α-Actinin-4 Is Selectively Required for Insulin-induced GLUT4 Translocation
2008; Elsevier BV; Volume: 283; Issue: 37 Linguagem: Inglês
10.1074/jbc.m801750200
ISSN1083-351X
AutoresIlana Talior‐Volodarsky, Varinder K. Randhawa, Hilal Zaid, Amira Klip,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoInsulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64–75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of α-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton. Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64–75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of α-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton. Insulin-regulated glucose transporter 4 (GLUT4) 4The abbreviations used are: GLUT4glucose transporter 4SILACstable isotope labeling by amino acids in cell cultureACTNα-actininPDGFplatelet-derived growth factorDNPdinitrophenolsiRNAsmall interfering RNAsiACTN4ACTN-4-specific siRNAsiNRnon-related control siRNANTnon-transfected cellsAktprotein kinase BPI3Kphosphoinositide 3-kinaseVAMPvesicle-associated membrane proteinGFPgreen fluorescent proteinGSTglutathione S-transferasePBSphosphate-buffered saline. 4The abbreviations used are: GLUT4glucose transporter 4SILACstable isotope labeling by amino acids in cell cultureACTNα-actininPDGFplatelet-derived growth factorDNPdinitrophenolsiRNAsmall interfering RNAsiACTN4ACTN-4-specific siRNAsiNRnon-related control siRNANTnon-transfected cellsAktprotein kinase BPI3Kphosphoinositide 3-kinaseVAMPvesicle-associated membrane proteinGFPgreen fluorescent proteinGSTglutathione S-transferasePBSphosphate-buffered saline. is a member of the SLC2A facilitative glucose transporter family (2Joost H.G. Bell G.I. Best J.D. Birnbaum M.J. Charron M.J. Chen Y.T. Doege H. James D.E. Lodish H.F. Moley K.H. Moley J.F. Mueckler M. Rogers S. Schurmann A. Seino S. Thorens B. Am. J. Physiol. Endocrinol. Metab. 2002; 282: 974-976Crossref PubMed Scopus (326) Google Scholar) and is responsible for glucose entry into muscle and fat tissues (3Herman M.A. Kahn B.B. J. Clin. Investig. 2006; 116: 1767-1775Crossref PubMed Scopus (263) Google Scholar, 4Tsao T.S. Stenbit A.E. Factor S.M. Chen W. Rossetti L. Charron M.J. Diabetes. 1999; 48: 775-782Crossref PubMed Scopus (48) Google Scholar, 5Rudich A. Konrad D. Torok D. Ben-Romano R. Huang C. Niu W. Garg R.R. Wijesekara N. Germinario R.J. Bilan P.J. Klip A. Diabetologia. 2003; 46: 649-658Crossref PubMed Scopus (97) Google Scholar). GLUT4 continuously cycles to/from the cell membrane through a series of endosomal compartments. 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Rev. 2004; 25: 177-204Crossref PubMed Scopus (350) Google Scholar, 23Rudich A. Klip A. Acta Physiol. Scand. 2003; 178: 297-308Crossref PubMed Scopus (61) Google Scholar), the gain in surface GLUT4 elicited by platelet-derived growth factor (PDGF), depolarization, or mitochondrial uncouplers is independent of actin dynamics (24Taha C. Tsakiridis T. McCall A. Klip A. Am. J. Physiol. 1997; 273: E68-E76PubMed Google Scholar, 25Torok D. Patel N. Jebailey L. Thong F.S. Randhawa V.K. Klip A. Rudich A. J. Cell Sci. 2004; 117: 5447-5455Crossref PubMed Scopus (45) Google Scholar, 26Tsakiridis T. Vranic M. Klip A. Biochem. J. 1995; 309: 1-5Crossref PubMed Scopus (63) Google Scholar).Intensive research has recently focused on identifying the individual mechanisms participating in GLUT4 traffic and the specific events regulated by insulin (27van Dam E.M. Govers R. James D.E. Mol. Endocrinol. 2005; 19: 1067-1077Crossref PubMed Scopus (88) Google Scholar, 28Gonzalez E. McGraw T.E. Mol. Biol. Cell. 2006; 17: 4484-4493Crossref PubMed Scopus (160) Google Scholar, 29Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (143) Google Scholar, 30Bai L. Wang Y. Fan J. Chen Y. Ji W. Qu A. Xu P. James D.E. Xu T. Cell Metab. 2007; 5: 47-57Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 31Koumanov F. Jin B. Yang J. Holman G.D. Cell Metab. 