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• W2058061580 abstract "To identify potential proteins interacting with the insulin-responsive glucose transporter (GLUT4), we generated fusion proteins of glutathione S-transferase (GST) and the final 30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of these carboxyl-terminal fusion proteins with adipocyte cell extracts revealed a specific interaction of GLUT4 with fructose 1,6-bisphosphate aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in intact 3T3L1 adipocytes, which decreased following insulin stimulation. Introduction into permeabilized 3T3L1 adipocytes of fructose 1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents that disrupt the interaction between aldolase and actin, inhibited insulin-stimulated GLUT4 exocytosis without affecting GLUT4 endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These data suggest that aldolase functions as a scaffolding protein for GLUT4 and that glucose metabolism may provide a negative feedback signal for the regulation of glucose transport by insulin. To identify potential proteins interacting with the insulin-responsive glucose transporter (GLUT4), we generated fusion proteins of glutathione S-transferase (GST) and the final 30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of these carboxyl-terminal fusion proteins with adipocyte cell extracts revealed a specific interaction of GLUT4 with fructose 1,6-bisphosphate aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in intact 3T3L1 adipocytes, which decreased following insulin stimulation. Introduction into permeabilized 3T3L1 adipocytes of fructose 1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents that disrupt the interaction between aldolase and actin, inhibited insulin-stimulated GLUT4 exocytosis without affecting GLUT4 endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These data suggest that aldolase functions as a scaffolding protein for GLUT4 and that glucose metabolism may provide a negative feedback signal for the regulation of glucose transport by insulin. The insulin-responsive glucose transporter GLUT4 is expressed primarily in adipose tissue, skeletal, and cardiac muscle (1Birnbaum M.J. Cell. 1989; 57: 305-315Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 2Charron M.J. Brosius F.C.d. Alper S.L. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2535-2539Crossref PubMed Scopus (327) Google Scholar, 3James D.E. Strube M. Mueckler M. Nature. 1989; 338: 83-87Crossref PubMed Scopus (667) Google Scholar, 4Fukumoto H. Kayano T. Buse J.B. Edwards Y. Pilch P.F. Bell G.I. Seino S. J. Biol. Chem. 1989; 264: 7776-7779Abstract Full Text PDF PubMed Google Scholar). Under basal conditions, GLUT4 slowly recycles between poorly defined intracellular compartments and the plasma membrane with the vast majority sequestered in these intracellular storage sites. Insulin stimulates a large increase in the rate of GLUT4 exocytosis concomitant with a smaller decrease in the rate of GLUT4 endocytosis (5Jhun B.H. Rampal A.L. Liu H. Lachaal M. Jung C.Y. J. Biol. Chem. 1992; 267: 17710-17715Abstract Full Text PDF PubMed Google Scholar, 6Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 7Czech M.P. Buxton J.M. J. Biol. Chem. 1993; 268: 9187-9190Abstract Full Text PDF PubMed Google Scholar). The overall insulin-induced changes in GLUT4 trafficking kinetics result in a 10–20-fold increase in the number of cell surface GLUT4 proteins that accounts for the majority of insulin-stimulated increases in glucose transport activity (8Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, 9Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Recently, several laboratories have begun to examine the subcellular distribution of GLUT4 to identify the mechanism responsible for the intracellular sequestration of the GLUT4 protein. Steady-state and kinetic analysis of various expressed GLUT4 chimeric proteins have indicated that both the amino- and carboxyl-terminal domains are important in GLUT4 internalization from the plasma membrane (10Marshall B.A. Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 1993; 268: 26193-26199Abstract Full Text PDF PubMed Google Scholar, 11Piper R.C. Tai C. Kulesza P. Pang S. Warnock D. Baenziger J. Slot J.W. Geuze H.J. Puri C. James D.E. J. Cell Biol. 1993; 121: 1221-1232Crossref PubMed Scopus (97) Google Scholar, 12Verhey K.J. Hausdorff S.F. Birnbaum M.J. J. Cell Biol. 1993; 123: 137-147Crossref PubMed Scopus (65) Google Scholar, 13Czech M.P. Chawla A. Woon C.W. Buxton J. Armoni M. Tang W. Joly M. Corvera S. J. Cell Biol. 1993; 123: 127-135Crossref PubMed Scopus (70) Google Scholar, 14Garippa R.J. Judge T.W. James D.E. McGraw T.E. J. Cell Biol. 1994; 124: 705-715Crossref PubMed Scopus (69) Google Scholar, 15Garippa R.J. Johnson A. Park J. Petrush R.L. McGraw T.E. J. Biol. Chem. 1996; 271: 20660-20668Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In particular, the carboxyl-terminal dileucine motif (SLL) was found to substantially alter GLUT4 trafficking kinetics and steady-state localization (16Corvera S. Chawla A. Chakrabarti R. Joly M. Buxton J. Czech M.P. J. Cell Biol. 1994; 126: 979-989Crossref PubMed Scopus (103) Google Scholar, 17Verhey K.J. Birnbaum M.J. J. Biol. Chem. 1994; 269: 2353-2356Abstract Full Text PDF PubMed Google Scholar). Although a specific GLUT4 sequence responsible for intracellular localization has yet to be identified, the presence of a carboxyl-terminal retention signal is consistent with the observation that expression of the GLUT4 carboxyl-terminal domain results in the translocation of the endogenous GLUT4 protein to the plasma membrane (18Lee W. Jung C.Y. J. Biol. Chem. 1997; 272: 21427-21431Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In addition, the presence of a GLUT4 carboxyl-terminal binding protein can also account for the apparent increase in GLUT4 carboxyl-terminal antibody immunoreactivity following insulin stimulation (19Smith R.M. Charron M.J. Shah N. Lodish H.F. Jarett L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6893-6897Crossref PubMed Scopus (134) Google Scholar, 20Wang W. Hansen P.A. Marshall B.A. Holloszy J.O. Mueckler M. J. Cell Biol. 1996; 135: 415-430Crossref PubMed Scopus (99) Google Scholar). To address these issues, we have used GST fusion proteins to identify and characterize GLUT4 carboxyl-terminal specific binding proteins. In this manuscript, we demonstrate that the carboxyl-terminal domain of GLUT4 can specifically associate with the bifunctional glycolytic enzyme fructose-1,6-bisphosphate aldolase. This in vitrointeraction appears to be physiologically important as glycolytic intermediates that disrupt aldolase-actin interactions also inhibit insulin-stimulated GLUT4 translocation. DNA fragments encoding the carboxyl-terminal sequences of GLUT1 (G1), IASGFRQGGASQSAKTPEELPHPLGADSQV; GLUT4 (G4), SATFRRTPSLLEQEVKPSTELEYLGPDEND; and GLUT4* (G4*), SATFRRTPSASEQEVKPSTELEYLGPDEND were provided by Dr. Morris Birnbaum and subcloned into pGEX-KG vector. The resultant glutathioneS-transferase (GST) 1The abbreviations used are: GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; 2DG, 2-deoxyglucose; MBP, maltose-binding protein; FBP, fructose 1,6-bisphosphate; G3P, glyceraldehyde 3-phosphate; SLO, Streptolysin-O. fusion proteins were purified by glutathione affinity chromatography as described by the manufacturer (Amersham Pharmacia Biotech). 3T3L1 fibroblasts were differentiated into adipocytes as described (21Olson A.L. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (206) Google Scholar). In labeling experiments, 3T3L1 adipocytes were incubated with 10 μCi/ml Tran35S label (ICN) for 24 h before isolation of cell extracts in 50 mm Hepes, pH 7.4, 1% Triton X-100, 10% glycerol. Insoluble material was separated by microcentrifugation and extracts precleared by incubation with glutathione-Sepharose beads for 1 h at 48 °C followed by a second 1 h incubation with the GST fusion protein. The beads were then washed three times with 50 mm Hepes, pH 7.4, 0.1% Triton-100, 10% glycerol, bound proteins eluted using Laemmli sample buffer and samples subjected to SDS-PAGE, and autoradiography or aldolase immunoblotting. For large scale preparation of the GST-GLUT4 binding proteins, rat epididymal fat pads were isolated from 50 rats. Cell extracts were incubated with the GST fusion proteins, eluants transferred onto polyvinylidene difluoride membrane and N-terminal sequences determined using an automated protein sequencer at the University of Michigan Protein Core