Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 96: 621-627, 2004. First published October 10, 2003; doi:10.1152/japplphysiol.00830.2003
8750-7587/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
96/2/621    most recent
00830.2003v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuo, C.-H.
Right arrow Articles by Ivy, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuo, C.-H.
Right arrow Articles by Ivy, J. L.

Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle

Chia-Hua Kuo, Hyonson Hwang, Man-Cheong Lee, Arthur L. Castle, and John L. Ivy

Exercise Physiology and Metabolism Laboratory, Department of Kinesiology and Health Education, University of Texas, Austin, Texas 78712

Submitted 5 August 2003 ; accepted in final form 6 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation. Male Sprague-Dawley rats were randomly assigned to one of six treatment groups: sedentary control (Con), Con with streptozocin (Stz/C), immediately postexercise (Ex0), Ex0 with Stz (Stz/Ex0), 5-h postexercise (Ex5), and Ex5 with Stz (Stz/Ex5). Rats were exercised by swimming (2 bouts of 3 h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% wt/vol). Stz was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT-4 protein of Ex0 rats was 30% above Con in fast-twitch (FT) red and 21% above Con in FT white muscle. In Ex5, GLUT-4 protein was 52% above Con in FT red and 47% above Con in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above Con in Ex5 rats. Neither GLUT-4 protein nor muscle glycogen was increased above Con in Stz/Ex0 or Stz/Ex5 rats. GLUT-4 mRNA in FT red muscle of Ex0 rats was 61% above Con but only 33% above Con in Ex5 rats. GLUT-4 mRNA in FT red muscle of Stz/C and Stz/Ex0 rats was similar but significantly elevated in Ex5/Stz rats. These results suggest that insulin is essential for the increase in GLUT-4 protein expression following postexercise carbohydrate supplementation.

glucose transporter isoform-4 messenger ribonucleic acid; gene transcription; translation; glucose; streptozocin

AN ACUTE EXERCISE BOUT CAN significantly improve the action of insulin on glucose clearance. This increased glucose clearance appears to be related to the restoration of the muscle glycogen stores. After a glycogen-depleting exercise, two phases of glycogen resynthesis were observed (10, 13, 23, 24). On completion of exercise, an initial rapid phase of glycogen repletion back to near preexercise levels is followed by a prolonged phase of glycogen synthesis to supercompensation levels in skeletal muscle. The first phase of glycogen resynthesis is insulin independent, but the second phase of glycogen resynthesis is insulin dependent (13, 20, 24).

Possibly accounting for the increase in glycogen synthesis during the insulin-independent phase is an increase in the permeability of the muscle to glucose (11). As this condition fades, the muscle demonstrates a marked increase in the sensitivity and responsiveness of glucose transport and glycogen synthesis to insulin (4, 10). The magnitude of this increased insulin action can be extremely high and does not appear to reverse completely until glycogen supercompensation has occurred (4, 26).

An adaptation that may contribute to the increase in insulin action and, therefore, increase in glycogen synthesis postexercise is an increase in GLUT-4 protein expression (16, 25). Our laboratory has reported that GLUT-4 mRNA in the skeletal muscle of the rat is significantly increased immediately after prolonged strenuous exercise and that during recovery there is a progressive increase in GLUT-4 protein (16, 17). The increase in GLUT-4 protein, however, was influenced by carbohydrate availability (17). Providing a carbohydrate supplement immediately after exercise caused a downregulation of the GLUT-4 mRNA, but acceleration in GLUT-4 protein expression. Conversely, postexercise fasting resulted in an increase in GLUT-4 mRNA but a slower GLUT-4 protein expression.

The mechanism by which carbohydrate supplementation postexercise downregulates GLUT-4 mRNA and stimulates an increase in GLUT-4 protein expression is unknown but may be mediated by an increase in circulating plasma insulin levels. Insulin has been found to downregulate GLUT-4 mRNA in mammalian cells (8) and to activate mRNA translation via the mitogen-activated protein kinase pathway (15, 18). Therefore, the purpose of the present study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats weighing between 120 and 135 g were housed in a room maintained on a 0700-1900 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), except when indicated. 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 the US Department of Health and Human Services.