2005; 2: 179-189Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Hypothesizing that GLUT4 traffic may be regulated by interaction with partner proteins, it is of fundamental and clinical interest to identify such proteins. Accordingly, we recently applied the novel SILAC (stable isotope labeling by amino acids in cell culture) approach (32Ong S.E. Mann M. Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (670) Google Scholar) to search for proteins that associate with GLUT4 in an insulin-regulated manner (1Foster L.J. Rudich A. Talior I. Patel N. Huang X. Furtado L.M. Bilan P.J. Mann M. Klip A. J. Proteome Res. 2006; 5: 64-75Crossref PubMed Scopus (94) Google Scholar). The study took advantage of the stable expression in L6 muscle cells of GLUT4 encoding an myc tag that faces the extracellular or luminal spaces. Immunoprecipitation via the myc epitope avoided interference of the antibody with endogenous partners of GLUT4. Through this strategy we identified a discrete cohort of proteins that were more abundant in immunoprecipitates from insulin-stimulated than control cells (1Foster L.J. Rudich A. Talior I. Patel N. Huang X. Furtado L.M. Bilan P.J. Mann M. Klip A. J. Proteome Res. 2006; 5: 64-75Crossref PubMed Scopus (94) Google Scholar). One of the most responsive proteins was α-actinin-4 (ACTN4). ACTN4 is an ≅100-kDa protein containing four spectrin-like repeats and a globular N-terminal actin binding domain that forms an anti-parallel homodimer and can cross-link actin filaments or link membrane proteins to F-actin (33Djinovic-Carugo K. Young P. Gautel M. Saraste M. Cell. 1999; 98: 537-546Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar).Here we hypothesize that ACTN4 might contribute to GLUT4 translocation toward the cell surface, providing a functional link between the transporter and actin filaments. This postulate was tested using the L6 muscle cells stably expressing GLUT4myc, which exhibits GLUT4 cycling and regulation by insulin, PDGF, membrane depolarization, and mitochondrial uncouplers (18Wijesekara N. Tung A. Thong F. Klip A. Am. J. Physiol. Endocrinol. Metab. 2006; 290: 1276-1286Crossref PubMed Scopus (69) Google Scholar, 25Torok D. Patel N. Jebailey L. Thong F.S. Randhawa V.K. Klip A. Rudich A. J. Cell Sci. 2004; 117: 5447-5455Crossref PubMed Scopus (45) Google Scholar, 34Kanai F. Nishioka Y. Hayashi H. Kamohara S. Todaka M. Ebina Y. J. Biol. Chem. 1993; 268: 14523-14526Abstract Full Text PDF PubMed Google Scholar, 35Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar, 36Khayat Z.A. Patel N. Klip A. Can J. Appl. Physiol. 2002; 27: 129-151Crossref PubMed Scopus (26) Google Scholar). We applied siRNA-mediated ACTN4 knockdown coupled to GLUT4myc immunolocalization by confocal fluorescence microscopy, quantification of surface exposure of the myc epitope and glucose uptake measurements, and conclude that ACTN4 is required to gather GLUT4 beneath the cell membrane exclusively in response to insulin.EXPERIMENTAL PROCEDURESReagents, Constructs, and siRNAs—o-Phenylenediamine dihydrochloride, monoclonal anti-ACTN1 (mouse IgM, clone BM-75.2 (37Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (388) Google Scholar)), anti-β-actin or polyclonal anti-myc antibodies, rat PDGF-BB, iron-saturated human transferrin, and LY249002 were from Sigma-Aldrich. 9E10 monoclonal antibody affinity matrix (AFC-150P) was from Covance Research Products (Berkeley, CA). Monoclonal (9E10) and polyclonal (A-14) anti-myc antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal ACTN4 antibody was from Alexis Biochemicals (San Diego, CA). Polyclonal anti-Akt and anti-phospho Akt (serine 473) antibodies were from Cell Signaling Technology (Beverly, MA). Indocarbocyanine (Cy3)-, Cy5-, or horseradish peroxidase-conjugated goat-anti-mouse and anti-rabbit IgG antibodies or donkey anti-mouse IgM were from Jackson ImmunoResearch Laboratories (West Grove, PA). Isopropylthiogalactopyranoside, rhodamine-bound phalloidin, and Alexa 488/Alexa 647-conjugated goat anti-mouse and anti-rabbit IgG antibodies were from Invitrogen. d-[2-deoxy-3H]glucose and 125I-labeled transferrin were from PerkinElmer Life Sciences. siRNAs targeted against ACTN4 (siACTN4, UCA ACG AAC UGG ACU ACU AUU), ACTN1 (siACTN1, CAC UUA UCU UCG ACA AUA A), or non-related control (siNR, AUU CUA UCA CUA GCG UGA CUU) were from Qiagen (Valencia, CA). Enhanced green fluorescent protein-tagged human ACTN4 (ACTN4-GFP) cDNA, previously described (38Hara T. Honda K. Shitashige M. Ono M. Matsuyama H. Naito K. Hirohashi S. Yamada T. Mol. Cell. Proteomics. 