help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1167
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/1/27    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, L.
Right arrow Articles by Saltiel, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, L.
Right arrow Articles by Saltiel, A. R.
Endocrinology Vol. 148, No. 1 27-33
Copyright © 2007 by The Endocrine Society

TC10{alpha} Is Required for Insulin-Stimulated Glucose Uptake in Adipocytes

Louise Chang, Shian-Huey Chiang and Alan R. Saltiel

Department of Internal Medicine and Physiology (A.R.S.) and Life Sciences Institute (L.C., S.-H.C., A.R.S.), University of Michigan Medical Center, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Alan R. Saltiel, Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216. E-mail: saltiel{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have suggested that activation of the Rho family member GTPase TC10 is necessary but not sufficient for the stimulation of glucose transport by insulin. We show here that endogenous TC10{alpha} is rapidly activated in response to insulin in 3T3L1 adipocytes in a phosphatidylinositol 3-kinase-independent manner, whereas platelet-derived growth factor was without effect. Knockdown of TC10{alpha} but not TC10ß by RNA interference inhibited insulin-stimulated glucose uptake as well as the translocation of the insulin-sensitive glucose transporter GLUT4 from intracellular sites to the plasma membrane. In contrast, loss of TC10{alpha} had no effect on the stimulation of Akt by insulin. Additionally, knockdown of TC10{alpha} inhibited insulin-stimulated translocation of its effector CIP4. These data indicate that TC10{alpha} is specifically required for insulin-stimulated glucose uptake in adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RHO FAMILY GTPASES are molecular switches involved in multiple signal transduction pathways (1, 2). These GTPases cycle between an inactive (GDP bound) form and an active (GTP bound) form. In the active form, GTPases interact with specific downstream effectors, thereby regulating diverse cellular functions such as cytoskeletal and microtubular dynamics, cell polarity, migration, and motility as well as gene expression (3). These proteins are also known to regulate vesicular trafficking (4).

The Rho family member GTPase TC10 has been shown to play a role in insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 in 3T3L1 adipocytes (5, 6). In this signaling cascade, the insulin receptor and TC10 reside constitutively in lipid raft microdomains of the plasma membrane. Upon binding to insulin, the insulin receptor catalyzes the tyrosine phosphorylation of the c-Cbl and Cbl-b protooncogenes after their recruitment via the adapter proteins APS and CAP (adapter protein containing PH and Src homology 2 domains and c-Cbl-associated protein, respectively) (7, 8, 9, 10, 11, 12). Once phosphorylated, Cbl interacts with the adapter protein CT10-related kinase (Crk)II through the Src homology 2 domain of the latter protein. Crk is constitutively associated with the nucleotide exchange factor C3G. Thus, binding of Crk to Cbl brings C3G into proximity with TC10 in lipid rafts, producing the activation of the G protein. Once activated, TC10 can interact with multiple downstream effectors including Exo70, CIP4/2, Par6B, and TCGAP (13, 14, 15, 16, 17, 18). Some of these effectors, such as CIP4, Par6, and TCGAP, interact with both Cdc42 as well as TC10, whereas Exo70 specifically interacts with TC10.

TC10 exists in two related isoforms, TC10{alpha} and TC10ß (5, 19). The two TC10 genes share identical sequences throughout the five Ras canonical boxes (G1-G5) as well as the effector domain, whereas the main differences are clustered in the amino and carboxyl termini. Both have a unique CXXCCAAX box, a presumed signal for palmityolation and prenylation, and colocalize with caveolin-enriched fractions (20, 21). Moreover, both are activated in response to insulin, but only TC10{alpha} can block glucose uptake when its dominant-negative form is expressed in adipocytes (5). Likewise, overexpression of TC10{alpha} has profound effects on remodeling of the actin cytoskeleton, whereas the ß-isoform does not. Creation of chimeras between the two proteins narrowed down the amino terminal sequences as responsible for this effect (22).

To explore the role of TC10 isoforms in insulin signaling, we sought to characterize the endogenous protein and determine whether it is essential for insulin action in the insulin-responsive 3T3L1 adipocyte cell line. We show here that endogenous TC10{alpha} is activated after insulin stimulation. Moreover, knockdown of the protein in adipocytes inhibits insulin-stimulated GLUT4 translocation and glucose uptake and prevents insulin-stimulated translocation of the downstream effector CIP4 without affecting other signaling events.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All chemicals and reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. The anti-AKT and anti-pAKT (S473) rabbit polyclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-pTyr antibody (4G10) was purchased from Upstate Cell Signaling Solutions (Charlottesville, VA). Antimyc monoclonal antibody (9E10), anti-insulin receptor ß, and antiinsulin receptor substrate antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Alexa (594) goat antimouse IgG antibodies and Alexa (488) conjugated Phalloidin were purchased from Invitrogen (Carlsbad, CA).

