Idea Transcript
Role of glucose transport in glycogen supercompensation in reweighted rat skeletal ERIK J. HENRIKSEN, CRAIG S. STUMP, THANH-HANG Muscle Metabolism Laboratory, Department of Physiology, University of Arizona College of Medicine, Rcson, Arizona Henriksen, Erik J., Craig S. Stump, Thanh-Hang T. Trinh, and Sean D. Beaty. Role of glucose transport in glycogen supercompensation in reweighted rat skeletal muscle. J. Appl. Physiol. 80(5): 1540-1546, 1996.-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-[P,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 t 2 to 16 t 1 nmol/mg muscle; P < 0.05) during the first 2 h of reweighting, glycogen accumulated at an average rate of 1.4 nmolmg-l h-l up to 18 h, reaching an apex of 38 t 1 nmol/mg. During this same reweighting period, insulinindependent, but not insulin-dependent, glucose transport activity was significantly enhanced (P < 0.05 vs. weightbearing 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. l
soleus muscle; weight 2-[1,2-3H]deoxy-D-glucose nase; citrate synthase
bearing; simulated uptake; GLUT-4
weightlessness; protein; hexoki-
SUSPENSION OF RODENTS is a frequently used and generally well accepted model of simulated weightlessnessand has been employed to investigate a variety of metabolic adaptations of skeletal muscle during reduced weight bearing (termed unweighting) (for a review, see Ref. 38). Less thoroughly studied are the metabolic responses to the return of hindlimb weight bearing (termed reweighting) after hindlimb suspension. We have previously demonstrated that reweighting of the soleus muscle after short-term (3 days) unweighting results in a dramatic, but transient, increase in soleus glycogen concentration (la>. This glycogen supercompensation could not be adequately explained by the activities of the primary regulatory enzymes of glycogen metabolism, glycogen synthase and phosphorylase. Therefore, we hypothesized that alterations in the transport and metabolism of glucose HINDLIMB
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in the reweighted muscle could play a role in this glycogenic response to reweighting (12). Glucose transport into skeletal muscle is acutely regulated by both insulin and by contractile activity (10, 27) thr ough the translocation of a regulatable glucose transporter isoform, the GLUT-4 protein (8, 17). The capacity for glucose transport is associated with the muscle content of GLUT-4 protein (10,24), and both insulin-stimulated glucose transport activity (2, 15,16,37) and GLUT-4 protein (14,22) are increased in unweighted rat soleus muscle. However, how these variables in the unweighted soleus muscle respond to the reintroduction of weight bearing is currently unknown. Hexokinase and citrate synthase are muscle enzymes that function in the cytosolic phosphorylation and mitochondrial oxidation, respectively, of glucose transported into the cell. Evidence has accumulated that the regulation of the muscle level of GLUT-4 may be coregulated with hexokinase (18, 25) and citrate synthase (6,ll) under conditions of increased neuromuscular activity. While it is known that these enzymes are increased in the soleus muscle of suspended animals (14, 33), their response to the relative increase in neuromuscular activity during reweighting has not been reported. In the context of this information, the primary goal of the present study was to test the hypothesis that enhanced glucose transport activity mediated by an increase in GLUT-4 protein is an important contributing factor in the glycogen supercompensation observed during reweighting of the soleus muscle. In addition, a secondary goal was to examine whether the reintroduction of weight bearing to a previously unweighted muscle would alter the cellular levels of hexokinase and citrate synthase. METHODS
Deatment of animals. Male Wistar rats (Harlan, Indianapolis, IN) weighing - 120 g were tranquilized with an intramuscular injection (10 ul/lOO g body wt) of Innovar-Vet (PitmanMoore, Mundelein, IL). The experimental groups were tail casted by using Hexalite orthopedic tape and Dow Corning Silastic 382 medical-grade elastomer (Factor II, Lakeside, AZ) and suspended for 3 days. Weight-bearing control animals underwent tranquilization but were not tail casted. Suspended animals were released from the apparatus and allowed to bear weight on their hindlimbs for the periods from 2 to 48 h. Food and water were available at all times. Between 8:00 and 10:00 A.M., animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (5 mg/lOO g body wt) and the soleus muscles were removed. Muscles were either clamp frozen in liquid nitrogen for use in tissue
