INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IT WAS SUGGESTED by Ren et al. (20) that a rapid increase in the insulin-regulated glucose transporter GLUT-4 in skeletal muscle is an early adaptation to exercise that facilitates replenishment of the muscle glycogen stores. This was based on the observations of a 50% increase in GLUT-4 protein in rat skeletal muscle 16 h after a prolonged exercise session and a 100% increase 16 h after a second exercise session the following day. The increase in GLUT-4 protein was accompanied by a proportional increase in insulin-stimulated glucose transport and glycogen synthesis in vitro.

To better characterize the effects of acute exercise on GLUT-4 protein expression and to better define the role of GLUT-4 in the regulation of muscle glycogen synthesis, we previously followed the changes in GLUT-4 protein and mRNA concentrations in rat skeletal muscle after an exercise plus carbohydrate supplementation regimen (13). We observed a progressive increase in GLUT-4 protein that was evident within 1.5 h of recovery in the fast-twitch red muscle and 5.0 h in the fast-twitch white muscle. Muscle GLUT-4 mRNA was significantly increased immediately postexercise, but returned to the preexercise level within 1.5 h of recovery. Total GLUT-4 mRNA remained at this level for a minimum of 5 h, during which time there was a substantial increase in GLUT-4 protein. The cause for this rapid downregulation of GLUT-4 mRNA and increase in translation efficiency during the recovery period was not evident, but it was noted that these cellular events followed the glucose supplement provided the rats on completion of the exercise session. Therefore, the present study was designed to investigate the effects of glucose supplementation on skeletal muscle GLUT-4 protein and mRNA concentrations after glycogen-depleting exercise.

The results indicate that carbohydrate supplemention provided in association with exercise lowers the GLUT-4 mRNA concentration while increasing GLUT-4 mRNA translation efficiency. Furthermore, this effect of carbohydrate supplementation is only evident after exercise. Thus carbohydrate supplementation appears to exert a modifying rather than a main stimulatory effect on GLUT-4 protein expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. GLUT-4 cDNA was generously donated by Dr. Morris J. Birnbaum (University of Pennsylvania). The 927-bp DNA fragment between EcoR I and Bgl II was gel purified as a template for production of a cDNA probe by the random priming labeling method. A 28S ribosomal RNA-specific probe was purchased from CLONTECH (Palo Alto, CA). Rat GLUT-4 antiserum was obtained from Dr. Samuel W. Cushman (NIH). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit antibody was purchased from Amersham (Arlington Heights, IL). Radioactively labeled [³²P]CTP and [³²P]ATP were purchased from DuPont-New England Nuclear (Wilmington, DE). Neutral nylon membrane and random primer DNA-labeling kit were purchased from Stratagene (La Jolla, CA). Amyloglucosidase for the glycogen assay was purchased from Boehringer Mannheim (Indianapolis, IN). All other chemicals were molecular biology grade and purchased from Sigma Chemical (St. Louis, MO) and Fisher (Pittsburgh, PA).

Animals. Male Sprague-Dawley rats weighing 230-250 g were housed in a room maintained on a 0700-to-1900 light cycle and at a temperature of 21°C. The rats were allowed free access to water and chow (Purina chow, Ralston Purina, St. Louis, MO). All procedures were approved by the Animal Care and Use Committee of the University of Texas and conformed to guidelines for the use of laboratory animals published by US Department of Health and Human Services.

Experimental procedure. Rats were randomly assigned to one of five treatment groups: exercise-fast (Ex-Fast, n = 12), exercise-carbohydrate (Ex-Cho, n = 6), sedentary-fast (Sed-Fast, n = 6), sedentary-carbohydrate (Sed-Cho, n = 6), and sedentary-control (Sed-Con, n = 6). The exercise protocol consisted of two 3-h swimming intervals with a 45-min rest period between intervals (20). The temperature of the water was maintained between 33 and 34°C. All rats swam 10 min/day for 2 days before the start of the experiment to get familiarized with the water.

Glycogen assay. About 50 mg of muscle were dissolved in 1 N KOH at 70°C for 30 min. Dissolved homogenate was neutralized by glacial acetic acid and incubated overnight in acetate buffer (0.3 M sodium acetate, pH to 4.8) containing amyloglucosidase (4). The reaction mixture was neutralized with 1 N NaOH. Samples were then analyzed by measuring glycosyl units by the Trinder reaction (Sigma Chemical).

