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• W2058110898 abstract "ADP-ribosylation factors (ARFs) play important roles in both constitutive and regulated membrane trafficking to the plasma membrane in other cells. Here we have examined their role in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. These cells express ARF5 and ARF6. ARF5 was identified in the soluble protein and intracellular membranes; in response to insulin some ARF5 was observed to re-locate to the plasma membrane. In contrast, ARF6 was predominantly localized to the plasma membrane and did not redistribute in response to insulin. We employed myristoylated peptides corresponding to the NH2 termini of ARF5 and ARF6 to investigate the function of these proteins. Myr-ARF6 peptide inhibited insulin-stimulated glucose transport and GLUT4 translocation by ∼50% in permeabilized adipocytes. In contrast, myr-ARF1 and myr-ARF5 peptides were without effect. Myr-ARF5 peptide also inhibited the insulin stimulated increase in cell surface levels of GLUT1 and transferrin receptors. Myr-ARF6 peptide significantly decreased cell surface levels of these proteins in both basal and insulin-stimulated states, but did not inhibit the fold increase in response to insulin. These data suggest an important role for ARF6 in regulating cell surface levels of GLUT4 in adipocytes, and argue for a role for both ARF5 and ARF6 in the regulation of membrane trafficking to the plasma membrane. ADP-ribosylation factors (ARFs) play important roles in both constitutive and regulated membrane trafficking to the plasma membrane in other cells. Here we have examined their role in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. These cells express ARF5 and ARF6. ARF5 was identified in the soluble protein and intracellular membranes; in response to insulin some ARF5 was observed to re-locate to the plasma membrane. In contrast, ARF6 was predominantly localized to the plasma membrane and did not redistribute in response to insulin. We employed myristoylated peptides corresponding to the NH2 termini of ARF5 and ARF6 to investigate the function of these proteins. Myr-ARF6 peptide inhibited insulin-stimulated glucose transport and GLUT4 translocation by ∼50% in permeabilized adipocytes. In contrast, myr-ARF1 and myr-ARF5 peptides were without effect. Myr-ARF5 peptide also inhibited the insulin stimulated increase in cell surface levels of GLUT1 and transferrin receptors. Myr-ARF6 peptide significantly decreased cell surface levels of these proteins in both basal and insulin-stimulated states, but did not inhibit the fold increase in response to insulin. These data suggest an important role for ARF6 in regulating cell surface levels of GLUT4 in adipocytes, and argue for a role for both ARF5 and ARF6 in the regulation of membrane trafficking to the plasma membrane. Insulin stimulates glucose disposal in peripheral tissues by virtue of the expression of the GLUT4 glucose transporter isoform (1Holman G.D. Cushman S.W. BioEssays. 1994; 16: 753-759Crossref PubMed Scopus (135) Google Scholar, 2Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, 3Zorzano A. Munoz P. Camps M. Mora C. Testar X. Palacin M. Diabetes. 1996; 45: S70-S81Crossref PubMed Google Scholar). In the absence of insulin, this transporter is intracellularly sequestered within the elements of the endosomal system, thetrans Golgi network and a specialized storage compartment (4Ploug T. van Deurs B. Cushman S.W. Ralston E. J. Cell Biol. 1998; 142: 1429-1446Crossref PubMed Scopus (240) Google Scholar, 5Slot J.W. Geuze H.J. Gigengack S. James D.E. Lienhard G.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7815-7819Crossref PubMed Scopus (353) Google Scholar, 6Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (715) Google Scholar, 7Slot J.W. Garruti G. Martin S. Oorschot V. Posthuma G. Kraegen E.W. Laybutt R. Thibault G. James D.E. J. Cell Biol. 1997; 137: 1243-1254Crossref PubMed Scopus (63) Google Scholar). Upon insulin stimulation or muscle contraction, GLUT4 is re-distributed from these intracellular locations to the plasma membrane, resulting in a dramatic increase in the rate of glucose entry into these tissues (4Ploug T. van Deurs B. Cushman S.W. Ralston E. J. Cell Biol. 1998; 142: 1429-1446Crossref PubMed Scopus (240) Google Scholar, 5Slot J.W. Geuze H.J. Gigengack S. James D.E. Lienhard G.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7815-7819Crossref PubMed Scopus (353) Google Scholar, 6Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (715) Google Scholar, 7Slot J.W. Garruti G. Martin S. Oorschot V. Posthuma G. Kraegen E.W. Laybutt R. Thibault G. James D.E. J. Cell Biol. 1997; 137: 1243-1254Crossref PubMed Scopus (63) Google Scholar). Several studies have suggested that the insulin-stimulated translocation of GLUT4 to the plasma membrane is mechanistically akin to the fusion of small synaptic vesicles with the neuronal plasma membrane (reviewed in Ref. 2Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). This has been supported by the identification of a morphologically similar GLUT4 storage compartment within adipocytes, and by the identification of solubleN-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) 1The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; ARF, ADP-ribosylation factor; GLUT, glucose transporter; deoxy-Glc, 2-deoxy-d-glucose; TfR, transferrin receptor; Tf, transferrin. located in GLUT4 vesicles (the v-SNAREs cellubrevin and vesicle-associated membrane protein 2) (8Cheatham B. Volchuk A. Kahn C.R. Wang L. Rhodes C. Klip A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15169-15173Crossref PubMed Scopus (162) Google Scholar, 9Martin L.B. Shewan A. Millar C.A. Gould G.