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

Contraction stimulates translocation of transporter GLUT4 in through a mechanism distinct from that of

S. LUND*t, G. D. HOLMANt, 0. SCHMITZ*, AND 0. PEDERSEN§ *Medical Research Laboratory, Aarhus Kommunehospital and Medical Department M (Endocrinology and ), Kommunehospitalet, Aarhus University Hospital, 8000 Aarhus C, Denmark; tDepartment of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom; and §Steno Diabetes 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 insulin exposure to insulin stimulate glucose transport in skeletal on the glucose transport and GLUT4 muscle through identical or different intracellular processing, translocation were investigated in rat soleus muscles by using though it has been assumed that two pools of glucose trans- a 3-O-methylglucose transport assay and the sensitive exofa- porters are present in muscle: one that is sensitive to insulin cial labeling technique with the impermeant photoaffinity and one that is activated by exercise (6, 15, 16). One study (17) reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D- applying a subcellular fractionation technique has shown an mannose-4-yloxy)-2-propylamine (ATB-BMPA), respectively. additive effect of maximal insulin stimulation and contraction Addition of wortmannin, which inhibits phosphatidylinositol on translocation of glucose transporters in muscle, whereas 3-kinase, reduced insulin-stimulated glucose transport (8.8 ± other studies (9, 16, 18) using nearly identical techniques have 0.5 ,lmol per ml per h vs. 1.4 ± 0.1 ,umol per ml per h) and not. GLUT4 translocation [2.79 ± 0.20 pmol/g (wet muscle The purposes of the present study were (i) to examine the weight) vs. 0.49 ± 0.05 pmol/g (wet muscle weight)]. In effect of contraction on GLUT4 translocation by means of the contrast, even at a high concentration (1 ,uM), wortmannin sensitive exofacial labeling technique using the impermeant had no effect on contraction-mediated (4.4 ± photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)ben- 0.1 ,imol per ml per h vs. 4.1 ± 0.2 ,umol per ml per h) and zoyl-1,3-bis(D-mannose-4-yloxy)-2-propylamine (ATB- GLUT4 cell surface content [1.75 ± 0.16 pmol/g (wet muscle BMPA) (19-21), (ii) to assess whether this translocation weight) vs. 1.52 ± 0.16 pmol/g (wet muscle weight)]. Con- accounts fully for the increase in glucose uptake, (iii) to traction-mediated translocation ofthe GLUT4 transporters to estimate whether insulin and contraction exhibit additive the cell surface was closely correlated with the glucose trans- effects on translocation of the glucose transporter, and finally port activity and could account fully for the increment in (iv) to determine whether the translocation of glucose trans- glucose uptake after contraction. The combined effects of porters induced by contraction is dependent upon the activa- contraction and maximal insulin stimulation were greater tion of wortmannin-sensitive signaling molecules, e.g., the than either stimulation alone on glucose transport activity phosphatidylinositol (Ptdlns) 3-kinases (22-25). (11.5 + 0.4 ,umol per ml per h vs. 5.6 + 0.2 ,umol per ml per h 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 ± 0.25 MATERIALS AND METHODS pmol/g (wet muscle weight) and 3.15 + 0.18 pmol/g (wet Materials. ATB-[2-3H]BMPA (specific activity 10 Ci/ muscle weight)]. The results provide evidence that contraction mmol; 1 Ci = 37 GBq) was prepared as described (26). stimulates translocation ofGLUT4 in skeletal muscle through 3-0-[3H]Methylglucose ([3H]MeGlc) and [14C]mannitol were a mechanism distinct from that of insulin. purchased from DuPont/NEN. A-Sepharose CL-4B, wortmannin, and bovine serum albumin (radioimmunoassay A major step in the