Anti-TC10 antibody
The anti-TC10 rabbit polyclonal antibody was a generous gift from Affinity Bioreagents (Golden, CO) and was generated against peptide DPKTLARLNDMKEKPIC, which correspond to amino acids 136–152 in human TC10, the human ortholog of TC10{alpha}. This anti-TC10 antibody detects by immunoblot analysis overexpressed hemagglutinin-TC10 and hemagglutinin-TC10{alpha} in COS-1 cells as well as endogenous mouse TC10{alpha} expressed in mouse tissues and 3T3L1 adipcoytes (data not shown). The antihuman TC10 antibody did not recognize overexpressed mouse TC10ß by either immunoprecipitation or immunoblot analysis (data not shown). Using small interfering RNA oligos specific for TC10{alpha}, protein levels of TC10{alpha} were reduced in 3T3L1 adipocytes (see Fig. 2BGo). Therefore, these data demonstrate that the anti-TC10 antibody is specific for TC10{alpha} and does not cross-react with TC10ß.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 2. TC10{alpha} knockdown inhibits insulin-stimulated glucose uptake. A, Differentiated 3T3L1 adipocytes were transfected with scrambled control or TC10{alpha}- or TC10ß-specific RNAi. Sc, Scrambled control; KD, knockdown. Four days later, the cells were serum starved and treated with or without 100 nM insulin for 30 min. The rate of 2-DG uptake was determined. Results are the mean ± SD of triplicate determinations and were reproduced five times. *, Significant difference, P < 0.02. Efficiency knockdown was confirmed by immunoblotting (B) or qPCR (C). Significant difference, *, P < 0.01, **, P < 001. A.U., arbitrary units.

 
Cell culture
Mouse 3T3L1 preadipocytes were maintained in DMEM containing 10% calf serum, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (Invitrogen). To induce differentiation of 3T3L1 fibroblast cells to adipocytes, 3T3L1 fibroblasts 3 d after confluency were treated with 100 µg/ml insulin, 1 µM dexamethasone, and 0.5 mM isobutyl-1-methylzanthine for 3 d and followed by 100 µg/ml insulin for 2 d as previously described (23, 24).

Preparation of plasma membrane fraction
Lipid raft-enriched plasma membrane fractions were prepared as described (5, 25). Briefly, 3T3L1 adipocytes were washed twice in Dulbecco’s PBS and then homogenized in a buffer of 20 mM HEPES (pH 7.5), 250 mM sucrose, and 1 mM EDTA containing protease inhibitors (Roche Applied Sciences, Indianapolis, IN) with a Dounce homogenizer. Homogenized cells were centrifuged at 3,000 x g and the supernatant was collected and centrifuged at 20,000 x g, producing a crude plasma membrane fraction. The resulting plasma membrane containing pellet was resuspended in 1.0 ml binding buffer [25 mM Tris (pH 7.5), 1 mM dithiothreitol (DTT), 10 mM MgCl2, 40 mM NaCl, 0.5% Nonidet P-40 (NP-40)] for p21 activated kinase (PAK)-1 pull-down assays (see below).

Assay of GTPase activity using glutathione-S-transferase (GST)-PAK1 p21 activated kinase 1 binding domain (PBD)
To measure in vivo levels of activated GTPase, PAK1 pull-downs using GST-PBD conjugated to agarose were performed as described previously (5, 25). A lipid raft-enriched plasma membrane fraction was resuspended in 1.0 ml of binding buffer [25 mM Tris (pH 7.5), 1 mM DTT, 10 mM MgCl2, 40 mM NaCl, 0.5% Nonidet P-40] in the presence of 7 µg of GST-PBD-agarose (Cytoskeleton, Inc., Denver, CO) for 1 h at 4 C. The beads were washed three times with wash buffer [25 mM Tris (pH 7.5), 1 mM DTT, 10 mM MgCl2, 40 mM NaCl, and 1% NP-40] and once with the wash buffer without NP-40. The beads were resuspended in 20 µl sodium dodecyl sulfate sample buffer, subjected to 4–20% SDS-PAGE, and analyzed by immunoblot analysis.

