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

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 (PI), 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.

Role of glucose transport in glycogen supercompensation in reweighted rat skeletal muscle.

Hindlimb weight bearing after a 3-day period of hindlimb suspension (reweighting) of juvenile rats results in a marked transient elevation in soleus glycogen concentration that cannot be explained on the basis of the activities of glycogen synthase and phosphorylase. We have hypothesized that enhanced glucose transport activity could underlie this response. We directly tested this hypothesis by assessing the response of insulin-dependent and insulin-independent glucose transport activity (in vitro 2-[1,2-3H]deoxy-D-glucose uptake) as well as glucose transporter (GLUT-4) protein levels during a 48-h reweighting period. After a net glycogen loss (from 29 +/- 2 to 16 +/- 1 nmol/mg muscle; P < 0.05) during the first 2 h of reweighting, glycogen accumulated at an average rate of 1.4 nmol.mg-1.h-1 up to 18 h, reaching an apex of 38 +/- 1 nmol/mg. During this same reweighting period, insulin-independent, but not insulin-dependent, glucose transport activity was significantly enhanced (P < 0.05 vs. weight-bearing control values) and was associated with an elevated level of GLUT-4 protein and the specific activity of total hexokinase. The specific activity of citrate synthase was also increased. By 24 h of reweighting, although insulin-independent glucose transport activity and GLUT-4 protein remained elevated, glycogen accumulation had ceased, likely due to enhanced phosphorylase activity at this time point. These results are consistent with the interpretation that the glycogen supercompensation seen during reweighting of the rat soleus may be regulated in part by an enhanced glucose flux arising from an increase in insulin-independent glucose transport activity and hexokinase activity.

Regulation of skeletal alpha-actin promoter in young chickens during hypertrophy caused by stretch overload.

Anterior latissimus dorsi (ALD) muscles of 3-wk-old male chickens were injected with plasmids containing various lengths of the chicken skeletal alpha-actin promoter (ranging from -2,090 to -77 relative to the transcription start site) driving luciferase. Hypertrophy of the left ALD muscle was induced by attaching a weight (11% of body wt) to the left wing of each chicken, with the unweighted contralateral wing serving the control. Six days of stretch overload significantly increased muscle mass 110%. Luciferase activity from the -2,090 actin-luciferase chimeric gene increased 127% compared with the contralateral control ALD muscle. Luciferase activities driven by the -424, -202, and -99 actin promoters were 179, 134, and 378% higher, respectively, in the stretched ALD muscle than in the contralateral control ALD muscle. Luciferase activity from the -77 deletion construct was not different between stretched and control muscles. These data indicate that the gene region responding to stretch is downstream of -99 and imply, but do not conclusively prove, that the region between -99 and -77, which contains serum response element 1, contributes to the stretch-induced increase in skeletal alpha-actin promoter activity in the ALD muscle.

Elevated interstitial fluid volume in soleus muscles unweighted by spaceflight or suspension.

Recent evidence by Kandarian et al. (J. Appl. Physiol. 71: 910-914, 1991) indicates that prolonged (28 days) unweighting of the rat soleus by hindlimb suspension results in a substantial increase in interstitial fluid volume (IFV), as defined by the inulin space. The lack of any significant difference in absolute IFV values between unweighted and control groups suggested that this elevated IFV was a consequence of muscle atrophy. Using young female rats, we directly tested this hypothesis by comparing the early responses of soleus muscle weight and IFV with unweighting by tail-cast suspension or actual exposure to microgravity during spaceflight. Significant differences from control were first observed after 3 days of suspension unweighting for soleus wet weight (-14%; P < 0.01) and IFV (+35%; P < 0.01) and increased further after 6 days (-32% and +53%, respectively; both P < 0.001). After 5.4 days of spaceflight, soleus wet weight was 38% less and IFV was 52% greater than control (both P < 0.001). A highly significant negative correlation between soleus wet weight and IFV for all groups was observed (r = -0.70, P < 0.001). These data indicate that elevated soleus IFV develops at an early time point during unweighting and that there is a direct relationship between the magnitude of this increase in IFV and the extent of muscle atrophy. This relationship also exists in soleus muscles unweighted by exposure to a microgravity environment.

Muscle glucose uptake in the rat after suspension with single hindlimb weight bearing.

