Exercise-induced translocation of skeletal muscle glucose transporters.

Exercise-induced translocation of skeletal muscle glucose tranmorters LAURIE J. GOODYEAR, MICHAEL F. HIRSHMAN, AND EDWARD S. HORTON Metabolic Unit, Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont 05405

GOODYEAR, LAURIE J., MICHAEL F. HIRSHMAN, AND EDWARD S. HORTON. Exercise-induced translocation of skeletal muscle glucose transporters. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E795-E799, 1991.-Skeletal muscle contractile activity results in increased rates of glucose transport that are associated with an increase in the number and activity of plasma membrane glucose transporters. In the current study it was determined whether exercise causes a translocation of glucose transporters from an intracellular pool to the plasma membrane and whether exercise and insulin stimulate the same glucose transporter protein. Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased from 10.1 + 0.73 to 15.0 t 1.4 pmol/mg protein (P < 0.01) in muscle of exercised rats, whereas microsomal membrane transporters decreased significantly from 6.0 t 0.7 to 4.2 t 0.4 pmol/ mg protein (P < 0.05). Western blot analysis using the monoclonal antibody mAb lF8 (specific for GLUT-4) demonstrated a 45% increase in plasma membrane GLUT-4 from exercised skeletal muscle compared with controls, whereas microsomal membranes from the exercised muscle had a concomitant 25% decrease in GLUT-4 protein. These data suggest that exercise recruits transporters to the plasma membrane from an intracellular microsomal pool, similar to the translocation of transporters that occurs with insulin stimulation. Furthermore, both exercise and insulin stimulate the translocation of GLUT-4 in skeletal muscle, while GLUT-l is not altered. male Sprague-Dawley rats; GLUT-l; GLUT-4; membrane pool; plasma membrane; cytochalasin

intracellular B binding

along with some reports that the stimulation of glucose transport in isolated muscle by contractile activity and maximal insulin stimulation can be additive or partially additive (17, 18, 23), suggest that exercise and insulin stimulate different “pools” of glucose transporters. However, Fushiki et al. (7), using the cytochalasin B binding assay, demonstrated that exercise increases plasma membrane glucose transporter number concomitant with a decrease in intracellular transporter number, suggesting that insulin and exercise may affect the same pool of transporters. To shed further light on this question, we have studied plasma membrane and intracellular glucose transporters using a different fractionation technique (12) from that used by Douen et al. (5) and Fushiki et al. (7) and we have performed immunological studies using the monoclonal antibody (lF8) to GLUT-4 (muscle/fat insulinregulatable glucose transporter) (14) and the polyclonal antibody (A379) to GLUT-l (erythrocyte/brain transporter)( 11). Our data, furnished by either the cytochalasin B binding technique or by Western blot analysis, suggest that exercise, similar to insulin, causes a translocation of glucose transporters from an intracellular pool to the plasma membrane. In addition, both insulin and exercise appear to translocate the same glucose transporter isoform. MATERIALS

INSULIN AND FIBER contraction increase rates of glucose uptake into skeletal muscle. Recent studies of insulin-stimulated skeletal muscle suggest that insulin causes a translocation and redistribution of glucose transporters from an intracellular pool to the plasma membrane (12, 16), a phenomenon that has been described previously in adipose cells (3, 21, 22). Additional studies in muscle have shown that exercise in vivo (5, 7, 8, 13, 15) and contractile activity in situ (10) are associated with an increase in the number (5, 7, 8, 10, 13, 15) and/or intrinsic activity (8, 10, 15, 20) of rat skeletal muscle plasma membrane glucose transporters. Furthermore, Douen et al. (5) have measured “intracellular” glucose transporters and concluded that, although both exercise and insulin cause an increase in plasma membrane glucose transporters, a concomitant decrease in the number of intracellular transporters occurs in the membranes from the insulin-stimulated animals but not in membranes from exercised animals. This finding, BOTH

