liquid nitrogen. Tension-time product was calculated by summation of force multiplied with stimulation time (200 ms). Glucose uptake. Glucose uptake was measured after the preincubation as described previously (2). In brief, the muscles were transferred to fresh buffer (containing 5.5 mm glucose) where 0.25 Ci/ml of 2-[1,2-3 H(N)]-deoxy-D-glucose (30.6 Ci/mmol; DuPont, NEN) and 0.1 Ci/ml of [1-14 C]-Dmannitol (54.5 mci/mmol; DuPont, NEN) were added. The muscle from the one leg was stimulated to contract isometrically for 30 min, whereas the other served as resting control. Some groups of muscles were stimulated with single pulses (square-wave pulses of 0.2-ms duration and 10-V amplitude) delivered at frequencies of 0.83, 1.33, 2, 3.33, 6.66, 10, and 15 Hz. Other groups of muscles were stimulated with 200-ms trains at a frequency of 100 Hz (square-wave pulses of 0.2-ms duration and 10-V amplitude) delivered at rates of 2, 1, 0.5, 0.2, and 0.1 Hz. After incubations, the muscles were removed from the contraction apparatus, blotted on filter paper, frozen in liquid nitrogen, freeze-dried, and weighed. For measurements of glucose uptake, muscles were digested in 600 l of 1 M KOH for 20 min at 70 C. Of the digest, 400 l were added to 3 ml of scintillation solution (High-ionic Flour, Packard) mixed, and counted for radioactivity (TRI-CARB 460C, Packard). Glucose uptake was determined from the intracellular accumulation of 2-[ 3 H]deoxy-Dglucose during 30 min of incubation. Glucose uptake is presented as millimoles glucose per kilogram dry weight (dw), assuming similar uptake kinetics for glucose and 2-deoxyglucose. Contraction-stimulated glucose uptake is calculated as the difference between the rested and stimulated muscle. Glycogen. For glycogen determination in muscles where glucose uptake was measured, 100 l of the 1 M KOH muscle digest were neutralized with 17 l glacial acetic acid, and 500 l 0.3 M acetate buffer containing 0.5% amyloglucosidase (Boehringer-Mannheim) were added to hydrolyze the glycogen. In the muscles where force was measured, muscle samples and glycogen were hydrolyzed in 500 l 1MHClfor2h at 100 C. Glucose was measured fluorometrically according to Lowry and Passonneau (26). Glycogen is presented as millimoles glucose per kilogram dry weight. Contractionstimulated glycogen breakdown is calculated as the difference between the rested and stimulated muscle. ATP, PCr, and lactate. Metabolites were extracted from freeze-dried muscle samples in 3 M HClO 4 for 20 min on ice and neutralized with KHCO 3. The neutralized extracts were centrifuged at 4 C and frozen for later analysis. ATP, PCr, and lactate concentrations were measured fluorometrically according to Lowry and Passonneau (26). Statistics. The data are presented as means SE. Glucose uptake, force reduction, and time-tension product in soleus and epitrochlearis during different stimulation protocols were compared by two-way analysis of variance with muscle and stimulation rate as factors. Analysis of variance with Fisher s least significant difference as post hoc test were used to compare effects of 200-ms trains delivered at different frequencies. Maximal contraction-stimulated glucose uptake in epitrochlearis and soleus was compared with Students t-test. Correlations were assessed by linear regression analysis. RESULTS Stimulation of epitrochlearis and split soleus muscles with 200-ms trains produced similar force initially [0.16 0.01 N (n 32) and 0.16 0.01 N (n 29) respectively]. Mean weights of split soleus and epitrochlearis muscles used for force measurements were also similar (5.38 0.28 and 5.20 0.21 mg dw,