This study was designed to examine the effect of non-weight-bearing conditions and the systemic influences of simulated microgravity on rat hindlimb muscles. For this purpose, rats were suspended (SUS) in a head-down position (45 degrees) with the left hindlimb non-weight bearing (NWB) and the right hindlimb bearing 20% of presuspension body mass (WB). Weight bearing by the SUS-WB limb was accomplished by using a platform connected to a rod in sleeve, cable, and pulley apparatus to which weight could be added. Rats (250-325 g) were assigned to SUS or cage control (CC) conditions for 14 days. The angle between the foot and leg for SUS-WB and CC remained similar (20-30 degrees) throughout the experiment while the SUS-NWB hindlimbs extended to approximately 140 degrees by day 12. On day 14, the soleus, plantaris, and gastrocnemius muscles from the SUS-NWB limbs exhibited significantly lower (P < or = 0.05) masses than presuspension mass values (29, 11, and 21%, respectively). Weight bearing by the SUS-WB limbs prevented the loss of mass by these muscles. In separate groups of SUS and CC rats, 2-deoxyglucose uptake during hindlimb perfusion was significantly higher in both SUS-NWB and SUS-WB hindlimbs at 24,000 microU/ml of insulin compared with CC for all the muscles examined (21-80%). In addition, extracellular space (ml/g) was significantly greater in the soleus muscles from both the SUS-NWB and SUS-WB hindlimbs (64%) compared with CC muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Spaceflight on STS-48 and earth-based unweighting produce similar effects on skeletal muscle of young rats.

Our knowledge of the effects of unweighting on skeletal muscle of juvenile rapidly growing rats has been obtained entirely by using hindlimb-suspension models. No spaceflight data on juvenile animals are available to validate these models of simulated weightlessness. Therefore, eight 26-day-old female Sprague-Dawley albino rats were exposed to 5.4 days of weightlessness aboard the space shuttle Discovery (mission STS-48, September 1991). An asynchronous ground control experiment mimicked the flight cage condition, ambient shuttle temperatures, and mission duration for a second group of rats. A third group of animals underwent hindlimb suspension for 5.4 days at ambient temperatures. Although all groups consumed food at a similar rate, flight animals gained a greater percentage of body mass per day (P < 0.05). Mass and protein data showed weight-bearing hindlimb muscles were most affected, with atrophy of the soleus and reduced growth of the plantaris and gastrocnemius in both the flight and suspended animals. In contrast, the non-weight-bearing extensor digitorum longus and tibialis anterior muscles grew normally. Earlier suspension studies showed that the soleus develops an increased sensitivity to insulin during unweighting atrophy, particularly for the uptake of 2-[1,2-3H]deoxyglucose. Therefore, this characteristic was studied in isolated muscles within 2 h after cessation of spaceflight or suspension. Insulin increased uptake 2.5- and 2.7-fold in soleus of flight and suspended animals, respectively, whereas it increased only 1.6-fold in control animals. In contrast, the effect of insulin was similar among the three groups for the extensor digitorum longus, which provides a control for potential systemic differences in the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin and exercise on rat hindlimb muscles after simulated microgravity.

This study was designed to examine insulin- and exercise-stimulated glucose uptake and metabolism in the hindlimb muscles of rats after conditions of simulated microgravity. To simulate microgravity, male Sprague-Dawley rats were suspended in a head-down (45 degrees) position with their hindlimbs non-weight bearing (SUS) for 14 days. In addition, rats were assigned to suspension followed by exercise (SUS-E), to cage control (CC), or to exercising control (CC-E) groups. Exercise consisted of five 10-min bouts of treadmill running at the same relative intensity for the CC-E and SUS-E rats (80-90% of maximum O2 consumption). Hindlimb perfusion results indicated that glucose uptake for the entire hindquarter at 24,000 microU/ml insulin (maximum stimulation) was significantly higher in the SUS (8.9 +/- 0.5 mumol.g-1.h-1) than in the CC (7.6 +/- 0.4 mumol.g-1.h-1) rats, signifying an increased insulin responsiveness. Glucose uptake at 90 microU/ml insulin was also significantly higher in the SUS (48 +/- 4; % of maximum stimulation over basal) than in the CC (21 +/- 4%) rats. In addition, exercise-induced increases in glucose uptake for the hindlimbs (133%) and glucose incorporation into glycogen for the plantaris (8.4-fold), extensor digitorum longus (5.4-fold), and white gastrocnemius (4.8-fold) muscles were greater for the SUS-E rats than for the CC-E rats (39% and 1.9-, 1.9-, and 3.0-fold, respectively). Therefore, suspension of the rat with hindlimbs non-weight bearing leads to enhanced muscle responses to insulin and exercise when they were applied separately. However, insulin action appeared to be impaired after exercise for the SUS-E rats, especially for the soleus muscle.

