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

Effects of insulin and exercise on rat hindlimb after simulated microgravity CRAIG

S. STUMP,

THOMAS

W. BALON,

AND

CHARLES

muscles

M. TIPTON

Department of Exercise and Sport Sciences, School of Health Related Professions, University of Arizona, Tucson, Arizona 85721; and Department of Exercise Science, University of Iowa, Iowa City, lowa 52242 STUMP, CRAIG S., THOMAS W. BALON, AND CHARLESM. TIPTON. Effects of insulin and exercise on rat hindlimb muscles after simulated microgravity. J. Appl. Physiol. 73( 5): 2044-2053, 1992.-This study was designedto examine insulin- and exercise-stimulatedglucoseuptake and metabolismin the hindlimb musclesof rats after conditions of simulated microgravity. To simulate microgravity, male Sprague-Dawley rats were suspended in a head-down (45") position with their hindlimbs non-weight bearing (SUS) for 14 days. In addition, rats were assignedto suspensionfollowed by exercise (SUS-E), to cage control (CC), or to exercising control (CC-E) groups. Exercise consistedof five lo-min bouts of treadmill running at the same relative intensity for the CC-E and SUS-E rats @O-90% of maximum 0, consumption). Hindlimb perfusion results indicated that glucoseuptake for the entire hindquarter at 24,000 ~U/ml insulin (maximum stimulation) wassignificantly higher in the SUS (8.9 t 0.5 pmol . g-l. h-‘) than in the CC (7.6 rf: 0.4 pmol . g-l . h-l) rats, signifying an increasedinsulin responsiveness.Glucoseuptake at 90 pU/ml insulin wasalsosignificantly higher in the SUS (48 + 4; % of maximum stimulation over basal) than in the CC (21 t 4%) rats, In addition, exercise-inducedincreasesin glucoseuptake for the hindlimbs (133%)and glucoseincorporation into glycogen for the plantaris (8.4-fold), extensor digitorum longus (5.4-fold), and white gastrocnemius @.$-fold) muscleswere greater for the SUS-E rats than for the CC-E rats (39%and 1.9-, 1.9-, and X0-fold, respectively). Therefore, suspensionof the rat with hindlimbs non-weight bearing leads to enhanced muscle responsesto insulin and exercise when they were applied separately. However, insulin action appeared to be impaired after exercisefor the SUS-E rats, especially for the soleusmuscle.

glucoseuptake; glycogen; treadmill running; hindlimb perfusion

THE SUSPENSIONof rats with their hindlimbs non-weight bearing, a model used to mimic the effects of microgravity, leads to the atrophy of antigravity hindlimb muscles

(l&19,36,37). This atrophy is accompanied by a number of metabolic changes, including increased glycogen concentrations (16, 36) and enhanced responses to insulin for glucose uptake and metabolism (3, 18, 19). The heightened muscle responses to insulin may be related to increased insulin receptor (3,19) and glucose transporter GLUT-4 concentrations (17) that have been observed in non-weight-bearing soleus (SOL) muscles. In addition, the activities of some glycolytic enzymes are increased in SOL muscle fibers after suspension (8). Interestingly, increased rates of glucose uptake and glycogen accumulation (12-48 h after reweighting) have been observed in 2044

SOL muscles after reexposure to weight bearing, suggesting that muscle responses to contractions may also be enhanced after periods of simulated microgravity (16, 18).

Previous studies that examined muscle responses to insulin after non-weight-bearing conditions used predominantly SOL muscles in isolated preparations. These studies were limited because only small muscles from juvenile animals (16, 19) or muscle strips (3) could be tested. Moreover, the slow oxidative SOL muscle is not representative of the total hindlimb musculature of the rat because the majority of the hindquarter consists of fast oxidative-glycolytic and fast glycolytic fibers (1). This may be of particular importance because there appears to be a considerable heterogeneity in response to exercise and insulin for muscles of different fiber-type composition (22). Therefore, this study was designed to examine the general response of the rat hindquarter to insulin and exercise for glucose uptake and metabolism after 2 wk of suspension while at the same time compar-

ing the responses of select muscles of various fiber type composition using a hindlimb perfusion technique. The muscles examined were the SOL (90% slow oxidative), plantaris (PL; 93% fast glycolytic and fast oxidative-glycolytic), white gastrocnemius (GW; 91% fast glycolytic), and extensor digitorum longus (EDL; 79% fast glycolytic) (1). We hypothesized that the increase in glucose utilization in response to insulin and exercise would be higher for the suspended rats than for the controls and that these responses would be most prevalent in the SOL and PL muscles that atrophy during suspension procedures (l&19,36,37). On the other hand, we predicted that the GW and EDL, which do not atrophy during suspension and are minimally recruited for normal weight-bearing purposes (15,32), would not demonstrate these effects. It was also hypothesized that increases in insulin action would be greater after exercise in the atrophied muscles of the suspended rats than in the muscles from control rats. METHODS

Animals. Young male Sprague-Dawley rats were obtained from a commercial dealer (Harlan Sprague Dawley, Indianapolis, IN) and maintained within the Exercise Physiology Laboratory animal care facility at the University of Arizona. This facility is maintained between 20-23OC with a 12:12-h light-dark cycle. The ani-

