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Am J Physiol Endocrinol Metab 291: E152-E158, 2006. First published February 14, 2006; doi:10.1152/ajpendo.00180.2005
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Regulation of Dishevelled and beta-catenin in rat skeletal muscle: an alternative exercise-induced GSK-3beta signaling pathway

William G. Aschenbach,1,* Richard C. Ho,1,* Kei Sakamoto,1 Nobuharu Fujii,1 Yangfeng Li,1 Young-Bum Kim,2 Michael F. Hirshman,1 and Laurie J. Goodyear1

1The Research Division, Joslin Diabetes Center and Department of Medicine; and 2Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Submitted 27 April 2005 ; accepted in final form 7 February 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
beta-catenin is a multifunctional protein involved in cell-cell adhesion and the Wnt signaling pathway. beta-Catenin is activated upon its dephosphorylation, an event triggered by Dishevelled (Dvl)-mediated phosphorylation and deactivation of glycogen synthase kinase-3beta (GSK-3beta). In skeletal muscle, both insulin and exercise decrease GSK-3beta activity, and we tested the hypothesis that these two stimuli regulate beta-catenin. Immunoblotting demonstrated that Dvl, Axin, GSK-3beta, and beta-catenin proteins are expressed in rat red and white gastrocnemius muscles. Treadmill running exercise in vivo significantly decreased beta-catenin phosphorylation in both muscle types, with complete dephosphorylation being elicited by maximal exercise. beta-Catenin dephosphorylation was intensity dependent, as dephosphorylation was highly correlated with muscle glycogen depletion during exercise (r2 = 0.84, P < 0.001). beta-Catenin dephosphorylation was accompanied by increases in GSK-3beta Ser9 phosphorylation and Dvl-GSK-3beta association. In contrast to exercise, maximal insulin treatment (1 U/kg body wt) had no effect on skeletal muscle beta-catenin phosphorylation or Dvl-GSK-3beta interaction. In conclusion, exercise in vivo, but not insulin, increases the association between Dvl and GSK-3beta in skeletal muscle, an event paralleled by beta-catenin dephosphorylation.

insulin; Wnt; Akt; protein kinase C; glycogen-synthase kinase-3beta

PHYSICAL EXERCISE elicits numerous cellular and molecular adaptations in skeletal muscle. These adaptations result, in part, from alterations of transcriptional processes that influence expression of exercise-responsive genes and, ultimately, proteins that collectively serve to enhance cellular metabolism, improve functional capacity, and reduce the risk of development of chronic diseases such as type 2 diabetes. Although a great deal of research within recent years has helped elucidate some of the cellular protein networks that transduce exercise signals in skeletal muscle, the biological consequences of these networks remain largely unknown. Furthermore, many exercise-induced adaptations in skeletal muscle are likely due to activation of cellular signaling networks that have yet to be identified.

Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of many cellular processes, including glycogen synthesis, protein synthesis, and gene transcription (6). On the basis of studies where insulin was used, the primary function of GSK-3 in skeletal muscle is thought to be the regulation of glycogen metabolism via phosphorylation and deactivation of glycogen synthase (16). Activated Akt can phosphorylate and deactivate GSK-3, relieving the inhibition of glycogen synthase and resulting in net glycogen synthesis. Similar to insulin, physical exercise also increases Akt activity and decreases GSK-3{alpha} and -beta activity, changes that coincide with the activation of glycogen synthase during exercise (19, 30, 31). However, unlike insulin, inhibition of contraction-induced Akt activation with the use of wortmannin does not fully block the decrease in GSK-3 activity (31), suggesting that there is another mechanism for GSK-3 deactivation in exercising muscle. Furthermore, under certain situations, GSK-3 deactivation with muscle contraction does not mirror changes in glycogen synthase phosphorylation on GSK-3 target residues (30). Taken together, these results led us to hypothesize that there are alternative upstream mechanisms leading to GSK-3 deactivation in contracting muscle and that the primary function of GSK-3 in contracting skeletal muscle may be unrelated to glycogen metabolism.

