Abstract

Article24 March 2005free access Role that phosphorylation of GSK3 plays in insulin and Wnt signalling defined by knockin analysis Edward J McManus Edward J McManus MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Kei Sakamoto Kei Sakamoto MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Laura J Armit Laura J Armit School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Leah Ronaldson Leah Ronaldson MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Natalia Shpiro Natalia Shpiro Division of Biological Chemistry and Molecular Mircrobiology, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Rodolfo Marquez Rodolfo Marquez Division of Biological Chemistry and Molecular Mircrobiology, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Dario R Alessi Corresponding Author Dario R Alessi MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Edward J McManus Edward J McManus MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Kei Sakamoto Kei Sakamoto MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Laura J Armit Laura J Armit School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Leah Ronaldson Leah Ronaldson MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Natalia Shpiro Natalia Shpiro Division of Biological Chemistry and Molecular Mircrobiology, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Rodolfo Marquez Rodolfo Marquez Division of Biological Chemistry and Molecular Mircrobiology, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Dario R Alessi Corresponding Author Dario R Alessi MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland Search for more papers by this author Author Information Edward J McManus1, Kei Sakamoto1, Laura J Armit2, Leah Ronaldson1, Natalia Shpiro3, Rodolfo Marquez3 and Dario R Alessi 1 1MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee, Scotland 2School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, Scotland 3Division of Biological Chemistry and Molecular Mircrobiology, MSI/WTB Complex, University of Dundee, Dundee, Scotland *Corresponding author. MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Tel.: +44 1382 344 241; Fax: +44 1382 223 778; E-mail: [email protected] The EMBO Journal (2005)24:1571-1583https://doi.org/10.1038/sj.emboj.7600633 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The inactivation of glycogen synthase kinase (GSK)3 has been proposed to play important roles in insulin and Wnt signalling. To define the role that inactivation of GSK3 plays, we generated homozygous knockin mice in which the protein kinase B phosphorylation sites on GSK3α (Ser21) and GSK3β (Ser9) were changed to Ala. The knockin mice were viable and were not diabetic. Using these mice we show that inactivation of GSK3β rather than GSK3α is the major route by which insulin activates muscle glycogen synthase. In contrast, we demonstrate that the activation of muscle glycogen synthase by contraction, the stimulation of muscle glucose uptake by insulin, or the activation of hepatic glycogen synthase by glucose do not require GSK3 phosphorylation on Ser21/Ser9. GSK3 also becomes inhibited in the Wnt-signalling pathway, by a poorly defined mechanism. In GSK3α/GSK3β homozygous knockin cells, Wnt3a induces normal inactivation of GSK3, as judged by the stabilisation of β-catenin and stimulation of Wnt-dependent transcription. These results establish the function of Ser21/Ser9 phosphorylation in several processes in which GSK3 inactivation has previously been implicated. Introduction Insulin promotes the conversion of glucose to glycogen in skeletal muscle by stimulating glucose uptake and activating glycogen synthase (Roach, 2002; Ferrer et al, 2003). Insulin activates glycogen synthase by inducing its dephosphorylation at a cluster of C-terminal residues (Ser641, Ser645, Ser649 and Ser653), which are phosphorylated by glycogen synthase kinase-3α (GSK3α) and GSK3β (Cohen, 1999). In muscle, insulin is thought to stimulate the dephosphorylation of glycogen synthase at these residues by inducing the inactivation of GSK3α and