Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise LAURIE J. GOODYEAR, MICHAEL F. HIRSHMAN, PATRICIA A. KING, ELIZABETH D. HORTON, CHARLOTTE M. THOMPSON, AND EDWARD S. HORTON Metabolic Unit, Department of Medicine, University of Vermont, Burlington, Vermont 05405 GOODYEAR, LAURIE J., MICHAEL F. HIRSHMAN, PATRICIA A. KING, ELIZABETH D. HORTON, CHARLOTTE M. THOMPSON, AND EDWARD S. HORTON. Skeletal muscle plasma membrane glucosetransport and glucosetransportersafter exercise,J. Appl. Physiol. 68(1): 193-198,1990.-Recent reports have shownthat immediately after an acute bout of exercisethe glucosetransport systemof rat skeletal muscleplasmamembranesis characterized by an increase in both glucosetransporter number and intrinsic activity. To determine the duration of the exercise responsewe examined the time courseof these changesafter completionof a singlebout of exercise.Male rats wereexercised on a treadmill for 1 h (20 m/min, 10% grade) or allowed to remain sedentary. Rats were killed either immediately or 0.5 or 2 h after exercise, and red gastrocnemiusmusclewas usedfor the preparation of plasmamembranes.Plasmamembraneglucosetransporter number was elevated 1.8- and 1.6-fold immediately and 30 min after exercise,although facilitated D-ghXOSe transport in plasmamembranevesicleswaselevated4- and 1.8fold immediately and 30 min after exercise, respectively. By 2 h after exerciseboth glucosetransporter number and transport activity had returned to nonexercisedcontrol values.Additional experimentsmeasuringglucoseuptake in perfusedhindquarter muscleproducedsimilar results. We concludethat the reversal of the increasein glucoseuptake by hindquarter skeletalmuscle after exerciseis correlated with a reversal of the increasein the glucosetransporter number and activity in the plasma membrane. The time course of the transport-to-transporter ratio suggeststhat the intrinsic activity responsereverses more rapidly than that involving transporter number.

(15) have reported that this increase in plasma membrane glucose transporter number is accompanied by an increase in the intrinsic activity of the transporter. An increase in glucose uptake produced by either exercise in vivo or electrical stimulation to produce muscle contractions in situ can persist for many hours after the cessation of exercise (4,5, 7,10,12). The duration of this effect appears to be variable depending on the duration and/or intensity of the exercise performed, the fed or fasted state of the animal, and whether rates of uptake were determined in the absence or presence of insulin. Some reports have shown an inverse relationship between glucose uptake and muscle glycogen concentrations (5, 7, 20, 26), suggesting a role for glycogen in the control of glucose uptake. More recent reports, however, have demonstrated that the reversal of an increase in glucose transport after exercise in mammalian muscle is not dependent on glycogen synthesis (18, 25, 27). The purpose of the current study was to determine the duration of the exercise-induced increase in plasma membrane vesicle glucose transport and glucose transporter number and to compare these changes with rates of glucose uptake in the perfused hindquarter preparation and with concentrations of muscle glycogen.

rat skeletal muscle; glucoseuptake; cytochalasin I3 binding; muscleglycogen;hindquarter perfusion

Animal care and exercise. Male Sprague-Dawley rats were obtained from Charles River Canada (Montreal, Quebec). Th e animals were maintained on a 12-h lightdark cycle (light from 0700 to 1900, dark from 1900 to 0700) and fed Purina lab chow and water ad libitum for 4-6 days before the experiment. Starting at 0700-0900, rats were either exercised on a Quinton model 42 tread-

GLUCOSETRANSPORT is stimulated severalfold by muscular exercise (cf. Ref. 9). As with insulin-stimulated

glucose transport, there is evidence to suggest that contraction-stimulated glucose transport in skeletal muscle follows saturation kinetics, and that exercise increases the maximal velocity ( Vmax) of transport without changing the Michaelis-Menten constant (Km) for glucose (9, 10, 15, 23). Because contraction-induced

transport fol-

lows saturation kinetics it must increase transport by increasing the number and/or activity of glucose transporters in the muscle plasma membrane. Recent reports from our laboratory support this hypothesis. Hirshman et al. (8) have demonstrated that immediately after an acute bout of treadmill exercise there is an increase in the number of glucose transporters present in the plasma membrane of skeletal muscle. More recently, King et al.