regulation of glucose uptake in skeletal grade) were from Sigma. Nonaethyleneglycol dodecyl was muscle is the transport of glucose across the cell membrane. from Boehringer Mannheim, GLUT4 monoclonal antibody Insulin and contraction, the latter induced in vivo by acute 1F8 was from Genzyme, and 1251-labeled sheep anti-mouse exercise or in vitro by electric stimulation, are able to mediate f(ab')2 fragment was from Amersham. glucose uptake in muscles. Much evidence indicates that Animals and Muscle Preparation. All experiments were muscle contractions promote glucose uptake even in the carried out with 3-week-old Wistar rats weighing 50-60 g. absence of insulin (1, 2). Animals were fasted overnight prior to the experiments and The mechanism by which the glucose transport is regulated killed by a blow to the neck followed by cervical dislocation. after contraction may involve an increase in the content of Intact soleus muscles (=20 mg) were dissected as described glucose transporters in the plasma membrane primarily via (20). recruitment (translocation) of glucose transporters from an Muscle Incubations. All muscles were initially preincu- intracellular pool to the plasma membrane (3-7) and by bated for 10 min in 5 ml of oxygenated Krebs-Henseleit changes in the turnover rate of the transporters (intrinsic bicarbonate buffer (KHB buffer, pH = 7.4) containing 2 mM activity) (8-10). pyruvate, 38 mM mannitol, and 0.1% bovine serum albumin It is unclear whether the effects of contraction and insulin (radioimmunoassay grade). The Ptdlns 3-kinase inhibitor, stimulation on glucose uptake in skeletal muscle are additive. wortmannin, was added to the KHB buffer at 1 ,tM (if not Some investigators (11-14) have reported an additive effect of otherwise stated) immediately before use. Muscles were then the two stimuli on the glucose transport, but others (9) have further incubated for 20 min in an identical medium in the not. Further, it needs to be elucidated whether contraction and Abbreviations: ATB-BMPA, 2-N-4-(1-azi-2,2,2-trifluoroethyl)ben- The publication costs of this article were defrayed in part by page charge zoyl-1,3-bis(D-mannose-4-yloxy)-2-propylamine; PtdIns, phosphati- payment. This article must therefore be hereby marked "advertisement" in dylinositol; MeGic, 3-0-methylglucose; mU, unit(s) x 10-3. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5817 Downloaded by guest on September 28, 2021 5818 Biochemistry: Lund et al Proc. Natl. Acad. Sci. USA 92 (1995) absence or presence of insulin [1 X 10-3 unit (mU)/ml] and following protease inhibitors: 1.0 mM pefabloc [4-(2- wortmannin at the same concentration as in the preincuba- aminoethyl)benzenesulfonyl fluoride], 1.0 mM benzamidine, tion medium. All incubations were carried out at 30°C under leupeptin (10 ,u/ml), pepstatin (10 ,tg/ml), aprotinin (10 continuous gassing with 95% 02/5% C02 in a shaking water ,g/ml), and antipain (10 ,u/ml). The samples were solubilized bath. for 60 min and then centrifuged for 30 min at 80,000 x gm.. Muscle Stimulation. For electrical stimulation, an experi- The resulting supernatant was then subjected to immunopre- mental setup was developed allowing simultaneous stimulation cipitation with an anti-peptide serum raised against the 13-aa of 12 isolated muscles. Each muscle was mounted on two C-terminal end of GLUT4 (28). The labeled were platinum electrodes positioned 3 mm apart and surrounding separated by gel electrophoresis and the radioactivity was the central part of the muscle. The ends of the muscle were not measured in 3-mm gel slices as described (20). fixed allowing the muscle to shorten during stimulation. The The level of radioactivity specifically associated with each mounted muscle was first immersed in 5 ml of oxygenated peak