2-[U-14C]deoxyglucose (2-DG) uptake assay
Glucose uptake assays were performed as described previously (25, 26). Briefly, 3T3L1 adipocytes were electroporated with either scrambled control or TC10-specific Stealth small interference RNA (siRNA) duplexes (Invitrogen) at a concentration of 1 nM siRNA double-stranded oligo/5 x 105 cells. Cells were then harvested four days after electroporation. The cells were serum-starved in DMEM containing 0.5% fetal bovine serum (FBS) for 3 h. Then the cells were washed twice with PBS and incubated in 0.45 ml of Krebs-buffered Ringer solution of 30 mM HEPES (pH 7.4), 10 mM NaHCO3, 120 mM NaCl, 4 mM KH2PO3, 1 mM MgSO4, and 1 mM CaCl2 in the presence or absence of 100 nM insulin for 30 min at 37 C. Glucose uptake was initiated by the addition of 50 µl of reaction mixture containing 0.1 µCi of 2-DG (PerkinElmer, Shelton, CT) buffer of Krebs-buffered Ringer solution containing 20 µM cold 2-DG. The cells were incubated for 5 min at room temperature, and then the reaction was stopped by the addition of 50 µl of 200 mM cold 2-DG into each well. The cells were washed three times with ice-cold PBS (pH 7.4) and solubilized in 0.5 ml of 0.1% sodium dodecyl sulfate at room temperature for 10 min. Ten microliters of each sample were removed to measure protein concentration. Samples were then assessed for radioactivity by scintillation counting in Ready Gel (Beckman Coulter, Fullerton, CA). Results were the mean ± SD of three individual determinations, and the experiment was reproduced five times.

Real-time quantitative PCR (qPCR)
To verify siRNA knockdown efficiency in adipocytes, RNA extractions were performed using RNAeasy kit according manufacturer’s instructions (QIAGEN, Valencia, CA). RT-PCR was performed using random primers and Stratascript polymerase according to manufacturer’s instructions (Stratagene, La Jolla, CA). cDNA was analyzed by real-time qPCR analysis was performed using SYBR green normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems 7200; Applied Biosystems Inc., Foster City, CA). PCR primers used were: GAPDH forward, 5'-TGAAGCAGGCATCTGAGGG-3'; GAPDH reverse 5'-CGAAGGTGGAAGAGTGGGAG-3'; TC10{alpha} forward, 5'-TGCTTCTCCGTGGTAAATCC; TC10{alpha} reverse, 5'-CCTTGTTCCACACAGACAGG; and TC10ß forward, 5'-CGGCTGCAATGGACATGAG; TC10ß reverse, 5'-GGCACGTATTCCTCTGGGAAG. Relative expression was assessed by comparative cycle threshold method correcting for amplification efficiency of the primers and performed in triplicate.

GLUT4 translocation
A retroviral construct expressing myc-GLUT4-enhanced green fluorescent protein (eGFP) (pMX-GLUT4myc7-GFP), containing a myc tag in the first exofacial loop of GLUT4 (a generous gift of Dr. Harvey Lodish, Massachusetts Institute of Technology, Boston, MA) was generated as described previously (27). 3T3L1 fibroblasts infected with retroviral myc-GLUT4-eGFP containing viral particles were selected by GFP intensity using flow cytometry. Cells expressing high levels of myc-GLUT4-eGFP as determined by GFP intensity were collected and then differentiated into adipocytes as described above. The assay was performed with slight modifications as described (27, 28). Briefly, after 3T3L1 adipocytes were electroporated with scrambled control or specific siRNA, cells were seeded on 96-well plates. Four days after electroporation, cells were serum starved in DMEM containing 0.5% FBS and treated with 100 nM insulin for the times indicated. The cells were fixed for 10 min at room temperature in 10% buffered neutral formalin (VWR International, West Chester, PA). One well was permeabilized with 0.5% Triton X-100 as a control to measure total Myc signal. After the fixation was quenched with 50 mM glycine for 5 min, cells were incubated overnight with blocking buffer [1% goat serum, 1% BSA, PBS (pH 7.4)]. Cells were incubated for 60 min with anti-Myc monoclonal antibody in blocking buffer [1% BSA, 1% ovalbumin, PBS (pH 7.4)]. Cells were incubated for 30 min with Alexa594 goat antimouse antibody in blocking buffer. After washing with PBS, fluorescence was measured with a Fluostar Optima plate reader (BMG LABTECH, Inc., Durham, NC) using the appropriate filter sets. The percentage of GLUT4 at the plasma membrane was calculated for each condition. GFP fluorescence was used to correct for variation in cell density in each well.

