Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5817-5821, June 1995 Biochemistry Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin S. LUND*t, G. D. HOLMANt, 0. SCHMITZ*, AND 0. PEDERSEN§ *Medical Research Laboratory, Aarhus Kommunehospital and Medical Department M (Endocrinology and Diabetes), 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 glucose transporter 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 glucose uptake (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. Protein 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 proteins 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).