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Role of RalA downstream of Rac1 in insulin-dependent glucose uptake in muscle cells
Cellular Signalling 24 (2012) 2111–2117
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Role of RalA downstream of Rac1 in insulin-dependent glucose uptake in muscle cells Shinsuke Nozaki a, Shuji Ueda a, Nobuyuki Takenaka b, Tohru Kataoka a, Takaya Satoh a, b,⁎ a b
Division of Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650‐0017, Japan Laboratory of Cell Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1‐1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599‐8531, Japan
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Article history: Received 7 July 2012 Accepted 16 July 2012 Available online 20 July 2012 Keywords: Glucose uptake GTPase Insulin Rac1 RalA Skeletal muscle
a b s t r a c t The small GTPase RalA has been implicated in glucose uptake in insulin-stimulated adipocytes, although it remains unclear whether RalA has a similar role in insulin signaling in other types of cells. Recently, we have demonstrated that the Rho family GTPase Rac1 has a critical role in insulin-dependent glucose uptake in myoblast culture and mouse skeletal muscle. However, the mechanisms underlying Rac1-dependent glucose uptake, mostly mediated by the plasma membrane translocation of the glucose transporter GLUT4, remain largely unknown. The purpose of this study is to examine the involvement of RalA in Rac1 regulation of the translocation of GLUT4 to the plasma membrane in muscle cells. Ectopic expression of a constitutively activated RalA mutant indeed stimulated GLUT4 translocation, suggesting an important role of RalA also in muscle cells. GLUT4 translocation induced by constitutively activated mutation of Rac1 or more physiologically by upstream Rac1 regulators, such as phosphoinositide 3 kinase and the guanine nucleotide exchange factor FLJ00068, was abrogated when the expression of RalA was downregulated by RNA interference. The expression of constitutively activated Rac1, on the other hand, caused GTP loading and subcellular redistribution of RalA. Collectively, we propose a novel mechanism involving RalA for Rac1-mediated GLUT4 translocation in skeletal muscle cells. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Insulin-dependent glucose uptake in fat and muscle cells is largely mediated by translocation of the glucose transporter GLUT4 from intracellular storage sites to the plasma membrane. Insulin enhances exocytosis of GLUT4 vesicles and suppresses their endocytosis, leading to the net accumulation of GLUT4 in the plasma membrane. Accumulating evidence reveals that diverse signaling molecules, including phosphoinositide 3 kinase (PI3K) and the serine/threonine kinase Akt2, exert important roles in insulin signaling. However, the mechanisms underlying insulin-dependent GLUT4 trafficking remain incompletely understood [1–4]. Rho family small GTPases regulate a wide variety of cellular responses such as cytoskeletal rearrangements [5,6]. We and others recently reported that the Rho family member Rac1 is involved in the regulation of GLUT4 translocation in skeletal muscle cells [7–9]. One critical role of Rac1 in GLUT4 trafficking is thought to be the regulation of the actin cytoskeleton. Insulin induces rapid and marked reorganization of the actin cytoskeleton in myocytes,
Abbreviations: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; PI3K, phosphoinositide 3 kinase; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate. ⁎ Corresponding author. Tel./fax: +81 72 254 7650. E-mail address: [email protected] (T. Satoh). 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2012.07.013
including the formation of membrane ruffles, in which diverse signaling molecules, such as PI3K and Akt, become accumulated [10,11]. This signaling is considered to be crucial for the induction of GLUT4 translocation, and is mediated by Rac1 [10,12]. A subsequent study further showed that Rac1-dependent actin cytoskeletal remodeling and submembrane tethering of GLUT4 vesicles are important for cell surface expression of GLUT4 [13]. However, the regulation of actin cytoskeletal remodeling may not be a sole function of Rac1. It is likely that Rac1 is also involved in a series of insulin-dependent events, such as GLUT4 vesicle trafficking and its docking/fusion to the cell membrane, taking into consideration that constitutively activated Rac1 alone is able to induce GLUT4 translocation in the absence of insulin without enhancing the activity of the serine/threonine kinase Akt [7,8]. The small GTPase RalA has been implicated in intracellular membrane trafficking, including polarized membrane delivery, neurosecretion, and endocytosis [14]. In adipocytes, RalA resides in GLUT4 vesicles, and interacts with the exocyst subunit Sec5 and the motor protein Myo1c, thereby regulating GLUT4 targeting to the plasma membrane in response to insulin [15]. However, it remains uncertain whether RalA is also involved in GLUT4 translocation in other types of cells, such as muscle cells. In particular, the role of RalA downstream of Rac1, a skeletal muscle-specific regulator of insulin signaling, is totally unknown. In this paper, we show that RalA in fact plays a pivotal role also in Rac1-mediated GLUT4 translocation in muscle cells. These results emphasize a RalA-dependent common mechanism underlying GLUT4
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trafficking in contrast to divergence in upstream signaling pathways in different cell types. 2. Materials and methods 2.1. Materials Antibodies against Myc (mouse monoclonal (sc-40)) and hemagglutinin (HA) (rat monoclonal (11 867 423 001)) epitope tags were purchased from Santa Cruz Biotechnology and Roche Applied Science, respectively. Antibodies against V5 (rabbit polyclonal (V8137)) and FLAG (rabbit polyclonal (F7425)) epitope tags and tubulin (mouse monoclonal (T4026)) were purchased from Sigma-Aldrich. Anti-RalA (mouse monoclonal (610221)) and anti-Rac1 (mouse monoclonal (610650)) antibodies were purchased from BD Biosciences. An anti-phospho-(Ser/Thr) Akt substrate (rabbit polyclonal (9611)) antibody was purchased from Cell Signaling Technology. Antimouse IgG, anti-rabbit IgG, and anti-rat IgG antibodies conjugated with Alexa Fluor® 488/546/647 were purchased from Life Technologies. Tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (P1951) was purchased from Sigma-Aldrich. Anti-mouse IgG (NA9310) and anti-rabbit IgG (NA9340) antibodies conjugated with horseradish peroxidase were purchased from GE Healthcare. 