Journal of Applied Physiology AJP: Cell Physiology
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J Appl Physiol 82: 772-775, 1997;
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Journal of Applied Physiology
Vol. 82, No. 3, pp. 772-775, March 1997
METABOLISM

Enlargement of glycogen store in rat liver and muscle by fructose-diet intake and exercise training

Taro Murakami1, Yoshiharu Shimomura1, Noriaki Fujitsuka1, Masahiro Sokabe2, Koji Okamura3, and Shuichi Sakamoto3

1 Department of Bioscience, Nagoya Institute of Technology, Gokiso, and 2 Department of Physiology, Nagoya University School of Medicine, Showa-Ku, Nagoya 466; and 3 Otsuka Pharmaceutical Co., Ltd., Kanzaki-Gun, Saga 842-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Murakami, Taro, Yoshiharu Shimomura, Noriaki Fujitsuka, Masahiro Sokabe, Koji Okamura, and Shuichi Sakamoto. Enlargement of glycogen store in rat liver and muscle by fructose-diet intake and exercise training. J. Appl. Physiol. 82(3): 772-775, 1997.---This study investigated the effect of long-term intake of a fructose diet and exercise training on glycogen content in liver and skeletal muscle in female rats. Thirty-six rats (8 wk old) were divided into two dietary groups and were fed with a control (chow) diet or fructose diet (containing 20% fructose) for 12 wk. During this period, one-half of the rats in each dietary group were trained by using a motor-driven treadmill (running speed of 25 m/min and duration of 90 min/day, 5 days/wk). The liver glycogen was increased by intake of a fructose diet and exercise training, and the content was in the following order: control-diet and sedentary rats < fructose-diet and sedentary rats <=  control-diet and trained rats < fructose-diet and trained rats in the ratio of 1:3.4:3.6:5.0. The glycogen content in gastrocnemius muscle showed the same trend as that in liver; the ratio was 1:1.3:1.3:1.6. These results indicate that both long-term intake of the fructose diet and exercise training synergistically increased glycogen in both tissues.

cytochrome oxidase; serum triglyceride; run training

INTRODUCTION

MANY STUDIES HAVE DEMONSTRATED that glycogen in liver and skeletal muscle is important to maintain physical performance during prolonged exercise (1, 19, 20). Therefore, athletes are continuously looking for ways to augment the glycogen contents in tissues before exercise and to spare glycogen during exercise. Although glucose is an important metabolic fuel during exercise, glucose intake before exercise is started results in hyperinsulinemia, which may cause an exercise-induced rapid decrease in blood glucose and promote greater depletion of liver and muscle glycogen (5, 15, 19). In contrast, it has been reported that fructose ingestion causes only modest hyperinsulinemia and no decline in plasma glucose level after exercise is started (13-15). Although effects of fructose on muscle glycogen were reported to be controversial, fructose replenishes liver glycogen more rapidly than does glucose (6). These facts suggest that fructose ingestion before submaximal exercise could promote increasing glycogen store and sparing it during exercise. However, these studies were limited to the effects of short-term feeding of fructose.

It is interesting to examine effects of long-term intake of the fructose diet on glycogen contents in the tissues, because the animals should be adapted to the diet by the long-term intake and the effects of the diet might be different from those of the short-term intake. Here we provide the evidence suggesting that the long-term intake of fructose diet augmented glycogen content in both tissues and that this effect of the fructose diet was further enhanced by exercise training.

MATERIALS AND METHODS

Animal care and experimental design. All procedures involving animals were approved by the experimental animal care committee of the Nagoya Institute of Technology. The rats were housed at 22°C with light from 0500 to 1700.