2007; 6: 479-491Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 39Honda K. Yamada T. Seike M. Hayashida Y. Idogawa M. Kondo T. Ino Y. Hirohashi S. Oncogene. 2004; 23: 5257-5262Crossref PubMed Scopus (47) Google Scholar), was a kind gift of Dr. Kazufumi Honda (National Cancer Center Research Institute, Tokyo, Japan). Enhanced green fluorescent protein-tagged C-terminal K-Ras tail (K-Ras tail-GFP) cDNA, as in Yeung et al. (40Yeung T. Terebiznik M. Yu L. Silvius J. Abidi W.M. Philips M. Levine T. Kapus A. Grinstein S. Science. 2006; 313: 347-351Crossref PubMed Scopus (250) Google Scholar), was a kind gift of Dr. Sergio Grinstein (Hospital for Sick Children, Toronto). All cDNA constructs were prepared by Qiagen Hi-Speed Maxi-prep kits according to the manufacturer's protocol.Cell Culture and Transfections—L6 rat myoblasts stably expressing GLUT4 with an exofacial myc epitope (L6-GLUT4myc) and L6 wild-type myoblasts were differentiated into myotubes as described (34Kanai F. Nishioka Y. Hayashi H. Kamohara S. Todaka M. Ebina Y. J. Biol. Chem. 1993; 268: 14523-14526Abstract Full Text PDF PubMed Google Scholar, 35Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar). Post-seeding day-2 myoblasts or day-4 myotubes were non-transfected (NT) or transfected with 100 nm siACTN4, siACTN1, or siNR using calcium phosphate (CellPhect Transfection kit; GE Healthcare). siRNA-calcium phosphate precipitates were removed 12 h after addition, and cells were maintained for 72 h until experimentation. siACTN4 and siACTN1 were each effective against their targets (see "Results"). K-Ras tail-GFP or ACTN4-GFP cDNAs were, respectively, transfected by calcium phosphate as above or Lipofectamine 2000® (Invitrogen).Cell Lysates and Immunoblotting—Cells were lysed as described (41Niu W. Huang C. Nawaz Z. Levy M. Somwar R. Li D. Bilan P.J. Klip A. J. Biol. Chem. 2003; 278: 17953-17962Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and equal protein samples were resolved by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Bio-Rad), and immunoblotted with anti-ACTN4 or anti-ACTN1 (1:2000), anti-β-actin (1:5000), anti-phospho-Ser473 Akt, phospho-Thr308 Akt, anti-phosphoextracellular signal-regulated kinase (1:1000), or anti-myc (1:1000) antibodies. Horseradish peroxidase-bound secondary antibodies were detected by Western Lightning™ chemiluminescence reagent plus (PerkinElmer Life Sciences).GLUT4myc Immunoprecipitation—L6-GLUT4myc myoblasts seeded in 10-cm dishes and grown to confluence or differentiated into myotubes were serum-deprived for 4 h before stimulation with 100 nm insulin for 20 min, 50 ng/ml PDGF for 7 min, or 25 μm LY249002 for 20 min at 37 °C. Lysates (1.5 mg) were immunoprecipitated with 40 μl of 9E10 Monoclonal Affinity Matrix as described (1Foster L.J. Rudich A. Talior I. Patel N. Huang X. Furtado L.M. Bilan P.J. Mann M. Klip A. J. Proteome Res. 2006; 5: 64-75Crossref PubMed Scopus (94) Google Scholar).GST Pulldown Assay—Fragments corresponding to the GLUT4 N′-terminal tail (amino acids 1–24), large central loop (amino acids 222–287), and C′-terminal tail (476–509) were fused together and ligated into the EcoRI and XhoR sites of pGEX-2T vector (Amersham Biosciences) and called cytoGLUT4-GST. The sequence coding for human ACTN4 was cut from the ACTN4-GFP construct and introduced into the Nde1 and Nhe1 sites of vector pET-27b (+) (here called ACTN4-His). The fusion proteins and GST were expressed in DH5α cells after induction with 0.3 mm isopropylthiogalactopyranoside and purified on glutathione-Sepharose (GE Healthcare) or nickel-nitrilotriacetic acid-agarose (Qiagen), respectively. Purified GST was used