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(The FASEB Journal. 1998;12:1701-1712.)
© 1998 FASEB

RESEARCH COMMUNICATION

Exercise-induced overexpression of key regulatory proteins involved in glucose uptake and metabolism in tetraplegic persons: molecular mechanism for improved glucose homeostasis

Nils Hjeltnesa, Dana Galuskab, Marie Björnholmb, Anne-kirsti Aksnesa, Anne Lannema, Juleen R. Zierathb and Harriet Wallberg-Henrikssonb,1

a Sunnaas Hospital, 1450 Nesoddtangen, Norway
b Department of Clinical Physiology, Karolinska Hospital, SE-171 76 Stockholm, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Complete spinal cord lesion leads to profound metabolic abnormalities and striking changes in muscle morphology. Here we assess the effects of electrically stimulated leg cycling (ESLC) on whole body insulin sensitivity, skeletal muscle glucose metabolism, and muscle fiber morphology in five tetraplegic subjects with complete C5-C7 lesions. Physical training (seven ESLC sessions/wk for 8 wk) increased whole body insulin-stimulated glucose uptake by 33±13%, concomitant with a 2.1-fold increase in insulin-stimulated (100 µU/ml) 3-O-methylglucose transport in isolated vastus lateralis muscle. Physical training led to a marked increase in protein expression of GLUT4 (378±85%), glycogen synthase (526±146%), and hexokinase II (204±47%) in vastus lateralis muscle, whereas phosphofructokinase expression (282±97%) was not significantly changed. Hexokinase II activity was significantly increased, whereas activity of phosphofructokinase, glycogen synthase, and citrate synthase was not changed after training. Muscle fiber type distribution and fiber area were markedly altered compared to able-bodied subjects before ESLC training, with no change noted in either parameter after ECSL training. In conclusion, muscle contraction improves insulin action on whole body and cellular glucose uptake in cervical cord-injured persons through a major increase in protein expression of key genes involved in the regulation of glucose metabolism. Furthermore, improvements in insulin action on glucose metabolism are independent of changes in muscle fiber type distribution.—Hjeltnes, N., Galuska, D., Björnholm, M., Aksnes, A.-K., Lannem, A., Zierath, J. R., Wallberg-Henriksson, H. Exercise-induced overexpression of key regulatory proteins involved in glucose uptake and metabolism in tetraplegic persons: molecular mechanism for improved glucose homeostasis. FASEB J. 12, 1701–1712 (1998)


Key Words: GLUT4 • glycogen synthase • hexokinase • phosphofructokinase • skeletal muscle fiber type • glucose transport • insulin resistance


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PEOPLE WITH SPINAL cord lesions are characterized by decreased whole body insulin sensitivity, extreme muscle wasting, and altered muscle morphology (1, 2). The loss of physical mobility is a devastating result of the spinal cord injury, and severely compromises the metabolic state of the inflicted person. With long-standing injury, glucose intolerance and peripheral insulin resistance develop (3, 4), and these metabolic disturbances are thought to contribute to the increased risk for cardiovascular disease in this patient group (5, 6).

Several lines of evidence link physical exercise and muscle contraction with improved glucose homeostasis and enhanced insulin sensitivity. Acute exercise in humans (7, 8) and rodents (9–11) or in vitro contraction of isolated skeletal muscle (10, 12–14) directly increases insulin sensitivity. Exercise training improves glucose tolerance and insulin action in insulin-resistant humans (15, 16) and obese insulin-resistant rodents (17). The molecular mechanism for enhanced glucose uptake with chronic exercise training may be related in part to increased expression and activity of key proteins involved in the regulation of glucose uptake and metabolism in skeletal muscle.