Immunofluorescence studies
3T3L1 adipocytes were electroporated with either scrambled control or TC10-specific RNA interference (RNAi). Four days after electroporation, cells were serum starved in DMEM containing 0.5% FBS and treated with or without 100 nM insulin for 30 min. The cells were fixed with 4% paraformaldehyde. Cells were incubated with 50 mM glycine in PBS (pH 7.4) to quench paraformaldehyde fixation. The cells were incubated in blocking buffer [1% BSA, 1% goat serum in PBS (pH 7.4)] for 1 h. To detect endogenous CIP4, cells were incubated with CIP4 monoclonal antibody at 1 µg/ml in blocking buffer followed by goat Alexa (594) goat antimouse IgG at 1 µg/ml in blocking buffer. When RNAi knockdown studies were conducted using adipocytes stably expressing myc-GLUT4-eGFP, the nonpermeabilized cells were incubated with anti-myc mouse monoclonal antibody (9E10) at 1 µg/ml in blocking buffer. To detect myc staining or actin, cells were incubated with Alexa594 goat antimouse IgG at 1 µg/ml and Alexa (488) Phalloidin at 1:500 in blocking buffer for 1 h. Images of the cells were captured using an FV300 scanning laser confocal microscope (Olympus America, Inc., Center Valley, PA). Efficiency knockdown by RNAi was confirmed by RT-PCR followed by real-time qPCR as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Previous studies evaluated the regulation of exogenously expressed mouse TC10 isoforms in 3T3L1 adipocytes (5, 6). We sought to confirm these results by examining the activation of endogenous TC10{alpha} by insulin. 3T3L1 adipocytes were treated with insulin for 0, 2, 5, and 10 min. Plasma membrane fractions were prepared, lysed, and subjected to pull down with GST-PBD, followed by immunoblot analysis using anti-TC10{alpha} antibody. Insulin produced a dramatic increase in TC10{alpha} activity as indicated by the precipitation with immobilized PAK GTPase binding domain (Fig. 1AGo). This increase was seen as early as 2 min and sustained through 10 min but declined thereafter, consistent with results observed previously with exogenous TC10{alpha} (6).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		FIG. 1. Insulin activates endogenous TC10. Plasma membrane fractions were prepared from 3T3L1 adipocytes treated with 100 nM insulin (Ins; A and B), 100 nM insulin and 100 nM wortmannin (WM; B), and 10 ng/ml PDGF (C) for the times indicated, and a GST-PBD pull-down was performed. GST-PBD pull-downs were analyzed by immunoblot antibodies with the antibodies indicated. Quantification of the GST-PBD pull-down bands is shown below. This experiment is representative of three independent experiments. TCL, Total cell lysate.

 
Exogenously expressed TC10 was previously shown to be activated by insulin in a phosphatidylinositol-3 kinase (PI 3-kinase)-independent manner (5, 6). To determine whether this was also true for endogenous TC10{alpha}, adipocytes were serum starved and pretreated with or without 100 nM wortmannin, a PI 3-kinase inhibitor, for 1 h before the addition of insulin (Fig. 1BGo). Plasma membrane fractions were prepared, and GST-PBD pull-down was performed as described above. Inhibition of PI 3-kinase activity with wortmannin did not inhibit insulin-stimulated activation of TC10 (Fig. 1BGo), although the stimulation of Akt phosphorylation was completely blocked under these conditions. In addition, we studied the effects of platelet-derived growth factor (PDGF) on TC10{alpha} activation (Fig. 1CGo). PDGF treatment did not activate endogenous TC10{alpha} in adipocytes. Thus, activation of endogenous TC10{alpha} in adipocytes is specific for insulin and is PI 3-kinase independent, consistent with previous studies (5, 6).

Previous studies revealed that overexpression of mutant forms of APS or CAP, both of which reside upstream of TC10, blocked the insulin stimulation of GLUT4 translocation or glucose uptake (9, 11). To study the role of TC10{alpha} and -ß in the regulation of glucose transport in adipocytes, their expression was reduced using specific RNAi oligos. Cells were electroporated with either scrambled control or TC10{alpha}-specific RNAi oligos. The TC10{alpha}-specific oligos knocked down expression of TC10{alpha} by approximately 75% as shown by Western blotting and qPCR (Fig. 2Go, B and C). TC10{alpha}-specific oligos did not affect the expression of TC10ß as determined by qPCR (Fig. 2CGo). Likewise, siRNA-mediated knockdown of TC10ß was specific and did not reduce levels of TC10{alpha} expression (Fig. 2CGo). Knockdown of TC10{alpha} inhibited insulin-stimulated glucose uptake by 35%, compared with cells transfected with scrambled RNAi oligo. However, knockdown of TC10ß in adipocytes was without effect on insulin-stimulated glucose uptake (Fig. 2AGo).

TC10{alpha} activation is independent of PI 3-kinase activation, and TC10{alpha} knockdown inhibits insulin-stimulated glucose uptake. To determine whether TC10{alpha} knockdown affected insulin receptor signaling through PI 3-kinase, adipocytes were electroporated with scrambled control or TC10{alpha}-specific or TC10ß-specific knockdown oligos and then treated with or without insulin (Fig. 3Go). Efficiency of knockdown was confirmed by qPCR (data not shown). Knockdown of TC10{alpha} or TC10ß in adipocytes had no effect on the stimulation of tyrosine phosphorylation or Akt phosphorylation by insulin (Fig. 3Go).