Experimental design and procedures. Rats were randomly assigned to one of six different treatment groups: sedentary control (Con, n = 6), Con with a prior streptozocin (Stz) treatment (Stz/C, n = 5), immediately postexercise (Ex0, n = 6), Ex0 with a prior Stz treatment (Stz/Ex0, n = 5), 5-h postexercise (Ex5, n = 7), and Ex5 with a prior Stz treatment (Stz/Ex5, n = 5). For all Stz-treated rats, 110 mg/kg of Stz were delivered by intraperitoneal injection, and exercise was performed 72 h after the Stz treatment. Stz was freshly dissolved (25 mg/ml) in a 50 mM citric acid solution before injection. To determine the effect of the Stz treatment on the rats, blood samples were taken from a tail vein and analyzed for glucose. All Stz-treated rats were confirmed as being hyperglycemic before being assigned to a treatment group. The Stz treatment was successful in ~70% of the rats. Rats not treated with Stz were injected with vehicle only.

The exercise protocol consisted of two 3-h swimming intervals with a 45-min rest between intervals (25). This protocol was selected because it had previously been found to rapidly increase the muscle GLUT-4 protein concentration (16, 17, 25). The temperature of the water was maintained between 33 and 34°C. All rats swam for 10 min/day for 2 days before the start of the experiment to familiarize them with the exercise environment. This familiarization protocol has no effect on muscle glycogen or GLUT-4 protein concentration (16).

Carbohydrate treatments were administered by oral intubation. Rats from all groups received 0.4 ml of a 50% (wt/vol) glucose solution when the first exercise interval was completed. After the second session of exercise, 1 ml of a 50% (wt/vol) glucose solution was given immediately to all rats, except for those in the Ex0 and Stz/Ex0 groups. Rat chow was continuously supplied ad libitum during the recovery period. Rats from the Ex0 and Stz/Ex0 groups were wrapped in a towel immediately postexercise, and a 0.5-ml blood sample was taken from a tail vein. The rats were then anesthetized in preparation for muscle sampling. Blood samples were taken from the Con, Stz/C, Ex5, and Stz/Ex5 rats 20 min after the final glucose intubation. They were then anesthetized 5 h after completion of the exercise protocol for muscle sampling. All rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (65.0 mg/kg body wt). After muscle sampling, rats were euthanized by cardiac injection of pentobarbital sodium.

Muscle sampling consisted of excising the gastrocnemius of both legs. Fast-twitch red and white sections were rapidly separated over ice and clamped frozen with tongs cooled in liquid N2. These muscle sections were used for glycogen and GLUT-4 protein determination. Fast-twitch red muscle was also used for GLUT-4 mRNA determination.

Plasma glucose and insulin assays. Blood samples were added to 0.1 ml EDTA (24 mg/ml, pH 7.4) and centrifuged for 10 min at 1,000 g, and the plasma was recovered. Plasma glucose was measured with a glucose analyzer (Yellow Springs Instruments 23A, Yellow Springs, OH), and plasma insulin was measured by RIA (Linco, St. Charles, MO).

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 (Boehringer Mannheim, Indianapolis, IN). The reaction mixture was neutralized with 1 N NaOH. Samples were then analyzed by measuring glucosyl units by the Trinder reaction (Sigma, St. Louis, MO).

Glycogen synthase assay. To best approximate the in vivo activity of glycogen synthase, its activity was determined at 30°C in the presence of 7 mmol/l ATP, 100 µmol/l UDP-1-glucose, and varying concentrations of glucose-6-phosphate (G-6-P), as described by Bloch et al. (2). Fast-twitch red gastrocnemius muscle samples were prepared for analysis by homogenization in 5 volumes of 60% glycerol, 50 mmol/l KF, and 20 mmol/l EDTA at pH 7.0 and were further diluted with 8 volumes of 50 mmol/l KF and 20 mmol/l EDTA. In addition to ATP, G-6-P, and UDP-glucose, the reaction buffer contained 50 mmol/l MOPS buffer, 25 mmol/l KF, 20 mmol/l EDTA, 10 mmol/l KH2PO4, and 10 mg/ml glycogen at pH 6.9. An eight-point dose-response curve was performed on four muscle samples from each treatment group, and nonlinear regression analysis was used to distinguish changes in sensitivity from changes in responsiveness under these conditions. Responsiveness was defined as the maximal obtainable G-6-P-stimulated reaction rate at physiological substrate and inhibitory nucleotide concentrations. Sensitivity was defined as the concentration of G-6-P at which half-maximal reaction rate occurred (A0.5). Once activity curves for glycogen synthase were constructed, the activities of glycogen synthase at the A0.5 and in the absence of G-6-P were estimated.

