Transgenic mice overexpressing GLUT-1 protein in muscle exhibit increased muscle glycogenesis after exercise

Jian-Ming Ren1,2, Nicole Barucci1, Bess A. Marshall3, Polly Hansen4, Mike M. Mueckler3, and Gerald I. Shulman1

1 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; 2 Metabolic Diseases Research, Bristol-Myers Squibb, Princeton, New Jersey 08543; 3 Departments of Cell Biology and Physiology and of 4 Medicine, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of the present study was to determine the rates of muscle glycogenolysis and glycogenesis during and after exercise in GLUT-1 transgenic mice and their age-matched littermates. Male transgenic mice (TG) expressing a high level of human GLUT-1 and their nontransgenic (NT) littermates underwent 3 h of swimming. Glycogen concentration was determined in gastrocnemius and extensor digitorum longus (EDL) muscles before exercise and at 0, 5, and 24 h postexercise, during which food (chow) and 10% glucose solution (as drinking water) were provided. Exercise resulted in ~90% reduction in muscle glycogen in both NT (from 11.2 ± 1.4 to 2.1 ± 1.3 µmol/g) and TG (from 99.3 ± 4.7 to 11.8 ± 4.3 µmol/g) in gastrocnemius muscle. During recovery from exercise, the glycogen concentration increased to 38.2 ± 7.3 (5 h postexercise) and 40.5 ± 2.8 µmol/g (24 h postexercise) in NT mice. In TG mice, however, the increase in muscle glycogen concentration during recovery was greater (to 57.5 ± 7.4 and 152.1 ± 15.7 µmol/g at 5 and 24 h postexercise, respectively). Similar results were obtained from EDL muscle. The rate of 2-deoxyglucose uptake measured in isolated EDL muscles was 7- to 10-fold higher in TG mice at rest and at 0 and 5 h postexercise. There was no difference in muscle glycogen synthase activation measured in gastrocnemius muscles between NT and TG mice immediately after exercise. These results demonstrate that the rate of muscle glycogen accumulation postexercise exhibits two phases in TG: 1) an early phase (0-5 h), with rapid glycogen accumulation similar to that of NT mice, and 2) a progressive increase in muscle glycogen concentration, which differs from that of NT mice, during the second phase (5-24 h). Our data suggest that the high level of steady-state muscle glycogen in TG mice is due to the increase in muscle glucose transport activity.

swimming; glucose transport; GLUT-1; glycogen


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE IS THE MAJOR TISSUE responsible for whole body glucose disposal during hyperinsulinemic clamp (21, 24). Under hyperinsulinemic-hyperglycemic conditions, the fate of glucose after entering muscle cells is glycogen synthesis (22). During prolonged intense exercise, muscle glycogen can be depleted (near zero) (1, 10), but it recovers after cessation of exercise if carbohydrate is provided. After exercise, high carbohydrate loading increases plasma glucose and insulin levels. As a result, muscle glycogen concentration exceeds preexercise level. This glycogen supercompensation phenomenon was initially observed by Bergstrom and Hultman (1) and subsequently comfirmed by a number of investigators (10).