METHODS

mill for 1 h at 20 m/min up a 10% grade or allowed to remain sedentary with no access to food.

Plasma membranepreparation. For experiments where plasma membranes were prepared, rats were studied either immediately or 0.5 or 2 h after exercise, while nonexercised control rats were studied at corresponding times. Animals were killed by decapitation and the blood

collected in heparinized tubes for subsequent analysis of plasma glucose and insulin concentrations (see below). For each plasma membrane preparation portions of red gastrocnemius muscle from both legs of a single rat were rapidly dissected, trimmed free of fat and connective tissue, and weighed. A small portion of red gastrocnemius

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muscle was also removed for muscle glycogen determimembrane suspension was taken from each preparation nation (see below). for subsequent protein determinations. Preparation of plasma membranes was performed as Uptake was measured at four time points for each previously described (8). Briefly, -1.5 g of muscle were preparation to determine true initial rates of transport. minced, polytroned, and homogenized in a buffer con- For the nonexercised rats, transport was measured at 0, 1, 3, and 5 s and for exercised animals fluxes were taining 20 mM N-2-hydroxyethylpiperazine-N’-2-ethmeasured at 0, 1,1.5, and 2 s. Transport was initiated by anesulfonic (HEPES) and 250 mM sucrose, pH 7.4. After combining an aliquot of the membranes with 80 pl of removal of an aliquot for marker enzyme and protein determinations, KC1 and Na+ pyrophosphate were added incubation medium (HEPES-buffered Krebs solution to final concentrations of 0.3 M and 25 mM, respectively, containing 6 &i of L-[3H(N)]glucose and 1.6 &i of Dand the homogenate was centrifuged at 227,000 g,,, for [ 14C(U) ] glucose). Transport was stopped by the addition 50 min. The supernatant was discarded and the pellet of 1 ml of ice-cold stop solution (185 mM NaCl, 5 mM resuspended in buffer and then incubated at 30°C for 1 KCl, 1.2 mM MgClz, 20 mM HEPES, pH 7.8) containing h in the presence of protease inhibitors (leupeptin, pep- 0.2 mM p-hydroxymercuribenzoate (Na salt) to the restatin, and aprotinin, final concn 2.5 pg/ml each) and action vessel. The membranes were rapidly filtered (MilDNase (final concn 2,000 kU/ml). The mixture was then lipore HA 0.45 pm), washed, and the filter and adhering counting by diluted, cooled, and centrifuged at 227,000 gtmax for 50 membranes analyzed by liquid scintillation min. The pellet was resuspended in 34% sucrose, layered using quench correction for the dual label. Zero time in a discontinuous gradient (45, 38, 34, 32, 30, 27, and measurements were determined by the addition of the 12% sucrose), and then centrifuged for 16 h at 68,000 stop solution to the Eppendorf tube before the addition $ max. Fractions were collected from the gradient at the of the membrane suspension. The initial rates of L- and 12/27 and 27/30 interfaces, and combined, diluted with D-ghme were obtained from the linear portion of a graph of influx vs. time. Facilitated transport was calwater, and centrifuged at 331,000 gmax for 60 min. The resulting pellet was resuspended and aliquots were re- culated by subtracting the initial rate of L-glucose influx moved for marker enzyme and protein determinations. from that of D-glucose. Hindquarter perfusion experiments. Either immediThe remaining sample was used for the cytochalasin B binding and glucose transport assays. All samples were ately or 1.5 h after exercise, rats were anesthetized with frozen and stored in liquid N2 until assayed. pentobarbital