Role of insulin-dependent cortical fodrin/spectrin remodeling in glucose transporter 4 translocation in rat adipocytes

Abstract

Fodrin or nonerythroid spectrin is an abundant component of the cortical cytoskeletal network in rat adipocytes. Fodrin has a highly punctate distribution in resting cells, and insulin causes a dramatic remodeling of fodrin to a more diffuse pattern. Insulin-mediated remodeling of actin occurs to a lesser extent than does that of fodrin. We show that fodrin interacts with the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 4, and this interaction is increased by insulin stimulation and decreased by prior latrunculin A treatment. Latrunculin A disrupts all actin filaments, inhibits glucose transporter 4 (GLUT4) translocation, and causes fodrin to partially redistribute from the plasma membrane to the cytosol. In contrast, cytochalasin D disrupts only the short actin filament signal, and cytochalasin D neither inhibits GLUT4 translocation nor fodrin redistribution in adipocytes. Together, our data suggest that insulin induces remodeling of the fodrin-actin network, which is required for the fusion of GLUT4 storage vesicles with the plasma membrane by permitting their access to the t-SNARE syntaxin 4.

Figure 1.

Insulin induces actin-fodrin remodeling in rat adipocytes. (A) Rat adipocytes treated with 100 nM insulin or not were fixed and subjected to immunostaining for actin (red) and fodrin (green). The three-dimensional images were reconstructed from serial confocal images taken along the z-axis as described in Materials and Methods. Bar, 10 μm in this and other confocal images. (B) Confocal images from a total of 100 cells each, basal and insulin treated, were graded according to the presence or absence of a clear punctate fodrin signal (left). The number of cells that retained a punctate signal after insulin exposure is shown on the right.

Figure 2.

Insulin has no effect on the association of actin and fodrin with the adipocyte plasma membrane. Subcellular fractionation (CY, cytosol) of rat adipocytes treated with 100 nM insulin or not was performed as described in Materials and Methods. Equal protein amounts of the fractions were separated by SDS-PAGE and analyzed by Western blot by using the antibodies to the proteins indicated. Detection was by enhanced chemiluminescence (ECL). The migration of marker proteins is indicated on the right in this and the other figures.

Figure 3.

Fodrin and syntaxin 4 colocalize and interact in rat adipocytes. (A) Rat adipocytes treated with 100 nM insulin or not were fixed and subjected to immunostaining for actin (gray scale), fodrin (green), and Syn4 (red). The three-dimensional images were reconstructed from serial confocal images taken along the z-axis as described in Materials and Methods. Bar, 10 μm. (B) Top left, PM lysates were prepared in Triton X-100, and imunoprecipitations were performed as described in Materials and Methods. In addition (top right), 100 μg of plasma membrane protein was solubilized in 60 mM octylglucoside for 2 h and then incubated with anti-monoclonal α-fodrin antibody (AA2) or nonspecific IgG (5 μg each) for an another 2 h. Protein A beads (20 μl) were added to the lysates and incubated for 60 min. The supernatant was removed, and the beads were washed three times with PBS containing 60 mM octylglucoside. Bound samples were solubilized in Laemmli sample buffer with 2% SDS, and equal portions were subjected to SDS-PAGE and analyzed by Western blotting by using anti-fodrin monoclonal and anti-syntaxin 4 polyclonal antibodies. The bands from the immunoblots were scanned, and the relative intensities were assessed by NIH Image software (bottom). The results are the mean ± SE of four independent determinations.

Figure 4.

Latrunculin A disrupts cortical actin and inhibits insulin-dependent fodrin remodeling in rat adipocytes. After incubation in the absence or presence of 25 μg/ml latrunculin A for 30 min at 37°C, adipocytes were incubated without or with 100 nM insulin for an additional 30 min. The cells were then fixed and immunostained with anti-fodrin, anti-Syn4 antibodies, and Alexa Fluor 594-phalloidin. The 3-D images were reconstructed from sequential confocal images taken at 1-μm intervals along the z-axis as described in Materials and Methods.

Figure 5.

Latrunculin A treatment causes redistribution of actin and fodrin from the plasma membrane to the cytosolic fraction. After incubation in the absence or presence of 25 μg/ml latrunculin A (L) for 30 min at 37°C, adipocytes were incubated without or with 100 nM insulin (I) for an additional 30 min. Subcellular fractionation (LHM, combined heavy and light microsomes) of cells was performed as described in Materials and Methods. Equal proportions of the fractions were separated by SDS-PAGE and analyzed by Western blot by using antibodies to the proteins indicated. Detection was by ECL.

Figure 6.

Interaction of syntaxin 4 and fodrin depends on the cortical actin network. Rat adipocytes were treated with or without 100 nM insulin for 15 min or in the presence or absence 25 μg/ml LatA as in Figures 4 and 5. Total cell lysates were prepared (see Materials and Methods) and anti-monoclonal fodrin antibody (AA2) or nonspecific IgG (each 5 μg) was incubated with 100 μg of lysate 2 h. Then, 20 μl of protein A beads was added to the lysate and incubated for 60 min. Thereafter, the beads were washed three times with 0.05% Triton X-100 in PBS and one time with PBS. Bound samples were solubilized in Laemmli sample buffer with 2% SDS, and equal portions were subjected to SDS-PAGE and analyzed by Western blotting by using anti-fodrin monoclonal and anti-syntaxin 4 polyclonal antibodies.

Figure 7.

Cytochalasin D has no effect on actin–fodrin distribution and insulin-induced GLUT4 translocation in rat adipocytes. After incubation in the absence or presence of 10 μM cytochalasin D (C) for 30 min at 37°C, adipocytes were incubated without or with 100 nM insulin (I) for an additional 30 min. (A) Subcellular fractionation was performed, and fractions were analyzed as described in previous figures in Materials and Methods. An equal proportion of each fraction was separated by SDS-PAGE and analyzed by Western blot by using antibodies to the proteins indicated. Detection was by ECL. (B) Cells were fixed and immunostained with Alexa Fluor 594-phalloidin. The 3-D images were reconstructed from sequential confocal images taken at 1-μm intervals along the z-axis as described in Materials and Methods. Enlarged images of representative cells are in right-hand panels.

Figure 8.

Fodrin remodeling is insensitive to latrunculin A after insulin treatment. After incubation in the presence of 100 nM insulin for 15 min at 37°C, adipocytes were incubated without or with 25 μg/ml latrunculin A for an additional 30 min. Subcellular fractionation was performed as described in Materials and Methods. Equal proportions of the fractions were separated by SDS-PAGE and analyzed by Western blot using the antibodies indicated. Detection was by ECL.

Figure 9.

Insulin stimulation increase the colocolization of fodrin and GLUT4 in rat adipocytes. Rat adipocytes treated with 100 nM insulin or not and were fixed and subjected to immunostaining for GLUT4 (right) and fodrin (left). The three-dimensional images were reconstructed from serial confocal images taken along the z-axis as described in Materials and Methods.

Figure 10.

Insulin-dependent fodrin remodeling. In basal state, the punctate fodrin signal and actin filaments forming a network beneath the plasma membrane. A portion of Syn4 can interact with fodrin. After insulin stimulation, fodrin–actin remodeling occurs, resulting in more diffuse signal for fodrin. The interaction between syn4 and fodrin increases, and fusion of GSVs with the plasma membrane occurs as a result of SNARE protein interactions.