Proc. Natl. Acad. Sci. USAVol. 92, pp. 5817-5821, June 1995Biochemistry

Contraction stimulates translocation of glucose transporterGLUT4 in skeletal muscle through a mechanism distinctfrom that of insulinS. LUND*t, G. D. HOLMANt, 0. SCHMITZ*, AND 0. PEDERSEN§*Medical Research Laboratory, Aarhus Kommunehospital and Medical Department M (Endocrinology and Diabetes), Kommunehospitalet, Aarhus UniversityHospital, 8000 Aarhus C, Denmark; tDepartment of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom; and §StenoDiabetes Center and Hagedorn Research Institute, 2820 Gentofte, Copenhagen, Denmark

Communicated by Rolf Luft, Karolinska Institute, Stockholm, Sweden, March 6, 1995 (received for review December 1, 1994)

ABSTRACT The acute effects of contraction and insulinon the glucose transport and GLUT4 glucose transportertranslocation were investigated in rat soleus muscles by usinga 3-O-methylglucose transport assay and the sensitive exofa-cial labeling technique with the impermeant photoaffinityreagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-BMPA), respectively.Addition of wortmannin, which inhibits phosphatidylinositol3-kinase, reduced insulin-stimulated glucose transport (8.8 ±0.5 ,lmol per ml per h vs. 1.4 ± 0.1 ,umol per ml per h) andGLUT4 translocation [2.79 ± 0.20 pmol/g (wet muscleweight) vs. 0.49 ± 0.05 pmol/g (wet muscle weight)]. Incontrast, even at a high concentration (1 ,uM), wortmanninhad no effect on contraction-mediated glucose uptake (4.4 ±0.1 ,imol per ml per h vs. 4.1 ± 0.2 ,umol per ml per h) andGLUT4 cell surface content [1.75 ± 0.16 pmol/g (wet muscleweight) vs. 1.52 ± 0.16 pmol/g (wet muscle weight)]. Con-traction-mediated translocation of the GLUT4 transporters tothe cell surface was closely correlated with the glucose trans-port activity and could account fully for the increment inglucose uptake after contraction. The combined effects ofcontraction and maximal insulin stimulation were greaterthan either stimulation alone on glucose transport activity(11.5 + 0.4 ,umol per ml per h vs. 5.6 + 0.2 ,umol per ml perh and 9.0 ± 0.2 ,umol per ml per h) and on GLUT4 translo-cation [4.10 + 0.20 pmol/g (wet muscle weight) vs. 1.75 ± 0.25pmol/g (wet muscle weight) and 3.15 + 0.18 pmol/g (wetmuscle weight)]. The results provide evidence that contractionstimulates translocation ofGLUT4 in skeletal muscle througha mechanism distinct from that of insulin.

A major step in the regulation of glucose uptake in skeletalmuscle is the transport of glucose across the cell membrane.Insulin and contraction, the latter induced in vivo by acuteexercise or in vitro by electric stimulation, are able to mediateglucose uptake in muscles. Much evidence indicates thatmuscle contractions promote glucose uptake even in theabsence of insulin (1, 2).The mechanism by which the glucose transport is regulated

after contraction may involve an increase in the content ofglucose transporters in the plasma membrane primarily viarecruitment (translocation) of glucose transporters from anintracellular pool to the plasma membrane (3-7) and bychanges in the turnover rate of the transporters (intrinsicactivity) (8-10).

It is unclear whether the effects of contraction and insulinstimulation on glucose uptake in skeletal muscle are additive.Some investigators (11-14) have reported an additive effect ofthe two stimuli on the glucose transport, but others (9) havenot. Further, it needs to be elucidated whether contraction and

exposure to insulin stimulate glucose transport in skeletalmuscle through identical or different intracellular processing,though it has been assumed that two pools of glucose trans-porters are present in muscle: one that is sensitive to insulinand one that is activated by exercise (6, 15, 16). One study (17)applying a subcellular fractionation technique has shown anadditive effect of maximal insulin stimulation and contractionon translocation of glucose transporters in muscle, whereasother studies (9, 16, 18) using nearly identical techniques havenot.The purposes of the present study were (i) to examine the

effect of contraction on GLUT4 translocation by means of thesensitive exofacial labeling technique using the impermeantphotoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)ben-zoyl-1,3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-BMPA) (19-21), (ii) to assess whether this translocationaccounts fully for the increase in glucose uptake, (iii) toestimate whether insulin and contraction exhibit additiveeffects on translocation of the glucose transporter, and finally(iv) to determine whether the translocation of glucose trans-porters induced by contraction is dependent upon the activa-tion of wortmannin-sensitive signaling molecules, e.g., thephosphatidylinositol (Ptdlns) 3-kinases (22-25).