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10Olson A.L. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (209) Google Scholar, 11Volchuk A. Mitsumoto Y. He L. Lui Z. Habermann E. Trimble W.S. Klip A. Biochem. J. 1994; 304: 139-145Crossref PubMed Scopus (69) Google Scholar) which bind in a highly specific manner to t-SNAREs located in the adipocyte plasma membrane (Syntaxin 4 and Syndet) (8Cheatham B. Volchuk A. Kahn C.R. Wang L. Rhodes C. Klip A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15169-15173Crossref PubMed Scopus (162) Google Scholar, 10Olson A.L. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (209) Google Scholar, 12Volchuk A. Wang Q. Ewart H. Liu Z. He L. Bennett M. Klip A. Mol. Biol. Cell. 1996; 7: 1075-1082Crossref PubMed Scopus (126) Google Scholar, 13Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 14Rea S. Martin L.B. McIntosh S. Macaulay S.L. Ramsdale T. Baldini G. James D.E. J. Biol. Chem. 1998; 273: 18784-18792Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Vesicle-associated membrane protein 2 has been shown to be the predominant v-SNARE that targets small synaptic vesicles to the pre-synaptic plasma membrane by interacting with the cognate t-SNAREs, syntaxin1, and synaptosome-associated protein of 25 kDa (SNAP-25) found on the target membrane (15Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar). Recent studies implicating vesicle-associated membrane protein 2 in insulin-stimulated GLUT4 translocation further strengthen the mechanistic parallels between regulated exocytosis of small synaptic vesicles and the insulin-stimulated movement of GLUT4-containing vesicles to the adipocyte cell surface (9Martin L.B. Shewan A. Millar C.A. Gould G.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 16Martin S. Tellman J. Livingstone C. Slot J.W. Gould G.W. James D.E. J. Cell Biol. 1996; 134: 625-635Crossref PubMed Scopus (180) Google Scholar). ADP-ribosylation factors (ARFs) are a family of GTP-binding proteins (17Moss J. Vaughan M. J. Biol. Chem. 1995; 270: 12327-12330Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 18Boman A.L. Kahn R.A. Trends Biochem. Sci. 1995; 20: 147-150Abstract Full Text PDF PubMed Scopus (237) Google Scholar). To date, 6 isoforms have been identified in mouse tissues (19Hosaka M. Toda K. Takatsu H. Torii S. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1996; 120: 813-819Crossref PubMed Scopus (63) Google Scholar) which fall within three groups, ARFs1, -2 and -3 constitute group I, ARFs4 and -5 group II and ARF6 is the sole member of group III identified to date. ARF proteins have been proposed to play several roles in the control of membrane traffic, including the formation of secretory vesicles at the trans Golgi network, regulating endosome-endosome fusion, and notably in regulating the fusion of secretory vesicles with the plasma membrane in bovine adrenal medulla cells (20Moss J. Vaughan M. J. Biol. Chem. 1998; 273: 21431-21434Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 21D'Souza-Schorey C. van Donselaar E. Hsu V.W. Yang C. Stahl P.D. Peters P.J. J. Cell Biol. 1998; 140: 603-616Crossref PubMed Scopus (197) Google Scholar, 22Galas M.-C. Helms J.B. Vitale N. Thiersé D. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 23Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (329) Google Scholar, 24Lenhard J.M. Kahn R.A. Stahl P.D. J. Biol. Chem. 1992; 267: 13047-13052Abstract Full Text PDF PubMed Google Scholar, 25Faundez V. Horng J.-T. Kelly R.B. J. Cell Biol. 1997; 138: 505-515Crossref PubMed Scopus (88) Google Scholar). Given the importance of ARF proteins in regulated membrane trafficking, we set out to identify which ARF isoforms were expressed in murine 3T3-L1 adipocytes, how their distribution was modulated by insulin, and whether they played a role in insulin-stimulated GLUT4 translocation. Here we show that adipocytes express ARF5 and ARF6, as determined by immunoblotting with ARF-specific antibodies. ARF5 was observed to exhibit modest re-distribution to the plasma membrane in response to insulin. In contrast, ARF6 was predominantly located within plasma membrane subcellular fractions, and its distribution was not altered by insulin treatment. Using myristoylated ARF NH2-terminal peptides to inhibit ARF action in permeabilized cells, we show that myristoylated ARF6 peptide partially inhibits insulin-stimulated glucose transport and GLUT4 translocation, whereas ARF1- and ARF5-myristoylated peptides were without effect. Insulin stimulates the movement of other proteins to the