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• W2029722068 abstract "To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin−1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. Transfection was accomplished by electroporation using an hemagglutinin (HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4 alone, insulin results in an ≈7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of HA-GLUT4-trafficking parallel those of the native GLUT4 and in addition, that newly synthesized HA-GLUT4 goes to the plasma membrane before being sorted into the insulin-responsive compartments. Short term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. Under conditions of maximal expression of dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose transporters. In contrast, expression of dynamin wild type decreases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. These data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin. To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin−1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. Transfection was accomplished by electroporation using an hemagglutinin (HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4 alone, insulin results in an ≈7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of HA-GLUT4-trafficking parallel those of the native GLUT4 and in addition, that newly synthesized HA-GLUT4 goes to the plasma membrane before being sorted into the insulin-responsive compartments. Short term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. Under conditions of maximal expression of dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose transporters. In contrast, expression of dynamin wild type decreases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. These data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin. In adipose cells, GLUT4 glucose transporters are constantly recycling between an intracellular compartment and the plasma membrane (1Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar, 2Holman G.D. Lo Leggio L. Cushman S.W. J. Biol. Chem. 1994; 269: 17516-17524Abstract Full Text PDF PubMed Google Scholar, 3Slot J.W. Gueze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (712) Google Scholar, 4Robinson L.J. Pang S. Hairns D.A. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar). In the basal state, where the rate of exocytosis is relatively low, the vast majority of the GLUT4 glucose transporters reside in an as yet poorly characterized intracellular compartment (1Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar, 3Slot J.W. Gueze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (712) Google Scholar, 5Malide D. Dwyer N.K. Blanchette-Mackie E.J. Cushman S.W. J. Histochem. Cytochem. 1997; 45: 1083-1096Crossref PubMed Scopus (81) Google Scholar). Stimulation of adipose cells with insulin leads to an increase in the rate of exocytosis of GLUT4-containing vesicles, resulting in a rapid shift in the steady state distribution of GLUT4 to the plasma membrane (1Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar, 2Holman G.D. Lo Leggio L. Cushman S.W. J. Biol. Chem. 1994; 269: 17516-17524Abstract Full Text PDF PubMed Google Scholar, 3Slot J.W. Gueze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (712) Google Scholar). After clearance of the hormone, the rate of GLUT4 exocytosis decreases and the steady state distribution shifts back to the intracellular compartment. The primary focus of recent investigations has been the identification and characterization of signaling molecules (e.g. p85/p110 phosphatidylinositol 3-kinase) and other cellular components (e.g. soluble NSF attachment protein receptors (SNAREs)) possibly involved in the regulated exocytosis of GLUT4 (6Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Ido Y. Ueda H. Stephens L. Jackson T. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (418) Google Scholar, 7Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 8Kotani K. Hara H. Kotani K. Yonezawa K. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 209: 343-348Crossref PubMed Scopus (144) Google Scholar, 9Timmers K.I. Clark A.E. Omatsu-Kanbe M Whiteheart S.W. Bennett M.K. Holman G.D. Cushman S.W. Biochem. J. 1996; 320: 429-436Crossref PubMed Scopus (55) Google Scholar, 10Cheatham B. Volchuk A. Kahn C.R. Wang L. Rhodes C.J. Klip A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15169-15173Crossref PubMed Scopus (162) Google Scholar, 11Olson A.L. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (209) Google Scholar). However, little is known about the mechanism of GLUT4 endocytosis. Previous reports provided indirect evidence that GLUT4 might be internalized by a mechanism involving clathrin-mediated endocytosis. Potassium depletion, known to disrupt formation of clathrin-coated vesicles (12Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G.W. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar), results in a decreased internalization of GLUT4 and mannose-6-phosphate receptors in rat adipose cells (13Nishimura H. Zarnowski M.J. Simpson I.A. J. Biol. Chem. 1993; 268: 19246-19253Abstract Full Text PDF PubMed Google Scholar). In 3T3-L1 adipocytes, GLUT4 has been shown to co-purify with clathrin-coated vesicles derived from the plasma membrane after treatment of the cells with the fungal toxin brefeldin A (14Chakrabarti R. Buxton J. Joly M. Corvera S. J. Biol. Chem. 1994; 269: 7926-7933Abstract Full Text PDF PubMed Google Scholar). Previous morphological analysis showed association of GLUT4 with clathrin-coated pits (4Robinson L.J. Pang S. Hairns D.A. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar), whereas little co-localization of GLUT4 with clathrin is observed in a recent study from our laboratory (5Malide D. Dwyer N.K. Blanchette-Mackie E.J. Cushman S.W. J. Histochem. Cytochem. 1997; 45: 1083-1096Crossref PubMed Scopus (81) Google Scholar). Since no functional studies involving components of clathrin-mediated endocytosis in insulin target cells have been reported, the mechanism of GLUT4 internalization still remains unclear. The dynamins belong to a family of 100-kDa GTPases that mediate the initial stages of endocytosis (15Obar R.A. Collins C.A. Hammarback J.A. Shpetner H.S. Vallee R.B. Nature. 1990; 347: 256-261Crossref PubMed Scopus (286) Google Scholar, 16Shpetner H.S. Vallee R.B. Nature. 1992; 355: 733-735Crossref PubMed Scopus (171) Google Scholar, 17Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 18van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.J. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (589) Google Scholar). To date, three mammalian dynamin genes (referred as dynamin−1 to dynamin−3) have been described (15Obar R.A. Collins C.A. Hammarback J.A. Shpetner H.S. Vallee R.B. Nature. 1990; 347: 256-261Crossref PubMed Scopus (286) Google Scholar, 19Cook T.A. Urrutia R. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 644-648Crossref PubMed Scopus (163) Google Scholar, 20Sontag J.-M. Fyske E.M. Ushkaryov Y. Liu J.-P. Robinson P.J. Südhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar, 21Nakata T. Takemura R. Hirokawa N. J. Cell Sci. 1993; 105: 1-5Crossref PubMed Google Scholar). Dynamin−1 is found in neurons, dynamin−2 is expressed ubiquitously, and dynamin−3 is enriched in testis (15Obar R.A. Collins C.A. Hammarback J.A. Shpetner H.S. Vallee R.B. Nature. 1990; 347: 256-261Crossref PubMed Scopus (286) Google Scholar,19Cook T.A. Urrutia R. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 644-648Crossref PubMed Scopus (163) Google Scholar, 20Sontag J.-M. Fyske E.M. Ushkaryov Y. Liu J.-P. Robinson P.J. Südhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar, 21Nakata T. Takemura R. Hirokawa N. J. Cell Sci. 1993; 105: 1-5Crossref PubMed Google Scholar). Although the distinct functions of these different dynamin proteins are not fully understood, considerable evidence now indicates that dynamin−1 participates in clathrin-mediated endocytosis. Dynamin−1 colocalizes with clathrin in intact cells on the light and electron microscopy levels (22Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar, 23Shpetner H.S. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and binds α-adaptin, a component of clathrin-coated pits, in vitro (24Wang L.H. Südhof T.C. Anderson R.G. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Furthermore, transfection of cultured mammalian cells with dominant-negative dynamin−1 mutants results in the accumulation of clathrin-coated pits at the plasma membrane (18van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.J. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (589) Google Scholar, 22Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar), whereas internalization of transferrin receptors, epidermal growth factor receptors, and β2-adrenergic receptors is inhibited (17Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 18van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.J. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (589) Google Scholar, 22Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar, 25Zhang J. Ferguson S.S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Even though a role of dynamin−2 in endocytosis remains unclear, a recent report suggests that dynamin−2 might also be localized to coated pits on the plasma membrane (26Warnock D.E. Baba T. Schmid S.L. Mol. Biol. Cell. 1997; 8: 2553-2562Crossref PubMed Scopus (92) Google Scholar). Thus, considering the high degree of amino acid sequence homology between dynamin−1 and dynamin−2 and the ability of dynamin−1 mutants to inhibit receptor-mediated endocytosis even in nonneuronal cells, both isoforms might act as functional homologues in endocytosis in nonneuronal cells (17Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 18van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.J. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (589) Google Scholar, 22Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar, 25Zhang J. Ferguson S.S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). However, the finding that dynamin−2 localizes to vesicles in the Golgi complex (27Henley J.R. McNiven M.A. J. Cell Biol. 1996; 133: 761-775Crossref PubMed Scopus (108) Google Scholar, 28Maier O. Knoblish M. Westermann P. Biochem. Biophys. Res. Commun. 1996; 223: 229-233Crossref PubMed Scopus (49) Google Scholar, 29Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (274) Google Scholar) implies additional functions of this isoform as well. To characterize the mechanism of GLUT4 endocytosis, we have overexpressed a dominant-negative mutant of dynamin−1 in isolated rat adipose cells. The effects of dynamin−1 on GLUT4- trafficking in vivo were monitored by utilizing a co-transfected recombinant GLUT4 containing an HA 1The abbreviations used are: HA, hemagglutinin; TGN, trans-Golgi network. epitope tag in the first exofacial loop (30Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar). All constructs were generated in the pCIS2 mammalian expression vector (a generous gift from Dr. C. Gorman). cDNAs for HA epitope-tagged dominant-negative K44A dynamin−1 and HA-tagged wild-type dynamin−1 (a generous gift from Drs. H. Damke and S. L. Schmid) were subcloned into the expression vector. Construction of the HA-tagged GLUT4 has been described previously (30Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar). For transfection experiments, the plasmids were purified in mg quantities using a maxiprep kit (Qiagen). Preparation of isolated rat epididymal adipose cells from male rats (CD strain, Charles River Breeding Laboratories, Inc.) was performed as described previously (31Weber T.M. Joost H.G. Simpson I.A. Cushman S.W. Kahn C.R. Harrison I.C. The Insulin Receptor. II. Alan R. Liss, Inc., New York1988: 171-187Google Scholar). Isolated cells were washed twice with Dulbecco's modified Eagle's medium containing 25 mm glucose, 25 mm HEPES, 4 mml-glutamine, 200 nm(−)-N6-(2-phenylisopropyl)-adenosine, and 75 μg/ml gentamycin, and resuspended to a cytocrit of 40% (≈5–6 × 106 cells/ml). 200 μl of the cell suspension were added to 200 μl of Dulbecco's modified Eagle's medium containing 100 μg of carrier DNA (sheared herring sperm DNA; Boehringer Mannheim) and expression plasmids as indicated. The total concentration of plasmid DNA in each cuvette was adjusted to 5 μg/cuvette with empty pCIS2. Electroporation was carried out in 0.4-cm gap-width cuvettes (Bio-Rad) using a T810 square wave pulse generator (BTX). After applying three pulses (12 ms, 200 V), the cells were washed once in Dulbecco's modified Eagle's medium, pooled in groups of 4–10 cuvettes, and cultured at 37 °C, 5% CO2in Dulbecco's modified Eagle's medium containing 3.5% bovine serum albumin. Rat adipose cells were harvested 3.5 or 20–24 h post-transfection and washed in Krebs-Ringer bicarbonate HEPES buffer, pH 7.4, 200 nm adenosine (KRBH buffer) containing 5% bovine serum albumin. Samples