Facility. Immunoblotting was performed as described (22Kao A.W. Ceresa B.P. Santeler S.R. Pessin J.E. J. Biol. Chem. 1998; 273: 25450-25457Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) using an aldolase antibody (kindly provided by Dr. Jurgen Bereiter-Hahn), IAO2 GLUT4-specific antibody (22Kao A.W. Ceresa B.P. Santeler S.R. Pessin J.E. J. Biol. Chem. 1998; 273: 25450-25457Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) or clathrin heavy chain antibody (Transduction Laboratories). Aldolase and GST fusion protein solutions were dialyzed against 10 mm imidazole, 10 mm KCl, 1 mmMgCl2, 0.1 mm dithiothreitol, pH 6.8, overnight at 48 °C and precleared by centrifugation at 45,000 rpm (TLA100 rotor, Beckman). 200 μg of rabbit skeletal muscle F-actin (Sigma) purified as described (23Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (959) Google Scholar) and 100 μl of the 20-μl aldolase solution were mixed for 30 min at room temperature following 5–10 strokes in a pestle homogenizer. The mixture was centrifuged and 300 μg/ml GST fusion protein was added to the pellet and suspended with a small plastic homogenizer rod. After incubation for 30 min at room temperature, the mixture was again centrifuged, and the supernatants and pellets subjected to SDS-PAGE. Proteins were detected by Coomassie Brilliant Blue staining. Fructose 1,6-bisphosphate (FBP) or glyceraldehyde 3-phosphate (G3P) were added to both the aldolase and the GST fusion proteins at a concentration of 50 μm as indicated. 3T3L1 adipocytes on glass coverslips were serum-starved for 3 h then left untreated or stimulated for 30 min with 100 nminsulin. Cells were fixed and permeabilized with 2% paraformaldehyde, 0.2% Triton X-100 in phosphate-buffered saline for 10 min and quenched in 100 mm glutamine/phosphate-buffered saline for 15 min. Cells were then washed and sequentially incubated in 5% donkey serum, 1% bovine serum albumin/phosphate-buffered saline blocking buffer, 1:200 IA02 anti-GLUT4 and 1:100 anti-aldolase (Biogenesis), and 12.5 μg/ml FITC-conjugated donkey anti-rabbit and 12.5 μg/ml Texas Red-conjugated donkey anti-goat antibodies (Jackson ImmunoResearch Laboratories). Coverslips were mounted onto glass slides with Vectashield mounting medium (Vector Labs) and viewed with a Bio-Rad laser confocal. Plasma membrane sheets were prepared from 3T3L1 adipocytes as described (24Chen D. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). For 2-deoxyglucose (2DG) treatment of intact 3T3L1 adipocytes, cells were serum starved and placed into glucose-free Dulbecco's modified Eagle's medium containing 5 mm pyruvate and 5 mm glutamine with or without 5 mm 2DG for 5 min before insulin stimulation. For fluorescence microscopy, the membrane sheets were fixed with 2% formaldehyde and sequentially incubated with 5% donkey serum, a 1:100 dilution of a rabbit anti-insulin regulatable glucose transporter antibody (East Acres) and 6.25 μg/ml of LRSC-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories). For immunoblotting experiments, cells were stimulated with 20 nm insulin for 20 min, membrane sheets were isolated and solubilized in a modified lysis buffer (22Kao A.W. Ceresa B.P. Santeler S.R. Pessin J.E. J. Biol. Chem. 1998; 273: 25450-25457Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) with 0.05% Triton X-100. A protein assay was performed, and lysates were placed into Laemmli sample buffer without boiling. The 3T3L1 adipocytes were permeabilized with Streptolysin-O (SLO; Murex Diagnostics Limited) as described (25Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (254) Google Scholar) with minor modifications. Briefly, adipocytes were washed 3 times with an intracellular buffer (20 mm HEPES, pH 7.2, 140 mml-glutamic acid, 5 mm EGTA, 7.5 mm MgCl2, 5 mm NaCl, 2 mm CaCl2) and then incubated for 5 min at 378 °C in intracellular buffer with 0.8 IU/ml Streptolysin-O and 10 mm MgATP. For immunofluorescence studies, cells were washed in buffer (10 mm MgATP, 1 mm dithiothreitol, and 0.1% bovine serum albumin) and treated for 5 min with either 5 mm 2DG, 25 mmFBP, or other aldolase substrates as indicated before insulin stimulation. 