Measurement of GLUT-4 protein concentration. Muscle samples were homogenized (1:20, red muscle and 1:14, white muscle) in 20 mM ice-cold HEPES, 1 mM EDTA, and 250 mM sucrose (HES) buffer (pH 7.4) with a Polytron (Brinkmann Instruments, Westbury, NY). Sample homogenates and standards were diluted 1:1 with Laemmli sample buffer (15). Muscle homogenates containing 75 µg (red gastrocnemius) or 100 µg (white gastrocnemius) of protein were then subjected to SDS-PAGE run under reducing conditions on a 12.5% resolving gel. Two GLUT-4 standards from rat heart, containing 15 and 30 µg of protein, were loaded in parallel with the muscle samples. Protein determinations were performed on each homogenate via the method of Bradford (3). Resolved proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA) as described previously (1). GLUT-4 antiserum diluted 1:500 was used for immunoblotting. GLUT-4 protein was visualized on hyperfilm by using the ECL Western blot detection kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions.

Measurement of GLUT-4 mRNA and ribosomal RNA concentrations. For RNA extraction, muscle tissues were homogenized in guanidium isothiocyanate--mercaptoethanol buffer with a Polytron. Total RNA was isolated from frozen tissue samples by the method of Chomczynski and Sacchi (5). For Northern blotting analysis, equal amounts of total RNA (30 µg) were denatured by heating at 60°C for 10 min and separated on 1% agarose-formaldehyde gels. Ethidium bromide staining of the formaldehyde gel and the transferred blots were used for determining the quality of the RNA sample. GLUT-4 mRNA was quantified by dot blotting analysis. Total RNA samples (4, 2, 1, 0.5, 0.25 µg) were applied directly to a nylon membrane by using a vacuum manifold (Bio-Rad, Richmond, CA) and were immobilized by ultraviolet cross-linking. Treatment groups were always analyzed in parallel. An Escherichia coli transfer RNA was used as a control to ensure specific binding of the probe. GLUT-4 mRNA level was determined by hybridization with radioactively labeled GLUT-4 cDNA as previously described (14). All dot blots were then stripped and reprobed with ³²P 5'end-labeled gene-specific 28S ribosomal RNA oligonucleotide probe and -actin cDNA. The amount of GLUT-4 mRNA present in each sample was determined by comparing the intensity of the sample dots with an external GLUT-4 heart standard run on each membrane.

Statistical analysis. A one-way analysis of variance among the experimental groups was performed on all variables. Fisher's protected least significance difference test, which holds the value of a type I error to 0.05 for each test, was used to distinguish significant differences between pairs of groups. A level of P < 0.05 was set for significance for all tests, and values are expressed as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

After 6 h of swimming, glycogen from both red and white sections of the gastrocnemius muscle was significantly reduced to ~50% of the preexercise level (Figs. 1 and 2). After a 16-h postexercise fast, red gastrocnemius muscle glycogen (Fig. 1) was similar to, but white gastrocnemius muscle glycogen was significantly lower than, sedentary control levels (Fig. 2). Exercise plus carbohydrate supplementation increased muscle glycogen above sedentary control levels by ~76% in red gastrocnemius muscle and ~42% in white gastrocnemius muscle. In contrast, carbohydrate supplementation in sedentary rats increased glycogen above sedentary control levels by only 40% in red gastrocnemius muscle and 15% in white gastrocnemius muscle. The differences in muscle glycogen between the exercised and sedentary carbohydrate-supplemented rats were significant for both red and white gastrocnemius.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of carbohydrate supplementation and fasting on glycogen concentration of red gastrocnemius muscle (RG) in sedentary and exercised rats. Exercise protocol consisted of two 3-h swimming intervals with a 45-min rest. When the exercised groups completed their 1st session of swimming, exercise-carbohydrate (Ex-Cho) and sedentary-carbohydrate (Sed-Cho) rats were intubated with 0.4 ml glucose (50% wt/vol). Rat chow was supplied to all groups during the 45-min rest period between intervals. When the exercised groups completed their 2nd session of exercise, rats from the Ex-Cho and Sed-Cho groups were intubated with 1 ml of glucose (50% wt/vol) and provided rat chow for 16 h. The sedentary control (Sed-Con) group received rat chow ad libitum until euthanized. Both the sedentary-fasted (Sed-Fast) and exercise-fasted (Ex-Fast) groups remained fasted for 16 h after the exercised groups had completed their second exercise session. RG muscles of Ex-Fast rats were sampled immediately (shaded part column) and 16 h (shaded plus open part of column) after exercise. Glycogen concentrations are expressed as µmol/g wet wt. Values are means ± SE. *P < 0.01, significantly different from each of sedentary groups. P < 0.01, significantly different from Sed-Con and Sed-Fast groups.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of carbohydrate supplementation and fasting on glycogen concentration of white gastrocnemius muscle (WG) in sedentary and exercised rats. Muscle samples from each group were collected and analyzed under the same experimental treatments as described in Fig. 1. Glycogen concentrations are expressed as µmol/g wet wt. Values are means ± SE. *P < 0.01, significantly different from each of the sedentary groups. P < 0.01, significantly different from Sed-Con and Sed-Fast groups. §P < 0.01, significantly different from Ex-Fast group.