Although GSK-3 has been implicated in the context of glycogen metabolism, it also plays a pivotal role in the regulation of the multifunctional protein beta-catenin. beta-Catenin is the mammalian homolog of the Drosophila armadillo protein (22), initially implicated in regulating embryonic development. beta-Catenin is now recognized to modulate two distinct cellular events: as a regulator of cell adhesion and as a primary component of the Wnt signaling pathway. Within the cell, beta-catenin exists as part of a large cytosolic complex containing GSK-3beta, the tumor suppressor adenomatous polyposis coli (APC), and Axin/conductin (37). In the basal state, the constitutively active GSK-3beta phosphorylates beta-catenin, marking it for degradation via the ubiquitin-proteosome pathway. As part of the traditional paradigm of Wnt signaling, the secreted glycoprotein Wnt activates a transmembrane receptor, Frizzled (Frz), which leads to activation of the intracellular protein Dishevelled (Dvl) and subsequent phosphorylation of an inhibitory serine residue (Ser9) on GSK-3beta that causes catalytic deactivation. Deactivation of GSK-3beta results in the accumulation of dephosphorylated beta-catenin (11, 23, 34), which allows beta-catenin to stabilize, accumulate in the cytoplasm, and translocate to the nucleus, where it regulates gene transcription. Although there is a paucity of data regarding beta-catenin regulation in skeletal muscle, it has been shown to regulate a host of myogenic proteins in cell culture systems (10, 13, 27, 36), some of which are known to be regulated by exercise in adult skeletal muscle (9, 26, 35). Recently, we have demonstrated in a preliminary study (29) that exercise in human skeletal muscle decreases beta-catenin phosphorylation.

The regulation of GSK-3beta, Dvl, and beta-catenin in the Wnt signaling network has been established in various cell systems, and here we demonstrate that components of this pathway are expressed in rat skeletal muscle. We found that treadmill running exercise increases the protein-protein interaction in rat skeletal muscle between Dvl and GSK-3beta, an intensity-dependent event paralleled by alterations in the phosphorylation state of beta-catenin. In contrast to exercise in vivo, insulin injection did not increase Dvl-GSK-3beta interaction or result in beta-catenin dephosphorylation. The beta-catenin system represents a novel mechanism by which contracting muscles transduce signals during physical exercise.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All antibodies used in this investigation were obtained from commercial sources. Rabbit polyclonal Axin, PKC{lambda}/{zeta}, and Dvl antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal beta-catenin, phospho-beta-catenin (Ser33/Ser37/Thr41), phospho-GSK-3{alpha}/beta (Ser21/Ser9), and phospho-Akt (Thr308) antibodies were purchased from Cell Signaling Technology (Beverly, MA); and a polyclonal GSK-3beta antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase-conjugated anti-mouse secondary antibodies were purchased from Amersham Biosciences (Piscataway, NJ), and anti-rabbit secondary antibody was from Zymed Laboratories, (San Francisco, CA).

Animals and housing. All protocols for animal use were approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and were in accordance with National Institutes of Health guidelines. Male Sprague-Dawley rats (60–80 g for in vitro studies and 175–200 g for in vivo studies) were obtained from Taconic (Germantown, NY), provided standard rodent chow and water ad libitum, and were housed under standard laboratory conditions (12:12-h light-dark cycle). Animals performed in vivo exercise under fed conditions but were fasted 12 h overnight prior to all in vitro muscle contraction experiments described below.

Treadmill exercise and in vivo insulin stimulation. Male rats (175–200 g) were familiarized with the treadmill (Quinton model 42) by running 5–10 min 2 days before the onset of experimentation. Animals were then subjected to one of two treadmill running protocols, either 60 min of steady-state exercise (22 m/min, 12% incline) or an exhaustive "ramp" protocol, as previously described (1). Briefly, the speed of the treadmill belt was kept constant at 0.7 mph, and the incline was increased by 1° every 2 min until rats could no longer remain on the belt. Immediately after exercise, rats were killed by cervical dislocation, and gastrocnemius muscles were quickly removed for separation of red (type I, 28%; type IIA, 63%; type IIB, 9%) and white (type I, 0%; type IIA, 13%; type IIB, 87%) portions (15) and snap-frozen in liquid nitrogen until subsequent processing and analyses. Nonexercised rats were also killed under basal conditions, or 10 min after being given a maximal dose of insulin by intraperitoneal injection (1 U/kg body wt).