GSK3β via phosphorylation of an N-terminal Ser residue (Ser21 in GSK3α and Ser9 in GSK3β), which is catalysed by protein kinase B (PKB, also known as Akt) (Cross et al, 1995; Shaw et al, 1997) and reversed by the muscle glycogen-associated protein phosphatase-1. Consistent with this model of regulation, small cell-permeable inhibitors of GSK3 with diverse structures stimulate glycogen synthase activity in cell lines or skeletal muscle (reviewed in Cohen and Goedert, 2004). However, it has also been reported that insulin stimulates the dephosphorylation of glycogen synthase at Ser7, which is not regulated by GSK3, and, furthermore, it has been suggested that GSK3 is not the only kinase which phosphorylates the residues between Ser641 and Ser653 (reviewed in Roach, 2002; Ferrer et al, 2003). Thus, the possibility that insulin may regulate glycogen synthase by a mechanism that is independent of GSK3 has not been excluded. It has also been proposed that insulin increases the activity of a glycogen-associated form of protein phosphatase-1 that dephosphorylates the residues in glycogen synthase that are phosphorylated by GSK3 (Suzuki et al, 2001; Delibegovic et al, 2003), but the mechanism or importance of this observation is unclear. The relative contributions made by the inactivation of GSK3 versus potential activation of protein phosphatase or regulation via other pathways to the activation of glycogen synthase is unknown. In the liver, hepatic glycogen synthase is also regulated by phosphorylation of the sites targeted by GSK3. In support of this, GSK3 inhibitors stimulate hepatic glycogen synthase (Coghlan et al, 2000; Cline et al, 2002). However, an important difference between muscle and liver glycogen synthase is that the latter is regulated by the level of blood glucose (Bollen et al, 1998). Glucose has been proposed to stimulate glycogen synthase by binding to phosphorylase-a, thereby promoting its conversion to phosphorylase-b, and so relieve the inhibition that phosphorylase-a exerts on the hepatic glycogen-associated form of protein phosphatase-1, termed PP1GL (Alemany and Cohen, 1986; Bollen et al, 1998). This allows protein phosphatase-1 to dephosphorylate and hence activate hepatic glycogen synthase, thereby stimulating glycogen synthesis. The relative importance of the insulin-induced inactivation of GSK3 versus the glucose-stimulated phosphatase activity in the regulation of hepatic glycogen synthase is unknown. In order for GSK3 to phosphorylate many of its substrates, including glycogen synthase, the substrate is required to be phosphorylated at a priming site, lying four residues C-terminal to the Ser/Thr residue phosphorylated by GSK3 (Frame and Cohen, 2001; Roach, 2002). In the case of glycogen synthase, CK2 is believed to act as a priming kinase by phosphorylating Ser657 (Roach, 2002; Ferrer et al, 2003). Recent studies have revealed that GSK3 possesses a specific phosphate recognition motif located within its kinase domain, which interacts specifically with the primed phosphorylated residue on the GSK3 substrate (Frame and Cohen, 2001). Following phosphorylation by PKB, the N-terminal phosphorylated sequence of GSK3 interacts with the phosphate recognition motif on the GSK3 kinase domain, thereby inhibiting GSK3 by competing for substrate binding. Mutation to Ala of the sites on GSK3α and GSK3β phosphorylated by PKB prevents the inhibition of GSK3 by insulin. The inactivation of GSK3 also plays an important role in the Wnt signalling pathway which is critical for embryonic development (Logan and Nusse, 2004). Wnt inhibits GSK3 by a poorly defined mechanism which is thought to involve disruption of a complex containing GSK3, axin, the adenomatous polyposis coli (APC) protein, and β-catenin. This leads to dephosphorylation and stabilisation of β-catenin, stimulating transcriptional processes controlled by β-catenin. In many forms of human colorectal cancer, mutations arise, mainly in APC and β-catenin, that induce the stabilisation of β-catenin, causing uncontrolled activation of the