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(The FASEB Journal. 1999;13:2246-2256.)
© 1999 FASEB

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice

JEFFREY W. RYDER*, YUICHI KAWANO*, DANA GALUSKA*, ROGER FAHLMAN*, HARRIET WALLBERG-HENRIKSSON*, MAUREEN J. CHARRON{dagger} and JULEEN R. ZIERATH*1

* Department of Clinical Physiology, Karolinska Hospital, S-171 76, Department of Physiology and Pharmacology, Karolinska Institute, S-171 77, Stockholm, Sweden; and the
{dagger} Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461-1602, USA

1Correspondence: Gustaf V’s Research Institute, Department of Clinical Physiology, Karolinska Hospital, S-171 76, Stockholm, Sweden. E-mail: jrz{at}klinfys.ks.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4-deficient and wild-type mice were studied after a 3 h swim exercise. In wild-type mice, insulin and swimming each increased 2-deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2-deoxyglucose glucose uptake in muscle from GLUT4-null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4-null mice, with no effect noted in fasted GLUT4-null mice. This exercise-associated 2-deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4-null muscle was increased 1.6-fold over basal levels after electrical stimulation. Contraction-induced glucose transport activity was fourfold greater in wild-type vs. GLUT4-null muscle. Glycogen content in gastrocnemius muscle was similar between wild-type and GLUT4-null mice and was reduced ~50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild-type, with no change in GLUT4-null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4-null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise-induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild-type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4-null mice were totally restored after 24 h carbohydrate refeeding.—Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg-Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.


Key Words: metabolism • glucose transport • glycogen synthase • electrical stimulation • physical exercise


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SKELETAL MUSCLE IS the major tissue responsible for glucose uptake and metabolism in the postprandial state (1) . Glucose transport is generally thought to be a rate-limiting step for glucose uptake and metabolism in insulin-sensitive tissue such as skeletal muscle (2 3 4 5 6) . In people with type 2 diabetes mellitus, reduced glucose transport in skeletal muscle appears to be a major factor responsible for reduced whole body glucose uptake (7 8 9) . Thus, intense interest is focused on understanding the molecular mechanism(s) that regulates glucose transport in an effort to develop strategies to improve glucose homeostasis in type 2 diabetic patients.

Insulin and muscle contractions are potent stimulators of the glucose transport process in skeletal muscle (2 , 10 11 12 13 14 15 16 17 18) . Insulin elicits an increase in glucose transport via a signaling mechanism involving signal transduction through the insulin receptor substrate (IRS)/phosphatidylinositol (PI) 3-kinase pathway (18 , 19) . Muscle contraction directly increases glucose transport and metabolism by an insulin-independent mechanism (14 15 16 17) ; however, the intracellular signaling mechanisms involved in this pathway are poorly defined. Insulin (20 , 21) and prior muscle contraction, through exercise (22) or electrical stimulation (23) , promote glucose uptake by a mechanism involving translocation of GLUT4, the predominant glucose transporter isoform expressed in skeletal muscle (24 , 25) , from an intracellular compartment to the plasma membrane and transverse tubules. We have used mice generated with the murine GLUT4 gene disrupted (GLUT4-null) (26) to provide direct evidence that GLUT4 is essential for insulin- (27, 28) and hypoxia- (29) stimulated glucose transport in extensor digitorum longus (EDL) muscle. GLUT1 is also expressed in skeletal muscle (30 , 31) , although it is generally believed to play a negligible role in glucose transport and does not appear to be involved in insulin- or exercise-mediated glucose transport in skeletal muscle (22) . Whether GLUT4 is essential for exercise-mediated glucose uptake and metabolism in skeletal muscle is not known at this time.

At rest or after exercise, once glucose is transported across the plasma membrane, its primary fate is conversion to glycogen through the glycogen synthesis pathway (32 , 33) . In addition to stimulation of glucose transport, insulin and prior muscle contraction lead to the stimulation of glycogen synthase, a key regulatory enzyme responsible for glycogen synthesis (2 , 12) . During exercise or muscle contraction induced by electrical stimulation, skeletal muscle primarily uses intramuscular glycogen stores to meet the energy demands of the working muscle (2 , 12 , 33 34 35) . Upon cessation of exercise, glucose transport and glycogen synthase activity are increased (2 , 10 11 12 13 14 15 16 17 , 34 , 35) and, provided adequate carbohydrate supplies are available, muscle glycogen stores are elevated to levels that exceed the fed sedentary state (33 , 34) . Glucose transport has been implicated to be rate-limiting for glycogen synthesis in skeletal muscle (6) , but it has been a challenge to dissect the effects of exercise on glucose transport from those on glucose metabolism, as these processes are closely linked. Thus, we have used GLUT4-null mice to determine whether GLUT4 is essential for postexercise glucose uptake and glycogen resynthesis in skeletal muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
GLUT4-null mice were generated and genotyped as described previously by Katz et al. (26) . Male GLUT4-null mice and C57Bl6/CBA F1 hybrids (wild-type) were housed in the animal facility at the Karolinska Hospital and given a standard rodent chow and water ad libitum. The local Animal Ethical Committee approved all protocols.

