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G Protein-Coupled Receptor Kinase 2 Mediates Endothelin-1-Induced Insulin Resistance via the Inhibition of Both Gq/11 and Insulin Receptor Substrate-1 Pathways in 3T3-L1 Adipocytes

The Department of Medicine (I.U., T.I., J.L.B., H.S., J.-C.L., C.J.H., J.M.O.), Division of Endocrinology and Metabolism, and the Biological Sciences Graduate Program (J.L.B.), University of California, San Diego, La Jolla, California 92093-0673

Address all correspondence and requests for reprints to: Jerrold M. Olefsky, MD., Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: jolefsky{at}ucsd.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 -subunit (Gq/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and Gq/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited Gq/11 signaling, including tyrosine phosphorylation of Gq/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of Gq/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ENDOTHELIN-1 (ET-1) IS a vascular polypeptide primarily secreted by endothelial cells (1). Elevated plasma ET-1 levels have been reported in patients with insulin resistance, such as type 2 diabetes (2, 3), obesity (4), and hypertension (5). We have recently reported that chronic in vitro ET-1 treatment leads to enhanced insulin receptor substrate (IRS)-1 degradation, decreased tyrosine phosphorylation of IRS-1 and G protein-q/11 -subunit (Gq/11), and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes (6). ET-1 initiates its actions by binding to the seven-transmembrane receptor (7TMR), endothelin type A receptor. The insulin receptor, on the other hand, is a receptor tyrosine kinase, which activates its signaling cascade by phosphorylating various intracellular substrates, including IRS-1, IRS-2, Shc, and Gq/11. We and others have reported that exogenous administration of ET-1 induces insulin resistance by decreasing muscle glucose disposal in vivo in rats (7) and in healthy human subjects (8), but the mechanisms of ET-1-induced heterologous desensitization of insulin signaling are incompletely understood.