cell surface, including the transferrin receptor (TfR) and GLUT1 (26Calderhead D.M. Kitagawa K. Tanner L.I. Holman G.D. Lienhard G.E. J. Biol. Chem. 1990; 265: 13801-13808Abstract Full Text PDF PubMed Google Scholar, 27Tanner L.I. Lienhard G.E. J. Biol. Chem. 1987; 262: 8975-8980Abstract Full Text PDF PubMed Google Scholar). Strikingly, we observed marked inhibition of insulin-stimulated TfR and GLUT1 movement to the cell surface in the presence of myristoylated ARF5 peptide, implying an important role for ARF5 in the stimulated delivery of recycling membrane proteins to the cell surface. Myristoylated ARF6 peptide decreased the cell surface abundance of TfR and GLUT1 in both the basal and insulin-stimulated states, but did not inhibit the ability of insulin to increase cell surface levels of these proteins. These data argue for an important role for ARF6 in regulating cell surface levels of GLUT4 in adipocytes, and provide evidence for a role for both ARF5 and ARF6 in the regulation of membrane trafficking to the plasma membrane. α-Toxin was from Calbiochem, United Kingdom, wortmannin from Sigma, UK, and 125I-transferrin and [14C]sucrose were from NEN Life Science Products Inc. and Amersham International, respectively. All other reagents were as described (16Martin S. Tellman J. Livingstone C. Slot J.W. Gould G.W. James D.E. J. Cell Biol. 1996; 134: 625-635Crossref PubMed Scopus (180) Google Scholar, 28Martin S. Reaves B. Banting G. Gould G.W. Biochem. J. 1994; 300: 743-749Crossref PubMed Scopus (47) Google Scholar). 3T3-L1 fibroblasts were grown and differentiated into adipocytes exactly as described in Refs. 16Martin S. Tellman J. Livingstone C. Slot J.W. Gould G.W. James D.E. J. Cell Biol. 1996; 134: 625-635Crossref PubMed Scopus (180) Google Scholar and 28Martin S. Reaves B. Banting G. Gould G.W. Biochem. J. 1994; 300: 743-749Crossref PubMed Scopus (47) Google Scholar. Cells were used between passages 3 and 10 in all experiments, and between days 8 and 13 after induction of differentiation. Prior to use, cells were incubated in serum-free Dulbecco's modified Eagle's medium for 2 h. Adipocytes were subjected to a differential centrifugation procedure as described previously (28Martin S. Reaves B. Banting G. Gould G.W. Biochem. J. 1994; 300: 743-749Crossref PubMed Scopus (47) Google Scholar, 29Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). Briefly, cells were scraped and homogenized in ice-cold HES (20 mm HEPES, 1 mm EDTA, 255 mm sucrose, pH 7.4, 5 ml/10-cm plate) containing protease inhibitors (1 μg/ml pepstatin A, 0.2 mm diisopropyl fluorophosphate, 20 μml-transepoxysuccinyl-leucylamido-4-guanidiniobutane, and 50 μm aprotinin). The homogenate was centrifuged at 19,000 × g for 20 min at 4 °C. The pellet from this spin was resuspended in 2 ml HES, layered onto 1 ml of 1.12m sucrose in HES, and centrifuged at 100,000 ×g for 1 h at 4 °C in a swing-out rotor. Plasma membranes were collected from the interface by careful aspiration, resuspended in HES, and collected by centrifugation at 41,000 ×g for 20 min at 4 °C. The supernatant from the 19,000 × g spin was re-centrifuged at 41,000 ×g to yield a high density microsomal pellet and the supernatant from this spin centrifuged at 180,000 × gfor 75 min at 4 °C to collect low density microsomes. All fractions were resuspended in equal volumes of HES buffer (cell equivalents), snap frozen in liquid nitrogen, and stored at −80 °C prior to use. 3T3-L1 adipocytes were washed twice with IC buffer (10 mm NaCl, 20 mm Hepes, 50 mm KCl, 2 mmK2HPO4, 90 mm potassium glutamate, 1 mm MgCl2, 4 mm EGTA, 2 mm CaCl2, pH 7.4) at 37 °C, then incubated in 500 μl of ICR buffer (IC buffer plus 4 mm MgATP, 3 mm sodium pyruvate, 100 μg/ml bovine serum albumin, pH 7.4) containing α-toxin at 250 hemolytic units/ml for 5 min to permeabilize the plasma membrane. The medium was removed and the cells covered with 500 μl of ICR buffer containing peptides, insulin, or vehicle as described in the figure legends. This methodology has been documented in Ref. 30Herbst J.J. Andrews G.C. Contillo L.G. Singleton D.H. Genereux P.E. Gibbs E.M. Lienhard G.E. J. Biol. Chem. 1995; 270: 26000-26005Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar. α-Toxin is a 34-kDa protein which inserts into the plasma membrane and oligomerizes to form a 3-nm aqueous pore that allows passage of molecules up to ∼5 kDa across the cell membrane. We therefore employed this experimental system to determine the role of ARF proteins on [3H]2-deoxy-d-glucose (deoxy-Glc) transport as has been described previously (30Herbst J.J. Andrews G.C. Contillo L.G. Singleton D.H. Genereux P.E. Gibbs E.M. Lienhard G.E. J. Biol. Chem. 1995; 270: 26000-26005Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). After permeabilization and incubation with peptides and insulin as described in the figure legends, 50 μl of radioisotope solution was added to each well of adipocytes such that the final concentration of deoxy-Glc was 50 μm and 0.5 μCi/well. Also included in this 50-μl aliquot was [14C]sucrose (final concentration 50 μm, 0.05 μCi per well) so as to allow estimation of the nonspecific association of sugar with the cells. The transport rates presented have been corrected for this calculation. Uptake was carried out for 3 min, then the cells were rapidly washed three times in ice-cold phosphate-buffered saline and air-dried. Cell associated radioactivity was determined by solubilizing the cells in 1% Triton X-100. Nonspecific association of radioactivity with the cells amounted to less than 20% of the specific uptake under these conditions. After experimental manipulations, coverslips of adipocytes were rapidly washed in ice-cold buffer for the preparation of plasma membrane lawns exactly as described in Ref. 9Martin L.B. Shewan A. Millar C.A. Gould G.