METABOLIC STRESS AND GLUCOSE UPTAKE IN SKELETAL MUSCLES 1239 Fig. 1. Force development in soleus (A) and epitrochlearis (B) muscles stimulated with 200-ms trains delivered at different rates for 30 min. Trains were delivered at rates of 0.1 Hz (F), 0.2 Hz (ƒ), 0.5 Hz ( ),1Hz(Œ),or2Hz(E). Values are means SE; n 6 8 for epitrochlearis and n 5 7 for soleus. respectively). The force decreased in both muscles during the 30 min of stimulation, and the decline was larger the shorter the rest periods were between the trains (Fig. 1, A and B). Two-way analysis of variance showed that the decreases in force were more pronounced in epitrochlearis than in soleus muscle (P 0.001). Tension-time products were higher in soleus than in epitrochlearis (P 0.0001) when stimulated with identical protocol. In contrast to force, tensiontime product tended to increase with increasing rate of stimulation (Tables 1 and 2). In epitrochlearis, however, there was a decrease in time-tension product at the highest rate of stimulation (Table. 1). Basal glucose uptake was 5.5 0.2 (n 104) and 4.1 0.1 (n 100) mmol kg dw 1 30 min 1 in soleus and epitrochlearis (P 0.00001), respectively. Glucose uptake increased with increasing rate of stimulation both when stimulated with short trains and with single pulses. The contraction-stimulated glucose uptake correlated with the total number of pulses in both soleus (r 0.89; P 0.0001) and epitrochlearis (r 0.64; P 0.03). In muscles stimulated with 200-ms trains, twoway analysis of variance showed differences between stimulation protocol and muscles (P 0.0001; Tables 1 and 2) and interaction (P 0.01). At a lower rate of stimulation, glucose uptake was increased more in epitrochlearis than in soleus (Tables 1 and 2). The highest glucose uptakes achieved when the muscles were stimulated with 200-ms trains were, however, similar in soleus and epitrochlearis (10.9 0.7 and 12.0 0.8 mmol kg dw 1 30 min 1, respectively). In muscles stimulated with single pulses, two-way analysis of variance showed differences between muscles and stimulation (Fig. 2; P 0.0001) but no interaction (P 0.25). Half-maximal glucose uptake was obtained at a stimulation frequency of 1.5 and 3 Hz in epitrochlearis and soleus muscles, respectively. This is in agreement with previous in vitro studies (27, 30). In soleus, the highest glucose uptake achieved when muscles were stimulated with single pulses was lower than when muscles were stimulated with 200-ms trains [10.9 0.7 (n 18) vs. 7.5 1.1 mmol kg dw 1 30 min 1 (n 9); P 0.02]. In resting soleus and epitrochlearis muscles, the glycogen concentration was 85.3 2.3 (n 104) and 153.9 3.4 dw (n 100), respectively. In muscles stimulated electrically, the glycogen concentration decreased with increasing rate of stimulation (Tables 1 and 2, Fig. 3). There was a high correlation Table 1. Tension-time products, contraction-stimulated glucose uptake, and concentration of glycogen, ATP, PCr, and lactate in soleus muscles stimulated 30 min with 200-ms trains delivered at different frequencies Stimulation Protocol Tension-Time Product, N s Contraction- Stimulated Glucose Uptake, mmol kg 1 30 min 1 Glycogen, ATP, PCr, Lactate, Rate of 200-ms trains, Hz 0.1 401 56*(5) 1.6 0.6*(13) 85.2 9.6 (13) 15.8 1.4 (5) 51.7 5.6 (5) 13.4 1.8* (5) 0.2 583 59*(6) 2.9 0.8*(13) 63.8 6.4*(13) 14.0 1.5 (6) 42.8 6.7 (6) 16.2 1.9*(6) 0.5 1,129 142 (5) 6.2 1.0 (8) 67.4 6.5(8) 12.0 1.0 (4) 36.3 3.5*(4) 20.4 3.6*(4) 1.0 1,136 105 (6) 8.9 0.7 (5) 46.6 8.0*(5) 12.5 0.9 (6) 40.9 3.2*(6) 25.0 4.6* (6) 2.0 945 84 (7) 10.9 0.7 (18) 46.9 3.7*(18) 10.8 0.4* (5) 33.3 3.0* (5) 37.3 6.9 (7) Values are means SE; no. of muscles is given in parentheses. Contraction-stimulated glucose uptake was calculated as the difference between rested and stimulated muscles. Basal glucose uptake was 5.1 0.2 mmol kg dry wt 1 30 min 1 (57). Concentrations of metabolites in control soleus muscles: glycogen 86.1 3.5 dry wt (57), ATP 15.3 0.6 dry wt (7), PCr 54.3 2.9 dry wt (7), and lactate 2.2 1.0 dry wt (8). PCr, phosphocreatine. *Significantly different from, P 0.05. Significantly different from, P 0.05.