sodium (45 mg/kg body wt) and prepared Protein and 5’-nucleotidase determinations. Protein for hindquarter perfusion experiments as described by was determined by the Coomassie brilliant blue method Ruderman et al. (21). The perfusion medium (175 ml), (Bio-Rad protein assay, Bio-Rad Laboratories, Richconsisting of Krebs-Henseleit bicarbonate buffer, 4% mond, CA) described by Bradford (2) using crystalline bovine serum albumin (fraction V, Armour Pharmaceubovine serum albumin as the standard. The specific 5’- ticals, Kankakee, IL), 6 mM glucose, and 40 pU/ml nucleotidase activities of the homogenate and plasma insulin was continuously gassed with a mixture of 95% membrane fractions was assayed as described by Avruch 02-5% COz. The first 25 ml that passed through the and Hoelzl-Sallach (1). animal were discarded followed by the recycling of the Plasma membrane glucose transporter determination. medium at a flow rate of 13 ml/min for 37.5 min. The Equilibrium D-glucose-inhibitable [ 3H]cytochalasin B first 7.5 min allowed for equilibration of the preparation binding was measured and the calculation of glucose followed by a 30-min perfusion period during which transporter number concentration was performed as de- glucose uptake was measured. Perfusate samples were scribed by Wardzala et al. (24). Briefly, Scatchard plots taken at 0,15, and 30 min of the perfusion and processed were generated from binding studies in which membranes for subsequent measurement of glucose and insulin conwere incubated with varying concentrations of cytochalcentrations. System pressure, temperature, and appearasin B in the presence or absence of D-glucose. Cytoance of the hindquarter were monitored continuously. chalasin E was added to decrease nonspecific binding. Glucose uptake was calculated as the difference in perover the 300min perfusion The total number of glucose transporters and the disso- fusate glucose concentration ciation constant (J&) were determined from a linear plot period and expressed as micromoles per gram perfused derived by subtraction along the radial axes of binding muscle per hour. The perfused muscle mass was esticurves generated in the presence of D-glucose from those mated as total body weight/6 based on previous experiments in which hindquarter muscles were perfused with generated in the absence of D-glucose. Glucose transport activity in plasma membrane vesicles. a marker dye, dissected, and weighed (unpublished data). Because anesthesia, surgery, and the equilibration period D-[14C]glucose and Lj3H]glucose uptake in plasma membrane vesicles was determined according to the procedure took ~28-32 min, rates of glucose uptake were measured at 0.5 and 2 h postexercise. Thus measurements of gluof King et al. (15). By using this method, flux measurements were performed under equilibrium exchange con- cose uptake in perfused hindlimb muscles could not be ditions using a rapid filtration technique at 25°C. Memobtained during the immediate postexercise period with branes were rapidly thawed and preequilibrated with a this technique. HEPES-buffered Krebs-Ringer solution (135 mM NaCl, Glucose, insulin, and glycogen analyses. Gastrocnemius 5 mM KCl, 1.2 mM MgCl,, and 20 mM HEPES, pH 7.6, muscle samples were frozen in liquid N, and stored at 25OC)containing 40 mM L- and D-&COSe. This glucose -80°C until analyzed for glycogen content by the proconcentration is two times the &+ An aliquot of the cedure of Hultman (11). Blood samples were collected in Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on November 4, 2018. Copyright © 1990 American Physiological Society. All rights reserved.