Measurement of GLUT-4 protein concentration. Muscle samples were homogenized (1:20, red muscle and 1:15, white muscle) in 20 mM ice-cold N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 mM EDTA, and 250 mM sucrose (hydroxyethyl starch) buffer (pH 7.4) with a Polytron (Brinkman Instruments, Westbury, NY). Sample homogenates and standards were diluted 1:1 with Laemmli sample buffer. Muscle homogenates containing 75 µg (red gastrocnemius) or 100 µg (white gastrocnemius) of protein were then subjected to SDS-polyacrylamide gel electrophoresis 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 (16). GLUT-4 antiserum (a gift from Dr. Samuel W. Cushman, NIH) diluted 1:500 was used for immunoblotting. GLUT-4 protein was visualized on hyperfilm using the enhanced chemiluminescence Western Blot Detection Kit (Amersham, Arlington Height, IL), according to the manufacturer's instructions, and the values were made relative to the 30-µg heart standard.

Measurement of GLUT-4 mRNA and ribosomal RNA concentrations. For RNA extraction, muscle tissues were homogenized in guanidium isothiocyanate-{beta}-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 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 (a gift from Dr. Morris J. Birnbaum, University of Pennsylvania), as previously described (17). All dot blots were then stripped and reprobed with [32P]5'-end-labeled gene-specific 28S ribosomal RNA oligonucleotide probe and {beta}-actin cDNA (ClonTech, Palo Alto, CA). 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. Radioactively labeled [{alpha}32-P]CTP and [{gamma}32-P]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).

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


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Seventy-two hours following the Stz injection, insulin responses of Stz-treated rats to glucose intubation were effectively eliminated (Fig. 1). This resulted in all Stz rats becoming substantially hyperglycemic (Fig. 2). Con and Ex5 rats displayed plasma insulin concentrations that were six- to sevenfold higher than those of the Stz/C and Stz/Ex5 rats. Conversely, the plasma glucose concentrations of the Stz/C and Stz/Ex5 rats were four- to fivefold higher than those of the Con and Ex5 rats. By design, the insulin response to a glucose load could not be determined in the Ex0 or Stz/Ex0 rats, but the Stz/Ex0 rats exhibited significantly higher plasma glucose and lower plasma insulin concentrations than Ex0 rats postexercise.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1. Plasma insulin concentration of the rats after streptozocin (Stz) treatment. Experimental treatments, detailed in MATERIALS AND METHODS, are for the following 6 groups: sedentary control (Con), Con with a prior Stz treatment (Stz/C), immediately postexercise (Ex0), Ex0 with a prior Stz treatment (Stz/Ex0), 5 h postexercise (Ex5), and Ex5 with a prior Stz treatment (Stz/Ex5). Glucose (1 ml of a 50% wt/vol solution) was intubated immediately after each exercise session in rats from the Con, Stz, Ex5, and Stz/Ex5 groups and immediately after the first exercise session in rats from the Ex0 and Stz/Ex0 groups. Values are means ± SE. *Significantly different from corresponding Stz-treatment group, P < 0.01.

 


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Blood glucose concentration of the rats after Stz treatment. Glucose (1 ml of a 50% wt/vol solution) was intubated immediately after each exercise session in rats from the Con, Stz, Ex5, and Stz/Ex5 groups and immediately after the first exercise session in rats from the Ex0 and Stz/Ex0 groups. All Stz-treated rats displayed hyperglycemia, regardless of when time samples were collected or whether rats were exercised. Values are means ± SE. *Significantly different from corresponding Stz-treatment group, P < 0.01.

