Journal of Applied Physiology
Vol. 82, No. 6, pp. 1875-1881, June 1997
METABOLISM

Muscle adaptations to hindlimb suspension in mature and old Fischer 344 rats

Craig S. Stump, Charles M. Tipton, and Erik J. Henriksen

Muscle Metabolism Laboratory, Department of Physiology, University of Arizona, Tucson, Arizona 85721-0093

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Stump, Craig S., Charles M. Tipton, and Erik J. Henriksen. Muscle adaptations to hindlimb suspension in mature and old Fischer 344 rats. J. Appl. Physiol. 82(6): 1875-1881, 1997.We examined skeletal and cardiac muscle responses of mature (8 mo) and old (23 mo) male Fischer 344 rats to 14 days of hindlimb suspension. Hexokinase (HK) and citrate synthase (CS) activities and GLUT-4 glucose transporter protein level, which are coregulated in many instances of altered neuromuscular activity, were analyzed in soleus (Sol), plantaris (Pl), tibialis anterior (TA), extensor digitorum longus (EDL), and left ventricle. Protein content was significantly (P < 0.05) lower in all four hindlimb muscles after suspension compared with controls in both mature (21-44%) and old (17-43%) rats. Old rats exhibited significantly lower CS activities than mature rats for the Sol, Pl, and TA. HK activities were significantly lower in the old rats for the Pl (19%) and TA (33%), and GLUT-4 levels were lower in the old rats for the TA (38%) and EDL (24%) compared with the mature rats. Old age was also associated with a decrease in CS activity (12%) and an increase in HK activity (14%) in cardiac muscle. CS activities were lower in the Sol (20%) and EDL (18%) muscles from mature suspended rats and in the Sol (25%), Pl (27%), and EDL (25%) muscles from old suspended rats compared with corresponding controls. However, suspension was associated with significantly higher HK activities for all four hindlimb muscles examined, in both old (16-57%) and mature (10-43%) rats, and higher GLUT-4 concentrations in the TA muscles of the old rats (68%) but not the mature rats. These results indicate that old age is associated with decreased CS and HK activities and GLUT-4 protein concentration for several rat hindlimb muscles, and these variables are not coregulated during suspension. Finally, old rat skeletal muscle appears to respond to suspension to a similar or greater degree than mature rat muscle responds.

age; soleus; plantaris; extensor digitorum longus; tibialis anterior; cardiac muscle; simulated weightlessness; weight-bearing; GLUT-4 protein; hexokinase; citrate synthase

INTRODUCTION

HINDLIMB SUSPENSION OF RATS is a model of simulated microgravity that has been used to investigate a variety of skeletal muscle adaptations during non-weight-bearing conditions (31). Particularly notable during suspension is the atrophy of antigravity hindlimb muscles, especially the soleus (Sol), which has a high percentage of slow oxidative fibers (17, 29-31). Alternatively, mass is affected less by hindlimb suspension in muscles that are not normally involved in weight bearing or are composed of predominantly fast-glycolytic fibers, such as the extensor digitorum longus (EDL). Previous animal studies using simulated (4, 11, 12, 16, 22, 29, 30) or actual (3, 32) microgravity have also suggested that the capacity of hindlimb muscles to take up, phosphorylate, and oxidize glucose is increased per unit mass. Evidence for this comes from studies utilizing hindlimb perfusion (29, 30) and isolated muscle incubation techniques (11-13, 32), as well as from the examination of muscle enzyme activities (4, 11, 12, 22) and GLUT-4 protein concentrations (11, 12, 16).

GLUT-4 is the predominant glucose transporter protein expressed in skeletal muscle, and its concentration is associated with glucose transport capacity (10, 19). GLUT-4 concentration has been shown to increase in Sol muscles from young rats after hindlimb suspension periods of 7 days or less (11, 12, 16). In addition, the ability to phosphorylate glucose in the cytosol as measured by hexokinase activity is increased in rat Sol muscles after hindlimb suspension (3, 11, 27) and spaceflight (3). Previous enzyme data also suggest an increase in the glycolytic capacity of individual fibers from Sol muscles (3, 4) after various periods of hindlimb suspension (4) and after spaceflight (3). Finally, Sol (4, 11, 12) and gastrocnemius (4) muscles appear to maintain or increase citrate synthase activity after non-weight-bearing conditions.