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AnimaZ care and exercise. Male Sprague-Dawley rats were received from Charles River Canada (Montreal, Quebec). Animals were housed in a room maintained at 23°C with a 12:12-h light-dark cycle and were allowed free access to food and water for a minimum of 4 days before the experiment. Beginning at 0700-0900 h, rats were either exercised on a treadmill (Quinton, model 42) for 1 h at 20 m/min up a 10% grade or allowed to remain sedentary with no accessto food. Membrane preparation. Immediately after exercise, rats were killed by a blow to the head and cervical dislocation. Approximately 6-7 g of mixed hindlimb muscle (gastrocnemius, soleus, and biceps femoris) were removed from both legs, cleaned free of fat and connective tissue, and then weighed. Plasma and microsomal membrane fractions were isolated by a procedure that has been described previously in detail (12). Protein and marker enzyme assays. Homogenate and subcellular membrane protein was determined for each

0 1991 the American

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preparation by the Coomassie brilliant blue method (BioRad Laboratories, Richmond, CA) described by Bradford (2) using crystalline bovine serum albumin as the standard. K+-stimulated-p-nitrophenol phosphatase (KpNPPase) specific activity, a plasma membrane marker, was assayed in the absence or presence of 20 mM K+ (1). UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase), an enzyme marker associated with the Golgi apparatus, was also measured in each fraction according to the method of Fleischer (6). Ca’+stimulated adenosinetriphosphatase (ATPase) activity, a sarcoplasmic reticulum enzyme marker, was assayed according to the procedure of Seiler and Fleischer (19). Cytochalasin B binding. Equilibrium D-glucose-inhibitable [3H]cytochalasin B binding was measured, and the concentration of glucose transporters was calculated by a modification of the method of Wardzala et al. (22) as previously described (12). Western blotting. Plasma and microsomal membrane protein (50 pg), along with molecular weight markers (Bio-Rad), were subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), which was run under nonreducing conditions using an 8% resolving gel as previously described (12). Briefly, resolved proteins were electrophoretically transferred from the gel to a nitrocellulose membrane. Incubations were carried out in tris(hydroxymethyl)aminomethane (Tris)buffered saline (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5 at 22°C) unless otherwise indicated. The nitrocellulose transfer membrane was blocked in TBS with 0.2% Tween 20 and 5% Carnation nonfat dry milk. To identify GLUT-4 proteins, the transfer membranes were incubated with 5 yg/ml mouse anti-rat glucose transporter monoclonal antibody (mAb lF8; Genzyme, Boston, MA). Antibody binding to the transfer membrane was visualized by incubating with anti-mouse immunoglobulin, 1251labeled species-specific F(ab’)2 fragment from sheep. To identify GLUT-l proteins, membranes were incubated with the polyclonal antibody A379 at a dilution of 1:200 (gift of S. Cushman, National Institutes of Health). A379 was produced from a synthetic peptide of a 12-amino acid COOH-terminal sequence from rat brain GLUT-l (11). Antibody binding to the transfer membrane was visualized by incubating with 1251-labeled protein A (Amersham). After incubation the transfer membranes were exposed to Kodak XAR-5 film at -80°C for 48 h. Autoradiograph bands were quantitated by video densitometry (Gel/Image Analysis Systems, Technology Resources, Nashville, TN). Statistical analysis. All data are reported as means t SE. The statistical analyses were performed using unpaired Student’s t test. P < 0.05 was considered to be statistically significant. RESULTS

Body weight, muscle weight, and protein recovery. The mean body weight of control and exercised animals was not significantly different (Table 1). The amount of muscle used in the membrane preparations was also the same for each group. Homogenate protein concentration and the total amount of protein recovered in the micro-

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TABLE 1. Body and muscle weights used in membrane preparations, and protein recovery Control

Body wt, g Muscle wt, g Total protein Homogenate, mg Plasma membrane, mg Microsomal membrane,

mg

Exercise

229t6 6.3220.04

228t5 6.27t0.03

761k29 3.30t0.24* 2.42t0.32

825t51 4.15t0.30* 2.24t0.27

Values are means t SE. Control, n = 10 animals; exercise, n = 9. Levels of significance are comparisons between control and exercise groups. Absence of superscripts indicates no significant difference. * P c 0.05.