2.2. Cell culture and RNA interference L6 and L6-GLUT4 rat myoblasts [7] were cultivated in minimum essential medium Eagle (M4655; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (Cell Culture Bioscience), 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 μg/ml streptomycin. A mixture of three small interfering RNA (siRNA) duplexes designed from the rat RalA cDNA sequence (RalA siRNA) (duplex-1, 5′-AAGAUGUCGAUCU GCACUUCCUCCC-3′; duplex-2, 5′-AAAUCUGUUCCCUGAAGUCCGCU GU-3′; duplex-3, 5′-CAUAGUUAACGUUCCACUGGUCAGC-3′) (Life Technologies) was used for down-regulation of RalA. The control siRNA (1022076) was purchased from Qiagen. Cells were transfected with siRNA (200 nM) using the Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer's instructions. 2.3. Induction of differentiation of L6 cells L6 cells were induced to differentiate into multinuclear myotubes by replacement of 10% (v/v) fetal bovine serum in the culture medium with 2% horse serum (GIBCO, 16050‐122) and subsequent incubation for 5 days. 2.4. Adenovirus infection Adenoviruses for the expression of 3 × HA-tagged Rac1 (WT), 3 × HA-tagged Rac1(G12V), V5-tagged RalA (WT), V5-tagged RalA (G23V), and FLAG-tagged FLJ68ΔN were generated and used to infect L6-GLUT4 cells by using the Adenovirus Cre/loxP kit (Takara Bio, Japan) according to the manufacturer's instructions. A recombinant adenovirus for the N-terminally myristoylated catalytic subunit of bovine PI3Kα (Myr-p110) was kindly provided by Dr. Wataru Ogawa (Kobe University Graduate School of Medicine) [16]. 2.5. Immunofluorescent microscopy and GLUT4 reporter assay The exofacial GLUT4 reporter GLUT4myc7‐green fluorescent protein (GFP) and an L6-derived cell line that stably expresses this reporter (termed L6-GLUT4) were previously described [7,17]. L6-GLUT4 cells were starved in serum-free medium for 2 h prior to measurement of GLUT4 translocation. For the detection of the cell surface-exposed GLUT4 reporter, formaldehyde-fixed cells were treated with an anti-Myc antibody before permeabilization. After
washing three times with phosphate-buffered saline (−), cells were permeabilized with 0.1% (v/v) Triton X-100 and then counterstained with another primary antibody for the identification of the cells that express a specific ectopically expressed or endogenous protein. Images were obtained using a 40 × oil objective on a confocal laser-scanning microscope (LSM 700; Carl Zeiss) and processed by the Zeiss LSM Image Browser, version 3.5. The relative amount of GLUT4myc7-GFP exposed at the plasma membrane was estimated from measurements of Myc and GFP fluorescence signals. Intensities of Myc and GFP fluorescence signals in regions of interest were quantified using ImageJ software, and ratios of Myc and GFP signals (Myc/GFP) were calculated [8]. 2.6. Immunoblotting Proteins were separated by SDS‐polyacrylamide gel electrophoresis and transferred on to a 0.45 μm pore size polyvinylidene difluoride membrane (GE Healthcare). The membrane was stained with the respective primary antibodies and a horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody, followed by visualization by enhanced chemiluminescence detection reagents (GE Healthcare). 2.7. Pull down assay for activated Rac1 and RalA Proteins were expressed in L6-GLUT4 cells through adenoviral infection. Cells were harvested with a pull down assay buffer [50 mM Tris–HCl (pH 7.4), 0.5% Nonidet P-40, 0.15 M NaCl, 20 mM MgCl2, and protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan)]. Lysates were centrifuged at 18,000×g for 1 min, and supernatants were added to glutathione S-transferase (GST)-Myc-PAK (67–150) (for Rac1) or GST-3× V5-Sec5 (1–99) (for RalA) immobilized on glutathione– Sepharose beads, and incubated at 4 °C for 30 min. Glutathione– Sepharose beads were washed twice with pull down assay buffer, and activated Rac1 or RalA bound to the beads was detected by immunoblotting using an anti-Rac1 or anti-RalA antibody. 2.8. Overlay assay (in situ detection) for activated Rac1 In situ detection of the activated form of Rac1 in paraformaldehydefixed cells were performed as described previously [7]. 3. Results As a step to assess the involvement of RalA in glucose uptake in muscle cells, a constitutively activated mutant of RalA (RalA (G23V)) was ectopically expressed in L6-derived myoblast (L6-GLUT4) cells, and its effect on GLUT4 translocation to the plasma membrane was examined (Fig. 1). The L6-GLUT4 cell line stably expresses an N-terminally GFP- and exofacially Myc epitope-tagged GLUT4 reporter that is detected by an anti-Myc antibody only when re-localized in the plasma membrane [7]. L6-GLUT4 cells expressing RalA(G23V) were highly positive for Myc staining whereas L6-GLUT4 cells without RalA(G23V) were not stained by an anti-Myc antibody (Fig. 1A). Fluorescent intensities of GFP and Myc staining in L6-GLUT4 cells positive or negative for RalA(G23V) expression were quantified, and the Myc/GFP ratio, which reflects the extent of cell surface translocation of GLUT4, was calculated (Fig. 1B). Ectopic expression of RalA(G23V) in fact induced an approximately 2.5-fold increase in the level of GLUT4 translocated to the plasma membrane. Therefore, RalA is likely to be implicated in signaling that leads to glucose uptake in muscle cells. We and others have demonstrated that the Rho family GTPase Rac1 plays an important role in insulin-dependent GLUT4 translocation to the plasma membrane in cultured muscle cells [7,9] and mouse skeletal muscle [8]. However, the mechanism by which Rac1-dependent signaling causes the plasma membrane translocation of GLUT4 remains obscure. By using the GLUT4 reporter assay
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Fig. 1. Cell surface translocation of GLUT4 induced by a constitutively activated mutant of RalA. (A) RalA (G23V) was ectopically expressed in L6-GLUT4 cells, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of RalA (G23V) was detected by immunostaining of the V5 epitope tag. Images were acquired from the focal plane as depicted in the schematic diagram. Scale bar, 50 μm. (B) Cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells in which RalA (G23V) was not expressed (“−”) and L6-GLUT4 cells in which RalA (G23V) was expressed (“RalA (G23V)”) was quantified. Data are shown as means±S.E. (n=6). *Pb 0.001.