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heparinized tubes, stored on ice, and centrifuged at 10,000 g for 10 min. Analysis of plasma samples for glucose and perfusate glucose concentrations was made with a glucose analyzer (model 24, Yellow Spring Instruments, Yellow Springs, OH). Insulin concentrations were determined by the radioimmunoassay procedure of Starr et al. (22) modified by use of polyethylene glycol (8,000 mol wt) to separate the bound from free insulin tracer. Statistt’cal analysis. Data were analyzed by using a oneway analysis of variance by the general linear models procedure with pairwise comparisons done by the least significant difference test (Statistical Analysis Systems). The least significant difference test controls the comparison-wise error rate at a 0.05 a-level. All data are reported as means t SE. RESULTS

GLUCOSE

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2. 5 ‘-Nucleotidase recovery and enrichment in the plasma membrane preparation

TABLE

Nonexercised

5’-Nucleotidase specific activity, recoveries, and enrichment. The plasma membrane enzyme, Wnucleotidase,

was used to determine purity of the plasma membrane preparation. The specific activities of 5 ’ -nucleotidase in the homogenate and plasma membrane fractions did not differ between the nonexercised and exercised muscle (Table 2). The recovery of 5’-nucleotidase activity in the plasma membrane fraction (total enzyme activity in the fraction expressed as a percent of the total activity in the homogenate) was -10% for both nonexercised and exercised groups. The plasma membrane 5’-nucleotidase enrichment relative to the enzyme activity of the homogenate, an indication of purity of the plasma membrane marker, was -Z&fold in both groups. Plasma glucose, insulin, and muscleglycogen concentrations. Plasma glucose and insulin concentrations are

shown in Fig. 1. Plasma glucose concentrations were elevated immediately after exercise (15%) but had re1. Rat body weights, red gastrocnemius weight, and homogenate and membrane protein concentrations TABLE

Nonexercised 18

Exercised 22

wt, g 23625 237k2.8 Muscle wt, g 1.5t0.02 1.5&0.01 214t6 211t4 Homogenate protein, mg Plasma membrane protein, pg 930&108 1,058+94 Values are means t SE; n, no. of preparations. Data from all nonexercised and exercised rats were pooled; hindlimb perfusion studies were excluded. iody

Exercised

18 22 ;fomogenate sp act, nmol 30 min-l k 112&5 111+4 mg-l Plasma membrane sp act, nmol 30 2,583+206 2,372t160 min-‘. mg-’ Plasma membrane enzyme recovery, % 9.8+1*1 10.1-+0.8 Enzyme fold enrichment 22*2 22-1-l Values are means & SE. Marker enzyme activities were measured in the original homogenates and plasma membrane fractions using procedures described in METHODS. Total 5’-nucleotidase activity of the homogenate and plasma membrane fraction was determined by multiplying their specific activities with their respective protein recoveries. Percent recovery was the result of dividing total enzyme activity of the plasma membrane by the total enzyme activity of the homogenate multiplied by 100. l

l

Body weight, muscle weight, and protein recoveries.

Body weight, weight of the portion of the red gastrocnemius muscle used in the membrane preparations, and homogenate and plasma membrane protein recoveries are shown in Table 1. Because there were no significant differences among data from exercised rats killed immediately and 30 min and 2 h after exercise or among data from nonexercised rats taken at the three time points, the results are pooled into two groups: nonexercised and exercised. Total homogenate protein represents ~14% of the total muscle used for each experiment and was not different between nonexercised and exercised rats. The recovery of plasma membrane protein was not affected by exercise.

195

GLUCOSE

(mg/dt)

+----160

------LB-

0

:

O--O

120

l

-*

:

non-exercised exercised

t

80 INSUtlN

I

6

40

0’

i

(@J/ml)

----O

Y?T6

l

I 0

I 0.5 TIVE

POST-EXFRCISE

I 2 (hr)

1. Plasma glucose and insulin concentrations of exercised rats (20 m/min, 10% grade, 1 h) immediately and 0.5 and 2 h postexercise and in nonexercised rats at corresponding times. Plasma glucose concentrations were significantly elevated at time 0 in exercised rats compared with nonexercised rats. Insulin concentrations were significantly different between time 0 and 2 h for both exercised and nonexercised rats. Each point is mean -i- SE of 6-9 animals. SE smaller than symbols are not shown. FIG.

turned to nonexercised base-line concentrations by 30 min postexercise. Although insulin concentrations were slightly lower in the exercised rats, these differences were not significant. Two hours after exercise insulin concentrations were increased in both the nonexercised and exercised animals compared with concentrations measured for each group at time 0. Gastrocnemius muscle glycogen concentrations were reduced by 56% immediately after exercise and had returned to concentrations in nonexercised animals by 30 min after exercise (Fig. 2). Glucose uptake, glucose transport, and glucose transporter measurements. Rates of hindquarter skeletal mus-

cle glucose uptake in the nonexercised rats were the same at the 0.5- and 2-h time points. Likewise, for the nonexercised rats there were no significant differences in glucose transporter number or plasma membrane vesicle transport activities between the 0, 0.5, and 2 h time points. Therefore these data have been pooled. In exercised animals, glucose uptake in the perfused hindquarter was increased by 62% 30 min after exercise

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3. Facilitated D-glucose transport activities, cytochalasin B binding sites, estimated total glucose transporters, dissociation constants, and transport- to- transporter ratios

TABLE

NON-EXERCISED

-

GLUCOSE

EXERCISED

Nonexercised

Glucose transport, nmol .

