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Molecular Endocrinology, doi:10.1210/me.2005-0455
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Molecular Endocrinology 20 (11): 2864-2875
Copyright © 2006 by The Endocrine Society

Enigma Interacts with Adaptor Protein with PH and SH2 Domains to Control Insulin-Induced Actin Cytoskeleton Remodeling and Glucose Transporter 4 Translocation

Romain Barrès, Thierry Grémeaux, Philippe Gual, Teresa Gonzalez, Jean Gugenheim, Albert Tran, Yannick Le Marchand-Brustel and Jean-François Tanti

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 568 (R.B., T.Gr., P.G., T.Go., Y.L.M.-B., J.-F.T.), F-06107 Nice, France; Université de Nice Sophia-Antipolis (R.B., T.Gr., P.G., T.Go., J.G., A.T.,Y.L.M.-B., J.-F.T.), Faculté de Médecine, F-06107, Nice, France; Centre Hospitalier Universitaire de Nice (J.G.), Service de Chirurgie Digestive et Centre de Transplantation Hépatique, Nice, France; and Centre Hospitalier Universitaire de Nice Fédération d’Hépatologie (A.T.), Nice, France

Address all correspondence and requests for reprints to: Jean-François Tanti, Institut National de la Santé et de la Recherche Medicale, Unité 568, Faculté de médecine, avenue de Valombrose, 06107 Nice Cedex 02, France. E-mail: tanti{at}unice.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN SIGNALING IS required for normal glucose homeostasis. Binding of insulin to its receptor results in the phosphorylation of several intracellular substrates, including the insulin receptor substrate (IRS) family, Shc, and APS (adaptor protein with PH and SH2 domains). IRS phosphorylation leads to the activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway that is critical for insulin-induced glucose transport and glucose transporter 4 (Glut 4) translocation. However, although necessary, the PI 3-kinase pathway does not seem to be sufficient, and recently, a new pathway, independent of PI 3-kinase activation, has been described (1). This pathway requires tyrosine phosphorylation of the adaptor protein APS by the insulin receptor (2). APS is the last identified member of a conserved family of SH2 adaptor molecules including Lnk and SH2-B (3). All members of this family contain a C-terminal SH2 domain, a central PH domain, and a N-terminal proline-rich region. Lnk is involved in T cell receptor activation whereas APS and SH2-B are involved in the signaling pathway of receptors for various growth factors including insulin and IGF-I. APS is highly expressed in insulin-responsive tissues, especially in adipocytes, and is the preferential target for the insulin receptor kinase in these cell types compared with SH2-B (3, 4). APS associates with phosphotyrosine residues situated within the activation loop of the insulin receptor via its SH2 domain (4, 5, 6) and undergoes insulin-stimulated tyrosine phosphorylation on tyrosine 618, allowing it to bind Cbl in complex with Cbl-associated protein (CAP) (7). Once phosphorylated on tyrosine residues, Cbl recruits the adapter protein CrkII in a complex with C3G, a GDP to GTP exchange factor for TC10, a Rho-family GTPase that regulates Glut 4 trafficking (1). Several data favor an important role of APS in Glut 4 translocation. Indeed, in 3T3-L1 adipocytes, expression of a Y618F mutant of APS that is not phosphorylated in response to insulin, or knock-down of APS protein expression by using short interfering RNA strategy inhibits insulin-induced Glut 4 translocation and glucose transport (2, 8). Studies of APS-deficient mice have led to contradictory results because one report describes an increase in whole body insulin sensitivity and insulin effect on glucose transport in adipocytes whereas a more recent study concludes that APS does not appear to modulate insulin regulation of glucose metabolism (9, 10). Thus, to bring about gain of insight into function of this protein, we searched for new partners of APS and identified Enigma as an APS-binding protein by yeast two-hybrid screening (11). Enigma belongs to a family of cytoplasmic proteins that, structurally, contains one amino-terminal PDZ domain and one to three carboxy-terminal LIM domains. Both PDZ and LIM domains are protein-protein interacting modules, and several LIM domain-containing proteins were found to be associated with the actin cytoskeleton, playing a role in signal transduction and organization of actin filaments during various cellular processes (12, 13, 14). The LIM domains of Enigma bind to protein kinases, whereas its PDZ domain was shown to interact with actin-binding proteins such as ß-tropomyosin (13, 14, 15). This family of proteins could be involved in targeting protein kinases to cytoskeleton elements. Our recent report showing that Enigma and APS are colocalized with F-actin in insulin-induced membrane ruffles (11) and the previous report that Enigma interacts directly with the insulin receptor (16) support a model in which the APS-Enigma complex could be a link between insulin signaling and actin cytoskeleton.

Several studies have revealed the importance of actin cytoskeletal organization in the regulation of insulin-induced Glut 4 translocation (1, 17) and implicated the TC10 pathway in this process (1). Because the APS-Enigma complex is involved in actin organization (11), this prompted us to study the potential role of Enigma in insulin-induced Glut 4 translocation in adipocytes.

In the present study, we showed that APS and Enigma were colocalized with cortical actin in 3T3-L1 adipocytes, and we propose that Enigma could play a role in insulin-induced Glut 4 translocation by regulating cortical actin dynamics.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
APS and Enigma are Colocalized with Cortical Actin Cytoskeleton in 3T3-L1 Adipocytes
We wanted to determine whether Enigma was expressed in 3T3-L1 cells and was associated with APS and actin cytoskeleton. Using a cDNA probe encoding the full-length sequence of Enigma, we detected, by Northern blot analysis, a transcript of 2.4 kb in both 3T3-L1 fibroblasts and fully differentiated 3T3-L1 adipocytes (Fig. 1Go). Enigma mRNA expression was not markedly modified upon differentiation in adipocytes. By contrast, APS mRNA expression was up-regulated in differentiated 3T3-L1 adipocytes (Fig. 1Go) in agreement with previously published data (4).