MATERIALS AND METHODSMaterials. ATB-[2-3H]BMPA (specific activity 10 Ci/

mmol; 1 Ci = 37 GBq) was prepared as described (26).3-0-[3H]Methylglucose ([3H]MeGlc) and [14C]mannitol werepurchased from DuPont/NEN. Protein A-Sepharose CL-4B,wortmannin, and bovine serum albumin (radioimmunoassaygrade) were from Sigma. Nonaethyleneglycol dodecyl wasfrom Boehringer Mannheim, GLUT4 monoclonal antibody1F8 was from Genzyme, and 1251-labeled sheep anti-mousef(ab')2 fragment was from Amersham.Animals and Muscle Preparation. All experiments were

carried out with 3-week-old Wistar rats weighing 50-60 g.Animals were fasted overnight prior to the experiments andkilled by a blow to the neck followed by cervical dislocation.Intact soleus muscles (=20 mg) were dissected as described(20).Muscle Incubations. All muscles were initially preincu-

bated for 10 min in 5 ml of oxygenated Krebs-Henseleitbicarbonate buffer (KHB buffer, pH = 7.4) containing 2mMpyruvate, 38 mM mannitol, and 0.1% bovine serum albumin(radioimmunoassay grade). The Ptdlns 3-kinase inhibitor,wortmannin, was added to the KHB buffer at 1 ,tM (if nototherwise stated) immediately before use. Muscles were thenfurther incubated for 20 min in an identical medium in the

Abbreviations: ATB-BMPA, 2-N-4-(1-azi-2,2,2-trifluoroethyl)ben-zoyl-1,3-bis(D-mannose-4-yloxy)-2-propylamine; PtdIns, phosphati-dylinositol; MeGic, 3-0-methylglucose; mU, unit(s) x 10-3.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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absence or presence of insulin [1 X 10-3 unit (mU)/ml] andwortmannin at the same concentration as in the preincuba-tion medium. All incubations were carried out at 30°C undercontinuous gassing with 95% 02/5% C02 in a shaking waterbath.Muscle Stimulation. For electrical stimulation, an experi-

mental setup was developed allowing simultaneous stimulationof 12 isolated muscles. Each muscle was mounted on twoplatinum electrodes positioned 3 mm apart and surroundingthe central part of the muscle. The ends of the muscle were notfixed allowing the muscle to shorten during stimulation. Themounted muscle was first immersed in 5 ml of oxygenatedKHB containing 2 mM pyruvate, 38 mM mannitol, and 0.1%bovine serum albumin in the absence or presence of 1 ,uMwortmannin for 10 min (preincubation medium). Muscles werethen further incubated for 20 min in an identical medium in theabsence or presence of insulin (1 mU/ml) and 1 ,uM wort-mannin. For the last 5 min of this incubation period, muscleswere stimulated to contract at various frequencies (2.5-50 Hz)with 0.5-ms square-wave 10-V pulses by using a pulse generatorbuilt at Aarhus Kommunehospital. All incubations were car-ried out at 30°C under continuous gassing with 95% 02/5%CO2 in a shaking water bath. This experimental setup ensuredthat all the muscles were exposed to wortmannin for exactly thesame length of time and at the same temperature.Measurement of MeGlc Transport into Muscle. Glucose