described in Fig. 1, we next tested the involvement of RalA downstream of Rac1 in signaling that mediates glucose uptake. The endogenous expression level of RalA, as evaluated by immunoblotting, was significantly downregulated with specific siRNA duplexes (Fig. 2A). The constitutively activated mutant Rac1 (G12V) was ectopically expressed in L6-GLUT4 cells pretreated with control or RalA-specific siRNA, and the translocation of the GLUT4 reporter was assessed (Fig. 2B and C). Rac1 (G12V) induced GLUT4 translocation in control siRNA-treated cells as previously described [7]. Rac1 (G12V)-induced GLUT4 translocation was largely suppressed in L6-GLUT4 cells in which the expression of RalA was reduced. Therefore, RalA exerts a critical function downstream of Rac1 although it remains unclear whether the residual level of induction of GLUT4 translocation by Rac1 (G12V) in RalA siRNA-treated cells is ascribed to incomplete knockdown of RalA or RalA-independent mechanisms. PI3K is a key component in insulin signaling in various types of cells as proposed from a variety of observations, such as the inhibitory effect of the specific inhibitor wortmannin [2]. Given that PI3K acts immediately downstream of the insulin receptor/insulin receptor substrate-1 complex, insulin-dependent activation of Rac1 is likely to be mediated by PI3K. Actually, we observed that insulin-induced Rac1 activation was sensitive to the inhibitory effect of wortmannin [7]. Here, we further examined the effect of wortmannin on insulin activation of Rac1 in detail by two distinct assays (Fig. 3). A pull-down assay for the activated form of Rac1 showed that insulin caused a rapid increase in the level of Rac1·GTP, which was sensitive to the inhibitory effect of wortmannin, suggesting the involvement of PI3K in insulin-dependent activation of Rac1 (Fig. 3A). This inhibition of Rac1 activation, however, was not complete, and therefore a small
Fig. 2. Inhibition of Rac1 (G12V)-induced cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP by RalA knockdown. (A) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and the expression of endogenous RalA was examined by immunoblotting. The expression levels of tubulin are also shown as a loading control. (B) Rac1 (G12V) was ectopically expressed in L6-GLUT4 cells treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of Rac1 (G12V) was detected by immunostaining of the HA epitope tag. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 1A. Scale bar, 50 μm. (C) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells in which Rac1 (G12V) was not expressed (“−”) and L6-GLUT4 cells in which Rac1 (G12V) was expressed (“Rac1 (G12V)”) was quantified. Data are shown as means±S.E. (n=6). *Pb 0.001.
population of Rac1 may become activated in response to insulin by a PI3K-independent mechanism (Fig. 3A). In fact, an overlay assay showed that wortmannin-sensitive Rac1 activation occurs mostly in the cell surface areas in which actin cytoskeletal rearrangements were observed, whereas wortmannin-insensitive activation seems to be induced inside the cell (Fig. 3B). PI3K-mediated Rac1 activation on the surface of insulin-stimulated cells may be critical for GLUT4 translocation because insulin-dependent GLUT4 translocation is totally sensitive to wortmannin treatment in these cells [7,9]. Rac1 activation triggered by an upstream regulatory component is physiologically more significant than that by mutation of Rac1 itself considering that a specific subpopulation of Rac1 may be involved in GLUT4 translocation as described above. Accordingly, we next tested the effect of RalA knockdown on GLUT4 translocation induced by Rac1 that is activated by its upstream regulators. A constitutively plasma
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Fig. 3. Subcellular region-specific activation of Rac1 in response to insulin. (A) L6 myotubes were treated with (white circles) or without (black circles) wortmannin (100 nM) for 30 min prior to insulin stimulation. Rac1·GTP was detected by a pull-down assay after stimulation with insulin (100 nM) for indicated times. Intensities of the bands in immunoblots (not shown) were quantified by ImageJ software, and fold induction of the Rac1·GTP level is shown. Data are shown as means±S.E. (n=3). (B) L6 myotubes were treated with or without wortmannin (100 nM) for 30 min prior to insulin stimulation. Rac1·GTP was detected by an overlay assay following stimulation with insulin (100 nM) for indicated times. F-actin was stained with TRITC-conjugated phalloidin. Thin dotted lines indicate the contour of the cell. Images were acquired from the focal plane as depicted in schematic diagrams. Green dots in schematic diagrams represent the position of Rac1·GTP. Scale bar, 20 μm.
membrane-localized and activated form of PI3K, termed Myr-p110 [16], is expressed in control and RalA-specific siRNA-treated L6-GLUT4 cells, and GLUT4 translocation to the plasma membrane was examined. Following the expression of Myr-p110, ruffling membranes were generated at the cell surface likely through the activation of Rac1, to which GLUT4 was in fact translocated in control siRNA-treated cells. This GLUT4 translocation was mostly diminished by downregulation of RalA (Fig. 4). The Dbl family guanine nucleotide exchange factor (GEF) FLJ00068 was characterized as a GEF responsible for PI3K-dependent activation of Rac1 in insulin signaling in L6-GLUT4 cells [7]. Thus, the effect of RalA knockdown on FLJ00068-induced GLUT4 translocation was also tested. N-terminally truncated FLJ00068, termed FLJ68ΔN, is a constitutively activated form of FLJ00068 [7], and FLJ68ΔN-induced GLUT4 translocation was again abrogated when RalA expression was suppressed (Fig. 5). RalA interacts with the exocyst complex in its GTP-bound form, and thus the formation of the GTP-bound form may be prerequisite for signaling. In fact, insulin increased the level of RalA·GTP in adipocytes [15]. The expression of the activated form of Rac1 also increased the level of RalA·GTP to an extent similar to that observed following 5 min-stimulation with insulin in L6-GLUT4 cells (Fig. 6). Moreover, RalA became localized in membrane ruffles generated after insulin treatment (Fig. 7A). Notably, the activated form of Rac1 also stimulated the
formation of membrane ruffles, in which both endogenous and ectopically expressed RalA co-localized with activated Rac1 (Fig. 7). Therefore, a subpopulation of Rac1 activated by insulin in the plasma membrane (as shown in Fig. 3B) may specifically direct the activation of RalA and its translocation to the membrane ruffling areas. 4. Discussion Here, we demonstrate that RalA is required for insulin-dependent GLUT4 translocation also in muscle cells. The notion that RalA is regulated downstream of another small GTPase Rac1 is of particular importance in muscle cell signaling. Currently, however, the mechanism by which the activity of RalA is regulated remains to be elucidated. In adipocytes, one possible link between Akt2 and RalA is a Ral GAP complex composed of RGC1 regulatory and RGC2 catalytic subunits [18]. Akt2-catalyzed phosphorylation of RGC2 in response to insulin inhibits its GAP activity, leading to the accumulation of the active form of RalA in the cell [18]. As shown in this study, RalA mediates PI3K-dependent signals for GLUT4 translocation also in muscle cells (Fig. 4). In these cells as well, PI3K leads to the activation of Akt2, which in turn may directly inactivate the Ral GAP complex. However, this may not account for the activation of RalA in response to insulin in muscle cells, because, specifically in these cells, insulin signaling is
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Fig. 4. Inhibition of Myr-p110-induced cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP by RalA knockdown. (A) L6-GLUT4 cells were treated with control or RalA-specific siRNA and infected with a mock (“–”) or Myr-p110-expressing (“Myr-p110”) recombinant adenovirus. Cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of Myr-p110 was detected by immunostaining of phosphorylated Akt substrates. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 1A. Scale bar, 50 μm. (B) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells infected with a mock (“–”) or Myr-p110-expressing (“Myr-p110”) recombinant adenovirus was quantified. Virtually all cells infected with a Myr-p110-expressing recombinant adenovirus were positive for staining of phosphorylated Akt substrates. Data are shown as means±S.E. (n=6). *Pb 0.001.