 

Glycogen concentrations of fast-twitch red and white muscle are presented in Fig. 3, A and B, respectively. The glycogen concentration of the fast-twitch red muscle, but not the fast-twitch white muscle, of the Stz/C rats was significantly lower than that of the Con rats. Exercise resulted in significant muscle glycogen depletion in both the Ex0 and Stz/Ex0 rats. Glycogen concentrations in red and white fast-twitch muscle were similar for Ex0 and Stz/Ex0 rats. Glycogen concentrations for the Ex5 and Stz/Ex5 rats were determined 5 h after glucose intubation. For the Ex5 rats, fast-twitch red and white muscle glycogen concentrations were 35 and 30% higher than fast-twitch red and white muscle of Con rats, respectively. For the Stz/Ex5 rats, the glycogen concentration of the fast-twitch red muscle was similar to that of the Stz/C rats but significantly lower than that of the Con or Ex5 rats. The glycogen concentration of the fast-twitch white muscle showed little response to glucose intubation in the Stz/Ex5 rats and was significantly lower in the Stz/Ex5 rats compared with the Con but not the Stz/C rats.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3. Glycogen concentrations of fast-twitch red (RG; A) and white gastrocnemius (WG; B) muscle. Muscle samples from each treatment group were collected and analyzed as described in MATERIALS AND METHODS. Glycogen concentrations are expressed as µmol/g wet weight. Values are means ± SE. *Significantly different from corresponding Stz-treatment group, P < 0.01. {dagger}Significantly different from the Con group, P < 0.01. §Significantly different from the Ex0 and Stz/Ex0 groups, P < 0.05.

 

Fast-twitch red (Fig. 4A) and white (Fig. 4B) muscle GLUT-4 protein concentrations were similar for Con and Stz/C rats, respectively, suggesting that the mild Stz treatment used had no immediate effect on the constitutive expression of muscle GLUT-4 protein. Immediately after exercise, the Ex0 rats exhibited a 30% increase in fast-twitch red and a 21% increase in fast-twitch white muscle GLUT-4 protein. Five hours after glucose intubation, GLUT-4 protein concentration of Ex5 rats was 53 and 47% above Con in fast-twitch red and white muscle, respectively. GLUT-4 protein in muscle of rats exposed to Stz showed no response to exercise (Stz/Ex0) or the combination of exercise and glucose intubation (Stz/Ex5).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. GLUT-4 protein concentrations of fast-twitch RG (A) and WG (B) muscle. Equal amounts of protein from each sample were loaded on a gel and immunoblotted as described in MATERIALS AND METHODS. Samples on the autoradiographs are expressed as %heart standard. Values are means ± SE. *Significantly different from corresponding Stz-treatment group, P < 0.01. {dagger}Significantly different from the Con group, P < 0.01. §Significantly different from the Ex0 group, P < 0.05.

 

Glycogen synthase activity and sensitivity to G-6-P was measured in fast-twitch red gastrocnemius muscle (Table 1). Although the Stz-treated rats tended to have a lower maximal glycogen synthase activity than rats not injected with Stz, there was no difference between groups. Furthermore, there was no effect of exercise on maximal activity. Immediately after exercise, the sensitivity of glycogen synthase to allosteric activation, as predicted by the G-6-P A0.5, was significantly increased in Stz-treated and nontreated rats, but there was no difference between groups. There was, however, a difference in the A0.5 between groups before exercise (Con < Stz/C) and 5 h after exercise (Ex5 < Stz/Ex5).


View this table:
[in this window]
[in a new window]
  		Table 1. Glycogen synthase activity of fast-twitch red gastrocnemius muscle determined under assay conditions that simulated normal ATP and UDP glucose cellular concentrations

 