was estimated by integrating the radioactivity under the KHB containing 2 mM pyruvate, 38 mM mannitol, and 0.1% peak curve and subtracting the average background activity of bovine serum albumin in the absence or presence of 1 ,uM slices on either side of the peak curve. wortmannin for 10 min (preincubation medium). Muscles were To test the effectiveness of the immunoprecipitation of then further incubated for 20 min in an identical medium in the photolabeled GLUT4 protein, supernatants were examined by absence or presence of insulin (1 mU/ml) and 1 ,uM wort- immunoblot analysis [with the monoclonal GLUT4 antibody mannin. For the last 5 min of this incubation period, muscles 1F8 and 1251-labeled sheep anti-mouse f(ab')2 fragment] be- were stimulated to contract at various frequencies (2.5-50 Hz) fore and after immunoprecipitation. In four experiments with 0.5-ms square-wave 10-V pulses byusing a pulse generator >92% of the GLUT4 protein was immunoprecipitated. The built at Aarhus Kommunehospital. All incubations were car- immunoprecipitation was specific since the GLUT4 anti- ried out at 30°C under continuous gassing with 95% 02/5% peptide serum did not immunoprecipitate photolabeled CO2 in a shaking water bath. This experimental setup ensured GLUT1 from human erythrocytes. In addition, immunopre- that all the muscles were exposed to wortmannin for exactly the cipitation with a preimmune serum did not produce a peak of same length of time and at the same temperature. GLUT4 from labeled rat soleus muscles. Measurement of MeGlc Transport into Muscle. Glucose The measured level of labeling of GLUT4 by ATB-BMPA transport activity was measured in the soleus muscles by using was converted from dpm/g (wet weight of muscle) into total the nonmetabolizable glucose analogue MeGlc as described by molar concentration of GLUT4 at the cell surface (Ptotal) Wallberg-Henriksson and Holloszy (27). Immediately after expressed as pmol/g (wet weight of muscle) by the following incubation with (orwithout) insulin (1 mU/ml) or contraction, equation for binding of ligands to macromolecules (29): Ptotal muscles were blotted on filter paper, moistened with KHB, and = P(Kd + A)/A, where A is the free ATB-BMPA concentra- incubated for 10 min in 3 ml of oxygenated KHB containing tion (100 ,uM in all experiments), Kd is the estimated dissoci- 8 mM [3H]MeGlc (437 ,uCi/mmol) and 32 mM [14C]mannitol ation constant of GLUT4 for the photolabel [250 ,uM (29)], (8 ,uCi/mmol) plus wortmannin if present during the previous and P is the level of labeled GLUT4 as measured by ATB- incubation periods and then at the same concentrations as BMPA in pmol/g (wet weight of muscle). during previous incubations. The incubation was carried out at Statistical Analysis. Results were analyzed statistically by 30°C. After incubation, the muscles were briefly blotted on filter Student's t test for unpaired data. All data are reported as paper, dampened with incubation medium, trimmed, and mean ± SEM. (In the experiment with MeGlc transport, n is frozen in liquid nitrogen. Frozen muscles were individually in the number of muscles, and in experiments with ATB-BMPA weighed, homogenized 10% (wt/vol) trichloroacetic acid, photolabeling, n is the number of and centrifuged at 1000 x g. Radioactivity in aliquots of the immunoprecipitations.) muscle extracts and of the incubation medium was measured with channels preset for simultaneous quantitation of 3H and RESULTS 14C. The amount of each isotope present in the samples was determined, and the concentration of MeGlc in the extracel- Effect of Contraction on MeGlc Uptake and Cell Surface lular and intracellular was calculated. Data are ex- Content of GLUT4. MeGlc uptake increased as a function of spaces the frequency of soleus muscle