0161-7567/92 $2.00 Copyright 0 1992 the AmericanPhysiological Society

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MUSCLE

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SIMULATED

mals were housed in groups of three to five until they reached 250-325 g body mass (9-12 wk in age) at which time they were randomly assigned to one of four experimental groups for 14 days. The four groups consisted of cage controls that did not exercise (CC), cage controls that did exercise (CC-E), suspended animals that did not exercise (SUS), and suspended animals that did exercise (SUS-E). Throughout the experimental period all rats were given water and were fed rat chow ad libitum (Wayne, Rodent Blox, Bartonville, IL). The rat chow was provided in paste as well as pellet form to encourage eating (36, 37). In addition, the rats were weighed and food and water consumption were measured after 1,7, and 14 days of the experiment. Institutional approval for the study was granted by the Committee on Animal Care at the University of Arizona. Procedures to simulate microgravity. A model similar to that previously described was used to simulate microgravity in the SUS and SUS-E rats (37). Briefly, the rats were restrained within a semicircular plastic tube with Velcro and elastic straps in a 45O head-down position so that their hindlimbs were non-weight bearing and fluids were shifted in a cephalic direction. To support the tail and prevent the rat from forward movement, four 2 X 0.5 cm Velcro tabs were glued to the rat’s tail and attached to a Velcro strip suspended above and posterior to the animal. The suspended rats were able to move in a 180° arc within their cages and occupied similar cage areas as the control rats (-430 cm2). Furthermore, each suspension apparatus was constructed and the laboratory balance was modified so that body mass could be determined without allowing the hindlimbs to be weight bearing. Treadmill running. All rats were familiarized to treadmill running before their assignment to experimental groups. The familiarization consisted of treadmill running for 12 min, three times a week, for 2 wk. The first 2 min were run at a 0’ grade at 16 m/min. This was followed by 2 min at the same speed but at a loo grade. The final 8 min were performed at the 10’ grade at 26.6 m/min. Maximal 0, consumption (Voz,,,) was also determined for each rat before group assignment as described previously, with slight modification (37). Briefly, the rats ran on a treadmill enclosed in an airtight Plexiglas chamber. Expired air was withdrawn from the front of the chamber by a vacuum pump, mixed by a fan, and analyzed for 0, (S-3AI Applied Electrochemistry, Ametek, Pittsburgh9 PA) and CO, (LB-2, Beckman, Anaheim, CA) percentages. All rats ran on the treadmill following a protocol of 3-min stages starting at 16 m/min and a 5O grade. The second stage was at the same speed but at a loo grade. This was followed by stages of increasing speed (21.3,24.0,26.6, and 31.9 m/min, all at a 10” grade) until the rat was unable or unwill@g to complete a stage at the prescribed speed and grade. VO, fnaXwas taken to be the highest VO, value measured during the test. The Vo2 values recorded for the last stage were 7.7 t 0.8% higher than the values recorded for the previous stage. On day 14 of the experiment, CC-E and SUS-E rats were removed from their respective cages and exposed to an acute session of treadmill running as described previously (10) but modified so that the rats ran at a similar

MICROGRAVITY

AND

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relative exercise intensity. The session consisted of five lo-min stages of running with 3-min rest periods between each stage. During the first stage the rats ran at increasing speeds and grade, in the same progression described above for the V02max test, until 8O-90% of estimated 00, m8X was attained. The rats continued to run at this speed and grade for each of the subsequent stages. VO, was monitored throughout the first and second stages. Because rats suspended for 14 days will exhibit a decrease in VO, m8Xby ~10% (37), the 80-90% target 60, for the SUS-E rats was estimated from a VO 2max value that was 10% less than the actual value obtained during the preexperimental V02max test. The target VO, for the CC-E rats was calculated from the actual preexperimentalo0, m8Xtest values, since Vo2 maX does not change significantly in control rats after 2 wk (37). Hindlimb perfusion. Immediately after the exercise session (CC-E, n = 29; and SUS-E, r~ = 25) or after removal from suspension (SUS, n = 32) or control (CC, n = 40) cages, the rats were injected with pentobarbital sodium (5 mg/lOO g body mass ip) and prepared for hindlimb perfusion as described previously (33, 38). All rats were in the fed state when removed from their cages for exercise and/or hindlimb perfusion. On completion of the surgery in which cannulas were placed in the abdominal aorta and vena cava, the rats were killed with an intracardiac injection of pentobarbital sodium. Perfusions of the SUS, SUS-E, CC, and CC-E hindlimbs at the three different insulin concentrations were performed randomly between 12:00 P.M. and 8:OO P.M. The rats’ hindlimbs were perfused with an initial perfusate volume of 150 ml. The first 25 ml of perfusate that flowed through the hindlimbs was discarded, and the remaining perfusate was recirculated at 12.5 ml/min at 37OC as described previously (38). The perfusate consisted of a Krebs-Henseleit buffer, containing aged-rejuvenated human erythrocytes added to obtain a hematocrit of 30%, 4% bovine serum albumin (Pentex, Kankakee, IL), 6 mM glucose, 0.15 mM pyruvate, and 1.5-2.0 mM lactate (originating from the erythrocytes). The perfusate was gassed continuously with 95% o,-5% Co,. In addition, porcine insulin (Sigma Chemical, St. Louis, MO) was added or withheld to obtain concentrations of 0, 90, or 24,000 ,tJJlml of cell-free perfusate. These concentrations represent no insulin, a submaximally stimulating concentration of insulin, and a maximally stimulating concentration of insulin. The rats’ hindlimbs were perfused for a 15-min equilibration period as described previously (10,38). Three minutes before the end of equilibration, 20 &i Of D- [ U-‘“cj glucose tracer (New England Nuclear, Boston, MA) was introduced to the medium. Three minutes was the approximate transit time necessary for the tracer to advance from the reservoir to the animal preparation at the flow rate utilized. At the end of the equilibration period (time 0) and at 45 min of perfusion, samples were obtained from the perfusate reservoir and placed in ice-cold tubes that were l

empty for glucose determinations or contained 2 vol of

ice-cold 6% (wt/vol) perchloric acid for lactate and pyruvate measurements. These samples were centrifuged (3,000 g, 4”C, 10 min), and the supernatant was removed