G protein-coupled receptor kinases (GRKs) are enzymes that phosphorylate agonist-activated 7TMRs, leading to 7TMR internalization and inhibition of further G protein activation (9, 10). GRKs also have other functions to regulate 7TMR signaling. Thus, GRKs directly bind to trimeric G protein -subunits, and inhibit G protein function (11, 12, 13, 14, 15, 16). We have recently demonstrated that one of the G proteins, Gq/11, is activated by the insulin receptor and can function in the process of glucose transport stimulation (17), and that GRK2 has an inhibitory role in insulin-stimulated glucose transport by decreasing activation of the Gq/11 pathway (18). In the current studies, we show that GRK2 contributes to chronic ET-1-induced insulin resistance by enhancing IRS-1 degradation and decreasing Gq/11 activation in 3T3-L1 adipocytes.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Microinjection of GRK2 Antibody or Short Interfering RNA (siRNA) Blocks ET-1-Induced Insulin Resistance
It is well established that GRK2 facilitates ET-1 signaling, and we have recently demonstrated that GRK2 can also function as a negative regulator of insulin action (18). Because chronic ET-1 treatment inhibits insulin-induced glucose transport (6), we investigated the role of GRK2 in ET-1-induced insulin resistance. Insulin-stimulated GLUT4 translocation was quantitated using two different methods. First, GLUT4 translocation was assessed by immunofluorescent staining of endogenous GLUT4, as previously described (19). In the basal state, most of the cells displayed staining of GLUT4 in the perinuclear region, whereas 63% of the cells displayed GLUT4 translocation to the plasma membrane after 1.7 nM insulin stimulation (Fig. 1A). When cells were pretreated with 10 nM ET-1 for 24 h, the effect of insulin was inhibited by 65%, consistent with our previous results (6). Microinjection of GRK2 antibody or GRK2 siRNA into the ET-1-treated cells largely rescued them from the ET-1-mediated decrease in GLUT4 translocation (Fig. 1, A and B). Microinjection of GRK5 or GRK6 antibody did not alter insulin-induced GLUT4 translocation, either in the absence or presence of ET-1 treatment (data not shown). Secondly, we adopted the method of McGraw and colleagues (20) by injecting an expression vector encoding hemagglutinin (HA)-GLUT4-green fluorescent protein (GFP). Because the HA tag is located in the first extracellular loop of GLUT4, this construct allows us to monitor the fusion of GLUT4 in the plasma membrane in nonpermeabilized cells by using an HA-directed antibody. At the same time, total expression of the GLUT4 construct is monitored by measuring total GFP fluorescence in individual cells. With this method, the ratio of cell surface HA staining to total GFP fluorescence provides a quantitative measure of GLUT4 translocation on a single cell bases. Both siRNA and HA-GLUT4-GFP expression vector were microinjected together into the cell nucleus of 3T3-L1 adipocytes. GFP fluorescence-positive cells were then analyzed by immunofluorescent microscopy to quantitate the ratio of cell surface HA-staining to total GLUT4-GFP fluorescence, as described in Materials and Methods. As can be seen in Fig. 1C, insulin-stimulated GLUT4 translocation was inhibited by chronic ET-1 treatment and this effect was prevented by injection of GRK2 siRNA. Thus, both methods used to quantitate GLUT4 translocation yielded comparable results.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Effects of Microinjection of Anti-GRK2 Antibody or GRK2-siRNA on GLUT4 Translocation in 3T3-L1 Adipocytes A, 3T3-L1 adipocytes on coverslips were treated with or without 10 nM ET-1 for 24 h, after microinjection with anti-GRK2 antibody or control IgG. B, Forty-eight hours after microinjection with siRNA against GKR2 or scrambled siRNA (Control), 3T3-L1 adipocytes were treated with or without 10 nM ET-1 for 24 h. including 4 h serum starvation. Cells were then stimulated with 1.7 nM insulin for 20 min. GLUT4 was immunostained as described in Materials and Methods. The percentage of cells positive for GLUT4 translocation (Ring assay) was calculated by counting at least 100 cells per condition. The data are the mean ± SE from three independent experiments. C, Twenty-four hours after nuclear microinjection of siRNA against GKR2 or scrambled siRNA (Control) together with HA-GLUT4-GFP expression vector, 3T3-L1 adipocytes were then pretreated with or without 10 nM ET-1 for 24 h, including 4 h serum starvation. Cells were stimulated with 17 nM insulin for 20 min. The cell surface HA-tag of exogenous HA-GLUT4-GFP was immunostained as described in Materials and Methods. The ratio of HA staining/GLUT4-GFP fluorescence was measured by C-Imaging Systems on at least 40 cells per condition.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Effect of WT- or KD-GRK2 Overexpression on Insulin Receptor Phosphorylation in 3T3-L1 Adipocytes A, 3T3-L1 adipocytes were infected with adenovirus encoding LacZ control (C), WT(W), or KD-GRK2 (K). Twenty-four hours after infection, these cells were treated with or without 10 nM ET-1 for 24 h, serum-starved for 16 h, stimulated with 17 nM insulin for 5 min, and lysed. Total cell lysates were analyzed by Western blotting using antiinsulin receptor (IR), PY20, or anti-GRK2 antibody, as described in Materials and Methods. Representative blots are shown from three independent experiments. B, The signal intensities were scanned and quantitated using NIH Image software. The ratio of tyrosine phosphorylation signal (middle panel in A)/insulin receptor protein level (top panel in A) was calculated and shown. The data are mean ± SE from three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Effect of WT- or KD-GRK2 Overexpression on Gq/11-cdc42-PI3-Kinase Pathway in 3T3-L1 Adipocytes A, 3T3-L1 adipocytes were infected with adenovirus expressing WT- (W) or KD-GRK2 (K) or control LacZ (C). Twenty-four hours after infection, these cells were treated with or without 10 nM endothelin-1 (ET-1) for 24 h, serum starved for 16 h, stimulated with 17 nM insulin for 10 min, and lysed. Samples were immunoprecipitated with anti-cdc42 antibody or control IgG (negative control; NC). PI3-kinase activity was measured as described in Materials and Methods. A representative film is shown and the graph represents the mean ± SE of three independent experiments. B, 3T3-L1 adipocytes were serum starved for 16 h, stimulated with 17 nM insulin or 10 nM endothelin-1 for the indicated time periods and lysed. Samples were immunoprecipitated with anti-GRK2 antibody or control IgG (negative control; NC). Immunoprecipitates and total cell lysate (TCL) were analyzed by Western blotting using anti-Gq/11 antibody as described in Materials and Methods. A representative blot is shown from three independent experiments. C, 3T3-L1 adipocytes were infected with adenovirus expressing WT (W) or KD-GRK2 (K) or control LacZ (C). Twenty-four hours after infection, these cells were serum-starved for 16 h, stimulated with 10 nM ET-1 or 17 nM insulin for 1 min, and lysed. Samples were immunoprecipitated with PY20 or control IgG (NC), and were analyzed by Western blotting using anti-Gq/11 antibody, as described in Materials and Methods. A representative blot is shown from three independent experiments.