contraction (Fig. 1). The pressed as ,&mol per ml of intracellular water space per h. Photolabeling ofRat Soleus Muscles, Immunoprecipitation, and Quantification of Photolabeled GLUT4 Protein. Muscles 12 were preincubated as described above, transferred to a dark room, and further incubated at 18°C for 8 min in KHB buffer containing 100 ,uM ATB-[3H]BMPA (1 mCi/ml) (20), insulin (1 mU/ml), and wortmannin if present during the previous incubation 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 emitting radiation of 300 nm. Muscles were manually turned over between irradiation intervals. After irradiation, muscles were immediately blotted on wet filter paper, trimmed, and frozen 0 in liquid nitrogen. The two frozen muscles from the same rat were pooled and weighed (-40 mg for the two muscles), and Basal 1.0 2.5 5.0 7.5 10.0 25.0 Insulin a total crude muscle membrane preparation was prepared by Contraction, Hz for 5 min homogenizing the muscles in an ice-cold sucrose buffer (25 mM Hepes/1 mM Na2EDTA/250 mM sucrose, pH 7.4) and FIG. 1. Effect of frequency of muscle contractions on MeGIc later centrifuging at 320,000 X gm. for 60 min. The resulting uptake in in vitro-incubated rat soleus muscles. The intact soleus pellet the total crude muscle membrane muscles were rapidly but carefully dissected, incubated, and then containing prepara- stimulated to contract for 5 minat the indi- tion was then and solubilized in the sucrose electrically frequencies resuspended buffer cated. Insulin-stimulated muscles were incubatedwith insulin (1 mU/ml). containing 2% (wt/vol) nonaethyleneglycol dodecyl, 0.5% The intracellular accumulation of MeGlc was measured for a 10-mi deoxycholic acid, and 0.1% SDS. All buffers contained the period at 30°C. Each bar shows the mean ± SEM (n = 8 to 16). Downloaded by guest on September 28, 2021 Biochemistry: Lund et al Proc. Natl. Acad. Sci. USA 92 (1995) 5819

-- 5.0 = 30.0 -. _b r.- C.) -= C)o: = 4.0 4 25.0

CI. 0 _1- C.) _ -o=: 3 20.0 to E0 3.0 :ou0 - C)t 3aC) D ._ 3 15.0 oe I 00E .= x -2.0 (U)0 oz C). u : 10- 0 cu0 -C: u = -1.0 E vE0 5.0 .0 0.0 _ 0.0 1 2 3 4

FIG. 2. Effect ofin vitro muscle contraction on cell surface GLUT4 FIG. 4. Effect of insulin, contraction, and the combined stimula- content in intact soleus muscles. Muscles were incubated, irradiated, tion with insulin and contraction on surface-accessible GLUT4. Ex- and solubilized, and GLUT4 was immunoprecipitated. Muscles were perimental conditions were as in Fig. 3. Bars: 1, basal; 2, insulin (1 stimulated electrically for 5 min to contract at the indicated frequen- mU/ml); 3, contractions at 10 Hz for 5 min; 4, insulin and contraction. cies. Insulin-stimulated muscles were incubated with insulin (1 mU/ Values are the mean + SEM (n = 6). *, P < 0.01 vs. insulin-stimulated ml). Values are the mean ± SEM (n = 6 to 8). Amount of photolabeled muscle. GLUT4 is given in both the observed level of labeling of GLUT4 by ATB-BMPA [dpm/g (wet muscle weight)] and in molar concentration insulin was '40% greater [3.05 ± 0.24 pmol per g (wet muscle of GLUT4 at the cell surface [pmol/g (wet muscle weight)]. Bars: 1, weight)] than that mediated by contraction (P < 0.01). basal; 2, contractions at 5 Hz for 5 min; 3, contractions at 10 Hz for Combined Effects of Insulin and Contraction on MeGIc 5 min; 4, insulin at 1 mU/ml. Uptake and GLUT4 Translocation. Maximal insulin stimula- maximal glucose transport activity was achieved at a contrac- tion (1.0 mU/ml) with high-frequency contractions of the tion frequency of 10 Hz for 5 min. The maximal contraction- soleus muscle (10 Hz for 5 min) resulted in a glucose uptake mediated glucose uptake represented '60% of the glucose significantly higher than that mediated by insulin or contrac- uptake induced by