Figure 3
View larger version (64K):
[in this window]
[in a new window]

  		FIG. 3. PI 3-kinase signaling in TC10{alpha}-knocked down adipocytes. Differentiated 3T3L1 adipocytes were transfected with scrambled control RNAi or TC10{alpha}-specific RNAi. Sc, Scrambled control; KD, knockdown; pTyr, phosphorylated tyrosine; IR, insulin receptor; IRS, insulin receptor substrate. Four days later, the cells were serum starved and treated with or without 100 nM insulin for 5 min. Total cell lysates were prepared and analyzed by immunoblot analysis with the indicated antibodies.

 
To determine the mechanism underlying the blockade of glucose uptake by knockdown of TC10{alpha}, we examined adipocytes stably expressing myc-GLUT4-eGFP. Adipocytes expressing myc-GLUT4-eGFP were transfected with scrambled control or TC10{alpha}-specific RNAi and treated with or without insulin. We analyzed the trafficking of the tagged GLUT4 protein by 96-well assay (Fig. 4AGo) or indirect immunofluorescence (Fig. 4BGo). In the myc-GLUT4-eGFP-expressing adipocytes, the myc epitope is located in the first exofacial loop, and the eGFP is fused to the C terminus of GLUT4. Upon GLUT4 vesicle fusion with the plasma membrane, the myc epitope is exposed to the extracellular space, allowing for quantification of GLUT4 fusion with the plasma membrane in nonpermeabilized cells using antibodies against the myc-epitope (27, 28, 29). In adipocytes transfected with scrambled control RNAi, myc-positive staining relative to total eGFP increased with insulin (Fig. 4AGo). TC10{alpha} knockdown blocked increased in myc staining after insulin treatment, indicating inhibition of GLUT4 vesicle trafficking and fusion. Consistent with these data, myc-positive cells and plasma membrane staining of myc-GLUT4-eGFP adipocytes was reduced in TC10{alpha} knockdown cells (Fig. 4BGo). In the adipocytes transfected with scrambled RNAi, almost all of the cells showed myc-positive staining and eGFP rim staining after insulin treatment (Fig. 4BGo).


Figure 4
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 4. TC10 knockdown inhibits insulin-stimulated GLUT4 translocation. Differentiated 3T3L1 adipocytes stably expressing myc-GLUT4-eGFP were transfected with scrambled control (Sc) or TC10{alpha}-specific RNAi (KD). Three days after transfection, insulin-stimulated myc-GLUT4-eGFP translocation was determined by 96-well assay (A) or immunofluorescence studies (B) as described in Materials and Methods. Bar, 20 µM. Cells were stimulated with insulin (Ins) at the indicated time points. In A, myc signal on the plasma membrane (PM) was normalized with eGFP signal. A.U., Arbitrary units.

 
TC10 has been shown to interact with several downstream effectors. Two of these TC10 effectors, CIP4 and Exo70, have been shown to translocate to the plasma membrane after insulin stimulation (13, 14, 26). To determine whether TC10 knockdown affected the localization of downstream effectors, TC10{alpha} was knocked down in adipocytes and the localization of CIP4 was examined (Fig. 5Go). In adipocytes electroporated with scrambled control TC10{alpha} RNAi oligo, CIP4 was translocated to the plasma membrane after insulin treatment for 5–10 min (Fig. 5Go). However, knockdown of TC10{alpha} with siRNA in adipocytes completely blocked the translocation of CIP4 to the plasma membrane after insulin stimulation. When TC10{alpha} was knocked down in adipocytes, there was also a noticeable decrease in actin staining with phalloidin (Fig. 5Go). Both TC10{alpha} and CIP4 have been shown to be involved in regulating actin structures (22, 30, 31, 32, 33, 34, 35, 36). Thus, whether this decrease in actin staining was an effect of TC10{alpha} knockdown or due to disruption of CIP4 function was not clear.


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 5. TC10 knockdown inhibits insulin-stimulated translocation of effector CIP4. Differentiated 3T3L1 adipocytes were transfected with scrambled control (Sc) or TC10{alpha}-specific RNAi (KD). Three days after transfection, cells were serum starved for 3 h and treated with 100 nM insulin (Ins) for the time indicated. Cells were then fixed and stained for actin or CIP4 as described in Materials and Methods. Bar, 20 µM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin stimulates glucose uptake in fat and muscle tissue via the translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane. This is a complex process that involves the sorting of the transporter into specialized storage vesicles, their trafficking to the plasma membrane, and targeting of the vesicles to specific sites at the membrane, followed by docking and fusion, resulting in the extracellular exposure of the transporter (37, 38). Whereas the precise steps in this process that are under hormonal control remain uncertain, numerous studies indicate that two pathways are required for insulin signaling. One involves activation of PI 3-kinase and downstream protein kinases, whereas the other involves activation of the Rho family GTPase TC10 (39, 40). This latter process occurs specifically in lipid raft subdomains of the plasma membrane (6, 21, 33, 34). Activation of TC10 by insulin is catalyzed by the exchange factor C3G, which translocates to lipid rafts with its binding partner CrkII, after Cbl tyrosine phosphorylation by the insulin receptor (6). Activation of TC10 depends on its localization in lipid raft subdomains of the plasma membrane (21).