Contraction experiments. In vitro contraction experiments were performed as previously described by our laboratory (31). Briefly, fasted rats weighing 60–80 g were killed by cervical dislocation, and soleus muscles were rapidly dissected. Both tendons were tied with silk suture, mounted on an incubation apparatus to maintain resting length, and preincubated for 20 min in 37°C oxygenated Krebs-Ringer bicarbonate buffer (117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 24.6 mM NaHCO3, pH 7.5) containing 5.5 mM glucose as an exogenous carbon source. After preincubation, muscles were transferred to a tissue support apparatus with stimulating electrodes (Harvard Apparatus, Holliston, MA) and incubated for an additional 10 min before the onset of contractions. Muscles were then stimulated for 10 min using a relatively intensive contraction protocol: train rate, 2/min; train duration, 10 s; pulse rate, 100 Hz; pulse duration, 0.1 ms at 100 V. Force production was monitored during the entire contraction protocol with an isometric force transducer (Kent Scientific, Litchfield, CT) and a chart recorder (Kipp & Zonen, Delft, The Netherlands). The in vitro contraction protocol represents an intense bout of exercise, glycogen is depleted by >50%, and in general, the contraction force by the end of the protocol is decreased to ~5–7% of initial force. The in vitro contraction protocol has been shown to maximally stimulate glucose transport and to maximally activate many exercise signaling proteins including AMP-activated protein kinase, ERK1/2, and p38. Immediately following contractions, muscles were removed from the buffer, dismounted, and snap-frozen in liquid nitrogen.

Preparation of skeletal muscle lysates. Frozen muscles were pulverized and Polytron homogenized (Brinkman Instruments) in ice-cold lysis buffer (20 mM Tris, 5 mM EDTA, 10 mM Na3PO4, 100 mM NaF, 2 mM Na3VO4, 1% Nonidet P-40, 10 µM leupeptin, 3 mM benzamidine, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5). Muscle homogenates were rotated end over end for 60 min at 4°C and centrifuged at 14,000 g for 10 min at 4°C. Protein concentrations of lysates were determined by the Bradford method with bovine serum albumin as a standard, using a kit from Bio-Rad Laboratories (Hercules, CA). Muscle lysates were frozen in liquid nitrogen and stored at –80°C until they were used for Western blotting and enzyme activity assays.

Immunoprecipitation. Whole cell lysates were prepared as described above. Skeletal muscle lysates (300 µg) were precleared with 40 µl of a 50% slurry of protein G-agarose beads (Amersham Biosciences) and incubated end over end at 4°C for 1 h. Polyclonal Dvl antibody (2 µg; Santa Cruz Biotechnology) was preincubated with 40 µl of a 50% slurry of protein G-agarose beads at 4°C for 1 h and then washed twice with lysis buffer. Precleared lysates were added to the Dvl antibody-protein G-agarose beads and incubated overnight at 4°C. Immunoprecipitates were washed four times with lysis buffer and resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with specific antibodies.

Western blot analyses. Forty to one hundred micrograms of lysate proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes (100 V for 60 min), and membranes were blocked for 60 min in Tris-buffered saline with 0.05–0.1% Tween-20 (TBST) and either 5% nonfat milk or bovine serum albumin. Proteins of interest were probed by incubating membranes in TBST containing 3–5% nonfat milk or bovine serum albumin and corresponding primary antibodies overnight on a rocker at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000) in TBST with 5% nonfat milk for 60 min at room temperature. Rat brain lysates were used for positive and internal controls. For loading controls, phosphor blots were stripped and reblotted for the specific total protein. When proteins were immunoprecipitated, that protein of interest was blotted, and it was determined that the pulldown was equal for all samples. Bands were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA), scanned, and quantitated by densitometry.

