Effect of denervation or unweighting protein in rat soleus muscle ERIK J. HENRIKSEN, KENNETH J. RODNICK, DAVID E. JAMES, AND JOHN 0. HOLLOSZY

CARL

on GLUT-4

E. MONDON,

Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110; Department of Medicine, Stanford University School of Medicine, and Geriatric Research, Education, and Clinical Center, Veterans Administration Medical Center, Palo Alto, California 94304

HENRIKSEN,ERIKJ., KENNETHJ. RODNICK$ARL E. MON- decline in both the basal and maximal rates of insulinDON,DAVIDE. JAMES,ANDJOHNO.HOLLOSZY.E~~~~~~~~~~~~-stimulated glucose transport in vitro (13), which coinvption or unweighting on GLUT-4 protein in rat soleus muscle. J. cides with a marked reduction in contractile activity as Appl. Physiol. 70(5): 2322-2327, 1991.-The purpose of this determined by electromyographic activity (1). However, study wasto test the hypothesisthat the decreasedcapacity for unweighting of longer duration (23 days) results in 1) the glucose transport in the denervated rat soleus and the inreturn of electromyographic activity to normal and then creasedcapacity for glucosetransport in the unweighted rat above-normal levels (1,25) and 2) an increase in the basoleusare related to changesin the expressionof the regulatsal rate of glucose transport to control levels and to the able glucosetransporter protein in skeletal muscle(GLUT-4). One day after sciatic nerve sectioning, when decreasesin the development of enhanced insulin action on this process stimulation of soleus2-deoxyglucose(2-DG) uptake by insulin (3, 12-14, 31). (-51%, P < O.OOl),contractions (-29%, P < 0.05), or insulin Recent studies have documented the existence of a and contractions in combination (-40%, P < 0.001) were ob- family of facilitative glucose transporters (GLUT-l to served,there wasa slight (-18%, NS) decreasein GLUT-4 pro- GLUT-5), the most characteristic feature of which is tein. By day 3 of denervation, stimulation of 2-DG uptake by their unique tissue distribution (reviewed in Ref. 2). insulin (-74%, P < O.OOl),contractions (-31%, P < O.OOl),or GLUT-4 is expressed only in tissues that exhibit insulinthe two stimuli in combination (-59%, P < O.OOl),as well as stimulated glucose transport (17) and is probably the GLUT-4 protein (-52%, P < O.OOl),was further reduced. Somajor glucose transporter isoform expressed in skeletal leus musclefrom hindlimb-suspendedrats, which developsan a strong enhanced capacity for insulin-stimulated glucose transport, muscle (2). We have recently demonstrated correlation between the rate of glucose transport stimushowedmuscle atrophy similar to denervated soleusbut, in contrast, displayed substantial increasesin GLUT-4 protein lated by insulin and contractile activity and the level of after 3 (+35%, P < 0.05) and 7 days (+107%, P < 0.001).These GLUT-4 expressed in skeletal muscles of sedentary rats results indicate that altered GLUT-4 expressionmay be a ma- (10). In addition, Rodnick et al. (26) have shown that the jor contributor to the changesin insulin-stimulated glucose level of GLUT-4 protein is increased in skeletal muscles transport that are observedwith denervation and unweighting. of exercise-trained animals, which correlates with an enWe conclude that muscleactivity is an important factor in the hancement of insulin-mediated glucose disposal (27). regulation of GLUT-4 expressionin skeletal muscle. muscleatrophy; glucosetransport; insulin; contractile activity; skeletal muscle IT IS WELL DOCUMENTEDthat both denervation,

by sectioning of the sciatic nerve, and unweighting, by hindlimb suspension, lead to significant atrophy (8, 30) as well as to marked alterations in glucose transport (3,4,7, 12-14,31,32) in the rat soleus. Within 24 h after denervation, stimulation of glucose transport by insulin is impaired (4, 32), and this insulin resistance is maintained during longer periods of denervation (7, 31, 32). Similarly, acute (524 h) unweighting of the soleus leads to a