transport activity was measured in the soleus muscles by usingthe nonmetabolizable glucose analogue MeGlc as described byWallberg-Henriksson and Holloszy (27). Immediately afterincubation with (or without) insulin (1 mU/ml) or contraction,muscles were blotted on filter paper, moistened with KHB, andincubated for 10 min in 3 ml of oxygenated KHB containing8 mM [3H]MeGlc (437 ,uCi/mmol) and 32 mM [14C]mannitol(8 ,uCi/mmol) plus wortmannin if present during the previousincubation periods and then at the same concentrations asduring previous incubations. The incubation was carried out at30°C. After incubation, the muscles were briefly blotted onfilter paper, dampened with incubation medium, trimmed, andfrozen in liquid nitrogen. Frozen muscles were individuallyweighed, homogenized in 10% (wt/vol) trichloroacetic acid,and centrifuged at 1000 x g. Radioactivity in aliquots of themuscle extracts and of the incubation medium was measuredwith channels preset for simultaneous quantitation of 3H and14C. The amount of each isotope present in the samples wasdetermined, and the concentration of MeGlc in the extracel-lular and intracellular spaces was calculated. Data are ex-pressed as ,&mol per ml of intracellular water space per h.

Photolabeling ofRat Soleus Muscles, Immunoprecipitation,and Quantification of Photolabeled GLUT4 Protein. Muscleswere preincubated as described above, transferred to a darkroom, and further incubated at 18°C for 8 min in KHB buffercontaining 100 ,uM ATB-[3H]BMPA (1 mCi/ml) (20), insulin(1 mU/ml), and wortmannin if present during the previousincubation periods and then at the same concentrations.Muscles were then irradiated twice for 3 min in a Rayonet(Southern Northeast New England Ultraviolet, Branford, CT)model RPR 100 photochemical reactor by using lamps emittingradiation of 300 nm. Muscles were manually turned overbetween irradiation intervals. After irradiation, muscles wereimmediately blotted on wet filter paper, trimmed, and frozenin liquid nitrogen. The two frozen muscles from the same ratwere pooled and weighed (-40 mg for the two muscles), anda total crude muscle membrane preparation was prepared byhomogenizing the muscles in an ice-cold sucrose buffer (25mM Hepes/1 mM Na2EDTA/250 mM sucrose, pH 7.4) andlater centrifuging at 320,000 X gm. for 60 min. The resultingpellet containing the total crude muscle membrane prepara-tion was then resuspended and solubilized in the sucrose buffercontaining 2% (wt/vol) nonaethyleneglycol dodecyl, 0.5%deoxycholic acid, and 0.1% SDS. All buffers contained the

following protease inhibitors: 1.0 mM pefabloc [4-(2-aminoethyl)benzenesulfonyl fluoride], 1.0 mM benzamidine,leupeptin (10 ,u/ml), pepstatin (10 ,tg/ml), aprotinin (10,g/ml), and antipain (10 ,u/ml). The samples were solubilizedfor 60 min and then centrifuged for 30 min at 80,000 x gm..The resulting supernatant was then subjected to immunopre-cipitation with an anti-peptide serum raised against the 13-aaC-terminal end of GLUT4 (28). The labeled proteins wereseparated by gel electrophoresis and the radioactivity wasmeasured in 3-mm gel slices as described (20).The level of radioactivity specifically associated with each

peak was estimated by integrating the radioactivity under thepeak curve and subtracting the average background activity ofslices on either side of the peak curve.To test the effectiveness of the immunoprecipitation of

photolabeled GLUT4 protein, supernatants were examined byimmunoblot analysis [with the monoclonal GLUT4 antibody1F8 and 1251-labeled sheep anti-mouse f(ab')2 fragment] be-fore and after immunoprecipitation. In four experiments>92% of the GLUT4 protein was immunoprecipitated. Theimmunoprecipitation was specific since the GLUT4 anti-peptide serum did not immunoprecipitate photolabeledGLUT1 from human erythrocytes. In addition, immunopre-cipitation with a preimmune serum did not produce a peak ofGLUT4 from labeled rat soleus muscles.The measured level of labeling of GLUT4 by ATB-BMPA

was converted from dpm/g (wet weight of muscle) into totalmolar concentration of GLUT4 at the cell surface (Ptotal)expressed as pmol/g (wet weight of muscle) by the followingequation for binding of ligands to macromolecules (29): Ptotal= P(Kd + A)/A, whereA is the free ATB-BMPA concentra-tion (100 ,uM in all experiments), Kd is the estimated dissoci-ation constant of GLUT4 for the photolabel [250 ,uM (29)],and P is the level of labeled GLUT4 as measured by ATB-BMPA in pmol/g (wet weight of muscle).