Activity assays. For determination of Akt activity, 300 µg of muscle lysate proteins were immunoprecipitated overnight at 4°C with 3 µg of Akt1/2 antibody coupled to protein G-Sepharose beads (Amersham Biosciences). Bead-immune complexes were washed with ice-cold lysis buffer, and Akt activity was determined by an in vitro kinase assay with the use of an Akt/serum- and glucocorticoid-regulated kinase peptide (PRRAATF, Upstate Biotechnology), as previously described by our laboratory (30, 33). GSK-3beta was immunoprecipitated from 500 µg of muscle lysate proteins with a polyclonal antibody (Upstate Biotechnology), and activity was determined with an in vitro kinase assay, as previously described by our laboratory (19). PKC{lambda}/{zeta} was immunoprecipitated from 500 µg of lysate protein with the use of 2 µg of a rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) coupled to protein A-sepharose beads. PKC{lambda}/{zeta} activities were determined in washed immunocomplexes, as previously described (14).

Glycogen determination. Glycogen content of frozen, pulverized muscle samples was determined by HCl hydrolysis followed by neutralization with NaOH, as previously described (18). The resulting concentration of free glucosyl residues was determined spectrophotometrically using a commercially available hexokinase-based assay kit (Sigma, St. Louis, MO).

Statistical analysis. Data are expressed as means ± SE (n = 6–8). Significance was determined a priori at P < 0.05. Data sets were checked for normality and homogeneity of variance. Student's t-test and analysis of variance with Tukey's post hoc methods were used for group comparisons.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
beta-Catenin-signaling proteins are expressed in skeletal muscle. As part of the canonical Wnt pathway, beta-catenin is sequestered by GSK-3beta, Axin, and APC in a destruction complex. To study beta-catenin regulation, we first determined that the proteins of the beta-catenin destruction complex, as well as the upstream regulator Dvl, were expressed in rat skeletal muscle. To this end, we immunoblotted lysates from red and white gastrocnemius muscle with antibodies directed against Dvl, Axin, beta-catenin, and GSK-3beta. As shown in Fig. 1, all four proteins were detected in both red and white skeletal muscle.


Figure 1
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  		Fig. 1. Representative immunoblots showing expression of beta-catenin signaling proteins in rat skeletal muscle. Red and white gastrocnemius muscles from Sprague-Dawley rats were processed, proteins (20–70 µg) were separated by SDS-PAGE (10%) and immunoblotted with anti-Dishevelled (Dvl), anti-Axin, anti-beta-catenin, and anti-glycogen synthase kinase (GSK)-3beta antibodies (shown in duplicate).

 
Regulation of GSK-3, Akt, and PKC by exercise in skeletal muscle. As shown in Fig. 2A, GSK-3 Ser9/21 phosphorylation was progressively increased from basal to steady-state submaximal and maximal exercise. This is consistent with previous data from our laboratory showing that exercise inhibits GSK-3beta catalytic activity (19, 30). Activities of two putative GSK-3 kinases, Akt and atypical PKC (PKC{lambda}/{zeta}), were also determined in muscle lysates with the use of in vitro kinase assays. Treadmill exercise increased Akt and PKC{lambda}/{zeta} activities in both red and white gastrocnemius muscle samples (Fig. 2, B and C, respectively). Additionally, the stimulation of Akt during exercise was associated with a robust phosphorylation of both Thr308 and Ser473 activation residues (data not shown).


Figure 2
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  		Fig. 2. GSK-3, Akt, and PKC{lambda}/{zeta} are regulated by exercise in vivo. GSK-3{alpha}/beta phosphorylation (A), Akt activity (B) and PKC{lambda}/{zeta} activity (C) were determined in muscle lysates obtained from rats under basal conditions after 60 min of steady-state exercise (Ex 60) or after a maximal (Max)-effort treadmill bout. p-, phosphorylated. *P < 0.05 vs. basal; #P = 0.07 vs. basal.

 
In vivo exercise regulates GSK-3beta association with Dvl. Dvl is a critical proximal signaling component in the regulation of GSK-3beta and beta-catenin in the canonical Wnt-signaling pathway. To determine whether the association between Dvl and GSK-3beta is regulated by exercise in skeletal muscle, we immunoprecipitated Dvl from red gastrocnemius muscle lysates obtained from rats under basal conditions immediately after 30- or 60-min bouts of submaximal steady-state and maximal exercise. Figure 3 shows that the association between Dvl and GSK-3beta progressively increases with exercise in a time- and intensity-dependent manner. In contrast to exercise in vivo, neither contraction of isolated muscles nor insulin treatment in vivo resulted in changes in the interaction between Dvl and GSK-3beta (Fig. 3).


