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Am J Physiol Endocrinol Metab 291: E1031-E1037, 2006. First published June 27, 2006; doi:10.1152/ajpendo.00204.2006
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Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle

Kei Sakamoto,* David E. Arnolds,* Nobuharu Fujii, Henning F. Kramer, Michael F. Hirshman, and Laurie J. Goodyear

The Research Division, Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts

Submitted 27 April 2006 ; accepted in final form 7 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr308 or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser9 phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr308 phosphorylation and GSK-3{alpha} Ser21 phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3{alpha} Ser21 phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.

exercise; glycogen; glycogen synthase kinase-3

INSULIN AND CONTRACTION are the major physiological stimuli that elicit glucose uptake and glycogen synthesis in skeletal muscle. Although insulin and contraction both cause the translocation of GLUT4 to the cell membrane to stimulate glucose uptake, they do so via distinct signaling pathways (16, 32). The insulin signaling pathway has been relatively well characterized and involves the rapid phosphorylation of the insulin receptor and insulin receptor substrate (IRS) proteins on tyrosine residues, followed by the activation of phosphatidylinositol 3-kinase (PI3K) (41). The essential role for PI3K in insulin-stimulated glucose uptake has been demonstrated through pharmacological approaches that utilize PI3K inhibitors such as wortmannin as well as through genetic approaches involving the overexpression of dominant-negative mutants of PI3K (38). In contrast to the insulin-stimulated pathway, contraction does not stimulate tyrosine phosphorylation of the insulin receptor or IRS-1 (15, 43), and wortmannin does not affect contraction-stimulated glucose uptake (2, 23, 25).

Akt, also known as protein kinase B (PKB), is a serine/threonine kinase that exists in three isoforms (Akt1/PKB{alpha}, Akt2/PKBbeta, and Akt3/PKB{gamma}), all of which are expressed in skeletal muscle (45). In addition to being activated by insulin, Akt is activated by a wide variety of growth factors in a PI3K-dependent manner (44). After the activation of PI3K, phosphatidylinositol 3,4,5-triphosphate (PIP3) is produced from phosphatidylinositol 4,5-bisphosphate (PIP2) and results in the recruitment of phosphoinositide-dependent kinase-1 (PDK1) and Akt to the plasma membrane. Akt is activated through phosphorylation on two regulatory sites, Thr308 in the activation loop and Ser473 in the hydrophobic COOH-terminal regulatory domain. PDK1 phosphorylates Akt at Thr308 (44), while recent data suggest that the mammalian target of rapamycin (mTOR)-rictor complex is the Akt Ser473 kinase (18, 37). On its activation, Akt phosphorylates numerous downstream substrates, including glycogen synthase kinase-3 (GSK-3), forkhead transcription factors, tuberous sclerosis complex (TSC)2, Bad, IRS-1, and AS160 (21, 22, 44). Thus, through its diverse array of substrates, Akt has been implicated as a key molecular mediator of such diverse cellular processes as growth, survival, transcription, and glucose/glycogen metabolism.

Despite their close sequence homology, knockout (KO) analysis has revealed distinct roles for each of the three Akt isoforms. Akt1 is essential for normal growth (4, 6), and Akt3 is essential for the development of normal brain size (12), whereas Akt2 is specifically involved in the maintenance of glucose homeostasis (5, 14, 27). Akt2 KO mice are characterized by insulin resistance involving fed-state hyperglycemia and hyperinsulinemia, beta-cell hypertrophy, and defects in insulin action in the liver and skeletal muscle (5). Importantly, isolated skeletal muscles from Akt2 KO mice have been shown to have marked decreases in insulin-stimulated glucose transport (5, 14, 27), clearly establishing that Akt2 is an essential mediator of insulin-stimulated glucose uptake in skeletal muscle.

Although early studies suggested that Akt was not activated by either contraction (2, 25, 39) or physical exercise (26, 46, 48), more recent data from our laboratory and those of others have convincingly shown that Akt is activated by exercise and contraction in both rodent (3, 28, 30, 34, 36) and human skeletal muscle (1, 7, 9, 33, 47). While intensity, time course, and fiber type composition all appear to influence the degree of Akt phosphorylation and activation with exercise, and thus may explain the disparity in findings concerning the activation of Akt with exercise and contraction, it is also possible that the recent positive findings concerning the activation of Akt are due to the availability of improved detection reagents that allow for the consistent resolution of the modest changes in Akt phosphorylation and activation that occur with exercise and contraction in skeletal muscle.