pathway, which leads to loss of cell polarity and increased proliferation (Sancho et al, 2004). Several groups have proposed that Wnt induces phosphorylation of GSK3 isoforms at the same sites phosphorylated by PKB (Yuan et al, 1999; Fukumoto et al, 2001); however, it has also been suggested that Wnt inactivates GSK3 by a distinct mechanism (Ding et al, 2000). Results Generation of GSK3 knockin mice We generated knockin mice in which the codon encoding Ser21 of GSK3α, and Ser9 of GSK3β, was changed to encode a nonphosphorylatable Ala residue, using the methodology described under Materials and methods and in Supplementary Figure 1. Single homozygous GSK3α21A/21A and GSK3β9A/9A, as well as double GSK3α/β21A/21A/9A/9A knockin mice, were bred using the strategy depicted in Table I. Throughout this study, wild-type (WT) littermate animals were employed in control experiments performed with the single GSK3α21A/21A and GSK3β9A/9A knockin mice. In the case of the double GSK3α/β21A/21A/9A/9A knockin mice, this was impractical, as WT littermate and double knockin mice can only be obtained at a ratio of 1 in 16 in the same cross (Table I). In order to breed GSK3α/β21A/21A/9A/9A and appropriate control mice, we interbred GSK3α/β21A/21A/9A/9A and GSK3α/β21A/21A/9A/9A as well as GSK3α/β+/+/+/+ and GSK3α/β+/+/+/+ littermates derived from a 1 in 16 ratio cross. Table 1. Mice matings reported in this study Cross Genotype Number (%) GSK3α(+/S21A)GSK3β(+/+) GSK3α(+/+) GSK3β(+/+) 50 (25.5%) GSK3α(+/S21A)GSK3β(+/+) GSK3α(+/S21A) GSK3β(+/+) 96 (49%) GSK3α(S21A/S21A) GSK3β(+/+) 50 (25.5%) GSK3α(S21A/S21A)GSK3β(+/+) GSK3α(S21A/S21A) GSK3β(+/+) 11 (100%) GSK3α(S21A/S21A)GSK3β(+/+) GSK3α(+/+)GSK3β(+/S9A) GSK3α(+/+) GSK3β(+/+) 48 (23%) GSK3α(+/+)GSK3β(+/S9A) GSK3α(+/+) GSK3β(+/S9A) 104 (50%) GSK3α(+/+) GSK3β(S9A/S9A) 56 (27%) GSK3α(+/+)GSK3β(S9A/S9A) GSK3α(+/+) GSK3β(S9A/S9A) 12 (100%) GSK3α(+/+)GSK3β(S9A/S9A) GSK3α(+/S21A)GSK3β(+/S9A) GSK3α(+/+) GSK3β(+/+) 3 (7.5%) GSK3α(+/S21A)GSK3β(+/S9A) GSK3α(+/+) GSK3β(+/S9A) 3 (7.5%) GSK3α(+/+) GSK3β(S9A/S9A) 2 (5%) GSK3α(+/S21A) GSK3β(+/+) 5 (12.5%) GSK3α(+/S21A) GSK3β(+/S9A) 19 (47.5%) GSK3α(+/S21A) GSK3β(S9A/S9A) 2 (5%) GSK3α(S21A/S21A) GSK3β(+/+) 1 (2.5%) GSK3α(S21A/S21A) GSK3β(+/ S9A) 3 (7.5%) GSK3α(S21A/S21A) GSK3β(S9A/S9A) 2 (5%) GSK3α(S21A/S21A)GSK3β(S9A/S9A) GSK3α(S21A/S21A) GSK3β(S9A/S9A) 150 (100%) GSK3α(S21A/S21A)GSK3β(S9A/S9A) The indicated matings were set up and the progeny genotyped as described in Materials and methods. The percentage of each genotype observed is indicated in parenthesis. GSK3 knockin mice develop and grow normally and are not diabetic The single GSK3α21A/21A, GSK3β9A/9A and double GSK3α/β21A/21A/9A/9A knockin mice were born at the expected Mendelian frequency (Table I) and displayed no overt phenotype. Growth curves from 4 to 16 weeks of age indicated that these animals were of normal size and weight (Figure 1A and Supplementary Figure 2). Glucose tolerance tests indicated that the single (Supplementary Figure 2) as well as double knockin mice (Figure 1B) were able to dispose of injected glucose at the same rate as WT controls, at 20 weeks of age. Double GSK3α/β21A/21A/9A/9A knockin mice also possessed normal fasted and fed glucose and insulin levels (Figure 1C). Figure 1.Growth and glucose metabolism in the double knockin mice. (A) The indicated male and female mice were weighed once a week between the ages of 4 and 16 weeks. Each point represents the mean±s.e.m., in which n is the number of mice analysed in each group. No statistical difference in the mean weights was observed for any group using the Student's t-test. (B) Glucose tolerance test of the indicated 20-week-old mice. Mice were injected intraperitoneally with 2 mg/g glucose solution and the blood glucose concentration was determined at the indicated times. n indicates the number of mice in each group. The data are presented as the mean±s.e.m. (C) The indicated mice were fasted overnight for 16 h (Fast) or fed ab libitum (Fed). The blood glucose and plasma insulin levels were measured. The data