Conversely, aging is associated with significant decreases in glycolytic capacity and in mitochondrial enzyme activities of several rat hindlimb muscles (2). Hexokinase activity has been shown to decrease with age in some studies (20, 26) but remained unchanged in others (2, 15) for a variety of muscles. Furthermore, GLUT-4 protein concentration decreases in skeletal muscles of rats over their life span. However, most of this decrease occurs during development from juvenile (1-3.5 mo) to young adult (10-13 mo) ages (2, 5).

Recent studies have suggested that the regulation of GLUT-4 protein levels may be coregulated with hexokinase (14, 21) and citrate synthase (9, 25) under conditions of increased neuromuscular activity. However, some dissociation in expression of these elements has been observed in Sol muscles from young rats with renewed weight bearing after hindlimb suspension (12). In addition, nearly every previous hindlimb suspension study examined rats <8 mo of age that had not attained peak muscle mass. Therefore, the purpose of this study was to compare mature (8 mo) with aged senescent (23 mo) male Fischer 344 rats for responses of hindlimb muscle GLUT-4 glucose transporter protein concentration and of hexokinase and citrate synthase enzyme activities to 14 days of hindlimb suspension. We hypothesized that GLUT-4 protein concentration and hexokinase and citrate synthase enzyme activities would increase in rat antigravity Sol and plantaris (Pl) muscles after hindlimb suspension. Furthermore, changes in GLUT-4 concentration and hexokinase and citrate synthase enzyme activities would be similar in magnitude for mature and old rats. We selected the antigravity Sol (90% slow oxidative) and Pl (53% fast glycolytic and 40% fast oxidative glycolytic) muscles, and the non-weight-bearing tibialis anterior (TA; 79% fast glycolytic) and EDL (79% fast glycolytic) muscles to compare differences based on fiber type composition (1) as well as on participation in weight-bearing function.

METHODS

RESULTS

Table  1.   Effect of old age and hindlimb suspension on muscle mass and protein concentration Group Sol Pl TA EDL Muscle mass, mg MC 136 ± 4  353 ± 12  632 ± 15  149 ± 4  MS 79 ± 3* 251 ± 5* 477 ± 10* 126 ± 2* OC 134 ± 3  347 ± 12  608 ± 14  146 ± 4  OS 84 ± 3* 238 ± 4* 476 ± 9* 128 ± 2* Muscle mass relative to body mass, mg/100 g MC 36.4 ± 0.4  94.4 ± 3.6  169.1 ± 1.7  40.0 ± 0.6  MS 26.9 ± 1.3* 85.5 ± 1.7* 162.0 ± 3.3  42.8 ± 0.8* OC 31.9 ± 0.5 82.7 ± 2.4 144.9 ± 3.1 34.8 ± 0.9 OS 23.8 ± 1.1* 67.7 ± 1.4* 135.5 ± 4.0* 36.5 ± 0.8  Muscle protein concentration, mg/g MC 175.3 ± 2.6  182.9 ± 4.8  200.8 ± 3.4  187.6 ± 3.5  MS 169.8 ± 2.8  174.2 ± 4.5  201.0 ± 2.8  174.5 ± 2.8* OC 170.7 ± 1.7  185.4 ± 4.3  199.7 ± 3.5  180.1 ± 2.4  OS 155.6 ± 3.3* 180.3 ± 2.2  199.0 ± 4.0  170.8 ± 6.2 Values are means ± SE. MC, mature control (8 mo); MS, mature suspended; OC, old control (23 mo); and OS, old suspended; Sol, soleus; Pl, plantaris; TA, tibialis anterior; and EDL, extensor digitorum longus. * Significantly different from corresponding control value; OC value significantly different from MC value, P < 0.05.

