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).

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 GSK3α^21A/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.

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 GSK3α^21A/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.

Analysis of liver glycogen synthase activity and glycogen levels in the knockin mice. (A) The indicated mice were fasted overnight and injected intraperitoneally with glucose (2 mg/g) or saline solution (for the 0 min time point), to anaesthetised mice. At the indicated time (10 min for saline control), the liver 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 liver isolated from three mice each assayed ±G6P in duplicate. Liver extracts from the mice were immunoblotted with the indicated antibodies. (B) The WT and knockin mice were fasted overnight for 16 h (Fast) or fed ab libitum (Fed), and liver glycogen content quantified as μmol of glycosyl units per gram of liver. The data are presented as the mean±s.e.m. for liver isolated from six mice in each group each assayed in triplicate.

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 GSK3α^21A/21A littermate controls are shown.

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.

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 GSK3α^21A/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.

Ability of Wnt to inactivate GSK3 in knockin ES cells. (A) The WT and double knockin GSK3α/β^{21A/21A/9A/9A} ES cells were incubated in medium containing Wnt3a or a control medium obtained from non-Wnt3a-expressing cells. At the indicated times, the phosphorylation state of β-catenin was assessed by immunoblot analysis employing an antibody that recognises β-catenin when phosphorylated on the three residues targeted by GSK3 (S33/37-P, T41-P). Quantitation of the total levels of β-catenin was performed by immunoblot analysis using LI-COR Odyssey infrared imaging system. The extracts were also immunoblotted with antibodies recognising the phosphorylated and total forms of GSK3. For each condition duplicate dishes were analysed and similar results were obtained in two separate experiments. (B) ES cells were co-transfected with a firefly luciferase reporter construct containing LEF/TCF-binding sites as well as a constitutive SV-40 Renilla luciferase construct, to control for transfection efficiency. At 24 h post-transfection, cells were incubated in medium containing Wnt3a or a control medium obtained from non-Wnt3a-expressing cells and luciferase activity was measured. As a control, cells were transfected with a firefly luciferase reportor construct in which the LEF/TCF-binding sites had been mutated (NEG) and were incubated with Wnt3a for 8 h. Luciferase activity was expressed in arbitrary units relative to the activity observed in unstimulated WT cells normalised for Renilla luciferase activity. Four dishes of cells were analysed for each condition and similar results were obtained in two separate experiments. Results shown are mean±s.d. for a single experiment.