2. K+-stimulated-p-nitrophenol phosphatase specific acNtivities, percent recoveries, and enrichments of subcellular membrane fractions from skeletal muscle of control and exercised rats TABLE

KpNPPase

Homogenate Specific activity, nmol mg-l Plasma membrane Specific activity, nmol . mg-l Recovery, % Fold enrichment Microsomal membranes Specific activity, nmol= mg-’ Recovery, % Fold enrichment l

Control

Exercise

30 min-l

222t12

197212

30 min-l

5,558+344 10.9t0.8 25.3t1.4

4,979+395 12.8kO.9 25.3rt1.3

-30 min-’

1,170+128

964k97

l

l

1.6t0.3 5.3t0.5

1.3t0.2 4.8kO.3

Values are means t SE. Control, n = 10; exercise, n = 9. KpNPPase, K+-stimulated-p-nitrophenol phosphatase. There were no significant differences between control and exercise groups.

somal membrane fractions were not different between the control and exercise groups. However, there was a greater protein recovery in the plasma membrane fraction from the exercised muscle (Table 1). In previous studies in which we isolated plasma membranes from skeletal muscle, the protein recovery was similar between exercised and control groups (8, 10, 13, 15). Therefore the current observation of increased plasma membrane protein may be due to random experimental variation. Marker enzymes. KpNPPase activity, a plasma membrane marker, was greatly enriched in the plasma membrane fraction in comparison with the crude homogenate (Table 2). In addition, KpNPPase specific activity was approximately fivefold greater in the plasma membranes than in microsomal membranes. Recovery of KpNPPase was -12% in the plasma membrane fraction, whereas -1.5% was in the microsomal fraction. Galactosyltransferase specific activity, an enzyme associated with the Golgi apparatus, was enriched in the microsomal fraction by approximately double that of the plasma membrane fraction, whereas the recovery of galactosyltransferase was -38% greater in the microsomal fraction compared with the plasma membrane fraction (Table 3). Ca2+stimulated ATPase activity, a sarcoplasmic reticulum enzyme marker, demonstrated that there was virtually no sarcoplasmic reticulum contamination of either the microsomal or plasma membrane fractions (Table 4). No significant differences in marker enzyme specific activity, enrichment, and recovery were observed in plasma membranes or microsomal membranes from exercised


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TABLE 3. UDP-galactose-N-acetylglucosamine galactosyltransferase specific activities, percent recoveries, and enrichments of subcellular membrane fractions from skeletal muscle of control and exercised rats

Control

Galactosyltransferase

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Insulin Exercise 501.19 5OP4 + + + + BPM IPM BMC IMC CPM EPM CMC EMC

Treatment Protein Membranes

Exercise

Homogenate

4.6kO.2 4.3kO.3 Specific activity, nmol. mg-’ .2 h-’ Plasma membrane 60.8k4.3 57.5k4.4 Specific activity, nmol . mg-’ .2 h-l 5.8kO.5 7.2kO.8 Recovery, % Fold enrichment 13.4kO.9 14.Ok1.3 Microsomal membranes Specific activity, nmol. mg-’ .2 h-i 137.1k16.2 149.2k14.9 Recovery, % 9.Okl.O 9.0+0.4 Fold enrichment 30.51-4.1 36.Ok3.7 Values are means + SE. Control, n = 10; exercise, n = 9. There were no significant differences between control and exercise groups. TABLE 4. Ca2+-stimulated ATPase specific activities, percent recoveries and enrichments of subcellular membrane fractions from skeletal muscle of control and exercised rats Ca’+-stimulated ATPase

Control

Exercise

Homogenate Specific activity, nmol. mg-’ +min-l 496k34 495245 Plasma membrane Specific activity, nmol. mg-’ . min-’ 62+18 62+13 Recovery, % 0.04+0.01 0.06+0.01 Fold enrichment 0.13+0.04 0.14+0.03 Microsomal membranes ND ND Specific activity, nmol .mg-’ . min-’ Recovery, % Fold enrichment Values are means + SE. Control, n = 10; exercise, n = 9. There were no significant differences between control and exercise groups. ND, not detectable.