1240 METABOLIC STRESS AND GLUCOSE UPTAKE IN SKELETAL MUSCLES Table 2. Tension-time products, contraction-stimulated glucose uptake, and concentration of glycogen, ATP, PCr, and lactate in epitrochlearis muscles stimulated 30 min with 200-ms trains delivered at different frequencies Stimulation Protocol Tension-Time Product, N s Contraction- Stimulated Glucose Uptake, mmol kg 1 30 min 1 Glycogen, ATP, PCr, Lactate, Rate of 200-ms trains, Hz 0.1 254 22*(6) 3.8 0.4* (11) 108.6 13.2 (11) 16.4 0.8 (6) 47.8 3.8(6) 33.7 6.9*(6) 0.2 475 46 (7) 8.5 0.6 (13) 81.1 6.7* (13) 14.6 0.9 (7) 49.2 7.4(7) 37.9 4.9*(7) 0.5 549 55 (6) 9.4 1.2 (7) 70.8 4.2*(7) 10.3 1.2*(6) 33.8 5.9(6) 42.0 8.8(6) 1.0 527 39 (8) 9.2 0.8 (6) 52.7 3.9* (6) 10.6 1.1*(7) 43.1 6.6(7) 58.6 8.5 (7) 2.0 394 52 (6) 12.0 0.8 (15) 63.3 6.2*(15) 11.1 0.9*(6) 44.1 5.6(6) 41.5 5.2(6) Values are means SE; no. of muscles is given in parentheses. Contraction-stimulated glucose uptake was calculated as the difference between rested and stimulated muscles. Basal glucose uptake was 3.9 0.2 mmol kg dry wt 1 30 min 1 (52). Concentrations of metabolites in control epitrochlearis: glycogen 148.1 5.1 dry wt (52), ATP 17.7 0.6 dry wt (9), PCr 66.8 3.8 dry wt (9), and lactate 5.9 1.9 dry wt (10). *Significantly different from, P 0.05. Significantly different from, P 0.05. between the glycogen content after the contraction and glucose uptake both in soleus (r 0.93, P 0.00001) and epitrochlearis (r 0.87, P 0.00001). For all stimulation protocols except 2 Hz, muscles from one leg were stimulated electrically, whereas contralateral muscles served as resting controls. Assuming that the initial glycogen concentration and basal glucose uptake were the same on both sides, contraction-stimulated glucose uptake and glycogen breakdown were calculated for each rat. There was a high correlation between contraction-stimulated glucose uptake and glycogen breakdown for both soleus (r 0.84, P 0.00001; Fig. 4A) and epitrochlearis (r 0.91, P 0.00001; Fig. 4B). Mean contraction-stimulated glucose uptake correlated with mean force reduction (Fig. 5; r 0.94, P 0.001). ATP and PCr also decreased during contractile activity and mean contraction-stimulated glucose uptake correlated with mean reduction in ATP (Fig. 6; r 0.84, P 0.003) and with mean PCr breakdown (r 0.70, P 0.03). Force reduction correlated with glycogen breakdown in soleus (r 0.70; P 0.0001) and epitrochlearis (r 0.71; P 0.0001). Reduction in ATP concentration also correlated with the reduction in force in epitrochlearis (r 0.67; P 0.0001) and soleus (r 0.57; P 0.002). DISCUSSION In the present study, we have made a thorough investigation of how different stimulation protocols stimulate glucose uptake in a slow-twitch oxidative (soleus) and fast-twitch glycolytic (epitrochlearis) muscle. Several conclusions can be made: 1) the highest glucose uptake achieved with tetanic contractions was similar in soleus and epitrochlearis, 2) higher glucose uptake could be obtained in soleus when the muscle was stimulated with short tetanic contractions compared with single pulses, 3) much higher glucose uptake was obtained in epitrochlearis than in soleus Fig. 2. Glucose uptake epitrochlearis (Œ) and soleus (F) muscles stimulated for 30 min with single pulses delivered at frequencies varying between 0.83 and 15 Hz. Contraction-stimulated glucose uptake was calculated as the difference between rested and stimulated muscles. dw, Dry weight. Values are means SE; n 5 18 for soleus and n 6 15 for epitrochlearis. Fig. 3. Glycogen concentration in epitrochlearis (Œ) and soleus (F) muscles after 30 min with single pulses delivered at frequencies varying between 0.83 and 15 Hz. Values are means SE; n 5 18 for soleus and n 6 15 for epitrochlearis.