Exercise protocol
Overnight fasted (16 h) or fed wild-type or GLUT4-null mice (10 to 14 wk old) were assigned to sedentary or exercised groups. The swimming protocol was a modification of the procedure used extensively in earlier studies with rats (14 , 34 , 35) . Six mice swam together in plastic barrels measuring 45 cm in diameter and filled to a depth of ~40 cm. Water temperature was maintained at 34–35°C. Mice swam for six 30 min intervals separated by 5 min rest periods. After the last swim interval, mice were dried and put back in their cages for 5 or 24 h or were studied immediately. At the onset of the 5 or 24 h recovery periods, mice received an intraperitoneal (i.p.) glucose injection (0.5 mg/g body weight) and subsequently were given free access to chow and water.

Muscle preparation
Mice were anesthetized via i.p. injection of 2.5% Avertin (0.02 ml/g body weight). EDL muscles were removed for in vitro incubation. All incubation media were prepared from a pregassed (95% O2/5% CO2) stock of Krebs Henseleit buffer (KHB) supplemented with 5 mM HEPES and 0.1% bovine serum albumin (BSA) (radioimmunoassay grade). EDL muscles were incubated in a shaking water bath (30°C) for 15 min in 1 ml KHB supplemented with 20 mM mannitol with or without a further addition of 12 nM insulin. The gas phase in the vial was maintained at 95% O2/5% CO2 for the duration of the study.

Assessment of glucose transport
Glucose transport was assessed using the glucose analog 2-deoxyglucose as described by Hansen and co-workers (36) . Muscles were transferred to vials containing 1 mM [3H]-2-deoxyglucose (2.5 µCi/ml) and 19 mM [14C]-mannitol (0.7 µCi/ml) and incubated in the absence or presence of 12 nM insulin for 20 min. Under these conditions, 2-deoxyglucose uptake directly reflects glucose transport, not metabolism, in mouse skeletal muscle. After incubation, muscles were digested in 0.5 M NaOH. Sample aliquots were used for protein determination using a commercially available kit (Pierce, Inc., Rockford, Ill.) based on the Bradford method (37) . Extracellular space and intracellular 2-deoxyglucose concentration were determined as described previously (38) . Glucose transport activity is expressed as nmol 2-deoxyglucose x mg protein-1 x 20 min-1.

In vitro muscle contraction procedure
EDL muscles were obtained from fed wild-type or GLUT4-null mice and incubated in KHB containing 5 mM glucose and 15 mM mannitol (recovery media) for 60 min. Muscles from individual mice were randomly assigned to contraction or basal (unstimulated) conditions. Muscles were mounted on a jeweler’s chain connected to a Research Grade Isometric Force Transducer (Harvard Apparatus, Inc., South Natick, Mass.). The muscle was positioned between two stainless steel electrodes and resting tension was adjusted to 0.25 g. The isometric tension development during contractions was recorded using a compact 2-Channel Student Oscillograph (Harvard Apparatus, Inc., South Natick, Mass.). The muscle was immersed in 2 ml recovery media (30°C) and isometric muscle contraction was achieved via electrical stimulation using a modification of a previously described protocol (39) . Muscles were stimulated with 200 ms impulse trains, at a frequency of 100 Hz (0.2 ms pulse duration, 10 V amplitude), delivered at a rate of 1 contraction per 2 s for 10 min. Pulses were generated by a Tektronix TM 503 Power Module (Beaverton, Oreg.) and amplified on a 4-Channel Power Amplifier (Somedic, Inc., Sollentuna, Sweden). Muscles were rinsed for 10 min in KHB containing 20 mM mannitol, and 2-deoxyglucose uptake was determined as described above.

Photolabeling of cell surface GLUT1
EDL muscles from sedentary (fed or fasted) or exercised (fed) GLUT4-null mice were incubated for 15 min in KHB, in the absence of insulin, as described above. Muscles were incubated for 8 min at 18°C in KHB containing 1 mCi/ml ATB[2-3H]BMPA (2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(d-mannose-4-yloxy)-2-propylamine) and subsequently irradiated with ultraviolet light for 2 x 3 min, as described (40) . Thereafter muscles were blotted, trimmed free of connective tissue, and frozen in liquid nitrogen. Muscles were weighed and processed to determine cell surface GLUT1 content (40 , 41) . GLUT1 was immunoprecipitated from solubilized crude membrane preparations using anti-carboxyl-terminal peptide GLUT1 anti-serum (a generous gift from Dr. Geoff Holman, University of Bath, Bath, U.K.). Level of radioactivity specifically associated with GLUT1 was determined as described (41) . Results are expressed as cpm x 100 mg wet weight muscle-1.