Figure 1
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  		Fig. 1. Expression of Enigma and APS in 3T3-L1 Adipocytes

Northern blot containing poly(A)+ RNA from 3T3-L1 fibroblasts or adipocytes was hybridized with Enigma, APS, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. The arrows on the right denote the position of the Enigma, APS, and GAPDH transcripts. A typical Northern blot representative of two independent experiments is shown.

 
To determine the localization of the two proteins, 3T3-L1 adipocytes were cotransfected with both hemagglutinin (HA)-Enigma and Myc-APS cDNAs, and the distribution of the two proteins was studied by immunofluorescence staining using anti-HA and anti-Myc antibodies, respectively, followed by confocal microscopy (Fig. 2Go, A–C). We used hemagglutinin (HA)-tagged Enigma because no commercial antibody was available against Enigma, and the antibody kindly provided by Dr. Gill (13) did not recognize the endogenous protein in immunofluorescence or immunoprecipitation studies. HA-Enigma was distributed at the plasma membrane and in intracellular compartments (Fig. 2AGo). Myc-APS was predominantly localized near the cell surface, as previously described (2), with lower levels detected in intracellular structures (Fig. 2BGo). An overlay of APS and Enigma staining revealed that the two proteins colocalized mainly at the plasma membrane (Fig. 2CGo) with partial colocalization inside the cells. APS and Enigma were previously shown to be associated with F-actin in membrane ruffles (11). In 3T3-L1 adipocytes, F-actin is mainly cortical, underlying the plasma membrane, with some F-actin present in the perinuclear region (1). We next wanted to determine whether Enigma and APS were localized with F-actin. Myc-APS or HA-Enigma cDNAs were transiently transfected in 3T3-L1 adipocytes, and F-actin structures were visualized after Texas-red phalloidin staining (Fig. 2Go, D–I). Phalloidin staining indicated that F-actin was mainly organized as cortical actin distributed at the periphery of the cells with some F-actin in the perinuclear region (Fig. 2Go, E and H). An overlay of both APS and Enigma staining with F-actin staining revealed a colocalization of APS and Enigma with cortical actin (Fig. 2Go, F and I).


Figure 2
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  		Fig. 2. APS and Enigma Are Colocalized with Cortical Actin Cytoskeleton in 3T3-L1 Adipocytes

3T3-L1 adipocytes were seeded on coverslips after transfection with plasmid coding for HA-Enigma or Myc-APS. After fixation and permeabilization, cells were costained with anti-Myc antibody to detect APS (panels B and D), anti-HA antibody to detect Enigma (panels A and G), and, Texas Red-conjugated phalloidin to detect F-actin (panels E and H) and analyzed by confocal immunofluorescence microscopy. The merge images are shown in panels C, F, and I. Representative cells from three to four independent experiments are shown.

 
The Interaction between APS and Enigma Is Involved in Glut 4 Translocation
APS is an essential component of the CAP/Cbl pathway in the control of insulin-induced glucose uptake (2). Moreover, insulin-stimulated Glut 4 translocation in adipocytes is dependent upon cortical actin remodeling (17). We investigated whether the interaction between Enigma and APS plays a role in insulin-induced Glut 4 translocation. To this aim we used an APS mutant lacking the NTPY motif (Myc-APS[APTA]) needed for Enigma binding in NIH-3T3 fibroblasts (11). We first verified that this APS mutant did not interact with Enigma in 3T3-L1 adipocytes. We compared the ability of wild-type Myc-APS and Myc-APS[APTA] to interact with HA-Enigma by coexpressing both proteins in 3T3-L1 adipocytes. As shown in Fig. 3Go, wild-type Myc-APS coprecipitated with HA-Enigma whereas Myc-APS[APTA] did not. Insulin did not modify the interaction between APS and Enigma (data not shown). We next examined whether expression of the Myc-APS[APTA] mutant modulates insulin-stimulated Glut 4 translocation. 3T3-L1 adipocytes were electroporated with plasmids coding for green fluorescent protein (GFP), GFP-APS, or GFP-APS[APTA] together with a construct coding for a DsRed protein fusion of Myc-Glut 4 (Myc-Glut 4-DsRed), and were treated without or with insulin. Myc-Glut 4-DsRed at the cell surface of cotransfected cells was detected after anti-Myc antibody and Cy5-conjugated secondary antibody binding as described (18). Only cells with a continuous Cy5 fluorescent rim were considered as positive for Glut 4 translocation. The use of a Glut 4 transporter with a Myc-epitope tag allowed us to determine the amount of Glut 4 that was properly inserted in the plasma membrane with the Myc-epitope toward the extracellular medium. In the basal state, the number of cells with Myc-Glut 4-DsRed at the cell surface was similar between the different conditions of transfection (Fig. 4AGo, left panel). The number of GFP or GFP-APS-expressing cells displaying a fluorescent rim at the cell surface was similarly increased after insulin stimulation. In contrast, the number of cells presenting a continuous fluorescent rim at the cell surface increased by 30% with the expression of GFP-APS[APTA] compared with adipocytes that expressed GFP or GFP-APS (Fig. 4AGo, left panel). We then determined the effect of GFP-APS[APTA]on insulin-induced glucose uptake. To this aim, 3T3-L1 adipocytes were transfected with 400 µg of vectors coding for GFP, GFP-APS, or GFP-APS[APTA]. In these conditions approximately 60–70% of cells were actually transfected as previously shown (19), allowing for the measurement of an effect of the different constructs on glucose uptake. Expression of GFP-APS[APTA] increased the insulin effect on glucose uptake compared with cells expressing GFP or GFP-APS (Fig. 4AGo, right panel). This effect was not due to a change in early steps in insulin signaling or to an increase in the amount of Glut 4 (Fig. 4BGo). These results indicate that Enigma binding to APS could play a role in the regulation of Glut 4 translocation and glucose uptake.