Statistical Analysis. Results were analyzed statistically byStudent's t test for unpaired data. All data are reported asmean ± SEM. (In the experiment with MeGlc transport, n isthe number of muscles, and in experiments with ATB-BMPAphotolabeling, n is the number of immunoprecipitations.)

RESULTSEffect of Contraction on MeGlc Uptake and Cell Surface

Content of GLUT4. MeGlc uptake increased as a function ofthe frequency of soleus muscle contraction (Fig. 1). The

12

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Basal 1.0 2.5 5.0 7.5 10.0 25.0 InsulinContraction, Hz for 5 min

FIG. 1. Effect of frequency of muscle contractions on MeGIcuptake in in vitro-incubated rat soleus muscles. The intact soleusmuscles were rapidly but carefully dissected, incubated, and thenstimulated electrically to contract for 5 minat the frequencies indi-cated. Insulin-stimulated muscles were incubatedwith insulin (1 mU/ml).The intracellular accumulation of MeGlc was measured for a 10-miperiod at 30°C. Each bar shows the mean ± SEM (n = 8 to 16).

5818 Biochemistry: Lund et al

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FIG. 2. Effect of in vitro muscle contraction on cell surface GLUT4content in intact soleus muscles. Muscles were incubated, irradiated,and solubilized, and GLUT4 was immunoprecipitated. Muscles werestimulated electrically for 5 min to contract at the indicated frequen-cies. Insulin-stimulated muscles were incubated with insulin (1 mU/ml). Values are the mean ± SEM (n = 6 to 8). Amount of photolabeledGLUT4 is given in both the observed level of labeling of GLUT4 byATB-BMPA [dpm/g (wet muscle weight)] and in molar concentrationof GLUT4 at the cell surface [pmol/g (wet muscle weight)]. Bars: 1,basal; 2, contractions at 5 Hz for 5 min; 3, contractions at 10 Hz for5 min; 4, insulin at 1 mU/ml.

maximal glucose transport activity was achieved at a contrac-tion frequency of 10 Hz for 5 min. The maximal contraction-mediated glucose uptake represented '60% of the glucoseuptake induced by insulin at a maximally stimulatory concen-tration of 1.0 mU/ml (6.6 ± 0.3 ,umol per ml per h vs. 10.6 ±0.5 gmol per ml per h; P < 0.01). The increment was 4- to5-fold the basal uptake of MeGlc (1.4 ± 0.1 ,tmol per ml perh, P < 0.01).Frequency of contraction above 10 Hz or contraction for

extended periods did not result in further glucose transportactivity; in contrast, muscles exposed to such high stimuli oftenremained contracted even after the cessation of the stimula-tion, a phenomenon that might be caused by hypoxia andaccumulation of Ca2+ in the muscle, thus shifting the rate-limiting step from transport over the membrane to diffusion ofMeGlc into the muscle.

Contraction significantly increased the amount of GLUT4on the muscle cell surface in a dose-dependent manner froma basal value of 0.44 ± 0.04 pmol/g (wet muscle weight) to apeak level of 1.75 ± 0.25 pmol/g (wet muscle weight) (P < 0.01)(Fig. 2). Again the maximal translocation ofGLUT4 provoked by

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FIG. 3. Effect of insulin at maximally stimulatory concentrations (1mU/ml), contraction at high frequency (10 Hz for S min), and thecombined effect of insulin and contraction on MeGlc uptake in soleusmuscles. Bars: 1, basal; 2, insulin (1 mU/ml); 3, contractions at 10 Hzfor 5 min; 4, insulin and contraction. Values are the mean + SEM (n= 8 to 12). *, P < 0.01 vs. insulin-stimulated muscle.