o 1996 the American
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say grade; Sigma Chemical) in the absence or presence of 2 mu/ml porcine insulin (Eli Lilly, Indianapolis, IN). Electrical stimulations. For electrical stimulation of muscle contractions, the distal tendon of the muscle strip was attached to a vertical Lucite rod containing two platinum electrodes (10, 20). The proximal end was clipped to a jeweler’s chain and attached to a Grass model FT03 isometric force transducer. The mounted muscle was immersed in 25 ml KHB containing 8 mM glucose and 32 mM mannitol and continuously oxygenated with 95% 02-5% CO2 at 37°C. The muscle was stimulated with supramaximal square-wave pulses of 0.2-ms duration by using a Grass Sll stimulator. One to 10 tetanic contractions were produced by stimulating the muscle at 50 Hz for 10 s at a rate of 1 contraction/min. GZucose transport activity. After either the initial incubation in the absence or presence of insulin or the electrical stimulation protocol, all muscles were rinsed for 10 min at 37°C in 2 ml KHB containing 40 mM mannitol and insulin, if present previously. Finally, the muscles were transferred to 1.5 ml KHB containing 1 mM 2-[l,2-3H]deoxy-D-glucose (2DG; 300 uCi/mmol, 39 mM D-[U-i4C]mannitol (0.8 pCi/mmol) (ICN Radiochemicals), and insulin, if present previously. After this final 20-min incubation, muscles were trimmed of connective tissue, clamp frozen in liquid nitrogen, weighed, and dissolved in 0.5 ml of 0.5 N NaOH. After solubilization, 5 ml of scintillant were added and samples were analyzed for radioactivity in the 3H and 14C channels. The specific uptake of 2-DG was calculated as described previously (13). All values for in vitro 2-DG uptake are expressed as picomoles of 2-DG per milligram muscle wet weight per 20 min. Statistical anaZysis. The significance of differences between two groups was determined by using an unpaired Student’s t-test. When multiple comparisons were made, the significance of differences between groups was assessed by Dunnett’s post hoc test, with the nonsuspended weightbearing control group being used as the reference group. A P value of < 0.05 was considered significant.
metabolite determinations or were prepared into strips weighing m 18 mg (10, 13) and used for in vitro incubations. Fresh-frozen muscle factors. For analysis of glycogen (9), the muscle was dissolved by heating in 1 ml of 5 N KOH. Glycogen was purified by ethanol precipitation and then hydrolyzed to glucose by heating for 3 h at 100°C in 2 N HCl. After cooling, the sample was neutralized to pH 6-8 with 4 N NaOH and 0.1 M triethanolamine HCl and was assayed spectrophotometrically for glucose (1). Muscles from a separate group of animals were homogenized in 40 volumes of ice-cold 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid buffer (pH 7.4) containing 1 mM EDTA and 250 mM sucrose. Total protein concentration was determined by using the bicinchoninic acid method (Sigma Chemical, St. Louis, MO). The homogenates were frozen at -70°C until analysis. GLUT-4 protein was then assayed essentially as described by Rodnick et al. (30). Briefly, 25 ug of protein from each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (26) by using a 12% polyacrylamide gel (Jule Laboratories, New Haven, CT) and transferred to nitrocellulose filter paper. The nitrocellulose papers were then blocked with 5% nonfat dry milk (Carnation, Los Angeles, CA) in phosphate-buffered saline (PBS; pH 7.4) containing 0.2% sodium azide overnight at 4°C. GLUT-4 protein was detected by incubating the nitrocellulose papers at 37°C for 1 h in PBS containing 1% powdered milk and a 1:250 dilution of an antiserum specific for the COOH-terminal peptide sequence (residues 498-509) of this protein (East-Acres Biologicals, Southbridge, MA). Thereafter, the papers were washed in PBS containing 1% Triton X-100 and then incubated with 0.30 uCi/ml goat anti-rabbit 1251-labeled immunoglobulin G (ICN Radiochemicals, Irvine, CA) in PBS for 1 h at 37°C. After the final wash, papers were dried and exposed to Kodak XAR-5 film at - 70°C for 24-48 h. Autoradiographs were analyzed by scanning densitometry (model GS300 with GS370v2.3 software, Hoefer, San