Glycogen synthase has been suggested to be the key rate-limiting enzyme for muscle glycogen synthesis. Evidence for this is derived from data that demonstrate a strong correlation between glycogen synthesis and glycogen synthase activation during hyperinsulinemic clamp and immediately after glycogen-depleting exercise (10, 13). Both insulin and exercise activate glycogen synthase by converting the inactive D form to the active I form through different molecular mechanisms in muscle cells (4). After exercise, glycogen concentration increases over time, whereas muscle glycogen synthase activity decreases (10). Despite the normalization of glycogen synthase activity, glycogen concentration continues to rise above the preexercise level (10). This suggests that glycogen supercompensation is due to factors other than high glycogen synthase activity after exercise. It has been suggested that an increase in muscle glucose transport activity after exercise may contribute to exercise-induced glycogen supercompensation (2). Because the increased glucose transport activity parallels the activation of glycogen synthase after exercise, it is difficult to assess the importance of glucose transport in glycogen synthesis after exercise. Transgenic mice overexpressing human GLUT-1 exhibit increased glucose transport activity and decreased glycogen synthase (17). Nevertheless, muscle glycogen concentration is approximately eightfold higher in GLUT-1 transgenic mice when compared with their nontransgenic littermates (17). The marked increase in muscle glycogen concentration in transgenic mice may be due to the high glucose transport activity. Previous study has also shown that the rate of glycogenolysis during exercise is glycogen concentration dependent (19). Therefore, the purpose of the present study was to determine the rate of glycogen breakdown during exercise and the rate of glycogen repletion after exercise in GLUT-1 transgenic mice and nontransgenic littermates. We found that the rate of muscle glycogenolysis during 3 h of swimming was greater in transgenic than in nontransgenic mice. Furthermore, muscle glycogenesis during the first 5 h of recovery from exercise was similar between transgenic and nontransgenic mice but was increased between 5 and 24 h of recovery only in TG mice. These results suggest that the early phase of rapid glycogen accumulation is largely due to a combination of glycogen synthase activation and increased glucose transport activity. However, the late phase of glycogen synthesis is due to a persistent increase in glucose transport activity in transgenic mice.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and experimental design. Generation of the transgenic mouse line in which the human GLUT-1 glucose transporter was overexpressed specifically in muscle has been described previously (14). Expression of the transgene is restricted to skeletal muscle and does not appreciably affect the expression of the GLUT-4 isoform (14). All animals used for experiments were littermates resulting from the breeding of a single line of GLUT-1 overexpressing mice carrying a single copy of the transgenic locus with nontransgenic C57BL/6XSJL F2 mice. Only age-matched male mice (8-10 wk old) were used in all experiments. Animals were housed in a room maintained at 23°C with a fixed 12:12-h light-dark cycle and were given free access to Purina chow and water ad libitum. All experiments were performed in the morning (10 AM). Mice were accustomed to swimming (water temperature at 35°C) for 10 min/day for 2 days, and then they were exercised using a modified protocol as described previously (18). Briefly, the animals swam for a total of 3 h. After 3 h of swimming, mice were either killed immediately or allowed to recover for up to 24 h, during which they had free access to food and 10% glucose as drinking water. After 5 or 24 h of recovery, all mice were killed. Gastrocnemius muscles were isolated from both sedentary and exercised mice at 0, 5, and 24 h of recovery to determine muscle glycogen concentration and glycogen synthase activity. Plasma samples were collected by cardiac puncture in anesthetized mice (ip injection of pentobarbital sodium 5 mg/g body wt).

Muscle incubation. Extensor digitorum longus (EDL) muscles were isolated from transgenic (TG) and nontransgenic (NT) mice. Muscles were incubated in 2 ml of oxygenated Krebs-Henseleit bicarbonate (KHB) buffer (11) containing 8 mM glucose and 32 mM mannitol.

Because TG mice exhibit increased glucose transport activity and decreased glycogen synthase activity (17), it is of interest to determine whether activating glycogen synthase could promote glycogenesis. LiCL2 is known to activate glycogen synthase but has no effect on glucose transport (23). To this end, 2-deoxyglucose uptake and glycogen synthase activity were measured in EDL muscles from nonexercised TG and NT mice in the presence and absence of 10 mM LiCL2. Moreover, the rate of 2-deoxyglucose uptake in EDL muscles from TG and NT mice was also determined before and after exercise to correlate glucose transport activity with the rate of glycogenesis. The flasks were shaken in a Dubnoff incubator, and muscles were processed and assayed for glycogen concentration and glycogen synthase activity.