FIG. 4. Effect of insulin, contraction, and the combined stimula-tion with insulin and contraction on surface-accessible GLUT4. Ex-perimental conditions were as in Fig. 3. Bars: 1, basal; 2, insulin (1mU/ml); 3, contractions at 10 Hz for 5 min; 4, insulin and contraction.Values are the mean + SEM (n = 6). *, P < 0.01 vs. insulin-stimulatedmuscle.

insulin was '40% greater [3.05 ± 0.24 pmol per g (wet muscleweight)] than that mediated by contraction (P < 0.01).Combined Effects of Insulin and Contraction on MeGIc

Uptake and GLUT4 Translocation. Maximal insulin stimula-tion (1.0 mU/ml) with high-frequency contractions of thesoleus muscle (10 Hz for 5 min) resulted in a glucose uptakesignificantly higher than that mediated by insulin or contrac-tion alone (11.5 ± 0.4 ,umol per ml per h vs. 9.0 ± 0.2 ,umolper ml per h or 5.6 ± 0.2 ,umol per ml per h; P < 0.01 in both)Fig. 3). The combined stimulation induced an increase thatapproached a level expected from the addition of the twoindividual stimuli. Equivalent observations were done whenexamining translocation of the GLUT4 transporter [4.10 ±

0.20 pmol/g (wet muscle weight) vs. 3.15 ± 0.18 pmol/g (wetmuscle weight) or 1.75 ± 0.25 pmol/g (wet muscle weight); P< 0.01 in both] (Fig. 4).

Effect of Wortmannin on Insulin- or Contraction-Stimulated MeGlc Uptake and GLUT4 Translocation. After

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1 2 3 4 5 6

FIG. 5. Comparison of the effect of 1 imM wortmannin on basal and

insulin- and contraction-stimulated glucose transport. Intact soleus

muscles were preincubated with or without 1 A.M wortmannin for 10

min and then for an additional 20 min in an identical medium (± 1 ALMwortmannin, as in preincubation medium). During this incubation,muscles were stimulated with insulin at 1 mU/ml or by contraction at

7.5 Hz for two 5-mmn periods with a 1-mmn rest in between for the last

11 min of this 20-mmn incubation. All muscles were exposed to 1 A&Mwortmannin for exactly the same time and at the same temperature.Bars: 1, basal; 2, basal and 1 ,Mwortmannin; 3, insulin (1 mu/mI);4, insulin and wortmannin; 5, contractions at 7.5 Hz for two 5-mmnperiods; 6, contraction and wortmannin. Values are the mean ± SEM

(n = 10 to 16). *, P < 0.01 vs. insulin-stimulated muscle.

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FIG. 6. Inhibition of insulin-stimulated glucose transport activityby wortmannin. Rat soleus muscles were preincubated for 10 min withthe indicated concentrations of wortmannin and then for a further 20min with insulin at 1 mU/ml and wortmannin at the same concen-tration as in the preincubation medium. o, Insulin-stimulated glucoseuptake without addition of wortmannin; *, basal glucose uptake (noinsulin and wortmannin). Values are the mean ± SEM (n = 6 to 8).

incubation with wortmannin, insulin-stimulated glucose up-take was almost abolished (8.8 ± 0.5 ,umol per ml per h vs. 1.4± 0.1 ,mol per ml per h; P < 0.01) (Fig. 5). The latter levelwas comparable to basal level, which was unaltered by wort-mannin exposure [1.3 ± 0.2 ,umol per ml per h (basal) and 1.2± 0.2 ,umol per ml per h (basal + 1 ,uM wortmannin)]. Fig. 6shows the inhibition of insulin-stimulated glucose uptake bywortmannin (IC50 = 3.6 ± 1.6 nM). In contrast, wortmannin,even at a high concentration (1 ,uM), failed to affect theglucose uptake induced by contraction (4.4 ± 0.1 ,umol per mlper h and 4.1 ± 0.2 ,umol per ml per h) (Fig. 5).

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FIG. 7. Effect of wortmannin on GLUT4 translocation detectedwith ATB-BMPA. Muscle cells were incubated in insulin (1 mU/ml)or stimulated to contract at 7.5 Hz for two 5-minperiods with a 1-minrest in between, and where indicated with addition of 10 nM or 1 ,uMwortmannin (see Fig. 5). The labeled transporters were solubilized andimmunoprecipitated with anti-C-terminal peptide antibodies againstGLUT4 and resolved by gel electrophoresis. The positions of molec-ular mass marker proteins are indicated. The results are from singlerepresentative experiments. (A) Insulin (1 mU/ml). (B) Insulin andwortmannin at 1 ,uM (0) or 10 nM (*). (C) Contraction (7.5 Hz fortwo 5-min periods). (D) Contraction and wortmannin at 1 ,uM.