METABOLIC STRESS AND GLUCOSE UPTAKE IN SKELETAL MUSCLES 1241 Fig. 5. Relationship between force reduction and contraction-stimulated glucose uptake. Soleus (F) and epitrochlearis (Œ) muscles were stimulated for 30 min with 200-ms trains delivered at different rates. Contraction-stimulated glucose uptake was calculated as the difference between rested and stimulated muscles. Values are means SE; (force reduction: n 4 7 for soleus and n 6 8 for epitrochlearis; glucose uptake: n 5 18 for soleus and n 6 15 for epitrochlearis). Our data, however, suggest that the highest glucose uptake achieved with short tetanic contractions is similar in fast- and slow-twitch muscles. Surprisingly, the importance of stimulation intensity has not been focused with major interest previously, and only a few protocols with tetanic contractions have been compared (9, 31). Our data clearly show that much higher glucose uptake was obtained in the glycolytic epitrochlearis muscle than in the soleus when stimulated with tetanic contraction at lower rates, whereas maximal contraction-stimulated glucose uptake was similar. Because metabolic stress has been suggested to mediate contraction-stimulated glucose uptake, the fatigue-re- Fig. 4. Relationship between glycogen breakdown and glucose uptake in soleus (A) and epitrochlearis (B) muscles. Muscles were stimulated for 30 min with different protocols, and the contractionstimulated glucose uptake and glycogen breakdown were calculated as the difference between control and stimulated muscles. F, Œ, Muscles stimulated with single pulses; E,, muscles stimulated with 200-ms impulse trains. Values are means SE; n 5 18 for each point. when muscles performed short tetanic contractions at low rates, and 4) contraction-stimulated glucose uptake correlated with glycogen breakdown as well as with other markers of fatigue (decrease in force, reduction ATP and PCr). The highest glucose uptake rates achieved with short tetanic contractions were similar in epitrochlearis and soleus muscles. Although soleus muscles have a higher total concentration of GLUT-4 (11, 15) and higher insulin-stimulated glucose uptake than epitrochlearis, (2, 11, 15), most studies have reported that contraction-stimulated glucose uptake is higher in fast-twitch than in slow-twitch muscles (11, 23, 31). Fig. 6. Relationship between reduction in ATP and contractionstimulated glucose uptake. Soleus (F) and epitrochlearis (Œ) muscles were stimulated for 30 min with 200 ms trains delivered at different rates. Contraction-stimulated glucose uptake was calculated as the difference between rested and stimulated muscles. Values are means SE (ATP: n 5 6 for soleus and n 6 7 for epitrochlearis; glucose uptake: n 5 18 for soleus and n 6 15 for epitrochlearis).

1242 METABOLIC STRESS AND GLUCOSE UPTAKE IN SKELETAL MUSCLES sistant soleus muscle may require more intense stimulation than epitrochlearis to achieve the metabolic perturbations that activate contraction-stimulated glucose uptake. A new finding of the present study is that lower glucose uptake can be achieved in soleus when muscles are stimulated with single pulses compared with short trains delivered at various rates. On the other hand, this is not the case in epitrochlearis muscles. Fast- and slow-twitch muscles, however, differ both in the frequency required for tetanic contraction and in the amount of sarcoplasmic reticulum (5, 14), which makes it difficult to compare intracellular effects of a stimulation protocol in different muscle fiber types. Our data clearly highlight that the stimulation protocol has to be considered carefully when the effect of contractile activity is compared in different muscles. In vivo, glucose uptake increases with increasing workload, indicating that energy consumption and energy status in the muscles are important for the regulation of glucose uptake (1). Most in vitro studies use isometric contractions where no external work is performed, and a thorough investigation of dynamic contractions should be performed to establish the relationship between work and glucose uptake during electrical stimulation. Holloszy and Narahara (18), however, compared glucose uptake in frog muscles stimulated with similar frequency while lifting different loads but found no relationship between work and glucose uptake. Ihlemann et al. (19) used a different procedure