Given that the activation of Akt by insulin clearly stimulates glucose uptake and glycogen synthesis, and that exercise activates Akt, we set out to determine whether Akt2, the Akt isoform specifically linked to glucose homeostasis, is essential for exercise-stimulated glucose uptake and glycogen synthesis. We report here that, although Akt2 is not essential for the exercise-induced increases in glucose uptake and glycogen synthase activity, it is necessary for the normal contraction-induced phosphorylation of GSK-3{alpha}, pointing to a role for Akt2 in the response to contraction in skeletal muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. All antibodies used in this study were obtained from commercial sources. Phospho-Akt Thr308 and phospho-GSK-3{alpha}/beta Ser21/9 antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Phospho-glycogen synthase Ser645/649/653/657 antibody was obtained from Calbiochem (San Diego, CA). Phospho-AMP-activated protein kinase (phospho-AMPK) Thr172 antibody was obtained from Biosource (Carlsbad, CA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was obtained from Amersham Bioscience (Piscataway, NJ).

Animals. Protocols for animal use were reviewed and approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and were in accordance with National Institutes of Health guidelines. Akt2+/– heterozygous mice on C57BL/6N background (5) were generously donated by Dr. Morris Birnbaum (Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA). These mice were mated to produce littermates that were homozygous for intact Akt2 allele (wildtype control) and heterozygous and homozygous for the null Akt2 allele (KO). All mice were housed on a 12:12-h light-dark cycle and fed standard laboratory chow and water ad libitum. Twelve- to fifteen-week-old mice were used in all experiments.

Treadmill exercise. Mice were familiarized with the treadmill (Quinton model no. 42) over 3 separate days before the experiment. During the familiarization protocol, mice ran for 10 min at 0.4 (day 1), 0.6 (day 2), and 0.8 (day 3) mph, with 0% grade. Three days later, mice were randomly assigned to basal or exercise groups. Exercised animals performed 60 min of exercise at 0.8 mph and a 15% grade, whereas basal animals remained sedentary. Mice were immediately killed by cervical dislocation at the end of the exercise protocol, and soleus muscles were rapidly dissected and either assayed for glucose transport or snap-frozen in liquid nitrogen and assayed for glycogen content.

Measurement of glucose transport in isolated muscle. Exercised or rested mice were killed by cervical dislocation, and soleus muscles were rapidly dissected, tied with surgical sutures, and mounted on an in vitro incubation apparatus as described (36). Isolated muscles were incubated for 15 min in Krebs-Ringer bicarbonate buffer (KRB; 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24.6 mM NaHCO3, pH 7.4) containing 2 mM pyruvate in the presence or absence of 50 mU/ml insulin at 37°C. After a 15-min incubation period, muscles were quickly transferred to KRB buffer containing 1 mM 2-deoxy-[1,2,3H]glucose (1.5 µCi/ml) and 7 mM [14C]mannitol (0.45 µCi/ml) (NEN, Boston, MA) and incubated at 30°C for 10 min. Insulin was present in the transport media if present during incubation. Both the KRB and transport buffer were continuously gassed with 95% O2-5% CO2. Glucose transport was terminated, and muscles were processed for the measurement of 2-deoxy-[1,2,3H]glucose uptake as described previously (17).

Glycogen determination. Glycogen content of frozen, pulverized muscle was determined by HCl hydrolysis, followed by neutralization with NaOH, as described (24). The resulting concentration of free glucosyl residues was determined spectrophotometrically using a commercially available hexokinase-based assay kit (Sigma, St. Louis, MO).

In situ muscle contraction. Mice were anesthetized with pentobarbital sodium (90 mg/kg body wt, ip), and hindlimb muscles were contracted in situ by electrical stimulation of peroneal or sciatic nerves, as described (36). One leg of each animal was stimulated to contract for 5 or 15 min, while the contralateral leg served as a sham-operated control. Mice were killed by cervical dislocation at the cessation of electrical stimulation, and the tibialis anterior muscle was rapidly dissected and frozen in liquid nitrogen.