To assess whether the observed changes in glucose transportactivity induced by wortmannin were associated with changesin translocation of the glucose transporter, the cell surfaceGLUT4 content was determined with the impermeant photo-label ATB-BMPA. As shown in Fig. 7 concomitant adminis-tration of wortmannin (10 nM or 1 ,M) reduced the level ofGLUT4 at the cell surface from 2.79 ± 0.20 pmol/g (wetmuscle weight) observed in insulin-stimulated muscles to 1.15± 0.14 pmol/g (wet muscle weight) (10 nM wortmannin; P <0.01, n = 5) and 0.49 ± 0.05 pmol/g (wet muscle weight) (1 ,tMwortmannin; P < 0.01, n = 5), whereas the translocation ofGLUT4 induced by contraction was unaltered by wortmanninexposure [1.75 ± 0.16 pmol/g (wet muscle weight) vs. 1.52 ±0.16 pmol/g (wet muscle weight) (1 ,uM wortmannin; notsignificant, n = 5)]. Administration of wortmannin had also noeffect on basal GLUT4 cell surface content [0.44 ± 0.08pmol/g (wet muscle weight) vs. 0.43 ± 0.07 pmol/g (wetmuscle weight) (1 ,uM wortmannin; not significant, n = 5)].

DISCUSSIONAn intriguing finding of the present study is that contraction-mediated increase in glucose uptake and GLUT4 translocationin skeletal muscle is not crucially dependent on a wortmannin-sensitive signaling pathway in contrast to that of insulin. Ourdata therefore provide direct evidence that contraction stim-ulates glucose uptake and translocation of GLUT4 at stepsmore distal than the wortmannin-sensitive molecules (e.g., thePtdIns 3-kinases) in the insulin signaling pathway or by stim-ulation through an entirely different pathway.Wortmannin and the LY29002 compound inhibit a number

of protein kinases at higher concentrations but are thought tobe specific for PtdIns 3-kinases at lower concentrations (forreview, see ref. 30). Recent studies in cultured cell systemssuggest, however, that other signaling proteins may also besubject to wortmannin inhibition at low doses (30). Studies in3T3-L1 and CHO cells have shown that PtdIns 3-kinases maybe key intermediates in the insulin action cascade leading toglucose transport stimulation. Addition ofwortmannin (22, 23,25) and LY294002 (24) hinders insulin-stimulated glucosetransport and translocation of GLUT4 in 3T3-L1 cells. Ourstudy in in vitro incubated rat soleus muscles confirms thatinhibition of wortmannin-sensitive signaling mechanisms re-sults in a suppression of the insulin-stimulated glucose trans-port activity and translocation of GLUT4 and that wortmanninis a potent inhibitor of insulin-stimulated glucose transporteven in nanomolar concentrations (IC5o = 3.6 ± 1.6 nM) (23).In contrast, the glucose uptake and the translocation ofGLUT4 mediated by contraction are not influenced by wort-mannin even at high concentrations (1 ,uM).

In line with other reports (31, 32), we demonstrate thatmaximal insulin-stimulated glucose uptake is =40% higherthan maximal contraction-stimulated uptake. Furthermore,the data substantiate that contraction induces translocation ofthe major glucose transporter of skeletal muscle (GLUT4) tothe plasma membrane. In contrast to previous reports usingthe subcellular fractionation technique on skeletal muscles(3-5, 8-10), the use of the impermeant photolabel ATB-BMPA allows us to circumvent drawbacks normally encoun-tered with subcellular fractionation. These include difficultiesin separating surface membranes from intracellular vesiclescontaining GLUT4 and a poor recovery. Our results clearlyindicate that the contraction-mediated translocation of theGLUT4 transporters to the cell surface is closely correlatedwith the glucose transport activity and accounts fully for theincrement in glucose uptake after contraction.The combined effect of contraction and maximal insulin

stimulation on glucose uptake was almost additive in the invitro-incubated soleus muscle. Similar data on the additiveeffect of contraction and maximal insulin stimulation have