The results of these studies support a relationship among the chronic level of muscle activity, expression of GLUT-4 protein, and glucose transport capacity. Therefore, in the present study we investigated the possibility that the changes in glucose transport activity in denervated or unweighted muscle may be associated with alterations in the expression of this glucose transporter isoform. In addition, we addressed the question of whether denervated muscle becomes resistant to the stimulation of glucose transport by contractile activity. MATERIALS AND METHODS Denervation studies. Male Wistar

NE) weighing -140

rats (Sasco, Omaha, g were anesthetized with pentobar-

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GLUT-4

PROTEIN

EXPRESSION

bital sodium (1.5 mg/lOO g body wt ip) supplemented with halothane during surgery. Denervation of one hindlimb was carried out by removal of a 3-mm section of the sciatic nerve just beneath the biceps femoris muscle. The sciatic nerve of the contralateral limb was left intact. Recovery from anesthesia was rapid (1-2 h), and animals resumed normal feeding and behavior patterns within 2-4 h. Denervation periods were 1 and 3 days. Food was restricted to 3 g after 5:00 P.M. of the evening before the experiment. Between 8 and 9 A.M., animals were anesthetized with pentobarbital sodium (5 mg/lOO g body wt ip) and the soleus muscles were removed. One strip (16-23 mg) from each soleus muscle was obtained as described previously (10) and used for in vitro measurements of glucose transport (see below). The remaining piece of muscle was clamp-frozen in liquid N, for subsequent determinations of GLUT-4 protein level and enzyme activities. Muscle incubations. Soleus muscles were incubated initially for 60 min in 2 ml of oxygenated Krebs-Henseleit bicarbonate buffer (KHB) (19) containing 8 mM glucose, 32 mM mannitol, and 0.1% bovine serum albumin (radioimmunoassay grade) in the absence or presence of the additions indicated in Fig. 1. The gas phase in the flasks was 95% O,-5% CO,. The flasks were shaken in a Dubnoff incubator at 35OC. Muscle stimulations. For electrical stimulation, the distal tendon of the muscle was attached to a vertical Lucite rod containing two platinum electrodes (16). 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 20 ml KHB containing the same additions as during the prior 60-min incubation and continuously oxygenated with 95% O,-5% CO, at 35OC. For activation of glucose transport activity by contractions, the muscles were stimulated with supramaxima1 square-wave pulses of 0.2ms duration by means of a Grass Sll stimulator. Ten tetanic contractions were produced by stimulation at 100 Hz for 10 s at a rate of one contraction per minute for 10 min. This stimulation protocol was found to elicit a maximal effect of muscle contractions on sugar transport, as evidenced by the finding that increasing the number of tetani from 10 to 15 had no further effect on glucose transport activity (E. J. Henriksen and J. 0. Holloszy, unpublished data). Measurements of glucose transport activity. Glucose transport activity was measured using the glucose analogue 2deoxyglucose (2-DG) and a modification (37) of the procedure used previously in frog sartorius muscle (16, 21). After the initial incubation periods or electrical stimulation, the muscles were rinsed in the absence of glucose for 10 min at 29OC in 2 ml of oxygenated KHB containing 40 mM mannitol and any additions present during the previous incubation period. The muscles were then incubated for 20 min at 29°C in 1.5 ml of KHB containing 1 mM 2-deoxy-[1,2-3H] glucose (1.5 mCi/ mmol) and 39 mM [ U-‘“Cl mannitol(8 &i/mmol) (ICN Radiochemicals, Irvine, CA) in the absence or presence of the additions indicated. The gas phase in the flasks during both the rinse and incubation periods was 95% O,-5% CO,. The muscles were then processed, and the extracellular space and intracellular 2-DG concentra-

IN MUSCLE 3

A 0

% VL 5

_ m

‘E 2

u

$ g g i Ed g 5 1 4 f w z

2323

ATROPHY

INV soleus l-day

DNV soleus.

3-day

DNV soleus.