Molecular candidates for improved glucose homeostasis in connection with exercise include the insulin/exercise-regulated glucose transporter GLUT42 (16, 18–25), hexokinase (HK) II (26–29), and glycogen synthase (GS) (31–33). A direct connection between increased GLUT4 protein expression and increased basal and insulin-stimulated glucose transport and metabolism has been made from GLUT4-transgenic mouse models (34, 35). Transgenic overexpression of GLUT4 in skeletal muscle prevents the impairment of glycemic control and the accompanying hyperglycemia caused by high fat feeding (36), and markedly improves whole body insulin action in streptozotocin-induced diabetic mice (37). Although glucose transport is generally believed to be rate-limiting for insulin-mediated glucose metabolism in muscle (38), under hyperglycemic or hyperinsulinemic conditions, the rate-limiting step may shift beyond transport (39). Indeed, transgenic overexpression of HK II, the predominant HK isoform in skeletal muscle, leads to increased basal and insulin-stimulated glucose uptake (29). Perturbations that alter neuromuscular activity patterns, including physical exercise or chronic electrical stimulation, are physiological means to achieve protein overexpression. Chronic electrical stimulation of the tibial nerve has been demonstrated to increase total GLUT4 protein expression in rat skeletal muscle (40). In humans, electrically stimulated leg cycling (ESLC) has been developed to create a means for individuals with both tetraplegia and paraplegia to exercise their paralyzed limbs (41). Since exercise has profound effects on glucose homeostasis, we hypothesized that exercise training may lead to increase whole body insulin sensitivity by enhancing key proteins involved in the regulation of skeletal muscle glucose transport and metabolism in tetraplegic persons.

Tetraplegic patients with long-standing injury display a striking reduction in muscle fiber area, an increased percentage of type IIb fibers (65 vs. 30% observed in the general population), and a complete loss of type I fibers (1). Since exercise training is known to increase muscle mass (42, 43), we considered whether muscle contraction through electrical stimulation could reverse the altered skeletal muscle morphology in tetraplegic patients.

The aim of this study was to determine whether ESLC improves whole body insulin sensitivity and normalizes altered muscle morphology in tetraplegic subjects. Five subjects with long-standing complete spinal cord injury were studied by means of an hyperinsulinemic euglycemic clamp and an open muscle biopsy procedure, before and after 8 wk of ESLC. Electrically stimulated leg cycling improved whole body insulin sensitivity and dramatically increased insulin action on glucose transport in isolated skeletal muscle. These metabolic adaptations are associated with a dramatic increase in protein expression of key genes involved in glucose metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Five male subjects with complete chronic lesion of the cervical spinal cord were studied. All study participants received a thorough clinical examination, including routine blood and urine chemistry analysis and X-rays of the chest, spinal column, and extremities. Clinical and anthropometric characteristics of the study participants are presented in Table 1. Condom drainage was used for bladder emptying in all subjects. Three subjects were treated for muscle spasms with baclofen (20–25 mg x 2–4) and one subject received additional treatment with diazepam (5 mg x 2). Subjects were informed of the nature, purpose, and possible risks of the study before voluntarily consenting to participate. The study protocol was reviewed and approved by the institution's ethics committee.


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  		Table 1. Physical characteristics of the tetraplegic subjects

Experimental design
All investigations were performed before noon, after an overnight fast, before and after an 8-wk physical training period of ESLC. The study period included a 2-wk adaptation phase and an 8-wk training period. The adaptation phase was included to perform base line testing procedures indicated above and to ensure that the subjects were familiarized with the training equipment. Training consisted of seven exercise sessions per week (one session per day for 3 days and two sessions per day for 2 days). Training bouts were performed on a computer-controlled, functional electrical stimulation exercise ergometer (ERGYS-I-Clinical Rehabilitation System, Therapeutic Alliances Inc., Fairborn, Ohio) (44, 45). A doctor and a physiotherapist supervised all ESLC sessions. Subjects did not perform ESLC bouts 48 h prior to physiological testing procedures.

Electrically stimulated leg cycling
Surface electrodes were placed over the motor neuron end plates (motor points) of the quadriceps, hamstrings, and gluteal muscle groups of each leg. Three electrodes (two active and one reference) were applied over each muscle group. Six separate channels connected to a computer-controlled closed loop system were used to apply sequential surface muscle stimulation during cycle ergometry. Each channel delivered monophasic rectangular pulses for 350 µs at 30 Hz to each of the two active electrodes. Stimulation intensities ranged from an initial threshold level (determined for each individual muscle group to elicit a palpable contraction of 28–44 mA) to a maximum level of 130 mA. A computerized pedal position sensor was used to continuously calculate velocity and to evaluate the stimulus amplitude required for the six muscle groups to maintain a smooth motion with a constant cranking frequency of 50 revolutions per minute.