Figure 3
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  		Fig. 3. Exercise increases the interaction of GSK-3beta with Dvl in skeletal muscle. Lysates (300 µg protein) from basal and exercised skeletal muscles were immunoprecipitated, using a polyclonal Dvl antibody, and immunoblotted for GSK-3beta. For comparison, muscle (–/+) contraction in vitro and (–/+) intraperitoneal injection of a maximal dose of insulin are also shown. *P < 0.001 vs. basal. #P < 0.05 vs. 30 min and 60 min exercise.

 
Exercise, but not insulin, regulates beta-catenin in an intensity-dependent manner. Maximal insulin treatment and 60 min of steady-state exercise both resulted in increases in Akt phosphorylation (Fig. 4A) and decreases in GSK-3beta activity (Fig. 4B). Despite similar trends in the regulation of GSK-3beta, only treadmill exercise resulted in significant decreases in beta-catenin Ser33/Ser37/Thr41 phosphorylation in red gastrocnemius muscle in vitro (Fig. 4C). Treadmill exercise for 30 and 60 min also decreased beta-catenin phosphorylation by approximately 36 (P < 0.06) and 39% (P < 0.05), respectively, in soleus muscle (data not shown), similar to the findings in red gastrocnemius. Steady-state exercise elicited significant reductions in beta-catenin Ser33/Ser37/Thr41 phosphorylation in red, but not white, skeletal muscle. We then hypothesized that the magnitude of beta-catenin regulation was dependent on the intensity of the treadmill exercise bout. Therefore, in addition to steady-state exercise, beta-catenin phosphorylation was also examined in muscle lysates obtained from rats immediately after an exhaustive incremental treadmill exercise protocol. Not only did maximal exercise result in beta-catenin Ser33/Ser37/Thr41 dephosphorylation in both red and white gastrocnemius muscles, but a nearly complete dephosphorylation was observed with this form of exercise (Fig. 5). Furthermore, as shown in Fig. 6, a striking correlation was observed between beta-catenin phosphorylation and muscle glycogen depletion during exercise (r2 = 0.84, P < 0.001). Because the degree of glycogen depletion is an index of exercise intensity, these results demonstrate that regulation of beta-catenin in contracting skeletal muscle is strongly related to exercise intensity.


Figure 4
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  		Fig. 4. Exercise, but not insulin, regulates beta-catenin in skeletal muscle. Rats were killed under basal conditions 10 min after being given a maximal dose of insulin by intraperitoneal injection (1 U/kg body wt) or after a 60-min submaximal exercise bout. Akt Thr308 phosphorylation [p-Akt (Thr308); A], GSK-3beta activity (B), and beta-catenin Ser37/Ser33/Thr41 phosphorylation [p-beta-catenin (Ser37/Ser33/Thr41); C] were determined in red gastrocnemius muscles. *P < 0.05 vs. basal.

 

Figure 5
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  		Fig. 5. Dephosphorylation of beta-catenin with exercise in vivo. To determine whether exercise-induced beta-catenin dephosphorylation is intensity dependent, rats performed either 60 min of steady-state exercise or an exhaustive "ramp" treadmill protocol for 60 min. To determine fiber type specificity, both red and white gastrocnemius muscles were removed and processed for determination of beta-catenin phosphorylation by SDS-PAGE and immunoblotting with an anti-phospho-beta-catenin antibody. *P < 0.05 vs. basal.

 

Figure 6
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  		Fig. 6. beta-Catenin dephosphorylation and muscle glycogen depletion during exercise are strongly related. Muscle glycogen content was determined in red (bullet) and white ({circ}) gastrocnemius samples after exercise and was strongly correlated with beta-catenin phosphorylation status, suggesting that beta-catenin dephosphorylation is related to exercise intensity.