7

l

0 No Additions

Insulin

(2000plJ/ml)

Vanadate (5 mw

4

z 5 g 8 $

F 3 *E g r’ 2 k 5’ ; E c; -d 0

Contractions

Insulin + Contractions FIG. 1. Effect of denervation on %deoxyglucose uptake in soleus after stimulation by insulin, vanadate, contractions, or insulin and contractions in combination. Glucose transport activity in l- and 3-day denervated (DNV) and contralateral innervated (INV) soleus muscles was measured after treatment with insulin or vanadate (A) or after electrically stimulated contractile activity alone or in combination with insulin (B), as described in MATERIALS AND METHODS. Each bar represents mean + SE for number of muscles shown at bottom of bar. *P < 0.05, **p < 0.01, *** P < 0.001 vs. innervated control.

tions were determined as described previously (37). 2-DG uptake under the se condi tions is linear and represents glucose transport activity (10) . Gl ucose transport activity is expressed as pmol 2-DG. ml intracellular water-l 20 min? Unweighting studies. Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing -180 g were used . This strain of rat was used in light of the vast database available from studies on the effects oIf unweighting on skeletal muscle metabolism in Sprague-Dawley rats (as reviewed in Ref. 30). Each animal was wrapped in a terry cloth towel with the tail exposed, and the tail was wash ed with a warm det ergent soluti .on, rinsed, and dried. Tincture of benzoin was then applied to the proximal 7 cm of the tail and allowed to dry to a slight tackiness (-5 min). A suspension hook was attached to the tail with a 1 X 1.6-cm strip of adhesive traction tape and secu red with three ligatures of 4-o surgical silk. The anima1 was then suspended at a 45’ angle in a 33 X 33 X 38-cm Plexi .glas cage by attaching the susp lension hook to a overhead wheel, the reby unweighting the hindlimb muscles. The animal was able to move in all directions with the forelimbs and had free access to food and water. l

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2324

GLUT-4

PROTEIN

EXPRESSION

IN

MUSCLE

ATROPHY

day of unweighting (-14%; Table 1), significant atrophy of the soleus was observed only after 3 days of unweighting (-26%), with a further loss of mass by day 7 (-38%), consistent with the findings of previous investigations (30). No significant alteration in the total protein concentration was caused by unweighting. Interestingly, unweighting resulted in significant increases in the activities of hexokinase (+30% at day 3) and citrate synthase (+45% at day 7) when expressed relative to muscle mass, in agreement with previous observations of increases in the specific activity of mitochondrial enzymes in singlefiber preparations from unweighted soleus (30). However, when the activities of these enzymes are expressed per whole muscle, the values for the l-day (citrate synthase, 2.36 t 0.17 pmol min-’ . muscle-‘; hexokinase, 0.095 t 0.007 pm01 min-l . muscle-‘), 3-day (2.34 t 0.19 and 0.085 t 0.009), and 7-day (2.70 t 0.13 and 0.085 t 0.005) unweighted soleus did not differ significantly from the weight-bearing control (2.78 t 0.17 and 0.106 t 0.007). Therefore the increases in specific activities of these enzymes were the result of no loss of total enzyme content coupled with a net loss of total muscle mass. Glucose transport capacity in denervated soleus. As shown in Fig. lA, basal glucose transport activity was significantly reduced after 1 (-28%) and 3 days (-29%) of denervation. Stimulation of glucose transport with a maximally effective concentration of insulin (2,000 pU/ ml) was reduced by 51% after 1 day of denervation and by 74% after 3 days of denervation. It has been suggested on the basis of insulin-binding studies (4, 7) that insulin resistance in the denervated soleus is the result of a postreceptor-binding defect. Vanadate, which activates glucose transport in skeletal muscle through the same pathway as insulin (11) but at a point distal to the binding of insulin to its receptor (9), was used to test this hypothesis (Fig. 1A). Denervation for 1 or 3 days caused reductions of 42 and 70%, respectively, in stimulation of glucose transport activity by a maximally effective concentration of vanadate (5 mM). These findings provide further support for the hypothesis that denervation-induced insulin resistance is caused by an alteration in some postbinding event. Glucose transport in isolated mammalian skeletal muscle can also be stimulated by an insulin-independent mechanism that is activated by contractions (5, 10, 23, 34). The maximal capacity for glucose transport is that rate achieved through the additive effects of both insulin-dependent and insulin-independent pathways (5,10, 23,34). We therefore examined whether denervation had a deleterious effect on contraction-stimulated glucose RESULTS transport as well as on the maximal capacity for glucose Body weight, muscle mass, total protein, and enzyme transport (Fig. 1B). After 1 day of denervation, contracactiuities. Body weights for the animals in the various tion-stimulated glucose transport was reduced by 29%. of the ability of the groups are shown in Table 1. Denervation for 1 day had This was not due to an impairment no effect on soleus mass relative to body weight (Table denervated soleus to contract in response to the electri1). However, by day 3 of denervation, the soleus mass was cal stimulus, inasmuch as maximal force production in 25% (P < 0.001) smaller than control. Denervation had this muscle was not different (1.17 t 0.12 g/mg wet wt) no significant effect on total protein concentration or on from that in the innervated control (1.01 t 0.13). Interthe activities of hexokinase and citrate synthase (Table estingly, 2 additional days of denervation did not further 1), indicating that muscle mass, total protein, and en- reduce this rate of glucose transport (31% less than conzymes decreased in parallel. trol), whereas force production was markedly diminished Although there was some loss of soleus mass after 1 (0.30 t 0.11, P < 0.001 vs. control).

