ADP-ribosylation Factor 6 (ARF6) Defines Two Insulin-regulated Secretory Pathways in Adipocytes

The results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation.

Abstract

ADP-ribosylation factor 6 (ARF6) appears to play an essential role in the endocytic/recycling pathway in several cell types. To determine whether ARF6 is involved in insulin-regulated exocytosis, 3T3-L1 adipocytes were infected with recombinant adenovirus expressing wild-type ARF6 or an ARF6 dominant negative mutant (D125N) that encodes a protein with nucleotide specificity modified from guanine to xanthine. Overexpression of these ARF6 proteins affected neither basal nor insulin-regulated glucose uptake in 3T3-L1 adipocytes, nor did it affect the subcellular distribution of Glut1 or Glut4. In contrast, the secretion of adipsin, a serine protease specifically expressed in adipocytes, was increased by the expression of wild-type ARF6 and was inhibited by the expression of D125N. These results indicate a requirement for ARF6 in basal and insulin-regulated adipsin secretion but not in glucose transport. Our results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation. ADP-ribosylation factor 6 (ARF6) appears to play an essential role in the endocytic/recycling pathway in several cell types. To determine whether ARF6 is involved in insulin-regulated exocytosis, 3T3-L1 adipocytes were infected with recombinant adenovirus expressing wild-type ARF6 or an ARF6 dominant negative mutant (D125N) that encodes a protein with nucleotide specificity modified from guanine to xanthine. Overexpression of these ARF6 proteins affected neither basal nor insulin-regulated glucose uptake in 3T3-L1 adipocytes, nor did it affect the subcellular distribution of Glut1 or Glut4. In contrast, the secretion of adipsin, a serine protease specifically expressed in adipocytes, was increased by the expression of wild-type ARF6 and was inhibited by the expression of D125N. These results indicate a requirement for ARF6 in basal and insulin-regulated adipsin secretion but not in glucose transport. Our results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation. ADP-ribosylation factor phospholipase D Dulbecco's modified Eagle's medium plasma membrane glucose transporter 1 glucose transporter 4 brefeldin A guanosine 5′-3-O-(thio) triphosphate ADP-ribosylation factors are members of the Ras superfamily of low molecular weight GTP-binding proteins. Although ARFs1 were identified as cofactors required for the cholera toxin-catalyzed ADP-ribosylation of heterotrimeric G protein, Gs (1Kahn R.A. Gilman A.G. J. Biol. Chem. 1986; 261: 7906-7911Abstract Full Text PDF PubMed Google Scholar), they have subsequently been shown to play an important role in numerous membrane trafficking events. In addition, ARFs also stimulate the activity of phospholipase D (PLD)in vitro (2Caumont A. Galas M. Vitale N. Aunis D. Bader M. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 3Kahn R.A. Yucel J.K. Malhotra V. Cell. 1993; 75: 1045-1048Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 4Liscovitch M. Cantley L.C. Cell. 1995; 81: 659-662Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 5Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2003) Google Scholar), suggesting that they may exert their effects at least in part by altering membrane phospholipid metabolism. ARF6, one member of the ARF family, has been suggested to play a role in vesicle trafficking and in cytoskeletal organization (2Caumont A. Galas M. Vitale N. Aunis D. Bader M. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 6D'Souza-Schorey C. Li G.P. Colombo M. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar, 7D'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 (196) Google Scholar, 8D'Souza-Schorey C. Boshans R. McDonough M. Stahl P.D. Van Aelst L. EMBO J. 1998; 16: 5445-5454Crossref Scopus (205) Google Scholar, 9Galas M. Helms J. Vitale N. Thierse D. Aunis D. Bader M. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 10Peters P. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar, 11Radhakrishna H. Klausner R. Donaldson J.G. J. Cell Biol. 1996; 134: 935-947Crossref PubMed Scopus (214) Google Scholar, 12Song J. Khachikian Z. Radhakrishna H. Donaldson J.G. J. Cell Sci. 1998; 111: 2257-2267Crossref PubMed Google Scholar, 13Zhang Q. Cox D. Tseng C. Donaldson J. Greenberg S. J. Biol. Chem. 1998; 273: 19977-19981Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The available data suggest that ARF6 has a cell type-dependent subcellular distribution (14Yang C. Heimberg H. D'Souza-Schorey C. Meukler M. Stahl P. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and cell type-dependent function. In Chinese hamster ovary (CHO) cells, endogenous ARF6 is exclusively localized on the plasma membrane (15Cavenagh M.M. Whiteney J.A. Carroll K. Zhang C.-J. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and overexpressed ARF6 is localized at the plasma membrane and in endosomes, suggesting a role for this protein in membrane trafficking along the endocytic pathway (6D'Souza-Schorey C. Li G.P. Colombo M. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar, 10Peters P. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). However, overexpressed ARF6 in HeLa cells is localized to the plasma membrane and to a tubulovesicular compartment that is distinct from transferrin-positive endosomes (11Radhakrishna H. Klausner R. Donaldson J.G. J. Cell Biol. 1996; 134: 935-947Crossref PubMed Scopus (214) Google Scholar, 16Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (417) Google Scholar). Others have found that ARF6 is localized to chromaffin granules and is involved in exocytosis during regulated secretion (2Caumont A. Galas M. Vitale N. Aunis D. Bader M. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Galas M. Helms J. Vitale N. Thierse D. Aunis D. Bader M. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Finally, in normal rat kidney (NRK) cells, endogenous and overexpressed ARF6 localizes to the plasma membrane and to juxtanuclear region and may play a role in modeling the plasma membrane and in cortical actin organization (12Song J. Khachikian Z. Radhakrishna H. Donaldson J.G. J. Cell Sci. 1998; 111: 2257-2267Crossref PubMed Google Scholar). In adipocytes, insulin affects several processes associated with intracellular membrane trafficking. Insulin enhances glucose transporter 4 (Glut4) translocation from intracellular compartments to the plasma membrane (17Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (951) Google Scholar), stimulates accumulation of transferrin receptors and insulin-like growth factor II receptors on the cell surface (18Tanner L.I. Lienhard G.E. J. Cell Biol. 1989; 108: 1537-1545Crossref PubMed Scopus (84) Google Scholar), and increases the secretion of several proteins (19Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar, 20Scherer P. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2733) Google Scholar). The molecular mechanism by which insulin induces intracellular redistribution of Glut4 is still unclear, but it probably stimulates exocytosis (21Rampal A.L. Jhun B.H. Kim S. Liu H. Manka M. Lachaal M. Spangler R.A. Jung C.Y. J. Biol. Chem. 1995; 270: 3938-3943Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and inhibits endocytosis of the transporter (22Holman G.D. Cushman S.W. Bioessays. 1994; 16: 753-759Crossref PubMed Scopus (135) Google Scholar). Adipsin, a serine protease specifically expressed in adipocytes, is constitutively secreted from adipocytes, and its secretion is augmented 2–3-fold by insulin treatment (19Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar, 20Scherer P. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2733) Google Scholar). In addition, insulin stimulates PLD activity in rat adipocytes (23Standaert M.L. Avignon A. Yamada K. Bandyopadhyay G. Farese R.V. Biochem. J. 1996; 313: 1039-1046Crossref PubMed Scopus (60) Google Scholar), which may be important for activation of signaling and targeting processes in the plasma membrane. Although it has been shown that ARF proteins are associated with the insulin receptor (24Shome K. Vasudevan C. Romero G. Curr. Biol. 1997; 7: 387-396Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), it is unknown whether ARF6 is required for insulin-stimulated membrane trafficking. In this study, we examined the role of ARF6 in two insulin-regulated processes in 3T3-L1 adipocytes: glucose transport and adipsin secretion. Our data strongly suggest the involvement of ARF6 in basal and insulin-regulated adipsin secretion but not in insulin-stimulated glucose transport. Insulin, dexamethasone, isobutylmethylxanthine, [3H]-2-deoxyglucose, and Dulbecco's modified Eagle's medium (DMEM) were from Sigma. The production of mouse anti-ARF6 antibody was described previously (14Yang C. Heimberg H. D'Souza-Schorey C. Meukler M. Stahl P. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Goat anti-adipsin antibody was kindly provided by Dr. Jess Miner (University of Nebraska). 3T3-L1 fibroblasts were grown to confluence and 2 days later were differentiated as described previously (25Tordjman K.M. Leingang K.A. James D.E. Mueckler M.M. Proc. Natl. Acad. Sci., U. S. A. 1989; 86: 7761-7765Crossref PubMed Scopus (116) Google Scholar). 3T3-L1 adipocytes were used for experiments between 10 and 14 days after differentiation. 293 cells were grown at 37 °C in DMEM with 10% fetal bovine serum in the presence of 50 units of penicillin per ml and 50 μg of streptomycin per ml and 5% CO2. The human ARF6 cDNA, provided by M. Vaughan and J. Moss (National Institutes of Health) (26Tsuchiya M. Price S.R. Tsai S. Moss J. Vaughan M. J. Biol. Chem. 1991; 266: 2772-2777Abstract Full Text PDF PubMed Google Scholar), was amplified using the polymerase chain reaction and primers containing Bgl II restriction sites. Wild-type ARF6 and its mutant D125N were subcloned into the Bam HI site of the adenovirus vector pACCMV (provided by C. Newgard, University of Texas Southwestern Medical Center, Dallas, TX) which was previously linearized by Bam HI digestion and treated with calf alkaline phosphatase. The ARF6 mutant, D125N, was constructed by the method of Kunkel (27Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4897) Google Scholar). The mutagenic oligonucleotide used was 5′-GCCAACAAGCAGAACCTGCCCGAT-3′. Recombinant viruses were generated as described previously (28Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (562) Google Scholar). Briefly, the plasmid pACCMV-ARF6 (1 μg) was cotransfected with the plasmid pJM17 (4 μg, provided by C. Newgard) into 293 cells by using the transfection kit from Stratagene (Transfection MBS, mammalian transfection kit). Cell lysis indicative of recombination occurred 1–2 weeks following cotransfection. Cell lysates were subjected to immunoblot analysis to assay for the expression of ARF6 protein. The recombinant viruses were amplified and purified as described (28Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (562) Google Scholar). Purified viruses (1–5 × 1010 plaque-forming units/ml) were stored at −80 °C in Tris-buffered saline containing 1% bovine serum albumin and 10% glycerol. 5 × 109 plaque-forming units of virus were used to infect 35-mm dishes of 3T3-L1 adipocytes overnight. The infected cells were fed with fresh DMEM containing 10% fetal bovine serum for an additional 48 h before use in experiments. 3T3-L1 adipocytes were serum-starved for at least 3 h, washed three times with Krebs-Ringer phosphate buffer and then treated or not with insulin (1 μm) for 20 min at 37 °C. [3H]-2 - Deoxyglucose uptake was measured as described previously (25Tordjman K.M. Leingang K.A. James D.E. Mueckler M.M. Proc. Natl. Acad. Sci., U. S. A. 1989; 86: 7761-7765Crossref PubMed Scopus (116) Google Scholar). Nonspecific background uptake in the presence of the inhibitor cytochalasin B (20 μm) was subtracted from all values. Glucose transport activity was normalized to protein concentration measured using the bicinchoninic acid assay (Pierce). 3T3-L1 adipocytes grown in 35-mm dishes were serum-starved and treated as described above. Plasma membrane (PM) lawns were prepared as described (29Elmendorf J.S. Chen D. Pessin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and solubilized in 150 μl of 1% SDS in PBS. The protein concentration was determined using the Pierce bicinchoninic acid assay kit. 1.6–2 μg of total protein was used for immunoblot analysis. 3T3-L1 adipocytes grown in 35-mm dishes were serum-starved for at least 3 h before the start of an experiment. Cells were treated or not with insulin (1 μm) for 30 min, and the cell culture media were collected and precipitated with 10% trichloroacetic acid. Precipitated proteins were used for immunoblot analysis with anti-adipsin antibody (30Spurlock M.E. Hahn K.J. Miner J.L. Physiol. Behav. 1996; 60: 1217-1221Crossref PubMed Scopus (20) Google Scholar). Polyacrylamide gel electrophoresis and immunoblot analysis of ARF6 were performed as described (14Yang C. Heimberg H. D'Souza-Schorey C. Meukler M. Stahl P. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Immunoblot analysis for Glut1 and Glut4 were performed as described by Hresko et al. (31Hresko R.C. Heimberg H. Chi M.M. Mueckler M. J. Biol. Chem. 1998; 273: 20658-20668Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). GTP-overlay blot was performed as described. Proteins were resolved on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose as described above (14Yang C. Heimberg H. D'Souza-Schorey C. Meukler M. Stahl P. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Briefly, nitrocellulose sheets were preincubated for 30 min at room in 50 containing 5 and with 1 was for 1 h in the were then washed times with the and at −80 °C. To determine whether ARF6 is involved in insulin-regulated membrane trafficking in 3T3-L1 adipocytes, we expressed wild-type or (D125N) mutant ARF6 proteins in 3T3-L1 adipocytes using an adenovirus expression of ARF6 proteins was by immunoblot analysis. shown in of ARF6 were in 3T3-L1 adipocytes infected with the recombinant with The use of that of the 3T3-L1 adipocytes were infected by the of virus expressing not The in a of the protein by to is to with the guanine nucleotide H. D. Nature. 1991; PubMed Scopus Google Scholar). and the are all GTP-binding proteins. examined the of the wild-type and the mutant to cell lysates were prepared from 3T3-L1 adipocytes that were not infected or infected with recombinant adenovirus expressing wild-type ARF6 and its mutant D125N, and the lysates were subjected to with anti-ARF6 antibody (14Yang C. Heimberg H. D'Souza-Schorey C. Meukler M. Stahl P. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). were then subjected to immunoblot or GTP-overlay blot analysis. shown in the mutant protein to to the wild-type ARF6 in the Insulin stimulates translocation of the glucose Glut1 and Glut4 from intracellular compartments to the cell surface and increases glucose uptake in differentiated 3T3-L1 To determine whether of ARF6 affects glucose 3T3-L1 adipocytes were not infected or infected with recombinant adenovirus expressing wild-type ARF6, or the ARF6 mutant, D125N. The or serum-starved 3T3-L1 adipocytes were treated or not with insulin for 30 min, and uptake was then shown in insulin-stimulated glucose uptake was not affected by the of wild-type or mutant with all did in a in basal glucose To determine whether ARF6 affects Glut1 or Glut4 lawns were prepared from and 3T3-L1 The collected lawns were used for immunoblot analysis using Glut1 or Glut4. 3 that of wild-type ARF6 or its mutant D125N did not the of Glut1 or Glut4 in the plasma membrane basal or insulin-stimulated to cells or cells that expressed The in basal glucose transport by may be of an in the activity of Glut1 or Glut4 in the or to a in the basal of transporter that not be by this has been shown that ARF6 is involved in regulated exocytosis in chromaffin cells (2Caumont A. Galas M. Vitale N. Aunis D. Bader M. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Galas M. Helms J. Vitale N. Thierse D. Aunis D. Bader M. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), we determined the of ARF6 on a regulated pathway in 3T3-L1 Adipsin, a serine protease specifically expressed in cells, is constitutively secreted into the media and its secretion is by 3T3-L1 adipocytes were not infected or infected with adenovirus expressing wild-type ARF6, or the D125N shown in the expression of wild-type ARF6 increased the release of adipsin into the medium basal and insulin-stimulated In contrast, the expression of the mutant inhibited with virus did not adipsin secretion. In this we that ARF6 is involved in basal and insulin-stimulated adipsin but that of ARF6 not affect the of Glut1 or Glut4 in the plasma membrane basal or insulin-stimulated Our results suggest the existence of at least two compartments or pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin secretion and one for the glucose Glut1 and Glut4 undergo endocytosis and J.E. D.E. J.S. K.J. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a the of Glut1 and Glut4 on the cell surface the of their and Insulin the accumulation of Glut1 and Glut4 on the plasma membrane by their exocytosis by the of their J.E. D.E. J.S. K.J. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have been in of the of the of Glut4. of the suggest a compartment for Glut4 in adipocytes and J.E. D.E. J.S. K.J. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the that ARF6 is involved in endocytosis and exocytosis of Glut1 and Glut4 in 3T3-L1 adipocytes, or negative on processes by ARF6 wild-type or its dominant negative mutant, D125N, not affect the of Glut1 or Glut4 on the plasma membrane basal or insulin-stimulated it is that ARF6 is involved in neither endocytosis nor exocytosis of Glut1 and Glut4 in 3T3-L1 To these of the of endocytosis and exocytosis for glucose in 3T3-L1 adipocytes be The available data indicate that ARF6 is not required for membrane trafficking endocytosis (6D'Souza-Schorey C. Li G.P. Colombo M. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar), membrane C. van Donselaar E. Hsu V.W. Yang C. Stahl P.D. Peters P.J. J. Cell Biol. 1998; 140: 603-616Crossref PubMed Scopus (196) Google Scholar, 16Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (417) Google Scholar), and regulated exocytosis M. Helms J. Vitale N. Thierse D. Aunis D. Bader M. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), but also an important role in cytoskeletal organization C. Boshans R. McDonough M. Stahl P.D. Van Aelst L. EMBO J. 1998; 16: 5445-5454Crossref Scopus (205) Google Scholar, 11Radhakrishna H. Klausner R. Donaldson J.G. J. Cell Biol. 1996; 134: 935-947Crossref PubMed Scopus (214) Google Scholar). In addition, a in chromaffin cells, ARF6 may in exocytosis by PLD activity on the plasma membrane (2Caumont A. Galas M. Vitale N. Aunis D. Bader M. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). results have been for and has been shown that and secretion in to cytoskeletal organization A.M. D. G. Biol. Cell. 1998; PubMed Scopus Google Scholar, Price R. A. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar, A. A. Biol. Cell. 1996; 7: PubMed Scopus (52) Google Scholar). it has been that of the of the of endocytosis C. L.J. Nature. 1996; PubMed Scopus Google Scholar) and that the actin is required for endocytosis C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The molecular between membrane cytoskeletal and PLD activity is still it has been that ARF6 with a protein, C. Boshans R. McDonough M. Stahl P.D. Van Aelst L. EMBO J. 1998; 16: 5445-5454Crossref Scopus (205) Google Scholar). The factor for ARF S. S. J.E. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and factor for M. Peters P.J. J. Donselaar A. D'Souza-Schorey C. P. EMBO J. PubMed Scopus Google Scholar), also be involved in membrane trafficking and the GTP-binding J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. A. Science. 1993; PubMed Scopus Google Scholar), S. R.J. N. J. Cell Biol. 1995; PubMed Scopus Google Scholar), V. M. K. I. C. R. M. Nature. 1996; PubMed Scopus Google Scholar, S. L. D.S. M. Biochem. 1995; PubMed Scopus Google Scholar), and the heterotrimeric G protein J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) have been to the in the D is to is to the which has been shown to a in guanine nucleotide and its dominant negative effects by the factor for A. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). A mechanism of has been for Ras dominant in this and P. A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, M. D. S. Cell. Biol. 1993; PubMed Scopus Google Scholar, S. E. T. J. D. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Although we not the mechanism by which a dominant negative on adipsin we that which is for to and in a in ARF6 factor which is essential for ARF6 to membrane trafficking. is that the D125N have a on ARF a to the of ARF In addition, the its dominant by the it may be a for the of these ARF6 is from members of the ARF by its to brefeldin A a has been shown that the organization of the T. K. S. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), from and L. M. M. A. Donaldson J.G. J. Klausner R.D. J.E. Cell. 1992; Scholar), and to of proteins to the To determine the of on the release of adipsin from 3T3-L1 adipocytes, adipsin secretion was measured in or 3T3-L1 adipocytes treated or with 10 for 30 of on basal or adipsin secretion was in experiments not with the role of ARF6 in this In addition, we did not of on basal or insulin-regulated glucose uptake not which is with previously results S. L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. M. S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). be of to determine whether ARF6 a role in of regulated exocytosis in to adipsin secretion. are the of ARF6 on insulin secretion in are also the of the mutant on glucose transport in 3T3-L1 adipocytes it appears that insulin and glucose transport Z. Yang and M. on J. Miner (University of for anti-adipsin antibody and C. Newgard (University of Texas Southwestern Medical for adenovirus vector pACCMV and also M. Vaughan and J. Moss (National Institutes of Health) for the ARF6