Preparation of skeletal muscle tissue lysates. Muscles were pulverized at the temperature of liquid nitrogen and then homogenized with a Polytron (Brinkman Instruments) on ice in lysis buffer (20 mM Tris, pH 7.5, 5 mM EDTA, 10 mM Na3PO4, 100 mM NaF, 2 mM NaVO4, 1% Nonidet P-40, 10 µM leupeptin, 3 mM benzamidine, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Homogenates were rotated end over end for 1 h at 4°C and then centrifuged at 14,000 g for 10 min at 4°C. The supernatants were collected, and the protein concentrations were determined by the Bradford method using a dye reagent from Bio-Rad (Hercules, CA). Muscle lysates were aliquoted, snap frozen in liquid nitrogen, and stored at –80°C for immunoblot analysis.

Immunoblotting. Equal amounts of protein (20–60 µg) were resolved by SDS-PAGE (8–10% polyacrylamide), transferred to nitrocellulose membranes, and blocked for 1 h at room temperature in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.8) containing 0.05% Tween-20 (TBS-T) and either 5% nonfat dry milk or 5% bovine serum albumin. Membranes were then incubated overnight at 4°C in the indicated primary antibodies. Membranes were probed with horseradish peroxidase-conjugated secondary antibody and visualized using an enhanced chemiluminescence system (Perkin Elmer, Wellesley, MA). Bands were scanned and quantified by densitometry.

Glycogen synthase activity assay. Pulverized muscle (10–20 mg) was homogenized 1:20 in ice-cold buffer (50 mM Tris·HCl, 5 mM EDTA, 100 mM NaF, pH 7.8). Glycogen synthase activity in the presence and absence of 6.7 mM glucose 6-phosphate was determined as previously described (42) and is expressed as the ratio of glycogen synthase activity in the absence of glucose 6-phosphate to that in the presence of glucose 6-phosphate.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using two-way ANOVA. When analysis of variance revealed significant differences, Tukey’s post hoc test for multiple comparisons was performed. Differences between groups were considered statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Akt2 deficiency does not impair exercise-stimulated glucose uptake. As an initial step in determining the role of Akt2 in exercise-stimulated glucose uptake in skeletal muscle, we first performed a preliminary study to determine whether Akt2-deficient mice could perform a moderate treadmill exercise protocol (60 min at 0.8 mph and a 15% incline). After our pilot study confirmed that Akt2-deficient mice could perform our moderate exercise protocol normally, we treadmill exercised Akt2-deficient and wildtype control animals and determined 2-deoxyglucose transport in isolated soleus muscle. As illustrated in Fig. 1A, treadmill exercise resulted in similar ~1.5-fold increases in glucose transport in both wildtype and Akt2 KO animals. Although two groups have reported that, in Akt2 KO mice, insulin-stimulated glucose uptake was significantly reduced at both submaximal (14, 27) and maximal (14) doses of insulin in isolated soleus muscle, a previous report found that insulin-stimulated glucose uptake is blunted in isolated extensor digitorum longus (EDL), but not soleus, of Akt2 KO animals (5). As such, it could be argued that our results are not specific to a nonessential role for Akt2 in exercise-stimulated glucose uptake but rather result from a generalized nonessential role for Akt2 in regulating glucose transport in the soleus muscle. To control for this possibility, we incubated isolated soleus muscle with maximal insulin (50 mU/ml) and assayed 2-deoxyglucose transport (Fig. 1B). While insulin increased glucose transport in both wildtype and Akt2 KO muscles, insulin-stimulated glucose transport was significantly lower in soleus muscles from Akt2 KO animals compared with muscles from wildtype animals (P < 0.01).


Figure 1
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  		Fig. 1. 2-Deoxyglucose uptake in response to treadmill exercise and in vitro insulin in wild-type (WT) and Akt2 knockout (KO) soleus muscle. 2-Deoxyglucose uptake was measured after 60 min of treadmill exercise at 0.8 mph and a 15% grade in isolated soleus muscle from WT and Akt2 KO animals (A). Treadmill exercise induced significant increases in 2-deoxyglucose transport in soleus muscle from both WT and Akt2 KO animals, with no difference between genotypes. Soleus muscles were assayed for 2-deoxyglucose transport after stimulation with 50 mU/ml insulin (B). Insulin induced significant increases in glucose transport in both genotypes, although the increase was significantly greater in soleus from WT animals. n = 5–10/group; *P < 0.01.