At the end of the suspension periods (1,3, or 7 days), the animals were anesthetized with thiamylal sodium (6 mgl 100 g body wt ip) and the soleus muscles were quickly removed and frozen between blocks of solid CO, covered with polyethylene. All muscles were stored at -9OOC until assayed. Total and GLUT-4 protein determinations. Muscles (lo-20 mg wet wt) were homogenized for 15 s in 40 vol of ice-cold 20 mM N-Z-hydroxyethylpiperazine-N’-Z-ethanesulfonic acid buffer (pH 7.4) containing 1 mM EDTA and 250 mM sucrose by use of a Tekmar tissue homogenizer set at 75% of maximum output. Total protein concentration was determined using the bicinchoninic acid method (Pierce, Rockford, IL). The homogenates were frozen at -2OOC until analysis. GLUT-4 protein was then assayed using the method of Rodnick et al. (26). Briefly, 25 pg of protein from each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (20) using a 10% polyacrylamide gel 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 using the antiserum R820, which is specific for the COOH-terminal peptide sequence (residues 498-509) of this protein (17). Nitrocellulose papers were exposed to 10 pglml R820 in PBS and 1% powdered milk for l. h at 37OC. Thereafter the blots were washed in PBS containing 1% Triton X-100, incubated with 2.5 PC1 125I-protein A (Amersham, Arlington Heights, IL) in 10 ml PBS for 1 h at 37”C, dried, and exposed to Kodak XAR-5 film at -7OOC. The labeled bands were traced on the nitrocellulose, cut out, and counted in a gamma counter (Beckman model 8000). The counts from each band were corrected for background radiation by counting nonlabeled areas of nitrocellulose. Results were corrected for standards to allow direct comparison of counts from different blots. The data are expressed as counts per minute per 25 pg protein. Enzyme actiuities. Hexokinase (33) and citrate synthase (29) activities were assayed spectrophotometritally on the same homogenates that were used for determination of GLUT-4 and total protein. Statistics. The significance of differences between two groups was assessed using Student’s t test. When multiple comparisons were made, a factorial analysis of variance was used, with post hoc analysis using Scheffe’s F test.

l

l

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2325

GLUT-4 PROTEIN EXPRESSION IN MUSCLE ATROPHY TABLE 1. Body weight, soleus weight, soleus total protein concentration, activities in denervated and suspended animals

Body Wt, Condition

g

and soleus hexokinase

and citrate synthase

Enzyme Activities, pm01 mg muscle-’ min-’

Soleus Wt, mg/lOO g body wt

Soleus Total Protein Concn, mg/g muscle

Hexokinase

l

l

Citrate

synthase

Innervated control 1-day denervated 3-day denervated

142t2 138t3 145+2

43.9kl.O 45.9t0.5 33.0+1.2?

142+5 133+6 155+8

0.74t0.07 ND 0.79kO.09

21.4t1.5 ND 23.5k3.4

Weight-bearing control l-day unweighted 3-day unweighted 7-day unweighted

192+4 181+4 185k2 205+2$

48.521.8 41.6tl.O 35.9a1.4t 30.2+1.9?

