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First published online 5 October 2004
doi: 10.1242/jcs.01421

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Insulin but not PDGF relies on actin remodeling and on VAMP2 for GLUT4 translocation in myoblasts

Dòra Török^1,*, Nish Patel^1,2,*, Lellean JeBailey^1,3, Farah S. L. Thong¹, Varinder K. Randhawa^1,3, Amira Klip^1,2,3, and Assaf Rudich^1,

¹ Programme in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada
² Department of Physiology, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada
³ Department of Biochemistry, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada

View larger version (23K): [in a new window]   Fig. 1. Insulin and PDGF increase the amount of GLUT4myc at the cell surface and stimulate glucose uptake with different time courses that correlate with Akt phosphorylation. Confluent GLUT4myc myoblasts were incubated with either 100 nM insulin or 50 ng ml–1 PDGF-BB at 37°C for the times indicated, and assayed for (A) GLUT4myc translocation or (B) 2-deoxyglucose uptake; *, P<0.05 against time 0. The means±s.e.m. of at least three independent experiments are shown per condition, each done in triplicate. (C) Total cell lysates were separated by 10% SDS-PAGE and immunoblotted with anti-phospho-Akt (Ser 473) antibody. The same membrane was stripped and reblotted for total Akt.

View larger version (48K): [in a new window]   Fig. 2. Time course of insulin- and PDGF-induced actin remodeling in myoblasts. Serum-deprived GLUT4myc myoblasts were left untreated (a,f) or stimulated with 100 nM insulin (b-e) or with 50 ng ml–1 PDGF-BB (g-j) for the times indicated. The cells were then fixed, permeabilized and labeled with rhodamine-conjugated phalloidin. Representative images are shown of a single focal plane near the dorsal surface of the cell. The images shown are representative of five experiments. Scale bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 3. Insulin and PDGF promote morphologically distinct actin structures. (A) Serum-deprived L6 GLUT4myc myoblasts were stimulated with either insulin or PDGF-BB for the times indicated in B, after which cells were fixed and stained for actin. Collapsed xy projections were obtained by confocal microscopy to reveal distinct filamentous actin structures such as stress fibers and long ruffles (a, arrows and asterisk, respectively), short ruffles (b, open arrowheads), and filopodia (c, closed arrowheads). (B) Images were obtained from three independent experiments and the presence of long and short actin ruffles was scored in at least 60 cells per condition. The means±s.e.m. are given of the proportion of cells exhibiting either of the two types of actin structure at each time point after insulin or PDGF stimulation. *, P<0.001 against time 0. (C) GLUT4myc proteins were stained with anti-Myc antibody followed by FITC-conjugated secondary antibody. Composite images of F-actin labeled with rhodamine-conjugated phalloidin and GLUT4myc protein staining are presented in cells that exhibit long ruffles (a), short ruffles (b) and filopodia (c); yellow indicates regions of co-localization. The images shown are representative of five individual experiments. Scale bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 4. The actin-disrupting agent cytochalasin D affects GLUT4myc translocation differently in response to insulin and PDGF. Serum-deprived GLUT4myc myoblasts were treated without (a-c) or with (d-f) 250 nM cytochalasin D for 20 minutes before and during incubation with either 50 ng ml–1 PDGF-BB or 100 nM insulin for 7 minutes at 37°C. The cells were then either (A) fixed, permeabilized and stained for filamentous actin with rhodamine-phalloidin or (B) left intact and reacted with an anti-Myc antibody and subjected to the colorimetric assay for cell surface GLUT4myc. The increase in surface GLUT4myc in response to each stimulus over basal (unstimulated) conditions was calculated and presented as the proportion of the response in the absence of cytochalasin D. The means±s.e.m. are shown of four independently performed experiments, each performed in triplicate. *, P<0.05 compared with the respective control in the absence of the drug. Scale bar, 10 µm.

View larger version (60K): [in a new window]   Fig. 5. Tetanus toxin (TeTx) reduces the insulin-dependent, but not the PDGF-dependent, gain in GLUT4myc at the surface. GLUT4myc myoblasts were co-transfected with 0.6 µg of eGFP-encoding cDNA and 0.9 µg of either pcDNA3 (empty vector) or TeTx-encoding cDNA, as indicated. (A) After 24 hours, cells were serum starved and then stimulated without insulin (a,b), with 100 nM insulin (c,d) or with 50 ng ml–1 PDGF (e,f). Cells co-expressing TeTx and eGFP are shown at the top, and the corresponding anti-Myc surface staining from the same field of view is shown at the bottom. All images are collapsed xy projections. (B) Quantification of cell-surface GLUT4myc labeling in empty vector and TeTx-transfected cells, showing the means±s.e.m. of three independent experiments. Scale bar, 10 µm.