mediated by the small GTPase Rac1, and insulin activation of Akt2 is not dependent on Rac1 [7,8]. Thus, the regulatory mechanism for RalA may be different among different types of cells, and the signaling molecules that regulate RalA specifically in muscle cells will be identified in future studies. Upon activation, RalA coordinates the exocyst complex and the motor protein Myo1c through direct interactions, leading to efficient trafficking of GLUT4 vesicles in adipocytes [15]. It is feasible that RalA exerts its function in muscle cells through a similar mechanism because the exocyst-mediated vesicle trafficking process is highly conserved [14]. GTP loading to RalA is required for specific interactions with downstream components, and in fact constitutively activated Rac1 induced the accumulation of the GTP-bound form of RalA (Fig. 6). This Rac1-dependent activation may occur only in a limited population of total RalA molecules, which may account for a small increase in the level of the GTP-bound form of RalA in stimulated cells (Fig. 6). Furthermore, we found that a GTPase-deficient RalA mutant ectopically expressed in muscle cells can induce GLUT4 translocation although less
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Fig. 5. Inhibition of FLJ68ΔN-induced cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP by RalA knockdown. (A) FLJ68ΔN was ectopically expressed in L6-GLUT4 cells treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of FLJ68ΔN was detected by immunostaining of the FLAG epitope tag. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 1A. Scale bar, 50 μm. (B) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells in which FLJ68ΔN was not expressed (“–”) and L6-GLUT4 cells in which FLJ68ΔN was expressed (“FLJ68ΔN”) was quantified. Data are shown as means ± S.E. (n = 4). *P b 0.001.
effectively than insulin (Fig. 1). Therefore, GTP hydrolysis-independent mechanisms for the dissociation of the RalA–exocyst complex, such as phosphorylation of Sec5 catalyzed by protein kinase C [19], may regulate vesicle trafficking also in muscle cells. Collectively, mechanisms
Fig. 6. Activation of RalA in response to Rac1 (G12V) expression. RalA·GTP in L6 myotubes was detected by a pull-down assay following stimulation with insulin (100 nM) for indicated times or ectopic expression of Rac1 (G12V). The expression of endogenous RalA and Rac1 (G12V) was detected by immunoblotting using antibodies against RalA and the HA epitope tag, respectively.
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Fig. 7. Redistribution of RalA in membrane ruffles induced by Rac1 (G12V). (A) RalA (WT) and Rac1 (G12V) were ectopically expressed in L6 myotubes. These cells were stimulated with insulin (100 nM) for indicated times and subcellular localization of RalA (WT) was determined by immunostaining of the V5 epitope tag. The expression of Rac1 (G12V) was detected by immunostaining of the HA epitope tag. F-actin was stained with TRITC-conjugated phalloidin. Thin dotted lines indicate the contour of the cell. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 3B. Scale bar, 20 μm. (B) Rac1 (G12V) was ectopically expressed in L6 myotubes, and subcellular localization of endogenous RalA was determined by immunostaining. The expression of Rac1 (G12V) was detected by immunostaining of the HA epitope tag. F-actin was stained with TRITC-conjugated phalloidin. Thin dotted lines indicate the contour of the cell. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 3B. Scale bar, 20 μm.
underlying GLUT4 vesicle trafficking downstream of RalA may be common, at least in part, in adipocytes and muscle cells, although different regulatory proteins for RalA are likely to be involved. In this study, we also show that insulin activates Rac1 by PI3K -dependent and independent mechanisms (Fig. 3). Given that the translocation of GLUT4 to the plasma membrane in response to insulin is mediated by PI3K, PI3K-dependent Rac1 activation, which occurs at the cell periphery, may be necessary for GLUT4 translocation. The constitutively activated form of PI3K or the GEF FLJ00068 is expected to stimulate only a specific subpopulation of the intracellular pool of the Rac1 molecule that is responsible for GLUT4 translocation. GLUT4 translocation induced by these regulatory components upstream of Rac1 was also sensitive to RalA downregulation, further supporting the notion that RalA acts downstream of Rac1 in insulin signaling (Figs. 4 and 5). Rac1-dependent actin cytoskeletal remodeling on the cell surface has been considered to be an important step for the induction of GLUT4 translocation [9,20]. In this study, we found that RalA plays a pivotal role downstream of Rac1, and constitutively activated Rac1 induced the re-localization of RalA in the plasma membrane (Figs. 2, 4, 5, and 7). Therefore, it is feasible that Rac1 is responsible not only for cytoskeletal remodeling, but also for other cellular processes, including RalA-mediated signaling, in the induction of glucose uptake. Further studies are required to fully address still unknown functions of Rac1 in insulin-dependent GLUT4 translocation. Acknowledgements We thank Wataru Ogawa for a recombinant adenovirus for constitutively activated PI3K. This work was supported by Grants-in-Aid for
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[17] J.S. Bogan, A.E. McKee, H.F. Lodish, Molecular and Cellular Biology 21 (2001) 4785–4806. Insulin-responsive compartments containing GLUT4 in 3T3-L1 and CHO cells: regulation by amino acid concentrations. [18] X.W. Chen, D. Leto, T. Xiong, G. Yu, A. Cheng, S. Decker, A.R. Saltiel, Molecular Biology of the Cell 22 (2011) 141–152. A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action. [19] X.W. Chen, D. Leto, J. Xiao, J. Goss, Q. Wang, J.A. Shavit, T. Xiong, G. Yu, D. Ginsburg, D. Toomre, Z. Xu, A.R. Saltiel, Nature Cell Biology 13 (2011) 580–588. Exocyst function is regulated by effector phosphorylation. [20] J.A. Lopez, J.G. Burchfield, D.H. Blair, K. Mele, Y. Ng, P. Vallotton, D.E. James, W.E. Hughes, Molecular Biology of the Cell 20 (2009) 3918–3929. Identification of a distal GLUT4 trafficking event controlled by actin polymerization.
