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Endocrinology Vol. 147, No. 7 3333-3343
Copyright © 2006 by The Endocrine Society

Reduced Expression of Focal Adhesion Kinase Disrupts Insulin Action in Skeletal Muscle Cells

Danshan Huang, Michelle Khoe, Dusko Ilic and Michael Bryer-Ash

West Los Angeles Veterans Administration Medical Center and UCLA Gonda (Goldschmied) Diabetes Center (D.H., M.K., M.B.), Division of Endocrinology, Diabetes and Hypertension, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095; and Department of Stomatology and Anatomy (D.I.), University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Michael Bryer-Ash, M.D., UCLA Division of Endocrinology, Diabetes and Hypertension; 900 Veteran Avenue, Warren Hall Room 24-130; Los Angeles, California 90095. E-mail: mbryerash{at}mednet.ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Integrins mediate interactions between cells and extracellular matrix proteins that modulate growth factor signaling. Focal adhesion kinase (FAK) is a key multifunctional integrin pathway protein. We recently reported that disruption of FAK impairs insulin-mediated glycogen synthesis in hepatocytes. To test the hypothesis that FAK regulates skeletal muscle insulin action, we reduced FAK expression in L6 myotubes using FAK antisense. In untransfected myotubes, insulin stimulated both FAK tyrosine phosphorylation and kinase activity. Cells treated with antisense FAK showed 78 and 53% reductions in FAK mRNA and FAK protein, respectively, whereas insulin receptor substrate 1/2 and paxillin abundance were unaffected. Insulin-stimulated U-14C-glucose incorporation into glycogen was abolished by FAK antisense, and 2-deoxy-glucose uptake and glucose transporter 4 (GLUT4) translocation were both markedly attenuated. Antisense FAK did not alter GLUT1 or GLUT3 protein abundance. Immunofluorescence staining showed decreased FAK Tyr397 phosphorylation and reduced actin stress fibers. Thus, in skeletal myotubes, FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. Integrin signaling may play an important regulatory role in muscle insulin action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOCAL ADHESION KINASE (FAK) is a nonreceptor tyrosine kinase that preferentially localizes at focal adhesions (1). It is activated by integrin-mediated engagement and serves as a signaling protein within cytoskeleton-associated networks (1). FAK expression regulates the arrangement of focal adhesions and the cytoskeleton, which results in the formation of a motile cell phenotype (2). It has been reported previously that integrin engagement stimulates both insulin receptor substrate (IRS)-1-associated phosphatidylinositol-3 (PI3)-kinase activity and Akt/protein kinase B (Akt/PKB) activity (3), which are important intermediaries in the action of insulin to stimulate glucose transport and glycogen synthesis. Moreover, the expression of IRS-1 mRNA is abolished in FAK knockout mouse fibroblast cells (4). These findings indicate the potential importance of cross talk between integrin and insulin signaling pathways in the regulation of insulin action.

Insulin has diverse effects on cells, including stimulation of glucose transport, gene expression, and alteration of cell morphology (5, 6, 7). These effects are mediated by the activation of signaling pathways, which leads to a rapid reorganization of actin filaments that coincides with plasma membrane ruffling and intense accumulation of pinocytic vesicles at the cell membrane (7). The actin cytoskeleton facilitates propagation of the morphological, metabolic, and nuclear effects of insulin by regulating the appropriate subcellular distribution of the molecules that participate in the insulin signaling pathway (7). It has been reported that disassembly of filamentous actin (F-actin) abolishes insulin-stimulated glucose transport in L6 myotubes without affecting basal transport activity (8). This suggests that the actin skeleton may provide the mechanical support that stabilizes the glucose transporter at the appropriate location, making it accessible to incoming signals and available for insulin-induced translocation to the plasma membrane.

We previously showed that in the liver of healthy Sprague Dawley rats, FAK rapidly undergoes tyrosine phosphorylation upon insulin stimulation under euglycemic conditions in vivo (9). Moreover, we found that, when insulin resistance was ameliorated in obese Zucker rats by administration of an adenoviral construct encoding a soluble inhibitor of tumor necrosis factor-{alpha}, hepatic FAK tyrosine phosphorylation was markedly increased under insulin-stimulated conditions (9). We recently overexpressed mutant FAK constructs in a HepG2 hepatoma cell line and showed that both the focal adhesion targeting property and tyrosine kinase activity of FAK are essential for insulin-mediated stimulation of glycogen synthesis and that FAK regulates insulin action downstream of PI3-kinase, by specific interactions with both Akt/PKB and glycogen synthase kinase (GSK)-3ß, and thus may play an important role in hepatic insulin action (10).

