Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes

Abstract

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.

FIGURE 1.

P-Rex1 expression and localization in 3T3-L1 adipocytes. A, P-Rex1 domain structure and localization of peptide used for antibody generation. P-Rex1 polyclonal antibodies were raised to the indicated peptide sequence (33). B, 50 μg of cell lysate (LYS), cytosolic (CYT), or plasma membrane (PM) fractions prepared from differentiated 3T3-L1 adipocytes were immunoblotted with affinity-purified P-Rex1 anti-peptide antibodies. C, resting or insulin-stimulated (100 nm) 3T3-L1 adipocytes expressing GFP-GLUT4 (green) were fixed, permeabilized, and stained with P-Rex1 anti-peptide antibodies (red). Yellow regions in merged images indicate areas of colocalization (see arrows). Bar, 2 μm.

FIGURE 2.

P-Rex1 promotes membrane ruffling in differentiated 3T3-L1 adipocytes. A and B, resting or insulin-stimulated (100 nm) vector or wild-type HA-P-Rex1-expressing 3T3-L1 adipocytes were stained with Texas Red phalloidin to detect F-actin (A and B) or DNase I to detect monomeric G-actin (B). Single sections from the middle (A) or the base of each cell (A and B) are shown. The arrows indicate membrane ruffling at the cell base (bar, 2 μm). Inset regions are magnified in zoom images (B). C, HA-tagged P-Rex1 lacking the DH domain (ΔDH), the N-terminal domains (ΔN), or the 4-phosphatase homology region (Δ4P) and a mutant with a C1583S substitution in the 4-phosphatase homology region were generated. D, detergent-soluble lysates of 3T3-L1 adipocytes (50 μg) transfected with wild type HA-P-Rex1 or mutant constructs were immunoblotted with HA antibodies (lane 1, HA-P-Rex1; lane 2, HA-P-Rex1(ΔN), lane 3, HA-P-Rex1(ΔDH); lane 4, HA-P-Rex1(Δ4P); lane 5, HA-P-Rex1(C1583S)). E, cells expressing HA-P-Rex1 or P-Rex1 mutants were serum-starved and treated with insulin and scored for membrane ruffling using Texas-Red phalloidin (0 nm (white bars), 1 nm (striped bars), 10 nm (gray bars), or 100 nm insulin (black bars)). For each insulin concentration, 50 cells were scored. Results represent the mean ± S.E. of three independent transfections per construct (*, p < 0.05).

FIGURE 3.

P-Rex1 expression in 3T3-L1 adipocytes enhances insulin-induced GLUT4 plasma membrane association in a PI3K- and actin-dependent manner. A, 3T3-L1 adipocytes co-transfected with GFP-GLUT4 and vector (solid line) or HA-P-Rex1 (dashed line) were serum-starved and stimulated with 0.1, 1, 10, or 100 nm insulin for 30 min, and 50 cells were scored for evidence of plasma membrane GFP-GLUT4. Representative images of transfected cells stimulated with 1 nm insulin are shown. B, 3T3-L1 adipocytes, co-transfected with GFP-GLUT4 and vector (solid lines), or HA-P-Rex1 (dashed lines), were serum-starved and left untreated (square boxes), or pretreated with 25 nm LY294002 for 45 min (crosses), prior to 0.1, 1, 10, or 100 nm insulin treatment for 30 min. C, 3T3-L1 adipocytes co-transfected with GFP-GLUT4 and HA-P-Rex1 were serum-starved and pretreated with either 50 nm cytochalasin D (Cyto D) or 25 mm latrunculin A (Lat A) prior to stimulation with insulin (1 nm). For each condition, 50 cells were scored for GFP-GLUT4 plasma membrane (PM) localization. Means ± S.E. (error bars) of three independent experiments are shown (*, p < 0.05; **, p < 0.01). White bars, 0 nm insulin; striped bars, 1 nm insulin.

FIGURE 4.