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Table  2.   Effect of old age and hindlimb suspension on cardiac muscle from left ventricle Group Protein Concentration, mg/g GLUT-4 Concentration, units/µg protein Hexokinase Activity, nmol · mg1 · min1 Citrate Synthase Activity, nmol · mg1 · min1 MC 171.1 ± 5.1  100 ± 3  30.0 ± 0.4  824 ± 27  MS 180.5 ± 3.1  93 ± 3  28.8 ± 1.6  853 ± 15  OC 174.3 ± 2.7  94 ± 3  34.1 ± 0.9* 723 ± 17* OS 175.7 ± 3.1  92 ± 2  34.3 ± 1.0  775 ± 19 Values are means ± SE. * Statistically significant difference between MC and OC groups, P < 0.05.

DISCUSSION

To our knowledge, this is the first study to examine skeletal muscle adaptations to hindlimb suspension comparing aged senescent rats (23 mo) with mature rats (8 mo). These ages were selected to examine the effects of old age separate from the developmental period. Previously, the vast majority of studies using hindlimb suspension have examined animals <6 mo of age. However, one study has compared developing young (3 mo) with old (23 mo) rats for various skeletal muscle enzymatic adaptations (27). The present study measured GLUT-4 glucose transporter protein concentrations and the activities of hexokinase and citrate synthase enzymes in a variety of rat hindlimb muscles. These factors represent indexes for the transport, phosphorylation, and oxidation steps of glucose metabolism. Fischer 344 rats were selected because they do not become obese with age; therefore, the effects of age per se can be more readily evaluated (33). Furthermore, the animals were hindlimb suspended for 14 days so that significant changes in the Pl, EDL, and TA might be observed. Previous studies have shown that significant changes in Sol mass occur within the first 3 days of suspension (11, 17, 31), but changes in other hindlimb muscles may not become apparent until after 1 wk or longer (17, 29-31).

In this study, old age was associated with a decrease in GLUT-4 concentration for the TA and EDL muscles but not for the Sol or Pl (Fig. 2). Previous studies comparing 10- to 17-mo-old rats with 24- to 29-mo-old rats did not show a significant difference in GLUT-4 concentration for any of the muscles examined (2, 5, 6, 18). The reasons for the discrepancy between the results of those studies and the findings of the present study are not clear. However, the TA was not examined previously, and the only study that examined the EDL showed a trend toward a decrease in GLUT-4 concentration when comparing 6- to 8-mo-old with 27- to 29-mo-old rats (18). The GLUT-4 results for the Sol and Pl muscles in the present study are consistent with Gulve et al. (5).

Surprisingly, non-weight-bearing-induced atrophy of the Sol and Pl (Table 1, Fig. 1) did not result in significant changes in GLUT-4 concentration for either the 8-mo-old rats or the 23-mo-old rats (Fig. 2). Previous studies using young rats have consistently shown increased Sol muscle GLUT-4 concentrations (33-107%) after 3-7 days of hindlimb suspension (10, 11, 16), the greatest increase being observed after 7 days (11). Furthermore, insulin-stimulated glucose transport activity in Sol muscle increases to a similar extent after comparable non-weight-bearing periods (11, 13). This is consistent with other studies showing a strong correlation between GLUT-4 concentration and glucose transport capacity (10, 19). The reason GLUT-4 concentration was not increased in the Sol and Pl with hindlimb suspension in this study is obscure. It may be that the increases in GLUT-4 after non-weight-bearing-induced atrophy are transient, reaching a peak at 7 days but normalizing by 14 days. However, Stump et al. (30) demonstrated that glucose uptake remains elevated at maximally stimulating insulin concentrations after 14 days of unilateral hindlimb suspension in both the Sol and Pl muscles from 3-mo-old rats. This result may imply a dissociation between muscle GLUT-4 concentration and glucose transport or may be due to a lesser response to hindlimb suspension in mature and aged rats. Two studies reported that muscle GLUT-4 protein concentration was not increased in old rats (25-29 mo) with exercise training but was increased in mature rats (6-17 mo) (6, 18). This finding may indicate that muscles from senescent animals are generally less plastic in terms of upregulating GLUT-4 protein content. Conversely, a recent study reported a 55% increase in rat hindlimb muscle GLUT-4 concentration in 24-mo-old Fischer 344 rats after 8 wk of exercise training (33). Furthermore, a decrease in senescent muscle plasticity would not explain the lack of an increase in GLUT-4 concentration for the 8-mo-old rats after suspension in the present study.