Red and white gastrocnemius GLUT-4 protein concentrations were increased significantly in both the Ex-Fast and the Ex-Cho rats 16 h after exercise. In the postexercise fasted rats, red gastrocnemius GLUT-4 protein concentration increased by 43% (Fig. 3), and white gastrocnemius muscle GLUT-4 protein concentration increased by 43% (Fig. 4) above sedentary control levels. Exercise plus carbohydrate supplementation, however, increased red gastrocnemius GLUT-4 protein concentration 88% above sedentary control levels. This increase was significantly higher than that which occurred after postexercise fasting. In white gastrocnemius muscle, GLUT-4 protein concentration following exercise with carbohydrate supplementation increased 68% above sedentary control levels and 17% above the postexercise fasting level. In the sedentary rats, neither carbohydrate supplementation nor fasting significantly affected the muscle GLUT-4 protein concentration.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of carbohydrate supplementation and fasting on the GLUT-4 protein of RG muscle in sedentary and exercised rats. Representative autoradiogram bands showing labeled GLUT-4 protein appear above the appropriate column for each treatment group. Muscle samples from each group were collected and analyzed under the same experimental treatments as described in Fig. 1. Equal amounts (75 µg) of protein from each sample were loaded on a gel and immunoblotted by method described in MATERIALS AND METHODS. Samples on autoradiographs are expressed as % heart standard (30 µg). Values are means ± SE. *P < 0.01, significantly different from each of sedentary groups. §P < 0.01, significantly different from Ex-Fast group.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of carbohydrate supplementation and fasting on the GLUT-4 protein of WG muscle in sedentary and exercised rats. Representative autoradiogram bands showing labeled GLUT-4 protein appear above the appropriate column for each treatment group. Muscle samples from each treatment group were collected and analyzed under the same experimental treatments as described in Fig. 1. Equal amounts of protein (100 µg) from each muscle sample were loaded on a gel and immunoblotted as described in MATERIALS AND METHODS. Samples on autoradiographs are expressed as %heart standard (30 µg). Values are means ± SE. *P < 0.01, significantly different from each of the sedentary groups. §P < 0.01, significantly different from Ex-Fast group.

Irrespective of fiber type or activity status, muscle glycogen and GLUT-4 protein concentrations were found to be positively correlated 16 h after carbohydrate supplementation (r = 0.81) (Fig. 5). That is, the higher the GLUT-4 protein concentration of the muscle sample, the greater the glycogen concentration.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Correlation between the muscle GLUT-4 protein concentration and glycogen concentration after a carbohydrate supplement. Muscles from Sed-Cho and Ex-Cho rats were excised 16 h after carbohydrate supplement for glycogen determination. Both fast-twitch RG and fast-twitch WG sections of muscle were used for statistical analysis.

GLUT-4 mRNA concentration in red gastrocnemius muscle was significantly increased 16 h after exercise in both the carbohydrate-supplemented and fasted rats (Fig. 6). After the 16-h postexercise fasting, red gastrocnemius muscle GLUT-4 mRNA concentration increased to ~80% above sedentary control levels. In contrast, GLUT-4 mRNA was only 40% above sedentary control levels after exercise with carbohydrate supplementation. The difference in muscle GLUT-4 mRNA levels of Ex-Fast and Ex-Cho rats was significant. Neither carbohydrate supplementation nor fasting had an effect on the muscle GLUT-4 mRNA levels of sedentary rats.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of carbohydrate supplementation and fasting on the GLUT-4 mRNA of RG muscle in sedentary and exercised rats. Representative autoradiogram bands showing labeled GLUT-4 mRNA appear above the appropriate column for each treatment group. Muscle samples from each treatment group were collected and analyzed under the same experimental treatments as described in Fig. 1. Equal amounts of RNA (8 µg) from each muscle sample were loaded on dot blots for quantification by method described in MATERIALS AND METHODS. Samples on autoradiographs are expressed as %heart standard. Values are means ± SE. *P < 0.01, significantly different from each of the sedentary groups. §P < 0.01, significantly different from the Ex-Fast group.