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar. After fixation in paraformaldehyde, plasma membrane lawns were incubated with anti-GLUT-4 (1:100 dilution) antibodies for 1 h at room temperature (9Martin L.B. Shewan A. Millar C.A. Gould G.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). After washing, the coverslips were then incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG, washed and mounted on glass coverslips. Coversips were viewed using a × 40 objective lens on a Zeiss Axiovert microscope operated in Laser Scanning Confocal mode. Samples were illuminated at 488 nm and the signal at 510 nm collected. Duplicate coverslips were prepared at each experimental condition, and 10 random images of plasma membrane lawns collected from each. These were quantified using MetaMorph (Universal Imaging, CA) software on a DAN PC (Noran Instruments, Surrey, UK). Similar methods were employed to assay GLUT1 levels in plasma membranes. Transferrin receptors present at the cell surface were quantified as outlined in Refs. 27Tanner L.I. Lienhard G.E. J. Biol. Chem. 1987; 262: 8975-8980Abstract Full Text PDF PubMed Google Scholar and 31Jess T.J. Belham C.M. Thomson F.J. Scott P.H. Plevin R.J. Gould G.W. Cell. Signalling. 1996; 8: 297-304Crossref PubMed Scopus (36) Google Scholar. After experimental manipulations, cells were rapidly chilled by three washes in ice-cold KRP buffer (136 mm NaCl, 4.7 mm KCl, 1.25 mm MgSO4, 1.25 mmCaCl2, 5 mm NaH2PO4, pH 7.4) containing 1 mg/ml bovine serum albumin. Thereafter, cells were incubated in the same buffer containing ∼3 nm125I-transferrin for 2 h on ice. After this time, the media was aspirated and the monolayers washed three times with 1 ml of KRP/bovine serum albumin for 1 min. Cells were then solubilized in 1m NaOH and the radioactivity associated with each well determined by γ-counting. For each condition, duplicates plates were incubated exactly as above, but in the presence of 1 μmtransferrin; the radioactivity associated with each well under these conditions was the value of nonspecific binding at each condition, and was found to vary between 5 and 10% of the total counts per well. The peptides employed in this study were prepared either by Thistle Research (Glasgow, UK) or as outlined in Ref. 22Galas M.-C. Helms J.B. Vitale N. Thiersé D. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar. The sequences of the peptides used is shown in TableI. Antibodies to ARF5 were provided by Dr. J. Moss (NHLBI, National Institutes of Health, Bethesda, MD) and Dr. R. A. Kahn (Atlanta, GA) (17Moss J. Vaughan M. J. Biol. Chem. 1995; 270: 12327-12330Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 32Mumby S.M. Kahn R.A. Manning D.R. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 265-269Crossref PubMed Scopus (267) Google Scholar). Antibodies to ARF6 were provided by Dr. J. Donaldson (NHLBI, National Institutes of Health, Bethesda, MD) (33Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (421) Google Scholar). Anti-GLUT1 was generously provided by Professor Geoff Holman (University of Bath).Table ISequence alignment of ARF proteins at the amino terminusARF1aaMGNIFANLFKGLFGKKEMRIARF2aaMGNVFEKLFKSLFGKKEMRIARF3aaMGNIFGNLLKSLIGKKEMRIARF4aaMGLTISSLFSRLFGKKQMRIARF5aaMGLTVSALFSRIFGKKQMRIARF6aaMGKVLSK—-IFGNKEMRIShown is a sequence alignment of the murine isoforms of the ARF family (19Hosaka M. Toda K. Takatsu H. Torii S. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1996; 120: 813-819Crossref PubMed Scopus (63) Google Scholar). The peptides used in this study are underlined, and in the case of ARF5 and ARF6 were synthesized with or without a myristoyl group at position Gly-2. Open table in a new tab Shown is a sequence alignment of the murine isoforms of the ARF family (19Hosaka M. Toda K. Takatsu H. Torii S. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1996; 120: 813-819Crossref PubMed Scopus (63) Google Scholar). The peptides used in this study are underlined, and in the case of ARF5 and ARF6 were synthesized with or without a myristoyl group at position Gly-2. We have used a panel of ARF-specific antibodies to examine the expression and subcellular distribution of ARFs 5 and 6 in 3T3-L1 adipocytes (Fig. 1). In the basal (non-stimulated) state, ARF5 was predominantly localized to the soluble protein fraction of adipocytes, with some association with intracellular membranes. In response to insulin treatment, ARF5 levels at the plasma membrane were observed to increase with a concomitant decrease chiefly from the soluble protein fraction of the cells. Similar results were obtained using two different anti-ARF5 antibodies. In the same experiments, GLUT4 was observed to translocate to the plasma membrane in response to insulin from intracellular membrane fractions as has been extensively reported (1Holman G.D. Cushman S.W. BioEssays. 