corresponding to the cells from one cuvette were distributed into 1.5-ml microcentrifuge tubes. After stimulation with 67 nm (1 × 104 microunits/ml) insulin for 30 min at 37 °C, subcellular trafficking of GLUT4 was stopped by the addition of 2 mm KCN (32Kono T. Suzuki K. Dansey L.E. Robinson F.W. Blevins T.L. J. Biol. Chem. 1981; 256: 6400-6407Abstract Full Text PDF PubMed Google Scholar). All of the following steps were performed at room temperature. A monoclonal anti-HA antibody (HA.11, Berkeley Antibody Co.) was added at a dilution of 1:1000, and the cells were incubated for 1 h. Excess antibody was removed by washing the cells three times with KRBH, 5% bovine serum albumin. Then 0.1 μCi of 125I-sheep anti-mouse antibody (Amersham Pharmacia Biotech) was added to each reaction, and the cells were incubated for 1 h. Finally, the cells were spun through dinonylphtalate oil to remove the unbound antibody (31Weber T.M. Joost H.G. Simpson I.A. Cushman S.W. Kahn C.R. Harrison I.C. The Insulin Receptor. II. Alan R. Liss, Inc., New York1988: 171-187Google Scholar), and the cell surface-associated radioactivity was counted in a γ-counter. The resulting counts were normalized to the lipid weight of the samples (31Weber T.M. Joost H.G. Simpson I.A. Cushman S.W. Kahn C.R. Harrison I.C. The Insulin Receptor. II. Alan R. Liss, Inc., New York1988: 171-187Google Scholar). Unless stated otherwise, the values obtained for pCIS-transfected cells were subtracted from all other values to correct for nonspecific antibody binding. Antibody binding assays were performed in duplicate or quadruplicate. To increase the insulin response of rat adipose cells transfected with epitope-tagged GLUT4 above that observed with the original technique (30Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar), we tested the experimental procedure as follows. Rat adipose cells were transfected with various amounts of HA-GLUT4 expression plasmid and analyzed at different time points for basal and insulin-stimulated cell surface HA-GLUT4 using the anti-HA antibody binding assay. The tagged glucose transporters become detectable at the cell surface in response to insulin as early as 2 h post-transfection. Synthesis of HA-GLUT4 continues until about 16 h post-transfection, at which time its level stays relatively constant and maximal to 24 h post-transfection (data not shown). As judged by immunohistochemistry using the same monoclonal anti-HA antibody and nonpermeabilized, insulin-stimulated cells, the electroporation procedure yields ≈10% HA-positive cells (Ref. 33Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar and data not shown). As shown in Fig. 1, after culturing the transfected rat adipose cells for 3.5 h, acute (30 min) insulin typically resulted in a 7–10-fold increase in cell surface anti-HA antibody binding after correction for transfection with the empty vector alone (Fig. 1 A). Prolonged expression of the tagged GLUT4 (24 h) resulted in higher amounts of HA-GLUT4 at the plasma membrane in both the basal and insulin-stimulated states (Fig. 1 B). Compared with 3.5 h of expression, the amount of insulin-stimulated cell surface HA-GLUT4 with 24 h of expression was increased about 4–5-fold. However, because the -fold increase in basal cell surface HA-GLUT4 from 3.5 to 24 h of expression was greater than that observed in the insulin-stimulated state, insulin stimulation resulted only in a 2–3-fold increase in cell surface glucose transporters. As a compromise between signal strength and insulin response, we selected transfection using 0.5 μg of plasmid/cuvette and 3.5 h of protein expression as our standard conditions. To determine the extent of overexpression of GLUT4, total membrane fractions from adipose cells transfected with 0.1 to 5 μg of HA-GLUT4 plasmid/cuvette and cultured for 24 h were analyzed by Western blotting (data not shown). Both endogenous and recombinant GLUT4 were detected using the same polyclonal antibody against the C terminus of GLUT4. Quantitation of the blots shows that the amount of GLUT4 was increased by 1.1–2-fold compared with pCIS-transfected cells. Thus, taking into account that only ≈10% of the cells are transfected (33Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar), overexpression of GLUT4 in transfected cells ranged from <2- to 