The concentration of -actin mRNA in red gastrocnemius muscle across treatments was not different, indicating specificity of response for the GLUT-4 mRNA. There was also no significant difference in 28S ribosomal RNA detected among treatments (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results of this study suggest a complex interaction between exercise with carbohydrate supplementation and GLUT-4 protein expression in skeletal muscle. In agreement with previous findings, we demonstrated that an acute exercise session could initiate an increase in muscle GLUT-4 protein concentration (13, 20). In addition, we found that this exercise-induced increase in GLUT-4 protein could be enhanced by carbohydrate supplementation, although addition of the supplement resulted in a reduced muscle GLUT-4 mRNA concentration. We also observed a high correlation between the level of muscle GLUT-4 protein and glycogen concentration after a carbohydrate supplement.

The increase in GLUT-4 protein after exercise appeared to be regulated by both pretranslational and translational events. When rats were fasted for 16 h after exercise, GLUT-4 mRNA increased by 80% above control, whereas GLUT-4 protein increased only 43% above control. These results indicate that under the postexercise fasted condition muscle GLUT-4 expression was regulated by predominately pretranslational mechanisms. Furthermore, the relatively low increase in GLUT-4 protein in the presence of a relatively high GLUT-4 mRNA concentration suggests a low translational efficiency of GLUT-4 mRNA in the exercise-fasted state. In contrast to the exercise with fasting, carbohydrate supplementation plus exercise resulted in an 88% increase in GLUT-4 protein, whereas the increase in GLUT-4 mRNA was only 40%. Thus translational efficiency of GLUT-4 mRNA during the 16-h exercise-recovery period appeared to be greatly improved with carbohydrate supplementation.

In a prior study from our laboratory (13), we found that, immediately after prolonged aerobic exercise, muscle GLUT-4 mRNA was increased ~50% above control levels. When carbohydrate was provided, total GLUT-4 mRNA returned to control levels within 1.5 h and remained at this level for a minimum of 5 h. During this period, there was a substantial increase in GLUT-4 mRNA translation as GLUT-4 protein increased ~40% above the control level. An explanation for the increase in translation came in the finding that despite the decline in total GLUT-4 mRNA there was an increase in GLUT-4 mRNA-polysome association. An increase in GLUT-4 mRNA-polysome association is generally considered translationally active mRNA and therefore implies that there was an activation of the translation process during this period of recovery. The cause for this shift in translational efficiency was not evident, although it was observed to occur after the carbohydrate supplement. On the basis of the present findings, however, we can now propose that exercise plus carbohydrate supplement results in a downregulation of GLUT-4 mRNA and a conversion of the exercise-induced GLUT-4 expression from predominately pretranslational to predominately translational events.

This cellular strategy for control of GLUT-4 protein synthesis may have important physiological relevance. An increase in GLUT-4 mRNA during prolonged exercise would prepare the muscle for rapid translation of GLUT-4 mRNA once sufficient glucose is made available for restoration of glycogen stores. The increase in GLUT-4 protein would thus promote an increase in the rate of glucose uptake and would stimulate glycogen synthesis (8, 18, 21). However, if the translational process of GLUT-4 protein was directly driven by pretranslational activation without regulation, the GLUT-4 protein production would be directly increased after the initial GLUT-4 mRNA elevation after exercise-induced glycogen depletion. Under this condition, a protracted contraction-stimulated GLUT-4 protein translocation (7, 17) would continuously forward the newly synthesized GLUT-4 protein to the plasma membrane. This could lead to a rapid clearance of blood glucose and acute hypoglycemia. Therefore, after prolonged aerobic exercise, regulation of muscle GLUT-4 protein expression at the translational level can ensure that tissues other than skeletal muscle receive appropriate amounts of glucose when blood glucose and carbohydrate reserves are low.