Training protocol
A 1-min passive warm-up and a 1-min acceleration to a predetermined target load initiated each exercise session. Thereafter, full resistance was administered until the subject reached fatigue. Fatigue was defined as a decrease in cycling rate below 35 rpm, at the maximal stimulation intensity (130 mA). Electrical stimulation was automatically terminated upon fatigue. The first exercise load was set at the inherent resistance of the ergometer (~6 watts), and the increment in load resistance was increased from one exercise bout to the next by 6.1 watts. A 2-min passive cooldown followed each active ESLC bout. A 5-min pause separated each exercise bout and cooldown period. During the training sessions, the ergometer power output was set at a level low enough for subjects to ideally work 30 min per bout. However, if fatigue occurred, a modified protocol was used and subjects were asked to perform up to five exercise bouts, each separated by a 5-min pause, for a total of 30 min of exercise. When 30 min of continuous exercise was achieved at 6.1 watts, resistance was increased by an additional ~6.1 watts and subsequent sessions were conducted at the greater power output. After 30 min of continuous pedaling at the higher power output, resistance was increased by 6.1 watts during the subsequent session. Power output was increased in this manner throughout the training period.

Euglycemic clamp procedure
Peripheral insulin sensitivity was evaluated by the euglycemic-hyperinsulinemic clamp technique as described previously (46). Fasting plasma glucose concentration was maintained by a variable glucose infusion. Blood glucose was determined at 5-min intervals throughout the duration of the insulin infusion (2 min at 17.6 nmol x kg-1 x min-1, followed by a continuous infusion of 4.4 nmol x kg-1 x min-1). Whole body glucose utilization was calculated during the last 80-min period of the steady-state insulin infusion.

Muscle biopsy procedure
On a separate occasion, within 1 wk of the euglycemic-hyperinsulinemic clamp procedure, skeletal muscle (250–300 mg) was obtained under local anesthesia (mepivacaine chloride 5 mg/ml; Carbocaine, Södertälje, Sweden) from the vastus lateralis portion of the quadriceps femoris using an open biopsy technique (8). Approximately 8–10 smaller muscle strips (~20 mg) were prepared for in vitro incubations as described previously (8). A second muscle biopsy (80 mg) was obtained and frozen in liquid nitrogen for enzyme analysis and protein expression studies. A third muscle specimen (25 mg) was mounted in embedding medium, frozen in isopentane cooled to its freezing point with liquid nitrogen, and stored at -80°C for histochemical analysis.

Muscle incubation and 3-O-methylglucose transport determinations
Glucose transport was assessed using 3-O-methylglucose by a modification of the method described for rat epitrochlearis muscle (47). Muscle specimens were incubated for 10 min at 35°C in Krebs-Henseleit's bicarbonate buffer (KHB) supplemented with 5 mM HEPES (hydroxyethylpiperazine-N-2-ethanesulfonic acid), 18 mM mannitol, 2 mM pyruvate, and 0.1% bovine serum albumin (BSA). The concentrations of HEPES and BSA were maintained in all incubation media. The gas phase in the incubation flasks was kept at 95% 02/5% CO2. Muscle specimens were incubated with or without 100 µU/ml insulin for 30 min in KHB containing 20 mM mannitol. Thereafter, 3-O-methylglucose transport was assessed after a 20-min exposure to KHB, which contained 5 mM 3-O-[3H]methylglucose (437 µCi/mM) and 15 mM [14C]mannitol (33 µCi/mM). Muscle specimens were processed as described (47). 3-O-methylglucose transport activity is expressed per milliliter of intracellular water per hour.

Enzymatic analysis
Muscle biopsies were homogenized as described (48) and used to directly assess phosphofructokinase (PFK) activity. Aliquots of the remaining sample were stored in at -80°C for subsequent measurement of citrate synthase (CS) and HK activity. PFK was measured spectrophotometrically as described by Opie and Newsholm (49). Homogenates were diluted in assay buffer (50 mM Tris, 6 mM MgCl2, 200 mM KCl, pH 8.2; 1 mM ATP, 2 mM AMP, 0.17 mM NADH, 2.5 µg/ml antimycin, 50 µg/ml aldolase (from rabbit muscle), and 5 µg/ml glyceraldehydrogenase/triosephosphate isomerase (from rabbit muscle). The reaction was started by addition of 3 mM (final concentration) fructose-6-phosphate.