are presented as the mean±s.e.m., with the number, n, of animals in each group indicated. Approximate equal numbers of male and female mice were used in (B) and (C). Download figure Download PowerPoint Analysis of PKB and GSK3 in the muscle of knockin mice Mice were fasted overnight and injected with insulin. In order to verify whether the PI 3-kinase signalling pathway was normally active in the knockin mice, PKBα activity was measured. In the skeletal muscle of GSK3α21A/21A, GSK3β9A/9A and double GSK3α/β21A/21A/9A/9A knockin mice, insulin induced a robust activation of PKB, as well as phosphorylation at one of its activating residues (Ser473), similar to that seen in WT control mice (Figure 2A). The levels of GSK3α and GSK3β protein in the skeletal muscle of GSK3α21A/21A, GSK3β9A/9A and double GSK3α/β21A/21A/9A/9A knockin mice were normal (Figure 2B). We also found that the level of tyrosine phosphorylation in the ‘T-loop’ of GSK3 isoforms was similar in WT and knockin mice and not affected by insulin treatment (Figure 2B). Consistent with this, the specific activities of GSK3α derived from muscle extracts of WT and GSK3α21A/21A knockin mice not injected with insulin were similar (Figure 2B). The GSK3β activity in muscle extracts from GSK3β9A/9A knockin mice that had not been injected with insulin was slightly higher than that of WT mice (Figure 2B). A low level of phosphorylation of GSK3β at Ser9, in WT non-insulin-stimulated muscle, could explain this. Insulin was found to induce a marked phosphorylation of both GSK3 isoforms in WT control mice (Figure 2B). In the GSK3α21A/21A knockin mice, no phosphorylation of GSK3α at Ser21 was observed, while phosphorylation of GSK3β at Ser9 was normal. Similarly, in the GSK3β9A/9A knockin mice, no phosphorylation of GSK3β at Ser9 was detected, while phosphorylation of GSK3α at Ser21 occurred normally. In the double knockin GSK3α/β21A/21A/9A/9A mice, phosphorylation of neither isoform of GSK3 was observed. In muscles isolated from WT mice, insulin induced ∼40% inhibition of GSK3α and GSK3β activity, which was maintained for 40 min (Figure 2B). Dephosphorylation of immunoprecipitated GSK3 with protein phosphatase-1γ (PP1γ) largely restored the specific activities of GSK3α and GSK3β to those observed in unstimulated muscle after phosphatase treatment, indicating that inhibition of GSK3 results from Ser/Thr phosphorylation (data not shown). GSK3β in the GSK3α21A/21A muscle and GSK3α in the GSK3β9A/9A muscle were normally inactivated by insulin (Figure 2B). In contrast, insulin did not induce any inactivation of the S21A or S9A mutants of GSK3 in the knockin mice (Figure 2B), nor was the activity of the immunoprecipitated GSK3 increased by treatment with PP1γ (data not shown). Figure 2.PKB and GSK3 activity in GSK3 knockin mice. (A) The indicated mice were fasted overnight and injected intraperitoneally with insulin (150 mU/g) or saline solution (for the 0 min time point), to anaesthetised mice. At the indicated time (10 min for saline control), skeletal muscle was rapidly extracted and snap frozen in liquid nitrogen. PKBα activity was measured following its immunoprecipitation as described in Materials and methods. The results shown at each time point represent the mean±s.e.m. for three mice each assayed in duplicate for PKBα. The total levels and phosphorylation of PKBα at Ser473 were monitored by immunoblot analysis of tissue extracts. For each time point muscle samples from three separate mice are shown. (B) As above, except that GSK3α and GSK3β were immunoprecipitated from the indicated insulin-stimulated tissues and their activity was measured. The data are presented as the mean±s.e.m. for muscle isolated from three mice each assayed in triplicate. Muscle extracts from the indicated mice were immunoblotted with the indicated antibodies and samples from three separate mice are shown for each time point. The analysis with the three sets of knockin mice was each compared to WT littermate control mice in (A) and (B). Since these data from the sets of WT controls