1.920.19

mg-’ .s-l R-0, pmwmg

4.7kO.4

Estimated total plasma membrane transporters, pmol/g muscle

30t3

Kd, nM

69t5

TIME POST EXERCISE (hr) FIG. 2. Gastrocnemius muscle glycogen concentrations of exercised rats (20 m/min, 10% grade, 1 h) immediately and 0.5 and 2 h postexercise and in nonexercised rats at corresponding times. *Different from nonexercised time 0. Each bar is mean t SE of 6-9 animals.

6

,^

i

0 NONEXERCISED

0.5 TIME

POST-EXERCISE

2 (hr)

FIG. 3. Hindquarter skeletal muscle rates of glucose uptake in exercised rats (20 m/min, 10% grade, 1 h) 0.5 and 2 h after exercise and in nonexercised rats. *Different from nonexercised rats. Each bar is mean A SE of 6-8 animals.

and had returned to base-line levels by 2 h after exercise (Fig. 3). Exercise resulted in a near doubling in the number of plasma membrane cytochalasin B binding sites per milligram of plasma membrane protein both immediately and 30 min after exercise (Table 3). At 2 h postexercise the number of cytochalasin B binding sites was not different from controls. Likewise, when the number of plasma membrane glucose transporters per gram of muscle was estimated after adjusting for 5’-nucleotidase recovery, exercise was associated with an increased number of transporters immediately and 30 min postexercise, but not at 2 h postexercise (Table 3). There was an increase in the Kd immediately postexercise, but no change at 0.5 or 2 h postexercise (Table 3). Two previous reports from our laboratory have shown slightly higher, but not statistically significant, elevations in the I& postexercise, and significance in the current study may be a function of the larger sample size (8, 15). Because of the inconsistency between the studies, we are hesitant to speculate

Transport/transporter

0.47zkO.08

Time Postexercise, h

0 0.5 2 0 0.5 2 0 0.5 2 0 0.5 2 0 0.5 2

Exercised 7.8&0.8* 3.45to.4* 2*0&O. 1 8.6*0.2* 7,4t1.0* 3a8kO.4 49t,8* 53&8* 3321 101*7* 81zklO 73*9 0.91~0.10” 0.51~0*07 0.61-I-0.08

Values are means & SE. Facilitated glucose transport (40 mM glucose) and cytochalasin B binding sites were measured in the plasma membrane fractions as described in METHODS. Total plasma membrane transporters is an estimate of the number of glucose transporters per gram of muscle determined by adjusting for the total 5’-nucleotidase recovery in the plasma membrane fractions. For all postexercise and nonexercised glucose transport data, n = 6-9 expts/group. Transportto-transporter ratio is calculated only for preparations where both transport and transporters were measured (n = 6-8 expts/group). Nonexercised total no. of glucose transporters (R,), total transporter, and Kd data, n = 19 expts. * Significant difference from nonexercised.

further or assign much significance to this finding without further study. Plasma membrane glucose transport in the exercised rats was elevated 4- and 1%fold immediately and 30 min after exercise, respectively (Table 3). By 2 h postexercise glucose transport had returned to rates identical with those observed in nonexercised muscles. For the membrane preparations in which both transport rate and transporter number were measured, these two parameters were compared to obtain an estimate of transporter intrinsic activity, expressed as the ratio of transport to transporter number (Table 3). The single bout of exercise resulted in a significant increase in the glucose transport-to-glucose transporter ratio immediately after exercise, followed by a return of the ratio to near-control levels by 30 min postexercise (Table 3). At 2 h postexercise the transport-to-transporter ratio was not significantly elevated above the ratio in plasma membranes from nonexercised muscles. DISCUSSION,

This study confirms our previous findings that 1 h of treadmill running produces an increase in the number of plasma membrane glucose transporters and transport activity of hindlimb skeletal muscle immediately after exercise (8, 15). These data are in agreement with the reports of Douen et al. (3) and Fushiki et al. (6), who demonstrated a significant increase in plasma membrane glucose transporters in treadmill-exercised rats, but disagree with Sternlicht et al. (23), who have reported that acute exercise results in an increase in transporter in-