1994; 16: 753-759Crossref PubMed Scopus (135) Google Scholar, 2Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, 3Zorzano A. Munoz P. Camps M. Mora C. Testar X. Palacin M. Diabetes. 1996; 45: S70-S81Crossref PubMed Google Scholar). In contrast, ARF6 was observed chiefly in the plasma membrane fraction of 3T3-L1 adipocytes (34Yang C.-Z. Heimberg H. D'Souza-Schorey C. Mueckler M.M. Stahl P.D. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and did not exhibit appreciable alteration in subcellular distribution in response to insulin (Fig. 1). The plasma membrane localization of ARF6 is in agreement with studies in a range of cell types (34Yang C.-Z. Heimberg H. D'Souza-Schorey C. Mueckler M.M. Stahl P.D. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 35Zhang Q. Cox D. Tseng C.-C. Donaldson J.G. Greenberg S. J. Biol. Chem. 1998; 273: 19977-19981Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Although insulin did not appear to modulate the subcellular distribution of ARF6, it is possible that insulin may modify the ARF6-GDP/ARF6-GTP ratio at the plasma membrane, indeed several studies have suggested that unlike other members of the ARF family, ARF6 remains membrane associated even in its GDP-bound state (34Yang C.-Z. Heimberg H. D'Souza-Schorey C. Mueckler M.M. Stahl P.D. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 35Zhang Q. Cox D. Tseng C.-C. Donaldson J.G. Greenberg S. J. Biol. Chem. 1998; 273: 19977-19981Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Alternatively, insulin may modulate the rate of turnover from membrane to cytosolic states of ARF6 without an apparent alteration in distribution between these fractions. Hence the apparent lack of altered subcellular distribution of ARF6 in response to insulin does not preclude an important role for this protein in insulin action. Myristoylated peptides corresponding to the amino terminus of ARF proteins have been widely used in many laboratories to probe the function of ARF proteins in intracellular trafficking, including endoplasmic reticulum to Golgi transport, intra-Golgi transport, and endocytic vesicle fusion (36Barr F.A. Huttner W.B. FEBS Lett. 1996; 348: 65-70Crossref Scopus (36) Google Scholar, 37Donaldson J.G. Casse L.D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 38Kahn R.A. Randazzo P. Serafini T. Weiss O. Rulka C. Clark J. Amherdt M. Roller P. Orci L. Rothman J.E. J. Biol. Chem. 1992; 267: 13039-13046Abstract Full Text PDF PubMed Google Scholar). Myristoylated peptides corresponding to residues 2 through 13 of the NH2 terminus of ARF6 have also been shown to inhibit regulated exocytosis in permeabilized chromaffin cells (22Galas M.-C. Helms J.B. Vitale N. Thiersé D. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and to inhibit stimulated phospholipase D activity in these cells in response to agents which stimulate secretion (39Caumont A.-S. Galas M.-C. Vitale N. Aunis D. Bader M.-F. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In contrast, a corresponding peptide lacking the myristoyl group at Gly-2, or the cognate myristoylated peptide from ARF1 were without effect (22Galas M.-C. Helms J.B. Vitale N. Thiersé D. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). We therefore chose to adopt similar methodology to examine the role of ARF5 or ARF6 in insulin-stimulated glucose transport in α-toxin-permeabilized 3T3-L1 adipocytes. We synthesized peptides corresponding to residues 2 through 16 of murine ARF5 (with or without a myristoyl group at position Gly-2), and employed myristoylated ARF1 and ARF6 peptides described by one of us previously (22Galas M.-C. Helms J.B. Vitale N. Thiersé D. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Permeabilized adipocytes were incubated with the peptides for 10 min, then stimulated with 1 μm insulin for a further 20 min. At the end of this time, deoxy-Glc uptake was measured as described under “Experimental Procedures.” The results of a typical experiment are presented in Fig. 2 A. We consistently observed a diminution in the rate of insulin-stimulated deoxy-Glc uptake in cells incubated with myristoylated ARF6 peptide. In three experiments of this type, the extent of inhibition of insulin-stimulated deoxy-Glc transport was 47 ± 11%. In contrast, neither myristoylated ARF1 nor myristoylated ARF5 peptides inhibited insulin-stimulated deoxy-Glc uptake over the same concentration range. In control experiments (not shown) the addition of the peptide to intact 3T3-L1 adipocytes was without effect on either basal or insulin-stimulated deoxy-Glc uptake. This data argues that ARF6 may play a role in the regulation of plasma membrane GLUT4 levels in response to insulin, as GLUT4 is responsible for the