Figure 3
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  		Fig. 3. Deletion of the APTA motif of APS Prevents the Interaction with Enigma in 3T3-L1 Adipocytes

3T3-L1 adipocytes were electroporated with Myc-APSwt alone or cotransfected with HA-Enigma and Myc-APS or Myc-APS[APTA]. HA-Enigma was immunoprecipitated from cell lysates with anti-HA antibody, and immunoprecipitates were blotted with anti-Myc antibody. The level of expression of HA-Enigma and Myc-APS or Myc-APS[APTA] was controlled after immunoblotting with anti-HA antibody or anti-Myc antibody, respectively. Representative autoradiographs from three independent experiments are shown. IB, Immunoblotting; IP, immunoprecipitation.

 

Figure 4
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  		Fig. 4. Overexpression of APS[APTA] Increases Insulin-Stimulated Glut 4 Translocation and Glucose Uptake

A (Left panel), 3T3-L1 adipocytes were electroporated with 75 µg of Myc-Glut 4-DsRed cDNA together with 75 µg of GFP, GFP-APS, or GFP-APS[APTA] cDNAs. The cells were allowed to recover for 20 h, and then were serum starved for 2 h and in each of subsequently treated without or with 100 nM insulin for 45 min at 37 C. Cells were fixed and incubated with anti-Myc antibody and antimouse Cy5-conjugated antibody. Cotransfected cells were analyzed for the presence of a visually detectable plasma membrane (PM) fluorescent rim as described in Materials and Methods. These data represent scoring of at least 100 cells in each of four independent experiments. Each bar represents an average number of cotransfected cells displaying cell surface fluorescence ± SEM (*, P < 0.05). A (Right panel), 3T3-L1 adipocytes were electroporated with 400 µg of GFP, GFP-APS, or GFP-APS[APTA] cDNAs. Cells were allowed to recover and left untreated or stimulated with insulin (100 nM) for 20 min at 37 C. Uptake of [2-3H]deoxyglucose (2-DOG) was measured during a 3-min period as described in Materials and Methods. Means ± SEM from three independent experiments are shown (*, P < 0.01). B, 3T3-L1 adipocytes were electroporated as described above and left untreated or stimulated with insulin (100 nM) for 5 min at 37 C. Western blot shows 3T3-L1 adipocyte lysates blotted with phosphotyrosine, phospho-PKB (Ser473), phospho-ERK (T202/Y204), or Glut 4 antibodies. GFP-APS or GFP-APS[APTA] expression was visualized with anti-GFP antibody. Representative autoradiographs from three independent experiments are shown. IB, Immunoblotting; wt, wild type.

 
Overexpression of Enigma Decreases Insulin-Induced Glut 4 Translocation and Glucose Uptake in 3T3-L1 Adipocytes
The fact that the disruption of APS and Enigma interaction favored the insulin-induced Glut 4 translocation prompted us to investigate the role of Enigma in this process. 3T3-L1 adipocytes were electroporated with plasmids coding for GFP or GFP-Enigma together with Myc-Glut 4-DsRed. The number of cells with Myc-Glut 4-DsRed at the cell surface was quantified, as described above. In the basal state, the number of cells with Myc-Glut 4-DsRed at the cell surface was similar to that with the expression of GFP or GFP-Enigma. Importantly, after insulin stimulation, the number of cells with Myc-DsRed-Glut 4 at the cell surface was reduced by 40% with the expression of GFP-Enigma (Fig. 5AGo, left panel). LIM domains act as modular protein-binding domains that play an important role in the functions of LIM proteins. To determine whether LIM domains of Enigma were involved in its inhibitory effect on Glut 4 translocation, we expressed GFP-Enigma without the LIM domains (GFP-ENG {Delta}LIM1,2,3) together with Myc-Glut 4-DsRed. The expression of GFP-ENG {Delta}LIM1,2,3 did not decrease insulin-induced Myc-Glut 4-DsRed localization to the plasma membrane, suggesting that LIM domains are necessary for the inhibitory effect of Enigma (Fig. 5AGo, left panel). We then assessed whether the inhibition of insulin-induced Glut 4 translocation in cells overexpressing Enigma reduced the uptake of glucose. Insulin induced a 4-fold increase in glucose uptake in control cells, and Enigma expression reduced by 40% the insulin effect (Fig. 5AGo, right panel). We next examined whether Enigma expression altered the expression of Glut 4 and /or the early insulin receptor signal transduction. As shown in Fig. 5BGo, Enigma expression did not significantly modify the expression of Glut 4, the insulin receptor ß-subunit autophosphorylation and IRS1 tyrosine phosphorylation, or protein kinase B (PKB) and ERK activation. We also verified that Enigma expression did not modify the localization of Myc-Glut 4-DsRed in unstimulated adipocytes because inhibition of insulin-induced Myc-Glut 4-DsRed translocation in Enigma-expressing adipocytes might be due to mislocalization of this protein. Thus, we compared, by direct fluorescence and confocal microscopy, the subcellular localization of Myc-Glut 4-DsRed in unstimulated adipocytes that expressed GFP or GFP-Enigma. As shown in reconstituted fields (Fig. 6Go) Myc-Glut 4-DsRed was localized mainly in the perinuclear region both in GFP and GFP-Enigma transfected adipocytes. These results indicated that Enigma expression decreased insulin-induced glucose uptake probably by inhibiting endogenous Glut 4 translocation.