Effects of long-term feeding of fructose diet and exercise training (experiment 1). Thirty-six female Sprague-Dawley rats (7 wk old) were obtained from CLEA Japan (Tokyo). One week before initiation of the experiment, all of the rats were run on a motor-driven treadmill (model KN-73, Natsume-Seisakusho, Tokyo, Japan) designed for the mouse and rat at 10 m/min of speed for 10 min a day for 4 days to accustom animals to the treadmill. Then the animals (at 8 wk old) were randomly divided into two dietary groups: a control-diet (CE-2, CLEA Japan) group and a fructose-diet group. Fructose (20%, wt/wt) replaced starch in the control diet for the fructose diet. We used starch, but not glucose, as a control carbohydrate for studying long-term intake of fructose because starch is less insulinogenic than glucose (7) and is fully digested. Each dietary group was subdivided into two groups: a sedentary group (n = 9) and an exercise-trained group (n = 9). Rats were fed with free access to the appropriate diet and water for 12 wk. During this period, rats in the trained groups were run on the motor-driven treadmill between 1400 and 1700; running speed was 25 m/min and duration was 90 min/day, 5 days/wk (18). On the final day of the experiment, food was withheld from all rats from 1200 through 1700; then the rats were anesthetized with ether, blood was collected with a syringe from the inferior vena cava for preparation of serum, and then immediately a portion of the largest lobe of liver and the gastrocnemius muscles of both legs were removed, freeze-clamped at liquid nitrogen temperature, and stored at -80°C until used. The rats in the trained group were not exercised 24 h before they were killed.

Effects of short-term feeding of the fructose diet and acute exercise (experiment 2). In this experiment, effects of relatively short-term intake of the fructose diet and a single bout of exercise on the glycogen contents in liver and skeletal muscle were examined by using 32 rats (8 wk old). Rats were fed with free access to the fructose or control diet (n = 16 per each diet group) for 3 wk. In the last week of the experiment, all of the rats were accustomed to the treadmill by the same protocol as described in Effects of long-term feeding of fructose diet and exercise training (experiment 1). One day before they were killed, one-half of rats (n = 8) in each diet group were run on the treadmill at 25 m/min of speed for 90 min. On the final day of the experiment, rats were killed and tissues were treated by the same procedures as described in Effects of long-term feeding of fructose diet and exercise training (experiment 1).

Analyses. The serum triglyceride concentration was determined by the method of Fletcher (9). Extraction of cytochrome oxidase from gastrocnemius muscle was carried out as described previously (18), and the enzyme activity was measured at 25°C by the method of Smith (21). The glycogen contents in liver and gastrocnemius muscle were determined by the method of Lo et al. (16).

Statistics. Data are expressed as means ± SE. To evaluate the differences between groups, data were analyzed by two-way analysis of variance and post hoc (Fisher's protected least significant difference) test. P < 0.05 was defined as statistically significant.

RESULTS

Weights of body, liver, and gastrocnemius muscle and food intake (experiment 1). After the 12 wk, body weight was not different among the four groups (Table 1). Also, the food intake during the last week of the experiment was not different among the groups. These data suggest that control and fructose diets were equally digested and absorbed and that rat growth was not affected by the fructose diet. In contrast to body weight and food intake, liver weight was increased by long-term intake of the fructose diet and by endurance training (Table 1). Gastrocnemius muscle weight in trained rats was slightly greater in the fructose-diet group than in the control-diet group (Table 1).

Table 1. Effect of long-term intake of fructose diet and endurance training on weights of body, liver, and gastrocnemius muscle and on food intake

Control Diet
Fructose Diet
Sedentary Trained Sedentary Trained
Body weight, g 285 ± 7  286 ± 6  287 ± 5  285 ± 6 
Liver weight, g 7.2 ± 0.2  8.1 ± 0.3dagger 9.3 ± 0.4* 10.1 ± 0.3*dagger
Gastrocnemius muscle weight, g 3.5 ± 0.1  3.6 ± 0.1  3.7 ± 0.1  3.9 ± 0.1*
Food intake, g/wk 119 ± 3  128 ± 5  128 ± 6  134 ± 3
Values are means ± SE for 9 rats in each group. Food intake was measured in last week of experiment. * P < 0.05 between dietary groups. dagger P < 0.05 between sedentary and trained groups.