GLUT-4 protein concentration increased in the TA muscles of the old rats after hindlimb suspension compared with controls, reaching levels not significantly different from those in the mature rats' TA muscles (Fig. 2). Interestingly, suspension of the mature animals did not have a significant effect on TA GLUT-4 concentration. GLUT-4 protein content of the TA has not been measured previously after hindlimb suspension. However, glucose transport activity of the EDL, another anterior leg compartment muscle of similar fiber type composition, has been examined. Whereas no change in insulin-stimulated glucose transport was observed in the EDL of juvenile rats (1 mo) with 6 days of hindlimb suspension (13), evidence for increased glucose uptake has been observed in 3-mo-old rats after 14 days of hindlimb suspension (29, 30). The results of the latter studies have been postulated to be caused by either the increased stretch on the anterior leg compartment as the rats' hindlimbs assume exaggerated plantar flexion after 4 days of suspension (24, 29) or by systemic factors (30). The increase in GLUT-4 in the TA muscles from the OS rats in the present study may be the result of similar mechanisms. Alternatively, the TA muscle from the OS rats was significantly less in mass relative to body mass compared with the OC rats (Table 1). This trend was not observed for the mature rats' TA muscles or for the EDL muscles from either the mature or old rats, indicating that 14 days of hindlimb suspension may have resulted in selective atrophy of the TA muscle in the old rats. Henriksen et al. (11) have shown that GLUT-4 concentrations are increased in Sol muscles only after significant atrophy has occurred. However, as noted above, the fact that, in the present study, GLUT-4 concentrations did not change for the mature or old rats after atrophy of the Sol and Pl muscles indicates that this is not the only factor to be considered.

Hexokinase phosphorylates glucose as it enters the muscle cell and, therefore, represents the first step in glucose metabolism. Surprisingly, the effects of old age on hexokinase activity have been inconsistent in previous studies. Some studies have reported no change in hexokinase activity with aging for a variety of muscles (2, 15). In addition, Klitgaard et al. (20) did not observe a difference in hexokinase activity in rat Sol and Pl muscles when comparing 9-mo-old with 24-mo-old rats. However, a significant decrease was noted at 29 mo of age. In addition, when comparing 6-mo-old with 24-mo-old rats, Sanchez et al. (26) demonstrated a decrease in hexokinase activity in Sol and EDL muscles by 20 and 8%, respectively. Interestingly, the TA and Pl muscles were 33 and 19% lower in hexokinase activity after 23 mo compared with 8 mo in the present study, whereas the Sol and EDL muscles only exhibited a nonsignificant trend toward a decrease (Fig. 3). Conversely, hindlimb suspension resulted in significant increases in hexokinase in all four muscles examined for both the mature (10-43%) and old (16-31%) rats (Fig. 3). Similar increases in hexokinase activity have been observed in Sol (3, 27), gastrocnemius (27), and TA (3, 27) muscles from young (3-5 mo) rats after various periods of hindlimb suspension (3, 27) and spaceflight (3). In addition, hexokinase activity has been shown to increase in Sol muscles from 22-mo-old rats after 3 wk of hindlimb suspension, but increases in the gastrocnemius muscles were not statistically significant (27). Collectively, these results indicate that old age is associated with no change or a decrease in hexokinase activity, depending on the muscles examined and the ages selected. Conditions of simulated or actual microgravity consistently cause increases in hexokinase activity in most hindlimb muscles.