majority of insulin-stimulated glucose uptake in adipocytes. In order to address this directly, we measured insulin-stimulated GLUT4 translocation to the cell surface using the plasma membrane lawn technique. Permeabilized adipocytes were incubated with myristoylated ARF1, ARF5, and ARF6 peptides prior to insulin stimulation and assessment of plasma membrane GLUT4 levels. Data from a typical experiment are presented in Fig. 2 B, and the results of three experiments of this type presented in Fig. 2 C. We observed marked inhibition of GLUT4 translocation in the presence of 100 μm myristoylated ARF6 peptide (42 ± 3%;n = 3). A modest inhibition of GLUT4 translocation was also observed in the presence of 100 μm myristoylated ARF5 peptide, but the effect was considerably less than that induced by the ARF6 peptide and did not reach significance in every experiment. 100 μm Myristoylated ARF1 peptide was without effect in this assay. A dose-response curve for these peptides on GLUT4 translocation is presented in Fig. 2 D. No significant effect on basal (unstimulated) plasma membrane levels of GLUT4 were observed after incubation with the peptides (Fig. 2 B), but low signal precludes detailed quantification of the level of GLUT4 at the plasma membrane in the absence of insulin. Insulin treatment of adipocytes results in the movement of other proteins to the plasma membrane, including the TfR, the IGF-II/cation-independent mannose-6-phosphate receptor (27Tanner L.I. Lienhard G.E. J. Biol. Chem. 1987; 262: 8975-8980Abstract Full Text PDF PubMed Google Scholar, 40Tanner L.I. Lienhard G.E. J. Cell Biol. 1989; 108: 1537-1545Crossref PubMed Scopus (84) Google Scholar) and GLUT1 (26Calderhead D.M. Kitagawa K. Tanner L.I. Holman G.D. Lienhard G.E. J. Biol. Chem. 1990; 265: 13801-13808Abstract Full Text PDF PubMed Google Scholar), albeit to a much lesser extent than is observed for GLUT4 (typically ∼2-fold compared with 12–15-fold). This is probably due to movement of these proteins from the recycling endosomal system to the plasma membrane. We wished to determine whether the effect of the myristoylated ARF6 peptide to inhibit insulin-stimulated GLUT4 translocation was specific for GLUT4, or whether other proteins which traffic between intracellular membranes and the cell surface in an insulin-regulated manner were also effected. We therefore examined the effect of insulin on cell surface levels of TfR and GLUT1 in permeabilized adipocytes incubated with myristoylated ARF peptides (Fig. 3). Insulin stimulation of permeabilized adipocytes results in a ∼2-fold increase in plasma membrane TfR levels, in agreement with published studies (27Tanner L.I. Lienhard G.E. J. Biol. Chem. 1987; 262: 8975-8980Abstract Full Text PDF PubMed Google Scholar, 40Tanner L.I. Lienhard G.E. J. Cell Biol. 1989; 108: 1537-1545Crossref PubMed Scopus (84) Google Scholar). Prior incubation with myristoylated ARF1 peptide did not reduce the magnitude of this response (Fig. 3 A). In contrast, prior incubation of 3T3-L1 adipocytes with myristoylated ARF5 peptide significantly inhibited the ability of insulin to stimulate TfR levels at the cell surface with no effect on the basal (unstimulated) TfR levels. In contrast, myristoylated ARF6 peptide reduced cell surface TfR levels in both basal and insulin-stimulated cells significantly compared with control cells, without significantly reducing the fold increase in cell surface TfR levels observed in response to insulin (Fig. 3 A). Similar data were also observed for GLUT1 (Fig. 3 B), with the exception that the reduction in plasma membrane GLUT1 levels in the presence of the ARF6 peptide were not as extensive as those observed for TfR (Fig.3 B). Consistent with this, we observed a diminution of basal (unstimulated) deoxy-Glc uptake in cells incubated with the ARF6 peptide (∼15% inhibition), but this effect did not reach statistical significance, presumably because the rate of basal transport is low (data not shown). Nevertheless, these data argue that both ARF5 and ARF6 are intimately involved in the trafficking of membrane proteins between the plasma membrane and the recycling endosomal system in this cell type. Here we have shown that insulin stimulation of 3T3-L1 adipocytes results in the redistribution of ARF5 from the soluble protein (cytosolic) fraction to the plasma membrane. We hypothesize that ARF5 recycles between the plasma membrane and intracellular (cytosolic) fractions presumably as a consequence of GDP/GTP exchange, and that insulin stimulates GTP loading of this protein. Despite this insulin-stimulated translocation of ARF5, our data is not consistent with a role for this protein in insulin-stimulated GLUT4 translocation. Rather, we suggest that ARF5 plays a role in the insulin-stimulated trafficking of membrane proteins between the recycling endosomal system and the plasma membrane, as evidenced by the ability of myristoylated ARF5 peptides to inhibit insulin-stimulated TfR and GLUT1 movement to the cell surface (Fig. 3). To our knowledge, this is the first demonstration of a functional role for ARF5 in membrane trafficking. In contrast to the