to vary energy turnover. By mounting muscles at different length, force development was varied and glucose uptake was related to force development and energy turnover (19, 20). In the present study, a similar force was developed initially during the repeated tetanic contractions. The calculated tensiontime product did not show any clear relationship between tension-time product and glucose uptake. In epitrochlearis, for example, the highest glucose uptake was achieved when muscles were stimulated with 200-ms trains at a frequency of 2 Hz, whereas higher tension-time products were obtained when trains were delivered at frequencies of 1 and 0.5 Hz. Tension-time products were also much higher in soleus than in epitrochlearis during stimulation protocols, whereas higher glucose uptake was obtained in epitrochlearis. Time-tension product, however, does not necessarily reflect energy turnover because a major part of ATP is used for Ca 2 handling, which furthermore differs between muscle fiber types (5, 38). Metabolic stress seems to be involved in contractionstimulated glucose uptake, and AMP-activated protein kinase has recently been suggested to mediate contraction-stimulated glucose uptake in skeletal muscle (12, 20, 13). The AMP-activated protein kinase is a fuel gauge, and the kinase is activated by reduction in ATP/AMP and PCr/Cr ratios and in ph (12, 33). In the present study, contraction-stimulated glucose uptake correlated with the reduction in force, suggesting that metabolic stress plays a role. Contraction-stimulated glucose also correlated with the reduction in ATP and PCr, which supports the hypothesis that activation of AMP-activated protein kinase could mediate contraction-stimulated glucose uptake. However, the role of AMP-activated protein kinase for contraction-stimulated glucose uptake has recently been questioned by Derave et al. (8), who reported that high glycogen concentration inhibits contraction-stimulated activation of AMP-activated protein kinase without affecting glucose uptake. In the present study, we show a correlation between glucose uptake and glycogen breakdown when muscles are stimulated with different intensities for a fixed period of time. Surprisingly, this correlation has not been established, although a great number of studies have suggested that glycogen participates in the regulation of glucose uptake in skeletal muscles (16, 17, 21, 22). Our laboratory has previously studied the relationship between glucose uptake and the intensity of electrical stimulation in the anaesthetized rat (23). In that study, glucose uptake was positively correlated to glycogen breakdown for all muscles (E. Jóhannsson, J. Jensen, and H. A. Dahl, unpublished observation). Kawanaka and co-workers (24) have also reported a correlation between glucose uptake and glycogen concentration after activity, both when the period of time for muscle activity was varied during in vitro contraction and during swimming (25). Furthermore, the fact that contraction-stimulated glucose uptake is reduced in muscles where the initial glycogen concentration is increased (9, 17) also supports the hypothesis that glycogen breakdown participates in the regulation of contraction-stimulated glucose uptake. A correlation between glycogen breakdown and glucose uptake does not prove a causal relationship. Glycogen seems, however, to have actions other than as an energy source for ATP production. Stephenson et al. (37) reported that excitation-contraction coupling and Ca 2 release was reduced in skinned muscle fibers where glycogen concentration was reduced, although the concentrations of ATP and PCr were maintained high. In agreement with previous studies we also find a correlation between glycogen breakdown and decrease in force (4). Because glycogen seems to be so important for excitation-contraction coupling and contractile activity (37), glycogen may also be the link to stimulation of glucose uptake. The glycogen molecule is associated with the enzymes that regulate its own metabolism in a glycogen-protein sarcoplasmic reticulum complex (10), and a major part of GLUT-4-containing vesicles are localized in the same area and translocated to the transverse tubules during contractile activity (32). It has been suggested that the GLUT-4- containing vesicles are physically attached to the glycogen particle and are released as the glycogen molecules diminish (7), but this theory has not been confirmed. An alternative explanation may be that during glycogen breakdown, phosphorylase kinase, phosphorylase, phosphatase, glycogen synthase, and other enzymes are released from the glycogen particle into the cytosol (10, 28) and that one of these enzymes