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been reported in incubated muscles (11, 13, 14, 33). The failureto find that the combined stimulation is completely additivemight be caused by some efflux of MeGlc occurring under theconditions with very high rates of MeGlc transport, therebyresulting in a slight underestimation of the glucose transportactivity (32).The possibility that the effects of maximal insulin stimula-

tion and contraction are additive on the translocation ofglucose transporters to the surface membrane has been ex-amined previously but with conflicting results. Some studiesusing subcellular fractionation techniques to isolate plasmamembranes have led to the hypothesis that a combined stim-ulation with contraction and insulin increases the intrinsicactivity of the glucose transporters in the plasma membrane (9,16, 18). In contrast, a study (17) in which GLUT4 in membranefractions was assessed by Western blot analysis suggests thatthe combined effects of insulin and contraction stimulation areadditive on GLUT4 translocation.By using the more sensitive surface labeling technique (20),

the present results show that the combined stimulation with amaximal insulin level and contraction results in a furtherincrease in labeled glucose transporters (GLUT4) on the cellsurface to a level significantly higher than that mediated byinsulin or contraction alone. Our studies therefore extend andcorroborate the findings obtained with the subcellular frac-tionation technique (17) that the combined stimulation isnear-additive on the translocation of glucose transporter.The observation that maximal insulin stimulation and con-

traction have additive effects on translocation of GLUT4 inmuscle might suggest the presence of two pools of glucosetransporters. One pool may be accessible for translocation viainsulin but not through exercise, whereas the other may beavailable for translocation during exercise but not affected byinsulin (6, 16).Another possibility could be that intracellular glucose trans-

porters form a more or less uniform pool that is maintained bya balance between the exocytosis and endocytosis of GLUT4(34, 35). According to this scenario, contraction may mainlyreduce the rate of endocytosis of the GLUT4, in contrast toinsulin, which apparently accelerates the rate of exocytosis ofthe glucose transporters (34) and to some extent also reducesthe rate of endocytosis (36, 37). To our knowledge, there is nodata to distinguish between these potential mechanisms butidentification of GLUT4 in several subcellular locations inmuscle, including the t-tubulus and subsarcolemma regions,suggests the mechanism involving translocation from separatepools is most likely.

We acknowledge J. Lyhne for construction of the pulse generator.H. Petersen, M. M0ller, and E. Hornemann are thanked for excellenttechnical assistance. Prof. H. 0rskov is thanked for stimulatingdiscussions. This study was supported by grants from Institute ofExperimental Clinical Research, University of Aarhus, Danish Med-ical Research Council, Danish Diabetes Association, Hafnia Fonden,Torben Frimodt og Alice Frimodts Fond, Else og Mongens Wedell-Wedellsborgs Fond, Novo Nordisk Foundation, and Aage Louis-Hansens Mindefond.

1. Wallberg-Henriksson, H. & Holloszy, J. 0. (1984) J. Appl.Physiol. 57, 1045-1049.

2. Ploug, T., Galbo, H. & Richter, E. A. (1984) Am. J. Physio. 247,E726-E731.

3. Hirshman, M. F., Wallberg-Henriksson, H., Wardzala, L. J., Hor-ton, E. D. & Horton, E. S. (1988) FEBS Lett. 238, 235-239.

4. Douen, A. G., Ramlal, T., Klip, A., Young, D. A., Cartee, G. D.& Holloszy, J. 0. (1989) Endocrinology 124, 449-454.

5. Fushiki, T., Wells, J. A., Tapscott, E. B. & Dohm, G. L. (1989)Am. J. Physiol. 256, E580-E587.

6. Douen, A. G., Ramlal, T., Rastogi, S., Bilan, P. J., Cartee, G. D.,Vranic, M., Holloszy, J. 0. & Klip, A. (1990) J. Biol. Chem. 265,13427-13430.

7. Hirshman, M. F., Goodyear, L. J., Wardzala, L. J., Horton, E. D.& Horton, E. S. (1990) J. Bio. Chem. 265, 987-991.

8. King, P. A., Hirshman, M. F., Horton, E. D. & Horton, E. S.(1989) Am. J. Physiol. 257, C1128-C1134.

9. Goodyear, L. J., King, P. A., Hirshman, M. F., Thompson, C. M.,Horton, E. D. & Horton, E. S. (1990) Am. J. Physio!. 258,E667-E672.