GRK2 Directly Binds to Gq/11 and Inhibits Insulin-Induced Activation of the Gq/11 Pathway before and after ET-1 Treatment

To further assess the mechanisms of inhibition of Gq/11 by GRK2, we measured the association of endogenous GRK2 and Gq/11 before and after insulin or ET-1 stimulation (Fig. 3B). Association of endogenous GRK2 and Gq/11 was not detected in the basal state, but was markedly enhanced by either ET-1 or insulin with a maximal response at 5 min, and decreased to basal levels by 60 min. As seen in Fig. 3C, both WT- and KD-GRK2 overexpression inhibited Gq/11 tyrosine phosphorylation stimulated by ET-1 or insulin. These results suggest that GRK2 inhibits the activation of Gq/11 and its downstream actions through a direct association of GRK2 and Gq/11, independent of GRK2 kinase activity.

GRK2 Is Involved in ET-1-Stimulated Serine Phosphorylation of IRS-1 and Degradation of IRS-1 Protein
We next examined the effect of WT- or KD-GRK2 expression on the IRS-1 pathway in the presence of chronic ET-1 treatment (Fig. 4, A and B). As reported previously (6), chronic ET-1 treatment leads to degradation of IRS-1, inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation, and decreased association of IRS-1 and p85. Overexpression of WT-GRK2 had no effect on these alterations in the IRS-1 pathway caused by chronic ET-1 treatment. In contrast, overexpression of KD-GRK2 inhibited ET-1-induced IRS-1 degradation, suggesting that the kinase activity of GRK2 is involved in this process. Similarly, KD-GRK2 rescued the chronic ET-1-induced inhibition of IRS-1 tyrosine phosphorylation (Fig. 4A) and PI3-kinase association (Fig. 4B). These results led us to assess the association of endogenous GRK2 with IRS-1 before and after insulin or ET-1 stimulation (Fig. 4C). Binding of endogenous GRK2 and IRS-1 was not detected in the basal state but was markedly enhanced after ET-1 treatment. Insulin treatment did not induce the association of these proteins. We then examined whether GRK2 was involved in IRS-1 degradation after chronic insulin treatment (Fig. 4D). Insulin treatment for 16 h, as well as ET-1 treatment for 24 h, caused IRS-1 degradation, as reported previously (22, 23). Although overexpression of KD-GRK2 inhibited ET-1-induced IRS-1 degradation, it did not inhibit insulin-induced IRS-1 degradation.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Effect of WT- or KD-GRK2 Overexpression on IRS-1-PI3-Kinase Pathway in 3T3-L1 Adipocytes A, 3T3-L1 adipocytes were infected with adenovirus expressing WT- (W) or KD-GRK2 (K) or control LacZ (C). Twenty-four hours after infection, these cells were treated with or without 10 nM ET-1 for 24 h, serum-starved for 16 h, stimulated with 17 nM insulin for 5 min (for IRS-1 and PY20 blots) or 10 min (for p85 blot), and lysed. Samples were then immunoprecipitated with anti-IRS-1 antibody. Immunoprecipitates and total cell lysates were analyzed by Western blotting using anti-IRS-1, PY20, or anti-p85 antibody as described in Materials and Methods. B, 3T3-L1 adipocytes were infected with adenovirus expressing WT- (W) or KD-GRK2 (K) or control LacZ (C). Twenty-four hours after infection, these cells were treated with or without 10 nM ET-1 for 24 h, serum-starved for 16 h, stimulated with 17 nM insulin for 10 min, and lysed. Samples were immunoprecipitated with or without (negative control; NC) anti-IRS-1 antibody. PI3-kinase activity was measured as described in Materials and Methods. The graph represents the mean ± SE of three independent experiments. C, 3T3-L1 adipocytes were serum-starved for 16 h, stimulated with 17 nM insulin or 10 nM ET-1 for the indicated time periods, and lysed. Samples were immunoprecipitated with anti-GRK2 antibody or control IgG (negative control; NC). Immunoprecipitates and total cell lysate (TCL) were analyzed by Western blotting using anti-IRS-1 as described in Materials and Methods. D, 3T3-L1 adipocytes were infected with adenovirus expressing WT- (W) or KD-GRK2 (K) or control LacZ (C). Twenty-four hours after infection, these cells were treated with 10 nM ET-1 for 24 h or 17 nM insulin for 16 h, and lysed. Total cell lysates were analyzed by Western blotting using anti-IRS-1 antibody as described in Materials and Methods. In all cases, representative images are shown from three independent experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Effect of WT- or KD-GRK2 Overexpression on ET-1-Induced Serine Phosphorylation of IRS-1 A, 3T3-L1 adipocytes were serum starved for 16 h, stimulated with 10 nM ET-1 for the indicated time periods and lysed. Total cell lysate were analyzed by Western blotting using antiphospho-specific IRS-1 antibody against serine 307, 612, and 636, as described in Materials and Methods. A representative blot is shown from three independent experiments. B, 3T3-L1 adipocytes were infected with adenovirus expressing WT- (W) or KD-GRK2 (K) or control LacZ (C). Twenty-four hours after infection, these cells were serum-starved for 16 h, stimulated with 10 nM ET-1 or 17 nM insulin for 5 min, and lysed. Whole cell lysates were analyzed by Western blotting using antiphospho-specific IRS-1 antibody against serine 307, 612, and 636. Representative blots are shown from three independent experiments. C, The signal intensity of IRS-1 Ser612-phosphorylation was scanned and quantitated using NIH Image software. The data are the mean ± SE from three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