Blood glucose measurements
Blood glucose was sampled via the tail vein from fed and fasted sedentary wild-type or GLUT4-null mice and analyzed using a One Touch glucose monitor (Lifescan, Inc., Milpitas, Calif.). Blood glucose levels were measured immediately after the final swimming bout and 0.5, 1, 2, 5, and 24 h after a postexercise glucose injection (0.5 mg/g body weight).

Biochemical assays
Gastrocnemius muscle and liver were removed from anesthetized mice as described above and immediately frozen in liquid nitrogen. Glycogen content was measured fluorometrically on HCl extracts of muscle or liver as described (42) . Results are expressed as µmol glucose x g wet weight-1. Glycogen synthase activity was assessed in gastrocnemius muscle as described (43) . Gastrocnemius muscle was homogenized (1:50 dilution) in 50 mM MOPS, 25 mM NaFl, 20 mM EDTA, and 0.1% BSA. Glycogen synthase activity was measured in the presence of 0.17 mM UDP-glucose and either 0.2 mM (active form) or 4 mM (total activity) glucose-6-phosphate. Glycogen synthase activity is reported as fractional activity of the enzyme (active/total), with total activity expressed as µmol x g wet weight-1 x min-1.

Glycogen synthase protein expression
Gastrocnemius muscle from fed mice was homogenized in 20 mM Tris (pH 8.0), 135 mM NaCl, 2.7 mM KCl, 1 mM MgCl, 0.5 mM Na3Vo4, 1% Triton X 100, 10% glycerol, 1 µg/ml leupeptin, 0.2 mM phenylmethanesulfonyl fluoride, and 10 mM NaF. Homogenates were centrifuged at 12,000 x g at 4°C for 10 min. Aliquots (50 µg) were solubilized in Laemmli buffer (44) , separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Glycogen synthase protein content was determined by immunoblot analysis (45) using a polyclonal antibody directed against the COOH terminus of glycogen synthase (generous gift from Dr. Oluf Pedersen, Steno Memorial Hospital, Gentofte, Denmark). Proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, Ill.) and quantified by densitometry. Results are presented as arbitrary units.

Statistics
Data are presented as mean ± SE. Analysis of differences between two treatments within a genotype was performed using a paired Student’s t test. An unpaired Student’s t test was used to assess differences between wild-type and GLUT4-null mice. All other differences were determined using a one-way analysis of variance with Fischer’s LSD post hoc analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Basal and insulin-stimulated glucose transport activity
2-Deoxyglucose glucose uptake was assessed in EDL muscle isolated from fasted (Fig. 1A ) or fed (Fig. 1B ) wild-type or GLUT4-null mice. Basal and insulin-stimulated 2-deoxyglucose glucose uptake is severely blunted in EDL muscle from GLUT4-null mice vs. wild-type mice (27 , 28) . Basal 2-deoxyglucose glucose uptake was 82% and 42% lower in fasted and fed GLUT4-null mice, respectively, compared to wild-type mice. Although insulin (12 nM) induced a twofold increase in 2-deoxyglucose uptake in muscle from both fasted and fed wild-type mice, no effect was noted in GLUT4-null muscle.



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  		Figure 1. Effects of insulin, exercise, and refeeding on glucose transport activity in EDL muscle from wild-type (open bars) or GLUT4-null (filled bars) mice. 2-Deoxyglucose uptake was assessed under basal or insulin-stimulated (12 nM) conditions in EDL muscle in from sedentary (Sed), exercised (Swim), or 5 h carbohydrate refed exercised (Rec) mice. Sedentary and exercised mice were studied under fasted (A) or fed (B) conditions. Results are expressed as nmol x mg protein-1 x 20 min-1. Values are mean ± SE for n = 5–10 mice per group. *P < 0.01 and **P < 0.0005 vs. basal (non-insulin-stimulated) muscle from wild-type or GLUT4-null sedentary mice.