 
Akt2 deficiency does not affect muscle glycogen content after exercise. Given that skeletal muscle glucose uptake is closely linked to muscle glycogen content, and that Akt activation is linked to glycogen synthesis through its ability to phosphorylate and inactivate GSK-3 (11), we next determined whether Akt2 deficiency affects glycogen content in the soleus muscle in either the basal state or following the same treadmill exercise protocol used in determining glucose uptake in Fig. 1. As shown in Fig. 2, the lack of Akt2 does not affect glycogen content in the soleus muscle in either the basal or exercised state.


Figure 2
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  		Fig. 2. Glycogen content in the basal and exercised state in WT and Akt2 KO soleus muscle. Glycogen content was determined in soleus muscle from WT and Akt2 KO mice in the basal state and after 60 min of treadmill exercise. Exercise induced significant decreases in muscle glycogen content, with no differences between genotypes. n = 6–8/group; *P < 0.05.

 
Contraction-stimulated Akt-GSK-3 signaling is disrupted in the absence of Akt2. We next investigated contraction-stimulated signaling in the absence of Akt2. For these experiments, we subjected tibialis anterior muscles to 5 min of in situ contraction. We first measured T-loop Akt phosphorylation (Thr308) in sham or contraction-stimulated tibialis anterior muscle and found that contraction significantly stimulated Akt Thr308 phosphorylation only in wildtype animals (Fig. 3A). This suggests that Akt2 may be the dominantly activated isoform in response to in situ muscle contraction in mouse.


Figure 3
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  		Fig. 3. Contraction-stimulated Akt phosphorylation. Akt Thr308 and pAMPK(Thr172) phosphorylation was measured in tibialis anterior muscle after 5 min of in situ contraction. Contraction induced significant increases in Akt Thr308 phosphorylation in tibialis anterior muscle from WT but not Akt2 KO animals. A: contraction equally phosphorylated AMPK in both WT and Akt2 KO animals. B: pAMPK, phosphorylated AMP-activated protein kinase; pAkt, phosphorylated Akt; Contr, contraction. n = 5–6 group; *P < 0.05.

 
GSK-3 is a canonical Akt substrate that becomes inactivated after phosphorylation at Ser21 (GSK-3{alpha}) and Ser9 (GSK-3beta). To determine whether the lack of Akt2 results in deficiencies in contraction-stimulated phosphorylation and inactivation of GSK-3, we immunoblotted basal and contraction-stimulated muscle lysates for phospho-GSK-3{alpha}/beta Ser21/9 and found that phosphorylation of GSK-3{alpha} (Fig. 4A), but not GSK-3beta (Fig. 4B), was significantly decreased in Akt2 KO animals, with two-way ANOVA revealing a main effect of genotype. To rule out the possibility that GSK-3{alpha} expression was reduced in Akt2-deficient muscle, we immunoblotted for total GSK-3 and found that there was no difference between Akt2 KO and wildtype muscles (data not shown). To control for the possibility that Akt2 deficiency affected muscle contractile activity, we immunoblotted basal and contracted muscle lysates for AMPK Thr172 phosphorylation; we have previously reported that AMPK activity increases in a dose/intensity-dependent manner with in vivo exercise and in vitro contractions (29). We found contraction to equally activate AMPK in both wildtype and Akt2 KO animals (Fig. 3B). This suggests that the reduction in GSK-3{alpha} phosphorylation is specific to Akt2 deficiency, but we cannot completely rule out the possibility that lack of Akt2 may affect muscle contractile activity.


Figure 4
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  		Fig. 4. Contraction-stimulated glycogen synthase kinase-3 (GSK-3) phosphorylation. GSK-3{alpha} Ser21 phosphorylation was measured in tibialis anterior muscle after 5 min of in situ contraction. Contraction caused significant increases in GSK-3 phosphorylation in tibialis anterior from both WT and Akt2 KO animals, although GSK-3{alpha} Ser21 phosphorylation was significantly reduced in Akt2 KO animals, with a main effect of genotype. (A). Contraction caused similar increases in GSK-3beta Ser9 phosphorylation in tibialis anterior muscle from WT and Akt2 KO animals (B). n = 5–6/group; *P < 0.05.