GRKs are a well-known family of kinases that phosphorylate ligand-bound 7TMRs. The phosphorylation of 7TMRs by GRKs leads to 7TMR internalization and uncoupling of the 7TMR from further G protein activation (9, 10). Recent papers have reported that GRK2 directly binds to active Gq/11 through its RGS domain, inhibiting Gq/11 activity (27). Consistent with these reports, our recent study demonstrated that GRK2 inhibits insulin-stimulated glucose transport by interacting with the Gq/11/ cdc42/ PI3-kinase pathway at the Gq/11 step (18). Because GRK2 functions in ET-1 action, we hypothesized that GRK2 is activated by ET-1 and plays a role in the desensitization of insulin’s biologic effects.

Our results show that inhibition of GRK2 rescued insulin signaling to GLUT4 translocation in the presence of chronic ET-1 treatment (Fig. 1). We have found that both insulin and ET-1 enhanced the association of GRK2 with Gq/11, and that chronic ET-1 treatment inhibited insulin activation of the Gq/11 pathway, which was further inhibited by overexpression of either WT- or KD-GRK2 (Fig. 3). These results suggest that the decreased activity of Gq/11 and its downstream effectors is mediated by the association of Gq/11 and GRK2. We also conclude that the kinase activity of GRK2 is not required for its inhibitory effects on Gq/11, consistent with our previous report showing that the inhibitory effect of GRK2 was mediated through the RGS domain and was independent of GRK kinase activity (18).