In this report, we show for the first time that insulin activates endogenous TC10 in adipocytes, consistent with previous studies on exogenously expressed TC10 (5, 6). This activation occurs over a time course identical with that reported for the exogenously expressed protein. We also show that activation of the G protein is specific for insulin and is not seen with PDGF. Additionally, insulin activation of TC10 is unaffected by wortmannin, demonstrating that TC10 activation by insulin is PI 3-kinase independent.

There are two gene products encoding TC10 proteins, TC10{alpha} and TC10ß (5, 19). To evaluate the importance of the TC10 pathway insulin action and determine which gene product might be relevant to insulin action, we knocked down both TC10{alpha} and -ß, followed by assay of insulin-stimulated glucose uptake and GLUT4 translocation. Interestingly, reduction of TC10{alpha} and not TC10ß protein levels in adipocytes attenuated insulin-stimulated glucose transport. This inhibition was due to a defect in GLUT4 translocation. As a control, neither insulin-stimulated general tyrosine phosphorylation nor Akt phosphorylation was affected on knockdown of either TC10 isoform in adipocytes.

There have been conflicting reports about the role of the TC10/CAP/C3G pathway in the stimulation of glucose transport by insulin in adipocytes (41, 42, 43). Whereas Czech’s (43) laboratory found that reduction of CAP protein with siRNA had no effect on glucose uptake in 3T3L1 adipocytes, Ahn et al. (41) used siRNA to knock down CAP and APS in human and mouse adipocytes and found that both gene products were essential for insulin action. Whereas the reason for the discrepancy between these findings is not clear, our recent studies (Chiang, S.-H. and A. R. Saltiel, unpublished observations) suggest that these differences might be explained by different levels of GLUT1 protein in cells (44), which is unaffected by CAP knockdown. Moreover, the effect of TC10 knockdown in experiments described here are consistent with a role for the CAP/Cbl pathway in the regulation of glucose transport by insulin.

The extent of inhibition of insulin-stimulated glucose uptake seen with TC10{alpha} knockdown is similar to the level of inhibition observed with knockdown of either Akt1 or Akt2 alone, essential components of PI 3-kinase signaling (45). Moreover, similar effects of siRNA knockdown have been observed for the components of the exocyst complex, which assembles in response to activation of TC10 by insulin (26). Together, these data indicate that activation of both TC10 and PI 3-kinase are essential for insulin-stimulated glucose transport.


   Acknowledgments
 
We thank the members of the Saltiel laboratory for helpful discussions, Dr. Stuart Decker for insightful discussions, and Dr. Carey Lumeng for technical assistance with real-time qPCR.


   Footnotes
 
This work was supported by Grants DK61618 and DK60591 from the National Institutes of Health (to A.R.S.).

Disclosure statement: L.C. and S.-H.C. have nothing to disclose. A.R.S. was previously a member of the editorial board of Endocrinology.

First Published Online September 28, 2006

Abbreviations: APS, adapter protein containing PH and Src homology 2 domains; CAP, c-Cbl-associated protein; Crk, CT10-related kinase; 2-DG, 2-[U-14C]deoxyglucose; eGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; NP-40, Nonidet P-40; PAK, p21 activated kinase; PBD, p21 activated kinase 1 binding domain; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol-3 kinase; qPCR, quantitative PCR; RNAi, RNA interference; siRNA, small interference RNA.

Received August 25, 2006.