Citrate synthase activity was analyzed in muscle homogenates as described by Alp et al. (50). Homogenates were diluted in assay buffer (50 mM Tris, 0.2 mM 5,5 dithio-bis-2-nitrobenzoic acid, pH 8.1, 1 mM acetyl CoA 0.1, and 0.05% Triton-X-100, final concentrations) and citrate synthase activity was assessed spectrophotometrically. Oxaloacetate (0.35 mM final concentration) was used as substrate.

Hexokinase activity was determined spectrophotometrically, according to Zammit and Newsholme (51). Skeletal muscle (10 mg) was homogenized in ice-cold buffer (1:50 dilution) containing 50 mM triethanolamine, 1 mM EDTA, and 2 mM MgCl2 (pH 7.5). Muscle extracts were subjected to centrifugation (5000 g x 5 min at 4°C) and the supernatant was collected for enzyme analysis. To determine heat-stable HK activity (HKI), one aliquot of the supernatant was heated at 45°C for 45 min in the absence of glucose. Heat treatment results in a loss of 95% of HKII activity and 10% of HKI activity (52). Since activity measured in the heated sample largely represents that of HKI, HKII activity was estimated as the difference between HK and HKI activity. Samples were diluted in buffer containing 75 mM triethanolamine, 7.5 mM MgCl2, 0.8 mM EDTA, 1.5 mM KCl, pH 7.5, 0.4 mM NADP, 2.5 mM ATP, 7.5 mM phosphocreatine, 0.1 mg/ml creatine phosphokinase (lyophilized, from rabbit muscle), and 5 µg/ml glucose 6-phosphate dehydrogenase (from yeast). HK activity was determined in the presence of 2 mM glucose.

Glycogen synthase activity was assayed by a modification of the method presented by Thomas et al. (53), based on the incorporation of [14C]UDPG into glycogen. Skeletal muscle (5–10 mg) was homogenized in ice-cold buffer (1:50) containing 50 mM 3-[N-morpholino]propanesulfonic acid buffer, 25 mM NaF, 20 mM EDTA, and 1 mg/ml BSA. Glycogen synthase activity was measured in the presence of 0.19 mM of UDPG. The active form of glycogen synthase was determined using 0.3 mM glucose-6-phosphate and total activity was assessed with saturating levels of glucose-6-phosphate (6 mM).

Protein expression studies
Muscle biopsies (20 mg) were homogenized at 4°C in buffer consisting of 25 mM HEPES (pH 7.4), 10 mM EDTA, 100 mM NaF, 1% (vol/vol) Triton X-100, and protease inhibitors (1 mM benzamide, 10 µg/ml aprotinin, and 2 mM phenylmethylsufonyl fluoride). Homogenates were subjected to centrifugation (2000 x g for 45 s) and the supernatant was collected for analysis of HKII, PFK, and GS protein expression. GLUT4 protein expression was determined in total membranes (plasma membranes and microsomes) as described (54). Protein was determined in total membranes and homogenates by the Bradford method (55), using a commercially available kit (Bio-Rad Chemical Division, Richmond, Calif.). Aliquots of muscle homogenate (40 µg) or total membranes (25 µg) were solubilized in Laemmli sample buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes as described (56). Protein expression of HKII, PFK, GS, and GLUT4 was assessed by immunoblotting with specific antibodies. The polyclonal antibodies used to detect PFK, GS, and HKII were generous gifts from Dr. Oluf Pedersen (Steno Memorial Hospital, Gentofte, Denmark). Each polyclonal antibody was generated by immunizing rabbits with a synthetic peptide homologous to the last nine amino acids in the COOH terminus the respective protein. (27, 32). GLUT4 protein expression was assessed using a polyclonal antibody raised against the COOH-terminal peptide of GLUT4 (Biogenesis, Poole, U.K.), diluted 1:1000 in phosphate-buffered saline (pH 7.4) containing 1% milk. Proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, lll.) and quantified by densitometry. All samples were processed on the same gel to avoid gel-to-gel variation.

Histochemical analysis
Muscle fiber typing procedures have been previously described (1). Serial transverse sections (10 µm) were cut at -20°C using a microtome. Sections were stained for myofibrillar ATPase activity after preincubation at different pH in acidic (pH 4.3, or 4.6) or alkaline (pH 10.3) buffer. On the basis of the myofibrillar ATPase staining characteristics, an area containing at least 200 fibers was typed as type I (slow-twitch) or type II (fast-twitch), and into subgroups IIa and IIb.