insulin at a maximally stimulatory concen- tion alone (11.5 ± 0.4 ,umol per ml per h vs. 9.0 ± 0.2 ,umol per ml per h or 5.6 ± 0.2 ,umol per ml per h; P < 0.01 in tration of 1.0 mU/ml (6.6 ± 0.3 ,umol per ml per h vs. 10.6 ± both) Fig. 3). The combined stimulation induced an that 0.5 gmol per ml per h; P < 0.01). The increment was 4- to increase approached a level expected from the addition of the two 5-fold the basal uptake of MeGlc (1.4 ± 0.1 ,tmol per ml per h, P < 0.01). individual stimuli. Equivalent observations were done when Frequency of contraction above 10 Hz or contraction for examining translocation of the GLUT4 transporter [4.10 ± extended periods did not result in further glucose transport 0.20 pmol/g (wet muscle weight) vs. 3.15 ± 0.18 pmol/g (wet activity; in contrast, muscles exposed to such high stimuli often muscle weight) or 1.75 ± 0.25 pmol/g (wet muscle weight); P remained contracted even after the cessation of the stimula- < 0.01 in both] (Fig. 4). tion, a phenomenon that might be caused by hypoxia and Effect of Wortmannin on Insulin- or Contraction- accumulation of Ca2+ in the muscle, thus shifting the rate- Stimulated MeGlc Uptake and GLUT4 Translocation. After limiting step from transport over the membrane to diffusion of MeGlc into the muscle. 12 - Contraction significantly increased the amount of GLUT4 on the muscle cell surface in a dose-dependent manner from 0L. a basal value of 0.44 ± 0.04 pmol/g (wet muscle weight) to a peak level of 1.75 ± 0.25 pmol/g (wet muscle weight) (P < 0.01) (Fig. 2). Again the maximal translocation ofGLUT4 provoked by

16 -

.0 * 12-

0 0 a. - -6 E 8-

1 2 3 4 5 6 Q. FIG. 5. Comparison of the effect of 1 imM wortmannin on basal and = 4 - 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 ALM wortmannin, as in preincubation medium). During this incubation, 0 l 4 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 FIG. 3. Effect of insulin at maximally stimulatory concentrations (1 11 min of this 20-mmn incubation. All muscles were exposed to 1 A&M mU/ml), contraction at high frequency (10 Hz for S min), and the wortmannin for exactly the same time and at the same temperature. combined effect of insulin and contraction on MeGlc uptake in soleus Bars: 1, basal; 2, basal and 1 ,Mwortmannin; 3, insulin (1 mu/mI); muscles. Bars: 1, basal; 2, insulin (1 mU/ml); 3, contractions at 10 Hz 4, insulin and wortmannin; 5, contractions at 7.5 Hz for two 5-mmn for 5 min; 4, insulin and contraction. Values are the mean + SEM (n periods; 6, contraction and wortmannin. Values are the mean ± SEM = 8 to 12). *, P < 0.01 vs. insulin-stimulated muscle. (n = 10 to 16). *, P < 0.01 vs. insulin-stimulated muscle. Downloaded by guest on September 28, 2021 5820 Biochemistry: Lund et aL Proc. Natl. Acad. Sci. USA 92 (1995)

10- To assess whether the observed changes in glucose transport activity induced by wortmannin were associated with changes 0.) in translocation of the glucose transporter, the cell surface 8- GLUT4 content was determined with the impermeant photo- 04 label ATB-BMPA. As shown in Fig. 7 concomitant adminis- 6- tration of wortmannin (10 nM or 1 ,M) reduced the level of GLUT4 at the cell surface from 2.79 ± 0.20 pmol/g (wet 0. muscle weight) observed in insulin-stimulated muscles to 1.15 a 4- ± 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 ,tM 2- wortmannin; P < 0.01, n = 5), whereas the translocation of -4 I GLUT4 induced by contraction was unaltered by wortmannin exposure [1.75 ± 0.16 pmol/g (wet muscle weight) vs. 1.52 ± ou i/ 0.16 pmol/g (wet muscle weight) (1 ,uM wortmannin; not 0 1 10 100 1000 Basal significant, n = 5)]. Administration ofwortmannin had also no Wortmannin, nM effect on basal GLUT4 cell surface content [0.44 ± 0.08 FIG. 6. Inhibition of