Exercise-induced glucose transport
To determine the role of GLUT4 in exercise-induced glucose transport, we assessed 2-deoxyglucose transport activity in EDL muscle obtained from fasted or fed wild-type or GLUT4-null mice immediately after 3 h (6x30 min intervals) of swimming. Acute swimming led to a twofold increase in 2-deoxyglucose uptake in fasted (Fig. 1A ) and fed (Fig. 1B ) wild-type mice, respectively. The exercise-induced increase in 2-deoxyglucose uptake activity was similar to that observed in muscle incubated with a maximally effective concentration of insulin (12 nM). Insulin and prior exercise have an additive effect on glucose transport in skeletal muscle (14 , 34 , 46) . In wild-type mice, insulin and swimming elicited a 20% greater rate of glucose transport than the absolute effect of either stimulus alone. In fasted GLUT4-null mice (Fig. 1A ), exercise did not lead to a significant increase in 2-deoxyglucose uptake. However, the combined treatment of exercise and insulin resulted in a 2.1-fold increase (P<0.01) in glucose transport compared to basal nonexercised conditions (Fig. 1A ). In fed GLUT4-null mice, exercise led to a twofold increase in glucose transport in EDL muscle, with no further increase under insulin-stimulated conditions.

Effects of carbohydrate refeeding on glucose transport
Glucose transport activity was measured in EDL muscle from wild-type and GLUT4-null mice that had been fed carbohydrates for 5 h after an acute swimming bout (Fig. 1) . In wild-type mice, the acute effect of exercise on 2-deoxyglucose uptake was reversed after 5 h of carbohydrate refeeding. The partial additive effect of insulin and exercise on 2-deoxyglucose uptake in EDL muscle observed in fasted wild-type mice subjected to exercise was reversed after 5 h carbohydrate refeeding. In contrast, in fed mice subjected to exercise, insulin responsiveness remained enhanced in EDL muscle after 5 h carbohydrate refeeding. In fasted GLUT4-null mice, carbohydrate feeding for 5 h after acute swimming was associated with a transient increase in 2-deoxyglucose uptake in EDL muscle, which was threefold greater than pre-exercised levels. Insulin did not increase 2-deoxyglucose uptake in EDL muscle from GLUT4-null mice fed carbohydrate for 5 h after exercise. In fed GLUT4-null mice, the exercise-associated increase in 2-deoxyglucose uptake was completely reversed after carbohydrate refeeding for 5 h postexercise. The reason for the discrepancy between the response of 2-deoxyglucose uptake to exercise in fasted vs. fed GLUT4-null mice is not clear. Relative to wild-type mice, exercise-induced glucose transport activity in fed GLUT4-null mice was markedly lower and reached a level comparable to basal 2-deoxyglucose uptake activity in wild-type mice.

Tension development during muscle contraction
We assessed tension development and force reduction during in vitro contraction of isolated EDL muscle from fed wild-type and GLUT4-null mice. The peak tension in the muscle during the initial phase of the electrical stimulation protocol was 3.8 ± 1.2 and 3.9 ± 0.3 g for wild-type and GLUT4-null muscle, respectively (not significant). The time for 50% reduction in peak tension was 53 ± 12 and 52 ± 10 s for wild-type and GLUT4-null muscle, respectively (N.S.). Furthermore, the overall pattern of tension development throughout the electrical stimulation procedure was similar between wild-type and GLUT4-null muscle.

Contraction-induced glucose transport activity
The effect of in vitro muscle contraction on 2-deoxyglucose uptake activity was determined in isolated EDL muscle from fed wild-type and GLUT4-null mice (Fig. 2 ). Isometric muscle contraction was achieved by electrical stimulation. Muscle contraction led to a 4.1-fold increase (P<0.01) in 2-deoxyglucose uptake in EDL muscle from fed wild-type mice. In GLUT4-null mice, muscle contraction led to a 1.6-fold increase (P<0.01) in 2-deoxyglucose uptake. Nevertheless, the contraction-induced 2-deoxyglucose uptake in GLUT4-null muscle was not significantly greater than the rate of basal (unstimulated) glucose transport in wild-type muscle. Furthermore, contraction-induced 2-deoxyglucose uptake was 4.1-fold greater in wild-type vs. GLUT4-null muscle (P<0.05).



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  		Figure 2. Effect of in vitro muscle contraction on 2-deoxyglucose uptake in EDL muscle from fed wild-type or GLUT4-null mice. EDL muscles were isolated and incubated for 60 min in recovery media as described in Materials and Methods. Paired muscles were randomly assigned to a basal (unstimulated; open bar) or stimulated (electrically stimulated muscle contraction; filled bar) group. Muscles were stimulated to contract (see Materials and Methods). Results are expressed as nmol x mg protein-1 x 20 min-1. Values are mean ± SE for n = 4 paired observations for wild-type mice and n = 8 paired observations for GLUT4-null mice. *P < 0.01 vs. basal and {dagger}P < 0.05 vs. wild-type mice under identical conditions.