Cytochrome oxidase activity (experiment 1). Cytochrome oxidase is one of the key enzymes in the mitochondrial electron-transport chain, and the activity is used as a marker of the mitochondrial oxidative capacity. To confirm the training effect, we measured this enzyme activity in gastrocnemius muscle. The cytochrome oxidase activity in the muscle was significantly increased by training in both diet groups (1.6- and 1.4-fold for control- and fructose-diet groups, respectively; Fig. 1).
Fig. 1. Cytochrome oxidase activity in gastrocnemius muscle. Values are means ± SE for 9 rats in each group. Open bars, sedentary; filled bars, trained. One unit of cytochrome oxidase catalyzed oxidation of 1 µmol of reduced cytochrome c/min. *P < 0.05.
[View Larger Version of this Image (15K GIF file)]

Serum triglyceride concentration (experiment 1). The serum triglyceride concentration was significantly increased (3.4-fold) by intake of the fructose diet (Fig. 2). However, the concentration in trained rats was not significantly different between two diet groups (Fig. 2), indicating that the fructose-induced increase in serum triglyceride was abolished by endurance training.
Fig. 2. Serum triglyceride concentration. Values are means ± SE for 9 rats in each group. Open bars, sedentary; filled bars, trained. * P < 0.05.
[View Larger Version of this Image (15K GIF file)]

Glycogen contents of liver and gastrocnemius muscle (experiment 1). The glycogen content in liver was significantly increased by 3.7-fold by the fructose diet and by 3.6-fold by training (Fig. 3A). The content in liver was further increased by the combination of fructose-diet intake and endurance training (Fig. 3A). The glycogen content in gastrocnemius muscle showed the same trend as that in liver, although the effects of diet and training were less compared with those observed in liver (Fig. 3B).
Fig. 3. Glycogen content in liver (A) and gastrocnemius muscle (B) after long-term (12-wk) intake of fructose diet and endurance training. Values are means ± SE for 9 rats in each group. Open bars, sedentary; filled bars, trained. * P < 0.05.
[View Larger Version of this Image (19K GIF file)]

Effects of short-term feeding of fructose diet and acute exercise on glycogen contents of liver and gastrocnemius muscle (experiment 2). To determine whether the regulation of glycogen contents in liver and gastrocnemius muscle is dependent on the feeding period and training state, the effects of 3 wk of feeding of the fructose diet and a single bout of exercise on glycogen contents in liver and gastrocnemius muscle were examined. The glycogen content in liver was not altered by a 3-wk intake of the fructose diet (Fig. 4A). On the other hand, the content in liver was significantly increased by acute exercise by 4.0- and 3.9-fold in rats fed control and fructose diets, respectively. However, the synergistic increase in hepatic glycogen content induced by both long-term feeding of fructose and endurance training (Fig. 3A) was not observed in this experiment. The glycogen content in gastrocnemius muscle was not altered by short-term feeding of the fructose diet and acute exercise (Fig. 4B).
Fig. 4. Glycogen content in liver (A) and gastrocnemius muscle (B) after short-term (3-wk) intake of fructose diet and a single bout of exercise. Values are means ± SE for 8 rats in each group. Open bars, sedentary; filled bars, exercised. * P < 0.05.
[View Larger Version of this Image (17K GIF file)]

DISCUSSION

It is well known that the oxidative capacity in skeletal muscle is increased by physical training (3, 10, 11). In this study, the activity of skeletal muscle cytochrome oxidase was elevated by exercise training in both control-diet and fructose-diet groups but was not affected by the diets in either sedentary or trained rats. The training effect was also clearly observed in serum triglyceride concentration; the triglyceride concentration in sedentary rats was elevated threefold by the fructose diet but showed no significant elevation in trained rats. Because liver triglyceride concentration (~20 mg/g wet weight) was not affected by either diet or training in this study, the fructose diet resulted in increased serum triglyceride concentration by increasing triglyceride release from liver, and exercise training ameliorated fructose-induced hypertriglycemia, probably by suppressing conversion of fructose into triglyceride in liver and/or promoting triglyceride utilization in muscle. The latter is the more possible mechanism for suppressing fructose-induced hypertriglycemia, because training increases enzymes responsible for uptake, activation, and oxidation of fatty acids in muscle tissue (2, 17).