data relating to ARF5, we show that a myristoylated peptide corresponding to the amino terminus of ARF6 partially inhibits insulin-stimulated GLUT4 translocation and deoxy-Glc transport in α-toxin permeabilized 3T3-L1 adipocytes (Fig. 2), implicating ARF6 as a key component of this response. Furthermore, we show that incubation of 3T3-L1 adipocytes with myristoylated ARF6 peptide reduces TfR number at the cell surface both in the basal state and after insulin stimulation, without decreasing the fold increase in cell surface levels in response to insulin (Fig. 3 A); similar results were observed for GLUT1, except the reduction in plasma membrane GLUT1 levels were not as great (Fig. 3 B). Collectively, these data argue that ARF6 plays a fundamental role in trafficking between intracellular membranes and the cell surface, indeed ARF6 has been proposed to mediate the targeting of recycling vesicles to the plasma membrane, either from a perinuclear compartment (CHO cells (21D'Souza-Schorey C. van Donselaar E. Hsu V.W. Yang C. Stahl P.D. Peters P.J. J. Cell Biol. 1998; 140: 603-616Crossref PubMed Scopus (197) Google Scholar)) or from a unique tubular-vesicular compartment (HeLa cells (33Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (421) Google Scholar)). With this in mind, several models may be proposed to explain the inhibitory effect of myristoylated ARF6 peptides on insulin-stimulated GLUT4 translocation. GLUT4 has been proposed to populate at least two distinct intracellular compartments, one of which corresponds to the recycling endosomal system, the other a specialized intracellular storage compartment referred to as GLUT4 storage vesicles (reviewed in Refs. 1Holman G.D. Cushman S.W. BioEssays. 1994; 16: 753-759Crossref PubMed Scopus (135) Google Scholar, 2Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, and 41Holman G.D. Leggio L.L. Cushman S.W. J. Biol. Chem. 1994; 269: 17516-17524Abstract Full Text PDF PubMed Google Scholar). Hence, the partial inhibition of insulin-stimulated GLUT4 translocation by myristoylated ARF6 peptides may be explained by the selective inhibition of translocation of one of the proposed multiple intracellular GLUT4 pools. This is consistent with previous studies which implicate ARF6 in the exocytosis of secretory vesicles in other cell types (22Galas M.-C. Helms J.B. Vitale N. Thiersé D. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). A second model, however, should also be considered which may also explain the experimental data presented here. Previous studies of ARF6 function in other cell types have shown that mutants of ARF6 disrupt receptor-mediated endocytosis (42D'Souza-Schorey C. Li G. Columbo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). Overexpression of ARF6 redistributed TfR to the cell surface, while a dominant negative mutant of ARF6 was shown to redistribute TfR to intracellular membranes and inhibit TfR recycling to the cell surface, suggesting that ARF6 is an integral component of the endocytic apparatus and that its GTP cycle/nucleotide status regulate progression through the endocytic pathway (42D'Souza-Schorey C. Li G. Columbo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). In agreement with these studies, we have shown that myristoylated ARF6 peptide decreases the cell surface levels of TfR both in the presence and absence of insulin without affecting the magnitude of the insulin-dependent increase in TfR at the cell surface (Fig. 3 A), consistent with ARF6 regulating progression of TfRs through the endocytic pathway. Hence, an alternative explanation of the data presented here is that the myristoylated ARF6 peptide functions in a fashion similar to a dominant negative ARF6, resulting in a change in the steady-state distribution of TfR such that the intracellular/plasma membrane ratio is increased. It is possible that after insulin stimulation when cell surface GLUT4 levels are increased, ARF6 function regulates the internalization/recycling of GLUT4 in a similar manner to that of the TfR. When ARF6 function is disrupted by the myristoylated peptide, intracellular levels of GLUT4 and TfR are increased, resulting in decreased plasma membrane levels of both of these proteins. This effect is only manifest for GLUT4 in the insulin-stimulated state as plasma membrane levels of GLUT4 in the absence of insulin are already very low. Hence, in this model, the role of ARF6 is not specific for any particular GLUT4 compartment, but rather is manifest at the level of GLUT4 recycling between the plasma membrane and intracellular compartments, which is known to occur even in the presence of insulin (1Holman G.D. Cushman S.W. BioEssays. 