P-Rex1 enhances GLUT4 association with the plasma membrane. A, serum-starved 3T3-L1 adipocytes, co-transfected with exofacial Myc-GLUT4-GFP and HA-P-Rex1 (black bars) or vector control (white bars), were stimulated with 1 or 100 nm insulin for 30 min. Intact (non-permeabilized) cells were scored for Myc immunofluorescence on the cell surface. Results shown are the means ± S.E. (error bars) of three separate experiments in which 50 cells were scored per transfection (**, p < 0.01; *, p < 0.05). B, representative images of 3T3-L1 adipocytes co-expressing vector, or HA-P-Rex1 with Myc-GLUT4-GFP in resting or insulin-stimulated cells. Bar, 2 μm.

FIGURE 5.

P-Rex1-induced insulin-stimulated GLUT4 trafficking is Rac1-specific. A, serum-starved 3T3-L1 adipocytes, co-transfected with the indicated P-Rex1 constructs and GFP-GLUT4, were scored for plasma membrane fluorescence following stimulation with 1 or 100 nm insulin. Results are the mean ± S.E. (error bars) of three separate experiments in which 50 cells were scored per condition per experiment. B, data in A are represented as -fold increase over unstimulated values (*, p < 0.05). White bars, 0 nm insulin; striped bars, 1 nm insulin; black bars, 100 nm insulin. C, 3T3-L1 adipocytes co-transfected with constitutively active RhoGTPases and GFP-GLUT4 were scored for plasma membrane fluorescence (n = 3; *, p < 0.05). D, cells co-expressing vector or HA-P-Rex1(ΔDH) with GFP-GLUT4 and either constitutively active T7-tagged Rac1(V12), or T7-tagged Cdc42(V12), were scored for plasma membrane GFP-GLUT4 fluorescence (n = 3; *, p < 0.05). E, cells co-expressing vector or HA-P-Rex1 with GFP-GLUT4 and either Myc-tagged dominant negative Rac1 or Myc-tagged dominant negative Cdc42 were scored for GFP-GLUT4 at the plasma membrane. For A–C, 50 cells were scored for the presence of plasma membrane GFP-GLUT4 for each transfection. Results are the mean ± S.E. of three independent transfections (*, p < 0.05). White bars, 0 nm insulin; striped bars, 1 nm insulin; gray bars, 10 nm insulin; black bars, 100 nm insulin). Representative images showing the subcellular localization of HA-P-Rex1(ΔDH), GFP-GLUT4, and T7-Rac1V12 (top) and HA-P-Rex1, GFP-GLUT4, and Myc-Rac1V12N17 (bottom) in both quiescent and insulin-stimulated (100 nm) cells.

FIGURE 6.

Dominant negative P-Rex1 or RNAi-mediated depletion of P-Rex1 inhibits insulin-stimulated glucose uptake in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were transfected with either vector or HA-P-Rex1(ΔN), serum-starved (white bars), and treated with 10 nm insulin (gray bars). Following stimulation, cells were washed three times and then incubated for 20 min in 100 μm 2-[¹⁴C]DOG (7.5 μCi/ml) in assay buffer at 37 °C. Cells were washed and assayed in replicates of three per experiment (*, p < 0.05). B, 250 pmol of siRNA oligonucleotides specific for P-Rex1 or a nonspecific scrambled sequence (control) were transfected using Lipofectamine 2000 reagent into 3T3-L1 adipocytes for 72 h. Cells were immunoblotted for P-Rex1 expression using anti-P-Rex1 antibodies. Duplicate samples were immunoblotted with anti-actin antibodies to confirm equal protein loading. C, 3T3-L1 adipocytes were transfected with 250 pmol of either control or P-Rex1 siRNA oligonucleotides and seeded into 24-well plates. 72 h post-transfection, cells were serum-starved and insulin-stimulated (striped bars, 1 nm insulin; gray bars, 10 nm insulin) prior to incubation with 100 μm 2-[³H]DOG (7.5 μCi/ml) in 2-DOG assay buffer for 20 min at 37 °C and assayed in replicates of three per experiment. Data is displayed as a ratio of insulin-stimulated 2-[³H]deoxyglucose uptake over basal levels of 2-deoxyglucose uptake for each siRNA transfection. Over three separate experiments (*, p < 0.05).