Cell surface GLUT1 content
Since 2-deoxyglucose uptake was increased in GLUT4-null muscle after acute swimming, it seemed possible that the exercise effect on glucose transport was mediated by an increase in cell surface GLUT1 content. GLUT1 protein expression in EDL is similar between wild-type and GLUT4-null mice (27) . To test whether exercise-induced glucose uptake in GLUT4-null muscle was mediated by GLUT1, we used the bis-mannose derivative ATB-[2-3H]BMPA to label GLUT1 at the cell surface in EDL muscle from sedentary vs. exercised GLUT4-null mice. Cell surface GLUT1 labeling was similar in EDL muscle from sedentary and exercised GLUT4-null mice (Fig. 3 ). Thus, the increase in 2-deoxyglucose uptake observed in EDL muscle from fed GLUT4-null mice between basal and exercise-stimulated conditions cannot be accounted for by increased cell surface GLUT1 content.



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  		Figure 3. Cell surface GLUT1 content in EDL muscle from sedentary or exercised GLUT4-null mice. Cell surface GLUT1 was determined using the bis-mannose exofacial photolabeling compound ATB-BMPA as described in Materials and Methods. A) Representative gel profile of cell surface GLUT1 content in EDL muscle from sedentary (open circles) or exercised (filled circles) GLUT4-null mice. Arrow indicates molecular mass marker at 51.2 kDa. B) Cell surface GLUT1 content in sedentary (Sed) or exercised (Swim) mice is reported as mean ± SE for n = 4 muscle preparations for each group. Three muscles were pooled for each preparation. Values are cpm x mg wet weight-1.

Effects of fasting and feeding on cell surface GLUT1 content
The contribution of GLUT1 to glucose transport under basal or insulin-stimulated conditions has generally thought to be minimal (22 , 30 , 31 , 40 , 47) . Since basal 2-deoxyglucose uptake was increased in EDL muscle from GLUT4-null mice between fasting and 5 h carbohydrate refed conditions (0.49±0.04 vs. 1.51±0.09 nmolxmg protein-1x20 min-1 for fasted vs. 5 h carbohydrate feeding; P<0.001), we considered the possibility that GLUT1 mediated this effect. Cell surface GLUT1 labeling of EDL muscle obtained from fed GLUT4-null mice was not increased (Fig. 4 ) despite a 3.1-fold increase in basal glucose transport activity. Thus, glucose transport activity in EDL muscle from fed GLUT4-null mice does not appear to be mediated by an increase in cell surface GLUT1 content.



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  		Figure 4. Cell surface GLUT1 in EDL muscle from fed or fasted GLUT4-null mice. Cell surface GLUT1 was determined using the bis-mannose exofacial photolabeling compound ATB-BMPA as described in Materials and Methods. A) Representative gel profile of cell surface GLUT1 content in EDL muscle from fed (open circles) or fasted (filled circles) GLUT4-null mice. Arrow indicates molecular mass marker at 49.5 kDa. B) Cell surface GLUT1 content in fed or fasted mice is reported as mean ± SE for n = 3 muscle preparations for each group. Each preparation contained three muscles. Cell surface GLUT1 content is reported as described in Fig. 3 .

Blood glucose concentration
Blood glucose was determined in fed and fasted sedentary mice, as well as in exercised mice, prior to and after a postexercise glucose injection. Blood glucose levels were similar between fed sedentary wild-type and GLUT4-null mice (Table 1 ). After an overnight fast, blood glucose levels were reduced in wild-type and GLUT4-null mice compared to the respective fed level. A greater reduction in blood glucose level was noted in GLUT4-null mice with fasting. These results are consistent with previous observations in GLUT4-null mice (26) . In contrast, blood glucose levels were lower in wild-type mice compared to GLUT4-null after exercise (3.3±0.3 vs. 4.9±0.1 mM, P>0.01). Blood glucose levels were measured in exercised GLUT4-null and wild-type mice after an i.p. glucose injection and free access to standard rodent chow and water. Similar blood glucose levels were observed in wild-type and GLUT-null mice at all time points between 0.5 and 1 h into the carbohydrate refeeding period. Two hours after carbohydrate refeeding, however, blood glucose levels were significantly higher in GLUT4-null mice (10.5±0.7 vs. 16.9±1.3 mM for wild-type and GLUT4-null mice, respectively; P<0.01). After 5 h carbohydrate refeeding postexercise, blood glucose levels were similar between genotypes and restored to the respective fed levels.