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Role of RalA downstream of Rac1 in insulin-dependent glucose uptake in muscle cells Shinsuke Nozaki a, Shuji Ueda a, Nobuyuki Takenaka b, Tohru Kataoka a, Takaya Satoh a, b,⁎ a b
Division of Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650‐0017, Japan Laboratory of Cell Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1‐1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599‐8531, Japan
a r t i c l e
i n f o
Article history: Received 7 July 2012 Accepted 16 July 2012 Available online 20 July 2012 Keywords: Glucose uptake GTPase Insulin Rac1 RalA Skeletal muscle
a b s t r a c t The small GTPase RalA has been implicated in glucose uptake in insulin-stimulated adipocytes, although it remains unclear whether RalA has a similar role in insulin signaling in other types of cells. Recently, we have demonstrated that the Rho family GTPase Rac1 has a critical role in insulin-dependent glucose uptake in myoblast culture and mouse skeletal muscle. However, the mechanisms underlying Rac1-dependent glucose uptake, mostly mediated by the plasma membrane translocation of the glucose transporter GLUT4, remain largely unknown. The purpose of this study is to examine the involvement of RalA in Rac1 regulation of the translocation of GLUT4 to the plasma membrane in muscle cells. Ectopic expression of a constitutively activated RalA mutant indeed stimulated GLUT4 translocation, suggesting an important role of RalA also in muscle cells. GLUT4 translocation induced by constitutively activated mutation of Rac1 or more physiologically by upstream Rac1 regulators, such as phosphoinositide 3 kinase and the guanine nucleotide exchange factor FLJ00068, was abrogated when the expression of RalA was downregulated by RNA interference. The expression of constitutively activated Rac1, on the other hand, caused GTP loading and subcellular redistribution of RalA. Collectively, we propose a novel mechanism involving RalA for Rac1-mediated GLUT4 translocation in skeletal muscle cells. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Insulin-dependent glucose uptake in fat and muscle cells is largely mediated by translocation of the glucose transporter GLUT4 from intracellular storage sites to the plasma membrane. Insulin enhances exocytosis of GLUT4 vesicles and suppresses their endocytosis, leading to the net accumulation of GLUT4 in the plasma membrane. Accumulating evidence reveals that diverse signaling molecules, including phosphoinositide 3 kinase (PI3K) and the serine/threonine kinase Akt2, exert important roles in insulin signaling. However, the mechanisms underlying insulin-dependent GLUT4 trafficking remain incompletely understood [1–4]. Rho family small GTPases regulate a wide variety of cellular responses such as cytoskeletal rearrangements [5,6]. We and others recently reported that the Rho family member Rac1 is involved in the regulation of GLUT4 translocation in skeletal muscle cells [7–9]. One critical role of Rac1 in GLUT4 trafficking is thought to be the regulation of the actin cytoskeleton. Insulin induces rapid and marked reorganization of the actin cytoskeleton in myocytes,
Abbreviations: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; PI3K, phosphoinositide 3 kinase; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate. ⁎ Corresponding author. Tel./fax: +81 72 254 7650. E-mail address: [email protected] (T. Satoh). 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2012.07.013
including the formation of membrane ruffles, in which diverse signaling molecules, such as PI3K and Akt, become accumulated [10,11]. This signaling is considered to be crucial for the induction of GLUT4 translocation, and is mediated by Rac1 [10,12]. A subsequent study further showed that Rac1-dependent actin cytoskeletal remodeling and submembrane tethering of GLUT4 vesicles are important for cell surface expression of GLUT4 [13]. However, the regulation of actin cytoskeletal remodeling may not be a sole function of Rac1. It is likely that Rac1 is also involved in a series of insulin-dependent events, such as GLUT4 vesicle trafficking and its docking/fusion to the cell membrane, taking into consideration that constitutively activated Rac1 alone is able to induce GLUT4 translocation in the absence of insulin without enhancing the activity of the serine/threonine kinase Akt [7,8]. The small GTPase RalA has been implicated in intracellular membrane trafficking, including polarized membrane delivery, neurosecretion, and endocytosis [14]. In adipocytes, RalA resides in GLUT4 vesicles, and interacts with the exocyst subunit Sec5 and the motor protein Myo1c, thereby regulating GLUT4 targeting to the plasma membrane in response to insulin [15]. However, it remains uncertain whether RalA is also involved in GLUT4 translocation in other types of cells, such as muscle cells. In particular, the role of RalA downstream of Rac1, a skeletal muscle-specific regulator of insulin signaling, is totally unknown. In this paper, we show that RalA in fact plays a pivotal role also in Rac1-mediated GLUT4 translocation in muscle cells. These results emphasize a RalA-dependent common mechanism underlying GLUT4
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trafficking in contrast to divergence in upstream signaling pathways in different cell types. 2. Materials and methods 2.1. Materials Antibodies against Myc (mouse monoclonal (sc-40)) and hemagglutinin (HA) (rat monoclonal (11 867 423 001)) epitope tags were purchased from Santa Cruz Biotechnology and Roche Applied Science, respectively. Antibodies against V5 (rabbit polyclonal (V8137)) and FLAG (rabbit polyclonal (F7425)) epitope tags and tubulin (mouse monoclonal (T4026)) were purchased from Sigma-Aldrich. Anti-RalA (mouse monoclonal (610221)) and anti-Rac1 (mouse monoclonal (610650)) antibodies were purchased from BD Biosciences. An anti-phospho-(Ser/Thr) Akt substrate (rabbit polyclonal (9611)) antibody was purchased from Cell Signaling Technology. Antimouse IgG, anti-rabbit IgG, and anti-rat IgG antibodies conjugated with Alexa Fluor® 488/546/647 were purchased from Life Technologies. Tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (P1951) was purchased from Sigma-Aldrich. Anti-mouse IgG (NA9310) and anti-rabbit IgG (NA9340) antibodies conjugated with horseradish peroxidase were purchased from GE Healthcare. 