150t5 153t6 159t5 141+2

1.16t0.08 1.26kO.09 1.51+0.07* 1.38t0.12

30.2kl.l 31.7k1.3 33.5k1.8 43.8+1.8t

Values are means + SEfor 4-13 animals or muscles per group. ND, not determined. * P < 0.05, t P < 0.001 vs. control. $ P < 0.001 vs. l-day and 3-day unweighted groups.

The increases above basal in glucose transport activity induced by insulin (Fig. 1A) and by contractions (Fig. 1B) were additive in the control and l- and 3-day denervated groups (Fig. 1B). This maximal rate of stimulated glucose transport was reduced by 40 and 59% after 1 and 3 days of denervation, respectively. These data indicate that the denervation-induced resistance to maximal stimulation of glucose transport is the result of defects in both the insulin- and the contraction-dependent pathways.

extended these previous findings in the following ways. First, we have demonstrated that acute denervation of the soleus causes a significant impairment in contraction-stimulated glucose transport (Fig. 1B). This suggests that the defect associated with denervation is not specific to the insulin-signaling pathway but involves a step common to both contraction and insulin pathways. Second, by showing that denervation also causes resistance to vanadate (Fig. lA), which acts distally to the insulin-binding step (9), we have provided further supGLUT-4 protein in denervated or unweighted soleus. port for a postbinding step as the defect causing the obUnlike denervation, which caused a marked insulin resis- served insulin resistance (4, 7). The glucose transporter tance of the glucose transport process (Fig. lA), un- itself would be a good candidate to account for these weighting of the soleus has been shown previously to pro- changes in glucose transport, and it is of considerable gressively enhance insulin action on this process (3, 12- interest that GLUT-4 levels are significantly reduced in 14, 31). We therefore measured the level of GLUT-4 denervated soleus muscle (Fig. 2). protein in denervated and unweighted soleus muscles to An experimental model that produces opposite alterdetermine whether these alterations in glucose transport ations in glucose metabolism to denervation is that of may be associated with changes in the expression of this hindlimb unweighting (3, 12-14, 31). In the unweighted protein. As shown in Fig. 2, after 1 day of denervation the soleus, insulin-stimulated glucose transport is enhanced level of GLUT-4 protein was 18% less than control, al- (3, 12-14, 31). In the present study, we showed that though this decline did not achieve statistical signifiGLUT-4 levels are increased with unweighting (Fig. Z), cance (P > 0.05). However, after 3 days of denervation, providing a functional basis for the changes observed in GLUT-4 protein was reduced by 52% (P < 0.001) relative glucose transport. Previous studies have demonstrated to control. that insulin resistance may not be related to altered levAfter 1 day of unweighting, soleus GLUT-4 protein els of GLUT-4 protein in skeletal muscle. In both genetiwas increased by 22% relative to control (Fig. Z), but this cally obese mice (18) and non-insulin-dependent diabetic difference did not achieve statistical significance. A pro- humans (24), GLUT-4 protein levels in skeletal muscle gressive increase in the level of GLUT-4 protein was ob- are not different from control. However, on the basis of served with unweighting of longer duration, with in- the present results concerning GLUT-4 protein exprescreases of 35 and 107% after 3 and 7 days, respectively sion in denervated and unweighted muscle (Fig. 2) and (Fig. 2). When expressed on a whole-muscle basis, previous reports of altered GLUT-4 protein expression GLUT-4 protein in the l-day (1.34 t 0.07 X lo5 cpm/ in muscles of different fiber type compositions (10) and muscle) and 3-day (1.40 t 0.10 X 105) unweighted soleus after exercise training (26), it is evident that neural input did not differ from that in weight-bearing control soleus and/or contractile activity may be an important modula(1.30 t 0.12 X 105). However, 7 days of unweighting re- tor of GLUT-4 protein expression in skeletal muscle. sulted in a net increase in GLUT-4 protein (1.73 t 0.12 X Increasing the chronic level of muscle activity, through 105; P < 0.05). training of animals by either treadmill or wheel running or by chronic low-frequency electrical stimulation, leads to increases both in enzymes involved in glucose phosDISCUSSION phorylation (15, 28, 35) and oxidation (15, 27,28) and in In the present study, we have confirmed that denervaGLUT-4 protein levels (26). This parallel regulation of tion of the soleus induces severe insulin resistance of the glucose transporter expression and the capacity to metabglucose transport process (Fig. 1A) (4, 7,32). Insulin re- olize glucose also appear to be functional under condisistance was evident within 24 h of denervation and be- tions of unweighting and denervation. Unweighting of came more pronounced over a 3-day period. We have also the soleus, which eventually results in normal or aboveDownloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 4, 2018. Copyright © 1991 American Physiological Society. All rights reserved.