Figure 5 illustrates the GLUT-4 mRNA level in fast-twitch red muscle. There was no difference in the GLUT-4 mRNA concentrations of Con and Stz/C rats. Exercise significantly increased muscle GLUT-4 mRNA by ~61% in the Ex0 rats. This increase in GLUT-4 mRNA, however, was not observed in the muscle of the Stz/Ex0 rats. Five hours after glucose intubation, the GLUT-4 mRNA concentration of the Ex5 rats was significantly lower than that of the Ex0 rats but 33% above that of the Con rats. In the Stz/Ex5 rats, a slight but significant increase in muscle GLUT-4 mRNA concentration was observed (25% above Stz/C). No differences in 28S ribosomal or {beta}-actin RNA were detected among treatments (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5. GLUT-4 mRNA concentration in fast-twitch RG muscle. Equal amounts of RNA (8 µg) from each sample were loaded on dot blots for quantification, as described in MATERIALS AND METHODS. Samples on the autoradiographs are expressed as %heart standard. Values are means ± SE. *Significantly different from corresponding Stz-treatment group, P < 0.01. {dagger}Significantly different from the Con group, P < 0.01. §Significantly different from the Ex0 group, P < 0.05.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
After prolonged exercise that results in glycogen depletion, there is an increase in the muscle GLUT-4 protein concentration, but the rate of increase can be influenced by substrate availability. In this regard, our laboratory previously found that muscle GLUT-4 protein of the rat was significantly increased shortly after exercise and that the rate of increase was enhanced by carbohydrate supplementation (17). Muscle GLUT-4 mRNA was significantly increased immediately postexercise but rapidly returned to the preexercise level on supplementation. Muscle glycogen storage during recovery was highly correlated with the increase in GLUT-4 protein, indicating that the rate of glycogen storage after exercise is controlled, in part, by GLUT-4 protein expression. Insulin is known to be secreted on carbohydrate ingestion and to regulate both gene transcription and translation (8, 15, 18, 21). Therefore, we tested the hypothesis that the effects of carbohydrate supplementation on GLUT-4 mRNA transcription and GLUT-4 protein expression are induced by the insulin response to the supplement when provided postexercise. We also evaluated the effect of insulin on muscle glycogen storage.

To investigate the effects of insulin on GLUT-4 expression, we limited pancreatic insulin secretion by injection of Stz. The injection of Stz resulted in a severely depressed fasting insulin level and the inability of the pancreas to respond to a carbohydrate challenge. Without the normal rise in insulin following carbohydrate supplementation, the fasting blood glucose level was increased fourfold above normal. It was lowered significantly by exercise but still remained approximately threefold above Con. Blood glucose was approximately fivefold above Con 5 h after supplementation.

The inability of the pancreas to maintain a normal insulin concentration had a dramatic effect on exercise-responsive GLUT-4 protein expression. In accordance with our laboratory's earlier research, we found that an acute bout of exercise increased the muscle GLUT-4 protein concentration. The increase in protein was significant immediately postexercise and continued to increase during the 5 h following carbohydrate supplementation. GLUT-4 mRNA was significantly increased immediately after exercise but had declined significantly by 5 h after carbohydrate supplementation. The GLUT-4 response patterns for both fast-twitch red and white muscle were similar. In contrast to the non-Stz rats, rats injected with Stz showed no increase in GLUT-4 mRNA or GLUT-4 protein immediately postexercise. During the 5 h following supplementation, there was a significant rise in GLUT-4 mRNA, but there was still no increase in GLUT-4 protein expression. These results indicate that GLUT-4 protein expression can be rapidly increased by an acute bout of exercise, but that this adaptation requires a physiological insulin concentration.

Our finding that GLUT-4 protein was increasing during the time in which GLUT-4 mRNA was declining suggests that the effect of insulin on GLUT-4 protein expression is regulated in part at the level of translation (17). Insulin has been found 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 (18). It has also been found to downregulate GLUT-4 mRNA by inhibiting GLUT-4 gene transcription and reducing GLUT-4 mRNA stability (8), which may explain the downregulation of the GLUT-4 mRNA. However, the finding that the increase in GLUT-4 mRNA in muscle of exercised Stz-treated rats was significantly delayed suggests that insulin has at least a permissive role in the gene transcription process. This could involve the regulation or synthesis of transcription factors required for exercise-induced GLUT-4 gene transcription.

Full expression of the GLUT-4 gene appears to require the concerted interaction of the transcription factors MyoD, myocyte enhancer factor-2 (MEF-2), and the thyroid receptor-{alpha}1 (7). Recently, Mora and Pessin (21) reported that nuclear extracts from insulin-deficient rats had reduced binding to the MEF-2 binding site compared with extracts from non-insulin-deficient rats and that insulin deficiency resulted in the down-regulation of the MEF-2 isoform MEF-2A. Replacement of MEF-2A to nuclear extracts from insulin-deficient rats fully restored binding activity of the MEF-2 element. In addition, Zheng et al. (27) reported that the adenosine analog 5-amino-imidazole-4-carboxamide ribonucleoside activates the GLUT-4 gene similar to exercise and that 5-amino-imidazole-4-carboxamide ribonucleoside increased MEF-2 binding to its DNA binding site. It is interesting to note that simply acutely raising plasma insulin levels by carbohydrate supplementation will not result in an increase in GLUT-4 mRNA or protein expression (17). Taken together, this information suggests that insulin deficiency may result in the downregulation of crucial transcription factors such as MEF-2A, which are essential for both exercise and hormonal regulation of GLUT-4 gene expression.