3T3L1 adipocytes were placed into L-15 medium and microinjected using an Eppendorf Micromanipulator 5171 and Transjector 5246. Injection buffer consisted of 100 mm KCl and 5 mm sodium phosphate, pH 7.2, with 1.0 mg/ml FITC-dextran (Sigma), 1.2 mg/ml maltose-binding protein (MBP)-Ras CAAX, and 2.5 mg/ml either aldolase antibody (Biogenesis) or pre-immune serum (Research Genetics). Microinjected cells were placed in serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin for 1.5 h, treated with or without 20 nm insulin for 20 min, and plasma membrane sheets isolated. Sheets were incubated with a 1:1,000 dilution of sheep anti-MBP antibody (generously provided by Dr. Morris Birnbaum) and 1:400 dilution of IAO2 GLUT4 antibody, followed by incubation in 12.5 mg/ml FITC-conjugated donkey anti-sheep and Cy5-conjugated donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories). In each experiment, 20–40 injected cells positive for FITC-MBP were scored for GLUT4-specific immunofluorescence by two individuals blinded to treatment. The results of two or three independent experiments were analyzed using the analysis program InStat 2.0. To identify proteins that interact with the GLUT4 carboxyl-terminal domain, we expressed the GLUT1 and GLUT4 carboxyl-terminal 30 amino acids as GST fusions, GST-G1 and GST-G4, respectively. An additional fusion construct of the GLUT4 carboxyl terminus was generated containing a substitution of two residues shown to be important in GLUT4 endocytosis, leucines 488 and 489, with alanine and serine (GST-G4*), respectively (15Garippa R.J. Johnson A. Park J. Petrush R.L. McGraw T.E. J. Biol. Chem. 1996; 271: 20660-20668Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Corvera S. Chawla A. Chakrabarti R. Joly M. Buxton J. Czech M.P. J. Cell Biol. 1994; 126: 979-989Crossref PubMed Scopus (103) Google Scholar, 17Verhey K.J. Birnbaum M.J. J. Biol. Chem. 1994; 269: 2353-2356Abstract Full Text PDF PubMed Google Scholar). Incubation of GST and GST-G4 with extracts from [35S]methionine-labeled 3T3L1 adipocytes resulted in the binding of multiple proteins. However, there were two faint bands at 40 and 43 kDa that appeared to specifically precipitate with GST-G4 and GST-G4* but not with GST or GST-G1 (Fig. 1 A). Pretreatment of 3T3L1 adipocytes with insulin before isolation of the cell extracts had no significant effect on the in vitro association of the 40 and 43 kDa protein with GST-G4 and GST-G4* (Fig. 1 A). Cell extracts were also prepared from primary isolated rat adipocytes and incubated with the GST fusion proteins (Fig. 1 B). Similar to 3T3L1 adipocytes, Coomassie Blue staining of these precipitates demonstrated that the 40- and 43-kDa proteins were specifically precipitated with GST-G4 and GST-G4* but not by GST or GST-G1. Although other more intense bands were detected on the Coomassie Blue stained gel, these were also seen in the absence of lysate and were therefore nonspecific. We could not obtain a defined amino acid sequence following purification of the 43-kDa protein; however, the 40-kDa protein was partially sequenced and identified as fructose 1,6-bisphosphate aldolase. To confirm that the 40-kDa protein that bound GST-G4 and GST-G4* was aldolase, immunoblotting with an aldolase-specific antibody was performed (Fig. 1 C). Incubation of cell extracts with GST-G4 and GST-G4* resulted in the co-precipitation of a 40-kDa protein that demonstrated specific immunoreactivity with an aldolase antibody. In contrast, no immunoreactive proteins were detected in the GST or GST-G1 precipitates. Furthermore, as observed in the [35S]methionine-labeled 3T3L1 adipocytes, cell extracts from insulin-stimulated cells also precipitated aldolase immunoreactivity in the presence of GST-G4 and GST-G4* but not GST or GST-G1. These data demonstrate that the 40-kDa protein that specifically binds the carboxyl-terminal domain of GLUT4 is fructose 1,6-bisphosphate aldolase. Aldolase is an unusual protein in that it not only possesses enzymatic activity but also plays a structural role in the assembly of the actin cytoskeleton (26O'Reilly G. Clarke F. FEBS Lett. 1993; 321: 69-72Crossref PubMed Scopus (54) Google Scholar). The interaction of aldolase with actin can be modulated by aldolase substrates and products (27Wang J. Morris A.J. Tolan D.R. Pagliaro L. J. Biol. Chem. 1996; 271: 6861-6865Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 28Kusakabe T. Motoki K. Hori K. Arch. Biochem. Biophys. 