 
Akt2 deficiency does not affect contraction-stimulated glycogen synthase phosphorylation or activity. GSK-3 inactivates glycogen synthase by phosphorylating a cluster of serine residues located at its COOH terminus (Ser641/645/649/653); phosphorylation and inactivation of GSK-3 can thus lead to decreases in glycogen synthase phosphorylation and increases in glycogen synthase activity (8). To determine whether the decreases in contraction-stimulated GSK-3{alpha} Ser21 phosphorylation resulted in changes in contraction-stimulated glycogen synthase phosphorylation at GSK-3 target residues, we immunoblotted basal and contraction-stimulated muscle lysates for phospho-glycogen synthase (Ser645/649/653/657). Five minutes of in situ contraction induced significant, similar decreases in glycogen synthase phosphorylation in tibialis anterior muscles from both wildtype and Akt2 KO animals (Fig. 5A). Glycogen synthase expression was not altered by Akt2 deficiency (data not shown). To determine whether Akt2 deficiency results in changes in glycogen synthase activity independently of changes in glycogen synthase phosphorylation, we measured glycogen synthase activity in tibialis anterior muscles from wildtype and Akt2 KO animals subjected to 15 min of in situ contraction. Consistent with the results from glycogen synthase phosphorylation, contraction induced normal increases in glycogen synthase activity in the absence of Akt2 (Fig. 5B).


Figure 5
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  		Fig. 5. Contraction-stimulated glycogen synthase (GS) phosphorylation and activity. Glycogen synthase Ser645/649/653/657 phosphorylation was measured in tibialis anterior muscle in response to 5 min of situ contraction (A). Contraction caused significant decreases in glycogen synthase phosphorylation in tibialis anterior muscle from both WT and Akt2 KO animals. Glycogen synthase activity was measured in tibialis anterior muscle in the basal state or after 15 min of in situ contraction (B). Glycogen synthase activity was significantly increased with contraction in tibialis anterior muscle from both WT and Akt2 KO animals, with no difference between genotypes. n = 5–6/group; *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The cellular response to exercise involves multiple alterations in the transcriptional and metabolic profile of contracting muscle (35). Understanding the pathways that govern these effects has been the subject of intensive research and holds the promise of furthering our understanding of this complex physiological phenomenon, as well as uncovering pathways that will hold relevance for the treatment of metabolic disorders such as diabetes. Chief among the metabolic responses to exercise in skeletal muscle are increases in glucose uptake and glycogen synthesis. In this investigation, we explored the possibility that Akt2, which is activated by contraction and is required for normal insulin-stimulated glucose uptake, could contribute to exercise/contraction-stimulated glucose uptake and glycogen synthesis in skeletal muscle.

The molecular mechanisms underlying contraction-stimulated glucose transport are complex and appear to involve a high level of redundancy (19, 32). We found that, despite its essential role in insulin-stimulated glucose uptake, Akt2 is not necessary for exercise-stimulated increases in glucose transport in skeletal muscle in vivo. This finding is consistent with previous studies pointing to distinct mechanisms for insulin- and contraction-stimulated glucose transport. While these pathways are likely to converge before GLUT4 translocation, potentially at the level of AS160 (3), our findings make clear that Akt2 is necessary for the stimulation of glucose uptake in skeletal muscle by insulin, but not exercise. Previous studies using wortmannin found that in vitro contraction-stimulated glucose uptake is not dependent on PI3K and Akt activation (2, 25). However, these studies have been conducted in vitro, and until now it has remained unknown whether Akt2 is necessary for in vivo exercise-stimulated glucose uptake in skeletal muscle. This study thus provides novel, direct, genetic evidence to exclude Akt2 as an obligate intermediate in exercise-stimulated glucose uptake. We are unable to discern, however, whether this is because Akt2 is not involved in contraction-stimulated glucose uptake, or whether Akt2 normally participates in contraction-stimulated glucose uptake, but redundant signaling mechanisms activated by contraction are able to compensate for the loss of Akt2 in the KO animals employed in this study. A large body of evidence, for example, suggests that AMPK and/or the calcium-mediated signaling pathway is a major contributor to contraction-stimulated glucose uptake (19, 32).