Francisco, CA). GLUT-4 protein levels in muscle samples from reweighted muscles were expressed relative to the average of nonsuspended weight-bearing control samples (arbitrarily set at 1.0) run on the same gel. Citrate synthase (34) and hexokinase (39) activities were assayed spectrophotometrically on the same homogenates that were used for determination of GLUT-4 and total protein. Insulin treatment. Muscles were incubated in the flaccid state at 37°C for 20 min in stoppered Erlenmeyer flasks containing 2 ml of oxygenated Krebs-Henseleit bicarbonate buffer (KHB) supplemented with 8 mM glucose, 32 mM mannitol, and 0.1% bovine serum albumin (radioimmunoas-
RESULTS
Response of body weight and soleus muscle wet weight and total protein to reweighting. There were no significant differences in final body weights among the various groups (Table 1). The 3-day unweighting period resulted in a 30% reduction (P < 0.05) in soleus wet weight and total mixed protein content, with no alteration in protein concentration. Soleus wet weight and protein content displayed a slow return toward weight-
Table 1. Body weights, soleus muscle wet weights, total protein contents, and total protein concentrations Final
Group
Weight-bearing 3 day suspended reweighting, 0 2 4 8 12 18 24 48 Values
are means
control + h
-t SE for 5-10
Body
Weight,
g
Soleus
Wet Weight,
mg
Soleus
Protein
Content,
mg
Soleus Protein Concentration, mg/g
129t2
45.3 + 1.0
7.4 IL 0.2
16423
12421 128k2 118 + 3 12124 132+3 128+4 12223 13054
31.7? 37.3 38.8 35.8 39.8 37.5 39.8 40.4
5.2 5.4 5.4 5.2 5.5 5.2 6.5 6.8
16657 143 + 139 k 145 t 140 + 137 2 163+3 169+2
animals
per group.
? + + + + t k
1.1* 1.6* 1.2* 2.2* 1.1* 1.7* 1.9* 1.9
*P < 0.05 vs. weight-bearing
control
value.
k + ? + + + t +
0.2* 0.3* 0.1* 0.3* 0.2* 0.3* 0.4 0.4
3* 4* 6* 4* 3*
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bearing control values over the passive reweighting period. Protein content recovered -60% of the deficit by 24 h, and this value was not significantly different from the weight-bearing control value by 48 h of reweighting. The protein concentration of the soleus was significantly reduced (-12 to 16%; P < 0.05) compared with weight-bearing control values during the first 18 h of recovery but had returned to control values by 24 h of reweighting. Response of soleus glycogen concentration to reweighting. As we have shown previously (15, 16), glycogen concentration in the 3-day unweighted soleus was 40% greater (P < 0.05) than in soleus from weight-bearing control animals (Fig. 1). After 2 h of passive reweighting, soleus glycogen concentration was reduced by 44% compared with the unweighted soleus value. However, during the next 16 h, glycogen concentration increased at an average rate of 1.4 nmol mg-l h-l so that by 18 h of recovery the concentration of this polysaccharide (37 8 +- 0 . 5 nmol./mg) was 225% greater than the 2-h value and 77% greater than the normal weight-bearing value. The period of most rapid glycogenesis appeared to be from 4 to 12 h of recovery. During the period of recovery from 24 to 48 h, glycogen levels in the soleus returned to control values, with no further change at 72 h (data not shown). This observation of soleus glycogen supercompensation during recovery from unweighting confirms our previous findings (12). This same pattern of changes in glycogen concentration was observed when the data are expressed relative to muscle protein content (data not shown). Response of soleus glucose transport activity and GLUT-4 protein to reweighting. As shown in Fig. 2, the net effect of insulin on the stimulation of glucose transport activity above basal was 75% greater (P < 0.05) in the 3-day unweighted soleus compared with the weight-bearing control muscle (2,202 t 94 vs. 1,255 t 131 pmolmg-l 20 min). However, this enl
IN REWEIGHTED
Control
1
SOLEUS
0
2
4
8
12
18
24
48
1
Hours of reweighting
l
Fig. 2. Glucose transport activity in soleus muscles of weightbearing control and suspension-reweighting groups. Open section of bars, 2- [ 1,2-3H] deoxy-D-glucose (2-deoxyglucose) uptake in absence of insulin; solid section of bars, net effect of insulin (2,000 yU/ml) above basal on 2-deoxyglucose uptake. Values are means + SE for 5-10 animals. *P < 0.05 vs. control value for insulin-independent glucose transport activity. **P < 0.05 vs. control value for total glucose transport activity (insulin independent + insulin dependent).