as negative control. Myoblasts were lysed in PBS with 1% Triton X-100 and protease inhibitors. Freshly prepared cytoGLUT4-GST and GST bound to beads were mixed with 1 mg of cell lysate or ACTN4-His, incubated overnight at 4 °C, harvested by centrifugation, and washed in PBS buffer containing Triton X-100. Bound proteins were eluted in sample buffer and visualized by immunoblotting.Glucose Uptake and Cell-surface GLUT4myc Detection—Cells grown in 24-well plates and serum-starved for 3–5 h were treated without or with 100 nm insulin for 20 min, and 2-deoxyglucose uptake was measured as described (5Rudich A. Konrad D. Torok D. Ben-Romano R. Huang C. Niu W. Garg R.R. Wijesekara N. Germinario R.J. Bilan P.J. Klip A. Diabetologia. 2003; 46: 649-658Crossref PubMed Scopus (97) Google Scholar) using 10 μm [2-deoxy-3H]glucose. Cell-surface GLUT4myc was detected as described (35Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar, 42Wang Q. Khayat Z. Kishi K. Ebina Y. Klip A. FEBS Lett. 1998; 427: 193-197Crossref PubMed Scopus (185) Google Scholar). Briefly, serum-deprived cells left untreated or treated for 7 min with 50 ng/ml PDGF or for 20 min with 100 nm insulin, 0.5 mm DNP, or 120 mm K+ at 37 °C as in Wijesekara et al. (18Wijesekara N. Tung A. Thong F. Klip A. Am. J. Physiol. Endocrinol. Metab. 2006; 290: 1276-1286Crossref PubMed Scopus (69) Google Scholar) and Torok et al. (25Torok D. Patel N. Jebailey L. Thong F.S. Randhawa V.K. Klip A. Rudich A. J. Cell Sci. 2004; 117: 5447-5455Crossref PubMed Scopus (45) Google Scholar) were washed twice with ice-cold PBS, blocked 10 min with 3% (v/v) goat serum, and reacted with polyclonal anti-myc antibody (1:200) for 1 h at 4 °C. Cells were fixed for 10 min with 3% paraformaldehyde, reacted with horseradish peroxidase-bound goat anti-rabbit secondary antibody (1:2000) for 1 h at 4 °C, washed 6 times with PBS, and incubated with 1 ml o-phenylenediamine dihydrochloride reagent and allowed to develop for 20–30 min in the linear range in the dark at room temperature. The reaction was stopped with 1 ml/well of 3 n HCl. Supernatants were collected and absorbance was measured at 492 nm. Background absorbance obtained in the absence of anti-myc antibody was subtracted from all values.Immunofluorescence Microscopy, Image Acquisition, and Analyses—After insulin treatment, cells were fixed and permeabilized in 0.1% (v/v) Triton X-100 for 3 min at 4 °C to preserve actin morphology. Labeling of actin filaments with rhodaminebound phalloidin and antigen-specific immunostaining was as noted (43Khayat Z.A. Tong P. Yaworsky K. Bloch R.J. Klip A. J. Cell Sci. 2000; 113: 279-290Crossref PubMed Google Scholar), using monoclonal anti-myc 1:100 and polyclonal anti-ACTN4 1:200 in 0.1% (w/v) bovine serum albumin in PBS, together with fluorophore-coupled secondary antibodies for 1 h at room temperature. To prevent artifactual co-localization, fluorophores of maximal spectral separation were used. To reduce possible fluorophore spectra crossover, cells were imaged by multichannel scanning with a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). Acquisition parameters were adjusted to exclude saturation of the signal.For rounded-up L6-GLUT4myc myoblasts, cells detached from the substratum with Ca2+- and Mg2+-free PBS at 37 °C were left untreated or treated with 100 nm insulin for 20 min at 37 °C on re-attachment to glass coverslips as noted. Fixed cells were processed intact for myc epitope detection or permeabilized for staining of the myc epitope, ACTN4 or actin, as above. Images were obtained with a Zeiss Axiovert 100M laser scanning confocal microscope at room temperature using a 60× or 100× oil objective at the same gain setting unless indicated otherwise. The gain detector was set using cells labeled with only secondary antibody. Rounded-up myoblasts with peripheral GLUT4myc signal (>50 cells per condition) were scored "blindly," and the number of positive cells expressed as a percentage of cells counted.125I-Labeled Transferrin Recycling—Transferrin recycling was examined