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  		Table 1. Blood glucose levels in Wild-type and GLUT4-null micea

Muscle glycogen content
Glycogen content was determined in gastrocnemius skeletal muscle from wild-type and GLUT4-null mice under fed or fasting conditions or after 3 h of swimming. Fed muscle glycogen levels were comparable between wild-type and GLUT4-null mice (Fig. 5A ). Fasting led to a ~30% decrease in muscle glycogen content in wild-type and GLUT4-null mice. Fasting levels of muscle glycogen were similar between wild-type and GLUT4-null mice. To determine whether GLUT4 is essential for muscle glycogen resynthesis after exercise, glycogen content was assessed in gastrocnemius skeletal muscle obtained immediately after 3 h of swimming or after 5 or 24 h of carbohydrate refeeding (recovery). Exercise led to a comparable ~50% decrease in muscle glycogen content in wild-type and GLUT4-null mice, with similar postexercise levels noted between genotypes. In wild-type mice, carbohydrate refeeding for 5 h after exercise led to a 60% increase in muscle glycogen content above fed levels (21.2±1.0 vs. 34.2±3.2 µmolxg wet wt-1 for fed vs. 5 h refeeding, respectively). Muscle glycogen content was not further increased in wild-type mice after 24 h of carbohydrate refeeding. In contrast, no glycogen resynthesis occurred in GLUT4-null mice after 5 h of carbohydrate refeeding (6.7±1.1 vs. 6.2±1.0 µmolxg wet wt-1 for swim vs. 5 h refeeding, respectively). After 24 h of carbohydrate refeeding, glycogen content in GLUT4-null mice was restored to fed levels.



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  		Figure 5. Muscle and liver glycogen content in wild-type (open bars) and GLUT4-null (filled bars) mice. Gastrocnemius muscle (A) and liver (B) were obtained from fed or fasted sedentary mice, from fasted exercised (swimming) mice, or from fasted exercised mice after 5 or 24 h of carbohydrate feeding. Results are expressed as µmol x g wet weight-1. Values are mean ± SE for n = 6–9 mice per group. *P < 0.05 and **P < 0.0005 vs. fed sedentary for each genotype. {dagger}P < 0.05 and {ddagger}P < 0.005 vs. wild-type under identical conditions.

Liver glycogen content
Liver glycogen content was determined in wild-type or GLUT4-null mice under fed or fasted conditions or after 3 h of swimming. The fed liver glycogen level was 2.5-fold greater in GLUT4-null vs. wild-type mice (Fig. 5B ). Fasting led to a dramatic reduction of liver glycogen content in both genotypes—to a level 5–15% of the respective fed value. Exercise led to a further depletion of fasting liver glycogen content in wild-type but not in GLUT4-null mice. Carbohydrate feeding for 5 h after exercise resulted in a profound increase in liver glycogen content irrespective of genotype. In wild-type mice, carbohydrate feeding after exercise led to a threefold increase in liver glycogen content compared to fed levels. In GLUT4-null mice, liver glycogen was restored to fed levels after 5 h of carbohydrate feeding. The level of liver glycogen after 5 h carbohydrate feeding was comparable between genotypes. A further increase in liver glycogen content was observed in GLUT4-null mice 24 h after exercise. In contrast, no further glycogen accumulation occurred in liver of wild-type mice 24 h after exercise.

Glycogen synthase activity and protein expression
Glycogen synthase (fractional) activity in gastrocnemius muscle from wild-type mice was not significantly altered by swimming or postexercise carbohydrate feeding (Table 2 ). However, fractional activity of glycogen synthase was greater in skeletal muscle from GLUT4-null vs. wild-type mice after 5 and 24 h recovery. Total glycogen synthase activity was similar between GLUT4-null and wild-type muscle (1.17± 0.09 and 1.11±0.06 µmolxg wet weight-1xmin-1 for GLUT4-null vs. wild-type mice, respectively), with no difference between treatment groups. Immunoblot analysis revealed that glycogen synthase protein expression is unaltered by GLUT4 ablation in gastrocnemius muscle (Fig. 6 ; 88±6 vs. 100±14 arbi-trary units for GLUT4-null vs. wild-type mice, respectively). These results are consistent with a previous report indicating that glycogen synthase protein expression reflects total glycogen synthase activity (48) .


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  		Table 2. Glycogen synthase activity in gastrocnemius musclea



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  		Figure 6. Western blot analysis of glycogen synthase protein expression in gastrocnemius muscle from fed sedentary wild-type (W) or GLUT4-null (N) mice. A) Representative autoradiograph of GS protein expression. B) Glycogen synthase protein expression reported as mean ± SE for n = 8 mice per group. Values are expressed as arbitrary units. Results are expressed as percent of wild-type values.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
GLUT4 is the predominant glucose transporter expressed in skeletal muscle (24 , 25) and is a major regulator of glucose transport in response to stimuli such as insulin and prior muscle contraction or exercise (16 , 20 21 22 23) . Here we provide new evidence that 2-deoxyglucose uptake is increased in glycolytic skeletal muscle from GLUT4-null mice after acute (3 h) exercise. Thus, a component of the exercise-induced increase in glucose uptake in skeletal muscle appears to be mediated via a GLUT4-independent mechanism. The greatest effect of exercise on glucose transport in GLUT4-null skeletal muscle was observed when animals were studied in the fed state. 2-deoxyglucose uptake activity was also increased in isolated EDL muscle after in vitro electrical stimulation; thus, local rather than systemic factors are likely to mediate the exercise-induced increase in glucose uptake in GLUT4-null and wild-type mice. The transport mechanism by which exercise stimulates glucose uptake in GLUT4-null muscle remains unclear, but the effect is not mediated by an increase in cell surface GLUT1. Nevertheless, we show that contraction-induced 2-deoxyglucose uptake was fourfold greater in wild-type vs. GLUT4-null muscle. Thus, we provide direct evidence that GLUT4 clearly is the primary mediator of exercise-induced glucose uptake in skeletal muscle.