2326

GLUT-4

PROTEIN

EXPRESSION

IN

MUSCLE

ATROPHY

FIG. 2. Effect of unweighting or denervation on expression of GLUT-4 protein in soleus. Top: representative autoradiogram of immunoblot of GLUT-4 protein in unweighted and denervated soleus muscles and their respective controls. Protein (25 pg) from each muscle was analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with antiserum R820 against GLUT-4 protein. Immunolabeled bands were then visualized with ‘ZSI-labeled protein A. Molecular weight of the visualized protein was -43,000. Bottom: bands from individual immunoblots were counted for radioactivity as described in MATERIALS AND METHODS. Each bar represents mean + SE for number of individual samples shown at bottom of bar. *P < 0.05, **P < 0.01 vs. control.

Contrd

ldav

3dav

Unweighted

T-day

Control

l-day

I-day

Denervated

normal electromyographic activity (1, 25), causes concomitant increases in GLUT-4 protein and hexokinase and citrate synthase specific activities (Table 1) (30). Denervation of the soleus, which eliminates neural control of contractile activity, leads to decreases in GLUT-4 protein (Fig. 2) and, in investigations using a longer treatment period than in the present study, also leads to decreasesin a number of oxidative enzymes (22,36). Therefore, we believe that, as is the case for hexokinase (15), it may be appropriate to consider the GLUT-4 protein as one of the important proteins that constitute the oxidative machinery of the muscle. This entire subset of proteins (GLUT-4, hexokinase, oxidative enzymes) presumably serve critical roles during prolonged states of increased activity, during which the ability of working muscle to take up and utilize blood-borne glucose is important for the maintenance of energy production. During the initial 24 h of unweighting, the alterations in GLUT-4 protein expression (Fig. 2) did not reflect the change in insulin-stimulated glucose transport (13). In addition, while insulin-stimulated glucose transport in the l-day denervated soleus had decreased 51% (Fig. l), the GLUT-4 protein level had decreased only 18% (Fig. 2). Although this decrease in GLUT-4 protein likely contributed to the decrease in glucose transport in the l-day denervated soleus, other factors must also be involved in this insulin resistance. One possible explanation for the initial reduced capacity for glucose transport under both experimental conditions is an altered subcellular distribution of existent glucose transporters, with a reduced

ability of the muscle to translocate transporters to the plasma membrane. This possibility remains to be tested experimentally. It has been shown that in addition to the GLUT-4 isoform the GLUT-l isoform of the glucose transporter is also expressed in skeletal muscle, although it appears to be confined to the plasma membrane (6). We have not observed any alterations in the expression of GLUT-l protein in homogenates of denervated, unweighted, or exercise-trained soleus muscles (unpublished results). This indicates that these activity-mediated changes are specific to the GLUT-4 isoform. In summary, we have demonstrated that the changes in the expression of GLUT-4 protein in soleus muscle cannot completely explain the acute (~24 h) responses of insulin-stimulated glucose transport to either denervation or unweighting. However, there appears to be an association between the decreased level of GLUT-4 protein and the increasing insulin resistance in 3-day denervated soleus and between the increasing level of this protein and the progressively enhanced action of insulin on glucose transport reported in the unweighted soleus. The expression of GLUT-4 protein in soleus may be dependent on the habitual level of contractile activity performed by the muscle. We thank May Chen and Lori A. Nolte for excellent technical assistance. This work was supported by National Institutes of Health Grants AG-00425 (J. 0. Holloszy), DK-42503 (D. E. James), and Institutional National Research Service Award AG-00078 (E. J. Henriksen and K. J.

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GLUT-4

PROTEIN

Rodnick) and by National Aeronautics and Space Administration Grant MONO002109 (C. E. Mondon). Address for reprint requests: E. J. Henriksen, Dept. of Exercise and Sport Sciences, McKale Center 228A, University of Arizona, Tucson, AZ 85721. Received 20 November 1990; accepted in final form 5 February 1991.