While it is likely that the repressed muscle GLUT-4 protein expression in the Stz rats was due to a reduced insulin concentration, other possibilities exist. First, the Stz rats were hyperglycemic, and, second, they were in all likelihood hyperlipidemic, both of which can cause insulin resistance. However, it is unlikely that the hyperglycemia would have had an effect on GLUT-4 protein expression under the present experimental paradigm. The glucose toxicity that results from hyperglycemia requires a profuse cellular glucose uptake and, therefore, should not have had an effect under the present conditions in which plasma insulin and muscle glucose uptake were suppressed. Similarly, it is unlikely that a high plasma lipid concentration could account for the slow increase in GLUT-4 mRNA and inability of the Stz rats to increase their muscle GLUT-4 protein levels. Animal models that are severely hyperlipidemic, such as the fatty Zucker rat (fa/fa), have normal skeletal muscle GLUT-4 protein concentrations, and their skeletal muscle GLUT-4 protein response to exercise training is normal (1, 9).

With regard to muscle glycogen, Stz treatment caused a reduction in fast-twitch red muscle glycogen but not in fast-twitch white muscle glycogen. After exercise, the difference in muscle glycogen stores between Stz-treated and nontreated rats was eliminated. In Stz-treated rats, carbohydrate supplementation was able to partially return the muscle glycogen stores back to preexercise levels, but only in the nontreated Stz rats was muscle glycogen increased above preexercise levels (glycogen supercompensation). These results agree with earlier studies in which it was reported that insulin is not required for initial restoration of skeletal muscle glycogen stores after a glycogen-depleting exercise but that it is essential for glycogen supercompensation (10, 13, 24).

After a glycogen-depleting exercise, two phases of glycogen resynthesis are typically observed in the rat (10, 13). Immediately after exercise, there is a rapid initial phase of glycogen storage to near normal levels, which can occur in the absence of insulin. This increase in glycogen storage is facilitated by an increase in membrane permeability to glucose due to a protracted increase in glucose transporters incorporated into the plasma membrane during contraction (11, 19) and activation of glycogen synthase as a result of glycogen depletion (6). Thus the increase in glycogen stores in the Stz-treated rats after exercise was likely due to the protracted increase in glucose transport and increase in glycogen synthase activity.

After the insulin-independent effect of exercise on glucose transport subsides, it is replaced by a marked increase in the sensitivity and responsiveness of muscle to insulin (10, 26). This increase in insulin action can be sustained for a prolonged period and does not seem to reverse completely until glycogen supercompensation has occurred (4, 26). The inability of Stztreated rats to glycogen supercompensate was not due to an inability to activate muscle glycogen synthase. Postexercise glycogen synthase activity, as measured by its activity ratio, was significantly increased in both Stz-treated and nontreated rats. Furthermore, the increase in sensitivity of glycogen synthase to activation by G-6-P immediately postexercise was similar in muscle of Stz-treated and nontreated rats and was significantly higher in Stz-treated rats 5 h after carbohydrate supplementation. These results, therefore, suggest that substrate availability was rate limiting for glycogen storage in the Stz-treated rats. Because blood glucose was in excess of 400 mg% in Stz-treated rats after carbohydrate supplementation, substrate availability was likely limited by the inability of the Stz-treated rats to maintain an active muscle glucose transport after the insulin-independent phase of glycogen storage. It may also have been limited by the inability of the Stz-treated rats to increase GLUT-4 protein expression.

In the present study, we observed a rapid increase in GLUT-4 protein expression in skeletal muscle of the rat, which was insulin dependent. Insulin-stimulated muscle glucose transport is increased with increasing GLUT-4 protein concentration (12, 14), which appears to augment the rate and magnitude of glycogen storage after carbohydrate supplementation. In support of this premise is the observation that trained rats, which have an overexpression of muscle GLUT-4 protein, are more capable of storing muscle glycogen than untrained rats (22). Moreover, our laboratory previously found that 16 h after exercise-carbohydrate supplementation, compared with carbohydrate supplementation alone, muscle glycogen of the rat was significantly increased. Exercised rats were found to have a higher muscle GLUT-4 protein concentration, and this difference was found to account for 65% of the variance in glycogen storage (17). These results imply that, during an exercise-carbohydrate supplementation regimen, muscle GLUT-4 expression may directly control the magnitude of glycogen storage by elevating the glucose transport response to insulin stimulation.