In light of the aforementioned findings, we undertook the present study to test the hypothesis that FAK also regulates skeletal muscle insulin signaling and insulin action and that these are mediated via alteration of the actin cytoskeleton. Herein, we use an adenoviral antisense FAK construct to reduce FAK abundance in L6 myotubes and we show that FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. We conclude that the integrin signaling pathway may potentially play an important regulatory role in muscle insulin action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Antibody (Ab) to FAK, and FAK pY397 were purchased from BD Biosciences (San Diego, CA). Anti-caspase-3 Ab, Ab to Akt, Ser473, Thr308, and the Akt activity assay kit (catalog no. 9840) were from Cell Signaling Technology (Beverly, MA). Ab to Py99 and ß-actin, IRS-1, as well as IRS-2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-glucose transporter 4 (GLUT4) Ab was from Abcam Inc. (Cambridge, MA) Chemiluminescence kit, protein A/G-conjugated agarose beads, and secondary Ab were all from Pierce (Rockford, IL). Goat antirabbit fluorescein isothiocyanate (FITC) Ab and rhodamine-labeled phalloidin were purchased from Molecular Probes (Eugene, OR). All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Nitrocellulose membrane was from Schleicher & Schuell (Riviera Beach, FL). [N-3H]Deoxy-D-glucose was obtained from NEN Life Science Products (Boston, MA). [U-14C]Glucose (303 mCi/mmol) and [{alpha}-32P]dCTP (3000 Ci/mmol) were from ICN (Costa Mesa, CA). ß-Galactosidase (ß-gal) producing (LacZ-expressing) adenovirus and Adeno-X-EGFP [enhanced green fluorescence protein (EGFP)-expressing] adenovirus were from Clontech Laboratories (Palo Alto, CA). Anti-{alpha}-actin Ab and all other chemicals were purchased from Sigma unless otherwise stated. L6 myoblasts were from ATCC Inc. (Manassas, VA).

Generation of FAK adenoviral constructs
Adenovirus constructs were generated according to the method of Hardy et al. (11). The antisense FAK vector (pcDNA3 antisense FAK) was the gift of Drs. Y. Takeuchi and M. Suzawa from Kyoto University (Kyoto, Japan). Ndel-BamHI fragments of pcDNA3 antisense FAK were inserted into the pAdlox shuttle vector. Adenovirus expressing FAK antisense oligonucleotide (AdAS-FAK) was plaque-purified (1 x 1010 pfu/ml) and isolated as previously described (11, 12).

Cell culture, adenovirus infection, and insulin stimulation
The ATCC L6 myoblast cell line was used to seed 10-cm diameter dishes at 1.5 x 104 cells/cm2. Spontaneously fusing L6 cells were grown and maintained in {alpha}-MEM containing 2% (vol/vol) fetal bovine serum and 1% (vol/vol) antibiotic solution and incubated at 37 C in 5% CO2/95% air for 7 d. Differentiated L6 myotubes were then infected with AdAS-FAK by way of multiple infections of the virus. Subsequent experiments were performed 72 h after initial addition of virus. Before experiments, cells were starved in {alpha}-MEM containing 0.1% BSA for 5 h. For insulin stimulation, recombinant human insulin (Humulin; Eli Lilly Inc.) was added in a final concentration of 100 nM. After the indicated treatment times, cells were washed twice with ice-cold PBS and liquid N2 was added. One milliliter of lysis buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM Na3VO4, 10 mM sodium ß-glycerophosphate, 50 mM NaF, 5 mM Na4P2O7, 1 µM microcysin-LR, 0.27 M sucrose, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) was then added. Clarified supernatants were aliquoted with lysis buffer to equal protein content (Bradford Protein Assay reagent; Bio-Rad, Hercules, CA).

Northern blotting
Total RNA was extracted using RNA STAT-60 reagent (Tel-Test, Inc., Friendswood, TX). Thirty micrograms total RNA were denatured with formaldehyde/formamide and resolved by electrophoresis on 1% agarose gel containing formaldehyde. RNA was transferred onto a Hybond-N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ), that was hybridized at 65 C for 18 h. Probes used were cDNA fragments of FAK (3.3 kb) and GAPDH (1.3 kDa) (Sigma, St. Louis, MO) and were labeled with [{alpha}-32P]dCTP using a random primer labeling kit (Amersham Pharmacia Biotech).

Immunoprecipitation and Western blotting
Immunoblotting and immunoprecipitation were performed as previously described (10). All experiments were repeated three times, and results are shown in the figures. For each sample, a total of 30 µg of protein was loaded.

Glycogen synthesis measurement
Transfected L6 myotubes were incubated in serum- and glucose-deprived buffer B (HEPES-buffered saline containing 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, 1.0 mM CaCl2, and 0.1% BSA at pH 7.4) for 3 h. Medium was replaced with incubation medium containing 5.5 mM glucose and 0.33 µCi/ml [U-14C]D-glucose in the presence or absence of 100 nM insulin for 2 h at 37 C. Measurement of [U-14C]D-glucose incorporation into glycogen was performed by methods previously described (10, 13).

2-Deoxy-glucose (2-DOG) uptake
2-DOG uptake measurements were made according to the technique of Klip et al. (14). Transfected L6 myotubes were incubated in serum- and glucose-deprived buffer B for 4 h. The medium was replaced with an incubation medium containing 10 µM 2-DOG and 0.5 µCi/ml [N-3H]-2-DOG in the absence or presence of 100 nM insulin for 5 min. Carrier-mediated uptake was determined by quantitating cell-associated radioactivity in presence of 10 µM cytochalasin B. The radioactive medium was aspirated rapidly followed by three washes with ice-cold isotonic saline solution (0.9% NaCl wt/vol) before lysis in 0.05 M NaOH.

