Tissue-specific role of glycogen synthase kinase 3beta in glucose homeostasis and insulin action

Abstract

Dysregulation of the protein kinase glycogen synthase kinase 3 (GSK-3) has been implicated in the development of type 2 diabetes mellitus. GSK-3 protein expression and kinase activity are elevated in diabetes, while selective GSK-3 inhibitors have shown promise as modulators of glucose metabolism and insulin sensitivity. There are two GSK-3 isoforms in mammals, GSK-3alpha and GSK-3beta. Mice engineered to lack GSK-3beta die in late embryogenesis from liver apoptosis, whereas mice engineered to lack GSK-3alpha are viable and exhibit improved insulin sensitivity and hepatic glucose homeostasis. To assess the potential role of GSK-3beta in insulin function, a conditional gene-targeting approach whereby mice in which expression of GSK-3beta was specifically ablated within insulin-sensitive tissues were generated was undertaken. Liver-specific GSK-3beta knockout mice are viable and glucose and insulin tolerant and display "normal" metabolic characteristics and insulin signaling. Mice lacking expression of GSK-3beta in skeletal muscle are also viable but, in contrast to the liver-deleted animals, display improved glucose tolerance that is coupled with enhanced insulin-stimulated glycogen synthase regulation and glycogen deposition. These data indicate that there are not only distinct roles for GSK-3alpha and GSK-3beta within the adult but also tissue-specific phenotypes associated with each of these isoforms.

FIG. 1.

Generation of conditional GSK-3β-floxed (FL), targeted mice. (A) Schematic outline describing the targeting strategy for generating conditional GSK-3β-floxed (FL) mice and the subsequent tissue-specific KO mice. After homologous recombination with the targeting vector, exon 2 (E2) of the GSK-3β gene was replaced with a LoxP-flanked (floxed) exon 2 and an FRT-flanked neomycin resistance cassette. Mice carrying the GSK-3β-floxed allele were bred to FLPe recombinase-expressing mice to remove the neo cassette. Deletion of exon 2, and hence deletion of GSK-3β expression, is achieved by crossing GSK-3β FL mice with strains expressing Cre recombinase under the control of tissue-specific promoters. Triangles, Lox P recombination sites; ovals, FRT recombination sites; B, BglII. Asterisks represent hybridization sites for Southern blot probes. (B) Southern blot analysis. ES cells and mouse tail genomic DNA were digested with BglII and probed with the 5′ GSK-3β or neomycin DNA probe (*), confirming proper targeting of the GSK-3β-floxed allele in R1 mouse ES cell (mES) clones 1B9 and 2D12 and germ line transmission of the GSK-3β-floxed allele (by use of the 1B9 clone) in mice. The 11.8-kb band corresponds to the WT GSK-3β allele, whereas the 13.6-kb band corresponds to the correctly targeted GSK-3β FL allele. (C) PCR genotype analysis of genomic DNA isolated from the progeny of the GSK-3β⁺/FL F1 cross. The WT GSK-3β band is 886 bp, whereas the GSK-3β FL band is 1,095 bp.

FIG. 2.

Generation and characterization of liver-specific GSK-3β KO (LβKO) mice. (A) Weight analysis. Male GSK-3β FL/FL (termed FL/FLβ) control and GSK-3β FL/FL AlbCre⁺ (termed LβKO) mice were weighed every 2 weeks between the ages of 4 and 24 weeks. Each point represents the mean ± standard error of the mean (SEM), with n indicating the number analyzed in each group. No statistical difference in the mean weights was observed between the groups by Student's t test. (B) PCR analysis. Genomic DNA was isolated from the tissues stated and analyzed by PCR for Cre-mediated excision of GSK-3β. PCR for GSK-3β (top) demonstrates that exon 2 of the GSK-3β FL allele is excised only in the livers of the LβKO mice, not in those of the control FL/FLβ or the WT AlbCre^+/+ (littermate control) mice. The bottom panel displays genomic detection of the Cre transgene. (C) Western blot analysis. Tissue extracts from liver, muscle (quadricep) and brain were prepared from 8-week-old male mice of the genotypes indicated and immunoblotted with an antibody that specifically recognizes GSK-3α and GSK-3β. Equal loading of the lanes was assessed using an anti-GAPDH antibody, and the blots shown are representative of three independent experiments. (D) Quantification of GSK-3. The densitometries of GSK-3α and GSK-3β were measured and normalized to GAPDH levels, and the expression of each isoform is displayed relative to the total (GSK-3α plus GSK-3β) expression level in the respective WT (or AlbCre⁺) tissue.

FIG. 3.