Effects of prior exercise on the action of insulin-like growth factor I in skeletal muscle.

Prior exercise increases insulin sensitivity for glucose and system A neutral amino acid transport activities in skeletal muscle. Insulin-like growth factor I (IGF-I) also activates these transport processes in resting muscle. It is not known, however, whether prior exercise increases IGF-I action in muscle. Therefore we determined the effect of a single exhausting bout of swim exercise on IGF-I-stimulated glucose transport activity [assessed by 2-deoxy-D-glucose (2-DG) uptake] and system A activity [assessed by alpha-(methylamino)isobutyric acid (MeAIB) uptake] in the isolated rat epitrochlearis muscle. When measured 3.5 h after exercise, the responses to a submaximal concentration (0.2 nM), but not a maximal concentration (13.3 nM), of insulin for activation of 2-DG uptake and MeAIB uptake were enhanced. In contrast, prior exercise increased markedly both the submaximal (5 nM) and maximal (20 nM) responses to IGF-I for activation of 2-DG uptake, whereas only the submaximal response to IGF-I (3 nM) for MeAIB uptake was enhanced after exercise. We conclude that 1) prior exercise significantly enhances the response to a submaximal concentration of IGF-I for activation of the glucose transport and system A neutral amino acid transport systems in skeletal muscle and 2) the enhanced maximal response for IGF-I action after exercise is restricted to the signaling pathway for activation of the glucose transport system.

Influence of exercise training on the oxidative capacity of rat abdominal muscles.

Our purpose was to determine if endurance exercise training would increase the oxidative capacity of the abdominal expiratory muscles of the rat. Accordingly, 9 male rats were subjected to an endurance training protocol (1 h/day, 6 days/week, 9 weeks) and 9 litter-mates served as controls. Citrate synthase (CS) activity was used as an index of oxidative capacity, and was determined in the following muscles: soleus, plantaris, costal diaphragm, crural diaphragm, and in all four abdominal muscles: rectus abdominis, transversus abdominis, external oblique, and internal oblique. Compared to their non-trained litter-mates, the trained rats had higher peak whole body oxygen consumption rates (+ 16%) and CS activities in plantaris (+34%) and soleus (+36%) muscles. Thus, the training program caused substantial systemic and locomotor muscle adaptations. The CS activity of costal diaphragm was 20% greater in the trained animals, but no difference was observed in crural diaphragm. The CS activity in the abdominal muscles was less than one-half of that in locomotor and diaphragm muscles, and there were no significant changes with training except in the rectus abdominis where a 26% increase was observed. The increase in rectus abdominis CS activity may reflect its role in postural support and/or locomotion, as none of the primary expiratory pumping muscles adapted to the training protocol. The relatively low levels of CS activity in the abdominal muscles suggests that they are not recruited frequently at rest, and the lack of an increase with training indicates that these muscles do not contribute significantly to the increased ventilatory activity accompanying exercise in the rat.

Influences of chemical sympathectomy, demedullation, and hindlimb suspension on the VO2max of rats.

Results from previous studies have shown that the reduction in maximal oxygen consumption (VO2max) with simulated microgravity is attenuated in chemically sympathectomized rats. To determine the contributions of the catecholamines from the adrenal medulla in this process, investigations were conducted with 65 saline injected (SAL) and chemically sympathectomized (SX) female rats that were either surgically demedullated (DM), or intact (IN). Microgravity conditions were simulated by head-down suspension (HDS) while controls were assigned to individual cages (CC). The experimental period was 14 d. The rats were tested for VO2max, treadmill run time (RT), and submaximal mechanical efficiency (ME) prior to suspension and on days 7 and 14. Saline injected rats that had intact adrenal medullas (SAL-IN) exhibited significantly reduced measures of VO2max after 7 and 14 d by 15% and 21%, respectively. No significant reduction in VO2max was observed with HDS in the SX-IN animals. Sympathectomized rats that were demedullated (SX-DM) also exhibited a significant reduction in VO2max (12%). In addition, HDS was associated with a marked and significant reduction in RT in all groups. ME for submaximal exercise was significantly reduced after HDS in SAL-IN rats but not in the SX-IN rats. SX-DM rats experienced significant reductions in ME similar in magnitude to the SAL-IN rats. These results confirm that chemical sympathectomy attenuates the expected decrease in VO2max with HDS and suggests that circulating epinephrine contributes to this response.

Effect of 29 days of simulated microgravity on maximal oxygen consumption and fat-free mass of rats.