2046

MUSCLE

RESPONSES

TO SIMULATED

and frozen for later analysis. In addition, perfusate samples were collected from the arterial and venous lines at 15 and 40 min for the determination of hindlimb 14C0, production, which was used to estimate glucose oxidation. After the 45min perfusion period, the SOL, the PL, the EDL, and a portion (superficial white) of the gastrocnemius muscle were removed, rinsed in ice-cold saline, weighed, and frozen in liquid nitrogen for the determination of [ 14C] glucose incorporation into glycogen. Analyses of hindlimb perfusion samples. Glucose uptake was calculated as the decrease in glucose concentration over the 45min perfusion period multiplied by the perfusate volume and expressed as micromoles per gram perfused muscle per hour. Likewise, net lactate and pyruvate uptake or release were calculated as the change in concentration over the 45-min perfusion period multiplied by the perfusate volume and expressed as micromoles per gram perfused muscle per hour. Perfusate glucose was measured using a YSI model 23A glucose analyzer (Yellow Springs Instrument, Yellow Springs, OH), whereas lactate (14) and pyruvate (6) were determined using standard enzymatic procedures. Perfused muscle mass was considered to be ‘15% of body mass based on data obtained from separate groups of SUS (n = 6) and CC(n= 6) animals that were perfused as indicated above except that instead of [14C]glucose, Evans blue dye (20 mg/ml) was added to the perfusate. 14C02 production by the hindlimbs was determined as described by Ivy et al. (21). Briefly, 1.5 ml perfusate samples were injected into flasks containing 1.5 ml of 0.5 M acetic acid. The CO, released from the perfusate was trapped in 0.4 ml of Protosol (New England Nuclear) located in a hanging center well. The Protosol was transferred into vials containing Econofluor (New England Nuclear) and counted in a scintillation counter. Glucose oxidation was calculated using arterial-venous differences in 14C0, measured at 15 and 40 min into perfusion and at a flow rate of 12.5 ml/min, with the values expressed as nanomoles per gram perfused muscle per hour. In accordance with the methods of Seider et al. (35), the incorporation of [14C]glucose into glycogen was determined by solubilizing muscle samples (50-200 mg) in 0.5 ml of 1 N NaOH containing 10 mg/ml of carrier glycogen. Glycogen was precipitated by adding 1.6 ml of absolute ethanol and placing the samples in a deep freeze for 3 h at -7OOC. The precipitates were washed twice with 1 ml of 66% ethanol and dissolved in 0.5 ml of water. The resulting solution was added to scintillation vials containing scintillant for aqueous samples and counted. Muscle mass,glycogen, and glucose 6-phosphate (G-6-P) measurements. In a separate group of experiments, rats

were weight matched and assigned to exercising and nonexercising groups, CC (n = lo), CC-E (n = lo), SUS (n = lo), and SUS-E (n = lo), for 14 days. After the experimental period and/or exercise bouts, the rats were injected with pentobarbital sodium (5 mg/iOO g body mass ip), and the SOL, PL, EDL, and the GW muscles were removed from the right hindlimb, rinsed in ice-cold saline, weighed, frozen in liquid nitrogen, and stored (-70°C). The SOL, PL, and EDL masses were compared with muscles obtained from a group of rats killed before

MICROGRAVITY

AND EXERCISE

the experimental period (n = 9) to evaluate changes from the precondition. The frozen muscle samples were analyzed for glycogen concentration using a phenol-sulfuric acid calorimetric method for small tissue samples (36). In addition, the SOL and a portion of the GW from the left hindlimb were freeze-clamped with aluminum tongs cooled in liquid nitrogen, stored (-7O”C), and analyzed for G-6-P concentrations (25). These muscles were selected for G-6-P analysis because they are very different in fiber-type composition and can be rapidly isolated and freeze-clamped. Furthermore, &ml blood samples were collected from the SUS and CC rats via the abdominal aorta and were used to determine plasma insulin concentration using a double-antibody radioimmunoassay (Incstar, Stillwater, MN). Two other groups of cage control and suspended rats were also exercised on the treadmill and anesthetized as described above, but their hindlimb muscles were not removed and frozen until 30 min after the end of the exercise session (CC-30, n = 7; and SUS-30, n = 8, respectively). These muscles were analyzed for glycogen and G-6-P to determine their concentrations at the time hindlimb perfusion would begin. Approximately 30 min were required to complete the surgery and equilibration before the 45-min perfusion period began. Statistics. All values are expressed as means t SE. Analysis of variance procedures were used to evaluate differences between mean values from more than two groups or more than two conditions. Post hoc tests were performed using the Duncan’s multiple-range test. When only two mean values were being compared, independent t tests were employed. Statistical significance was set at the 0.05 probability level. RESULTS

Body mass and food and utater consumption. Body mass for the CC rats increased during the 14 days of the experiment by 20% when compared with preexperimental conditions. The SUS rats decreased 12% in body mass by day 7 but on day 14 were only 9% lower than the presuspension value. The loss of body mass by the SUS rats during the early days of the experiment may be due, in part, to decreases in food and water consumption. During the first 24 h, the SUS rats ate 49% less kilocalories and drank 51% less water than the CC rats (P 5 0.05). However, after this initial period, caloric and water consumption were similar between groups. In fact, if expressed as kilocalories per day per 100 g, the SUS rats ate significantly more (13-20%) than the CC rats during the second week of the experiment despite a lower rate of body mass gain during that time. Muscle mass. Changes in SOL, PL, and EDL muscle mass are shown in Table 1. During the 2-wk treatment period, the CC rats exhibited 21,27, and 23% increases in mass for the SOL, PL, and EDL respectively. On the other hand, significant (P 5 0.05) atrophy for the SOL (15%) and the PL (12%) was observed after 2-wk of SUS when compared with rats killed before the experiment. The EDL muscle did not change in mass during the SUS conditions. Furthermore, although the SUS rats had significantly less perfused muscle mass than the CC rats


MUSCLE

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TO SIMULATED

1. Influence of suspension and control conditions on the muss of select hindlimb muscles

TABLE

Mass, g Before Co,ntrol or Suspension

Muscle SOL

After 14 Days of Control

lllt3 302t14 11524

PL EDL

After 14 Days of Suspension

134t2* 385*9* 141t3*

9624” 267&F lllt2

Values are means + SE. SOL, soleus; PL, plantaris; EDL, extensor digitorum longus. (P 5 0.05).