We previously determined the consequence of genetic ablation of GLUT4 on hypoxia-stimulated glucose uptake (29) . Hypoxia is thought to activate glucose transport by mimicking the effects of muscle contraction on calcium release from the sarcoplasmic reticulum (49) . Incubation of wild-type EDL muscle under hypoxic conditions increased 2-deoxyglucose uptake by twofold, whereas GLUT4 ablation prevented the increase in glucose uptake after 45 min in vitro exposure to hypoxia (29) . Transgenic complementation of GLUT4 in EDL muscle was sufficient to restore the hypoxia-mediated glucose uptake (29) . Our initial report provided evidence that GLUT4 is essential for hypoxia-induced glucose uptake in skeletal muscle. In the present study, exercise and in vitro muscle contraction led to a small but significant increase in 2-deoxyglucose uptake in EDL muscle from fed, but not fasted, GLUT4-null mice. Our results in GLUT4-null mice were unexpected, since hypoxia and muscle contractions are believed to share a common pathway(s) in the activation of glucose transport. One possible explanation for the differences between hypoxia and muscle contraction on glucose uptake in GLUT4-null mice may be related to the degree of metabolic or cellular stress exerted on the muscle. Longer exposure to hypoxic media may invoke a glucose uptake response in GLUT4-null muscle via a mechanism that would be dependent on a certain threshold decrease in glycogen content. Increased cell surface GLUT1 does not account for the contraction-induced increase in the GLUT4-null muscle. Thus, we provide new evidence that a component of the contraction-induced glucose uptake cannot be attributed to GLUT4 or to increased cell surface GLUT1 levels. This effect may be masked when normal levels of GLUT4 are present.

Studies using transgenic mice have demonstrated that glucose transport plays a vital role in glycogen deposition in skeletal muscle. Transgenic overexpression of GLUT1 in skeletal muscle leads to a 7-fold increase in basal glucose uptake and a 10-fold increase in glycogen content, despite lower levels of glycogen synthase activity, compared to wild-type mice (6) . Transgenic overexpression of GLUT4 in skeletal muscle also leads to increased insulin-stimulated glucose transport and elevated glycogen content in skeletal muscle (50 51 52) . These studies have linked increased glucose flux to the level of glycogen accumulation. Physical training through exercise is one physiological means to achieve increased GLUT4 levels in skeletal muscle (53 , 54) . The training-induced increase in GLUT4 expression may be linked to enhanced postexercise muscle glycogen resynthesis in trained vs. untrained muscle in response to acute exercise (53 , 54) . Therefore, we tested the hypothesis that decreased glucose transport due to GLUT4 ablation would result in a decreased rate of glycogen accumulation in glycolytic skeletal muscle after acute exercise. Whereas muscle glycogen levels in wild-type mice were completely restored and even further elevated compared to fed levels 5 h after exercise, no significant glycogen accumulation was observed in skeletal muscle from GLUT4-null mice. Thus, GLUT4 is necessary for immediate glycogen resynthesis after acute exercise. Furthermore, GLUT4 ablation prevented an increase in muscle glycogen accumulation above the resting carbohydrate fed level. This phenomenon is referred to as glycogen supercompensation and is commonly observed after an exercise bout that substantially depletes muscle glycogen stores (33) . GLUT4 appears to be necessary to achieve glycogen supercompensation. Glycogen stores in gastrocnemius muscle from GLUT4-null mice were completely restored after 24 h carbohydrate refeeding postexercise, providing evidence that GLUT4 is not essential for glycogen resynthesis. Thus, although GLUT4 facilitates immediate postexercise glycogen resynthesis, it is not required for long-term glycogen resynthesis postexercise.