Blood biochemistry
Blood glucose concentration was determined by the glucose dehydrogenase method (57). Plasma insulin level was measured by radioimmunoassay (Insulin RIA 100 kit; Pharmacia, Uppsala, Sweden).

Statistics
Data are presented as mean ±SEM. Student's paired t test was used when median and mean values were similar or when the values appeared to be normally distributed (euglycemic-hyperinsulinemic clamp, 3-O-methylglucose). When multiple variables were compared, a two-way analysis of variance was used to evaluate statistical difference between pre- and posttraining. When analysis of variance resulted in a significant F-value, Newman-Keuls post hoc test was used to identify difference between the means. Nonparametric statistics (Sign test) were used for values not normally distributed (enzyme analysis, protein expression studies, and muscle fiber typing).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluation of ESLC training
During each bout of ESLC, subjects exercised until fatigue. The amount of ESLC training performed each week is presented in Table 2, and is reported in kilojoule (kJ = watt x time (s)/1000). Training level gradually increased in all subjects for the first 6 wk. During the last 2 wk of training, intercurrent urinary tract infection (subjects 1, 3, and 4) and preparation for final testing procedures (all subjects) led to a reduced level of training. The maximal amount of work sustained during one bout of exercise per week increased more than 10-fold during the training period ( Table 3).


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  		Table 2. Total amount of work (in kJ) for each subject during the ESLC training period2tfa


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  		Table 3. The maximal work sustained during one bout of exercise (in kJ) for each tetraplegic subject during ESLC training3tfa

Effects of ESLC training on whole body insulin-mediated glucose uptake
After ESLC training, insulin-mediated glucose disposal was increased by 33±13% (24.9±3.7 vs. 31.9±2.8 µmol x kg-1 x min-1 for pre- vs. posttraining; P<0.05; Fig. 1). Mean steady-state insulin (97±1 vs. 94±1 µU/ml) and glucose (5.8±0.4 vs. 5.9±0.4 mM) levels achieved during the last 80 min of the clamp were similar between pre- and posttraining.



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  		Figure 1. Effect of ESLC training on insulin-mediated whole body uptake. Rate of insulin-mediated whole body glucose utilization was measured during steady-state conditions for 80 min, at a plasma glucose concentration of 5.8 ± 0.4 mmol/l before and 5.9 ± 0.4 mmol/l after the 8-wk ESLC training period (NS). The mean steady-state serum insulin level was 97 ± 1 µU/ml before and 94 ± 1 µU/ml after the training (NS). Glucose uptake is expressed as mmol x kg-1 x min-1. Results are presented as mean ±SEM for five subjects. *P < 0.05 vs. pretraining.

Effects of ESLC training on basal and insulin-stimulated 3-O-methylglucose transport
Insulin (100 µU/ml) induced a 1.8-fold (P<0.05) increase in 3-O-methylglucose transport in incubated skeletal muscle specimens before training (0.81±0.16 vs. 1.42±0.26 µmol x ml-1 x h-1, for basal and insulin-stimulated muscle, respectively; Fig. 2). The pretraining basal and insulin-stimulated glucose transport values are similar to age-matched control subjects (1). After 8 wk of ESLC, basal and insulin-stimulated glucose transport activity were increased by 1.6 and 2.1-fold, respectively, compared to pretraining levels (P<0.05). Insulin induced a 2.3-fold (P<0.01) increase in skeletal muscle 3-O-methylglucose transport posttraining (1.32±0.17 vs. 3.00±0.46 µmol x ml-1 x h-1, basal vs. insulin, respectively).



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  		Figure 2. Effect of ESLC training on skeletal muscle glucose transport capacity. Basal and insulin-stimulated 3-O-methylglucose transport activity was assessed in skeletal muscle obtained from tetraplegic patients before and after 8 wk of ESLC training. Muscle specimens were incubated for 30 min in KHB medium, in the absence (open bar) or presence of 100 µU/ml insulin (filled bar). Thereafter, 3-O-methylglucose transport was assessed. Glucose transport is expressed per ml of intracellular water. Values are presented as mean ±SEM. *P < 0.05 vs. respective pre- or postbasal glucose transport activity.