* Significantly

different

from

precondition

mean

(38 t 3 and 50 t 5 g, respectively), when expressed as a percentage of body mass the values were nearly identical (15%). Plasma insulin concentrations. Plasma insulin concentration was slightly lower for the SUS rats (5.4 t 2.5 rig/ml) compared with the CC rats (6.7 t 2.7 rig/ml) at the time of death, but the difference was not statistically different (P > 0.05). I& during treadmill running. During the acute bouts of treadmill running the suspended rats #US-E and SUS-30) ran at 86.2 t 1.2% of 00, m8x, which was not significantly different, than the cage control rats (CC-E and CC-30) who ran at 88.3 t 1.1% of vo2,,,. However, when expressed in absolute terms the cage consrol rats (24.4 t 0.4 ml/min) ran at a significantly higher VO, than the suspended rats (22.4 t 0.4 ml/min). The respiratory exchange ratio, measured during the second lo-min stage of running, was not significantly different between the suspended (0.96 t 0.01) and control (0.94 t 0.01) animals. Although 12 of 43 suspended and 10 of 46 cage control rats were unable to complete all five stages at the prescribed speed and grade, the data from these animals were included in the statistical analyses. Muscle glycogen concentrations. Glycogen concentrations (mg/g) and contents (mg/muscle) for the SOL, PL, GW, and EDL muscles from rats at rest and after exercise are shown in Table 2. The SUS rats’ SOL (65%) and PL (56%) muscles had significantly higher glycogen concentrations compared with the CC rats’ muscles. However, there were no significant differences in glycogen TABLE

2. Hindlimb

muscle glycogen immediately

cc

CC-E

(n = 10)

(n = 10)

PL GW FJDL

PL EDL

cc-30 (n = 7)

1.81+0.42* 2.30tO.43* 3.32t0.50* 2.44k0.47”

2.40t0.41 2.89+0.51* 2.63t0.40” 2.70t0.48”

5.10t0.06 1.98t0.21 0.76+0.08

0.23t0.05* 0.82t0.13* 0.33tO.O6*

0.32kO.05” 1.13kO.53” 0.37tO.06”

,

sus (n = 10)

concentration,

3.73iz0.42 4.91kO.48 5.07kO.28 5.21kO.53

0

content,

* Mean value significantly value (P 5 0.05).

different

from

no-exercise

condition;

T

l 17

.

SUS-E fn = 10)

sus-30 (n = 8)

mglg

6.15+0.66’f 7.67kl.Olt 5.44t0.49 6.58kO.69

2.75tO.35* 1.43tO.31” 1.09+0.32*t 1.75tO.30”

2.67tO.35” 2.43t0.57* 1.30+0.30*~ 1.84tO.29*

0.57a0.07 1.85kO.26 0.68t0.08

0.26tO.03” 0.41_to.09* 0.20tO.O3*

0.27tO.05* 0.67&O. 15* 0.22tO.O3*

mg

Values are means t SE; n, no. of rats. CC, control rats at rest; CC-E, control rats immediately exercise; SUS, suspended rats at rest; SUS-E, suspended rats immediately after exercise; SUS-30, gastrocnemius. CC-E, or CC-30

2047

concentration between SUS and CC conditions for the GW or EDL muscles. In addition, glycogen concentrations were significantly lower for the SOL, PL, GW, and EDL immediately after exercise for both the SUS-E (5581%) and CC-E (35~54%) rats compared with the muscles from the SUS and CC rats, respectively. Lower glycogen contents were also observed for the SOL, PL, and EDL muscles immediately after exercise in the SUS-E (54-78%) and CC-E (55-59%) rats compared with the muscles from their nonexercising counterparts. Furthermore, when muscle glycogen was measured 30 min after exercise, the content remained significantly lower for the SOL, PL, and EDL muscles in both the SUS-30 and CC30 rats compared with the muscles from the nonexercised rats (Table. 2), and glycogen concentrations remained significantly lower in all muscles except the SOL from the CC-30 rats. Glycogen concentrations were also significantly lower in the GW muscles from the SUS-E and SUS-30 rats compared with the muscles from the CC-E and CC-30 rats, respectively. G-6-P. G-6-P concentrations (nmol/mg protein) were measured for the suspended and control rats’ SOL and GW muscles immediately or 30 min after running on a treadmill or after removal from suspension or control cages (Table 3). No significant differences in G-6-P concentration were observed between the CC and SUS groups at rest for either the SOL or GW. Trends toward lower G-6-P concentrations in the GW muscles immediately after exercise and higher concentrations in SOL and GW muscles 30 min after exercise were observed for both the suspended and the cage control rats. However, only the value for the SOL muscles from the SUS-30 animals was significantly higher. Hindlimb glucose uptake. Glucose uptake rates by perfused rat hindlimbs either at rest or after acute exercise are shown in Table 4, top. Glucose uptake was significantly higher (15%) for the SUS rats compared with the CC rats at 24,000 &J/ml insulin, whereas no difference in glucose uptake was observed in the absence of insulin. Glucose uptake was also significantly higher (77%) in the SUS rats at the submaximally stimulating insulin concentration of 90 pU/ml. In addition, when the glucose uptake values at 90 pU/ml insulin are expressed as a percentage of maximal stimulation (24,000 pU/ml insul

Glycogen

SOL

AND EXERCISE

. f. and 30 mzn uTter antense treaamw runntng

Glycogen

SOL

MICROGRAVITY

t mean

value

after exercise; CC-30, control rats 30 min after suspended rats 30 min after exercise; GW, white significantly different from corresponding CC,


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MUSCLE

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TABLE 3. Glucose 6-phosphate concentration in soleus and white gustrocnemius muscles after suspension and treadmill running nmol/mg

Group

SOL

Cage control

GW

No exercise

45 min

2.46kO.38

2.36t0.43

(11)

(10)

Suspension

2.47t0.63

Cage control

8.79t1.61 (11)

5.69k2.14 (10)

7.56t1.72 (9)

5.18t1.10 00)

Suspension

2,68+0.46

(9)