Interestingly, ET-1 stimulation increases the kinase activity of GRK2, leading to phosphorylation of the endothelin type A (ETA) receptor (16). This led us to hypothesize that ET-1-induced activation of GRK2 kinase activity might be involved in ET-1-induced IRS-1 Ser/Thr phosphorylation and degradation. Overexpression of KD-GRK2 clearly inhibited ET-1-induced IRS-1 degradation, whereas WT-GRK2 did not, demonstrating a potential role for GRK2 kinase activity in this mechanism. Among the numerous Ser/Thr phosphorylation sites in IRS-1, we found that serine 307, 612, and 636 phosphorylation were stimulated by ET-1, and that phosphorylation of serine 612/636 was dependent on the kinase activity of GRK2. The decreased expression and tyrosine phosphorylation of IRS-1 were associated with diminished downstream insulin action. These results indicate that with chronic ET-1 treatment, GRK2 is involved in decreasing the activity of both Gq/11 and IRS-1, but by different mechanisms.

The current study is the first report showing that GRK2 is a serine/threonine kinase that, upon ET-1 stimulation, can associate with IRS-1 and promote ET-1-mediated IRS-1 serine phosphorylation and degradation. Several serine/ threonine kinases can phosphorylate IRS proteins, including MAPK (28), protein kinase C (29), Janus kinase (30, 31), mammalian target of rapamycin (22, 32), glycogen synthase kinase-3 (33), and IB kinase (34), and certain IRS serine/ threonine phosphorylation events are necessary for IRS degradation. Because these kinases do not operate simultaneously, it is possible that their role in IRS phosphorylation and degradation may vary across cell types, depending on which ligand is providing the stimulatory event. Like the other serine kinases that are activated by ligands other than insulin, the mechanism by which ET-1-stimulated GRK2 recognizes IRS-1 as a substrate remains unclear. Furthermore, in a previous study (6), we have shown that wortmannin, a PI3-kinase inhibitor, blocks ET-1-induced IRS-1 degradation, suggesting that PI3-kinase, or a downstream kinase, such as mammalian target of rapamycin, is also involved in the degradation process. At this point, it is unknown whether GRK2 and PI3-kinase interact with each other in the ET-1 signaling pathway or whether GRK2 and PI3-kinase provide separate inputs into the process of IRS-1 phosphorylation and degradation.

In summary, the current study has demonstrated a novel role for GRK2 in chronic ET-1-induced cellular insulin resistance. In addition to inhibiting the Gq/11 pathway, GRK2 kinase activity inhibits the IRS-1 pathway by enhancing degradation of IRS-1 after chronic ET-1 treatment.

	MATERIALS AND METHODS

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Materials
Mouse monoclonal anti-cdc42 antibody, rabbit polyclonal anti-p85 (N-SH2), antiphospho-IRS-1-against Ser307, anti-IRS-1 antibodies, cdc42 assay kit, and protein A agarose were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit polyclonal antiphospho-IRS-1-against Ser612 and –636 antibodies were from Cell Signaling Technology (Beverly, MA). Mouse monoclonal antiphosphotyrosine (PY20) antibody was from Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-GLUT4 antibody was purchased from Chemicon International Inc. (Temecula, CA). Rabbit polyclonal anti-GRK2, anti-GRK3, anti-GRK5, anti-GRK6, anti-Gq/11, and anti-cdc42 (P1) antibodies, and horseradish peroxidase-linked antirabbit and antimouse antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep IgG and fluorescein isothiocyanate (FITC)-conjugated and tetramethyl rhodamine isothiocyanate-conjugated antirabbit and antimouse IgG antibodies were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). siRNA against GRK2 was described previously (18). DMEM and fetal bovine serum were purchased from Life Technologies (Grand Island, NY). Plasmid vectors encoding WT- and KD- (K220R) GRK2 were kindly provided by Robert J. Lefkowitz (Duke University, Durham, NC). Radioisotope was from ICN (Costa Mesa, CA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Generation of Adenovirus Vectors
Adenoviruses were constructed as described previously (23) using Adenovirus Expression Vector Kit (Takara, Osaka, Japan) according to the manufacturer’s instructions. The recombinant adenoviruses were amplified in 293 cells and viral stock solutions with the viral titer >10⁸ pfu/mi were prepared.