In summary, the present findings indicate that the exercise-induced increase in GLUT-4 protein expression requires insulin. Insulin appears to assert its effect at the level of transcription and translation. Although insulin-deficient rats are capable of restoring their muscle glycogen stores to near normal levels in response to carbohydrate feeding postexercise, they are incapable of glycogen supercompensation. This suggests that the exercise-induced increase in GLUT-4 protein expression may be partially responsible for elevated glycogen stores that follow an exercise-carbohydrate supplementation regimen.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This research was supported by an American Diabetes Association research grant.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We recognize the excellent technical assistance provided by Joe Hancock and Zhenping Ding.

Present address of C.-H. Kuo: Taipei Physical Education College, Taipei, Taiwan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Ivy, Dept. of Kinesiology and Health Education, Bellmont Hall 222, Univ. of Texas at Austin, Austin, TX 78712 (E-mail: johnivy{at}mail.utexas.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Banks EA, Brozinick JT Jr, Yaspelkis BB III, Kang HY, and Ivy JL. Muscle glucose transport, GLUT-4 content, and degree of exercise training in obese Zucker rats. Am J Physiol Endocrinol Metab 263: E1010-E1015, 1992.[Abstract/Free Full Text]
  2. Bloch G, Chase JR, Meyer DB, Avison MJ, Shulman GI, and Shulman RG. In vivo regulation of rat muscle glycogen resynthesis after intense exercise. Am J Physiol Endocrinol Metab 266: E85-E91, 1994.[Abstract/Free Full Text]
  3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976.[CrossRef][ISI][Medline]
  4. Cartee GD, Young DA, Sleeper MD, Zierath J, Wallberg-Henriksson H, and Holloszy JO. Prolonged increase in insulin-stimulated glucose transport in muscle after exercise. Am J Physiol Endocrinol Metab 256: E494-E499, 1989.[Abstract/Free Full Text]
  5. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987.[ISI][Medline]
  6. Danforth WH. Glycogen synthetase activity in skeletal muscle: interconversion of two forms and control of glycogen synthesis. J Biol Chem 240: 588-593, 1965.[Free Full Text]
  7. Dohm GL. Invited Review: Regulation of skeletal muscle GLUT-4 expression by exercise. J Appl Physiol 93: 782-787, 2002.[Abstract/Free Full Text]
  8. Flores-Riveros JR, McLenithan JC, Ezaki O, and Lane MD. Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover. Proc Natl Acad Sci USA 90: 512-516, 1993.[Abstract/Free Full Text]
  9. Friedman JE, Sherman WM, Reed MJ, Elton CW, and Dohm GL. Exercise training increases glucose transporter protein GLUT-4 in skeletal muscle of obese Zucker (fa/fa) rats. FEBS Lett 268: 13-16, 1990.[CrossRef][ISI][Medline]
  10. Garetto LP, Richter EA, Goodman MN, and Ruderman NB. Enhanced muscle glucose metabolism after exercise in the rat: the two phases. Am J Physiol Endocrinol Metab 246: E471-E475, 1984.[Abstract/Free Full Text]
  11. Goodyear LJ, Hirshman MF, King PA, Horton ED, Thompson CM, and Horton ES. Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise. J Appl Physiol 68: 193-198, 1990.[Abstract/Free Full Text]
  12. Henriksen EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, and Holloszy JO. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol Endocrinol Metab 259: E593-E598, 1990.[Abstract/Free Full Text]
  13. Ivy JL. Role of insulin during exercise-induced glycogenesis in muscle: effect on cyclic AMP. Am J Physiol Endocrinol Metab Gastrointest Physiol 233: E509-E513, 1977.[Abstract/Free Full Text]
  14. Kern M, Wells JA, Stephens JM, Elton CW, Friedman JE, Tapscott EG, Pekala PH, and Dohm GL. Insulin responsiveness in skeletal muscle is determined by glucose transporter (GLUT4) protein level. Biochem J 270: 397-400, 1990.[ISI][Medline]