as noted (44Yan Q. Sun W. Kujala P. Lotfi Y. Vida T.A. Bean A.J. Mol. Biol. Cell. 2005; 16: 2470-2482Crossref PubMed Scopus (98) Google Scholar). Cells were loaded with 125I-labeled transferrin (1 μg/ml) for 30 min at 37 °C and washed once each with cold medium, acidic solution (0.15 m NaCl, 0.1 m glycine, pH 3.0) and again with medium. Cells were incubated with growth medium for 2, 4, 8, or 12 min at 37 °C, and media were collected for each time point. Cells were scraped into 1 m NaOH, and protein in the media was precipitated with 20% trichloroacetic acid. Radioactivity content in the cell extract (with internalized transferrin) or media pellet (with externalized transferrin) was counted. For each time point collected in triplicate, recycled 125I-labeled transferrin was calculated as the ratio of externalized versus internalized 125I-labeled transferrin. Data were corrected for nonspecific cell-associated 125I-labeled transferrin, determined from parallel cell cultures incubated with 20 μg/ml unlabeled transferrin during loading.Statistical Analysis—Results are shown as -fold values relative to the indicated control conditions in each figure. Statistical analysis was performed using Student's t test; p < 0.05 was considered to be statistically significant.RESULTSInsulin Specifically Enhances ACTN4 Interaction with GLUT4—Recently we reported an interaction between ACTN4 and GLUT4 in a yeast two-hybrid screen of a 3T3-L1 adipocyte cDNA library using a hybrid protein encoding only the cytosolic regions of GLUT4 (1Foster L.J. Rudich A. Talior I. Patel N. Huang X. Furtado L.M. Bilan P.J. Mann M. Klip A. J. Proteome Res. 2006; 5: 64-75Crossref PubMed Scopus (94) Google Scholar). ACNT4 is also expressed in skeletal muscle tissue, confirmed by immunoblot analysis of oxidative (soleus) and glycolytic (extensor digitorum longus) mouse muscles (data not shown). To further understand the functional significance of the interaction between ACTN4 and GLUT4, we first explored the specificity of insulin action on this association. In L6-GLUT4myc myotubes, ACTN4 was notably detectable in GLUT4myc immunoprecipitates in response to insulin in comparison with the basal state (Fig. 1A, top), relative to total amounts of GLUT4myc immunoprecipitated in each condition. Neither GLUT4myc nor ACTN4 was immunoprecipitated by anti-myc antibody from L6 wild-type cell lysates devoid of GLUT4myc (Fig. 1A, bottom).Like insulin, acute stimulation with PDGF also promotes GLUT4myc externalization and glucose transport in L6 myoblasts (25Torok D. Patel N. Jebailey L. Thong F.S. Randhawa V.K. Klip A. Rudich A. J. Cell Sci. 2004; 117: 5447-5455Crossref PubMed Scopus (45) Google Scholar). However, this response is insensitive to actin disruption by latrunculin B or to VAMP2 inactivation by tetanus toxin, hallmarks of insulin action (25Torok D. Patel N. Jebailey L. Thong F.S. Randhawa V.K. Klip A. Rudich A. J. Cell Sci. 2004; 117: 5447-5455Crossref PubMed Scopus (45) Google Scholar, 45Thong F.S. Bilan P.J. Klip A. Diabetes. 2007; 56: 414-423Crossref PubMed Scopus (187) Google Scholar). Therefore, we explored whether PDGF would affect the GLUT4-ACTN4 interaction. Interestingly, PDGF did not increase the binding of ACTN4 to GLUT4myc (Fig. 1B), highlighting the specificity of insulin on this association. These experiments were tested in myoblasts, as the PDGF receptor is only expressed at this stage of the myogenic process.Because PI3K is required for insulin-mediated recruitment of GLUT4 (46Thong F.S. Dugani C.B. Klip A. Physiology (Bethesda). 