Membrane preparation from L6 myotubes
Transfected L6 myotubes were incubated in serum- and glucose-deprived buffer B for 4 h. Serum-starved cells were exposed to insulin (100 nM) for 30 min. Cells were fractionated into plasma and light membrane components by an adaptation of the method of Zhou et al. (15). Cells were washed with PBS and immediately homogenized on ice using a PowerGen homogenizer (Fisher Scientific Inc., Pittsburgh, PA) set at 13,000 rpm in a buffer containing 0.25 M sucrose, 1 mM EDTA, 20 mM HEPES, 5 mM benzamidine, 1 µM aprotinin A, 1 µM leupeptin, and 1 µM phenylmethylsulfonyl fluoride, pH 7.4, at 4 C. The homogenate was then centrifuged at 9000 x g for 20 min. The supernatant was centrifuged at 180,000 x g for 90 min. The 180,000 x g supernatant comprised the light membrane fraction. The 180,000 x g pellet was resuspended in PBS with protease inhibitors, loaded on 10–30% (wt/wt) continuous sucrose gradient (3–4 mg protein per 5 ml gradient), and centrifuged at 48,000 rpm for 55 min in a SW-50.1 rotor. The pellet comprised the plasma membrane fraction. Membrane fractions and light membrane c proteins were resuspended in buffer A at a final protein concentration of 1 mg/ml. Both light membrane proteins and membrane fractions were analyzed by Western immunoblot.

In vitro Akt/PKB kinase assay
Transfected L6 myotubes were incubated in serum- and glucose-deprived buffer B for 4 h. Serum-starved cells were exposed to insulin (100 nM) for 30 min. Cells were washed with PBS and immediately homogenized on ice with cell lysis buffer. The solution was centrifuged at 14,000 x g, and the pellet was discarded, whereas the supernatant (whole-cell extraction) was taken for kinase assay. Twenty microliters of immobilized Akt primary Ab bead slurry was added to 200 µl whole-cell extract (150 µg protein) overnight at 4 C. The Akt Ab pellet was washed with cell lysis buffer and resuspended in 50 µl kinase buffer, supplemented with 1 µl of 10 mM ATP and 1 µg GSK-3 fusion protein, which was allowed to incubate for 30 min at 30 C. The reaction was terminated with 25 µl of 3x SDS sample buffer. The solution was mixed with a vortex mixer and then centrifuged. The supernatant was used for Western blot detection of phosphorylated GSK-3 fusion protein. The membrane was then reblotted with Akt Ab to confirm that an equal amount of Akt protein was put down.

Immunofluorescence microscopy
Transfected L6 myotubes in chamber slides were fixed with 3.7% formaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 15 min. Cells were stained with rhodamine-conjugated phalloidin for 30 min (Molecular Probes), followed by treatment with FITC-conjugated anti-paxillin Ab (Transduction Laboratories, Lexington, KY) or with the primary Ab anti-FAK397 phosphoAb (Biosource, Camarillo, CA) for 60 min. Primary Ab detection was performed with FITC-conjugated goat antirabbit IgG (Molecular Probes). In controls, primary Ab was omitted. Samples were examined using a Zeiss Axiophot microscope (Zeiss Inc., Oberkochen, Germany).

Statistical analysis
Statistical comparisons were conducted using Student’s two-tailed t test for paired or unpaired samples as appropriate, with appropriate correction for multiple comparisons (SigmaStat statistical software version 2.0; SPSS Inc., Chicago, IL). Data are reported as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FAK mRNA is decreased in antisense FAK-treated cells and this effect is specific for FAK
L6 myotubes were successfully infected with adenovirus, giving an approximately 80% Lac-Z control efficiency of infection, indicated by ß-gal immunostaining, as shown in Fig. 1AGo. FAK mRNA was analyzed by Northern blotting, and the results are presented in Fig. 1BGo. Seventy-two hours after infection, the level of FAK mRNA [normalized to amounts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] decreased by 78%, as measured by densitometry analysis in Fig. 1BGo. In Fig. 1CGo, immunoblot analysis shows that the effect of adenovirus antisense on protein expression was specific for FAK. As shown in Fig. 1BGo. FAK protein expression was reduced by about 53% compared with untransfected (NON) and Lac-Z adenovirus-transfected (ß-gal) cells. The abundance of the FAK substrate, paxillin, and the housekeeping protein, ß-actin, were unaffected by either Lac-Z control or adenovirus antisense FAK (AdAS-FAK) treatment (Fig. 1CGo, a, middle and lower bands). This suggests that there was a specific effect of AdAS-FAK on FAK protein expression and not an overall reduction in protein synthesis through general effects on cell metabolism, or nonspecific induction of cell death.


Figure 1
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  		FIG. 1. Effect of AdAS-FAK on FAK mRNA expression and protein abundance. A, Light microscopy of L6 myotubes untreated (NON, a) or treated with adenoviral Lac-Z constructs (b) or AdAS-FAK (c) and exposed to X-gal. B, Representative autoradiogram of Northern blot (a). Mean ± SEM of OD units from three experiments are shown in b. *, P < 0.01 for AdAS-FAK (0.30 ± 0.13) vs. NON (1.06 ± 0.08) and ß-gal-treated cells (0.96 ± 0.21). C, Representative immunoblots for FAK, paxillin, and ß-actin protein. Mean OD units ± SEM from six separate experiments is shown in b. *, P < 0.01 for AdAS-FAK (0.12 ± 0.01) vs. NON (0.26 ± 0.01) and ß-gal-treated cells (0.26 ± 0.01).

 
Reduced expression of FAK does not alter abundance of IRS-1 or IRS-2, nor their insulin-stimulated phosphorylation
As shown in Fig. 2Go, A and B, the level of IRS-1 or IRS-2 tyrosine phosphorylation and protein abundance was similar among NON, ß-gal, and AdAS-FAK-transfected myotubes after insulin stimulation, suggesting that reduction of FAK by antisense methodology does not affect IRS-1 or IRS-2 protein expression nor insulin-stimulated IRS-1 or IRS-2 phosphorylation.


Figure 2
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  		FIG. 2. Effect of AdAS-FAK on abundance and phosphorylation of IRS-1 and IRS-2. Immunoblot of L6 whole cell lysate showing abundance of IRS-1 (A, upper panel) or IRS-2 (B, upper panel), and separate experiments in which IRS-1 or IRS-2 were immunoprecipitated and immunoblotted with PY99 Ab to reveal insulin-stimulated tyrosine phosphorylation of IRS-1 (A, middle panel) or IRS-2 (B, middle panel). The membrane was then reblotted with anti-IRS-1 (A, lower panel) or IRS-2 (B, lower panel) Ab.

 
Insulin fails to stimulate glycogen synthesis in L6 myotubes transfected with AdAS-FAK constructs
As shown in Fig. 3Go, in NON and ß-gal-transfected L6 myotubes, insulin stimulated a 150–200% increase in [U-14C]glucose incorporation into glycogen, which was abolished in AdAS-FAK-treated myotubes, indicating that FAK is required for insulin-stimulated glycogen synthesis. Basal levels of glycogen synthesis did not differ among the three groups. Mean values were: basal, 162 ± 31, 201 ± 48, and 195 ± 31; poststimulation, 308 ± 76, 391 ± 68, and 170 ± 48 pmol/hr/mg protein ± SEM in NON, ß-gal, and AdAS-FAK, respectively.


Figure 3
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  		FIG. 3. Effect of AdAS-FAK on glycogen synthesis. Basal and insulin-stimulated glucose incorporation into glycogen were measured in L6 myotubes. Assays were performed as described in Materials and Methods. *, P < 0.01 for AdAS-FAK (basal 194.7 ± 31.2; insulin 170.0 ± 47.8 pmol/h/mg protein) vs. NON (162.1 ± 30.5; 307.4 ± 76.4) and ß-gal-treated cells (200.9 ± 47.9; 391.0 ± 68.3) during insulin stimulation. Data are from five separate experiments.

 
Treatment of L6 myotubes with AdAS-FAK constructs decreases insulin-stimulated glucose uptake
We examined whether FAK was essential for insulin-mediated glucose uptake in skeletal muscle cells. L6 myotubes were transfected with AdAS-FAK and then incubated in serum-free medium. Subsequently, rates of hexose uptake in the basal state and after an acute (5 min) insulin challenge were determined. The results are shown in Fig. 4Go. Although basal glucose transport appeared to be higher in AdAS-FAK-treated cells, this difference was not statistically significant. In both NON and ß-gal-transfected L6 myotubes, acute insulin stimulation augmented 2-DOG uptake by 2.1-fold (basal, 30 ± 3 and 27 ± 4; insulin, 64 ± 15 and 55 ± 15 pmol/min/mg protein ± SEM, respectively). In contrast, myotubes treated with AdAS-FAK had a significantly lower incremental response to insulin, showing only a 1.2-fold increase in 2-DOG uptake (basal, 39 ± 6; insulin, 50 ± 11 pmol/min/mg protein, increment = P < 0.044 vs. NON and ß-gal), indicating that normal expression of FAK is also necessary for insulin-mediated glucose uptake in skeletal muscle cells.


Figure 4
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  		FIG. 4. Effect of AdAS-FAK on glucose uptake. 2-DOG uptake in L6 myotubes. The assay was performed as described in Materials and Methods. *, P < 0.04 for the insulin-mediated increment in AdAS-FAK (basal 38.46 ± 5.76; insulin 49.66 ± 11.35 pmol/min/mg protein) vs. NON (30.27 ± 3.38; 64.04 ± 14.82) and ß-gal-treated cells (26.50 ± 3.80; 54.73 ± 13.40). Data are from six separate experiments.

 
Treatment of L6 myoblasts overexpressing GLUT4 with AdAS-FAK constructs decreases insulin-stimulated GLUT4 glucose transporter translocation
To determine whether FAK is necessary for optimal GLUT4 movement to the cell surface membrane in response to insulin, GLUT4 glucose transporter levels in plasma membrane fractions were measured. L6 myoblasts overexpressing GLUT4 were transfected with AdAS-FAK and then incubated in serum-free medium. Subsequently, plasma membrane fractions were isolated using an adaptation of a method described by Zhou et al. (15) in the basal state and after a 30-min insulin challenge. As shown in Fig. 5A-aGo (PM), there was a marked increase in GLUT4 translocation to the plasma membrane fractions in response to insulin in untreated and ß-gal-treated L6 myoblasts overexpressing GLUT4. Data from PM were quantitated by densitometry in Fig. 5A-bGo. Meanwhile, in the light membrane, GLUT4 levels were correspondingly decreased after insulin stimulation as shown in Fig. 5A-aGo (LM). Although basal glucose uptake appeared to be higher in AdAS-FAK-treated cells, this difference was not statistically significant. Moreover, as shown in Fig. 5BGo the protein abundance of glucose transporter 1 and 3 (GLUT1 and GLUT3) in these cells was unchanged. However, insulin-stimulated incremental GLUT4 translocation was significantly impaired in myoblasts treated with AdAS-FAK. This result thus parallels insulin-mediated glucose uptake in these cells.


Figure 5
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  		FIG. 5. Effect of AdAS-FAK on translocation of GLUT4. A-a, GLUT4 levels in the light membrane (LM) and membrane fractions (PM) measured by immunoblot in L6 myotubes. The analysis was performed as described in Materials and Methods. A-b, Results from PM were derived from three separate experiments and quantitated by densitometry. *, P < 0.02 for basal vs. insulin-treated cells. NON (basal 0.15 ± 0.02; insulin 0.23 ± 0.01 OD units), ß-gal (0.15 ± 0.01; 0.23 ± 0.02), AdAS (0.15 ± 0.02; 0.18 ± 0.02). B, GLUT1 and GLUT3 levels in whole cell lysates measured by immunoblot in L6 myotubes. Results were repeated three times.

 
Treatment of L6 myotubes with AdAS-FAK causes abnormalities of actin cytoskeletal organization and focal adhesion complex formation
It has been reported that FAK associates with, and thereby influences activation of the GTP-ase, Rho. This leads to morphological changes in the actin cytoskeleton (16). In view of this, and the fact that disassembly of F-actin abolishes insulin-stimulated glucose transport in L6 myotubes (8), we further examined the morphology of the actin cytoskeleton in L6 myotubes treated with AdAS-FAK (Fig. 6Go). In NON and ß-gal-transfected myotubes, double staining with rhodamine-labeled phalloidin (red) and paxillin antibodies (green) revealed actin microfilament bundles of variable thickness spanning the cell longitudinally (F-actin) and normal distribution of focal adhesion complexes, respectively. However, double staining in cells treated with AdAS-FAK showed fewer stress fibers and exhibited a less polarized morphology with nonfilamentous bundles of actin and scarce focal adhesion complexes (Fig. 6AGo). Figure 6BGo shows the same cell cultures double stained with phalloidin and an anti-FAK397 phospho-Ab (green). In NON and ß-gal-transfected myotubes, normal formation of actin microfilaments and localization of FAK to focal adhesion complexes were observed. In cells infected with AdAS-FAK, however, there was a disruption of cortical actin structure and an absence of FAK localization to focal adhesion complexes (Fig. 6BGo, dashed arrows). To confirm that the indicated cells were indeed infected with adenovirus, additional control experiments were performed in which cells were coinfected with an adenoviral green fluorescent protein (EGFP)-construct and either Lac-Z or AdAS-FAK adenovirus and stained with rhodamine-labeled phalloidin (Fig. 6CGo). The results confirm that the abnormal cytoskeletal architecture seen in the presence of AdAS-FAK is only seen in cells staining positive for EGFP. Adenoviral EGFP expression alone or coinfection with adenoviral EGFP with Lac-Z did not alter stress fiber formation.


Figure 6
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  		FIG. 6. Effect of AdAS-FAK on cytoskeletal structure. Actin stress fiber structure and focal adhesion formation visualized by overlay images as described in Materials and Methods. A, Phalloidin-rhodamine-red conjugate was used to view filamentous actin. Antibodies against paxillin (green) revealed the localization of the protein to focal adhesion complexes. B, Actin filaments were again viewed with phalloidin-rhodamine-red conjugate but instead cells were double-stained with phospho-Tyr397-FAK (green) to reveal focal adhesion complexes. C, Cells were infected with Ad-EGFP or coinfected with both Ad-EGFP and Ad-Lac-Z or Ad-EGFP and AdAS-FAK and stained for actin with rhodamine-red-phalloidin. EGFP serves as an indicator of adenovirus infection.

 
Insulin-mediated Akt/PKB-Ser473 and Akt/PKB-Thr308 phosphorylation as well as Akt activity are impaired in L6 myotubes treated with AdAS-FAK
Previous studies have shown that early insulin signaling events do not require cytoskeletal participation (8, 17). Activation of IRS-1/PI3-kinase complexes occurs at the plasma membrane in an actin-dependent fashion (7) by recruitment of Akt/PKB to intracellular GLUT4-containing vesicles and phosphorylation of protein targets thereon, thus facilitating intracellular glucose transport (18). Therefore, using a phosphospecific Ab, we analyzed the phosphorylation state of serine 473 (Ser473) and threonine 308 (Thr308), key residues in Akt/PKB, which undergo insulin-stimulated phosphorylation (19). As shown in Fig. 7Go, A and B, there was a marked increase in phosphorylation of Ser473 and Thr308 of Akt/PKB in response to insulin in untreated or ß-gal-treated L6 myotubes. Insulin-stimulated incremental Akt/PKB Ser473 phosphorylation was significantly impaired in myotubes treated with AdAS-FAK. A similar trend was observed for Akt/PKB and Thr308 phosphorylation, which marginally failed to reach statistical significance (P < 0.06). There appeared to be a modest decrease in basal Akt/PKB Ser473 phosphorylation in the presence of AdAS-FAK, but this did not reach statistical significance. Moreover, as previously shown, basal glycogen synthesis and glucose transport were unchanged. Levels of Akt/PKB protein did not differ among the three groups. The data are shown quantitatively in Fig. 7Go, A-b and B-b. In addition, these findings are supported by measurement Akt/PKB kinase activity, as there was significant stimulation of activity. Insulin-stimulated Akt/PKB kinase activity was impaired in L6 myotubes transfected with AdAS-FAK, (P < 05) as shown in Fig. 7CGo.


Figure 7
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  		FIG. 7. Effect of AdAS-FAK on downstream insulin signaling. Immunoblot of whole cell lysate showing Akt/PKB abundance (A and B, a, upper bands) and a separate experiment in which Akt/PKB was immunoprecipitated and immunoblotted with anti-pSer473-Akt/PKB or with anti-pThr308-Akt/PKB Ab (A and B, a, middle band). The membrane was reblotted with anti-Akt/PKB Ab (A and B, a, lower band). Akt/PKB Ser473 or Thr308 phosphorylation was quantitated by densitometry and is shown in A and B, b. Results are mean ± SEM for three experiments. *, P < 0.03 for AdAS-FAK (basal 0.19 ± 0.05; insulin 0.14 ± 0.06 OD units) vs. NON (0.13 ± 0.02; 0.24 ± 0.00) and ß-gal-treated cells (0.195 ± 0.04; 0.22 ± 0.07) during insulin stimulation. Akt/PKB activity in L6 myotubes was measured as described in Materials and Methods. Results are shown in C and are mean ± SEM for five experiments (percent incremental over basal: NON 5.40 ± 1.29, ß-gal 3.04 ± 0.34, AdAS-FAK 1.98 ± 0.25, P < 0.05).

 
Insulin-stimulated GSK-3{alpha}/ß phosphorylation is impaired in L6 myotubes treated with AdAS-FAK
To determine whether the impairment of insulin stimulated glycogen synthesis observed in the presence of FAK antisense is mediated via GSK-3, insulin-mediated GSK-3{alpha}/ß phosphorylation in L6 myotubes was measured by Western immunoblot. As shown in Fig. 8Go, phosphorylation of Ser9/21 of GSK-3{alpha}/ß increased after exposure to insulin in untreated or ß-gal-treated L6 myotubes, but this effect was significantly attenuated in myotubes treated with AdAS-FAK.


Figure 8
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  		FIG. 8. Effect of AdAS-FAK on abundance and phosphorylation of GSK-{alpha}/ß. Immunoblot of whole cell lysate (30 µg/lane) showing GSK-3ß abundance (upper bands) and a separate experiment using anti-pSer9- and pSer21-GSK Ab (lower band). GSK-3 Ser9/21 phosphorylation was quantitated by densitometry and is shown in the lower panel as the mean ± SEM of four experiments (percent increment over basal: NON 5.35 ± 0.74, ß-gal 5.99 ± 1.48, AdAS-FAK 1.99 ± 0.40; *, P < 0.01).

 
Treatment of L6 myotubes with AdAS-FAK does not promote apoptosis
Several reports (20, 21, 22) have described the protective role of FAK in various models of apoptosis. To determine whether reduced expression of FAK in this system leads to apoptosis, we measured levels of caspase-3 and its mature fragment in L6 myotubes treated with AdAS-FAK. Caspase-3 is synthesized as a 32-kDa inactive precursor, which is proteolytically cleaved to produce the corresponding mature fragments when the apoptotic pathway is activated (23). Caspase-3 remained uncleaved, and no mature fragments were observed in NON, ß-gal-, or AdAS-FAK-transfected myotubes, either under basal conditions or after insulin stimulation (data not shown). Thus, the impairment of insulin action observed in the presence of reduced abundance of FAK in this system did not result from up-regulation of apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A reduced capacity for insulin to stimulate glucose uptake and glycogen synthesis in target tissues such as skeletal muscle is a defining feature of insulin resistance in obesity and type 2 diabetes (24). Skeletal muscle is a primary determinant of glycemia in vivo and defective insulin signal transduction and GLUT4 glucose transporter translocation are present in skeletal muscle from persons with type 2 diabetes (25, 26, 27, 28).

Insulin stimulates glucose transport into skeletal muscle by causing the redistribution of the insulin-responsive glucose transporter, GLUT4, from its intracellular location to cell surface membranes (29). An intact cytoskeleton is required for propagation of the insulin signaling cascade by facilitating proper intracellular distribution of signaling molecules (17, 30) and actin filaments appear to facilitate insulin stimulation of glucose transport (7). Recently, Kanzaki and Pessin (31), using the actin-depolymerizing agent cytochalasin D, or the actin monomer-binding Red Sea sponge toxin, latrunculin, demonstrated that cortical F-actin plays an important regulatory role in insulin-stimulated GLUT4 translocation in adipocytes. Similarly, Jiang et al. (32) reported that expression of mutant neural Wiskott-Aldrich syndrome protein in adipocytes attenuated insulin-mediated cortical F-actin rearrangements and GLUT4 translocation, which occurs via a PI3-kinase-independent mechanism.

Because integrins are sites of anchorage for the actin cytoskeleton, it is plausible that an interaction between integrins and growth factor signaling pathways may confer cytoskeletal dependence to growth factor signaling. Recent studies have suggested roles for integrin signaling via FAK in the regulation of cell survival (22, 33), cell proliferation (34, 35), cell spreading (36), and cell migration (34, 37, 38). In skeletal muscle cells, FAK and PI3-kinase are important components of a pathway that regulates insulin-stimulated skeletal muscle cell spreading and differentiation (39, 40). FAK regulates the arrangement of focal adhesions and the cytoskeleton, leading to a motile phenotype (2). The selective Src-PTK inhibitor, PP2, prevents not only optimal phosphorylation of FAK at tyrosine 397 (Tyr397), but also interrupts integrin-mediated rearrangement of the actin cytoskeleton and formation of focal adhesions (41). Moreover, pharmacologic inhibition of FAK kinase activity or expression of kinase-deficient FAK, mutated by substituting Phe for Tyr397, inhibits transforming growth factor ß1-induced {alpha}-actin expression, stress fiber formation and cellular hypertrophy (42). We also recently overexpressed mutant FAK constructs in HepG2 hepatoma cells and showed that both the focal adhesion targeting property and tyrosine kinase activity of FAK are essential for normal insulin-mediated stimulation of glycogen synthesis (10). Moreover, expression of IRS-1 mRNA was found to be absent in FAK knockout mouse fibroblast cells (4). These data underscore the involvement of integrins, acting via FAK, in the regulation of insulin signaling pathways.

Therefore, clarification of the role of FAK in the regulation of insulin action and its relationship with the actin cytoskeleton will increase our understanding of the molecular basis of normal insulin action and acquired insulin resistance in skeletal muscle tissue. The aim of this study was to test the hypothesis that reduction of FAK abundance by treatment of cultured skeletal muscle cells with AdAS-FAK would lead to abnormal signaling protein activation and disruption of the architecture of the actin cytoskeleton, thereby impairing insulin action.

We demonstrated that L6 myotubes treated with AdAS-FAK exhibit impaired insulin-stimulated 2-DOG uptake, GLUT4 glucose transporter translocation and glycogen synthesis, whereas basal rates were unaffected. In a previous study, using two mutant FAK constructs in HepG2 hepatocytes, we also found that basal rates of glycogen synthesis were unaffected, whereas incremental insulin-stimulated glycogen synthesis was virtually abolished (10). To clarify the mechanism of this defect, in the present study, we measured abundance and both basal and insulin-stimulated phosphorylation of IRS-1, IRS-2, Akt/PKB, and GSK-3{alpha}/ß. We found that protein abundance is unchanged, but we observed a 35% reduction in insulin-mediated Akt phosphorylation at Ser473 as well as Thr308 and also confirmed that Akt activity was impaired. IRS-1 and IRS-2 phosphorylation was unchanged in both basal and insulin-stimulated states. These results suggest that FAK regulates insulin action downstream of the IRSs, possibly via interaction with Akt/PKB and GSK-3, a key molecule in the insulin signaling cascade, as we previously reported in HepG2 cells (10). Additional support comes from the work of Tsiani et al. (43), who observed that stimulation of glucose transport by the tyrosine phosphatase inhibitors vanadate and pervanadate is independent of PI3-kinase activation, but requires an intact cytoskeleton. Similarly, Tsakiridis et al. (7) reported that translocation of GLUT4 vesicles from the intracellular storage pool to the plasma membrane is mediated by interaction with actin filaments, whereas the basal rate of glucose transport does not seem to rely on actin filament architecture (8). However, it has also been reported that integrin modulates the insulin and insulin-like growth factor-1 signaling pathways by regulating cellular IRS-1 expression and that FAK-mediated signaling to c-Jun N terminal kinase appears to be involved in this process (4), although we found no alteration in IRS-1 protein abundance in this study. Moreover, Stenbit and colleagues (44) developed transgenic mice with the GLUT4 gene disrupted. The GLUT3 isoform has not been evaluated in these animals. Also GLUT1 protein was not increased in muscle of the GLUT4 null mice (45). Similarly, we find that L6 myotubes transfected with AdAS-FAK exhibited no change in GLUT1 or GLUT3 expression. Stuart et al. (46) previously reported that fasting hyperinsulinemia in subjects with acanthosis nigricans was associated with reduced expression of GLUT3 mRNA and protein along with an increase of GLUT1 mRNA without a concomitant increase protein in skeletal muscle. In our study, transfection of L6 myotubes with AdAS FAK did not affect GLUT3 nor GLUT1 expression, possibly due to our use of normal physiologic glucose and insulin concentrations in the culture media. Similar to our findings, both human and animal studies (47, 48, 49) have shown that, under basal conditions, levels of GLUT4 were increased in subjects with diabetes or insulin resistance and that insulin failed to further increase the plasma membrane content of GLUT4. This indicates that a primary defect in the normal muscle basal glucose uptake mechanism leads to redistribution of GLUT4, which in turn blunts the increment in glucose uptake in response to insulin.

In L6 myotubes transfected with AdAS-FAK, we observed a less polarized morphology of the actin cytoskeleton with bundles of actin around the periphery of the cells, suggesting that there may be disruption of downstream signaling to the actin cytoskeleton. Organization of actin filaments is controlled by the Rho family of small GTPases, which includes Rho, Rac, and Cdc42 (50). Rho is required for an adhesion-induced increase in F-actin and Cdc42 regulates adhesion-associated Akt and Erk2 activation (51). FAK has been shown to influence Rho activation through its association with the GTPase regulator associated with FAK (GRAF), leading to changes in the actin cytoskeleton (16). Therefore, it is possible that reduced levels of FAK in antisense-treated cells may lead to changes in a FAK/Rho signaling pathway that result in the observed loss of stress fibers.

There have been several reports describing the anti-apoptotic role of FAK in various apoptosis-inducing systems. Frisch et al. (33) found that constitutively activated FAK protected MDCK cells from apoptosis consequent upon the loss of matrix contact. Furthermore, Xu et al. (21) reported that attenuation of FAK expression leads to apoptosis in tumor cells. However, Ridyard et al. (2) noted that reduced expression of FAK protein by treatment with an antisense oligonucleotide of FAK did not induce a significant increase in the rate of cell death in chick embryo epiblast cells. Similarly, we found that a reduction in the level of FAK did not increase the apoptotic activity in cells treated with antisense to FAK. However, it is possible that the low levels of FAK protein still present in the antisense-treated cells may still have been sufficient to prevent apoptosis.

In conclusion, we have shown that reduction in FAK levels in skeletal muscle leads to a defect in acute insulin-stimulated glycogen synthesis and 2-DOG uptake, with defective insulin signal transduction through the Akt/PKB pathway and an accompanying reduction in stress fiber formation and impaired insulin mediated GLUT4 translocation. This is not attributable to nonspecific alteration of cellular proteins or increased apoptosis. Thus, it appears that FAK can regulate signaling pathways at multiple levels. In skeletal muscle cells, FAK appears to be essential for normal insulin-stimulated glucose transport and glycogen synthesis, by maintenance of actin cytoskeletal integrity. These findings in skeletal muscle cells parallel our previous data obtained in liver cells (10). Liver and skeletal muscle are two important target tissues of insulin in the regulation of glucose homeostasis and our data suggest that the integrin signaling pathway may play an important role in its regulation. Studies in vivo will be necessary to confirm the physiological importance of these findings.


   Acknowledgments
 
We thank Dr. Steven Young, Dr. Jim Sinnett-Smith, and Dr. Susan Phillips for helpful advice. Also, we thank Dr. Ira Goldfine and Betty Maddux, who kindly provided supplies and technical advice on GLUT4 experiments.


   Footnotes
 
M.B.A. was supported in part by a Merit Review Award from the Veterans’ Administration Research Service and by funds from the Gonda Family Endowment.

Present address for D.I.: StemLifeLine, Inc., 1300 Industrial Road #13, San Carlos, California 94114.

Danshan Huang, M.D., has nothing to declare. Michelle Khoe, M.D., has nothing to declare. Dusko Ilic, M.D., Ph.D., has nothing to declare. Michael Bryer-Ash., M.D., has nothing to declare. The authors declare that there is no conflict of interest that would prejudice the impartiality of this work.

First Published Online March 30, 2006

Abbreviations: Ab, Antibody; AdAS-FAK, adenovirus expressing FAK antisense oligonucleotide; 2-DOG, 2-deoxy-glucose; EGFP, enhanced green fluorescent protein; F-actin, filamentous actin; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate; ß-gal, ß-galactosidase; GLUT1, glucose transporter 1; GLUT3, glucose transporter 3; GLUT4, glucose transporter 4; GSK, glycogen synthase kinase; IRS, insulin receptor substrate; NON, untransfected; PI3, phosphatidylinositol-3; PKB, protein kinase B.

Received April 1, 2005.

Accepted for publication March 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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