E. K., R. R. ROY, J. A. HODGSON, AND V. R. EDGERTON. Electromyography of rat soleus, medial gastrocnemius, and tibialis anterior during hindlimb suspension. E=cp. Neural. 96: 635-649, 1987. BELL, G. I., T. KAYANO, J. B. BUSE, C. F. BURANT, J. TAKEDA, D. LIN, H. FUKUMOTO, AND S. SEINO. Molecular biology of mammalian glucose transporters. Diabetes Care 13: 198-208, 1990. BONEN, A., G. C. B. ELDER, AND M. H. TAN. Hindlimb suspension increases insulin binding and glucose metabolism. J. AppZ. Physiol. 65: 1833-1839, 1988. BURANT, C. F., S. K. LEMMON, M. K. TREUTELAAR, AND M. G. BUSE. Insulin resistance of denervated rat muscle: a model for impaired receptor-function coupling. Am. J. Physiol. 247 (Endocrinol. Metab. 10): E657-E666, 1984. CONSTABLE, S. H., R. J. FAVIER, G. D. CARTEE, D. A. YOUNG, AND J. 0. HOLLOSZY. Muscle glucose transport: interactions of in vitro contractions, insulin, and exercise. J. Appl. Physiol. 64: 2329-2332, 1988. DOUEN, A. G., T. RAMLAL, S. RASTOGI, P. J. BILAN, G. D. CARTEE, M. VRANIC, J. 0. HOLLOSZY, AND A. KLIP. Exercise induces recruitment of the “insulin-responsive glucose transporter.” J. BioZ. Chem. 265: 13427-13430,199O. FORSAYETH, J. R., AND M. K. GOULD. Inhibition of insulin-stimulated xylose uptake in denervated rat soleus muscle: a post-receptor effect. Diabetologia 23: 511-516, 1982. GOLDSPINK, D. F. The effects of denervation on protein turnover of rat skeletal muscle. Biochem. J. 156: 71-80, 1976. GREEN, A. The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin receptor level. Biothem. J. 238: 663-669, 1986. HENRIKSEN, E. J., R. E. BOUREY, K. J. RODNICK, L. KORANYI, M. A. PERMUTT, AND J. 0. HOLLOSZY. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E593-E598, 1990. HENRIKSEN, E. J., M. D. SLEEPER, J. R. ZIERATH, AND J. 0. HOLLOSZY. Polymyxin B inhibits stimulation of glucose transport in muscle by hypoxia or contractions. Am. J. Physiol. 256 (Endocrinol. Metab. 19): E662-E667, 1989. HENRIKSEN, E. J., AND M. E. TISCHLER. Time course of the response of carbohydrate metabolism to unloading of the soleus. Metabolism 37: 201-208, 1988. HENRIKSEN, E. J., AND M. E. TISCHLER. Glucose uptake in the rat soleus: effect of unloading and subsequent reloading. J. AppZ. Physiol. 64: 1428-1432, 1988. HENRIKSEN, E. J., M. E. TISCHLER, AND D. G. JOHNSON. Increased response to insulin of glucose metabolism in the six-day unloaded rat soleus muscle. J. BioZ. Chem. 261: 10707-10712, 1986. HOLLOSZY, J. O., AND F. W. BOOTH. Biochemical adaptations to endurance exercise in muscle. Annu. Reu. Physiol. 38: 273-291, 1976. HOLLOSZY, J. O., AND H. T. NARAHARA. Studies of tissue permeability. X. Changes in permeability to &methyl-glucose associated with contraction of frog muscle. J. BioZ. Chem. 240: 3493-3500,

1. ALFORD,

3.

4.

5.

6.

7.

8. 9.

10.

11.

12. 13. 14. 15.

16.

17. JAMES,

D. E., M. STRUBE, AND M. MUECKLER.

ATROPHY

2327

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Effect of denervation or unweighting on GLUT-4 protein in rat soleus muscle.

The purpose of this study was to test the hypothesis that the decreased capacity for glucose transport in the denervated rat soleus and the increased ...
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