Effects of ESLC training on protein expression of GLUT4, GS, HKII, and PFK
Protein expression of GLUT4, GS, HKII, and PFK was assessed in total membrane fractions or muscle homogenate by Western blot analysis ( Figs. 3–6). ESLC training led to a profound increase in protein expression of GLUT4 (378±85%; Fig. 3), glycogen synthase (526±146%; Fig. 4), and HKII (204±47; Fig. 5) in vastus lateralis muscle. In four of five subjects, PFK protein expression was increased 1.2- to 5.6-fold after training ( Fig. 6). Nevertheless, posttraining PFK levels were 50% lower in one subject, consequently, the increase in PFK expression observed after ESLC training did not reach statistical significance (282±97%; NS).



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  		Figure 3. Effect of ESLC training on GLUT4 protein expression in vastus lateralis skeletal muscle obtained from tetraplegic subjects. A) Primary experimental results of GLUT4 protein expression in skeletal muscle obtained from tetraplegic subjects before and after 8 wk of ESLC training. Crude membranes were prepared from skeletal muscle biopsies and proteins were separated by SDS-PAGE. GLUT4 was detected by immunoblotting. B) Skeletal muscle GLUT4 protein levels in tetraplegic subjects reported in arbitrary densitometric units. Results are presented as individual observations and as a group mean (open bars).



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  		Figure 4. Effect of ESLC training on glycogen synthase protein expression in vastus lateralis skeletal muscle obtained from tetraplegic subjects. A) Primary experimental results of glycogen synthase protein expression in skeletal muscle obtained from tetraplegic subjects as described in Fig. 3. Muscle homogenate was prepared from skeletal muscle biopsies and proteins were separated by SDS-PAGE. Glycogen synthase was detected by immunoblotting. B) Skeletal muscle glycogen synthase protein levels in tetraplegic subjects reported in arbitrary densitometric units. Results are presented as individual observations and as a group mean (open bars). Individual subjects are represented by the same symbol as in Fig. 3.



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  		Figure 5. Effect of ESLC training on hexokinase II protein expression in vastus lateralis skeletal muscle obtained from tetraplegic subjects. A) Primary experimental results of hexokinase II protein expression in skeletal muscle obtained from tetraplegic subjects as described in Fig. 3. Muscle homogenate was prepared from skeletal muscle biopsies and proteins were separated by SDS-PAGE. Hexokinase II was detected by immunoblotting. B) Skeletal muscle hexokinase II protein levels in tetraplegic subjects reported in arbitrary densitometric units. Results are presented as individual observations and as a group mean (open bars). Individual subjects are represented by the same symbol as presented in Fig. 3.



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  		Figure 6. Effect of ESLC training on phosphofructokinase protein expression in vastus lateralis skeletal muscle obtained from tetraplegic subjects. A) Primary experimental results of phosphofructokinase protein expression in skeletal muscle obtained from tetraplegic subjects as described in Fig. 3. Muscle homogenate was prepared from skeletal muscle biopsies and proteins were separated by SDS-PAGE. Phosphofructokinase was detected by immunoblotting. B) Skeletal muscle phosphofructokinase protein levels in tetraplegic subjects reported in arbitrary densitometric units. Results are presented as individual observations and as a group mean (open bars). Individual subjects are represented by the same symbol as in Fig. 3.

Effects of ESLC training on muscle enzyme profile and glycogen content
The effects of ESLC training on muscle enzyme activities were compared in biopsies obtained from tetraplegic subjects pre-and posttraining. Basal (non-insulin-stimulated) glycogen synthase activity ratio was similar pre- vs. posttraining ( Table 4). HKII activity, as a percentage of total activity, increased 25% after ESLC training (P<0.05). Enzyme activities of PFK and CS were not altered after ESLC training. Pretraining muscle glycogen levels were similar to able-bodied humans (D. Galuska, J. R. Zierath, and H. Wallberg-Henriksson, unpublished observation). ESLC training lead to a 68% increase in muscle glycogen content (80.8±26.7 vs. 135.9±15.4 mmol x g-1 wet weight for pre- vs. posttraining).