METABOLIC STRESS AND GLUCOSE UPTAKE IN SKELETAL MUSCLES 1243 in the non-glycogen-bound state starts a signaling pathway for translocation of GLUT-4. In conclusion, soleus muscles require more intense electrical stimulation than epitrochlearis to achieve high rates of glucose uptake. The highest glucose uptake rates achieved with tetanic contractions are, however, similar in the slow-twitch soleus muscle and the fast-twitch epitrochlearis muscle when stimulated with short tetanic contractions. When muscles are stimulated with different protocols for a fixed period of time, contraction-stimulated glucose uptake correlates with breakdown of ATP, PCr, and glycogen as well as with reduction in force. The present findings suggest that metabolic stress mediates the effect of contraction on glucose uptake. Whether activation of AMP-activated protein kinase or reduced glycogen concentration mediates the effect has yet to be determined. We thank Ada Ingvaldsen and Jorid T. Stuenæs for excellent technical assistance. This study was supported by the Research Council of Norway and the Nordic Academy for Advanced Study. REFERENCES 1. Ahlborg G, Felig P, Hagenfeldt L, Hendler R, and Wahren J. Substrate turnover during prolonged exercise in man. Splanchnic and leg metabolism of glucose, free fatty acids, and amino acids. J Clin Invest 53: 1080 1090, 1974. 2. Aslesen R and Jensen J. Effects of epinephrine on glucose metabolism in contracting rat skeletal muscle. Am J Physiol Endocrinol Metab 275: E448 E456, 1998. 3. Chauveau MA and Kaufmann M. Experiences pour la determination du coefficient de l activite nutritive et respiratoire des muscles en repos et en travail. C R Acad Sci 104: 1126 1132, 1887. 4. Chin ER and Allen DG. Effects of reduced muscle glycogen concentration on force, Ca 2 release and contractile protein function in intact mouse skeletal muscle. J Physiol (Lond) 498: 17 29, 1997. 5. Clausen T, van Hardeveld C, and Everts E. Significance of cation transport in control of energy metabolism and thermogenesis. Physiol Rev 733 774, 1991. 6. Coderre L, Kandror KV, Vallega G, and Pilch PF. Identification and characterization of an exercise-sensitive pool of glucose transporters in skeletal muscle. J Biol Chem 270: 27584 27588, 1995. 7. Coderre L, Vallega G, and Pilch PF. Association of GLUT-4 vesicles with glycogen particles in skeletal muscles, identification of a contraction-sensitive pool (Abstract). Diabetes 43: 159A, 1994. 8. Derave W, Ai H, Ihlemann J, Witters LA, Kristiansen S, Richter EA, and Ploug T. Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slowtwitch muscle. Diabetes 49: 1281 1287, 2000. 9. Derave W, Lund S, Holman GD, Wojtaszewski J, Pedersen O, and Richter EA. Contraction-stimulated muscle glucose transport and GLUT-4 surface content are dependent on glycogen concentration. Am J Physiol Endocrinol Metab 277: E1103 E1110, 1999. 10. Entman ML, Keslensky SS, Chu A, and Van Winkle WB. The sarcoplasmic reticulum-glycogenolytic complex in mammalian fast twitch skeletal muscle. J Biol Chem 255: 6245 6252, 1980. 11. Etgen GJ, Wilson CM, Jensen J, Cushman SW, and Ivy JL. Glucose transport and cell surface GLUT-4 protein in skeletal muscle of the obese Zucker rat. Am J Physiol Endocrinol Metab 271: E294 E301, 1996. 12. Hardie DG and Carling D. The AMP-activated protein kinase fuel gauge of the mammalian cell? Eur J Biochem 246: 259 273, 1997. 13. Hayashi H, Hirshman MF, Kurth EJ, Winder WW, and Goodyear LJ. Evidence for 5 AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47: 1369 1373, 1998. 14. Hennig R and Lømo T. Firing patterns of motor units in normal rats. Nature 314: 164 166, 1985. 15. Henriksen EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, and Holloszy JO. Glucose transporter protein content and glucose transport capacity in rat skeletal muscle. Am J Physiol Endocrinol Metab 259: E593 E598, 1990. 16. Henriksen EJ and Halseth AE. Early alterations in soleus GLUT-4, glucose transport, and glycogen in voluntary running rats. J Appl Physiol 76: 1862 1867, 1994. 