Figure 5
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  		Fig. 5. Enigma Overexpression Inhibits Insulin-Stimulated Glut 4 Translocation and Glucose Transport in 3T3-L1 Adipocytes

A (Left panel), 3T3-L1 adipocytes were electroporated with 75 µg of Myc-Glut 4-DsRed cDNA together with 75 µg of GFP, GFP-Enigma, or GFP-Enigma{Delta}LIM1,2,3 cDNAs. The cells were allowed to recover for 20 h, and then were serum starved for 2 h and subsequently treated without or with 100 nM insulin (Ins) for 40 min at 37 C. Cells were fixed and incubated with anti-Myc antibody and antimouse Cy5-conjugated antibody. Transfected cells displaying visually detectable plasma membrane (PM) rim fluorescence were detected by indirect immunofluorescence. These data represent scoring of at least 100 cells from four to five independent experiments. Each bar represents an average number of cotransfected cells displaying cell surface fluorescence ± SEM (*, P < 0.05). A (Right panel), 3T3-L1 adipocytes were electroporated with 400 µg of GFP or with 400 µg GFP-Enigma cDNA. Cells were allowed to recover and left untreated or stimulated with insulin (100 nM) for 20 min at 37 C. Uptake of [2-3H]deoxyglucose (2-DOG) was measured during a 3-min period as described in Materials and Methods. Means ± SEM from three independent experiments are shown (*, P < 0.01). B, 3T3-L1 adipocytes were electroporated with 400 µg of GFP or GFP-Enigma cDNAs, and cells were allowed to recover for 20 h and then incubated in serum-free media for 4 h. Cells were subsequently left untreated or stimulated with insulin (1 or 100 nM) for 5 min at 37 C. Proteins from cell lysate were subjected to Western blotting with monoclonal phosphotyrosine antibody to analyze IRS and insulin receptor tyrosine phosphorylation. PKB and ERK activation was analyzed with anti-phospho-PKB (Ser473) and anti-phospho-ERK (T202/Y204), respectively. Expression of Glut 4 and GFP-Enigma was visualized with anti-Glut 4 or anti-GFP antibody, respectively. Representative autoradiographs from three independent experiments are shown. IB, Immunoblotting; ENG wt, Enigma wild type.

 

Figure 6
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  		Fig. 6. Myc-Glut 4-DsRed Localization in 3T3-L1 Adipocytes Expressing Enigma

3T3-L1 adipocytes were transfected with Myc-Glut 4-DsRed and GFP or GFP-Enigma cDNAs. Cells were fixed and localization of Myc-Glut 4-DsRed in cotransfected cells was analyzed by direct fluorescence and confocal microscopy. Reconstituted fields are shown.

 
Enigma Overexpression Blocks Insulin-Induced Actin Remodeling
Insulin stimulates cortical actin rearrangement in 3T3-L1 adipocytes that is necessary for Glut 4 translocation (17). Enigma family members have been shown to regulate actin dynamics (20, 21, 22, 23), and Enigma overexpression induces loss of stress fibers in fibroblasts (11). The colocalization of Enigma with cortical actin in 3T3-L1 adipocytes suggests that it could play a role in actin remodeling. Because cortical actin structures in adipocytes are not as pronounced as stress fibers in fibroblasts, it is difficult to detect changes in actin organization in adipocytes after fixation. Therefore, to determine whether Enigma expression can modulate insulin-induced actin rearrangement in vivo, we examined the dynamics of actin rearrangements by fluorescent time-lapse microscopy in cells coexpressing a GFP-actin protein together with DsRed or Enigma expressed in fusion with DsRed. In each condition, DsRed or DsRed-Enigma expression was verified (data not shown), and cells with the same level of GFP-actin were examined. As shown in Fig. 7Go and in the time-lapse video [see supplemental data (supplemental Movies 1–3) published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org], insulin stimulation of the DsRed expressing cells increased actin reorganization, and large actin protrusions were formed at the plasma membrane (arrows in Fig. 7AGo and supplemental Movie 1). By contrast, in cells expressing DsRed-Enigma, clusters of GFP-actin were observed, and insulin stimulation did not lead to the formation of these actin protrusions, suggesting that GFP-actin dynamic was blocked (Fig. 7BGo and supplemental Movie 2). Thus, an inhibition of insulin-induced actin remodeling in cells expressing Enigma due to an alteration in actin organization could explain the inhibition of Glut 4 translocation and glucose uptake. To further investigate this hypothesis, we studied the effect of Enigma {Delta}LIM1,2,3, which did not inhibit insulin-induced Glut 4 translocation, on actin dynamic. As shown in Fig. 7CGo and supplemental Movie 3, expression of Enigma {Delta}LIM1,2,3 did lead to the formation of actin clusters and did not alter insulin-stimulated GFP-actin dynamics. Taken together, these data suggest that Enigma could play a role in glucose transport by regulating the dynamic of actin cytoskeleton.


Figure 7
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  		Fig. 7. Enigma Expression Altered Insulin-Induced Actin Rearrangements

3T3-L1 adipocytes were transfected with a plasmid encoding for GFP-actin together with DsRed (panel A and supplemental Movie 1), DsRed-Enigma (panel B and supplemental Movie 2), or DsRed-Enigma{Delta}LIM1,2,3 (panel C and supplemental Movie 3) cDNAs. The cells were then continuously visualized by time-lapse fluorescent microscopy. Selected time images before (0 min) and subsequent to the addition of insulin (10–180 min) are presented. The complete time-lapse films are provided in the supplemental data. This is a representation of insulin-stimulated time course of GFP-actin observed in three individual experiments.