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  		Table 4. Effect of electrical stimulation on enzyme activities in skeletal muscle from tetraplegic subjects pre and post 8 weeks of electrical stimulation

Effects of ESLC training on muscle fiber type distribution
Mean muscle fiber-type composition was determined before and after training. We have previously reported that muscle fiber type composition in able-bodied men is approximately 33% type I, 45% type IIa, and 32% type IIb fibers (1). The tetraplegic subjects in this study presented 2±1% type I, 35±7% type IIa, and 64±8% type IIb muscle fibers before training. In three of five subjects, types I muscle fibers were identified. After training, mean fiber type distribution was 5±2% type I, 55±8% type IIa, and 39±5% type IIb muscle fibers. Mean group values of fiber cross-sectional area could not be calculated due to large individual differences between posttraining values. The individual histochemical profiles for skeletal muscle myofibrillar APTase activity are presented for each subject ( Fig. 7). Although mean muscle fiber type composition was not significantly altered by physical training, a small increase in percentage type I muscle fibers and signs of transformation from type IIb to IIa muscle fibers were noted in four of five subjects.



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  		Figure 7. Histochemical staining of skeletal muscle obtained from tetraplegic patient before and after the ESLC training period. Muscles were cut in serial transverse sections (10 µm) with a microtome (-20°C). Staining was performed for myofibrillar ATPase activity to visualize the relative distribution of type I muscle (dark), type IIa (light), or type IIb (intermediate) muscle fibers.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we provide evidence for a molecular mechanism to account for increased insulin action on whole body glucose uptake after physical training. Eight weeks of daily physical training through electrical stimulated bicycle exercise led to a marked increase in the expression of key proteins known to regulate glucose metabolism in skeletal muscle. The training-induced overexpression of GLUT4, HKII, and glycogen synthase was directly coupled to increased skeletal muscle glucose transport and increased glycogen content, as well as enhanced whole body insulin-mediated glucose uptake in tetraplegic subjects. These training-induced adaptations preceded changes in muscle fiber type composition. To our knowledge, this is the first report to directly link overexpression of GLUT4, HKII, and glycogen synthase through exercise to enhanced glucose transport and metabolism in human skeletal muscle.

Several groups have demonstrated that overexpression of GLUT4 in skeletal muscle can prevent impaired whole body glucose homeostasis associated with various states of insulin resistance. For example, modest overexpression (twofold) of GLUT4 in heart, skeletal muscle, and adipose tissue in transgenic mice prevented the impairment of glycemic control and the accompanying hyperglycemia caused by high-fat feeding (36). Furthermore, GLUT4 overexpression in skeletal muscle markedly improved whole body insulin action and reduced blood glucose levels in streptozotocin-induced diabetic (37) and homozygous genetically predisposed diabetic (db/db) (58) mice. In human beings, physical training leads to a 1.6- to 2.6-fold increase in GLUT4 protein expression in skeletal muscle (16, 21, 59), and improves glucose tolerance and insulin action (15, 16, 21, 23, 59). Collectively, these studies underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Since GLUT4 is such an attractive candidate for gene therapy strategies aimed to enhance whole body insulin action, it is important to ascertain whether increased GLUT4 content can be directly coupled to increased glucose transport activity in human skeletal muscle. In the present study, we assessed glucose transport in skeletal muscle using the glucose analog 3-O-methylglucose, which reflects only glucose transport and not glucose phosphorylation. The fourfold increase in skeletal muscle GLUT4 content posttraining was associated with increased basal and insulin-stimulated glucose transport. Thus, our results provide the first direct evidence to couple an exercise training-induced increase in GLUT4 protein content with increased glucose transport activity in human skeletal muscle.

Recently, we have reported that total muscle mass is increased by 2% in this same group of subjects after physical training by electrical stimulation (2). This change corresponds to a 1.5 kg increase in muscle mass and may partly contribute to the increase in whole body insulin-stimulated glucose disposal. More important, however, here we show that the cellular glucose transport capacity was markedly increased per unit muscle mass, reaching values significantly above those of able-bodied subjects (1). Consequently, the direct increase in muscle glucose transport activity is likely to have a greater effect on whole body glucose uptake than the increased muscle mass noted in these subjects after ESLC.

Glycogen synthase and PFK are rate-limiting enzymes for glycogen synthesis and glycolysis, respectively. Exercise training led to a profound increase in glycogen synthase protein expression in skeletal muscle, with no effect noted on the basal (non-insulin-stimulated) GS activity ratio. Nevertheless, muscle glycogen content increased by 70% posttraining. Since insulin-stimulated whole body glucose uptake, skeletal muscle glucose transport, and muscle glycogen content were all increased with training, insulin-mediated glycogen storage was likely to be markedly improved in the tetraplegic subjects. Thus, our results provide evidence that electrical stimulation induced de novo protein synthesis of GS. PFK protein expression was increased in four of five subjects after ESLC training, yet no change was noted in PFK enzyme activity. However, the reduced training hours during the last 2 wk of the study may have affected the PFK enzyme activity levels. Nevertheless, our results suggest that this type of intense exercise training mainly induced de novo protein synthesis of GS and PFK rather than increasing the activity of these enzymes.