did not differ significantly from each other, only the results obtained for the WT GSK3α21A/21A littermate controls are shown. Download figure Download PowerPoint Effect of insulin on glycogen synthase in GSK3 knockin muscle We next assessed the level and activity of glycogen synthase in skeletal muscle extracts. Glycogen synthase protein levels were normal in the muscles of knockin mice (Figure 3A). We measured glycogen synthase activity in muscle extracts by assaying the incorporation of radioactive UDP-glucose into glycogen in the presence or absence of the allosteric activator glucose 6-phosphate (G6P) (Thomas et al, 1968). In WT mice, insulin activated glycogen synthase ∼2-fold, which was sustained for up to 40 min (Figure 3A). This is the normal degree of activation of muscle glycogen synthase that is induced by insulin. Activation was accompanied by dephosphorylation of Ser641 and Ser645, two of the residues phosphorylated by GSK3 that play critical roles in regulating its activity (Roach, 2002; Ferrer et al, 2003). In the single GSK3α21A/21A knockin mice, the basal level of glycogen synthase activity was normal, and insulin still induced an ∼2-fold activation after 10 min that was also accompanied by the dephosphorylation at the sites phosphorylated by GSK3. The activation of glycogen synthase in the GSK3α21A/21A knockin mice was less sustained than in control WT animals (Figure 3A). Although this effect was moderate, it was observed in two independent experiments with three mice being analysed for each time point. In the single GSK3β9A/9A, as well as the double GSK3α/β21A/21A/9A/9A knockin mice, the basal activity and phosphorylation of glycogen synthase at Ser641/Ser645 was comparable to control mice, but, strikingly, insulin failed to induce a significant activation or dephosphorylation of Ser641/Ser645 (Figure 3A). Figure 3.Effect of insulin on glycogen synthase in knockin mice. (A) The indicated mice were fasted overnight and injected intraperitoneally with insulin (150 mU/g) or saline solution (for the 0 min time point), to anaesthetised mice. At the indicated time (10 min for saline control), skeletal muscle was rapidly extracted and snap frozen in liquid nitrogen. Glycogen synthase activity was measured in the absence and presence of G6P. The data are presented as the mean±s.e.m. for muscle isolated from three mice each assayed ±G6P in duplicate. Muscle extracts from the indicated mice were immunoblotted with the indicated antibodies recognising phosphorylated and total glycogen synthase. As for Figure 2, only the WT controls for the GSK3α21A/21A are shown. (B) Soleus muscle was isolated and incubated in the presence or absence of insulin or AR-A014418. After 1 h of incubation, the muscle was rapidly frozen in liquid nitrogen. Glycogen synthase activity was assessed as in (A) above. The results are presented as the mean±s.e.m. for four muscles for each condition. Download figure Download PowerPoint We next investigated whether activation of glycogen synthase in the knockin mice could be induced by treatment of muscle with AR-A014418, a specific ATP competitive inhibitor of GSK3α and GSK3β (Bhat et al, 2003; Murray et al, 2004). We treated isolated soleus muscle from WT or the double GSK3α/β21A/21A/9A/9A knockin mice with either insulin or AR-A014418. Insulin failed to induce a significant activation of glycogen synthase in the GSK3α/β21A/21A/9A/9A knockin muscle under conditions that resulted in two-fold activation in WT muscle (Figure 3B). In contrast, AR-A014418 induced nearly five-fold activation of glycogen synthase in both the WT and double GSK3α/β21A/21A/9A/9A knockin muscle (Figure 3B). Relative expression of GSK3α and GSK3β in murine and human muscle The finding that insulin was unable to stimulate glycogen synthase in the single GSK3β9A/9A knockin mice suggested that GSK3β is the principal isoform phosphorylating glycogen synthase in murine muscle. This observation could be explained if GSK3β was expressed at a higher level than GSK3α in skeletal muscle. To investigate whether