Measurement of 2-deoxyglucose uptake into muscle. EDL muscles from sedentary and exercised (immediately after exercise and 5 h postexercise) TG and NT mice were isolated and incubated in vitro. After the initial incubation period, muscles were rinsed in the absence of glucose for 10 min at 29°C in 2 ml of oxygenated KHB containing 40 mM mannitol. Glucose uptake was measured using the nonmetabolizable glucose analog 2-deoxyglucose and a modification (25) of the procedure used previously in frog muscle (7, 16). The muscles were transferred to flasks and incubated with 1.5 ml of KHB containing 1 mM 2-deoxy-[1,2-3H]glucose (1.5 mCi/mmol) and 39 mM [14C]mannitol (3.9 µCi/mmol) for 20 min at 29°C. The gas phase in all flasks during both the rinse and incubation periods was 95% O2-5% CO2. The muscles were then processed, and the extracellular space and intracellular 2-deoxyglucose concentration were determined as described previously (25). All values for glucose uptake are expressed as micromoles of 2-deoxyglucose per milliliter of intracellular water per 20 minutes.

Measurements of muscle glycogen synthase activity. Gastrocnemius muscles from nonexercised and exercised mice were homogenized in 30 vol/wt of ice-cold buffer containing 20 mM HEPES, 1 mM EDTA, and 250 mM sucrose (pH 7.4) by use of a glass Potter-Elvehjem homogenizer immersed in ice water. Glycogen synthase activity was measured in the homogenate as described previously (17). The latter was measured in the presence and absence of 10 mM glucose 6-phosphate (G-6-P) and expressed as independent activity as a percentage of total activity, or I/I+D × 100%.

Measurement of plasma insulin, glucose, and free fatty acid levels. Plasma glucose levels were measured using a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA), and plasma free fatty acid (FFA) levels were determined using a diagnostic kit from Wako Chemicals (Richmond, VA). Plasma insulin levels were measured by an insulin RIA kit (Linco Research, St. Louis, MO).

Statistical analysis. Data in the text and figures are given as means ± SE. Multiple comparisons were performed by two-way ANOVA with nonrepeated measures. Differences between the two groups were determined using the Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle glycogen content and glycogen synthase activity in NT and TG mice after 3 h of swimming. In sedentary TG and NT mice, gastrocnemius muscle glycogen concentrations were 99.3 ± 4.7 and 11.2 ± 1.4 µmol/g, respectively (Fig. 1). Immediately after 3 h of swimming, glycogen concentration decreased by ~90% in both TG and NT mice (P < 0.05). During recovery with glucose loading, glycogen concentration increased from 2.1 ± 1.3 to 38.2 ± 7.3 µmol/g (P < 0.05) in NT mice after 5 h of recovery. There was no further increase in glycogen level from 5 to 24 h in NT mice (P > 0.05). In TG mice, muscle glycogen concentration increased from 11.8 ± 4.3 to 57.5 ± 7.4 µmol/g (P < 0.05) after 5 h of recovery. A further pronounced increase in glycogen concentration was noted from 5 to 24 h of recovery (P < 0.05), when it reached the level of 152.1 ± 15.7 µmol/g (Fig. 1). Similar results were obtained from EDL muscles from TG and NT mice before and after exercise (Table 1). Glycogen synthase activity measured in gastrocnemius muscle was lower in TG than in NT mice (P < 0.05) in the nonexercised state (Fig. 2). It increased from 24.7 ± 1.6 to 52.1 ± 3.8% (P < 0.05) in NT and from 9.8 ± 1.3 to 37.6 ± 3.1% (P < 0.05) in TG mice immediately after 3 h of swimming. During recovery, glycogen synthase activity decreased to 31.7 ± 5.2 and 32.1 ± 3.3% in NT mice 5 and 24 h after exercise. Similar decrease in glycogen synthase activity was observed in TG mice (to 30.2 ± 3.6 and 11.5 ± 1.1% 5 and 24 h after exercise; Fig. 2).


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Fig. 1.   Muscle glycogen concentration (µmol/g) in gastrocnemius muscles of nontransgenic (NT) and transgenic (TG) mice in response to 3 h of swimming exercise (EX). open circle , NT mice; , TG mice. Values are means ± SE; n = 6 for each time point. * P < 0.05, TG vs. NT. # P < 0.05, vs. non-EX.


                              
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Table 1.   Glycogen concentration in EDL muscles of TG and NT mice during and after 3 h of swimming



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Fig. 2.   Muscle glycogen synthase activity (I/I+D 100%, see text) in gastrocnemius muscles of NT and TG mice in response to 3 h of swimming. open circle , NT mice; , TG mice. Values are means ± SE; n = 6 for each time point. * P < 0.05, TG vs. NT.