2.2. Cell culture and RNA interference L6 and L6-GLUT4 rat myoblasts [7] were cultivated in minimum essential medium Eagle (M4655; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (Cell Culture Bioscience), 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 μg/ml streptomycin. A mixture of three small interfering RNA (siRNA) duplexes designed from the rat RalA cDNA sequence (RalA siRNA) (duplex-1, 5′-AAGAUGUCGAUCU GCACUUCCUCCC-3′; duplex-2, 5′-AAAUCUGUUCCCUGAAGUCCGCU GU-3′; duplex-3, 5′-CAUAGUUAACGUUCCACUGGUCAGC-3′) (Life Technologies) was used for down-regulation of RalA. The control siRNA (1022076) was purchased from Qiagen. Cells were transfected with siRNA (200 nM) using the Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer's instructions. 2.3. Induction of differentiation of L6 cells L6 cells were induced to differentiate into multinuclear myotubes by replacement of 10% (v/v) fetal bovine serum in the culture medium with 2% horse serum (GIBCO, 16050‐122) and subsequent incubation for 5 days. 2.4. Adenovirus infection Adenoviruses for the expression of 3 × HA-tagged Rac1 (WT), 3 × HA-tagged Rac1(G12V), V5-tagged RalA (WT), V5-tagged RalA (G23V), and FLAG-tagged FLJ68ΔN were generated and used to infect L6-GLUT4 cells by using the Adenovirus Cre/loxP kit (Takara Bio, Japan) according to the manufacturer's instructions. A recombinant adenovirus for the N-terminally myristoylated catalytic subunit of bovine PI3Kα (Myr-p110) was kindly provided by Dr. Wataru Ogawa (Kobe University Graduate School of Medicine) [16]. 2.5. Immunofluorescent microscopy and GLUT4 reporter assay The exofacial GLUT4 reporter GLUT4myc7‐green fluorescent protein (GFP) and an L6-derived cell line that stably expresses this reporter (termed L6-GLUT4) were previously described [7,17]. L6-GLUT4 cells were starved in serum-free medium for 2 h prior to measurement of GLUT4 translocation. For the detection of the cell surface-exposed GLUT4 reporter, formaldehyde-fixed cells were treated with an anti-Myc antibody before permeabilization. After
washing three times with phosphate-buffered saline (−), cells were permeabilized with 0.1% (v/v) Triton X-100 and then counterstained with another primary antibody for the identification of the cells that express a specific ectopically expressed or endogenous protein. Images were obtained using a 40 × oil objective on a confocal laser-scanning microscope (LSM 700; Carl Zeiss) and processed by the Zeiss LSM Image Browser, version 3.5. The relative amount of GLUT4myc7-GFP exposed at the plasma membrane was estimated from measurements of Myc and GFP fluorescence signals. Intensities of Myc and GFP fluorescence signals in regions of interest were quantified using ImageJ software, and ratios of Myc and GFP signals (Myc/GFP) were calculated [8]. 2.6. Immunoblotting Proteins were separated by SDS‐polyacrylamide gel electrophoresis and transferred on to a 0.45 μm pore size polyvinylidene difluoride membrane (GE Healthcare). The membrane was stained with the respective primary antibodies and a horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody, followed by visualization by enhanced chemiluminescence detection reagents (GE Healthcare). 2.7. Pull down assay for activated Rac1 and RalA Proteins were expressed in L6-GLUT4 cells through adenoviral infection. Cells were harvested with a pull down assay buffer [50 mM Tris–HCl (pH 7.4), 0.5% Nonidet P-40, 0.15 M NaCl, 20 mM MgCl2, and protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan)]. Lysates were centrifuged at 18,000×g for 1 min, and supernatants were added to glutathione S-transferase (GST)-Myc-PAK (67–150) (for Rac1) or GST-3× V5-Sec5 (1–99) (for RalA) immobilized on glutathione– Sepharose beads, and incubated at 4 °C for 30 min. Glutathione– Sepharose beads were washed twice with pull down assay buffer, and activated Rac1 or RalA bound to the beads was detected by immunoblotting using an anti-Rac1 or anti-RalA antibody. 2.8. Overlay assay (in situ detection) for activated Rac1 In situ detection of the activated form of Rac1 in paraformaldehydefixed cells were performed as described previously [7]. 3. Results As a step to assess the involvement of RalA in glucose uptake in muscle cells, a constitutively activated mutant of RalA (RalA (G23V)) was ectopically expressed in L6-derived myoblast (L6-GLUT4) cells, and its effect on GLUT4 translocation to the plasma membrane was examined (Fig. 1). The L6-GLUT4 cell line stably expresses an N-terminally GFP- and exofacially Myc epitope-tagged GLUT4 reporter that is detected by an anti-Myc antibody only when re-localized in the plasma membrane [7]. L6-GLUT4 cells expressing RalA(G23V) were highly positive for Myc staining whereas L6-GLUT4 cells without RalA(G23V) were not stained by an anti-Myc antibody (Fig. 1A). Fluorescent intensities of GFP and Myc staining in L6-GLUT4 cells positive or negative for RalA(G23V) expression were quantified, and the Myc/GFP ratio, which reflects the extent of cell surface translocation of GLUT4, was calculated (Fig. 1B). Ectopic expression of RalA(G23V) in fact induced an approximately 2.5-fold increase in the level of GLUT4 translocated to the plasma membrane. Therefore, RalA is likely to be implicated in signaling that leads to glucose uptake in muscle cells. We and others have demonstrated that the Rho family GTPase Rac1 plays an important role in insulin-dependent GLUT4 translocation to the plasma membrane in cultured muscle cells [7,9] and mouse skeletal muscle [8]. However, the mechanism by which Rac1-dependent signaling causes the plasma membrane translocation of GLUT4 remains obscure. By using the GLUT4 reporter assay
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Fig. 1. Cell surface translocation of GLUT4 induced by a constitutively activated mutant of RalA. (A) RalA (G23V) was ectopically expressed in L6-GLUT4 cells, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of RalA (G23V) was detected by immunostaining of the V5 epitope tag. Images were acquired from the focal plane as depicted in the schematic diagram. Scale bar, 50 μm. (B) Cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells in which RalA (G23V) was not expressed (“−”) and L6-GLUT4 cells in which RalA (G23V) was expressed (“RalA (G23V)”) was quantified. Data are shown as means±S.E. (n=6). *Pb 0.001.