Hindlimb Suspension

protein After

Muscle

AND EXERCISE

w

exercise 30 min 4.25t1.13

(7) 5.11tO.56*

(8)

10.39t2.26 0 8.17+1.37 (8)

0

EDL SOL PL GW Glucose Incorporation Into Giycogen

Values are means + SE; no. of rats in each group is in parentheses. * Mean value that is significantly different from no-exercise condition (P 22 0.05).

lin) over basal (0 ,&J/ml insulin), the value is still significantly higher in the SUS rats (48 t 4%) than in the CC rats (21 t 4%). The CC-E rats exhibited a significantly higher rate of glucose uptake (35%) at the submaximally stimulating insulin concentration (90 pU/ml) than the CC rats. However, the glucose uptake values at 0 and 24,000 pU/ml insulin were not significantly different between the CC and CC-E conditions. On the other hand, glucose uptake rates for the SUS-E rats were significantly higher than for the SUS rats at both the 0 and 90 pU/ml insulin concentrations (133 and Z4%, respectively) and significantly higher than the CC-E values at all the insulin concentrations examined (17-96%). 14C0, production by the hindlimbs. The production of l*CO, by the rat hindlimbs after 15 min of perfusion is

FIG. 1. Insulin sensitivity as indicated by glucose incorporation into glycogen at 90 pU/ml insulin expressed as a percentage of maximum over basal for soleus (SOL), plantaris (PL), white gastrocnemius (GW), and extensor digitorum longus (EDL) muscles. Values are means t SE. * Significantly different from cage control value (P 22 0.05).

shown in Table 4, bottom. Like glucose uptake, 14C02 production was significantly greater for the SUS rats compared with the CC rats at both 90 and 24,000 pU/ml insulin (78 and 86%, respectively). There was no significant difference between the two groups in the absence of insulin. In addition, there were no significant differences between the CC and CC-E rats in 14C0, production at any of the insulin concentrations examined. No significant differences in 14C0, production rates were observed between the SUS and SUS-E rats at the 0 and 90 pU/ml insulin concentrations; however, the SUS-E rats exhibited a rate that was only 42% of the SUS value (P 5 0.05) at 24,000 pU/ml insulin Similar 14C0, production results TABLE 4. Influence of suspension and treadmill running were observed after 40 min of perfusion except that the on glucose uptake and oxidation during hindlimb perfusion SUS and SUS-E values were not significantly different at any of the insulin concentrations examined. Insulin Concentration, $J/ml [14C]glucoseincorporation into glycogen. The values for 90 24,000 Group 0 [‘“Cl glucose incorporation into glycogen during hindlimb perfusion were not significantly different between Glucose uptake, pmol 9g-’ 9h-’ the SUS and CC rats in the presence of 24,000 pU/ml cc l&O.1 3.1t0.3 8.0t0.2 insulin or in the absence of insulin for any of the muscles (17) (12) (10) examined. However, at 90 PUlml insulin, glucose incorpoCC-E 2.5t0.2 4.2t0.3* 7.6t0.4 ration into glycogen was significantly greater for the (11) (10) (7) sus 2.1t0.3 5.5+0.3f9.2&0.4-f SOL (2.4 t 0.4 prnol. g-l . h-l) and EDL (3.6 t 0.4 (13) (11) 03) pmol g-l . h-l) m uscles from the SUS rats compared SUS-E 4.9+0.4*t 6.8kO.6*t 8.9+0.5t with those from the CC rats (0.9 t 0.2 and 0.9 t 0.1 (8) 17) (9) pmol. g-l h-l, respectively). The PL mean from the Glucose uxidation, nmol 8’ - h-l SUS rats also exhibited the same trend, but the differ51t6 8329 124t33 cc ence between the SUS and CC muscles was not statisti(10) (9) (1% cally significant. When rates for glucose incorporation CC-E 43+5 59t7 144t20 into glycogen at 90 ,&J/ml insulin were expressed as a (11) (10) (7) sus 56k6 148216-f 231+30-f percentage of maximal stimulation over basal, the SUS (13) (11) (5) rats exhibited significantly higher values than the CC 76t12 132tll 133t27* SUS-E rats for the SOL, PL, and EDL (Fig. 1). (8) (7) (9) Glucose incorporation into glycogen rates for the SOL, Values are means k SE; no. of rats in each group is in parentheses. PL, GW, and EDL were also compared between the exerSee Table 2 for group abbreviations. Values for glucose oxidation are cised and nonexercised conditions for the suspended and for 15 min into the perfusion period. * Value significantly different control rats (Fig. 2). Glucose incorporation into glycogen than no-exercise condition; t mean value significantly different from was significantly higher for the CC-E rats when comcorresponding CC or CC-E value (P 5 0.05). l

l

l


MUSCLE

A

RESPONSES

TO SIMULATED

Control

MICROGRAVITY

B

AND EXERCISE

2049

Suspension

8

6

GW

6

0

90

24,000

Insulin 2. Influence of suspension and intense treadmill running on [14C]glucose incorporation into glycogen in muscles from control (A) and suspended (B) rats. Values are means k SE; n = 6-18 muscles/data point. * Significantly different from no-exercise condition (P 5 0.05). FIG.

pared with CC rats (2.7- to M-fold) in all four muscles at the submaximally stimulating insulin concentration (90 &Vml). However, there were no significant differences between the CC-E and CC rats for glucose incorporation into glycogen in the absence of insulin or at the maximally stimulating insulin concentration (24,000 PI-J/ml)

for any of the muscles examined, The rates for glucose incorporation into glycogen at the submaximally stimulating insulin concentration were also significantly higher in the SUS-E rats than in the SUS rats for the PL, GW, and EDL muscles (3.8-, 7.L, and 3.8-fold, respectively) but not for the SOL. However, unlike the control