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trinsic activity with no change in plasma membrane glucose transporter number. An explanation for this discrepancy is not readily apparent; however, it may be related to the intensity or duration of exercise used, differences in the plasma membrane preparations or cytochalasin B binding assays, or to other factors such as the sex of the animals studied. Of particular interest in examining our data are the different time courses for changes in plasma membrane glucose transporter number and plasma membrane glucose transport after exercise. Immediately after exercise both glucose transporter number and glucose transport are elevated. However, at 30 min postexercise there is a significant decrease (56%) in glucose transport but no significant change in the number of glucose transporters present in the plasma membrane. This demonstrates that the time course for reversal of the elevation in glucose transporters and glucose transport activity after exercise is different. This is in contrast to studies showing that the reversal times of insulin-stimulated glucose transport and glucose transporter number in adipose cell plasma membranes are closely correlated (14). King et al. (15) and Sternlicht et al. (23) have demonstrated that immediately postexercise there is a significant increase in glucose transporter intrinsic activity. The present study confirms those findings because immediately postexercise the glucose transport-to-glucose transporter ratio, an indication of transporter intrinsic activity, was significantly increased above control values. However, at 30 min postexercise, transporter intrinsic activity, but not transporter number, had returned to control levels and at 2 h postexercise both transporter intrinsic activity and number were close to basal levels. These data suggest a more rapid reversal of the exerciseinduced increase in transporter intrinsic activity than of transporter number. A change in total transporter intrinsic activity could result from recruitment of a more active transporter or from the activation of transporters (basal and/or recruited). The separation of the time courses for the reversal of transporter number vs. activity suggests that the increase in intrinsic activity is not the result of recruiting a transporter characterized by a greater (and stable) intrinsic activity, The more transient nature of the intrinsic activity changes suggests that transporters are activated, but the nature of the activation or whether all or some of the transporters are involved is unknown. These different possibilities raise the question of whether exercise recruits a different form of glucose transporter from that which is present in the plasma membrane in the resting state. Such a possibility seems reasonable in light of a recent report showing that the intracellular insulin-sensitive glucose transporter of rat adipose cells is immunologically distinguishable from that of the basal plasma membrane glucose transporter (13). Previous studies have demonstrated that exercise in vivo and electrical stimulation to produce muscle contractions in situ will result in an increase in glucose uptake that can persist for many hours after the cessation of exercise (4, 7, 10, 12, 19, 25, 26). In the current study both plasma membrane glucose transport and hind-

GLUCOSE

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197

quarter skeletal muscle glucose uptake were elevated 30 min postexercise but had returned to nonexercised baseline rates by 2 h postexercise. Our glucose uptake data differ from that of Richter et al. (19) and Garetto et al. (7), who found that moderate or intense treadmill running increases insulin-stimulated glucose uptake at 2, 2.5, and 4 h postexercise. Although these experiments were similar to ours in regard to exercise protocol and degree of gastrocnemius glycogen depletion, in the previous studies rates of glucose uptake were measured with perfusate insulin concentrations of 75 pU/ml whereas in the current, study perfusate insulin concentration was 40 pU/ml to mimic in vivo concentrations that were determined at the three time points postexercise. It is possible that an exercise-induced increase in glucose uptake is observed for a longer period of time when muscle is perfused at an insulin concentration that is higher than it is exposed to in vivo. This explanation for the differences between our results and those previously reported seems reasonable in light of the fact that insulin-stimulated, but not basal, rates of glucose uptake in the perfused hindquarter have been reported to be elevated 2 h after exercise (7). The reversal of the exercise-induced increase in muscle permeability to glucose has been believed to be associated with muscle glycogen concentrations. Evidence for this comes from studies showing that repletion of glycogen stores is associated with the loss of an exercise-induced elevation in glucose uptake (5, 7, 20) and that carbohydrate feeding that results in increased glycogen concentrations accelerates, and carbohydrate restriction that maintains glycogen depletion slows, the reversal process (26). On the other hand, additional reports in isolated epitrochlearis muscles (25, 27) and individual tissues from perfused hindquarter muscle (18) have shown that the reversal of glucose transport after exercise in rat skeletal muscle is not dependent on glycogen synthesis. In those experiments glycogen concentrations were maintained at a low level (depleted); however, in all studies 3-0-methylglucose transport returned to baseline or near-base-line values. In the present report, an inverse relationship between glucose transporters and transport and glycogen is observed immediately postexercise, because both glucose transport and transporters are increased concomitantly with a 56% decrease in red gastrocnemius glycogen concentrations. On the other hand, 30 min after exercise both the number of plasma membrane glucose transporters and glucose transport activity remain elevated (1.6- and l&fold above base line, respectively), although glycogen concentraCons have returned to base-line concentrations. These data suggest that if glycogen synthesis and/or glycogen levels are involved in regulating the reversal of glucose transport, the time courses are not tightly coupled. Young et al. (25) and Wallberg-Henriksson et al. (27) studying in vitro isolated muscle preparations have proposed that the persistent increase in glucose uptake after exercise requires the presence of a minimal concentration of insulin, hypothesizing that a small amount of insulin is necessary to slow the return translocation of glucose transporters back to the intracellular pool, thus keeping