1994; 16: 753-759Crossref PubMed Scopus (135) Google Scholar, 41Holman G.D. Leggio L.L. Cushman S.W. J. Biol. Chem. 1994; 269: 17516-17524Abstract Full Text PDF PubMed Google Scholar, 43Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar). Distinguishing between these (or other) models for ARF6 action represents an important goal. These data also offer further insight into the ability of insulin to regulate membrane trafficking in adipocytes. We have shown that myristoylated ARF5 peptide inhibits insulin-stimulated GLUT1 and TfR translocation to the plasma membrane without significantly inhibiting GLUT4 translocation. This result implies that the pathways by which these two proteins reach the plasma membrane after insulin stimulation are distinct. Several studies have identified a GLUT4 compartment which is relatively devoid of TfR in both adipocytes and muscle (4Ploug T. van Deurs B. Cushman S.W. Ralston E. J. Cell Biol. 1998; 142: 1429-1446Crossref PubMed Scopus (240) Google Scholar, 44Ralston E. Ploug T. J. Cell Sci. 1996; 109: 2967-2978Crossref PubMed Google Scholar, 45Malide D. Dwyer N.K. Blanchette-Mackie E.J. Cushman S.W. J. Histochem. Cytochem. 1997; 45: 1083-1095Crossref PubMed Scopus (81) Google Scholar, 46Livingstone C. James D.E. Rice J.E. Hanpeter D. Gould G.W. Biochem. J. 1996; 315: 487-495Crossref PubMed Scopus (130) Google Scholar). This (GLUT4 storage vesicles) compartment has been suggested to be a GLUT4 storage compartment which is rapidly mobilized in response to insulin (2Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). Although there is ample data implicating some overlap between GLUT4 and the TfR in endosomes, it is possible that this overlap is mainly a consequence of these proteins sharing the same components of the endocytic arm of the recycling pathway. Indeed studies using mutant dynamin molecules has suggested that the slowly recycling (GLUT1/TfR positive) GLUT4 compartment contributes minimally to insulin-stimulated GLUT4 translocation (47Kao 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). In response to insulin when traffic through the recycling endosomal system is clearly increased, some GLUT4 will move to the cell surface from this location. We would suggest that this represents a modest proportion of the total insulin-stimulated GLUT4 translocation, as evidenced here by the lack of significant inhibition of GLUT4 translocation under conditions when GLUT1 and TfR translocation are significantly compromised (i.e. in the presence of ARF5 peptide). Hence we suggest that the main effect of insulin is to recruit GLUT4 from the post-endosomal storage compartment (GLUT4 storage vesicless), and that unlike insulin-stimulated movement through the recycling endosomes, this is independent of ARF5 function. In conclusion, we suggest that both ARF5 and ARF6 regulate trafficking of membrane proteins to and from the cell surface, and show that ARF6 plays an important regulatory role in the steady-state levels of GLUT4 at the adipocyte cell surface after insulin stimulation. We speculate that the actions of ARF5 and ARF6 are chiefly mediated by effects on the recycling endosomal system, at least in this insulin-responsive cell type. We thank Drs. Donaldson, Holman, Kahn, and Moss for their generous provision of antibodies used in this study and Dr. Francis Barr for constructive comments on this manuscript." @default.
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• W2058110898 title "Evidence for a Role for ADP-ribosylation Factor 6 in Insulin-stimulated Glucose Transporter-4 (GLUT4) Trafficking in 3T3-L1 Adipocytes" @default.
• W2058110898 cites W140498290 @default.
• W2058110898 cites W1482458531 @default.
• W2058110898 cites W1498459265 @default.
• W2058110898 cites W1526233070 @default.
• W2058110898 cites W1529589684 @default.
• W2058110898 cites W1583005540 @default.
• W2058110898 cites W1586115077 @default.
• W2058110898 cites W1967107611 @default.
• W2058110898 cites W1969766541 @default.
• W2058110898 cites W1975559877 @default.
• W2058110898 cites W1979365820 @default.
• W2058110898 cites W1983437370 @default.
• W2058110898 cites W1984474514 @default.
• W2058110898 cites W1997862023 @default.
• W2058110898 cites W2001049512 @default.
• W2058110898 cites W2008046589 @default.
• W2058110898 cites W2008597859 @default.
• W2058110898 cites W2024290580 @default.
• W2058110898 cites W2032941398 @default.
• W2058110898 cites W2033575261 @default.
• W2058110898 cites W2036467191 @default.
• W2058110898 cites W2038454505 @default.
• W2058110898 cites W2039180413 @default.
• W2058110898 cites W2044162798 @default.
• W2058110898 cites W2050051643 @default.
• W2058110898 cites W2051416430 @default.
• W2058110898 cites W2053533223 @default.
• W2058110898 cites W2059424055 @default.
• W2058110898 cites W2089617170 @default.
• W2058110898 cites W2097595798 @default.
• W2058110898 cites W2099942500 @default.
• W2058110898 cites W2107616801 @default.
• W2058110898 cites W2110905842 @default.
• W2058110898 cites W2115569175 @default.
• W2058110898 cites W2117483382 @default.
• W2058110898 cites W2119123632 @default.
• W2058110898 cites W2134406040 @default.
• W2058110898 cites W2139868649 @default.
• W2058110898 cites W2140752038 @default.
• W2058110898 cites W2150032835 @default.
• W2058110898 cites W2150158001 @default.
• W2058110898 cites W2163909866 @default.
• W2058110898 cites W2168648417 @default.
• W2058110898 cites W2184631394 @default.
• W2058110898 cites W2316810571 @default.
• W2058110898 cites W2328895879 @default.
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