hanced insulin effect was quickly lost during the recovery period (1,612 it 199 at 2 h, 1,764 t 337 at 4 h, 1,463 t 205 at 8 h, 1,535 t 335 at 12 h, 1,322 t 178 at 18 h, 1,020 t 77 at 24 h, and 1,067 t 48 pmolmg-l.20 min-l at 48 h; all were not significant vs. weightbearing control value). In contrast, a significant increase (+105% vs. control value; P < 0.05) in insulinindependent glucose transport activity developed during this period, and reached its apex at 8 h of recovery (+196% vs. control value; P < 0.05). This insulinindependent glucose transport activity remained elevated through 24 h of recovery before returning to control values by 48 h. Soleus muscle from the 3-day suspended animals displayed a 55% greater (P < 0.05) GLUT-4 protein level compared with the weight-bearing control group (Fig. 3). During the first 18 h of recovery, GLUT-4 protein remained 43-57% greater than control values and was still 28% greater than control values at 24 h of recovery (all P < 0.05). Only after 48 h of reweighting did GLUT-4 protein return to the weight-bearing control value. Response of soleus hexokinase and citrate synthase activities to reweighting. In these same muscle homogenates, the specific activities of total hexokinase (Fig. 4) and citrate synthase (Fig. 5) were assessed. Although hexokinase activity was not significantly elevated by unweighting itself, we observed a steady and substantial increase in hexokinase activity during the first 18 h of reweighting, at which time this variable was 82% 0 4 8 12 16 20 24 48 greater (P < 0.05) than weight-bearing control values. Hours of reweighting Even after 48 h of recovery, hexokinase remained 63% Fig. 1. Glycogen concentrations in weight-bearing control soleus greater (P < 0.05) than control values. muscles (open circle) and in soleus muscles from animals undergoing After 3 days of unweighting, soleus citrate synthase various reweighting periods after 3 days of hindlimb suspension (solid circles). Values are means -+ SE for 5-8 animals. *P < 0.05vs. activity was 26% greater (P < 0.05) than in the weight-bearing control value. weight-bearing control muscle. This variable remained l
I,
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Control Control
1 0
2
4
8
12
18
24
48
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10
2
4
8
12
18
24
48 1
1
Hours of reweighting Hours of reweighting Fig. 3. Effect of unweighting and reweighting on soleus GLUT-4 protein levels. Open bar, GLUT-4 level in weight-bearing control muscles, arbitrarily set at 1.0; solid bar, GLUT-4 level in 3-day unweighted soleus; stippled bars, GLUT-4 levels in soleus unweighted for 3 days and then reweighted for times indicated. Values are means 2 SE for 5 animals. *P < 0.05 vs. weight-bearing control value.