The mechanism by which carbohydrate supplement provided in association with exercise downregulates GLUT-4 mRNA while simultaneously increasing GLUT-4 mRNA translation cannot be determined from the available data. However, it is possible that these processes are mediated by changes in circulating insulin levels. This possibility is supported by the observation that insulin can stimulate the downregulation of GLUT-4 mRNA in mammalian cells by inhibiting GLUT-4 gene transcription and reducing GLUT-4 mRNA stability (6). Furthermore, insulin is known to activate mRNA translation via the mitogen-activated protein kinase pathway by initiating the formation of the 40S-initiation complex by phosphorylation of the protein PHAS-I (12, 16). Thus, during the time of GLUT-4 mRNa downregulation, the GLUT-4 mRNA in the translational cycle may be physically protected by the polysomes from the cellular degradation machinery. In contrast to the present findings, several studies have reported no change (19) or an increase (11) in GLUT-4 mRNA concentration after the infusion of glucose and insulin in sedentary rats. Thus, if insulin is involved in the downregulation of GLUT-4 mRNA as hypothesized, it may occur only in a postexercise state.

Carbohydrate supplementation had no direct effect on GLUT-4 mRNA transcription or translation. This is evidenced from the finding that sedentary rats provided a carbohydrate supplement failed to demonstrate any changes in the levels of their muscle GLUT-4 mRNA or protein. We can thus surmise that carbohydrate supplementation exerted a modifying rather than a main stimulatory effect on the exercise-induced GLUT-4 expression. This also appears to be true for fasting animals. Sedentary rats fasted for 16 h demonstrated no changes in muscle GLUT-4 mRNA or protein. As mentioned previously, GLUT-4 mRNA was significantly higher postexercise when rats were fasted vs. when they were supplemented with carbohydrate. Thus fasting postexercise may facilitate muscle GLUT-4 mRNA transcription and thus amplify GLUT-4 mRNA translation upon an increase in glucose availability. This possibility could help explain the early observations of Bergström et al. (2) that delaying carbohydrate consumption for several days after a glycogen-depleting exercise results in greater muscle glycogen storage than starting carbohydrate replacement the day of the exercise.

Finally, the effect of carbohydrate supplementation on glycogen storage in sedentary muscle was limited compared with that of exercised muscle. With the same amount of carbohydrate supplementation, the muscle glycogen level was only increased 40% above control in sedentary rats, whereas in the exercised rats it was increased 76% above control. In addition, there was a high correlation between the level of muscle GLUT-4 protein and glycogen concentration in rats provided the carbohydrate supplement. Although prior exercise can increase the glycogen storage response to a carbohydrate supplement without an increase in GLUT-4 protein (9), it is quite possible that the level of muscle GLUT-4 protein serves to set or limit the upper level of glycogen accumulation. Thus, the difference in glycogen storage between sedentary and exercised rats may have been due in part to a difference in muscle GLUT-4 protein content. In support of this premise is the observation that glycogen storage, as well as glucose transport, is enhanced in mice genetically engineered to overexpress the GLUT-4 protein in skeletal muscle (8, 21). Furthermore, Hickner et al. (10) recently found a significant relationship between the magnitude of increase in muscle glycogen over a 48- to 72-h postexercise period and muscle GLUT-4 protein concentration when comparing trained and untrained subjects.

In summary, we have found that carbohydrate supplementation has a modifying rather than a main activating effect on exercise-induced GLUT-4 protein expression in skeletal muscle. In exercised fasted muscle, GLUT-4 mRNA was dramatically increased with less GLUT-4 protein expression compared with the exercised carbohydrate-supplemented muscle. This result suggests a possible facilitation of muscle GLUT-4 mRNA transcription during postexercise fasting. The fact that translational efficiency of GLUT-4 mRNA appears to be enhanced if carbohydrate supplement is provided with exercise, indicates that carbohydrate supplementation switches the exercise-induced GLUT-4 expression from a predominantly pretranslational to a predominantly translational event. The role of GLUT-4 protein expression in insulin-dependent glycogen storage in skeletal muscle has been previously suggested (13, 20). In the present study, we have shown that the degree of glycogen storage after a carbohydrate load is highly correlated with the muscle GLUT-4 protein level. This correlation suggests that the level of GLUT-4 protein expression may limit skeletal muscle glycogen storage.