The predominant hexokinase isoform in skeletal muscle is hexokinase II, although hexokinase I is also expressed (60). Insulin (28) and muscle contractions (26, 30, 48, 61, 62) regulate hexokinase II expression and activity. ESLC training led to an increase in both hexokinase II protein expression and activity, but the magnitude of this increase was less than that observed for GS and GLUT4. Collectively, our results suggest that in response to muscle contraction, the expression of key regulatory steps in muscle glucose storage are up-regulated in a coordinated manner.

Although large differences in the microstructure of decentralized muscles below the injury level have been reported, a predominance of atrophic type IIb fibers and increased amounts of connective tissue have generally been observed in tetraplegic persons more than 1 year after injury (1, 63–68). Electrical stimulation can transform muscle fibers to a more normal distribution, depending on the stimulation intensity and duration. Recently, Mohr and co-workers (68) assessed muscle fiber type composition in para- and tetraplegic persons after ESLC training (30 min three times a week for 12 months) and noted a large-scale transformation of fibers from type IIb to type IIa, with no transformation to type I fibers. Here we noted a tendency for fiber type transformation from IIb to IIa dominance within 2 months of ESLC-training. Also, a small increase in type I fibers was observed in four of five subjects posttraining. With continued training, a pronounced dominance of type IIa fibers would have been expected. The present group of subjects were subjected to a twofold greater total stimulation load per week compared the group studied by Mohr and co-workers (68). This more intense stimulation pattern might be important for initiating a transition to type I fibers, which was noted in four of the subjects in the present study. Progressive electrical stimulation of tibialis anterior muscle of spinal cord injured subjects for 24 wk (from 15 min/day to 8 h/day), without load resistance, leads to enhanced muscle oxidative capacity, improved endurance properties, and increased type I fibers with no effect on muscle fiber size and strength (69). Since the duration of the ESLC training in the present study was short, our finding that muscle fiber type composition was not notably altered after ESLC training may not be surprising. Previous studies reporting fiber type transformation have followed training protocols of several months in duration (68–70).

In conclusion, daily electrical stimulation of denervated skeletal muscle in tetraplegic subjects for 8 wk normalized whole body insulin sensitivity. The intense electrical stimulatory procedure led to a marked increase in protein expression of key genes involved in the regulation of glucose metabolism, including GLUT4, glycogen synthase, and hexokinase II. Increased protein expression of GLUT4 was accompanied by a twofold increase in basal and insulin-stimulated glucose transport activity in skeletal muscle. Thus, electrical stimulation of paralyzed muscle may be a useful therapeutic tool to normalize glucose homeostasis and improve insulin sensitivity in people with spinal cord injury.


   ACKNOWLEDGMENTS
 
This study was supported by grants from the Novo-Nordisk Foundation, the Swedish National Center for Research in Sports, the Swedish Diabetes Association, Thurings Foundation, Tore Nilson's Foundation, Marcus and Amalia Wallenbergs Foundation, Foundation for Strategic Research, and the Swedish Medical Research Council (9517, 11945, 11823, and 12211).


   FOOTNOTES
 
1 Correspondence: Department of Clinical Physiology, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: hwh{at}klinfys.ks.se

2 Abbreviations: GLUT4, the insulin-responsive glucose transporter; HK, hexokinase; GS, glycogen synthase; ESLC, electrically stimulated leg cycling; KHB, Krebs-Henseleit buffer; HEPES, hydroxyethylpiperazine-N-2-ethanesulfonic acid; BSA, bovine serum albumin; PFK, phosphofructokinase; ATP, adenosine triphosphate; AMP, adenosine monophosphate; NADH, nicotinamide adenine dinucleotide (reduced); CS, citrate synthase; EDTA, ethylenediaminetetraacetic acid; UDPG, uridinediphosphoglucose.

Received for publication April 21, 1998. Revision received July 29, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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