elevated (+14-23%; P < 0.05) during the first 8 h of reweighting but was not significantly different from control values at 12 h of recovery. However, at 18 and 24 h of reweighting, citrate synthase was again significantly greater (+X5-22%; P < 0.05) than weightbearing control values before returning to control values at 48 h of recovery. Sensitivity of unweighted soleus muscle glucose transport to contractions. The data in Fig. 2 indicate that activation of insulin-independent glucose transport pathway in the previously unweighted soleus is achieved with reweighting. To investigate this phenomenon more rigorously, we determined the response of 2-DG uptake in weight-bearing and 3-day unweighted soleus muscles to various levels of electrically stimulated tetanic con(Fig. 6). Whereas five tetanic contractions
Fig. 5. Effect of unweighting and reweighting on soleus citrate synthase activity. Open bar, citrate synthase activity in weightbearing control muscles; solid bar, citrate synthase activity in 3-dayunweighted soleus; stippled bars, citrate synthase activities in soleus muscles unweighted for 3 days and then reweighted for times indicated. Values are means t SE for 5 animals. *P < 0.05 vs. weight-bearing control value.
were required to achieve maximal insulin-independent 2-DG uptake in normal weight-bearing muscle, in the 3-day unweighted soleus only a single tetanus was required to achieve this maximal response. The maximal rate of contraction-stimulated 2-DG uptake did not differ between weight-bearing and unweighted soleus muscle, in agreement with our previous findings (13). DISCUSSION
We have demonstrated previously that reweighting of the rat soleus muscle after a 3-day unweighting period results in a dramatic increase in glycogenesis (12). In that study, there was no association between this ranid rate of glycogen synthesis and an increased I
1000
800
600
0 Control
1 0
2
4
8
12
18
24
48
1
Hours of reweighting Fig. 4. Effect of unweighting and reweighting on soleus total hexokinase activity. Open bar, hexokinase activity in weight-bearing control muscles; solid bar, hexokinase activity in 3-day unweighted soleus; stippled bars, hexokinase activities in soleus muscles unweighted for 3 days and then reweighted for times indicated. Values are means t SE for 5 animals. *P < 0.05 vs. weight-bearing control value.
I
I
I
I
I
I
I
I
I
I
I
0
1
2
3
4
5
6
7
8
9
10
Number
of tetanic contractions
Fig. 6. Dose response of contraction-dependent glucose transport activity in soleus muscles of weight-bearing control and suspensionreweighting groups. Weight-bearing control soleus muscles (open circles) and 3-day unweighted soleus muscles (solid circles) were stimulated electrically to produce the indicated number of tetanic contractions. Glucose transport activity was then measured (see METHODS). Values are means + SE for 5 animals. *P < 0.05 vs. control muscles undergoing same number of tetanic contractions.
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activity of glycogen synth ase or a reduced activity of phosphorylase, indic ating that some other mechanis m was responsible for this metabolic response to reweighting. In the present study, we have found that a likely mechanism for this glycogenesis is an increase in insu lin-independent glucose transport activity because active during the period of this process is maximally most rapid glycogen syn thesis. This enhanced glucose transport activity would provide an increased number as glycogen. of glucose units available for deposition Furthermore, w e have presented additional new evidence supportive of functional roles of enhanced muscle levels of GLUT-4 protein and hexokinase in this glycogenie response to soleus reweighting. The evidence presented in this investigation is consistent with the hypothesis that the rate of glycogen synthesis in a muscle can be depend ent on the rate of glucose tranport i .nto the muscle. There are n umerous examples of thi .S type of autoregulation of skeletal muscle glucose metabolism. After a bout of glycogendepleting exercise, the recovery of glycogen stores in the epitrochlearis muscle is temporally associated with an increase in glucose transport activity (4). Additionally, in soleus and epitrochlearis muscles of transgenic mice overexpressing GLUT-l or GLUT-4 protein, both glucose