Lanes

3 4 5 6 7 8 9 2 1. Subcellular distribution of GLUT-4 analyzed by Western blot using mAb lF8. Representative autoradiograph of a Western blot in which mAb lF8 was used to identify GLUT-4 transporters as described in MATERIALS AND METHODS. Lanes 2-5 are plasma and microsomal membranes from skeletal muscle perfused in absence or presence of insulin [prepared during a previous investigation (12)], and lanes 6-9 are membranes from control or exercised muscle. Lanes 2 and 3 show increases in plasma membrane GLUT-4 density that occur with maximal insulin stimulation, whereas lanes 4 and 5 show corresponding decreases in microsomal membrane GLUT-4. Lanes 6 and 7 demonstrate increases in plasma membrane GLUT-4 density associated with acute exercise, and lanes 8 and 9 show corresponding decreases in microsomal membrane GLUT-4. PM, plasma membrane; MC, microsomal membrane; C, control; E, exercise; B, basal perfused; I, insulin-stimulated perfused. P 0.02 FIG.

175 150

P ( 0.05

TABLE 5. Glucose transporter number and dissociation constants of subcellular membrane fractions from skeletal muscle of control and exercised rats 25

Control

Exercise

I

0

Plasma membranes 10.07+0.73* 15.00+1.42* R, pmollmg 102.8k8.5 109.2+6.0 Kd, nM Microsomal membranes 6.00+0.69t 4.16f0.41.t %, pmol/mg 67.9k7.8 55.6k1.3 Kd, nM Values are means f SE. Control, n = 10; exercise, n = 9. R,, glucose transporter number; &, dissociation constant. Levels of significance are comparisons between control and exercise groups. Absence of superscripts indicates no significant difference. *P < 0.01. t P < 0.05.

and control rats. Cytochalasin B binding. Glucose transporter number (R,), as estimated from cytochalasin B binding studies, was increased 50% in the plasma membrane fraction of exercised rats. This increase occurred in the absence of a significant change in the dissociation constant (I&; Table 5). R, in the microsomal membrane fraction was decreased by 31% in the exercised rats, whereas the Kd for the exercise and control microsomal binding curves were not different (Table 5).

Control Plasma membranes

Exercise

Control

Exercise

Mlcrosomal membranes

FIG. 2. Video densitometry analysis of autoradiographs from Western blots of plasma and microsomal membrane GLUT-4 from exercised (n = 9) and control (n = 9) skeletal muscles. Data are expressed in arbitrary units as a percentage of mean densities of control and exercised plasma and microsomal membrane GLUT-4 from each set of experiments as described in MATERIALS AND METHODS.

Western blot analysis. Plasma and microsomal membranes prepared from exercised and control rats, along with basal and insulin-stimulated skeletal muscle subcellular membrane fractions that were prepared during a previous investigation (12), were analyzed by SDSPAGE to compare the distribution of GLUT-4 protein in muscle membrane fractions after stimulation with either insulin or exercise. Figure 1 is a representative autoradiograph of a Western blot in which mAb lF8 was used to identify GLUT-4 transporters. GLUT-4 appears as a double band with an approximate molecular mass of


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150 125 1

25 0 I-Control Plasma embranes

Exercise

Control

TRANSPORTERS

DISCUSSION

l-

1

GLUCOSE

Exercise

Microsomal membranes

FIG. 3. Video densitometry analysis of autoradiographs from Western blots of plasma and microsomal membrane GLUT-l from exercised (n = 9) and control (n = 9) skeletal muscles. Data are expressed in arbitrary units as a percentage of mean densities of control and exercised plasma and microsomal membrane GLUT-l from each set of experiments as described in MATERIALS AND METHODS. Plasma membrane GLUT-l protein was not significantly different between control and exercised preparations. Microsomal membrane GLUT-l protein was not significantly different between control and exercised preparations.