1997; 344: 184-193Crossref PubMed Scopus (58) Google Scholar). We therefore sought to determine the allosteric effect of these and related molecules on the binding of aldolase to GST-G4 (Fig.2). Dose-response relationships demonstrated that 1 mm FBP, a concentration similar to theK m of the enzyme for this substrate, completely inhibited the binding of aldolase to GST-G4 in vitro (Fig.2 A). The aldolase reaction product, G3P, also inhibited GST-G4 binding to aldolase, although at a significantly higher concentration, 10 mm (Fig. 2 B). In contrast, the aldolase product dihydroxyacetone phosphate, as well as two other structurally related sugars, fructose 1-phosphate and fructose 6-phosphate, had no effect on the interaction between GST-G4 and aldolase (Fig. 2, C, D, and E). Previous studies have demonstrated that aldolase is associated with the actin cytoskeleton and can cross-link actin fibers (27Wang J. Morris A.J. Tolan D.R. Pagliaro L. J. Biol. Chem. 1996; 271: 6861-6865Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 29Pagliaro L. Taylor D.L. J. Cell Biol. 1988; 107: 981-991Crossref PubMed Scopus (99) Google Scholar). Because the actin cytoskeleton has also been implicated in the regulation of GLUT4 trafficking (30Tsakiridis T. Vranic M. Klip A. J. Biol. Chem. 1994; 269: 29934-29942Abstract Full Text PDF PubMed Google Scholar, 31Wang Q. Bilan P.J. Tsakiridis T. Hinek A. Klip A. Biochem. J. 1998; 331: 917-928Crossref PubMed Scopus (139) Google Scholar), we next examined the ability of aldolase to function as a molecular scaffold linking GLUT4 to F-actin (Fig.3). F-actin was pre-incubated with aldolase, followed by a second incubation with GST-G1 or GST-G4. When the actin was pelleted by centrifugation, GST-G4 was found to be specifically associated with actin in the presence of aldolase (Fig. 3,lane 1). GST-G4 did not co-pellet with actin in the absence of aldolase, suggesting that the GLUT4 protein binds to actin indirectly (Fig. 3, lane 5). Likewise, the aldolase·GST-G4 complex was not pelleted by centrifugation in the absence of actin (Fig. 3, lane 7). GST-G1 did not associate with actin in either the absence or presence of aldolase (Fig. 3, lanes 4and 6). As previously observed in Fig. 2, FBP and G3P can inhibit the association of aldolase with GST-G4. FBP and G3P were also able to prevent the formation of the F-actin·aldolase·GST-G4 ternary complex (Fig. 3, lanes 2 and 3). These data indicate that in vitro, aldolase can function as a molecular scaffold linking the carboxyl-terminal domain of GLUT4 with filamentous actin. To determine whether aldolase and GLUT4 were co-localized in vivo, double immunofluorescence labeling was performed. 3T3L1 adipocytes were treated with or without insulin, fixed, and incubated with aldolase and GLUT4-specific antibodies followed by labeling with complimentary fluorescent-conjugated secondary antibodies (Fig.4). Under basal conditions, the membrane bound GLUT4 protein was found primarily in a peri-nuclear distribution and in small vesicles scattered throughout the cytoplasm (Fig. 4,panel A). Following insulin stimulation, GLUT4 moved to the cell surface generating a “rim”-like fluorescence (Fig. 4,panel D). Conversely, the soluble protein aldolase stained in a diffusely cytosolic pattern although it was also concentrated in the perinuclear region of the cell (Fig. 4, panels B andE). When their respective labels are superimposed, GLUT4 and aldolase exhibited a high degree of co-localization (Fig. 4,panels C and F), suggesting an in vivoassociation between aldolase and the GLUT4-containing compartments. To further investigate the in vivo significance of these findings, we took advantage of the glycolytic inhibitor 2DG, which induces the dissociation of perinuclear aldolase from the actin cytoskeleton (32Pagliaro L. Taylor D.L. J. Cell Biol. 1992; 118: 859-863Crossref PubMed Scopus (63) Google Scholar). In adipocytes, the insulin-induced translocation of GLUT4 to the plasma membrane can be detected by the fluorescent detection of a GLUT4 antibody on the cytoplasmic face of isolated plasma membrane sheets. Pretreatment of 3T3L1 adipocytes with 5 mm 2DG slightly reduced the amount of basal plasma membrane