described in Fig. 1, we next tested the involvement of RalA downstream of Rac1 in signaling that mediates glucose uptake. The endogenous expression level of RalA, as evaluated by immunoblotting, was significantly downregulated with specific siRNA duplexes (Fig. 2A). The constitutively activated mutant Rac1 (G12V) was ectopically expressed in L6-GLUT4 cells pretreated with control or RalA-specific siRNA, and the translocation of the GLUT4 reporter was assessed (Fig. 2B and C). Rac1 (G12V) induced GLUT4 translocation in control siRNA-treated cells as previously described [7]. Rac1 (G12V)-induced GLUT4 translocation was largely suppressed in L6-GLUT4 cells in which the expression of RalA was reduced. Therefore, RalA exerts a critical function downstream of Rac1 although it remains unclear whether the residual level of induction of GLUT4 translocation by Rac1 (G12V) in RalA siRNA-treated cells is ascribed to incomplete knockdown of RalA or RalA-independent mechanisms. PI3K is a key component in insulin signaling in various types of cells as proposed from a variety of observations, such as the inhibitory effect of the specific inhibitor wortmannin [2]. Given that PI3K acts immediately downstream of the insulin receptor/insulin receptor substrate-1 complex, insulin-dependent activation of Rac1 is likely to be mediated by PI3K. Actually, we observed that insulin-induced Rac1 activation was sensitive to the inhibitory effect of wortmannin [7]. Here, we further examined the effect of wortmannin on insulin activation of Rac1 in detail by two distinct assays (Fig. 3). A pull-down assay for the activated form of Rac1 showed that insulin caused a rapid increase in the level of Rac1·GTP, which was sensitive to the inhibitory effect of wortmannin, suggesting the involvement of PI3K in insulin-dependent activation of Rac1 (Fig. 3A). This inhibition of Rac1 activation, however, was not complete, and therefore a small
Fig. 2. Inhibition of Rac1 (G12V)-induced cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP by RalA knockdown. (A) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and the expression of endogenous RalA was examined by immunoblotting. The expression levels of tubulin are also shown as a loading control. (B) Rac1 (G12V) was ectopically expressed in L6-GLUT4 cells treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of Rac1 (G12V) was detected by immunostaining of the HA epitope tag. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 1A. Scale bar, 50 μm. (C) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells in which Rac1 (G12V) was not expressed (“−”) and L6-GLUT4 cells in which Rac1 (G12V) was expressed (“Rac1 (G12V)”) was quantified. Data are shown as means±S.E. (n=6). *Pb 0.001.
population of Rac1 may become activated in response to insulin by a PI3K-independent mechanism (Fig. 3A). In fact, an overlay assay showed that wortmannin-sensitive Rac1 activation occurs mostly in the cell surface areas in which actin cytoskeletal rearrangements were observed, whereas wortmannin-insensitive activation seems to be induced inside the cell (Fig. 3B). PI3K-mediated Rac1 activation on the surface of insulin-stimulated cells may be critical for GLUT4 translocation because insulin-dependent GLUT4 translocation is totally sensitive to wortmannin treatment in these cells [7,9]. Rac1 activation triggered by an upstream regulatory component is physiologically more significant than that by mutation of Rac1 itself considering that a specific subpopulation of Rac1 may be involved in GLUT4 translocation as described above. Accordingly, we next tested the effect of RalA knockdown on GLUT4 translocation induced by Rac1 that is activated by its upstream regulators. A constitutively plasma
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Fig. 3. Subcellular region-specific activation of Rac1 in response to insulin. (A) L6 myotubes were treated with (white circles) or without (black circles) wortmannin (100 nM) for 30 min prior to insulin stimulation. Rac1·GTP was detected by a pull-down assay after stimulation with insulin (100 nM) for indicated times. Intensities of the bands in immunoblots (not shown) were quantified by ImageJ software, and fold induction of the Rac1·GTP level is shown. Data are shown as means±S.E. (n=3). (B) L6 myotubes were treated with or without wortmannin (100 nM) for 30 min prior to insulin stimulation. Rac1·GTP was detected by an overlay assay following stimulation with insulin (100 nM) for indicated times. F-actin was stained with TRITC-conjugated phalloidin. Thin dotted lines indicate the contour of the cell. Images were acquired from the focal plane as depicted in schematic diagrams. Green dots in schematic diagrams represent the position of Rac1·GTP. Scale bar, 20 μm.
membrane-localized and activated form of PI3K, termed Myr-p110 [16], is expressed in control and RalA-specific siRNA-treated L6-GLUT4 cells, and GLUT4 translocation to the plasma membrane was examined. Following the expression of Myr-p110, ruffling membranes were generated at the cell surface likely through the activation of Rac1, to which GLUT4 was in fact translocated in control siRNA-treated cells. This GLUT4 translocation was mostly diminished by downregulation of RalA (Fig. 4). The Dbl family guanine nucleotide exchange factor (GEF) FLJ00068 was characterized as a GEF responsible for PI3K-dependent activation of Rac1 in insulin signaling in L6-GLUT4 cells [7]. Thus, the effect of RalA knockdown on FLJ00068-induced GLUT4 translocation was also tested. N-terminally truncated FLJ00068, termed FLJ68ΔN, is a constitutively activated form of FLJ00068 [7], and FLJ68ΔN-induced GLUT4 translocation was again abrogated when RalA expression was suppressed (Fig. 5). RalA interacts with the exocyst complex in its GTP-bound form, and thus the formation of the GTP-bound form may be prerequisite for signaling. In fact, insulin increased the level of RalA·GTP in adipocytes [15]. The expression of the activated form of Rac1 also increased the level of RalA·GTP to an extent similar to that observed following 5 min-stimulation with insulin in L6-GLUT4 cells (Fig. 6). Moreover, RalA became localized in membrane ruffles generated after insulin treatment (Fig. 7A). Notably, the activated form of Rac1 also stimulated the
formation of membrane ruffles, in which both endogenous and ectopically expressed RalA co-localized with activated Rac1 (Fig. 7). Therefore, a subpopulation of Rac1 activated by insulin in the plasma membrane (as shown in Fig. 3B) may specifically direct the activation of RalA and its translocation to the membrane ruffling areas. 4. Discussion Here, we demonstrate that RalA is required for insulin-dependent GLUT4 translocation also in muscle cells. The notion that RalA is regulated downstream of another small GTPase Rac1 is of particular importance in muscle cell signaling. Currently, however, the mechanism by which the activity of RalA is regulated remains to be elucidated. In adipocytes, one possible link between Akt2 and RalA is a Ral GAP complex composed of RGC1 regulatory and RGC2 catalytic subunits [18]. Akt2-catalyzed phosphorylation of RGC2 in response to insulin inhibits its GAP activity, leading to the accumulation of the active form of RalA in the cell [18]. As shown in this study, RalA mediates PI3K-dependent signals for GLUT4 translocation also in muscle cells (Fig. 4). In these cells as well, PI3K leads to the activation of Akt2, which in turn may directly inactivate the Ral GAP complex. However, this may not account for the activation of RalA in response to insulin in muscle cells, because, specifically in these cells, insulin signaling is
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Fig. 4. Inhibition of Myr-p110-induced cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP by RalA knockdown. (A) L6-GLUT4 cells were treated with control or RalA-specific siRNA and infected with a mock (“–”) or Myr-p110-expressing (“Myr-p110”) recombinant adenovirus. Cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of Myr-p110 was detected by immunostaining of phosphorylated Akt substrates. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 1A. Scale bar, 50 μm. (B) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells infected with a mock (“–”) or Myr-p110-expressing (“Myr-p110”) recombinant adenovirus was quantified. Virtually all cells infected with a Myr-p110-expressing recombinant adenovirus were positive for staining of phosphorylated Akt substrates. Data are shown as means±S.E. (n=6). *Pb 0.001.
mediated by the small GTPase Rac1, and insulin activation of Akt2 is not dependent on Rac1 [7,8]. Thus, the regulatory mechanism for RalA may be different among different types of cells, and the signaling molecules that regulate RalA specifically in muscle cells will be identified in future studies. Upon activation, RalA coordinates the exocyst complex and the motor protein Myo1c through direct interactions, leading to efficient trafficking of GLUT4 vesicles in adipocytes [15]. It is feasible that RalA exerts its function in muscle cells through a similar mechanism because the exocyst-mediated vesicle trafficking process is highly conserved [14]. GTP loading to RalA is required for specific interactions with downstream components, and in fact constitutively activated Rac1 induced the accumulation of the GTP-bound form of RalA (Fig. 6). This Rac1-dependent activation may occur only in a limited population of total RalA molecules, which may account for a small increase in the level of the GTP-bound form of RalA in stimulated cells (Fig. 6). Furthermore, we found that a GTPase-deficient RalA mutant ectopically expressed in muscle cells can induce GLUT4 translocation although less
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Fig. 5. Inhibition of FLJ68ΔN-induced cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP by RalA knockdown. (A) FLJ68ΔN was ectopically expressed in L6-GLUT4 cells treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP was assessed by immunostaining of the exofacial Myc epitope tag. The expression of FLJ68ΔN was detected by immunostaining of the FLAG epitope tag. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 1A. Scale bar, 50 μm. (B) L6-GLUT4 cells were treated with control or RalA-specific siRNA, and cell surface translocation of the GLUT4 reporter GLUT4myc7-GFP in L6-GLUT4 cells in which FLJ68ΔN was not expressed (“–”) and L6-GLUT4 cells in which FLJ68ΔN was expressed (“FLJ68ΔN”) was quantified. Data are shown as means ± S.E. (n = 4). *P b 0.001.
effectively than insulin (Fig. 1). Therefore, GTP hydrolysis-independent mechanisms for the dissociation of the RalA–exocyst complex, such as phosphorylation of Sec5 catalyzed by protein kinase C [19], may regulate vesicle trafficking also in muscle cells. Collectively, mechanisms
Fig. 6. Activation of RalA in response to Rac1 (G12V) expression. RalA·GTP in L6 myotubes was detected by a pull-down assay following stimulation with insulin (100 nM) for indicated times or ectopic expression of Rac1 (G12V). The expression of endogenous RalA and Rac1 (G12V) was detected by immunoblotting using antibodies against RalA and the HA epitope tag, respectively.
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Fig. 7. Redistribution of RalA in membrane ruffles induced by Rac1 (G12V). (A) RalA (WT) and Rac1 (G12V) were ectopically expressed in L6 myotubes. These cells were stimulated with insulin (100 nM) for indicated times and subcellular localization of RalA (WT) was determined by immunostaining of the V5 epitope tag. The expression of Rac1 (G12V) was detected by immunostaining of the HA epitope tag. F-actin was stained with TRITC-conjugated phalloidin. Thin dotted lines indicate the contour of the cell. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 3B. Scale bar, 20 μm. (B) Rac1 (G12V) was ectopically expressed in L6 myotubes, and subcellular localization of endogenous RalA was determined by immunostaining. The expression of Rac1 (G12V) was detected by immunostaining of the HA epitope tag. F-actin was stained with TRITC-conjugated phalloidin. Thin dotted lines indicate the contour of the cell. Images were acquired from the focal plane as depicted in the schematic diagram in Fig. 3B. Scale bar, 20 μm.
underlying GLUT4 vesicle trafficking downstream of RalA may be common, at least in part, in adipocytes and muscle cells, although different regulatory proteins for RalA are likely to be involved. In this study, we also show that insulin activates Rac1 by PI3K -dependent and independent mechanisms (Fig. 3). Given that the translocation of GLUT4 to the plasma membrane in response to insulin is mediated by PI3K, PI3K-dependent Rac1 activation, which occurs at the cell periphery, may be necessary for GLUT4 translocation. The constitutively activated form of PI3K or the GEF FLJ00068 is expected to stimulate only a specific subpopulation of the intracellular pool of the Rac1 molecule that is responsible for GLUT4 translocation. GLUT4 translocation induced by these regulatory components upstream of Rac1 was also sensitive to RalA downregulation, further supporting the notion that RalA acts downstream of Rac1 in insulin signaling (Figs. 4 and 5). Rac1-dependent actin cytoskeletal remodeling on the cell surface has been considered to be an important step for the induction of GLUT4 translocation [9,20]. In this study, we found that RalA plays a pivotal role downstream of Rac1, and constitutively activated Rac1 induced the re-localization of RalA in the plasma membrane (Figs. 2, 4, 5, and 7). Therefore, it is feasible that Rac1 is responsible not only for cytoskeletal remodeling, but also for other cellular processes, including RalA-mediated signaling, in the induction of glucose uptake. Further studies are required to fully address still unknown functions of Rac1 in insulin-dependent GLUT4 translocation. Acknowledgements We thank Wataru Ogawa for a recombinant adenovirus for constitutively activated PI3K. This work was supported by Grants-in-Aid for
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