this was so, we expressed mouse GSK3α and GSK3β with N-terminal glutathione S-transferase (GST) tags as expression standards for immunoblotting. We carried out a quantitative immunoblot analysis using LI-COR technology, in which equal amounts of recombinant GSK3α and GSK3β were analysed by immunoblotting with an anti-GST antibody and an anti-GSK3 antibody that recognises both GSK3 isoforms. This analysis permitted quantification of the relative abundance of GSK3α and GSK3β isoforms in murine skeletal muscle, and revealed ∼4-fold higher amounts of GSK3β than GSK3α (Figure 4A). Consistent with this, the specific activity of GSK3β is ∼5-fold higher than that of GSK3α in skeletal muscle (Figure 2B). As GSK3 inhibitors are being developed for the treatment of diabetes in humans, we performed a similar analysis employing human skeletal muscle derived from three lean and healthy donors. This revealed that, in human muscle of all three subjects, there was ∼3-fold excess of GSK3β over GSK3α (Figure 4B). Figure 4.Quantification of the relative levels of GSK3α and GSK3β in muscle. (A) The indicated amounts of the recombinant mouse GST-GSK3α and GST-GSK3β were subjected to immunoblot analysis with anti-GST antibody to verify equal loading of the isoforms or a Pan-GSK3 isoform antibody to determine the relative affinity of this antibody for GSK3α and GSK3β. Quantitation of the immunoblots was performed using LI-COR Odyssey infrared imaging system. Mouse skeletal muscle derived from three WT mice (40 μg total protein) was subjected to quantitative immunoblot analysis with the Pan-GSK3 isoform antibody and from the quantitation data the abundance of GSK3α relative to GSK3β is indicated. (B) As in (A), except that human GST-GSK3α and GST-GSK3β were employed as expression standards and skeletal muscle (30 μg total protein) from three healthy lean human subjects was analysed by immunoblot analysis. Download figure Download PowerPoint Analysis of glucose uptake and glycogen levels in GSK3 knockin muscle A major effect of insulin in muscle is to stimulate glucose uptake probably through the activation of PKB. Although the mechanism by which PKB stimulates glucose uptake is unknown, some studies have implicated the PKB-mediated inhibition of GSK3 in this process (Orena et al, 2000; Morfini et al, 2002). We therefore measured glucose uptake in isolated soleus muscle. These studies revealed that, in both the single as well as the double GSK3 knockin mice, insulin stimulated the uptake of glucose four-fold, similar to the effect observed in WT mice (Figure 5A). We next measured the total glycogen levels in the muscle of WT as well as the single and double GSK3 knockin mice. We observed that, in both mice fed ab libitum and fasted for 16 h, that there was no significant reduction in muscle glycogen levels in any of the GSK3 knockin mice compared to WT controls (Figure 5B). Figure 5.Analysis of glycogen levels and glucose uptake in muscle of the knockin mice. (A) Soleus muscle strips were isolated from the indicated mice and glucose transport activities were measured in the presence or absence of 100 nM insulin. The data are presented as the mean±s.e.m. for muscle isolated from five mice for each genotype. As for Figure 2, only the data for WT control for the GSK3α21A/21A are shown. (B) The indicated mice were fasted overnight for 16 h (Fast) or fed ab libitum (Fed). The mice were killed and the quadricep muscle was rapidly extracted and frozen in liquid nitrogen. The glycogen content in skeletal muscle is expressed as μmol of glycosyl units per gram of muscle. The data are presented as the mean±s.e.m. for muscle isolated from three mice in each group each assayed in triplicate. Download figure Download PowerPoint Effect of muscle contraction on glycogen synthase and glucose tolerance in GSK3 knockin muscle Muscle contraction potently activates glycogen synthase by a poorly defined mechanism (reviewed in Nielsen and Richter, 2003). To define whether phosphorylation of GSK3α at Ser21 and GSK3β at Ser9 plays a role in this