2050

MUSCLE

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rats, the suspended rats exhibited significantly higher rates of glucose incorporation into glycogen after exercise for the PL, GW, and EDL muscles (8.4-, 4.8-, and 5.4-fold, respectively) in the absence of insulin. In addition, glucose incorporation into glycogen rates were significantly higher for the SUS-E rats than for the CC-E rats at 90 pU/ml for the PL, GW, and EDL and in the absence of insulin for the PL and EDL. However, there were no significant differences between the SUS-E and SUS rats at the maximally stimulating insulin concentration for any of the muscles examined. Lactate and pyruvate uccumukxtion or removal. Lactate accumulation in the perfusate during hindlimb perfusion tended to be higher for the SUS and SUS-E rats than for the CC and CC-E rats, but no statistically significant differences were observed (P > 0.05; data not shown). In addition, no significant differences were observed for any group, condition, or insulin concentration for pyruvate uptake from the perfusate (data not shown). DISCUSSION

This study examined muscle responses to insulin and exercise in postjuvenile rats after 14 days of simulated microgravity. The most important findings were 1) exercise at the same relative intensity reduced PL, GW, and EDL glycogen concentrations to a greater extent in the suspended rats than in the control rats; 2) insulin stimulated hindlimb glucose uptake and oxidation, and glucose incorporation into glycogen for the SOL, PL, and EDL were higher for the SUS rats than for the CC rats; 3) hindlimb glucose uptake and its incorporation into glycogen in the PL, GW, and EDL were increased more after exercise in the suspended rats than in the control rats; and 4) some insulin-stimulated processes appeared to be impaired after exercise in the suspended rats’ muscles, especially in the SOL. The data presented in Table 2 indicate that the exercise protocol used in this study was effective in reducing glycogen in all four muscles examined (SOL, PL, GW, and EDL). However, the changes in glycogen concentration for these muscles during exercise were found to be significantly greater in the suspended rats than in the control rats (Fig. 3). This result is similar to previous studies showing higher glycogen depletion rates for SOL muscles immediately (~2 h) after reexposure to weight bearing (16). Increased rates of glycogenolysis after periods of non-weight bearing may be related to increases in muscle fiber glycolytic enzyme activities (8), higher resting muscle glycogen concentrations (Table 2) p lower hindlimb blood flow rates during contractions (27,29), or to increases in type II fiber recruitment as type I fiber tension capacity is diminished (9). Interestingly, the respiratory exchange ratio was not significantly different between the suspended and control rats during exercise, suggesting that they utilized similar percentages of carbohydrate. Hindlimb glucose uptake and oxidation (estimated from **CO, production) were significantly increased after suspension in the presence of both a submaximally stimulating insulin concentration (90 pU/ml) and a maximally stimulating concentration (24,000 pU/ml) compared

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A 8

SOL

EDL

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B

Cage

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0

I

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3. Reduction of glycogen in select hindlimb muscles during intense treadmill exercise. Values are means t SE of differences in glycogen between matched resting and postexercise conditions for cage control and suspended rats. Muscle glycogen concentration (A) and content (B) are shown. * Mean value that is significantly different from CC value (P 22 0.05). FIG.

with the control rats (Table 4). However, SUS and CC values were not significantly different in the absence of insulin (basal). Higher glucose uptake and metabolism rates at 24,000 pU/ml insulin after suspension suggests an increase in insulin responsiveness that is classically associated with changes in postreceptor mechanisms (23). Although a change in insulin sensitivity per se (shift in the insulin dose-response curve) cannot be identified conclusively with a single submaximal glucose uptake value and a coincident change in insulin responsiveness, it can be estimated by expressing submaximal responses as a percentage of the maximal response over basal values (26). When this approach was used, our data suggested that hindlimb muscle insulin sensitivity was greater for glucose uptake in the SUS rats (48 -t 4%) compared with the CC rats (21 t 4%). In addition, when glucose incorporation into glycogen was expressed as a


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percent of the maximum response over basal (Fig. 1), the SOL, PL, and EDL muscles all exhibited significantly higher values after SUS than CC. Interestingly, insulinstimulated glucose incorporation i nto glycogen increased for the SOL and PL muscles after SUS despite the fact that these muscles had elevated glycogen concentrations (Table 2). However, it is important to-point out that glucose incorporation into glycogen is not equivalent to glycogen synthesis because it reflects the difference between the rate of [ 14C]glucose conversion into glycogen and the rate of [14C]glycogen conversion into G-6-P. Nevertheless, it is influenced in a dose-dependent manner by insulin and is therefore a useful parameter for examining insulin action in muscle. Collectively, these findings suggest that the hindlimb musculature of the rat responds to insulin to a greater extent for several glucose uptake and metabolism parameters after conditions of simulated microgravity. This is in agreement with previous studies that examined whole SOL muscles from juvenile rats (l&19) and SOL muscle strips of both young and adult rats (3) after non-weightbearing conditions. The results for the SOL and PL in the present study may be related to an increase in insulin receptor density (3,19) or an increase in GLUT-4 transporter protein (17), which have been observed after nonweight-bearing-induced atrophy in the SOL. On the other hand, the increased EDL response to insulin for glucose incorporation into glycogen was surprising because it is not a weight-bearing muscle (15) and did not atrophy (Table 1). In addition, Henriksen et al. (19) have shown that EDL sensitivity to insulin for %deoxyglucose uptake was unchanged in juvenile rats after 6 days of suspension. One possible explanation for the increased EDL response in-the present study is that this muscle may be in a condition of chronic stretch when the rats’ hindlimbs assume exaggerated plantar flexion after 4 days of suspension (31). It is also possible that systemic factors are contributing. However, the lack of change in GW response makes this generalization somewhat tenuous. There are several systemic factors that may be influencing muscle responses to insulin after simulated microgravity. One is a reduction in food consumption, since starvation is known to increase muscle insulin sensitivity (4). However, this is an unlikely explanation for the present results because the SUS and CC rats had similar caloric consumption rates during the last 7 days of the experiment and comparable plasma insulin concentrations on day 14. A second possible systemic consideration is a decrease in body fat percentage that has been demonstrated in 14-day suspended rats (37). Third, a chronic exposure to epinephrine (~120 h; Ref, 5) or norepinephrine (10 days; Ref. 26) can cause an increase in insulin sensitivity. This is important because we have shown that rats have elevated plasma norepinephrine concentrations throughout suspensions lasting 14 days, whereas epinephrine is significantly increased for at least 7 days. Fourth, current and previous (36, 37) data for food consumption and changes in body mass suggest that resting metabolic rate may be increased during suspension, which may also influence glucose metabolism. Therefore the influence of systemic factors on muscle responses to