In this study, we describe an essential role for Akt2 in maximal insulin-stimulated glucose uptake in isolated soleus muscle. We believe that this is not due to impaired proximal insulin signaling, as intraperitoneal insulin injection resulted in normal IRS-1 phosphorylation and IRS-1-associated PI3K activation in the hindlimb muscles (unpublished observation). However, this finding is at odds with previous work using the same line of KO mice (5) and points to a greater role than previously recognized for Akt2 in the regulation of insulin-stimulated glucose uptake in skeletal muscle. We speculate that the differences between our findings and the previously published work may be due to differences in the in vitro muscle incubation protocol. In agreement with our findings, a previous study using the same line of Akt2-deficient mice reported that a submaximal dose of insulin that stimulated glucose uptake in wildtype EDL and soleus muscles failed to do so in Akt2 KO muscles (27). Furthermore, studies performed in Akt2-deficient mice on a different genetic background found reduced maximal insulin-stimulated glucose uptake in isolated soleus muscles (14).

We also found that Akt2 is not essential for the regulation of glycogen synthase activity in contracting skeletal muscle. Contraction-stimulated GSK-3beta phosphorylation was also unaffected by Akt2 deficiency, while contraction-stimulated GSK-3{alpha} phosphorylation was blunted in muscles from Akt2 KO animals. This implies that Akt2 is important for contraction-stimulated GSK-3{alpha} phosphorylation, but that other contraction-sensitive kinases are able to regulate GSK-3beta phosphorylation. p70 S6 kinase (10), mitogen-activated protein kinase kinase-activated kinase-1 (p90 RSK) (40), and PKA (13), for example, all have been reported to phosphorylate and inactivate GSK-3, and these molecules, or other Akt isoforms, could play a role in the Akt2-independent phosphorylation of GSK-3beta. Our results also support a model in which contraction-stimulated glycogen synthase phosphorylation and activity are independent of changes in GSK-3 phosphorylation and are consistent with recent findings from knockin mice with the Ser21/9 sites on GSK-3{alpha}/beta mutated to alanine, that GSK-3 phosphorylation is not required for contraction-regulated glycogen synthase activity (28).

Our finding that Akt2 is required for normal GSK-3{alpha} phosphorylation in contracting skeletal muscle confirms that Akt2 is essential for part of the physiological response to skeletal muscle contraction. Although much of the research concerning GSK-3 has focused on GSK-3beta, potentially because of its higher abundance (28), our results have consistently shown that contraction/exercise-stimulated increases in the phosphorylation of GSK-3{alpha} are considerably greater than increases in the phosphorylation of GSK-3beta in both rodents and humans (28, 33, 36), which suggests that the inhibition of GSK-3{alpha} may be an important molecular mediator of the effects of contraction in skeletal muscle. Furthermore, GSK-3 has been implicated in a wide variety of cellular processes beyond the control of glycogen metabolism (20), and GSK-3{alpha}-specific signaling has been reported (31), suggesting that further research concerning the role of contraction-stimulated GSK-3{alpha} inhibition will be fruitful in elucidating the molecular mechanisms underlying the complex effects of contraction in skeletal muscle.

In summary, our results show that Akt2, an essential mediator of insulin-stimulated glucose uptake in skeletal muscle, is not required for exercise-stimulated glucose uptake or glycogen synthesis. Our finding that Akt2 is necessary for normal exercise-induced increases in GSK-3{alpha} phosphorylation, however, suggests that Akt2 does play a role in the cellular response to contraction in skeletal muscle.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-68626 and AR-42238 (L. J. Goodyear).


   ACKNOWLEDGMENTS
 
We are grateful to Morris J. Birnbaum (Department of Medicine, University of Pennsylvania School of Medicine) for the generous donation of Akt2 KO mice.

Current address for K. Sakamoto: Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, Univ. of Dundee, Dow St., Dundee DD1 5EH, UK.


   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.

* K. Sakamoto and D. E. Arnolds contributed equally to this work. Back


   REFERENCES
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 DISCUSSION
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 REFERENCES
 

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