process, contraction of hind limb muscle was induced in anaesthetised mice in situ by electrical stimulation of the sciatic nerve in one leg, the other leg serving as the noncontracted control. In both the single as well as double GSK3 knockin mice, muscle contraction markedly stimulated the activity of glycogen synthase to the same extent found in the WT control mice (Figure 6A). Consistent with previous findings (Sakamoto et al, 2002, 2003), muscle contraction in WT mice induced a slight phosphorylation of GSK3α at Ser21 and GSK3β at Ser9 and also resulted in a marked dephosphorylation of glycogen synthase, especially at Ser641 and to a lesser extent at Ser645 (Figure 6B). Strikingly however, in both the single and double GSK3 knockin mice, although phosphorylation of the GSK3 knockin isoforms was abolished, exercise still led to dephosphorylation of glycogen synthase at Ser641 and Ser645 (Figure 6B). This does not result from inactivation of GSK3 isoforms by a mechanism distinct from the phosphorylation of Ser21 and Ser9, as the activity of GSK3α or GSK3β isoforms in the double GSK3α/β21A/21A/9A/9A knockin mice was not changed by muscle contraction (Figure 6C). In WT mice, contraction induced a modest inhibition of GSK3α activity consistent with its phosphorylation at Ser21. Contraction induced weak phosphorylation of PKB at Ser473 in WT and knockin mice, that was much weaker than that observed with insulin (Figure 6D). Muscle contraction also induced normal phosphorylation of the ERK1/ERK2 MAP kinases in the single and double GSK3 knockin mice. This could also contribute to the modest phosphorylation of GSK3 through the ERK-stimulated RSK. Figure 6.Effect of muscle contraction on glycogen synthase and glucose tolerance in the knockin mice. (A) One leg from anaesthetised WT and knockin mouse was subjected to in situ hindlimb muscle contraction (C, contraction) via sciatic nerve stimulation for 10 min, and the other leg served as noncontracted control (B, basal). Red and white gastrocnemius and extensor digitorum longus (EDL) muscles from both legs were rapidly extracted and snap frozen in liquid nitrogen. Glycogen synthase activity was measured in the absence and presence of G6P. The data are presented as the mean±s.e.m. for muscle isolated from four mice each assayed ±G6P in duplicate. (B) Muscle extracts from the indicated mice were immunoblotted with the indicated antibodies. As for Figure 2, only the WT controls for the GSK3α21A/21A are shown. (C) GSK3α and GSK3β were immunoprecipitated from WT and double knockin GSK3 muscle derived from (A), and activity was measured as a ratio of ±treatment with PP1γ phosphatase as described in Materials and methods. The data are presented as the mean±s.e.m. for muscle isolated from two mice, each assayed in triplicate. (D) Muscle extracts from the WT and knockin mice were immunoblotted with the indicated antibodies. The insulin-treated samples were derived from the muscle of mice that had been injected with insulin as described in Figure 2. In order to compare the levels of phosphorylation of PKB seen in response to insulin and muscle contraction, a short and long exposure of the immunoblot is shown. (E) The indicated mice were fasted overnight and blood glucose levels measured. The mice were then anaesthetised and after 30 min a glucose tolerance test was carried out after mice were injected intraperitoneally with 2 mg/g glucose solution and the blood glucose concentration was determined at the indicated times. n indicates the number of mice in each group. The data are presented as the mean±s.e.m. and similar numbers of female and male mice were present in each group. Download figure Download PowerPoint To investigate whether physical activity contributed the nondiabetic phenotype observed in the GSK3 knockin mice, as previously observed for mice lacking the muscle insulin receptor (Wojtaszewski et al, 1999), we performed a glucose tolerance test on anaesthetised control and GSK3α/β21A/21A/9A/9A animals. This revealed no significant differences between the WT and