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insulin during simulated microgravity warrants further investigation. It is well established that acute exercise or in vitro contractions stimulate glucose uptake in skeletal muscles (10, 11, 13, 22, 38) and that muscles become more sensitive to insulin after exercise (10, 38). Similarly, in this study, the CC-E rats exhibited significantly higher glucose uptake (Table 4) and glucose incorporation into glycogen (Fig. 2) rates than the CC rats at 90 pU/ml insulin. This was not observed at 0 or 24,000 pU/ml insulin, which was surprising and unlike previous studies using similar exercise protocols (10, 38). However, the effect of exercise on these parameters is transient (10,13), and some studies have not shown an increase in glucose or glucose analogue uptake after exercise or contractions in the absence of insulin or in the presence of a maximally stimulating concentration of insulin (11, 13). The present study also showed that exercise stimulated hindlimb muscle glucose uptake and its incorporation into glycogen in the absence of insulin to a greater extent in the suspended rats than in the cage control rats (Table 4 and Fig. 2). This finding was predicted because previous studies had demonstrated exaggerated glucose uptake (18) and glycogen accumulation (12-48 h after reweighting; Ref. 16) rates in atrophied SOL muscles after reexposure to weight bearing. What was unexpected was that the SOL did not show this effect (Fig. 2). A recent study demonstrated that non-weight bearing by the SOL is associated with increased GLUT-4 transporter protein concentrations (17), suggesting that glucose transport was not the limiting factor in the present study. This is supported by the finding that G-6-P was significantly elevated 30 min after exercise (Table 3). However, the lack of an increase in glucose incorporation into glycogen for the SOL may be related to the fact that it exhibited the least glycogen reduction during exercise of the muscles examined (Fig. 3). We hypothesized that insulin action would be increased to a greater extent after exercise in the suspended rats’ muscles. However, several observations from this study suggest that intense exercise by the SUSE rats may have impaired hindlimb muscle responses to insulin. First, glucose incorporation into glycogen in the SOL was not significantly different between the SUS and SUS-E rats at either 90 or 24,000 pU/ml insulin (Fig. 2). In addition, the PL muscles from the SUS-E rats reached maximal glucose incorporation into glycogen rates in the absence of insulin, and this value did not increase with the addition of insulin (Fig. 2). Moreover, the glucose incorporation into glycogen values for the SOL and PL muscles were somewhat lower for the SUS-E than for the SUS rats at the maximally stimulating insulin concentration; however, these differences were not statistically significant (Fig. 2). Finally, glucose oxidation after 15 min of perfusion at the maximally stimulating insulin concentration was significantly lower for the SUS-E rats than for the SUS rats (Table 4). The reason for this apparent decrease in insulin action is uncertain, but it is possible that more muscle fiber damage occurred in the SUS-E rats than in the CC-E rats during exercise, which resulted in impaired glycogen synthesis or glucose transport. Kasper et al. (24) showed that when rats were run


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trained after 28 days of suspension they exhibited more evidence of muscle damage than control or suspended rats that did not run during recovery. In another study, rat hindlimb muscles exhibited a significant number of necrotic fibers after 2 days of recovery from 12.5 days of actual microgravity (31). There is also evidence in humans that muscle damage induced by eccentric contractions will reduce muscle glycogen synthesis (7, 28). O’Reilly et al. (28) have hypothesized that the attenuation of glycogen repletion after eccentric contractions may be due to ultrastructural damage to the sarcolemma, which may impair glucose transport into the cell. There are several methodological and interpretive limitations that warrant further discussion. First, the production of ‘*CO2 during perfusion is only an estimate for glucose oxidation (Table 4), since it is uncertain how much [14C]glucose was first converted to [14C]lactate before oxidation and whether lactate metabolism significantly influenced 14C0, production. In addition, it is important to note that for this study glucose uptake values were greater than the sums of the glucose oxidation and the glucose incorporation into glycogen values especially at the 0 and 90 PUfml insulin concentrations. This is probably the result of comparing two different methodologies, the removal of a nonlabeled substrate from the perfusate and the appearance of a radiolabel as glycogen or CO,. There are several factors that would cause the glucose uptake values to be overestimated in this study: 1) -20% of the perfused hindlimb consists of skin and bones (33), 2) -10% of the muscle volume represents extracellular space in which glucose can equilibrate without being transported into muscle cells (38), and 3) the rat possesses a vertebral venous plexus, which is not excluded by the surgical procedure, to which a small amount of the perfusate may be lost to the recirculating system (33). Because these conditions are not influenced by insulin, they have a more significant influence at low insulin concentrations when glucose uptake rates are relatively small. Therefore, only qualitative comparisons can be made between the glucose uptake results and the 14C0, production and [ 14C]glucose incorporation into glycogen results. Another limitation in this study was that the suspended rats had less hindlimb muscle mass than the control rats, but the same perfusate flow rate was used. Therefore, the relative flow rate (ml. g-l 4min-I) during perfusion was greater for the suspended animals (24%). This is of particular importance at the maximally stimulating insulin concentration when glucose delivery to the muscles may be limiting. However, the literature is equivocal on this point, with some studies showing a direct correlation between blood flow and glucose uptake by skeletal muscles (I2,34) and others showing no relationship (2, 20). Nevertheless, the possibility exists that the higher glucose uptake rates (15~17%) for the SUS and SW-E hindlimbs and glucose oxidation rates (46%) for the SUS hindlimbs at 24,000 PUlml insulin may be due in part to a higher relative blood flow. In summary, increased insulin action was observed at a submaximally stimulating concentration for hindlimb glucose uptake and for glucose incorporation into glycogen in the SOL, PL, and EDL muscles. The finding that