Muscle 2-deoxyglucose uptake in NT and TG mice before and after 3 h of swimming. In sedentary TG and NT mice, EDL muscle 2-deoxyglucose uptake was 6.52 ± 0.72 and 0.97 ± 0.15 µmol · ml-1 · 20 min-1, respectively (Fig. 3). Three hours of exercise did not further increase the rate of 2-deoxyglucose uptake in either TG or NT mice.


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Fig. 3.   Rate of 2-deoxyglucose uptake into extensor digitorum longus (EDL) muscles of NT and TG mice before, and 0 and 5 h after swimming. open circle , NT mice; , TG mice. Values are means ± SE; n = 6 for each time point.

Plasma insulin, glucose, and FFA concentrations after 3 h of swimming. There was no significant difference in plasma insulin level between TG and NT mice before exercise (Table 2). Plasma insulin level decreased from 39.0 ± 3.8 to 20.0 ± 6.1 µU/ml in NT and from 31.7 ± 6.9 to 2.7 ± 0.2 µU/ml in TG mice immediately after 3 h of exercise (P < 0.05). During recovery, plasma insulin increased in both TG and NT mice, but the rise was significantly less in TG mice (P < 0.05). Plasma glucose level was not different between NT and TG mice before exercise (Table 1) (P > 0.05). In NT mice, plasma glucose and FFA levels were unchanged after 3 h of swimming. During recovery, plasma FFA decreased with no change in plasma glucose in NT mice. In TG mice, plasma glucose concentration decreased by 71% with no change in FFA level after 3 h of exercise. During recovery, plasma glucose level increased, whereas plasma FFA level decreased (P < 0.05).

                              
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Table 2.   Plasma glucose, insulin and FFA levels during recovery after 3 h of swimming in NT and TG

Effects of LiCL2 on muscle glycogen synthase activity and glycogenesis in vitro. Because TG mice exhibit increased glucose transport activity and decreased glycogen synthase activity (17), it is of interest to determine whether activating glycogen synthase could promote glycogenesis. LiCL2 is known to activate glycogen synthase but has no effect on glucose transport (23). We measured 2-deoxyglucose uptake and glycogen synthase activity in EDL and gastrocnemius muscles, respectively, from nonexercised TG and NT mice in the presence and absence of 10 mM LiCL2. As shown in Table 3, 3 h of incubation with 8 mM glucose and 10 mM LiCL2 resulted in a 30% increase in glycogen synthase and a twofold increase in glycogen concentration in NT mice. In TG mice, LiCL2 did not increase muscle glycogen synthase activity or glycogen concentration.

                              
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Table 3.   In vitro determination of 2DG uptake and glycogen synthesis in EDL muscles of sedentary TG and NT mice

Muscle high energy phosphate concentration in sedentary TG and NT mice. Quadriceps muscles from sedentary mice were excised for the measurements of high energy phosphate concentrations. As shown in Table 4, muscle ATP and phosphocreatine levels were lower in TG than in NT mice. There was no difference in muscle creatine and G-6-P concentrations between TG and NT mice (P > 0.05).

                              
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Table 4.   Glycogen and high energy phosphate concentrations in quadriceps muscles of sedentary TG and NT mice


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows that the high level of muscle glycogen content found in TG mice is the result of an increase in muscle glucose transport activity. Although glycogen synthase plays an important role in glycogen synthesis, glucose transporters serve as a gatekeeper to control the availability of glucose for glycogen synthesis.

There are two major glucose transporters expressed in skeletal muscle, GLUT-1 and GLUT-4 (8, 9, 15). In NT mice, GLUT-4 is expressed at a much higher level than GLUT-1 in muscle and resides primarily in the intracellular compartment (9, 15). During exercise and/or insulin stimulation, GLUT-4 undergoes a translocation process, moving from the intracellular compartment to the plasma membrane, where it is active (6). GLUT-1, on the other hand, resides only in the plasma membrane and is independent of any stimuli for activation (9, 15). It is expressed at very low levels in muscle and is responsible for basal glucose transport activity (9, 15). During exercise, muscle glycogen concentration decreases and glucose transport activity increases. After glycogen-depleting exercise, muscle glucose transport activity remains high in the absence of insulin for several hours (2) before returning to the baseline level. Glycogen synthase is maximally activated after exercise, when glycogen is near zero.