insulin-stimulated glucose transport activity pmol/g (wet muscle weight) vs. 0.43 ± 0.07 pmol/g (wet by wortmannin. Rat soleus muscles were preincubated for 10 min with muscle weight) (1 ,uM wortmannin; not significant, n = 5)]. the indicated concentrations of wortmannin and then for a further 20 min with insulin at 1 mU/ml and wortmannin at the same concen- DISCUSSION tration as in the preincubation medium. o, Insulin-stimulated glucose uptake without addition of wortmannin; *, basal glucose uptake (no An intriguing finding of the present study is that contraction- insulin and wortmannin). Values are the mean ± SEM (n = 6 to 8). mediated increase in glucose uptake and GLUT4 translocation in skeletal muscle is not crucially dependent on a wortmannin- incubation with wortmannin, insulin-stimulated glucose up- sensitive signaling pathway in contrast to that of insulin. Our take was almost abolished (8.8 ± 0.5 ,umol per ml per h vs. 1.4 data therefore provide direct evidence that contraction stim- ± 0.1 ,mol per ml per h; P < 0.01) (Fig. 5). The latter level ulates glucose uptake and translocation of GLUT4 at steps was comparable to basal level, which was unaltered by wort- more distal than the wortmannin-sensitive molecules (e.g., the mannin exposure [1.3 ± 0.2 ,umol per ml per h (basal) and 1.2 PtdIns 3-kinases) in the insulin signaling pathway or by stim- ± 0.2 ,umol per ml per h (basal + 1 ,uM wortmannin)]. Fig. 6 ulation through an entirely different pathway. glucose uptake Wortmannin and the LY29002 compound inhibit a number shows the inhibition of insulin-stimulated by of protein kinases at higher concentrations but are thought to wortmannin (IC50 = 3.6 ± 1.6 nM). In contrast, wortmannin, be specific for PtdIns 3-kinases at lower concentrations (for even at a high concentration (1 ,uM), failed to affect the review, see ref. 30). Recent studies in cultured cell systems glucose uptake induced by contraction (4.4 ± 0.1 ,umol per ml suggest, however, that other signaling proteins may also be per h and 4.1 ± 0.2 ,umol per ml per h) (Fig. 5). subject to wortmannin inhibition at low doses (30). Studies in 3T3-L1 and CHO cells have shown that PtdIns 3-kinases may (kDa) (kDa) be key intermediates in the insulin action cascade leading to 10680 49 32 28 18 10680 49 32 28 18 glucose transport stimulation. Addition ofwortmannin (22, 23, 1000 25) and LY294002 (24) hinders insulin-stimulated glucose - . transport and translocation of GLUT4 in 3T3-L1 cells. Our 4) 800- B study in in vitro incubated rat soleus muscles confirms that 600- inhibition of wortmannin-sensitive signaling mechanisms re-

I. sults in a suppression of the insulin-stimulated glucose trans- 0.) 400 - port activity and translocation of GLUT4 and that wortmannin a 200- is a potent inhibitor of insulin-stimulated glucose transport

0- even in nanomolar concentrations (IC5o = 3.6 ± 1.6 nM) (23). -l:; ) 2 4 6 8 10121416 In contrast, the glucose uptake and the translocation of Q. GLUT4 mediated by contraction are not influenced by wort- 0 1000- - 9 mannin even at high concentrations (1 ,uM). 1000 , , .. In line with other reports (31, 32), we demonstrate that 800- C 800- D maximal insulin-stimulated glucose uptake is =40% higher 600- 600- than maximal contraction-stimulated uptake. Furthermore, 400- the data substantiate that contraction induces translocation of 400- the major glucose transporter of skeletal muscle (GLUT4) to 200- 200- the plasma membrane. In contrast to previous reports using 0- the subcellular fractionation technique on skeletal muscles 0 2 4 6 8 10 1214 16 0 2 4 6 8 10121416 (3-5, 8-10), the use of the impermeant photolabel ATB-