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the EDL response to insulin was higher in the SUS rats was unexpected and may be the result of the chronic stretch imposed on this muscle or an unknown systemic factor. It remains uncertain whether these results are caused by the effects of hindlimb non-weight bearing during SUS or by changes in body composition, the sympathetic nervous system, blood flow, or other factors associated with the restraint or position of the animals. The results of this study also indicated that glycogen concentrations were reduced more by exercise in the suspended rats even though they ran at the same relative exercise intensity. In addition, exercise stimulated greater increases in hindlimb muscle glucose uptake and its incorporation into glycogen in the suspended rats than in the control rats. On the other hand, there was evidence that intense exercise may impair some insulinsensitive processes in the muscles of suspended rats. This defect in insulin action may be due to ultrastructural muscle damage incurred during exercise; however, this hypothesis remains untested. Finally, it will be important to begin to examine the effects of actual microgravity on skeletal muscle before the relevance of these findings can be applied to humans or animals during spaceflight. We thank Dr. E. J. Henriksen for helpful advice during the preparation of this manuscript. This study was supported in part by National Aeronautics and Space Administration (NASA) Grant NAG-2-392. C. S. Stump was supported by NASA Graduate Student Researchers Program Fellowship NGT-50493. Address for reprint requests: C. M. Tipton, Dept. of Exercise and Sport Sciences, Ina E. Gittings Bldg., University of Arizona, Tucson, AZ 85721. Received 10 February 1992; accepted in final form 8 June 1992. REFERENCES 1. ARMSTRONG, R. B., AND R. 0. PHELPS. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259-272, 1984. 2. BERGER, M., S. HAGG, AND N. B. RUDERMAN. Glucose metabolism in perfused skeletal muscle. &o&em. J. 146: 231-238, 1975. 3. BONEN, A., G. C. B. ELDER, AND M. H. TAN. Hindlimb suspension increases insulin binding and glucose metabolism. J. Appl. Physiol. 65: 1833-1839,1988. 4. BRADY, L. J.,M.N. GOODMAN, F.N. KALISH,ANDN. B. RUDERMAN. Insulin binding and sensitivity in rat skeletal muscle: effect of starvation. Am. J. Physiol. 240 (Endocrinol. Metab. 3): E184-E190, 1981. 5. BUDOHOSKI, L., R. A. J. CHALLISS, A. DUBANIEWICZ, H. KACIUBAUSCILKO, B, LEIGHTON, F. J. LOZEMAN, K. NMAR, E. A. NEWSHOLME, AND S. PORTA. Effects of prolonged elevation of plasma adrenaline concentration in vivo on insulin-sensitivity in soleus muscle of the rat. Biochem. J. 244: 655-660,1987. 6. CZOK, R., AND W. LAMPRECHT. Pyruvate, phosphoenolpyruvate, and D-glycerate-2-phosphate. In: Methods of Enzymatic Analysis (3rd ed.), edited by H. U. Bergmeyer. Deerfield Beach, FL: Verlag Chemie, 1981, p. 1446-1451. 7. DOYLE, J. A., AND W. M. SHERMAN. Eccentric exercise and glycogen synthesis (Abstract). Med. Sci. Sports Exercise 23: S98, 1991. A. HEYWOOD-COOKSEY, AND R.J. 8. FITTS, R. H.,C.J. BRIMMER, TIMMERMAN. Single muscle fiber enzyme shifts with hindlimb suspension and immobilization. Am. J. Physiol. 256 (CeZlPhysiol. 25): C1082-C1091,1989. 9. GARDETTO, P. R., J. M. SCHLUTER, AND R. H. FITTS. Contractile function of single muscle fibers after hindlimb suspension. J. Appl. Physiol. 66: 2739-2749, 1989. 10. GARETTO, L. P.,E. A. RICHTER, M.N. GOODMAN,ANDN. B.RuDERMAN. Enhanced muscle glucose metabolism after exercise in


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Concurrent exercise on a gravity-independent device during simulated microgravity.

Effects of exercise and food restriction on rat skeletal muscles.

Effects of simulated microgravity (HDT) on blood fluidity.

Animal model of simulated microgravity: a comparative study of hindlimb unloading via tail versus pelvic suspension.

Simulated microgravity-induced mitochondrial dysfunction in rat cerebral arteries.

Hindlimb unloading: rodent analog for microgravity.

Investigation of simulated microgravity effects on Streptococcus mutans physiology and global gene expression.

Effects of simulated microgravity on Streptococcus mutans physiology and biofilm structure.

Planarians sense simulated microgravity and hypergravity.

Effects of joint immobilization and hindlimb unloading on collagen fibers of soleus muscles in rats.

Effects of simulated microgravity by RCCS on the biological features of Candida albicans.

Effects of microgravity or simulated launch on testicular function in rats.

Effects Of treadmill training on hindlimb muscles of spinal cord-injured mice.

Cortical Effects on Ipsilateral Hindlimb Muscles Revealed with Stimulus-Triggered Averaging of EMG Activity.

Effect of hindlimb suspension simulation of microgravity on in vitro immunological responses.

Effects of treadmill exercise on the distribution of blood flow between the hindlimb muscles and abdominal viscera of the laying fowl.

The mechanism of anti-osteoporosis effects of 3-hydroxybutyrate and derivatives under simulated microgravity.

Upregulation of miR-223 in the rat liver inhibits proliferation of hepatocytes under simulated microgravity.

Adaptation in synergistic muscles to soleus and plantaris muscle removal in the rat hindlimb.

Movement-dependent after-effects in the firing of the spindle endings from the de-efferented muscles of the cat hindlimb.

Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility.

Effects of eccentric exercise in rehabilitation of phasic and tonic muscles after leg immobilization in rats.

Effects of insulin on diacylglycerol-protein kinase C signaling in rat diaphragm and soleus muscles and relationship to glucose transport.

Effects of short spaceflights on mechanical characteristics of rat muscles.