Citrate synthase activity was measured as an index of hindlimb muscle oxidative capacity for the mature and old rats (Fig. 4). Activities were lower (14-38%) in the 23-mo-old compared with the 8-mo-old rats for the Sol, Pl, and TA muscles after control conditions. This decrease is consistent with previous animal studies examining a variety of mitochondrial enzymes for several muscles and is probably secondary to a decrease in the total number of mitochondria (2). Hindlimb suspension in the present study had similar effects on citrate synthase activity for the mature and old animals (Fig. 4). Generally, there was a decrease in activity for the Sol and EDL muscles (18-27%). In addition, the Pl muscle was significantly lower in citrate synthase activity in the OS compared with the OC rats. Previous studies have found that, when muscle homogenates are examined, citrate synthase activity is decreased in Sol muscles after hindlimb suspension (31). In contrast, citrate synthase activity in individual Sol and gastrocnemius fibers was increased and not changed, respectively, after a 2- to 4-wk non-weight-bearing period (4). This discrepancy is apparently due to the increase in interstitial space relative to total mass that occurs in non-weight-bearing Sol muscles (30). However, this result has not been demonstrated for other hindlimb muscles (30). Our citrate synthase activity data were obtained from muscle homogenates and are expressed relative to total muscle protein. Furthermore, we believe that the decrease in cellular oxidative capacity after 14 days of hindlimb suspension in these animals has biological significance. Although increases in muscle interstitial space may have contributed to the decrease in activity observed in the Sol, it is unlikely that this effect would have masked a significant increase in cellular enzyme activity. By using similar muscle homogenate procedures, two studies have demonstrated in juvenile rats a significant increase in citrate synthase activity after 3-day (12) and 7-day (11) non-weight-bearing periods. Therefore, the decrease in citrate synthase activity after hindlimb suspension that was observed in the present study may be related to the age of the animals.

In the present study, the difference in myocardial GLUT-4 concentration with age was not statistically significant (Table 2). Cartee (2) reported a 12% decrease in GLUT-4 between 13 and 25 mo of age in Fischer 344/Brown Norway F1 hybrid rats, and Hall et al. (7) found a 27% decrease in myocardial GLUT-4 concentration when comparing 25-mo-old with 7-mo-old Fischer 344 rats. The old rats exhibited a 14% increase in hexokinase activity and a 12% decrease in citrate synthase activity in the present study (Table 2). Hansford and Castro (8) reported a generalized decrease in oxidative capacity in hearts from 24-mo-old rats compared with 6-mo-old rats, including decreases in citrate synthase activity and fatty acid oxidation. In addition, an increase in left ventricular hexokinase activity has been reported previously for old Fischer 344 rats (28). Collectively, these results suggest an increased cardiac muscle reliance on glycolytic energy sources and less on oxidative steps, especially fatty acid oxidation, with old age. Fourteen days of hindlimb suspension did not affect GLUT-4 concentration or hexokinase or citrate synthase activities (Table 2).

In summary, the results of this study indicate that hindlimb muscle GLUT-4 protein concentration, hexokinase activity, and citrate synthase activity tended toward lower values for the 23-mo-old compared with the 8-mo-old rats. However, the TA was the only muscle that was significantly lower for all three variables. This study also demonstrated that hindlimb muscles from mature and old rats responded similarly to suspension-induced non-weight-bearing. Decreases in muscle mass (Table 1) and total protein (Fig. 1) were nearly identical for the two age groups. In addition, hexokinase activities were increased after suspension for all four muscles examined, citrate synthase activities tended to decrease, and GLUT-4 protein levels were generally not different between the suspended and control animals for either age group. The exceptions, in which the old muscle responded to hindlimb suspension to a greater extent than the mature muscle, included a significant increase in GLUT-4 concentration, a greater increase in hexokinase activity, and a decrease in muscle mass relative to body mass for the TA. The enzyme results from this study are consistent with previous enzyme data (3, 4), suggesting that rat hindlimbs are more reliant on glycolytic metabolism after hindlimb suspension. The GLUT-4 concentration data are less convincing, although we do not have muscle glucose transport data for these animals nor do we know the transporter distribution or intrinsic activity. Finally, the results of this study did not indicate that GLUT-4 protein levels and hexokinase or citrate synthase activities were regulated together during 14 days of hindlimb suspension, as had been suggested previously with increased neuromuscular activity (9, 14, 21, 25).

ACKNOWLEDGEMENTS

We thank Lisa Sebastian and P. K. Edwards for excellent technical assistance.

FOOTNOTES

Address for reprint requests: E. J. Henriksen, Dept. of Physiology, Ina E. Gittings Bldg. no. 93, Univ. of Arizona, Tucson, AZ 85721-0093.

Received 28 October 1996; accepted in final form 20 February 1997.

REFERENCES