Slice Slice BMPA allows us to circumvent drawbacks normally encoun- tered with subcellular fractionation. These include difficulties FIG. 7. Effect of wortmannin on GLUT4 translocation detected in separating surface membranes from intracellular vesicles with ATB-BMPA. Muscle cells were incubated in insulin (1 mU/ml) containing GLUT4 and a poor recovery. Our results clearly or stimulated to contract at 7.5 Hz for two 5-minperiods with a 1-min indicate that the contraction-mediated translocation of the rest in between, and where indicated with addition of 10 nM or 1 ,uM GLUT4 transporters to the cell surface is closely correlated wortmannin (see Fig. 5). The labeled transporters were solubilized and with the glucose transport activity and accounts fully for the immunoprecipitated with anti-C-terminal peptide antibodies against increment in glucose uptake after contraction. GLUT4 and resolved by gel electrophoresis. The positions of molec- ular mass marker proteins are indicated. The results are from single The combined effect of contraction and maximal insulin representative experiments. (A) Insulin (1 mU/ml). (B) Insulin and stimulation on glucose uptake was almost additive in the in wortmannin at 1 ,uM (0) or 10 nM (*). (C) Contraction (7.5 Hz for vitro-incubated soleus muscle. Similar data on the additive two 5-min periods). (D) Contraction and wortmannin at 1 ,uM. effect of contraction and maximal insulin stimulation have Downloaded by guest on September 28, 2021 Biochemistry: Lund et aL Proc. Natl. Acad. Sci. USA 92 (1995) 5821 been reported in incubated muscles (11, 13, 14, 33). The failure 5. Fushiki, T., Wells, J. A., Tapscott, E. B. & Dohm, G. L. (1989) to find that the combined stimulation is completely additive Am. J. Physiol. 256, E580-E587. might be caused by some efflux of MeGlc occurring under the 6. Douen, A. G., Ramlal, T., Rastogi, S., Bilan, P. J., Cartee, G. D., conditions with very high rates of MeGlc transport, thereby Vranic, M., Holloszy, J. 0. & Klip, A. (1990) J. Biol. Chem. 265, resulting in a slight underestimation of the transport 13427-13430. glucose 7. Hirshman, M. F., Goodyear, L. J., Wardzala, L. J., Horton, E. D. activity (32). & Horton, E. S. (1990) J. Bio. Chem. 265, 987-991. The possibility that the effects of maximal insulin stimula- 8. King, P. A., Hirshman, M. F., Horton, E. D. & Horton, E. S. tion and contraction are additive on the translocation of (1989) Am. J. Physiol. 257, C1128-C1134. glucose transporters to the surface membrane has been ex- 9. Goodyear, L. J., King, P. A., Hirshman, M. F., Thompson, C. M., amined previously but with conflicting results. Some studies Horton, E. D. & Horton, E. S. (1990) Am. J. Physio!. 258, using subcellular fractionation techniques to isolate plasma E667-E672. membranes have led to the hypothesis that a combined stim- 10. Sternlicht, E., Barnard, R. J. & Grimditch, G. K. (1989) Am. J. ulation with contraction and insulin increases the intrinsic Physiol. 256, E227-E230. activity of the glucose transporters in the plasma membrane (9, 11. Wallberg-Henriksson, H., Constable, S. H., Young, D. A. & 16, 18). In contrast, a study (17) in which GLUT4 in membrane Holloszy, J. 0. (1988) J. Appl. Physiol. 65, 909-913. fractions was assessed by Western blot analysis suggests that 12. Ploug, T., Galbo, H., Vinten, J., Jorgensen, M. & Richter, E. A. the combined effects of insulin and contraction stimulation are (1987) Am. J. Physiol. 253, E12-E20. additive on GLUT4 translocation. 13. Zorzano, A., Balon, T. W., Goodman, M. N. & Ruderman, N. B. (1986) Am. J. Physiol. 251, E21-E26. By using the more sensitive surface labeling technique (20), 14. Nesher, R., Karl, I. E. & Kipnis, D. M. (1985)Am. J. Physiol. 249, the present results show that the combined stimulation with a C226-C232. maximal insulin level and contraction results in a further 15. Ploug, T., Wojtaszewski, J., Kristiansen, S., Hespel, P., Galbo, H. increase in labeled glucose transporters (GLUT4) on the cell & Richter, E. A. (1993) Am. J. Physiol. 264, E270-E278. surface to a level significantly higher than that mediated by 16. Brozinick, J. T., Jr., Etgen, G. J., Jr., Yaspelkis, B. B. & Ivy, J. L. insulin or contraction alone. Our studies therefore extend and (1994) Biochem. J. 297, 539-545. corroborate the findings obtained with the subcellular frac- 17. Gao, J., Ren, J., Gulve, E. A. & Holloszy, J. 0. (1994) J. Appl. tionation technique (17) that the combined stimulation is Physiol. 77, 1597-1601. near-additive on the translocation of glucose transporter. 18. Douen, A. G., Ramlal, T., Cartee, G. D. & Klip, A. (1990) FEBS The observation that maximal insulin stimulation and con- Lett. 261, 256-260. traction have additive effects on translocation of GLUT4 in 19. Wilson, C. M. & Cushman, S. W. (1992) Diabetes 40, 167. 20. Lund, S., Holman, G. D., Schmitz, 0. & Pedersen, 0. (1993) muscle might suggest the presence of two pools of glucose FEBS Let. 330, 312-318. transporters. One pool may be accessible for translocation via 21. Wilson, C. M. & Cushman, S. W. (1994) Biochem. J. 299, 755- insulin but not through exercise, whereas the other may be 759. available for translocation during exercise but not affected by 22. Okada, T., Kawano, Y., Sakakibara, T., Hazeki, 0. & Ui, M. insulin (6, 16). (1994) J. Biol. Chem. 269, 3568-3573. Another possibility could be that intracellular glucose trans- 23. Clarke, J. F., Young, P. W., Yonezawa, K., Kasuga, M. & Hol- porters form a more or less uniform pool that is maintained by man, G. D. (1994) Biochem. J. 300, 631-635. a balance between the and of GLUT4 24. Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J. (34, 35). According to this scenario, contraction may mainly & Kahn, C. R. (1994) Mol. Cell. Bio. 14, 4902-4911. reduce the rate of endocytosis of the GLUT4, in contrast to 25. Hara, K., Yonezawa, K., Sakaue, H., Ando, A., Kotani, K., insulin, which apparently accelerates the rate of exocytosis of Kitamura, T., Kitamura, Y., Ueda, H., Stephens, L., Jackson, the and to some extent T. R., Hawkins, P. T., Dhand, R., Clarke, A. E., Holman, G. D., glucose transporters (34) also reduces Waterfield, M. D. & Kasuga, M. (1994) Proc. Natl. Acad. Sci. the rate of endocytosis (36, 37). To our knowledge, there is no USA 91, 7415-7419. data to distinguish between these potential mechanisms but 26. Clark, A. E. & Holman, G. D. (1990) Biochem. J. 269, 615-622. identification of GLUT4 in several subcellular locations in 27. Wallberg-Henriksson, H. & Holloszy, J. 0. (1985)Am. J. Physiol. muscle, including the t-tubulus and subsarcolemma regions, 249, C233-C237. suggests the mechanism involving translocation from separate 28. Lund, S., Flyvbjerg, A., Holman, G. D., Larsen, F. S., Pedersen, pools is most likely. 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., We acknowledge J. Lyhne for construction of the pulse generator. Habberfield, A. D., Simpson, I. A. & Cushman, S. W. (1990) J. H. Petersen, M. M0ller, and E. Hornemann are thanked for excellent Bio. Chem. 265, 18172-18179. technical assistance. Prof. H. 0rskov is thanked for stimulating 30. Downward, J. (1994) Nature (London) 371, 378-379. discussions. This study was supported by grants from Institute of 31. Henriksen, E. J., Bourey, R. E., Rodnick, K. J., Koranyi, L., Experimental Clinical Research, University of Aarhus, Danish Med- Permutt, M. A. & Holloszy, J. 0. (1990) Am. J. Physiol. 259, ical Research Council, Danish Diabetes Association, Hafnia Fonden, E593-E598. Torben Frimodt og Alice Frimodts Fond, Else og Mongens Wedell- 32. Hansen, P. A., Gulve, E. A. & Holloszy, J. 0. (1994) J. Appl. 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