transport activity and glycogen deposition are markedly enhanced in parallel (5, 29). Finally, a defect in muscle glucose transport likely underlies the reduced rate of muscle glycogen synthesis observed in human subjects with non-insulin-dependent diabetes mellitus (3 1). The large and rapid decline in soleus glycogen with the initiation of reweighting (Fig. 1) confirms our previous finding of rapid (within 15 min and for up to 2 h) glycogenolysis with passive weight bearing by previmuscle (12) and is 1ikely ca used by ously unweighted of phosphorylase due to the muscle the activation contractions (12). These findings are consi stent with those of Stump et al. (36), who reported a greater reduction in glycogen concen tra tion duri ng a 30-min bout of in ten se exercise bout in skeletal muscle from 14-day-suspended rats compared with muscle from a weight-bearing control group. The plateauing and onset of the decline of glycogen concentration at 18-24 h of recovery (Fig. 1) are also in agreement with our previous study (12). The initial reduction in glycogenesis and the subsequent onset of net glycogen loss occurred despite the continued elevation in insulinindependent glucose transport activity (Fig. 2) and GLUT-4 levels (Fig. 3), likely due to our previous observation of increased phosphorylase activity at these time points (12). Both in vivo contractile activity (Fig. 2) and in vitro electrical stimulation of muscle contractions (Fig. 6) led to large increases in insulin-independent glucose transport activity in the previously unweighted soleus. The insulin-independent pathway for stimulation of glucose transport in muscle appears to be Ca2+ dependent (21, 40). One can therefore speculate that unweighting of the s oleus results in m .arked alterations in Ca2+ relea se from the sarcopla .smic reticu lum, the prim ary source of
IN REWEIGHTED
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intracellular Ca2+ in muscle cells, with depolarization and that this Ca2+ plays a functional metabolic role in enhancing glycogenolysis and glucose transport. Several lines of evidence support this contention. Stevens et al. (35) found that unweighted soleus muscle fibers display significant increases in the amounts of, and the time course for, Ca2+ uptake and release from the sarcoplasmic reticulum in response to caffeine-induced contractions. This may be related to the findings in unweighted soleus muscle of a marked upregulation of the fast isoform of the Ca2+-adenosinetriphosphatase pump protein by Schulte et al. (32) as well as of the expression of the dihydropyridine receptor gene by Kandarian et al. (23). Finally, unweighting of the soleus results in enhanced cooperativity in, and higher sensitivity of, Ca2+- activated muscle contraction (7, 35), possibly related to transitions in the isoforms of myosin heavy chain, troponin T, and troponin I (3). These various findings could provide a biochemical basis for our observation in the unweighted soleus of increased sensitivity of glucose transport activation by contractions (Fig. 6). We have previously shown that 3 days of soleus unweighting results in a 35% increase in GLUT-4 protein level (14), and our present results are in good agreement with this finding (Fig. 3). Chronically increased neuromuscular activity typically causes enhancement of GLUT-4 protein levels (see Ref. 11 and references therein). The reweighting of the soleus can also be considered a chronic increase in muscle activity relative to that experienced during the unweighting period. It is therefore not surprising that GLUT-4 protein levels were maintained at elevated levels during the first 24 h of reweighting. Further work is needed to identify the intracellular factors responsible for this adaptive response of GLUT-4 protein to increased neuromuscular activity. The continued increase in hexokinase activity as the reweighting period progressed (Fig. 4) suggests that glucose phosphorylation, even in the face of enhanced glucose transport into the cell at these time points (Fig. 2), was not rate limiting for glucose metabolism. This rapid increase in hexokinase activity in response to renewed muscle contractile activity is consistent with previous findings of increases in hexokinase protein levels after just 30 min of exercise (28). Little is known regarding