 
Muscle contractions in vitro do not regulate beta-catenin. To determine whether the effects of exercise to regulate beta-catenin were due to systemic factors or due to events initiated within muscle fibers, we isolated muscles from rats and contracted them in vitro, using a protocol that has been used extensively in our laboratory to examine regulation of numerous intracellular signaling proteins (31). Our in vivo data suggest that beta-catenin is regulated by exercise in both red (oxidative) and white (glycolytic) gastrocnemius muscles; however, because beta-catenin dephosphorylation is more sensitive to exercise in red gastrocnemius muscle, we isolated soleus (red) muscles to examine contraction in vitro. Consistent with our observations during in vivo exercise, as well as previous reports from our laboratory (31), Akt and GSK-3beta were both regulated by contractions, as evidenced by increased phosphorylation of Akt Thr308 (Fig. 7A) and GSK-3 Ser9/21 (Fig. 7B). However, despite regulation of these putative upstream molecules, beta-catenin Ser33/Ser37/Thr41 phosphorylation was not decreased by contractions (Fig. 7C). The lack of beta-catenin dephosphorylation could not be explained by reduced levels of the protein in soleus muscle, because immunoblotting with a pan-beta-catenin antibody revealed that beta-catenin is similarly expressed in soleus, although at about 19% lower levels compared with red gastrocnemius (98.3 ± 7.7 vs. 121.6 ± 8.5 arbitrary units for soleus and red gastrocnemius, respectively).


Figure 7
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  		Fig. 7. Skeletal muscle beta-catenin is regulated by in vivo exercise but not by contractions in vitro. Isolated soleus muscles were incubated in vitro under basal conditions (–) or contracted (+) for 10 min. Basal (–) and 60-min exercise in vivo (+) red gastrocnemius samples were used for comparison. Muscle proteins (70 µg) were separated by SDS-PAGE (10%) and immunoblotted with an anti-p-Akt (Thr308) antibody (A), an anti-p-GSK-3{alpha}/beta (Ser21/Ser9) antibody (B), or an anti-p-beta-catenin (Ser37/Ser33/Thr41) antibody (C).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Muscle contractile activity and physical exercise can increase GSK-3 phosphorylation and deactivation, as well as increase Akt activity (19, 30). However, GSK-3beta deactivation during exercise is partially due to Akt-independent mechanisms (31). Dvl is known to regulate GSK-3beta activity in cell systems as one of the key components in the canonical Wnt signaling pathway (17, 39). Here, we show that exercise in vivo results in time- and intensity-dependent increases in the physical association between Dvl and GSK-3beta in gastrocnemius skeletal muscle. The interaction between Dvl and GSK-3beta with treadmill exercise was paralleled by decreases in beta-catenin phosphorylation. This effect was dependent on exercise intensity, as evidenced by a strong correlation with muscle glycogen depletion. Taken together, these observations provide evidence that components of the canonical Wnt signaling network are activated in skeletal muscle during exercise in vivo and suggest a newly described signaling pathway by which exercise could induce the expression of a variety of genes and muscle regulatory factors. These data also highlight a potential alternative pathway leading to GSK-3beta inactivation in exercising skeletal muscle.

In contrast to exercise, insulin-mediated GSK-3beta phosphorylation is Akt-dependent because wortmannin treatment completely abolishes changes in both Akt and GSK-3beta activities (31). In line with this observation, insulin treatment did not result in increases in the association between Dvl and GSK-3beta. Furthermore, insulin had no effect on beta-catenin dephosphorylation. This is consistent with studies using a variety of cell lines that show that, although Wnt and insulin both inhibit GSK-3beta kinase activity, only Wnt increases free cytosolic beta-catenin levels (5). Thus insulin regulation of GSK-3beta in skeletal muscle is likely independent of Wnt signaling, whereas exercise regulation of GSK-3beta may involve this signaling network.