We examined whether decreased glycogen synthase protein expression or enzyme activity could account for the reduced rate of glycogen accumulation in GLUT4-null muscle. Neither glycogen synthase activity nor protein expression were reduced in gastrocnemius muscle from GLUT4-null mice; therefore, the reduced muscle glycogen content in GLUT4-null mice at 5 and 24 h postexercise was a consequence of reduced GLUT4-mediated glucose transport activity rather than a diminished capacity for glycogen synthesis. Because we used GLUT4-deficient mice, it is possible to separate the effects of exercise on glucose flux from glucose metabolism. Here we show that glycogen synthase activation after exercise occurs even when the absolute rate of glucose transport is not increased dramatically.

We measured blood glucose levels to confirm that the reduced muscle glycogen accumulation after exercise in GLUT4-null mice was not due to inadequate carbohydrate supply. Immediately after exercise, blood glucose levels were slightly lower in wild-type mice compared to GLUT4-null mice. Animals were given a glucose injection and free access to rodent chow and water after the first blood glucose value was obtained. In wild-type and GLUT4-null mice, blood glucose levels increased between 30 min and 1 h after exercise. In wild-type mice, the peak glucose level was observed 1 h into the refeeding period. In contrast, blood glucose values in GLUT4-null mice were further elevated to 16.9 mM 2 h after exercise, presumably due the diminished capacity of skeletal muscle for glucose uptake as a consequence of GLUT4 ablation. With 5 h refeeding, blood glucose levels were not different from the pre-exercised levels. Furthermore, 5 h after exercise, liver glycogen content was dramatically increased in wild-type and GLUT4-null mice, demonstrating that the reduced glycogen resynthesis in GLUT4-null muscle cannot be attributed to a diminished carbohydrate supply. Liver glycogen content was 2.3-fold greater in sedentary fed GLUT4-null vs. wild-type mice, and further increased 36% in GLUT4-null vs. wild-type mice 24 h after exercise. Glucose transport in liver is dependent on GLUT2 (55 , 56) rather than GLUT4. Since liver GLUT2 expression is ~twofold greater in GLUT4-null vs. wild-type mice (26) , the increased liver glycogen content is in accordance with previous studies showing increased glycogen deposition in skeletal muscle overexpressing glucose transporters (6 , 50 51 52) . Potentially, enhanced glycogen storage by the liver, presumably due to the increased GLUT2 expression, may be one major compensatory system to maintain normal glucose tolerance and euglycemia in GLUT4-null mice.

A striking observation made in the present study was the apparent influence of fasting and feeding on basal glucose transport activity in EDL muscle from GLUT4-null mice. Surprisingly, basal 2-deoxyglucose uptake was markedly increased in EDL muscle from fed vs. fasted GLUT4-null mice, whereas no difference was noted in wild-type mice. The elevated basal glucose uptake in EDL muscle from fed GLUT4-null mice was not accompanied by increased cell surface GLUT1 content. Furthermore, the reduced basal glucose uptake in EDL muscle from fasted, acutely exercised GLUT4-null mice was nearly normalized after 5 h carbohydrate refeeding. Nevertheless, the molecular mechanism(s) by which fasting and refeeding modulate glucose transport in skeletal muscle from GLUT4-null mice remain unknown. However, these observations highlight the potential influence of the nutritional status on glucose transport activity in skeletal muscle.

In conclusion, GLUT4 is the primary mediator of postexercise glucose uptake, although it appears that exercise and in vitro muscle contraction through electrical stimulation can influence the rate of glucose transport in mice lacking GLUT4 when studied in the fed state. Thus, under some conditions, muscle contraction can increase glucose uptake via GLUT4-independent mechanism(s). We have recently provided evidence that insulin-stimulated glucose uptake in skeletal muscle from GLUT4 mice is mediated by a facilitative transport process that is glucose and cytochalasin B inhibitable but not strongly labeled by ATB-BMPA (57) . However, the possibility of increased glucose flux through available GLUT1 cannot be completely ruled out. Here we provide the first direct evidence that GLUT4 is necessary to facilitate immediate muscle glycogen resynthesis after exercise (<5 h). Furthermore, our findings demonstrate that GLUT4 is not essential for glycogen repletion, since muscle glycogen levels in previously exercised GLUT4-null mice were totally restored after 24 h carbohydrate refeeding. Thus, exercise-induced activation of glycogen synthase is likely to be important for glycogenesis even when glucose uptake is not maximally activated.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Geoffrey D. Holman for generously providing the bis-mannose photolabeling reagent and GLUT1 antibodies. This work was supported by grants from the Swedish Medical Research Council (12211, 11823), the Swedish Diabetes Association, Novo Nordisk Insulin Foundation, The Marcus and Amalia Wallenberg’s Foundation, a Junior Individual Grant from the Foundation for Strategic Research to J.R.Z., the National Institutes of Health (DK47425, HL58119), and an Irma T. Hirschl Career Scientist award to M.J.C.


   FOOTNOTES
 
Received for publication April 1, 1999. Revised for publication August 3, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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