10-fold of endogenous GLUT4 levels, depending on the amount of expression plasmid used. To study the effects of dynamin overexpression on the subcellular distribution of epitope-tagged GLUT4, rat adipose cells were co-transfected with HA-GLUT4 and various concentrations of expression plasmids for wild-type and mutant dynamin. Fig. 2 illustrates the data when protein expression is carried out for 3.5 and 20 h. At 3.5 h post-transfection in dynamin-K44A-transfected cells, the basal cell surface level of HA-GLUT4 was increased as much as 2.6 ± 0.1-fold (mean ± S.D.) compared with cells transfected with HA-GLUT4 alone (Fig. 2 A). Likewise, a concomitant increase in cell surface HA-GLUT4 in the insulin-stimulated state was observed. (Fig. 2 A). In contrast, expression of wild-type dynamin decreased the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states to 51 ± 4 and 58 ± 13% that of the controls, respectively. After 20 h of expression of the dynamin mutant, the basal level of cell surface HA-GLUT4 equaled that for the insulin-stimulated state (Fig. 2 B). In addition, the absolute amount of cell surface HA-GLUT4 was increased by about 30–40% (37 ± 1% and 32 ± 12% in the absence and presence of insulin, respectively) compared with the insulin-stimulated control. Prolonged overexpression (20 h) of the wild-type dynamin decreased the amount of cell surface HA-GLUT4 in the insulin-stimulated state compared with both the control cells and the cells transfected with mutant dynamin and decreased the basal level compared with cells transfected with mutant dynamin (Fig. 2 B), as shown for 3.5 h of expression (Fig. 2 A). To verify that expression of the dynamins affects the endocytosis of GLUT4 in rat adipose cells, we analyzed the redistribution of cell surface HA-GLUT4 in the presence of the fungal metabolite wortmannin (7Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 34Kanai F. Ito K. Takoda M. Hayashi H. Kamohara S. Ishii K. Okada T. Hazeki O Ui M. Ebina Y. Biochem. Biophys. Res. Commun. 1993; 195: 762-768Crossref PubMed Scopus (266) Google Scholar, 35Clarke J.F. Young P.W. Yonezawa K. Kasuga M. Holman G.D. Biochem. J. 1994; 300: 631-635Crossref PubMed Scopus (334) Google Scholar). Acting as an inhibitor of the lipid kinase phosphatidylinositol 3-kinase (36Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N.F. Hsuan J.J. Courtneige S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (540) Google Scholar), wortmannin blocked the insulin-stimulated translocation of GLUT4 from its basal compartment to the plasma membrane (7Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 35Clarke J.F. Young P.W. Yonezawa K. Kasuga M. Holman G.D. Biochem. J. 1994; 300: 631-635Crossref PubMed Scopus (334) Google Scholar, 37Malide D. Cushman S.W. J. Cell Sci. 1997; 110: 2795-2806Crossref PubMed Google Scholar). When added simultaneously with insulin, wortmannin (100 nm) also blocked translocation of epitope-tagged HA-GLUT4 to the cell surface in transfected cells (data not shown). Previously, Holman and co-workers (38Yang J. Clarke J.F. Ester C.J. Young P.W. Kasuga M. Holman G.D. Biochem. J. 1996; 313: 125-132Crossref PubMed Scopus (104) Google Scholar) and a study from our laboratory (39Malide D. St-Denis J.F. Keller S.R. Cushman S.W. FEBS Lett. 1997; 409: 461-468Crossref PubMed Scopus (48) Google Scholar) showed that this inhibition of GLUT4 translocation does not affect the early steps of GLUT4 endocytosis. Thus, the addition of wortmannin to insulin-stimulated cells inhibited further translocation of GLUT4 from its intracellular compartment to the plasma membrane, thereby allowing measurements of the kinetics of GLUT4 endocytosis. Fig. 3demonstrates the results of such wortmannin experiments. In insulin-stimulated rat adipose cells transfected with HA-GLUT4 alone, the amount of recombinant glucose transporters present on the cell surface increased over time (Fig. 3 A). A similar time course of expression of luciferase activity was observed when cells were transfected with a pCIS2-luciferase construct (40Quon M.J. Chen H. Lin C.H. Zhou L.X. Ing B.L. Zarnowski M.J. Klinghoffer R. Kazlauskas A. Cushman S.W. Taylor S.I. Biochem. Biophys. Res. Commun. 