the intracellular signal responsible for increased hexokinase levels in response to contractions. However, in light of the above-mentioned alterations in Ca2+ fluxes during contractions performed by previously unweighted soleus muscle, one avenue of future research should be to investigate the role of increased intracellular Ca2+ release in the upregulation of this enzyme under conditions of increased muscle use. Interestingly, the response of citrate synthase in the soleus to the reintroduction of weight bearing appeared to be biphasic (Fig. 5). The first phase, observed during the first 8 h of reweighting, was characterized by a maintenance of the previously elevated enzyme activity, possibly due to the reintroduction of muscle contractile activity, because this is a powerful stimulus for mitochondrial biogenesis (19). By 12 h, however, citrate
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synthase had returned to control values. A second phase of elevated citrate synthase activity, observed through 24 h, was then evident. Henriksen and Halseth (11) and others (6) h ave previously observed an association between the upregulation of GLUT-4 protein and citrate synthase in skeletal muscle in response to contractile activity. While this association was maintained in the unweighted soleus and in the reweighted soleus up to 8 h (Figs. 3 and 5), this relationship was clearly nonexistent thereafter. This implies that during reweighting the regulation of the levels of these proteins is quite complex and that numerous regulatory factors may be involved. In conclusion, the data presented in the present study indicate that the rapid glycogenesis displayed by the reweighted soleus muscle is regulated, at least in part, by enhanced insulin-independent glucose transport activity associated with elevated GLUT-4 protein and hexokinase levels. The reweighted soleus muscle also experienced biphasic alterations in citrate synthase activity. The results provide further evidence that the recovery of muscle carbohydrate metabolism after unweighting is a complex process involving numerous regulatory events. We thank Katie Jolma, Mike Betlach, and Sean Anna for excellent technical assistance. This work was supported in part by National Aeronautics and Space Administration Grant NAG2-782 (to E. J. Henriksen); American Heart Association, Arizona Affiliate, Grant-in-Aid AZG-3-93 (to E. J. Henriksen); a Medical Student Fellowship from the American Diabetes Association (to C. S. Stump); and National Heart, Lung, and Blood Institute Grant T35-HL-07479 (to S. D. Beaty). Address for reprint requests: E. J. Henriksen, Dept. of Physiology, Ina E. Gittings Bldg. 93, Univ. ofArizona, Tucson, AZ 85721-0093. Received 1995.
27 September
1995;
accepted
in
final
form
5 December
REFERENCES 1. Bergmeyer, H. U., E. Bernt, F. Schmidt, and H. Stork. D-Glucose. In: Methods of’Enzymatic Analysis, edited by H. U. Bergmeyer. Deerfield Beach, FL: Verlag Chemie International, 1981, p. 1196-1201. 2. 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. 3. Campione, M., S. Ausoni, C. Y. Guezennec, and S. Schiaffino. Myosin and troponin changes in rat soleus muscle after hindlimb suspension. J. Appl. Physiol. 74: 1156-1160, 1993. 4. Cartee, G. D., D. A. Young, M. D. Sleeper, J. Zierath, H. Wallberg-Henriksson, and J. 0. Holloszy. Prolonged increase in insulin-stimulated glucose transport in muscle after exercise. Am. J. Physiol. 256 (Endocrinol. Metab. 19): E494-E499, 1989. 5. Deems, R. O., J. L. Evans, R. W. Deacon, C. M. Honer, D. T. Chu, K. Biirki, W. S. Fillers, D. K. Cohen, and D. A. Young. Expression of human GLUT-4 in mice results in increased insulin action. Diabetologia 37: 1097-1104,1994. 6. Etgen, G. J., R. P. Farrar, and J. L. Ivy. Effect of chronic electrical stimulation on GLUT-4 protein content in fast-twitch muscle. Am. J. Physiol. 264 (Regulatory Integrative Comp. Physiol. 33): R816-R819, 1993. 7. Gardetto, P. R., J. M. Schluter, and R. H. Fitts. Contractile function of single muscle fibers after hindlimb suspension. J. AppZ. Physiol. 66: 2739-2749, 1989. 8. Goodyear, L. J., M. F. Hirshman, and E. S. Horton. Exerciseinduced translocation of skeletal muscle glucose transporters. Am. J. Physiol. 261 (EndocrinoZ. Metab. 24): E795-E799, 1991.
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