To determine the time course of changes in maximal oxygen consumption (VO2max) and its relationships with body composition, a 29-d simulated microgravity study was initiated with 30 adult female rats (230-280 g) randomly assigned to one of three experimental groups; namely, head-down suspension (HDS) at 45 degrees, horizontal suspension (HS) at 0 degree, or cage control (CC). Five separate exercise performance tests with measurements related to VO2max, treadmill run time, and mechanical efficiency were conducted before suspension and on days 7, 14, 21, and 28. The animals were sacrificed and chemically analyzed for fat and fat-free free mass (FFM) between days 22 and 29. When oxygen consumption results were expressed on an absolute basis (ml.min-1), both suspension groups had significant decreases at day 7. Thereafter, the HDS rats remained significantly decreased, while the HS rats returned to values not significantly different than the presuspension values by day 28. When oxygen consumption was expressed on a relative basis (ml.kg-1.min-1), all groups exhibited significant reductions between 6-8%. Both suspended groups had significantly less fat than the CC rats but had similar values for FFM (p greater than 0.05). When VO2max was evaluated on a FFM basis, the HDS rats had a group mean that was 6% lower than the other two groups, but the difference was not statistically significant. Treadmill run time was significantly reduced in all groups after 28 d with the reduction from pre-suspension values being more pronounced with HDS animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Influences of chemical sympathectomy and simulated weightlessness on male and female rats.

Maximum oxygen consumption (VO2max) has been shown to be reduced after periods of simulated weightlessness. To assess the role of the sympathetic nervous system in these reductions, Sprague-Dawley rats were either chemically sympathectomized (SYMX) or injected with saline (SHAM) and assigned to head-down suspension (HDS), horizontal restraint with the hindlimbs weight bearing (HWB), or cage-control (CC) conditions. VO2max, run time (RT), and mechanical efficiency (ME) were measured before suspension and on days 7 and 14. Male and female SHAM HDS groups exhibited reduced measures of VO2max (12-13%) after 7 and 14 days, and this decrease was attenuated in the SYMX and HWB rats. HDS resulted in a significant reduction in RT (9-15%) in both the male and female rats, and ME was significantly reduced after HDS in male and female SYMX and male SHAM rats (23-33%) but not in the female SHAM rats. Lesser reductions in ME were observed in the HWB rats. HDS and HWB were associated with lower body, fat-free, and fat masses, which were similar in male and female rats as well as for the SHAM and SYMX conditions. In a related HDS experiment with normal rats, plasma norepinephrine and epinephrine were increased by 53 and 42% after 7 days, but only epinephrine returned to baseline after 14 days. It was concluded that chemical sympathectomy and/or a weight-bearing stimulus will attenuate the loss in VO2max associated with simulated weightlessness in rats despite similar changes in body mass and composition. The mechanism(s) remains unclear at this time.

Influence of single hindlimb support during simulated weightlessness in the rat.

Mature male rats (n = 16) were assigned to either 14 days of head-down suspension with one hindlimb supported (HDS) or to control cages (C) of similar dimensions. Hindlimb support during HDS preserved the muscle mass-to-body mass ratio (mg/100g) compared with C conditions for the soleus (48.3 +/- 1.0 to 41.7 +/- 1.0), plantaris (98.4 +/- 3.4 to 103.3 +/- 4.1), and gastrocnemius (484.7 +/- 18.5 to 507.2 +/- 13.9). However, the muscle mass-to-body mass ratio was significantly lower for the soleus (28.9 +/- 1.5), plantaris (83.9 +/- 3.6), and gastrocnemius (411.9 +/- 24.2) muscles from the freely hanging hindlimbs compared with the contralateral muscles from the supported hindlimbs or muscles from C animals. Citrate synthase activity (mumol.g-1.min-1) was significantly lower in soleus muscles from HDS rats in both the supported (19.4 +/- 2.3) and freely hanging (20.0 +/- 1.6) hindlimbs compared with C (28.5 +/- 3.1), whereas soleus muscle glycogen concentration (mg/g) was significantly higher in the freely hanging limbs from HDS rats (5.90 +/- 0.31) but not in the supported limbs (3.80 +/- 0.61) compared with C (4.34 +/- 0.50). Doppler flow probes were used to determine that iliac blood flow to freely hanging hindlimbs was significantly decreased after 48 (-19 +/- 5%) and 72 (-20 +/- 6%)h of HDS compared with presuspension values. In addition, iliac vascular resistance was significantly elevated at most time points during the 72 h of HDS in the freely hanging limbs but not the supported hindlimbs.(ABSTRACT TRUNCATED AT 250 WORDS)