 
APS and Enigma mRNAs Expression in Adipose Tissue of Obese Type 2 Diabetic Patients
Based on the above results, we hypothesized that in insulin-resistant states a change in the ratio between APS and Enigma could lead to the formation of nonproductive complex, leading to abnormal actin dynamics. To investigate this hypothesis, we measured APS and Enigma mRNAs expression by quantitative real-time PCR analysis in sc adipose tissue of patients with type 2 diabetes. Despite a trend toward an increase, APS gene expression was not significantly modified (P = 0.085) in adipose tissue of obese type 2 diabetic patients compared with control subjects (Fig. 8Go). By contrast, Enigma mRNA expression was 5-fold increased (P = 0.002) in adipose tissue of diabetic patients compared with control subjects (Fig. 8Go). These data indicate that the expression of APS and Enigma are differentially regulated in an insulin-resistant state.


Figure 8
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  		Fig. 8. Enigma mRNA Expression Is Increased in Adipose Tissue of Diabetic Patients

Total RNA was extracted from sc adipose tissue biopsies from control (empty bars) or obese and diabetic patients (black bars), and the amount of APS and Enigma mRNAs was quantified by real-time quantitative PCR. mRNA expression was normalized using RPLP0 RNA levels. Results are the means ± SEM of n = 7 for each group. *, P = 0.002, n.s., Nonsignificant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We previously identified the LIM and PDZ domains containing protein Enigma as a partner of APS (11). We recently demonstrated that the APS-Enigma complex is involved in actin organization in fibroblasts (11). APS is required for insulin-induced activation of the Cbl/CAP/TC10 pathway, which could be potentially involved in the regulation of actin cytoskeleton (2, 24). Cell cytoskeleton plays an important role in vesicular trafficking, and several reports strongly support a role of the actin cytoskeleton in insulin-induced Glut 4 translocation (1). In this study, we show that APS and Enigma colocalize with F-actin beneath the plasma membrane in 3T3-L1 adipocytes. These findings are in agreement with our previous report that Enigma binds to APS in fibroblasts and that the APS-Enigma complex colocalizes with F-actin. In fibroblasts or preadipocytes, F-actin is well known to form stress fibers, lamellipodia, and filipodia. However, in differentiated 3T3-L1 adipocytes, F-actin forms cortical actin, which lines the inner face of the plasma membrane (1). Thus, it is likely that the APS-Enigma complex is colocalized with cortical actin in adipocytes. A role of Enigma could thus be to target adaptor proteins or growth factor receptors to cytoskeleton, allowing the regulation of cell signaling. For instance, Enigma interacts with the Ret/ptc2 via its LIM2 domain, and this interaction is required for mitogenic signaling probably by targeting Ret/ptc2 to cytoskeletal elements beneath the plasma membrane (14, 25). In agreement with such a role, we found that the expression of an APS mutant unable to bind Enigma increased insulin-induced Glut 4 translocation whereas overexpression of Enigma alone markedly decreased both insulin-induced Glut 4 translocation and glucose uptake, suggesting that the interaction between Enigma and APS could be involved in the regulation of this important metabolic effect of insulin. Interestingly, using real-time imaging techniques, we show that the expression of Enigma altered actin organization with the formation of actin clusters and markedly reduced insulin-induced actin remodeling. A similar effect of Enigma on actin dynamics was reported in fibroblasts (11). Indeed, in fibroblastic cell lines, the expression of Enigma induced dramatic alteration in actin cytoskeleton morphology with loss of stress fibers and the appearance of F-actin clusters. Furthermore, in cells that overexpress RIL or FHL3, two other LIM-domain containing proteins, F-actin dynamics are markedly altered, suggesting that such a phenotype could be common to several LIM domain-containing proteins (22, 26).

De novo actin polymerization may play an important role in insulin-dependent Glut 4 translocation because latrunculin, which prevents actin polymerization, has an inhibitory effect on Glut 4 translocation (17, 27, 28). Moreover, jasplakinolide markedly increases actin polymerization at both cortical and perinuclear locations and inhibits insulin-induced actin dynamics and Glut 4 translocation (17). The involvement of actin dynamics in Glut 4 translocation is also suggested by the observation that unconventional myosin Myo1C is required for Glut 4 trafficking in response to insulin (29). Thus, the inhibition of insulin-induced actin remodeling induced by Enigma could explain its inhibitory effect on the translocation of Glut 4-containing vesicles in response to insulin. The precise mechanism involved in this effect is not clear at this moment and awaits further studies. However, our finding that Enigma protein deleted of its LIM domain did not inhibit Glut 4 translocation and actin remodeling suggests that proteins, which interact with the LIM domains, could take part in this inhibitory effect. The LIM domains of Enigma bind some protein kinase C isoforms (23) whereas its PDZ domain interacts with actin-binding protein such as ß-tropomyosin (13) or {alpha}-actinin (12), two proteins known to be involved in the regulation of actin organization. Thus, it is possible that an increase in Enigma expression leads to an abnormal regulation of a LIM domain-interacting protein that regulates F-actin turnover, resulting in a profound alteration in actin rearrangement. Interestingly, we found that the expression of Enigma is markedly increased in adipose tissue of diabetic patients whereas APS expression is not modified. This dysregulation in Enigma expression could contribute to the alteration in glucose transport in insulin-resistant state.

Actin cytoskeleton has been proposed to be necessary for several steps in insulin-induced glucose uptake. Indeed, the maintenance of Glut 4 storage compartment and the activation of signaling proteins involved in Glut 4 translocation are dependent on an intact actin structure (17, 27, 28, 30). Furthermore, insulin might modulate the cortical actin cytoskeleton in adipocytes to allow the tethering of Glut 4 vesicles to the plasma membrane before their docking and fusion (1, 31). Finally, it has been shown that insulin stimulated the formation of actin comet tails on Glut 4-containing vesicles that are necessary for the efficient Glut 4 translocation from its storage compartment to the plasma membrane (32). It is unlikely that inhibition of insulin signaling could explain the inhibitory effect of Enigma. Indeed, insulin-induced tyrosine phosphorylation of IRS1 and activation of PKB are not altered in Enigma-expressing cells. However, we cannot exclude that Enigma expression alters the activation of the TC10 signaling pathway, but disruption of F-actin was shown to be without effect on insulin-induced Cbl tyrosine phosphorylation (17). It is also unlikely that the inhibitory effect of Enigma is due to a disruption of Glut 4 sequestration compartments because both in control adipocytes and in adipocytes that express Enigma, Glut 4 is localized mainly in a perinuclear compartment that has been shown to correspond to its intracellular sequestration compartment. Thus, one possible explanation is that Enigma, by inhibiting insulin-induced actin remodeling, could alter the tethering of Glut 4-containing vesicles with the plasma membrane.

