Figure 1.
GSK-3 expression, body weight, plasma insulin concentration, glucose tolerance and insulin sensitivity in C57BL/6/129 GSK-3α muscle KO animals.
(A) Brain, heart, liver, testes and quad, gastroc, soleus and EDL muscles and (B) brain, heart, testes, liver and gastroc from eight week old MLC Cre − (−) and MLC Cre + (+) littermate control animals were extracted and lysed as described in Experimental Procedures and proteins resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against GSK-3α/β. Even loading was determined using antibodies to GAPDH. Blots are representative from three separate experiments. (B) Body weight of male MLC Cre − (open squares) and MLC Cre + (filled squares) littermate control mice was monitored weekly from age 4 to 22 weeks. Values are the mean ± SEM from eight separate animals. (C) Plasma insulin concentration was determined in eight week old male MLC Cre − (open bars) and MLC Cre + (filled bars) littermate control animals under fasted and fed conditions. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male MLC Cre − (open squares) and MLC Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (D) 2 mg/g glucose or (E) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from at least seven separate animals.
Figure 2.
Effect of GSK-3α muscle KO on insulin signaling.
(A) Quad, (B) gastroc and (C) liver from eight week old MLC Cre − (−) and MLC Cre + (+) littermate control animals was extracted following either an over night (16–18 h) fast alone or an overnight fast followed by i.p. administration of 150 mU/g insulin for 15 min. Twenty µg of the protein lysate was resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against phospho-PKB (pSer473), PKB, phospho-GSK-3β (pSer9), GSK-3β, phospho-GS (pSer641) and GS (lower left inset in C). Loading was determined using antibodies to either GAPDH or β-actin. Blots are representative from five separate experiments. (D) GSK-3 kinase activity was determined from muscle tissue extracted from male MLC Cre − control (open bars) and MLC Cre + (filled bars) mice as described in experimental procedures. GSK-3 kinase activity was determined using a quantitative peptide phosphorylation assay. GSK-3 kinase activity is expressed relative to the MLC Cre- control (which is set at 100%) and is the mean ± SEM of four different muscle samples with each assayed in triplicate. Genetic background was C57BL/6/129.
Figure 3.
Glycogen synthase activity in quad, gastroc and liver of GSK-3α muscle KO animals.
Glycogen synthase activity was determined in (A) quad, (B) gastroc and (C) liver of eight weeks old MLC Cre – (open bars) and MLC Cre + (filled bars) tissues by assaying incorporation of glucose from uridine diphospho-{6-3H)-D-glucose into glycogen and expressed as a ratio of activity in the absence divided by that in the presence of glucose-6-phosphate. Values are the mean ± SEM for at least five experiments carried out in duplicate. Glycogen content was measured in (D) quad, (E) gastroc and (F) liver from eight week old MLC Cre − (open bars) and MLC Cre + (filled bars) following either an overnight fast or random feeding. Tissues were extracted, acid-hydrolyzed and glycosyl units assayed using a glucose reagent hexokinase method (Amresco, Ohio) as described in Experimental Procedures. Glycogen content is expressed as µmol glucose/g tissue. Values are mean ± SEM from at least five separate animals with each assayed in triplicate. Genetic background of the animals was C57BL/6/129.
Figure 4.
GSK-3 expression, body weight, plasma insulin concentration, glucose tolerance and insulin sensitivity in GSK-3α liver KO animals.
Tissue extracts were prepared from eight wk old male mice of the genotypes indicated and immunoblotted with antibodies against (A) GSK-3α/β. Even loading was determined using antibodies to GAPDH. Blots are representative from three separate experiments. (B) Body weight of male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was monitored weekly from age 4 to 22 weeks. Values are the mean ± SEM from nine separate animals. (C) Plasma insulin concentration was determined in eight week old male Alb Cre − (open bars) and Alb Cre + (filled bars) littermate control animals under fasted and fed conditions. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (D) 2 mg/g glucose or (E) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from fourteen separate animals for Alb Cre – and nine separate animals for Alb Cre +. Genetic background was C57BL/6/129.
Figure 5.
Effect of GSK-3α liver KO on insulin signaling.
(A) liver, (B) muscle from eight week old Alb Cre − (−) and Alb Cre + (+) littermate control animals were extracted following either an overnight (16–18 h) fast alone or an overnight fast followed by i.p. administration of 150 mU/g insulin for 15 min. Twenty µg of the protein lysates was resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against phospho-PKB, PKB, phospho-GSK-3β, GSK-3β, phospho-GS and GS. Even loading was determined by blotting for either β-actin or GAPDH. Blots are representative from 4 separate experiments. (C) GSK-3 kinase activity was determined from liver tissue extracted from male Alb Cre − control (open bars) and Alb Cre + (filled bars) mice as described in experimental procedures. GSK-3 kinase activity was determined using a quantitative peptide phosphorylation assay. GSK-3 kinase activity is expressed relative to the Cre− control (which is set at 100%) and is the mean ± SEM of four different liver samples with each assayed in triplicate. Genetic background was C57BL/6/129.
Figure 6.
Glycogen synthase activity and glycogen content in muscle and liver of GSK-3 liver KO animals.
Glycogen synthase activity was determined in (A) liver and (B) quad of eight weeks old Alb Cre – (open bars) and Alb Cre + (filled bars) tissues by assaying incorporation of glucose from uridine diphospho-(6-3H)-D-glucose into glycogen and expressed as a ratio of activity in the absence divided by that in the presence of G-6-P. Values are the mean ± SEM for four separate experiments with each assayed in duplicate. Glycogen content was measured in liver (C) and quad (D) liver from eight week old ALB Cre − (open bars) and ALB Cre + (filled bars) following either an overnight fast or random feeding. Values are mean ± SEM from eight separate animals with each assayed in triplicate. For these experiments, the mouse background strain was C57BL/6/129.
Figure 7.
Glucose tolerance and insulin sensitivity in C57BL/6-GSK-3α global and ICR-GSK-3α liver KO animals.
Blood glucose concentration in eight week old male WT - (open squares) and C57BL/6-GSK-3α KO (filled squares) mice was measured at the indicated times following administration of (A) 2 mg/g glucose or (B) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male ICR-GSK-3α Alb Cre − (open squares) and ICR-GSK-3α Alb Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (C) 2 mg/g glucose or (D) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from nine separate animals for Alb Cre − and ten separate animals for Alb Cre +. Glycogen content was measured in liver (E) from ICR-GSK-3α Alb Cre − (open squares) and ICR-GSK-3α Alb Cre + (filled squares) following either an overnight fast or random feeding. Values are mean ± SEM from at least five separate animals with each assayed in triplicate.