46 and 43 kDa. These bands are identical to the bands we have previously identified in skeletal muscle and adipose cell membranes (12). Lanes 2-5 are plasma and microsomal membranes from skeletal muscle perfused in the absence or presence of insulin, and lanes 6-9 are the membranes from control or exercised muscle. Lanes 2 and 3 show the increase in plasma membrane GLUT-4 density, which occurs with maximal insulin stimulation, whereas lanes 4 and 5 show the decrease in microsomal membrane GLUT-4. Lanes 6 and 7 demonstrate the increase in plasma membrane GLUT- ,4 density associated with acute exercise, and lanes 8 and 9 sh .ow the decrease in microsomal membrane GLUT-4 due to exercise. Figure 2 represents the video densitometry analysis of autoradiographs from Western blots of exercised and control skeletal muscles. These data are expressed in arbitrary units as a percentage of the mean densities of control and exercised plasma and microsomal membrane GLUT-4 from each set of nine experiments. GLUT-4 in microsomal membranes was approximately twofold greater than in plasma membranes. GLUT-4 in plasma membranes from exercised skeletal muscle were increased -45% compared with controls. In microsomal membranes from exercised skeletal muscle, GLUT-4 protein decreased -25% compared with the microsomal membranes prepared from controls. Figure 3 represents the video densitometry analysis of autoradiographs using the GLUT-l antibody. The data are expressed in arbitrary units as a percentage of the mean densities of control and exercised plasma and microsomal membrane GLUT-l from each set of nine experiments. GLUT-l protein in microsomal membranes was slightly greater than in plasma membranes. GLUT1 in both plasma and microsom al membra nes was not significantly different between exercised and control preparations.

The present study demonstrates that exercise causes a redistribution of glucose transporters from an intracellular microsomal pool to the plasma membrane. Fushiki et al. (7), using the cytochalasin B binding assay, have also demonstrated that exercise increases plasma membrane glucose transporter number concomitant with a decrease in intracellular transporter number. Although Douen et al. (4,5) have also reported an exercise-induced increase in plasma membrane glucose transporter number @O-65%) and GLUT-4 protein (-M-fold), they found no change in glucose transporter number or GLUT-4 in an intracellular pool. In the current study we report that exercise results in a 50 and 45% increase in plasma membrane glucose transporter number and GLUT-4, respectively. However, we demonstrate that a 31 and 25% decrease in intracellular glucose transporter number and GLUT-4 protein, respectively, occurs after exercise. In comparing our methods with that of Douen et al. (5), it is clear that different techniques were used for the isolation of membrane fractions. However, it is interesting that both preparations have shown insulin stimulation to decrease intracellular glucose transporter number (12,16), whereas exercise decreased intracellular transporter number only using our preparation. These differences suggest that the intracellular fractions prepared using the two techniques may isolate a different mixture of membranes. Western blot experiments were done to determine whether the glucose transporters redistributed by exercise (as measured by cytochalasin B binding) would react with the monoclonal antibody (lF8) specific for GLUT4 or the polyclonal antibody A379 specific for GLUT-l. mAb lF8 has been shown to react with purified plasma membranes (12) and microsomal membranes (12, 14) from adipose cells, with crude microsomal fractions from heart, brown adipose tissue, and red and white skeletal muscle (14) and with purified plasma and microsomal membranes from basal and insulin-stimulated skeletal muscle (12) but not with non-insulin-sensitive tissues. Our immunoblotting data with mAb lF8 in subcellular fractions from exercised skeletal muscle clearly demonstrate that this insulin-sensitive glucose transporter is also sensitive to muscle contractions. Exercise did not have a significant effect on the GLUT-l protein. Douen et al. (4) have reported that insulin does not cause the redistribution of GLUT-l in rat skeletal muscle. Thus, although both exercise and insulin result in the redistribution of GLUT-4, neither stimulus appears to have a significant effect on the GLUT-l protein. These findings support the hypothesis that GLUT-4, but not GLUT-l, is responsive to insulin in skeletal muscle. In the current study we have not performed immunoblotting experiments on whole homogenates; however, we have recently reported that GLUT-4 and GLUT-l protein in red and white rat skeletal muscle homogenates are not increased by 30 min of maximal insulin stimulation or 1 h of acute exercise (9). This demonstrates that an exercise period of 1 h (as performed in the current study) is unlikely to alter the total glucose transporter