10. Sternlicht, E., Barnard, R. J. & Grimditch, G. K. (1989) Am. J.Physiol. 256, E227-E230.

11. Wallberg-Henriksson, H., Constable, S. H., Young, D. A. &Holloszy, J. 0. (1988) J. Appl. Physiol. 65, 909-913.

12. Ploug, T., Galbo, H., Vinten, J., Jorgensen, M. & Richter, E. A.(1987) Am. J. Physiol. 253, E12-E20.

13. Zorzano, A., Balon, T. W., Goodman, M. N. & Ruderman, N. B.(1986) Am. J. Physiol. 251, E21-E26.

14. Nesher, R., Karl, I. E. & Kipnis, D. M. (1985)Am. J. Physiol. 249,C226-C232.

15. Ploug, T., Wojtaszewski, J., Kristiansen, S., Hespel, P., Galbo, H.& Richter, E. A. (1993) Am. J. Physiol. 264, E270-E278.

16. Brozinick, J. T., Jr., Etgen, G. J., Jr., Yaspelkis, B. B. & Ivy, J. L.(1994) Biochem. J. 297, 539-545.

17. Gao, J., Ren, J., Gulve, E. A. & Holloszy, J. 0. (1994) J. Appl.Physiol. 77, 1597-1601.

18. Douen, A. G., Ramlal, T., Cartee, G. D. & Klip, A. (1990) FEBSLett. 261, 256-260.

19. Wilson, C. M. & Cushman, S. W. (1992) Diabetes 40, 167.20. Lund, S., Holman, G. D., Schmitz, 0. & Pedersen, 0. (1993)

FEBS Let. 330, 312-318.21. Wilson, C. M. & Cushman, S. W. (1994) Biochem. J. 299, 755-

759.22. Okada, T., Kawano, Y., Sakakibara, T., Hazeki, 0. & Ui, M.

(1994) J. Biol. Chem. 269, 3568-3573.23. Clarke, J. F., Young, P. W., Yonezawa, K., Kasuga, M. & Hol-

man, G. D. (1994) Biochem. J. 300, 631-635.24. Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J.

& Kahn, C. R. (1994) Mol. Cell. Bio. 14, 4902-4911.25. Hara, K., Yonezawa, K., Sakaue, H., Ando, A., Kotani, K.,

Kitamura, T., Kitamura, Y., Ueda, H., Stephens, L., Jackson,T. R., Hawkins, P. T., Dhand, R., Clarke, A. E., Holman, G. D.,Waterfield, M. D. & Kasuga, M. (1994) Proc. Natl. Acad. Sci.USA 91, 7415-7419.

26. Clark, A. E. & Holman, G. D. (1990) Biochem. J. 269, 615-622.27. Wallberg-Henriksson, H. & Holloszy, J. 0. (1985)Am. J. Physiol.

249, C233-C237.28. Lund, S., Flyvbjerg, A., Holman, G. D., Larsen, F. S., Pedersen,

0. & Schmitz, 0. (1994) Am. J. Physio. 267, E461-E466.29. Holman, G. D., Kozka, I. J., Clark, A. E., Flower, C. J., Saltis, J.,

Habberfield, A. D., Simpson, I. A. & Cushman, S. W. (1990) J.Bio. Chem. 265, 18172-18179.

30. Downward, J. (1994) Nature (London) 371, 378-379.31. Henriksen, E. J., Bourey, R. E., Rodnick, K. J., Koranyi, L.,

Permutt, M. A. & Holloszy, J. 0. (1990) Am. J. Physiol. 259,E593-E598.

32. Hansen, P. A., Gulve, E. A. & Holloszy, J. 0. (1994) J. Appl.Physio. 76, 979-985.

33. Enrich, C. & Gahmberg, C. G. (1985) Biochem. J. 227, 565-572.34. Satoh, S., Nishimura, H., Clark, A. E., Kozka, I. J., Vannucci,

S. J., Simpson, I. A., Quon, M. J., Cushman, S. W. & Holman,G. D. (1993) J. Bio. Chem. 268, 17820-17829.

35. Holman, G. D., Lo Leggio, L. & Cushman, S. W. (1994) J. Bio.Chem. 269, 17516-17524.

36. Jhun, B. H., Rampal, A. L., Liu, H., Lachaal, M. & Jung, C. Y.(1992) J. Biol. Chem. 267, 17710-17715.

37. Czech, M. P. & Buxton, J. M. (1993) J. Bio. Chem. 268, 9187-9190.

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