Accepted for publication September 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wennerberg K, Der CJ 2004 Rho-family GTPases: it’s not only Rac and Rho (and I like it). J Cell Sci 117:1301–1312[Abstract/Free Full Text]
  2. Etienne-Manneville S, Hall A 2002 Rho GTPases in cell biology. Nature 420:629–635[CrossRef][Medline]
  3. Jaffe AB, Hall A 2005 Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21:247–269[CrossRef][Medline]
  4. Symons M, Rusk N 2003 Control of vesicular trafficking by Rho GTPases. Curr Biol 13:R409–418
  5. Chiang SH, Hou JC, Hwang J, Pessin JE, Saltiel AR 2002 Cloning and functional characterization of related TC10 isoforms, a subfamily of Rho proteins involved in insulin-stimulated glucose transport. J Biol Chem 277:13067–13073[Abstract/Free Full Text]
  6. Chiang SH, Baumann CA, Kanzaki M, Thurmond DC, Watson RT, Neudauer CL, Macara IG, Pessin JE, Saltiel AR 2001 Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature 410:944–948[CrossRef][Medline]
  7. Ribon V, Printen JA, Hoffman NG, Kay BK, Saltiel AR 1998 A novel, multifunctional c-Cbl binding protein in insulin receptor signaling in 3T3–L1 adipocytes. Mol Cell Biol 18:872–879[Abstract/Free Full Text]
  8. Ribon V, Saltiel AR 1997 Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3–L1 adipocytes. Biochem J 324:839–845
  9. Liu J, Kimura A, Baumann CA, Saltiel AR 2002 APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3–L1 adipocytes. Mol Cell Biol 22:3599–3609[Abstract/Free Full Text]
  10. Liu J, DeYoung SM, Hwang JB, O’Leary EE, Saltiel AR 2003 The roles of Cbl-b and c-Cbl in insulin-stimulated glucose transport. J Biol Chem 278:36754–36762[Abstract/Free Full Text]
  11. Baumann CA, Ribon V, Kanzaki M, Thurmond DC, Mora S, Shigematsu S, Bickel PE, Pessin JE, Saltiel AR 2000 CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature 407:202–207[CrossRef][Medline]
  12. Ahmed Z, Smith BJ, Pillay TS 2000 The APS adapter protein couples the insulin receptor to the phosphorylation of c-Cbl and facilitates ligand-stimulated ubiquitination of the insulin receptor. FEBS Lett 475:31–34[CrossRef][Medline]
  13. Chang L, Adams RD, Saltiel AR 2002 The TC10-interacting protein CIP4/2 is required for insulin-stimulated Glut4 translocation in 3T3L1 adipocytes. Proc Natl Acad Sci USA 99:12835–12840[Abstract/Free Full Text]
  14. Inoue M, Chang L, Hwang J, Chiang SH, Saltiel AR 2003 The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422:629–633[CrossRef][Medline]
  15. Joberty G, Perlungher RR, Macara IG 1999 The Borgs, a new family of Cdc42 and TC10 GTPase-interacting proteins. Mol Cell Biol 19:6585–6597[Abstract/Free Full Text]
  16. Neudauer CL, Joberty G, Macara IG 2001 PIST: a novel PDZ/coiled-coil domain binding partner for the rho-family GTPase TC10. Biochem Biophys Res Commun 280:541–547[CrossRef][Medline]
  17. Chiang SH, Hwang J, Legendre M, Zhang M, Kimura A, Saltiel AR 2003 TCGAP, a multidomain Rho GTPase-activating protein involved in insulin-stimulated glucose transport. EMBO J 22:2679–2691[CrossRef][Medline]
  18. Kanzaki M, Mora S, Hwang JB, Saltiel AR, Pessin JE 2004 Atypical protein kinase C (PKC{zeta}/{lambda}) is a convergent downstream target of the insulin-stimulated phosphatidylinositol 3-kinase and TC10 signaling pathways. J Cell Biol 164:279–290[Abstract/Free Full Text]
  19. Vignal E, De Toledo M, Comunale F, Ladopoulou A, Gauthier-Rouviere C, Blangy A, Fort P 2000 Characterization of TCL, a new GTPase of the rho family related to TC10 and Ccdc42. J Biol Chem 275:36457–36464[Abstract/Free Full Text]
  20. Michaelson D, Silletti J, Murphy G, D’Eustachio P, Rush M, Philips MR 2001 Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J Cell Biol 152:111–126[Abstract/Free Full Text]
  21. Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, Saltiel AR, Pessin JE 2001 Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation. J Cell Biol 154:829–840[Abstract/Free Full Text]
  22. Chunqiu Hou J, Pessin JE 2003 Lipid raft targeting of the TC10 amino terminal domain is responsible for disruption of adipocyte cortical actin. Mol Biol Cell 14:3578–3591[Abstract/Free Full Text]
  23. Olson AL, Knight JB, Pessin JE 1997 Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes. Mol Cell Biol 17:2425–2435[Abstract]
  24. Brady MJ, Kartha PM, Aysola AA, Saltiel AR 1999 The role of glucose metabolites in the activation and translocation of glycogen synthase by insulin in 3T3–L1 adipocytes. J Biol Chem 274:27497–27504[Abstract/Free Full Text]
  25. Chiang SH, Chang L, Saltiel AR 2006 TC10 and insulin-stimulated glucose transport. Methods Enzymol 406:701–714[Medline]
  26. Inoue M, Chiang SH, Chang L, Chen XW, Saltiel AR 2006 Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering. Mol Biol Cell 17:2303–2311[Abstract/Free Full Text]