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content in rat skeletal muscle. We have recently reported that rates of plasma membrane vesicle glucose transport or plasma membrane glucose transporter number with the combination of contractile activity and maximal insulin stimulation are not additive (10). Similarly, Douen et al. (4), studying rats exposed to treadmill exercise followed by insulin injection, found no additive effect on plasma membrane GLUT-4 protein content or plasma membrane glucose transporter number (determined by cytochalasin B binding). Although not in agreement with some previous reports (17, 18, 23), our findings suggested that exercise and insulin stimulate the same transport system Results from the current study support this hypothesis, because exercise and insulin have been shown to translocate the same glucose transporter protein (GLUT-4). In conclusion, our data suggest that the stimulation of glucose transport in skeletal muscle by exercise is due, at least in part, to the translocation of glucose transporters from an intracellular pool to the plasma membrane. In addition, Western blot results with mAb lF8 in skeletal muscle subcellular membrane fractions demonstrate that insulin and exercise translocate the same glucose transporter (GLUT-4), whereas our work and that of Douen et al. (4) suggest that the distribution of GLUT-l in skeletal muscle is not significantly altered by insulin or exercise. We gratefully acknowledge the technical contributions to this study of Elizabeth Horton and Charlotte Thompson, and we thank Dr. Samuel Cushman for the gift of A379. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-26317. L. J. Goodyear was supported by an NIDDK postdoctoral research fellowship (T32 DK-07523). Present address of L. J. Goodyear: Metabolism Section, Joslin Research Laboratory, One Joslin Place, Boston, MA 02215. Address reprint requests to M. F. Hirshman. Received

1 March

1991; accepted

in final

form

16 July

GLUCOSE

1. BERS, D. M., K. D. PHILIPSON, AND A. Y. NISHIMOTO. Sodiumcalcium exchange and sidedness of isolated cardiac sarcolemmal vesicles. Biochim. Biophys. Actu 601: 358-371, 1980. 2. BRADFORD, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein-dye binding. Anal Biochem. 72: 248-254, 1976. 3. CUSHMAN, S. W., AND L. J. WARDZALA. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell: apparent translocation of intracellular transport systems to the plasma membrane. J. Biol. Chem. 255: 4758-4762,198O. 4. DOUEN, A. G., T. RAMLAL, G. D. CARTEE, AND A. KLIP. Exercise modulates the insulin-induced translocation of glucose transporters in rat skeletal muscle. FEBS Lett. 261: 256-260, 1990. 5. DOUEN, A. G., T. RAMLAL, A. KLIP, D. A. YOUNG, G. D. CARTEE, AND J. 0. HOLLOSZY. Exercise-induced increase in glucose transporters in plasma membranes of rat skeletal muscle. Endocrinology

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124: 449-454,1989. B. Isolation and characterization of Golgi apparatus and membranes from rat liver. Methods Enzymol. 31: 180-191, 1974. FUSHIKI, T., J. A. WELLS, E. B. TAPSCOTT, AND G. L. DOHM. Changes in glucose transporters in muscle in response to exercise. Am. J. Physiol. 256 (Endocrinol. Metub. 19): E580-E587, 1989. GOODYEAR, L. J., M. F. HIRSHMAN, P. A. KING, C. M. THOMPSON, E. D. HORTON, AND E. S. HORTON. Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise. J. AppZ. Physiol. 68: 193-198, 1990. GOODYEAR, L. J., M. F. HIRSHMAN, R. J. SMITH, AND E. S. HORTON. Glucose transporter number, activity, and isoform content in plasma membranes of red and white skeletal muscle. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E556-E561, 1991. GOODYEAR, L. J., P. A. KING, M. F. HIRSHMAN, C. M. THOMPSON, E. D. HORTON, AND E. S. HORTON. Contractile activity increases plasma membrane glucose transporters in absence of insulin. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E667-E672, 1990. HASPEL, H. C., M. G. ROSENFELD, AND 0. M. ROSEN. Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein. J. BioZ. Chem. 263: 398-403, 1988. HIRSHMAN, M. F., L. J. GOODYEAR, L. J. WARDZALA, E. D. HORTON, AND E. S. HORTON. Identification of an intracelular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle. J. BioZ. Chem. 265: 987-991, 1990. HIRSHMAN, M. F., H. WALLBERG-HENRIKSSON, L. J. WARDZALA, E. D. HORTON, AND E. S. HORTON. Acute exercise increases the number of plasma membrane glucose transporters in rat skeletal muscle. FEBS Lett. 238: 235-239, 1988. JAMES, D. E., R. BROWN, J. NAVARRO, AND P. PILCH. Insulinregulatable tissues express a unique insulin-sensitive glucose transport protein. Nature Lond. 333: 183-185, 1988. KING, P. A., M. F. HIRSHMAN, E. D. HORTON, AND E. S. HORTON. Glucose transport in skeletal muscle membrane vesicles from control and exercised rats. Am. J. Physiol. 257 (Cell Physiol. 26): Cll28-Cll34,1989. KLIP, A., T. RAMLAL, D. A. YOUNG, AND J. 0. HOLLOSZY. Insulin induced translocation of glucose transporters in rat hindlimb muscles. FEBS Lett. 244: 224-230, 1988. NESHER, R., I. E. KARL, AND D. M. KIPNIS. Dissociation of effects of insulin and contraction on glucose transport in rat epitrochlearis muscle. Am. J. Physiol. 249 (Cell Physiol. 18): C226-C232, 1985. PLOUG, T., H. GALBO, AND E. A. RICHTER. Increased muscle glucose uptake during contractions: no need for insulin. Am. J. Physiol. 247 (Endocrinol. Metub. 10): E726-E731, 1984. SEILER, S., AND S. FLEISCHER. Isolation of plasma membrane vesicle from rabbit skeletal muscle and their use in ion transport studies. J. BioZ. Chem. 257: 13862-13871, 1982. STERNLICHT, E., R. J. BARNARD, AND G. K. GRIMDITCH. Exercise and insulin stimulate skeletal muscle glucose transport through different mechanisms. Am. J. Physiol. 256 (Endocrinol. Metub. 19): E227-E230,1989. SUZUKI, K., AND T. KONO. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. NutZ. Acud. Sci. USA 77: 25422545,198O. WARDZALA, L. J., S. W. CUSHMAN, AND L. B. SALANS. Mechanism of insulin action on glucose transport in the isolated rat adipose cell. J. BioZ. Chem. 253: 8002-8005, 1978. ZORZANO, A., T. W. BALON, M. N. GOODMAN, AND N. B. RUDERMAN. Additive effects of prior exercise and insulin on glucose and AIB uptake by rat muscle. Am. J. Physiol. 251 (Endocrinol. Metub. 14): E21-E26, 1986. FLEISCHER,

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