The present study clearly demonstrated that glycogen contents in liver and skeletal muscle were increased by both long-term intake of the fructose diet and exercise training and were further increased by the combination of two conditions; i.e., both acted synergistically to increase glycogen store in the tissues. Furthermore, it was confirmed that relatively short-term intake of the fructose diet had no effect on the glycogen contents in the tissues and that the combination of the short-term intake of the fructose diet and a single bout of exercise had no synergistic effect on the glycogen contents, although a single bout of exercise significantly increased the glycogen content only in liver. This is a first report showing this synergistic increase in liver and muscle glycogen by the fructose diet and endurance training.

In the present study, relatively low values of hepatic glycogen contents were obtained. We withheld food from the rats for 5 h to adjust their metabolic conditions before they were killed at ~1700. Because diurnal variation of hepatic glycogen content has been reported (4), we examined effect of the fructose diet on hepatic glycogen content in untrained rats fed the experimetal diet for 3 wk. The hepatic glycogen content (mean ± SE for 4 rats) early in the light period (0700) was 59.0 ± 1.5 mg/g tissue for rats fed the control diet and 61.3 ± 4.4 mg/g tissue for rats fed the fructose diet, and the content at the end of the light period (1700) was decreased to 27.3 ± 3.5 and 26.5 ± 3.4, respectively, in rats fed each diet ad libitum. When rats were starved for 5 h before being killed at 1700, the content was further decreased to 9.6 ± 1.7 and 9.3 ± 1.0 mg/g tissue, respectively. These results indicate that hepatic glycogen content was affected by the time point for killing the rats and by feeding conditions. However, the diurnal variation was not affected by the fructose diet. In the present study, we killed rats at the time point that hepatic glycogen content was low. The same level of glycogen content was reported by other investigators who used the experimental conditions similar to ours (8). The effects of the fructose diet and exercise training on the glycogen content at the time point of high-glycogen content in a day remains to be examined.

It has been shown that fructose intake increases hepatic glycogen (22). Inhibition of glycogen phosphorylase by fructose 1-phosphate produced from fructose is suggested to be responsible for enhancing net glycogen store in liver (25) because a large part of dietary fructose is known to be taken up by liver (23). Exercise training has also been reported to increase hepatic glycogen content. However, the present study showed that a single bout of exercise, but not training, increased hepatic glycogen content to a level similar to that of the glycogen content increased by training. Therefore, the training effect on the hepatic glycogen store may be attributed to a function of the last bout of exercise in the training program. On the other hand, combination of the fructose diet and training had a synergistic effect to increase hepatic glycogen store. A mechanism responsible for the synergistic effect is not clear, but the inhibition of phosphorylase by fructose 1-phosphate may be involved in the mechanisms for increasing hepatic glycogen store in trained rats.

Whether dietary fructose has a direct effect on glycogen metabolism in skeletal muscle is not known. It has been shown that, after animals have been fed high-fructose diets (24), the ability of muscle to metabolize glucose is reduced, whereas its ability to oxidize fatty acids is increased. Endurance training has also been shown to increase fatty acid utilization in muscle tissue (2, 17). Furthermore, training increases glycogen synthase activity in the muscle (12). These effects of fructose and training could be involved in the mechanism for increasing muscle glycogen store in trained rats fed the fructose diet.

In conclusion, this study has shown that dietary fructose and endurance training acted synergistically to increase glycogen content in liver and skeletal muscle in the rat.

FOOTNOTES

Address for reprint requests: Y. Shimomura, Dept. of Bioscience, Nagoya Institute of Technology, Gokiso-Cho, Showa-Ku, Nagoya 466, Japan.

Received 25 September 1995; accepted in final form 25 November 1996.

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