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transport elevated. In the current study, in vivo insulin concentrations were relatively stable at both 30 min and 2 h postexercise (30-40 $J/ml), although glucose transport and glucose transporter number in skeletal muscle plasma membranes were decreased by 2 h postexercise. Thus, if this hypothesis is true, the low insulin concentrations (7.5 pU/ml) required to keep muscle glucose transport elevated under in vitro conditions (25) are probably not sufficient to delay the reversal of either glucose transport or glucose transporters in vivo. However, it is possible that a higher concentration of insulin would result in glucose transport and transporters remaining elevated for a more prolonged period of time. In conclusion, we found that 1 h of glycogen-depleting treadmill exercise in rats results in a short-term increase in glucose uptake in the perfused hindquarter when perfused with physiological concentrations of insulin (40 pU/ml), as uptake is increased at 30 min but not 2 h postexercise. The return to control rates of glucose uptake by hindquarter muscle at 2 h is correlated with a return in the number of glucose transporters and glucose transport activity in skeletal muscle plasma membranes to control values . Plasma membrane glucose transport data suggest that exercise 1ncreases glucose tra nsport by an increase in both the number of glucose transporters present in the plasma membrane and the intrinsic activity of exercised-recruited and/or basal glucose transporters. Furthermore the data demonstrate that the reversal of glucose transport and transporters after exercise follows a different time course. The authors gratefully acknowledge Rebecca Scaife and Stephanie DeBono for technical contributions to this study and Patricia Mead of Dr. David Robbins’ laboratory for performing the insulin assays. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant ROl-DK-26317. Address reprint requests to L. J. Goodyear. Received 24 April 1989; accepted in final form 28 August 1989. REFERENCES 1. AVRUCH, J., AND D. F. HOELZL~ALLACH. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim. Biophys. Actu 233: 334-347, 1971. 2. BRADFORD, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein, utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254,1976. 3. 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 124: 449-454,1989. 4. ELBRINK, J., AND B. A. PHIPPS. Studies on the persistence of enhanced monosaccharide transport in rat skeletal muscle following the cessation of the initial stimulus. Cell CaZcium 1: 349-358, 1980. 5. FELL, R. D., S. E. TERBLANCHE, J. L. IVY, J. C. YOUNG, AND J. 0. HOLLOSZY. Effect of muscle glycogen content on glucose uptake following exercise. J. AppZ. Physiol. 52: 434-437, 1982. 6. FUSHIKI, T., J. A. WELLS, E. B. TRAPSCOTT, AND G. L. DOHM. Changes in glucose transporters in muscle in response in exercise. Am. J. Physiol. 256 (Endocrinol. Metub. 19): E58O-E587, 1989. 7. GARETTO, L. P., E. A. RICHTER, M. N. GOODMAN, AND N. B. RUDERMAN. Enhanced muscle glucose uptake metabolism in the rat: the two phases. Am. J. Physiol. 246 (Endocrinol. Metab. 9): E47f-E475,1984.

GLUCOSE

TRANSPORTERS

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Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise.

Recent reports have shown that immediately after an acute bout of exercise the glucose transport system of rat skeletal muscle plasma membranes is cha...
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