  27. Bogan JS, McKee AE, Lodish HF 2001 Insulin-responsive compartments containing GLUT4 in 3T3–L1 and CHO cells: regulation by amino acid concentrations. Mol Cell Biol 21:4785–4806[Abstract/Free Full Text]
  28. Coster AC, Govers R, James DE 2004 Insulin stimulates the entry of GLUT4 into the endosomal recycling pathway by a quantal mechanism. Traffic 5:763–771[CrossRef][Medline]
  29. Jiang ZY, Chawla A, Bose A, Way M, Czech MP 2002 A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling. J Biol Chem 277:509–515[Abstract/Free Full Text]
  30. Aspenstrom P 1997 A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton. Curr Biol 7:479–487[CrossRef][Medline]
  31. Aspenstrom P, Fransson A, Saras J 2004 Rho GTPases have diverse effects on the organization of the actin filament system. Biochem J 377:327–337[CrossRef][Medline]
  32. Kanzaki M, Watson RT, Hou JC, Stamnes M, Saltiel AR, Pessin JE 2002 Small GTP-binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes. Mol Biol Cell 13:2334–2346[Abstract/Free Full Text]
  33. Kanzaki M, Pessin JE 2002 Caveolin-associated filamentous actin (Cav-actin) defines a novel F-actin structure in adipocytes. J Biol Chem 277:25867–25869[Abstract/Free Full Text]
  34. Kanzaki M, Pessin JE 2001 Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling. J Biol Chem 276:42436–42444[Abstract/Free Full Text]
  35. Murphy GA, Solski PA, Jillian SA, Perez de la Ossa P, D’Eustachio P, Der CJ, Rush MG 1999 Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth. Oncogene 18:3831–3845[CrossRef][Medline]
  36. Neudauer CL, Joberty G, Tatsis N, Macara IG 1998 Distinct cellular effects and interactions of the Rho-family GTPase TC10. Curr Biol 8:1151–1160[CrossRef][Medline]
  37. Dugani CB, Klip A 2005 Glucose transporter 4: cycling, compartments and controversies. EMBO Rep 6:1137–1142[CrossRef][Medline]
  38. Watson RT, Kanzaki M, Pessin JE 2004 Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes. Endocr Rev 25:177–204[Abstract/Free Full Text]
  39. Kanzaki M 2006 Insulin receptor signals regulating GLUT4 translocation and actin dynamics. Endocr J 53:267–293[CrossRef][Medline]
  40. Saltiel AR, Pessin JE 2003 Insulin signaling in microdomains of the plasma membrane. Traffic 4:711–716[CrossRef][Medline]
  41. Ahn MY, Katsanakis KD, Bheda F, Pillay TS 2004 Primary and essential role of the adaptor protein APS for recruitment of both c-Cbl and its associated protein CAP in insulin signaling. J Biol Chem 279:21526–21532[Abstract/Free Full Text]
  42. Mitra P, Zheng X, Czech MP 2004 RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin. J Biol Chem 279:37431–37435[Abstract/Free Full Text]
  43. Zhou QL, Park JG, Jiang ZY, Holik JJ, Mitra P, Semiz S, Guilherme A, Powelka AM, Tang X, Virbasius J, Czech MP 2004 Analysis of insulin signalling by RNAi-based gene silencing. Biochem Soc Trans 32:817–821[CrossRef][Medline]
  44. Liao W, Nguyen MT, Imamura T, Singer O, Verma IM, Olefsky JM 2006 Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3–L1 adipocytes. Endocrinology 147:2245–2252[Abstract/Free Full Text]
  45. Jiang ZY, Zhou QL, Coleman KA, Chouinard M, Boese Q, Czech MP 2003 Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc Natl Acad Sci USA 100:7569–7574[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
L. Bertrand, S. Horman, C. Beauloye, and J.-L. Vanoverschelde
Insulin signalling in the heart
Cardiovasc Res, July 15, 2008; 79(2): 238 - 248.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
I. J. Lodhi, D. Bridges, S.-H. Chiang, Y. Zhang, A. Cheng, L. M. Geletka, L. S. Weisman, and A. R. Saltiel
Insulin Stimulates Phosphatidylinositol 3-Phosphate Production via the Activation of Rab5
Mol. Biol. Cell, July 1, 2008; 19(7): 2718 - 2728.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
R. K. Petersen, L. Madsen, L. M. Pedersen, P. Hallenborg, H. Hagland, K. Viste, S. O. Doskeland, and K. Kristiansen
Cyclic AMP (cAMP)-Mediated Stimulation of Adipocyte Differentiation Requires the Synergistic Action of Epac- and cAMP-Dependent Protein Kinase-Dependent Processes
Mol. Cell. Biol., June 1, 2008; 28(11): 3804 - 3816.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Falasca, W. E. Hughes, V. Dominguez, G. Sala, F. Fostira, M. Q. Fang, R. Cazzolli, P. R. Shepherd, D. E. James, and T. Maffucci
The Role of Phosphoinositide 3-Kinase C2{alpha} in Insulin Signaling
J. Biol. Chem., September 21, 2007; 282(38): 28226 - 28236.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/1/27    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, L.
Right arrow Articles by Saltiel, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, L.
Right arrow Articles by Saltiel, A. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals