Calmodulin-Binding Domain of AS160 Regulates Contraction- but Not Insulin-Stimulated Glucose Uptake in Skeletal Muscle

Total AS160 was detected using anti-AS160 (Upstate), while AS160 phosphorylation was detected with a PAS antibody (Cell Signaling Technology) (10,11,16,17,19,20,22). A polyclonal anti-myc antibody (CST) was used to immunoprecipitate transfected AS160. Phosphorylation of AMPK-Thr¹⁷² (Biosource International) was used to evaluate muscle contraction efficacy. Horseradish peroxidase–conjugated anti-rabbit antibody (Amersham Biosciences) was used to bind and visually detect all primary antibodies.

Protocols for animal use were reviewed and approved by the Joslin Diabetes Center Institutional Animal Care and Use Committee and were in accordance with National Institutes of Health guidelines. Female ICR mice aged 8 weeks were purchased from Taconic (Hudson, NY). All mice were housed with a 12-h:12-h light:dark cycle and fed standard laboratory chow and water ad libitum. Mice were fasted overnight for 10 h before the morning of the experiment.

Human wild-type AS160 cDNA and four distinct mutant AS160 cDNA constructs have been characterized in 3T3-L1 cells as previously described (online appendix [available at http://dx.doi.org/10.2337/db07-0681]) (15,19). For in vivo gene transfer, AS160 cDNA was first subcloned into pCAGGS plasmids as previously described (online appendix) (22). Novel AS160 mutants were generated using the QuikChange XL kit from Stratagene (La Jolla, CA) and verified for accuracy using the Brigham and Women’s Hospital DNA sequencing lab (Boston, MA).

Purified AS160 plasmids were directly injected into mouse tibialis anterior muscles by a protocol described by Aihara and Miyazaki (33) and modified in our laboratory (7,22,34). For glucose bolus studies, individual mice were transfected with different cDNA constructs in each tibialis anterior. Seven days later, mice were anesthetized with 90 mg/kg pentobarbital sodium and administered saline or 20% glucose (1.0 g glucose/kg body wt) through the retroorbital sinus (online appendix). For contraction studies, mice were transfected with identical cDNA constructs in each tibialis anterior. Seven days after DNA injection, mice were anesthetized with intraperitoneal administration of 90 mg/kg body wt pentobarbital sodium. Peroneal nerves from both legs were surgically exposed for electrode placement and remained intact throughout the experiment. While one leg was left unstimulated (basal/sham), the other leg was subjected to electrical stimulation for 15 min of contractions (22).

Baseline blood samples were collected from the tail vein before intravenous delivery of [³H]-2-deoxyglucose. This tracer was combined with unlabeled glucose or saline in glucose injection experiments and occurred simultaneously with the onset of in situ peroneal nerve stimulation (as a saline solution) in contraction studies. For all treatments, blood samples were taken from the tail vein at 5, 10, 15, 25, 35, and 45 min postinjection for the determination of blood glucose and [³H]-2-deoxyglucose–specific activity. After collection of the final blood sample, animals were killed and tibialis anterior muscles removed and frozen in liquid nitrogen. Accumulation of [³H]-2-deoxyglucose in pulverized tissue was determined via a precipitation protocol adapted from Ferre et al. (35) using barium-hydroxide/zinc-sulfate and perchloric acid (22). Small amounts of the muscle were set aside for immunoblots.

Frozen muscle tissue was homogenized with a polytron (Brinkman Instruments) in chilled lysis buffer and processed for glucose uptake as previously described (22). Equal amounts of isolated skeletal muscle proteins (40–50 μg) were resolved by SDS-PAGE (36) for Western blot analysis (37). Antibody-bound proteins were visualized using enhanced chemiluminescence (Amersham Biosciences). Protein bands were scanned by ImageScanner (Amersham Biosciences) and quantitated by densitometry (Fluorchem 2.0; Alpha Innotech, San Leandro, CA).

Glycogen content of frozen, pulverized muscle was determined by HCl hydrolysis followed by NaOH neutralization. Resultant free glucosyl concentration was determined spectrophotometrically using a hexokinase-based assay kit (Sigma, St. Louis, MO).

Protein lysates (3–5 mg/ml) were placed in low-binding centrifuge tubes containing 1 μg anti-myc antibody (CST) and rotated end over end for 16 h (4°C). Washed protein G beads (20 μl) were then added and incubated for 2 h (4°C). Beads were washed three times in 500-1,000 μl lysis buffer (22). The pellet was finally aspirated, and 5 μl of 1 μg/μl BSA was added with 30 μl 4× Laemmli’s buffer and heated at 80°C for 3 min. Purified anti-myc–derived precipitates were used to determine calmodulin association with AS160 via immunoblotting.

Immunoprecipitates of AS160 were separated via SDS-PAGE and transferred to nitrocellulose. Membranes were blocked in Buffer-A (32) with 4% BSA and 0.5 mmol/l CaCl₂ for 2 h and then treated with 100 ng/ml biotinylated calmodulin (no. 208697; Calbiochem) in Buffer-A for 2 h. After incubation with calmodulin, membranes were washed in Buffer-A containing 0.05% Tween-20 (5 × 15 min) followed by incubation with streptavidin horseradish peroxidase (1:5,000) in Buffer-A for 1 h. Membranes were washed, and calmodulin association was detected using enhanced chemiluminescence. To determine total AS160 protein, membranes were subsequently stripped in Buffer-A containing EGTA (5 mmol/l) for 2 h and then incubated in the presence of anti-AS160 as previously described (10,22).

Data are expressed as means ± SE. Statistical analyses were performed using one- and two-way ANOVA. When ANOVA revealed significant differences, Tukey’s post hoc test for multiple comparisons was performed. P values <0.05 were considered statistically significant.

A previous study using recombinant AS160 and human AS160 immunoprecipitated from 3T3-L1 adipocytes showed that wild-type AS160, but not a CBD mutant, associates with calmodulin in the presence of Ca²⁺ (32). Here, we studied AS160 function by using an in vivo electroporation technique to express wild-type and CBD-mutant AS160 into the tibialis anterior muscle in mice. After 7 days, both wild-type and mutant AS160 expression was increased by over 14-fold compared with that in empty-vector controls (online appendix Fig. S1). Because of the differences in plasmid vehicle, transfection method, tissue type, and overall magnitude of expression between this system and the previous report in adipocytes (32), we first determined whether AS160 transfected and expressed in skeletal muscle could bind calmodulin.

Wild-type and CBD-mutant AS160 were immunoprecipitated from skeletal muscle lysates using the NH₂-terminal myc-epitope tag, separated via SDS-PAGE, and transferred to nitrocellulose. Membranes were then incubated with biotinylated calmodulin with 0.5 mmol/l CaCl₂. While wild-type AS160 strongly associated with biotinylated calmodulin, the CBD-mutant AS160 did not (Fig. 1A). Addition of EGTA fully prevented binding by wild-type AS160 (data not shown). Subsequently, membranes were stripped with EGTA and total AS160 expression assessed to account for differences in protein loading (Fig. 1B). These results demonstrate that wild-type AS160 derived from skeletal muscle transfections maintains the capacity to bind calmodulin. Furthermore, the point mutations in CBD-mutant AS160 cDNA were faithfully translated from gene to protein and prevented normal association with calmodulin.

We have previously shown that AS160 regulates insulin-stimulated glucose uptake in skeletal muscle tissue (22). Phosphorylation on key PAS motifs appears to be required to disengage the inhibitory effect of the Rab-GAP domain on glucose uptake, a mechanism also observed in various cell models (13–15). Although the initial characterization of the AS160 CBD in adipocytes showed that mutation of this region had no effect on insulin-stimulated GLUT4 translocation (32), there are currently no reports examining a role for the AS160 CBD on insulin-stimulated glucose uptake in adult skeletal muscle. Therefore, we administered intravenous glucose into fasted mice to induce hyperglycemia and an associated physiologic insulin response (22), while glucose uptake into injected muscles was measured using a ³H-2-deoxyglucose tracer in vivo. Since there were no significant differences in blood glucose values between transfected animals, glucose curves represent data from all electroporated mice (Fig. 2A). In empty-vector control muscles, endogenous PAS phosphorylation at 160 kDa was significantly increased in response to the glucose bolus (Fig. 2B). Both wild-type–and CBD-mutant–expressing muscles exhibited twofold increases in basal AS160 phosphorylation, and these values were similarly increased by intravenous glucose. The effects of wild-type and CBD-mutant AS160 on skeletal muscle glucose uptake are shown in Fig. 2C. There were no differences between empty-vector controls or muscles expressing wild-type or CBD-mutant AS160 in either basal or insulin-stimulated glucose uptake. Collectively, these data indicate that expression of the CBD-mutant AS160 does not alter insulin-stimulated phosphorylation of AS160 at PAS motifs or glucose uptake in vivo in adult skeletal muscle.

Increases in intracellular Ca²⁺ levels are a fundamental component of muscle contraction, and this rise in Ca²⁺ levels allows for the association of Ca²⁺ with calmodulin. Although AS160 has a functional CBD, it is not known whether this region regulates AS160 PAS phosphorylation events, AS160 Rab-GAP function, and/or contraction-stimulated glucose uptake.

To explore the effects of CBD-mutant AS160 on AS160 PAS phosphorylation and glucose uptake, mouse tibialis anterior muscles were transfected with wild-type and CBD-mutant AS160 and then stimulated to contract for 15 min (Fig. 3). Phosphorylation at PAS motifs was significantly increased by contraction in empty-vector control muscles and, despite their higher basal levels, further increased by contraction in wild-type and CBD-mutant AS160 transfected muscles (Fig. 3A). AMPK Thr¹⁷² phosphorylation was also increased similarly in control and transfected muscles in response to contraction (online appendix Fig. S2).

Despite a similar ability to increase AS160 PAS phosphorylation (Fig. 3A), muscles overexpressing CBD-mutant AS160 exhibited significant impairments in contraction-stimulated glucose uptake compared with that in empty-vector controls (∼45%) and wild-type AS160 (∼26%) (Fig. 3B). The reduction in contraction-stimulated glucose uptake due to wild-type AS160 overexpression is similar in magnitude to our previous findings in skeletal muscle (22). There were no differences observed between basal (sham) glucose uptake values among the various conditions. Blood glucose levels did not differ between mice injected with different constructs (online appendix Fig. S3). In addition, there were no alterations in glycogen metabolism pre- and postcontraction due to transfection with CBD-mutant AS160 (Fig. 3C). These results suggest that the AS160 CBD is essential for full contraction-stimulated glucose uptake in skeletal muscle and that this AS160-mediated inhibition of glucose uptake can occur independently of PAS phosphorylation status.

We have previously shown that a PAS (4P) mutant form of AS160 also inhibits contraction-stimulated glucose uptake in skeletal muscle (22). To determine whether expression of AS160 harboring both the CBD and 4P mutations inhibits contraction-stimulated glucose uptake in an additive manner, we generated a double-mutant (CBD + 4P) AS160 using site-directed mutagenesis. Mouse tibialis anterior muscles were then transfected with either 4P-mutant AS160, CBD-mutant AS160, or CBD + 4P–mutant AS160, and glucose uptake was subsequently evaluated following in situ contraction (Fig. 3D). Compared with empty-vector controls, each AS160 mutant clearly inhibited contraction-stimulated glucose uptake (47, 41, and 43% for 4P-mutant, CBD-mutant, and CBD + 4P–mutant AS160, respectively). No significant differences were observed between these groups, implying that glucose uptake is not further decreased by concomitant CBD + 4P mutations on AS160. Therefore, AS160 inhibitory action on glucose uptake may be maximally achieved by either the 4P or CBD mutations alone.

The AS160 CBD lies adjacent to the Rab-GAP domain. Therefore, it is possible that calmodulin association in this region physically hinders AS160 interaction with its target Rab(s) during contraction in skeletal muscle, which may then permit enhanced glucose uptake. To determine whether the inhibition in contraction-stimulated glucose uptake exerted by CBD-mutant AS160 was mediated by the Rab-GAP domain, we generated a double-mutant form of AS160 that contained point mutations in both the CBD and GAP regions of the protein. This resultant double-mutant CBD + R/K plasmid, as well as single CBD-mutant AS160 and empty pCAGGS control DNA, was then injected and electroporated into mouse skeletal muscle and examined following in situ contraction (Fig. 4). There were no significant differences in AS160 expression (online appendix Fig. S1) or PAS phosphorylation under basal or contraction conditions between CBD- and CBD + R/K–mutant AS160 (Fig. 4A).

Figure 4B shows that transfection with the double-mutant AS160 (CBD + R/K) reversed the CBD-mutant–induced decreases in glucose uptake. In fact, there was actually a significant increase (18%) in glucose uptake in muscles expressing the CBD + R/K–mutant AS160 compared with that in controls despite similar levels of AMPK phosphorylation (data not shown). Basal glucose uptake was also increased (61%) in muscles expressing CBD + R/K–mutant AS160 compared with that in control muscles and muscles expressing CBD-mutant AS160. These results are consistent with previous observations using R/K-mutant AS160 (22) and suggest that R/K mutants compete with endogenous AS160 at sites where the latter acts on Rabs and thus leads to Rab-GTP even in the absence of stimulus. Taken together, our findings indicate that the AS160 Rab-GAP domain acts as a restraint on basal and contraction-stimulated glucose uptake in vivo. Furthermore, an intact AS160 CBD appears to regulate AS160 GAP activity and is required for normal contraction-stimulated glucose uptake in mouse skeletal muscle.

Despite intensive research, the complete signal transduction networks mediating insulin- and contraction-stimulated glucose uptake in skeletal muscle have remained elusive. Both insulin and contraction clearly increase GLUT4 translocation; however, each stimulus utilizes independent proximal signaling cascades to achieve this effect. Our lab and others recently identified the Rab-GAP AS160 as a point of convergence for the distinct upstream signaling pathways activated by insulin and contraction (10,11,14,17). Furthermore, AS160 phosphorylation at critical PAS motifs appears to be necessary for full insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle (22).

Here, we explored the functional significance of the AS160 CBD on insulin- and contraction-stimulated glucose uptake. Using an in vivo gene transfer technique (7,22,34,38), we overexpressed wild-type and CBD-mutant AS160 constructs in adult mouse skeletal muscle. Our results indicate the following: 1) CBD-mutant AS160 inhibits contraction- but not insulin-stimulated glucose uptake, 2) inhibited contraction-stimulated glucose uptake by the CBD-mutant AS160 is restored when the AS160 Rab-GAP domain is simultaneously mutated, and 3) regulation of glucose metabolism by AS160 can occur through PAS-independent signaling mechanisms in skeletal muscle. Intriguingly, this reveals that although AS160 is indeed a convergence molecule for insulin and contraction signals, its regulation of glucose uptake is differentially manipulated in response to each stimulus.

AS160 was initially shown to contain a CBD exhibiting sequence homology with the Drosophila protein Pollux, possessing strong affinity for Ca²⁺/calmodulin (32). Expression of mutant AS160 incapable of binding calmodulin did not disrupt GLUT4 translocation in insulin-treated 3T3-L1 adipocytes. In the present study, we verified that wild-type AS160 strongly associated with calmodulin in the presence of Ca²⁺ when expressed in adult skeletal muscle. Conversely, this interaction was completely abolished by mutation of the AS160 CBD. It was not possible to capture and/or preserve evidence of calmodulin-AS160 interaction in vivo because of the fleeting nature of calcium transients in skeletal muscle. However, our biotinylated calmodulin binding experiments ex vivo coupled with the functional studies on glucose uptake in vivo coordinately show that these AS160-calmodulin interactions are likely to occur in vivo as well.

We found that expression of CBD-mutant AS160 did not alter insulin-stimulated AS160 PAS phosphorylation or glucose uptake in skeletal muscle. Note that since glucose injections caused a three- to fourfold increase in plasma glucose, the glucose uptake values should be interpreted in the context of stimulation by hyperglycemia and hyperinsulinemia rather than exclusively as insulin-stimulated glucose uptake. These results are consistent with the only previous work on the AS160 CBD in adipocytes (32) and suggest that calmodulin association with AS160 is not required for insulin signaling to glucose uptake. Some studies have reported an important role for Ca²⁺/calmodulin on insulin-stimulated glucose metabolism in adipocytes (39), L6 cells (21), and skeletal muscle tissue (40). However, these insulin-stimulated increases in Ca²⁺ are confined to a local microenvironment just below the plasma membrane (40) and likely would not regulate AS160 and/or GLUT4 translocation from deeper intracellular storage pools. Indeed, there are multiple nodes of insulin action on GLUT4 trafficking, including translocation, recruitment and/or docking, and fusion of GLUT4 to the plasma membrane (41,42). AS160 has been shown to regulate the rate at which GLUT4 vesicles dock at the plasma membrane, but its role in regulating fusion has not yet been directly determined (41,42). Therefore, local Ca²⁺/calmodulin activity at or about the plasma membrane might regulate an insulin-stimulated fusion of docked GLUT4 vesicles that did not involve AS160 (42).

In contrast, we show that contraction-stimulated glucose uptake is significantly inhibited by expression of CBD-mutant AS160. This inhibitory effect is not associated with deficient AS160 PAS phosphorylation and therefore represents a novel means of contraction-regulated AS160 signaling to glucose uptake. Interestingly, contraction in this system induces robust increases in glucose uptake due to increases in blood flow (22), which may have diluted some of the inhibition observed. Ca²⁺ transients are intrinsic to the mechanism of contraction in skeletal muscle, and there is strong evidence that Ca²⁺-encoded signals have metabolic implications as well (6,7,21). Here, we show that binding of calmodulin to AS160, in addition to PAS phosphorylation, is required for full contraction-stimulated glucose uptake in adult skeletal muscle. These two mechanisms of regulation have seemingly mutual effects, as concomitant mutation of AS160 at PAS sites and the CBD did not exert an additive inhibition on glucose uptake (Fig. 3C). It is noteworthy that AS160 mutated at PAS sites and/or the Rab-GAP domain strongly associates with calmodulin unless the CBD is mutated (online appendix Fig. S4), suggesting that these mutations do not impair the ability of AS160 to bind calmodulin.

AS160 is currently thought to be a restraint of GLUT4 translocation and glucose uptake in adipocytes, muscle cells, and skeletal muscle tissue (12,43). Multiple stimuli induce phosphorylation of AS160 (10,13–19), which removes the suppressive functions of the Rab-GAP domain and allows for normal glucose uptake (14,15,22). This study confirms that AS160 phosphorylation is necessary for full insulin-stimulated glucose uptake in mouse skeletal muscle. In contrast, based on these results and our previous study, contraction appears to require both phosphorylation and calmodulin-binding events for full stimulation of glucose uptake. It is unlikely that our results are artifactual or due to nonspecific effects because insulin-stimulated glucose uptake is normal in muscles expressing CBD-mutant AS160. Moreover, contraction-stimulated glucose uptake is completely restored when muscles express a double mutant containing disrupted CBD and GAP domains (CBD + R/K).

The AS160 Rab-GAP domain exhibits activity toward multiple Rabs in adipocytes (44). Phosphorylation of AS160 may sufficiently hinder its GAP activity for specific Rabs involved in the insulin signaling pathway (45). However, to disable AS160 GAP activity toward specific contraction-regulated Rabs, both AS160 phosphorylation and association with calmodulin may be required. The AS160 CBD lies adjacent to the GAP domain (32), making it possible that binding of calmodulin physically interferes and/or competes with AS160-targeted Rabs. These Rabs would therefore predominantly exist in their GTP-bound form and be free to mobilize GLUT4 vesicles for translocation to the cell surface. Reports have described different intracellular pools of GLUT4 mobilized by insulin and contraction (46,47). If true, it is tempting to consider that these respective pools may be regulated by distinct Rabs, especially since AS160 interacts with different Rabs in muscle (45) and fat (48) cells. Whereas insulin-sensitive GLUT4 pools may be adequately liberated by AS160 phosphorylation alone, contraction-sensitive pools would require both phosphorylation and an intact CBD to facilitate GLUT4 translocation.

We must also consider the possibility that AS160 phosphorylation is necessary but not sufficient by itself for full insulin-stimulated glucose uptake. For example, AS160 association with phospho-14–3-3 was shown to be necessary for GLUT4 translocation triggered by insulin in adipocytes (49). Whereas calmodulin may function as the contraction-regulated molecular inhibitor of AS160 GAP activity, 14–3-3 proteins may exert similar functions on insulin-stimulated glucose uptake in skeletal muscle. In this scenario, both insulin and contraction require AS160 PAS phosphorylation and an additional signaling input to fully ablate AS160 Rab-GAP function (Fig. 5). Future studies are warranted to elucidate the potential role of 14–3-3 proteins on insulin- and contraction-stimulated glucose uptake.

In conclusion, we have identified the AS160 CBD as a novel regulator of contraction-stimulated glucose uptake in adult skeletal muscle. Expression of CBD-mutant AS160 inhibited contraction- but not insulin-stimulated glucose uptake, an effect reversed by AS160 coexpressing CBD and Rab-GAP domain mutations. From these data as well as previous reports, an emerging model for AS160 function and regulation of glucose uptake in skeletal muscle is presented in Fig. 5. Clearly, both insulin and contraction signals converge on AS160 to regulate glucose uptake, but our findings reveal that contraction additionally requires the presence of the AS160 CBD for full stimulation of glucose uptake in skeletal muscle.

This research was supported by National Institutes of Health grants (AR42238 and AR45670 to L.J.G), Individual Kirschstein National Research Service awards (F32 DK075851 to E.B.T. and F32AR051663 to C.A.W.), and Institutional Predoctoral Fellowship T32 (Penn State University Graduate Program in Physiology, State Park, PA, and Joslin Diabetes Center, Boston, MA [to H.F.K.]).

Special thanks to Dr. Gustav Lienhard (Dartmouth Medical School) for the AS160 cDNA constructs and mutagenesis protocols, as well as his extensive helpful discussions, and to Dr. J. Miyazaki (Osaka University, Osaka, Japan) for donation of the pCAGGS expression vector.