  15. Kimball SR, Jefferson LS, Fadden P, Haystead TA, and Lawrence JC Jr. Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle. Am J Physiol Cell Physiol 270: C705-C709, 1996.[Abstract/Free Full Text]
  16. Kuo CH, Browning KS, and Ivy JL. Regulation of GLUT4 protein expression and glycogen storage after prolonged exercise. Acta Physiol Scand 165: 193-201, 1999.[CrossRef][ISI][Medline]
  17. Kuo CH, Hunt DG, Ding Z, and Ivy JL. Effect of carbohydrate supplementation on postexercise GLUT-4 protein expression in skeletal muscle. J Appl Physiol 87: 2290-2295, 1999.[Abstract/Free Full Text]
  18. Lin TA, Kong X, Haystead TA, Pause A, Belsham G, Sonenberg N, and Lawrence JC Jr. PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science 266: 653-656, 1994.[Abstract/Free Full Text]
  19. Lund S, Holman GD, Schmitz O, and Pedersen O. Contraction stimulates translocation of glucose transporter GLUT-4 in skeletal muscle through a mechanism distinct from that of insulin. Proc Natl Acad Sci USA 92: 5817-5821, 1995.[Abstract/Free Full Text]
  20. Mæhlum S, Høstmark AT, and Hermansen L. Synthesis of muscle glycogen during recovery after prolonged severe exercise in diabetic and non-diabetic subjects. Scand J Clin Lab Invest 37: 309-316, 1977.[ISI][Medline]
  21. Mora S and Pessin JE. The MEF2A isoform is required for striated muscle-specific expression of the insulin-responsive GLUT4 glucose transporter. J Biol Chem 275: 16323-16328, 2000.[Abstract/Free Full Text]
  22. Nakatani A, Han DH, Hansen PA, Nolte LA, Host HH, Hickner RC, and Holloszy JO. Effect of endurance exercise training on muscle glycogen supercompensation in rats. J Appl Physiol 82: 711-715, 1997.[Abstract/Free Full Text]
  23. Price TB, Perseghin G, Duleba A, Chen W, Chase J, Rothman DL, Shulman RG, and Shulman GI. NMR studies of muscle glycogen synthesis in insulin-resistant offspring of parents with non-insulin-dependent diabetes mellitus immediately after glycogen-depleting exercise. Proc Natl Acad Sci USA 93: 5329-5334, 1996.[Abstract/Free Full Text]
  24. Price TB, Rothman DL, Taylor R, Avison MJ, Shulman GI, and Shulman RG. Human muscle glycogen resynthesis after exercise: insulin-dependent and -independent phases. J Appl Physiol 76: 104-111, 1994.[Abstract/Free Full Text]
  25. Ren JM, Semenkovich CF, Gulve EA, Gao J, and Holloszy JO. Exercise induces rapid increases in GLUT4 expression, glucose transport capacity, and insulin-stimulated glycogen storage in muscle. J Biol Chem 269: 14396-14401, 1994.[Abstract/Free Full Text]
  26. Richter EA, Garetto LP, Goodman MN, and Ruderman NB. Enhanced muscle glucose metabolism after exercise: modulation by local factors. Am J Physiol Endocrinol Metab 246: E476-E482, 1984.[Abstract/Free Full Text]
  27. Zheng D, MacLean PS, Pohnert SC, Knight JB, Olson AL, Winder WW, and Dohm GL. Regulation of muscle GLUT-4 transcription by AMP-activated protein kinase. J Appl Physiol 91: 1073-1083, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. E. Civitarese, M. K. C. Hesselink, A. P. Russell, E. Ravussin, and P. Schrauwen
Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes
Am J Physiol Endocrinol Metab, December 1, 2005; 289(6): E1023 - E1029.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
L. J. Cluberton, S. L. McGee, R. M. Murphy, and M. Hargreaves
Effect of carbohydrate ingestion on exercise-induced alterations in metabolic gene expression
J Appl Physiol, October 1, 2005; 99(4): 1359 - 1363.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
96/2/621    most recent
00830.2003v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuo, C.-H.
Right arrow Articles by Ivy, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuo, C.-H.
Right arrow Articles by Ivy, J. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.