17. Hespel P and Richter EA. Glucose uptake and transport in contracting, perfused rat muscle with different pre-contraction glycogen concentrations. J Physiol (Lond) 427: 347 359, 1990. 18. Holloszy JO and Narahara HT. Studies of tissue permeability. X. Changes in permeability to 3-methylglucose associated with contraction of isolated frog muscle. J Biol Chem 240: 3493 3500, 1965. 19. Ihlemann J, Ploug T, Hellsten Y, and Galbo H. Effect of tension on contraction-induced glucose transport in rat skeletal muscle. Am J Physiol Endocrinol Metab 277: E208 E214, 1999. 20. Ihlemann J, Ploug T, Hellsten Y, and Galbo H. Effect of stimulation frequency on contraction-induced glucose transport in rat skeletal muscle. Am J Physiol Endocrinol Metab 279: E862 E867, 2000. 21. James DE, Kraegen EW, and Chisholm DJ. Muscle glucose metabolism in exercising rats: comparison with insulin stimulation. Am J Physiol Endocrinol Metab 248: E575 E580, 1985. 22. Jensen J, Aslesen R, Ivy JL, and Brørs O. Role of glycogen concentration and epinephrine on glucose uptake in rat epitrochlearis muscle. Am J Physiol Endocrinol Metab 272: E649 E655, 1997. 23. Jóhannsson E, Jensen J, Gundersen K, Dahl HA, and Bonen A. Effect of electrical stimulation patterns on glucose transport in rat muscles. Am J Physiol Regulatory Integrative Comp Physiol 271: R426 R431, 1996. 24. Kawanaka K, Tabata I, and Higuchi M. More tetanic contraction are required for activating glucose transport maximally in trained muscle. J Appl Physiol 83: 429 433, 1997. 25. Kawanaka K, Tabata I, Tanaka A, and Higuchi M. Effects of high-intensity intermittent swimming on glucose transport in rat epitrochlearis muscle. J Appl Physiol 84: 1852 1857, 1998. 26. Lowry OH and Passonneau JV. A collection of metabolite assays. In: A Flexible System of Enzymatic Analysis. New York: Academic, 1972, p. 147 218. 27. Lund S, Holman GD, Schmitz O, and Pedersen O. Contraction stimulates translocation of glucose transporter GLUT-4 in skeletal muscle through a mechanism distinct from that of insulin. Proc Natl Acad Sci USA 92: 5817 5821, 1995. 28. Meyer F, Heilmeyer LMG, Haschke RH, and Fischer EH. Control of phosphorylase activity in a muscle glycogen particle. J Biol Chem 245: 6642 6648, 1970. 29. Nesher R, Karl IE, Kaiser KE, and Kipnis DM. Epitrochlearis muscle. I. Mechanical performance, energetics, and fiber composition. Am J Physiol Endocrinol Metab 239: E454 E460, 1980. 30. Nesher R, Karl IE, and Kipnis DM. Dissociation of effects of insulin and contraction on glucose transport in rat epitrochlearis muscle. Am J Physiol Cell Physiol 249: C226 C232, 1985. 31. Ploug T, Galbo H, Vinten J, Jørgensen M, and Richter EA. Kinetics of glucose transport in rat muscle: effects of insulin and contractions. Am J Physiol Endocrinol Metab 253: E12 E20, 1987. 32. Ploug T, van Deurs B, Ai H, Cushman SW, and Ralston E. Analysis of GLUT-4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions. J Cell Biol 142: 1429 1446, 1998. 33. Ponticos M, Lu QL, Morgan JE, Hardie DG, Partridge TA, and Carling D. Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of

1244 METABOLIC STRESS AND GLUCOSE UPTAKE IN SKELETAL MUSCLES creatine kinase in skeletal muscle. EMBO J 17: 1688 1699, 1998. 34. Richter EA. Glucose utilization. In: Handbook of Physiology. Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Am. Physiol. Soc., 1996, sect. 12, chapt. 20, p. 912 951. 35. Richter EA, Garetto LP, Goodman MN, and Ruderman NB. Muscle glucose metabolism following exercise in the rat. Increased sensitivity to insulin. J Clin Invest 69: 785 793, 1982. 36. Richter EA, Garetto LP, Goodman MN, and Ruderman NB. Enhanced muscle glucose metabolism after exercise: modulation by local factors. Am J Physiol Endocrinol Metab 246: E476 E482, 1984. 37. Stephenson DG, Nguyen LT, and Stephenson GM. Glycogen content and excitation-contraction coupling in mechanically skinned muscle fibres of the cane toad. J Physiol (Lond) 519: 177 187, 1999. 38. Szentesi P, Zaremba R, van Mechelen W, and Stienen GJM. ATP utilization for calcium uptake and force production in different types of human skeletal muscle fibres. J Physiol (Lond) 531: 393 403, 2001. 39. Whitehead JP, Soos MA, Aslesen R, O Rahilly S, and Jensen J. Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not PKB activation or glucose uptake in rat muscle. Biochem J 349: 775 781, 2000.