GLUT4 immunofluorescence (Fig.5 A, panels 1 and3). In contrast, pretreatment with 2DG markedly attenuated the insulin-stimulated translocation of GLUT4 to the cell surface (Fig.5 A, panels 2 and 4). The 2DG inhibition of insulin-stimulated GLUT4 translocation occurred in a dose-dependent manner with an EC50 of approximately 100 mm (data not shown). This inhibition of GLUT4 translocation was also observed in GLUT4 immunoblots of isolated plasma membranes without any effect on the plasma membrane association of the clathrin heavy chain (Fig. 5 B). The blockade of insulin-stimulated GLUT4 translocation by 2DG could be due to either an inhibition of exocytosis or an increase in endocytosis. To determine whether 2DG affected the rate of GLUT4 endocytosis, we stimulated cells with insulin, removed the insulin by washing with an acidic buffer, and then assayed plasma membrane GLUT4 at various times after insulin removal in the presence of 2DG. Disappearance of GLUT4 from the cell surface after insulin removal was not inhibited by 2DG treatment but rather seemed to be slightly enhanced (data not shown). This small increase in the rate of GLUT4 endocytosis in the presence of 2DG likely reflected the inhibition of further GLUT4 exocytosis following insulin removal. This effect was similar to that reported for wortmannin, which also inhibits GLUT4 exocytosis but appears to enhance endocytosis due to the inhibition of residual exocytosis (33Yang J. Clarke J.F. Ester C.J. Young P.W. Kasuga M. Holman G.D. Biochem. J. 1996; 313: 125-131Crossref PubMed Scopus (103) Google Scholar). These data demonstrate that the rate of GLUT4 endocytosis was not significantly affected by 2DG pretreatment and is consistent with an inhibition of GLUT4 exocytosis. Although cells were incubated with glutamine and pyruvate as alternative energy sources during 2DG treatment, cellular ATP levels were found to decrease by 40% (data not shown). Because endocytosis and exocytosis may have different sensitivities to ATP depletion, the inhibition of insulin-stimulated GLUT4 exocytosis could have been due to this decrease in ATP. To ensure adequate cellular ATP levels while directly assessing the uncoupling of aldolase from GLUT4 in vivo, 3T3L1 adipocytes were permeabilized with SLO in the presence of 10 mm MgATP (Fig. 5, C and D). Under these experimental conditions, cellular metabolism did not deplete local concentrations of ATP (data not shown). In the SLO-permeabilized adipocytes, insulin induced an increase in the translocation of GLUT4 to the plasma membrane (Fig. 5 C,panels 1 and 2). As in the intact cells, preincubation with 2DG prevented GLUT4 translocation (data not shown). In addition, although preincubation with FBP slightly enhanced the basal amount of GLUT4 associated with the plasma membrane, FBP pretreatment markedly inhibited the insulin-stimulated translocation of GLUT4 (Fig. 5 C, panels 3 and 4). It should be noted that the small increase in plasma membrane-associated GLUT4 following SLO permeabilization has also been previously observed by others (25Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (254) Google Scholar). In agreement with the immunofluorescence analysis, immunoblotting of isolated plasma membranes demonstrated that FBP preincubation partially increased the basal plasma membrane-associated GLUT4 and inhibited the insulin-stimulated increase without affecting the plasma membrane association of the clathrin heavy chain (Fig.5 D). To examine the metabolite specificity for the inhibition of insulin-stimulated GLUT4 translocation, we compared the effects of several aldolase substrates, products and related sugars on GLUT4 translocation (data not shown). Consistent with their effect on aldolase-actin binding, pretreatment of SLO-permeabilized adipocytes with dihydroxyacetone phosphate, fructose 1-phosphate, fructose 6-phosphate, or glucose had no significant effect on insulin-stimulated GLUT4 translocation compared with control cells. To further confirm a specific requirement for aldolase in insulin-stimulated GLUT4 translocation, we next microinjected cells with control or aldolase antibodies and assessed single cell GLUT4 translocation (Fig.6). Specific identification of the microinjected cells was accomplished by co-injection