2005; 20: 271-284Crossref PubMed Scopus (175) Google Scholar), the interaction of GLUT4myc with ACTN4 was examined in the presence of the PI3K inhibitor LY294002. L6GLUT4myc myoblasts were pretreated with LY294002 for 20 min before and during insulin stimulation. This treatment prevented the insulin-stimulated association of GLUT4myc with ACTN4 (Fig. 1C), offering additional support for the specificity of this interaction in response to insulin.To verify that ACTN4 and GLUT4myc directly interact, as predicted from the two-hybrid analysis, we conducted pull-down assays of ACTN4 via cytoGLUT4-GST using cell extracts and pure recombinant proteins. As shown in Fig. 1D, cytoGLUT4-GST effectively pulled down endogenous ACTN4 from cell lysates and purified, recombinant ACTN4-His. In contrast, GST alone did not pull down ACTN4 in either assay.ACTN4 Knockdown Reduces Insulin-stimulated Glucose Transport and Surface GLUT4 Gain—To assess the functional relevance of ACTN4 to GLUT4 traffic, we explored the effects of silencing ACTN4 expression using a specific small interfering RNA oligonucleotide (siACTN4). Significant knockdown (60 ± 2%) of ACTN4 protein was achieved in siACTN4 (Fig. 2A) relative to NT or non-related control siRNA (siNR)-treated cells. In contrast, expression of GLUT4myc or the ACTN1 isoform was unaltered. ACTN4 knockdown significantly inhibited insulin-stimulated 2-deoxyglucose uptake (1.6 ± 0.2-fold) compared with the response to the hormone in NT (2.65 ± 0.12-fold) or siNR (2.6 ± 0.04-fold) cells. In contrast, ACTN4 knockdown did not affect hexose uptake in the unstimulated state (Fig. 2B). Importantly, siACTN4 did not perturb insulin signaling at the level of Akt phosphorylation on Ser-473 (Fig. 2C) or Thr-308 (data not shown).FIGURE 2ACTN4 knockdown reduces insulin-stimulated glucose transport. A–C, L6-GLUT4myc myotubes were either NT or transfected with 100 nm siNR or siACTN4. B and C, serum-starved cells were treated without or with 100 nm insulin for 20 min. B, 2-deoxyglucose uptake was measured at room temperature for 5 min. Shown are the means ± S.E. relative to basal NT cells from four independent experiments; *, p ≤ 0.05. A and C, lysates resolved by SDS-PAGE were immunoblotted with anti-ACTN4 or anti-ACTN1 (both ∼100 kDa), anti-myc (50 kDa) or anti-β-actin (45 kDa), and anti-Akt (65 kDa) or anti-phospho (p)-Akt (serine 473, 65 kDa) antibodies. Shown are representative blots from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Insulin-stimulated translocation of GLUT4myc to the cell surface was almost obliterated by siACTN4 without any consequence on basal cell surface GLUT4myc levels (Fig. 3A). These results suggest that the residual glucose uptake response observed in siACTN4-treated cells is likely ascribed to GLUT1. In contrast to insulin, ACTN4 knockdown had no inhibitory effect on the gain in surface GLUT4myc elicited by PDGF, K+-mediated depolarization, or DNP-mediated mitochondrial uncoupling (Figs. 3, A and B).FIGURE 3Insulin, but not PDGF, K+ depolarization or DNP-stimulated GLUT4 translocation is reduced by ACTN4 knockdown. L6-GLUT4myc myotubes were either NT or transfected with 100 nm siNR or siACTN4. Serum-depleted cells were treated without or with 100 nm insulin for 20 min and with 50 ng/ml PDGF for 7 min, and 120 mm K+ or 0.5 mm DNP for 20 min at 37 °C, and surface myc-tagged GLUT4 density was quantified using the antibody-coupled colorimetric assay. Shown are the means ± S.E. relative to basal NT cells from four independent experiments; *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ACTN1 Knockdown Does Not Affect GLUT4 Traffic—Because muscle cells also express ACTN1, we examined the effect of insulin on the association of this isoform with GLUT4. As shown on the Fig. 4A, insulin did not enhance the association of these proteins. Moreover, siRNA-mediated knockdown of ACTN1 (by 70%) was innocuous to basal or insulin-stimulated levels of surface GLUT4myc (Fig. 4, B and C) without affecting ACTN4 levels. Together, these results suggest that the interaction of ACTN4 with GLUT4 plays a functional and specific role in insulin-stimulated glucose uptake and GLUT4 translocation.FIGURE 4ACTN1 knockdown does not alter GLUT4 traffic. A, lysates of serum-starved L6-GLUT4myc or L6 wild-type myotubes were treated without or with 100 nm insulin for 20 min at 37 °C and immunoprecipitated (i.p.) with anti-myc antibody-bound beads (intraperitoneal myc). i.b., immunoblot. B and C, L6-GLUT4myc myotubes were
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