Cell Culture and Cell Treatment
3T3-L1 cells were cultured and differentiated as described previously (21). Differentiated 3T3-L1 adipocytes (d 14 after differentiation) were incubated with 10 nM ET-1 for 24 h before some assays. For adenovirus infection, 3T3-L1 adipocytes (d 11 after differentiation) were transduced for 16 h in DMEM high-glucose medium with 5% heat inactivated serum at a multiplicity of infection of 40 with either the recombinant adenovirus of WT-GRK2 or KD-GRK2 or a LacZ control. Transduced cells were incubated for 48 h at 37 C in 10% CO2 and DMEM high-glucose medium with 10% heat inactivated serum, followed by incubation in starvation media. The efficiency of adenovirus mediated gene transfer was above 90% as measured by histocytochemical staining of LacZ-infected cells with ß-galactosidase (data not shown).

Microinjection of Antibody or siRNA
Microinjection was carried out using a semiautomatic Eppendorf microinjection system (Eppendorf North America, Westbury, NY). Antibodies for microinjection were concentrated and dissolved at 5 mg/ml in microinjection buffer containing 5 mM sodium phosphate (pH 7.2), 100 mM KCl. Antibodies or control IgG at 5 mg/ml or 5 µM siRNA mixed with FITC-dextran were injected into the cytoplasm of 3T3-L1 adipocytes (d 12–14 after differentiation) for GLUT4 ring assay, as described previously (19). For the analysis of cell surface GLUT4-HA-epitope staining, HA-GLUT4-GFP expression vector DNA (0.1 mg/ml) was mixed together with 5 µM siRNA in microinjection buffer, and injected into the cell nucleus (d 10 after differentiation).

Immunostaining and Immunofluorescence Microscopy
Immunostaining of endogenous GLUT4 (ring assay) was performed essentially as described previously (17). 3T3-L1 adipocytes were stimulated with insulin for 20 min at 37 C and were fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 2% fetal calf serum in PBS for 10 min. The cells were then incubated with anti-GLUT4 antibody in PBS with 2% fetal calf serum overnight at 4 C. After washing, GLUT4 and injected IgG were detected by incubation with tetramethyl rhodamine isothiocyanate-conjugated donkey antirabbit IgG antibody and FITC-conjugated donkey antimouse or antisheep antibody, respectively, followed by observation under an immunofluorescence microscope. In all counting experiments, the observer was blinded to the experimental condition of each coverslip.

For the quantitative analysis of exogenous HA-GLUT4-GFP membrane fusion, we adapted the method of McGraw and colleagues (20) by microinjecting an expression vector encoding HA-GLUT4-GFP. The expression vector DNA (0.1 mg/ml) was mixed together with siRNA (5 µM), and microinjected into the cell nucleus of 3T3-L1 adipocytes (d 10). Twenty-four hours after microinjection, the cells were then treated with or without ET-1 for 24 h, including 4 h starvation, and stimulated with or without insulin for 20 min. Fixed cells were stained with anti-HA monoclonal antibody (Covance, Berkeley, CA) without permeabilization for 30 min at 37 C. Cells were then washed and stained with a secondary Cy3-conjugated antimouse antibody. GFP fluorescent-positive cells were imaged on a TE300 inverted microscope (Nikon, Tokyo, Japan). Images were captured and analyzed using Simple PCI software and a Hamamatsu Orca 12 bit charge-coupled device camera (C-Imaging Systems, Cranberry Township, PA). GLUT4 translocation and fusion with the plasma membrane was quantitated by taking the ratio of Cy3 (HA) to GFP (total) fluorescence. This measurement provides the ratio of the HA-GLUT4-GFP that has fused with the plasma membrane to the total amount of HA-GLUT4-GFP expressed in the cell, as described previously (20).

Western Blotting
Serum-starved 3T3-L1 adipocytes were stimulated with 17 nM insulin at 37 C for various time periods as indicated in each experiment. The cells were lysed in solubilizing buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40 (NP-40), 1 mM Na3VO4, 1 mM PMSF, and 10 mM NaF (pH 7.5) for 15 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. For Western blot analysis, whole cell lysates (20–50 µg protein) were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE. Gels were transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA) using Transblot apparatus (Bio-Rad, Hercules, CA). For immunoblotting, membranes were blocked and probed with specific antibodies. Blots were then incubated with horseradish peroxidase-linked secondary antibodies followed by chemiluminescence detection, according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL).