with a carboxyl-terminal domain of Ras fused to the MBP as a plasma membrane marker. In unstimulated cells, microinjection of either pre-immune IgG or the aldolase IgG had no effect on GLUT4 translocation, which remained at low basal levels (Fig. 6 A, panels 1-4). Microinjection of pre-immune IgG did not inhibit the insulin-stimulated translocation of GLUT4 (Fig. 6 B,panels 1 and 2). In contrast, the aldolase-specific IgG reduced the extent of GLUT4 translocation only in the cells that were microinjected but not in the surrounding nonmicroinjected cells (Fig. 6 B, panels 3 and4). Quantitation of these data demonstrated that insulin stimulated GLUT4 translocation in 49% of cells microinjected with pre-immune IgG and 29% of cells microinjected with aldolase-specific IgG (Fig. 6 C). Together, these data provide compelling evidence for a specific functional role of fructose 1,6-bisphosphate aldolase in the insulin stimulation of GLUT4 translocation in adipocytes. Previous studies have demonstrated that aldolase exhibits functional duality. In addition to its enzymatic activity, this protein plays a structural role in the binding and polymerization of actin (28Kusakabe T. Motoki K. Hori K. Arch. Biochem. Biophys. 1997; 344: 184-193Crossref PubMed Scopus (58) Google Scholar, 29Pagliaro L. Taylor D.L. J. Cell Biol. 1988; 107: 981-991Crossref PubMed Scopus (99) Google Scholar,34Chen-Zion M. Livnat T. Beitner R. Int. J. Biochem. 1992; 24: 821-826Crossref PubMed Scopus (34) Google Scholar). Depolymerization of the actin cytoskeleton with cytochalasin D or latrunculin B has been shown to attenuate insulin-stimulated GLUT4 translocation, suggesting that an intact actin cytoskeleton is required for insulin-stimulated GLUT4 translocation (30Tsakiridis T. Vranic M. Klip A. J. Biol. Chem. 1994; 269: 29934-29942Abstract Full Text PDF PubMed Google Scholar, 31Wang Q. Bilan P.J. Tsakiridis T. Hinek A. Klip A. Biochem. J. 1998; 331: 917-928Crossref PubMed Scopus (139) Google Scholar). Consistent with this model, our data demonstrate that aldolase, a functional tetramer, may also serve as a scaffolding protein linking GLUT4 and hence GLUT4 vesicles, to the actin cytoskeleton. In in vitroexperiments, aldolase not only specifically interacted with the carboxyl-terminal domain of GLUT4, but linked GLUT4 to polymerized actin. This interaction was disrupted by specific substrates of aldolase that inhibit GLUT4-aldolase or aldolase-actin interactions. These specific substrates not only prevented aldolase-actin interaction but also prevented GLUT4-aldolase binding in vitro and in a manner consistent with their ability to interfere with insulin-stimulated GLUT4 translocation in vivo. Furthermore, aldolase and GLUT4 exhibited an overlapping subcellular distribution, and microinjection of an aldolase-specific antibody reduced the insulin-stimulated translocation of GLUT4. Together, these data strongly suggest that aldolase plays a critical role in the dynamic association of GLUT4 vesicles with the actin cytoskeleton. The ability of aldolase substrates to disrupt the actin·aldolase·GLUT4 complex, as well as GLUT4 vesicle trafficking, suggests that the metabolism of glucose may provide a negative feedback signal to prevent further exocytosis of GLUT4 vesicles and potentially contribute to the mechanism of glucotoxicity. We thank Dr. Morris Birnbaum for providing glucose transporter, MBP-Ras cDNAs, and MBP antibody, Dr. Jurgen Bereiter-Hahn for supplying aldolase antibody, Dr. Jeffrey Elmendorf for assistance in analyzing microinjection results and Dr. Konstantin Kandror for helpful discussion and critical reading of the manuscript." @default.
• W2058061580 created "2016-06-24" @default.
• W2058061580 creator A5006844693 @default.
• W2058061580 creator A5011486327 @default.
• W2058061580 creator A5018176504 @default.
• W2058061580 creator A5030492395 @default.
• W2058061580 creator A5077728712 @default.
• W2058061580 date "1999-06-01" @default.
• W2058061580 modified "2023-10-03" @default.
• W2058061580 title "Aldolase Mediates the Association of F-actin with the Insulin-responsive Glucose Transporter GLUT4" @default.
• W2058061580 cites W1490846487 @default.
• W2058061580 cites W1530781869 @default.
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