1996; 226: 587-594Crossref PubMed Scopus (30) Google Scholar) under the same conditions (data not shown). Fig. 4illustrates that the addition of the protein synthesis inhibitor cycloheximide together with insulin prevents the increase of cell surface HA-GLUT4 with a lag time typical of the action of this reagent. A similar inhibitory effect of cycloheximide on cell surface HA-GLUT4 was observed in basal cells (data not shown). Thus, the increase of cell surface HA-GLUT4 with time apparently reflects the ongoing synthesis of recombinant glucose transporters during the assay. Nonetheless, addition of wortmannin to insulin-stimulated cells led to a decrease in cell surface HA-GLUT4 with a time course similar to that observed with native GLUT4 as previously reported (Fig. 3 B) (1Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar). Insulin-stimulated cells transfected with wild-type dynamin showed a time course of HA-GLUT4 clearance from the plasma membrane after wortmannin treatment that was similar to that observed in cells transfected with HA-GLUT4 alone (Fig. 3 D). However, cell surface HA-GLUT4 levels in dynamin-K44A cells were not decreased by the addition of wortmannin after insulin but continued to increase in the presence of the inhibitor (Fig. 3 C). Fig. 3 Eshows the time course data corrected for the synthesis of HA-GLUT4 during the assay. The addition of wortmannin to insulin-stimulated cells expressing dynamin-K44A did not change the cell surface level of the tagged glucose transporters; thus, all of the HA-GLUT4 appears to remain on the cell surface. Evidently the protein synthesis-associated increase in cell surface glucose transporters in rat adipose cells utilizes a wortmannin-insensitive trafficking pathway. As shown in Fig. 3 C, newly synthesized HA-GLUT4 still appeared on the cell surface in the presence of mutant dynamin and wortmannin. Under these conditions, the endocytosis of GLUT4 was inhibited by the dynamin mutant, leading to an accumulation of glucose transporters in the plasma membrane (Fig. 3 E). Likewise, the translocation of GLUT4 from the intracellular pool to the plasma membrane was inhibited by wortmannin. To further investigate the site at which the newly synthesized glucose transporters enter their recycling compartments, we studied the effects of wortmannin on the cell surface level of HA-GLUT4 under basal conditions. To increase the antibody binding signal, the incubation time with wortmannin was extended to 2 h. The results are illustrated in Fig. 5. In HA-GLUT4-transfected basal rat adipose cells, the amount of recombinant glucose transporter present on the cell surface increased 2.5-fold over 2 h of cell culture (Fig. 5 A). Similarly, a 3.5-fold increase in basal HA-GLUT4 levels was observed in dynamin-K44A-transfected cells (Fig. 5 B). In insulin-stimulated cells expressing HA-GLUT4 only, cell surface glucose transporters doubled within 2 h of incubation (Fig. 5 C). The addition of wortmannin to the latter cells leads to a decrease in cell surface HA-GLUT4 as described before (Fig. 3). In contrast, the addition of wortmannin to basal cells did not affect the protein synthesis-dependent increase in cell surface HA-GLUT4 either in the absence or presence of dynamin-K44A expression (Fig. 5, A and B, respectively). Thus, whereas wortmannin blocked GLUT4 translocation from the intracellular compartment to the plasma membrane, it had no effect on the observed protein synthesis-associated increase in cell surface glucose transporters. To study the subcellular trafficking of GLUT4 in an insulin target cell, we have transfected rat adipose cells with a recombinant glucose transporter containing an HA epitope tag in the first exofacial loop (30Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar). The HA-GLUT4 was detected on the cell surface of transfected cells by the binding of an antibody against the HA epitope. The observed insulin response of HA-GLUT4 translocation to the plasma membrane is markedly reduced when the amount of expression plasmid is increased and/or the time period of protein expression is extended (the latter reflecting the experimental conditions as described in the original protocol; cf. Ref. 30Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar). Thus, the magnitude of the insulin response is a function of the total amount of GLUT4 pr" @default.
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