PI3-Kinase Assay
3T3-L1 adipocytes were starved for 16 h and stimulated with insulin (17 nM) for 10 min, washed once with ice-cold PBS, lysed, and subjected to immunoprecipitation (300–500 µg total protein) with anti-cdc42 or anti-IRS-1 antibody for 4 h at 4 C. Immunocomplexes were precipitated with protein A-plus agarose (Upstate Biotechnology Inc.). The immunoprecipitates were washed three times with each of the following buffers: 1) PBS, containing 1% Nonidet P-40, 100 µM sodium orthovanadate (pH 7.4); 2) 100 mM Tris, 0.5 M LiCl, 100 µM sodium orthovanadate (pH 7.4); and 3) 10 mM Tris, 100 mM NaCl, 100 µM sodium orthovanadate (pH 7.4). The washed immunocomplexes were incubated with phosphatidylinositol for 5 min and then with [-³²P]ATP (3000 Ci/mmol) for 5 min at room temperature. Reactions were stopped with 20 µl of 8 N HCl, and mixed with 160 µl of CHCl3:methanol (1:1). Samples were centrifuged and the lower organic phase was applied to a silica gel thin-layer chromatography plate that had been coated with 1% potassium oxalate. thin-layer chromatography plates were developed in CHCl3:CH3OH:H₂O:NH4OH (60:47:11.3:2), dried, and exposed to an x-ray film. PI3-kinase activity was quantitated by scanning and analyzing the film using the NIH Image program (National Institutes of Health, Bethesda, MD).

cdc42 Assay
cdc42 activity was measured according to the manufacturer’s instructions (Upstate Biotechnology Inc.). 3T3-L1 adipocytes were starved for 16 h and stimulated with 17 nM insulin or 10 nM ET-1 for 1 min, washed once with ice-cold PBS and lysed with lysis buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl₂, 1 mM EDTA, 10% glycerol, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 25 mM NaF for 15 min at 4 C. Insoluble materials were removed by centrifugation. For a negative control, cell lysate was incubated with 1 mM GDP for 15 min at 30 C. Five micrograms of PAK-1 agarose beads, which specifically bind to active cdc42, were added to the cell lysates and incubated for 1 h at 4 C. Agarose beads were washed with lysis buffer three times, and boiled in 2x Laemmli sample buffer. Samples were resolved by SDS-PAGE and immunoblotted with anti-cdc42 antibody.

Statistical analysis
Data were analyzed by Student’s t test. P values < 0.05 were considered significant.

	ACKNOWLEDGMENTS

 
We thank Dr. Robert J. Lefkowitz (Howard Hughes Medical Institute at Duke University, Durham, NC) for providing cDNAs encoding WT- and KD- (K220R) GRK2, Dr. Timothy E. McGraw (Weill Cornell Medical College, New York, NY) for providing the HA-GLUT4-GFP expression vector, Ms. Jianying He (Toyama Medical and Pharmaceutical Industry, Toyama, Japan) for technical assistance, and Ms. Elizabeth J. Hansen (University of California San Diego, San Diego, CA) for editorial assistance.

	FOOTNOTES

 
Present address for I.U.: First Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.

This work was supported by a research grant from the National Institutes of Health (DK 33651) and the Whittier Institute for Diabetes. I.U. is supported through an American Diabetes Association Mentor-based Fellowship Award.

First Published Online June 30, 2005

Abbreviations: ET-1, Endothelin-1; FITC, fluorescein isothiocyanate; Gq/11, G protein-q/11 -subunit; GFP, green fluorescent protein; GLUT4, glucose transporter 4; GRK2, G protein-coupled receptor kinase 2; HA, hemagglutinin; KD, kinase deficient; PI3, phosphatidylinositol 3; PY20, antiphosphotyrosine; siRNA, short interfering RNA; 7TMRs, seven transmembrane receptors; WT, wild type.

Received for publication October 21, 2004. Accepted for publication June 17, 2005.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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