We propose that Enigma, by interacting with APS, acts as an inhibitor of cortical actin rearrangement and that insulin modifies the interaction between APS and Enigma or the interaction between Enigma and a protein associated with its LIM domains, allowing for actin reorganization and Glut 4 translocation. The later hypothesis is the more plausible because we did not detect a change in the association between APS and Enigma after insulin stimulation (data not shown). In cells that express a mutated APS unable to bind Enigma, the increase in Glut 4 translocation could be due to a more efficient cortical actin turnover that favors the insertion of Glut 4 in the plasma membrane. Thus, by interacting with Enigma, APS could be a link between insulin signaling toward glucose transport and actin reorganization in 3T3-L1 adipocytes. Studies in other cell types support a role of APS in regulation of actin cytoskeleton. Indeed, overexpressed APS colocalized with F-actin in stimulated B cells, and the increase in F-actin content after B-cell antigen receptor stimulation is more important in APS-overexpressing cells than in control cells (33). Conversely, in mast cells or B cells derived from APS-deficient mice the content in F-actin was decreased (34). Interestingly, insulin-stimulated glucose uptake in adipocytes isolated from APS–/– mice was significantly increased compared with APS +/+ mice. Although F-actin content in adipocytes from APS-deficient mice (9) has not been measured, the increase in insulin effect could be in favor of a modified cortical actin organization that facilitates insulin-induced Glut 4 insertion in the plasma membrane.

In conclusion, our study identifies Enigma as a partner of APS that is involved in F-actin dynamics in 3T3-L1 adipocytes and implies the interaction between APS and Enigma in the regulation of Glut 4 translocation in response to insulin. Thus, APS would act as a scaffolding protein that allows for the communication between different partners involved in the regulation of actin cytoskeleton organization. An imbalance between the amounts of these proteins can alter insulin-induced actin remodeling and lead to a decrease in glucose transport.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
DMEM, fetal calf serum, and calf serum were obtained from Invitrogen SARL (Cergy Pontoise, France). Polyvinylidene difluoride membranes were purchased from Millipore Corp. (Bedford, MA). BCA reagent was obtained from Pierce Biotechnology (Rockford, IL). Protease inhibitors were obtained from Roche Diagnostics (Mannheim, Germany). All other chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The anti-hemagglutinin (anti-HA) and anti-Myc antibodies were purchased from Roche Diagnostics and from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. APS and Glut 4 antibodies were purchased from Santa Cruz Biotechnology. Antiphospho-ERK and antiphospho-PKB were purchased from Cell Signaling Technology (Beverly, MA). Antiphosphotyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology, Inc. (Charlottesville, VA). Horseradish peroxidase-conjugated secondary antibodies and secondary antibodies coupled to fluorescein isothiocyanate (FITC) or Cy5 were obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Texas Red-phalloidin was purchased from Molecular Probes, Inc, (Eugene, OR). Enhanced chemiluminescence reagent was purchased from PerkinElmer Life Sciences (Boston, MA). Oligonucleotides were purchased from Eurogentec (Seraing, Belgium).

Plasmid Constructs
A plasmid encoding a C-terminal fusion protein DsRed with Myc-Glut 4 (Myc-Glut 4-DsRed) was obtained by subcloning a PCR amplification of Myc-Glut 4 [Glut 4 transporter with a Myc-epitope tag in its first extracellular loop (35)] into the SmaI restriction site of pDsRed-N (BD Biosciences Clontech, Palo Alto, CA) (18). The behavior of Myc-Glut 4-DsRed is similar to that of Glut 4-GFP (18, 36). Rat Myc-APS cDNA was kindly provided by Dr. D. Ginty (The Johns Hopkins University School of Medicine, Baltimore, MD). Human HA-Enigma cDNA was a gift from Dr. G. Gill (Department of Biology, University of California San Diego, La Jolla, CA).

Enigma {Delta}LIM1/2/3 and Myc-APS[APTA] constructs were generated as described previously (11). Full-length APS and Enigma cDNA sequences, as well as sequences encoding for various mutated forms, were amplified by PCR and subcloned in pEGFP-C1 or pDsRed-C1 (BD Biosciences Clontech). The plasmid DNAs were purified using a Qiagen purification kit (QIAGEN SA, Courtaboeuf, France).

Physical and Clinical Characteristics of the Study Population
Six female and one male severely obese diabetic patients [age 45 ± 6 yr; body mass index (BMI) 45 ± 8 kg/m2] were included in the present study. All patients were recruited through the department of surgery and liver transplantation where they were consulting for an elective bariatric surgery of their obesity. Diabetes was diagnosed before the bariatric surgery program (fasting plasma glucose >7mmol/liter or previously known diabetes). Before surgery, fasting blood samples were obtained and used for determination of glycemia (8 ± 3 mmol/liter) and insulinemia (11 ± 6 mIU/liter) (37). In addition, sc adipose tissue from seven lean women (age 51 ± 10 yr; BMI 22 ± 1 kg/m2), obtained while undergoing lipectomy for cosmetic purpose, was used as control adipose tissue (provided by Drs. Alessi and Casanova, Marseille, France). During surgery, a surgical biopsy of sc adipose tissues was collected. Samples of the biopsies were immediately frozen in liquid nitrogen and stored at –80 C. The experimental protocol was performed according to French legislation regarding ethics and human research (Huriet-Serusclat law, DGS 2003/0395). Informed consent was obtained from all obese subjects as well as from lean subjects.