We did not observe changes in Dvl-GSK-3beta interaction and beta-catenin dephosphorylation following contraction of isolated muscles in vitro despite robust changes with exercise in vivo. One explanation for this finding is that, because the association between Dvl and GSK-3beta is time- and intensity-dependent, it is likely that the 10-min contraction protocol was not sufficient to elicit activation of these signaling intermediates. Alternatively, these findings suggest that exercise regulation of this signaling system is due to systemic factors generated during the exercise bout. For example, exercise can regulate circulating IGF-I concentrations (25), muscle adrenergic receptors (3), and androgen receptor activation (32). These factors have been shown to regulate Wnt signaling and beta-catenin in cell systems in vitro and therefore represent candidates mediating systemic effects. Yet another possibility is that autocrine or paracrine factors regulate beta-catenin signaling. Although to our knowledge there have been no investigations to date, one could envision that exercise results in the secretion of Wnt glycoproteins from working muscle followed by activation of Frz receptors. This mechanism of activation would not be detected in our in vitro contraction system, because the local production of Wnt proteins operating through paracrine mechanisms would be washed out by the relatively high volume of incubation medium. Finally, for in vitro studies, small muscles are required to ensure adequate perfusion of the tissue; therefore, younger animals are used. It is possible that the differences in the regulation of beta-catenin in our in vivo and in vitro studies stem from age-related muscle biology.

Although a great deal of data, including the results of this study, support a role for GSK-3beta in Wnt signaling, the upstream events that act on GSK-3beta to subsequently lead to beta-catenin regulation are not fully understood. Several studies in cell systems have implicated two putative GSK-3 kinases, Akt (7) and atypical PKC (8, 12), in the deactivation of GSK-3beta in response to Wnt stimulation. Here, we show that exercise, like insulin, results in the activation of Akt and atypical PKC{lambda}/{zeta}; however, only exercise in vivo results in beta-catenin dephosphorylation. Therefore, activation of these kinases leading to phosphorylation and deactivation of GSK-3beta alone are not sufficient to liberate beta-catenin in skeletal muscle. Our observation that exercise, but not insulin, results in significant increases in the association between Dvl and GSK-3beta suggests that this interaction is necessary in the regulation of beta-catenin in skeletal muscle.

Although our understanding of how physical exercise regulates muscle remodeling has advanced significantly, the signaling mechanisms underlying changes in gene expression and plasticity in skeletal muscle are not well understood. Although the involvement of GSK-3 in glycogen metabolism has been studied extensively, our recent data suggest that it may play a marginal role in this process during exercise (1, 30). Multiple investigations have shown that GSK-3beta acts as a negative regulator of hypertrophy in C2C12 myocytes (28, 38) and in cardiomyocytes following surgically-induced pressure overload (2). Although these studies underscore the role of GSK-3beta in various muscle plastic responses, the distal targets of GSK-3beta action remain unclear. Nuclear accumulation of beta-catenin results in increases in the formation of complexes with lymphoid enhancer factors (LEF) and other T cell factor (TCF) family of transcription factors to affect the transcription of target genes (4, 20), representing an important component in skeletal muscle myogenesis (24) and muscle regeneration (21). Our data show that beta-catenin is regulated in an intensity-dependent manner, with the greatest change in beta-catenin dephosphorylation occurring in response to maximal exercise. Therefore, signaling through the beta-catenin pathway may be related to the degree of muscle damage or injury, which may provide the stimulus to recruit satellite cells and induce myogenesis. In cell systems, beta-catenin has been shown to regulate several myogenic proteins, such as MyoD, myogenin, and cyclin D1 (26). Interestingly, many of these same genes are also regulated in response to exercise in skeletal muscle. Thus exercise-induced inhibition of GSK-3beta and subsequent liberation of beta-catenin may turn out to play a central role in skeletal muscle remodeling, and this possibility is presently under investigation.

In conclusion, we show that key components of the canonical Wnt signaling pathway, including Dvl, GSK-3beta, and beta-catenin, are expressed in rat skeletal muscle and are regulated by exercise in vivo. The dephosphorylation of the multifunctional protein beta-catenin may require Dvl-mediated GSK-3beta inhibition. This modification of beta-catenin is known to increase its stability, promoting nuclear translocation and LEF/TCF-mediated gene transcription. Thus activation of the beta-catenin cascade may represent a novel exercise-induced signaling mechanism underlying skeletal muscle adaptations.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health grants R01-AR-42238 and R01-DK-68626 (L. J. Goodyear), an F32-DK-59769 Individual Kirschstein National Research Service Award (W. G. Aschenbach), and an F32-AR-049662 Individual Kirschstein National Research Service Award (R. C. Ho).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. J. Goodyear, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 (e-mail: laurie.goodyear{at}joslin.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* These authors contributed equally. Back


   REFERENCES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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 REFERENCES
 

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