In the present study, glucose was provided during recovery, which resulted in high plasma glucose and insulin levels in NT mice. As shown in Fig. 2, muscle glycogen synthase activity was lower 5 h after exercise than immediately after exercise (P < 0.05). This is consistent with the data published previously (10). Because of the inactivation of glycogen synthase and glucose transport activity, there was no appreciable increase in muscle glycogen concentration between 5 and 24 h of recovery in NT mice. These results suggest that the steady-state muscle glycogen concentration may be controlled by glucose transporter (GLUT-4) activity. In TG mice, muscle glycogen accumulation during the first 5 h of recovery is similar to that of NT mice. In contrast to that of NT mice, muscle glycogen concentration continued to increase between 5 and 24 h of recovery, reaching a level that was fourfold higher than that of NT mice. This marked increase in glycogen accumulation could be attributed to the high glucose transport activity in TG mice. The steady-state muscle glycogen level seen in TG mice may be controlled by glycogen synthase inactivation (Fig. 2). To investigate this possibility, we isolated EDL muscles from NT and TG mice in the nonexercised state. Muscles were incubated with 8 mM glucose in vitro in the presence and absence of 10 mM LiCL2. The latter activates glycogen synthase in muscle but has no effect on glucose transport activity (23). Three hours of incubation with LiCL2 resulted in a 30% increase in glycogen synthase activity and a twofold increase in glycogen concentration in NT mice. There was no change in glycogen level and glycogen synthase activity in TG mice after 3 h of incubation with LiCL2. These data further demonstrate that muscle glycogen synthase inactivation due to excessive glycogen accumulation helps to maintain steady-state glycogen level in TG mice.

The rate of muscle glycogenolysis during exercise is increased in TG mice. It has been shown that phosphorylase activation and substrate (inorganic phosphate and glycogen) availability dictate the rate of glycogen breakdown (3, 19). Because glycogen phosphorylase activation during exercise is transient, prolonged exercise such as 3 h of swimming results in inactivation of phosphorylase (5). Inorganic phosphate and glycogen concentrations become the rate-limiting step for glycogenolysis. The level of inorganic phosphate during exercise was not measured in this study. The high glycogen concentration in TG mice may be responsible for the greater glycogen breakdown in these mice during exercise. An alternative explanation for an increased rate of glycogenolysis in TG mice is that the high glucose transport activity in TG mice resulted in hypoglycemia during exercise. Hypoglycemia in turn would cause catecholamine release, which might promote muscle glycogenolysis (20, 22a). For the same reason, TG mice do not show an improvement in endurance during swimming, despite their high level of glycogen in skeletal muscle.

Plasma glucose level was higher in this study than reported previously in both NT and TG mice (14). The reason for this could be due to the anesthesia. Because a large sample volume was required in this study, blood samples were collected by cardiac puncture in anesthetized mice, which would cause an increase in plasma glucose level. As a result, there was no difference in plasma glucose level before exercise between NT and TG mice.

In summary, skeletal muscle glycogen concentration is markedly increased in TG mice, an increase that occurs within 24 h after exercise. The high muscle glycogen level may be due to a persistent increase in muscle glucose transport activity. Muscle glycogen synthase is tightly controlled by glycogen level, which feeds back to help maintain the steady-state muscle glycogen concentration.


    ACKNOWLEDGEMENTS

We thank Dr. John Holloszy for support in the preparation of this study.


    FOOTNOTES

The work was financially supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-40936.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J.-M. Ren, Bristol-Myers Squibb Co., Division of Metabolic Diseases, Route 206 and Provinceline Road, Princeton, NJ 08543 (E-mail: renj{at}bms.com).

Received 7 July 1999; accepted in final form 29 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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