Cell Culture
3T3-L1 fibroblasts were grown in 35- or 100-mm dishes in DMEM, 25 mM glucose, and 10% calf serum and induced to differentiate in adipocytes as described previously (38). Briefly, 2 d after confluence, medium was changed for DMEM, 25 mM glucose, 10% fetal calf serum (FCS) supplemented with isobutylmethylxanthine (0.25 mM), dexamethasone (0.25 mM), thiazolidinedione (10 µM), and insulin (5 µg/ml). The medium was removed after 2 d and replaced with DMEM, 25 mM glucose, 10% FCS supplemented with insulin for 2 additional days. The cells were then fed every 2 d with DMEM, 25 mM glucose, 10% FCS. 3T3-L1 adipocytes were used 8–15 d after the beginning of the differentiation protocol. The medium was changed 16 h before each experiment, to serum-free DMEM supplemented with 0.5% BSA.

Northern Blot
Northern blot analysis was performed on poly(A)+ RNA prepared from either 3T3-L1 fibroblasts or adipocytes. A [{alpha}-32P]dCTP-labeled probe corresponding to the coding sequences of APS or Enigma was generated by random priming, and hybridization was performed overnight at 60 C in 15% formamide, 1% BSA, 0.5 M sodium phosphate (pH 7.2), 7% sodium dodecyl sulfate, 100 µM EDTA, and 50 µg/ml denatured salmon sperm DNA and washed at a high stringency.

3T3-L1 Adipocytes Transfection and Glut 4 Translocation Assay
3T3-L1 adipocytes were transfected using a low-voltage electroporation technique as described previously (39, 40). Briefly, at d 6–7 after the beginning of the differentiation protocol, 3T3-L1 adipocytes grown on 10-cm tissue culture dishes were trypsinized and resuspended in DMEM at a concentration of 2 x 107 cells/ml. Cells were mixed with 75 µg of a plasmid coding for Myc-Glut 4-DsRed and empty plasmid (75 µg) or plasmid coding for GFP-Enigma wt, GFP-Enigma {Delta}LIM1,2,3, GFP-APS wt, or -APS[APTA] (final volume, 500 µl), and electroporation was performed with an electric pulse (160 V, 960 µF) using an Easyject electroporator system (Equibio, Ashford, UK). Cells were then plated on collagen IV-coated glass coverslips and allowed to recover for 20 h in complete medium. Thereafter, 3T3-L1 adipocytes were incubated for 2 h at 37 C in DMEM containing 0.1% fetal bovine serum and then either left untreated or stimulated with insulin (100 nM) for 45 min at 37 C. The cells were washed once with ice-cold PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 10 min at room temperature. After being washed, the cells were blocked with PBS, 5% BSA and incubated with anti-Myc antibodies for 90 min at 37 C. After washes, cells were incubated with Cy5-conjugated donkey secondary antibody (Jackson ImmunoResearch Laboratories) for 60 min at room temperature. Cells were then washed twice with ice-cold PBS and coverslips were mounted in Mowiol onto glass slides. Cells were examined using a Leica confocal microscope equipped with a Leica confocal laser scanning imaging. Quantification of the number of cells displaying a Cy5-fluorescent rim was determined from counting at least 100 cells that coexpressed both Myc-Glut 4-Dsred and the different GFP constructs from at least three independent experiments. Cotransfected cells with a continuous fluorescent rim are considered positive for Glut 4 translocation. By contrast, they are considered negative when the labeling is absent, discontinuous, or when it appears as punctuate structures (18).

Confocal Immunofluorescence Microscopy
3T3-L1 adipocytes were transfected by electroporation and plated on collagen IV-coated glass coverslips as described above. After 48 h, cells were washed with ice-cold PBS and fixed in 4% paraformaldehyde for 20 min at 4 C. After two washes with ice-cold PBS, cells were permeabilized with PBS containing 0.1% Triton X-100, and 1% BSA for 30 min at room temperature. After three washes with ice-cold PBS, cells were incubated for 2 h at room temperature with the appropriate primary antibodies in PBS/0.1% Triton X-100/1% BSA. After washes, cells were incubated with secondary antibodies coupled to FITC or Cy5 fluorochromes and Texas Red-phalloidin in PBS/0.1% Triton X-100/1% BSA for 30 min at room temperature. Cells were then washed twice with ice-cold PBS, and coverslips were mounted in Mowiol onto glass slides. Cells were examined using a Leica confocal microscope equipped with a Leica confocal laser scanning imaging. Cells were studied using a PL APO 63 x 1.40 oil immersion objective. Series of images were collected along the z-axis and examined by sequential excitation at 488 nm (FITC), 568 nm (Texas Red), and 647 nm (Cy5). Each experiment was repeated at least three times. The images were then analyzed using PHOTOSHOP software (Adobe Systems, Mountain View, CA).

2-Deoxyglucose Uptake
3T3-L1 adipocytes were transfected by electroporation with 400 µg of plasmids and plated on 12-well plates coated with collagen IV and allowed to recover for 20 h (39, 40). Thereafter, cells were serum-starved in DMEM supplemented with 0.1% fetal bovine serum for 2 h. Cells were then washed with Krebs-Ringer phosphate buffer (10 mM phosphate buffer, pH 7.4; 1 mM MgSO4; 1 mM CaCl2; 136 mM NaCl; 4.7 mM KCl) and incubated without or with insulin (100 nM) for 20 min in Krebs-Ringer phosphate buffer supplemented with 0.1% BSA. Glucose transport was determined by the addition of 2-[3H]deoxyglucose (0.1 mM, 0.5 µCi/ml) as described previously (41). The reaction was stopped after 3 min by aspiration, and cells were washed four times with ice-cold PBS. Cells were lysed in Krebs-Ringer phosphate, 1% Triton X-100, and glucose uptake was assessed by scintillation counting. Results were normalized for protein amount that was measured by BCA assay.

Immunoprecipitation and Immunoblot Analysis
Immunoprecipitation was performed as described (38). Cells were washed with ice-cold buffer (20 mM Tris, pH 7.4; 150 mM NaCl; 5 mM EDTA) before solubilization for 30 min at 4 C in lysis buffer (20 mM Tris, pH 7.4; 150 mM NaCl, 5 mM EDTA; protease inhibitors; and 1% Triton X-100). After centrifugation at 15,000 x g for 15 min at 4 C, cell lysates were incubated for 3 h at 4 C with 4 µg of appropriate antibodies preadsorbed on protein G-Sepharose. The beads were washed twice with high-salt buffer (20 mM Tris, pH 7.4; 500 mM NaCl; 5 mM EDTA; protease inhibitors; and 1% Triton X-100), and once with lysis buffer. Immune pellets were boiled for 5 min in Laemmli buffer. Proteins were resolved by SDS-PAGE using 7.5% or 10% gels and were transferred to polyvinylidene difluoride membrane. The membrane was blocked with saline buffer (10 mM Tris, pH 7.4; 140 mM NaCl) containing 5% (wt/vol) BSA and 0.1% Nonidet P-40 for 2 h at room temperature and was incubated overnight at 4 C with the indicated antibody. After incubation with horseradish peroxidase-conjugated secondary antibodies, proteins were detected by enhanced chemiluminescence.

Time-Lapse Microscopy
3T3-L1 adipocytes were transfected by electroporation with GFP-Actin with or without DsRed-Enigma or DsRed-Enigma {Delta}LIM1,2,3, plated on glass-bottom 35-mm microwell dishes (MatTek Corp., Ashland, MA), and allowed to recover for 20 h. Thereafter, cells were stimulated by 100 nM insulin, filmed for 2 h under constant conditions (5% CO2, 37 C), and observed by fluorescence using fully motorized Axiovert 200 microscope (Carl Zeiss, Göttingen, Germany) and a cooled, digital charge-coupled device camera (Roper Scientific, Evry, France) using a 20x lens. Images were recorded at one frame/min and processed using MetaMorph 2.0 image analysis software (Universal Imaging, Downingtown, PA) and Adobe Premiere pro software (Adobe Systems).

Analysis of mRNA Expression by Real-Time Quantitative PCR
Total RNAs from sc white adipose tissues were prepared using Trizol reagent (Invitrogen France, Cergy-Pontoise, France). The integrity of the RNA was confirmed by electrophoresis in ethidium bromide-containing agarose gels, and the RNA concentration was determined spectrophotometrically. cDNA was synthesized using Moloney murine leukemia virus transcriptase (Invitrogen) from 1 µg of total RNA. Real-time quantitative PCRs were performed starting with 5 µl of 1:10 dilution of the reverse transcribed cDNAs, in a final volume of 20 µl, with 0.5 µM of each primer using 1x TAQ MAN Master mix in an AbiPrism 7700 Sequence Detection System instrument and software (Applied Biosystems, Foster City, CA). The PCR conditions were: 2 min at 50 C, 10 min at 95 C followed by 40 cycles of a two-step PCR denaturation at 95 C for 15 sec and annealing extension at 60 C for 60 sec. A control without cDNA was performed for each experiment. The number of cycles (Ct) required for the fluorescence to reach a threshold limit was determined in duplicate for each sample. The relative amounts of the different mRNAs were quantified by using the second derivative maximum method. RPLP0 was used as an invariant control, and the relative quantification for a given gene was corrected to RPLP0 mRNA values. The results were expressed relative to the control condition, which was arbitrary assigned a value of 1. For each target an efficiency of the PCR method between 95% and 100%, a reproducibility of Ct values with an SE less than 3%, and a linear range of covering more than at least 7 log units were obtained. Amplification of specific transcripts was confirmed by melting curve profiles generated at the end of each run.

Predesigned primers were from Applied Biosystems with the following assay identification numbers.

RPLP0: Hs99999902_m1; APS: Hs00184134_m1; Enigma: hs00193775_m1.

Statistical Analysis
All data are presented as means ± SEM. The statistical comparison between experimental conditions was carried out using Student’s t test. P values < 0.05 were considered significant.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. D. D. Ginty and G. N. Gill for the gift of the different plasmids. We thank J. Vukmirica for critical reading of the manuscript and M. Cormont for helpful discussion of the data and for time-lapse videomicroscopy experiments.


   FOOTNOTES
 
This work was supported by grants from INSERM (France), the University of Nice, the Fondation Bettencourt-Schueller, the Fondation pour la Recherche Médicale, the Région Provence-Alpes Côte d’Azur, the Conseil Général des Alpes Maritimes, and the Association pour la Recherche Contre le Cancer (Grant 7449). J.-F.T. is supported by Centre National de la Recherche Scientifique (France). Y.L.M.B. was the recipient of an interface grant from Centre Hospitalier Universitaire of Nice. P.G. was the recipient of a French Research Ministry grant (ACI JC5327).

The authors have nothing to disclose.

First Published Online June 27, 2006

Abbreviations: APS, Adaptor protein with PH and SH2 domains; CAP, Cbl-associated protein; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; Glut 4, glucose transporter 4; HA, hemagglutinin; IRS, insulin receptor substrate; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B.

Received for publication November 14, 2005. Accepted for publication June 20, 2006.


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 DISCUSSION
 MATERIALS AND METHODS
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