LβKO mice display “normal” glucose homeostasis and insulin sensitivity. (A) Glucose metabolism profile. GTT and ITT were performed on 8-week- or 6-month-old male FL/FLβ control and LβKO mice as described in Materials and Methods. Values are the means ± SEMs, with n indicating the number of mice in each group. (B) Glycogen staining. Liver sections from fasted (overnight, 16 h) or fed (ad libitum) 8-week-old male FL/FLβ control and LβKO mice were stained with PAS to detect glycogen accumulation (dark purple staining; top) or hematoxylin and eosin (H+E) for general cell morphology (bottom). The images are representative of results from five animals. (C) Glycogen content in liver. Liver tissues were extracted from fasted (overnight, 16 h) or fed (ad libitum) 8-week-old male FL/FLβ control and LβKO mice, and glycogen content was determined by acid hydrolysis and is expressed as μmol of glucose units per gram of liver tissue. Data are presented as means ± SEMs, with each liver sample assayed in triplicate, and n indicates the number of mice in each group. NS, not significant.

FIG. 4.

Analysis of insulin signaling in LβKO mice. (A and E) Insulin signaling in liver (A) and muscle (quadricep) (E) samples. Eight-week-old male FL/FLβ control and LβKO mice were fasted overnight and injected i.p. with 100 mU/g insulin for 15 min or with 1 mg/g glucose for 20 min or were allowed to refeed ad libitum for 2 h. Following treatment, liver (A) and SM (E) tissues were extracted, lysed as described in Materials and Methods, and immunoblotted with the indicated antibodies. Equal loading of the lanes was assessed using an anti-GAPDH antibody, and a representative blot is shown (left) with the quantification of results from four independent experiments displayed (right). Data are presented as a bar graph showing signal intensities compared to that for fasted FL/FLβ mice, which is set as 1. NS, not significant. (B) Insulin signaling in hepatocytes. Primary hepatocytes were isolated from 10-week-old male FL/FLβ control and LβKO mice and serum starved overnight prior to incubation with 20 nM insulin. At the times indicated, cells were lysed and the lysates subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies. A representative blot is shown (left), with quantification of results from four independent experiments displayed (right). NS, not significant. (C) GSK-3 kinase assays. Liver tissues were extracted from 8-week-old male FL/FLβ control and LβKO mice and partially purified through a CM-Sepharose column as described in Materials and Methods. GSK-3 kinase activities were determined using a quantitative peptide phosphorylation assay and are expressed relative to that for the FL/FLβ control (which is set at 100%). Values are the means ± SEMs of results for five different livers, with each assayed in triplicate. (D) GS activity. Liver tissues were extracted from 8-week-old male FL/FLβ control and LβKO mice and homogenized, and GS activity was measured in the presence of 0.1 mM (low) or 10 mM (high) G6P as described in Materials and Methods. Values are the means ± SEMs of results from four different livers, with each assayed in duplicate. **, P < 0.01 (for comparison to fasted mice for each genotype); NS, not significant (for comparison to fasted FL/FLβ mice).

FIG. 5.

Generation of SM-specific GSK-3β KO (MβKO) mice. (A) Weight analysis. Male FL/FLβ control and GSK-3β FL/FL mlc1f Cre⁺ (termed MβKO) mice were weighed every week between the ages of 4 and 24 weeks. Each point represents the mean ± SEM, with n indicating the number analyzed in each group. No statistical differences in mean weight were observed between the groups by Student's t test. (B) PCR analysis. Genomic DNA was isolated from the tissues stated and analyzed by PCR for Cre-mediated excision of GSK-3β. PCR for GSK-3β (top) demonstrates that exon 2 of the GSK-3β FL allele was excised in all SM types of the MβKO mice but not the control FL/FLβ. The bottom panel indicates genomic detection of the Cre transgene. Gastroc, gastrocnemius. (C) Western blot analysis. Tissue extracts were prepared from 8-week-old male mice of the genotypes indicated and immunoblotted with an antibody that recognizes GSK-3α and GSK-3β. The loading of the lanes was assessed using an anti-GAPDH antibody, and the blots are representative of three independent experiments. Gastro, gastrocnemius.

FIG. 6.

MβKO mice display improved glucose homeostasis and insulin sensitivity. (A) Blood glucose profile. Six- or 12-week-old male FL/FLβ control and MβKO mice were either fasted overnight for 16 h (fasted) or fed ad libitum (random fed), and blood glucose levels were measured. The data are presented as means ± SEMs, with the number (n) of mice in each group indicated. (B) Glucose metabolism profile. GTT and ITT were performed on 6-week- or 12-week-old male FL/FLβ control and MβKO mice as described in Materials and Methods. The inset depicts the plasma insulin concentrations in the FL/FLβ and MβKO mice following a 15-min i.p. injection of glucose. Values are means ± SEMs, with n indicating the number of mice in each group. Asterisks signify statistically significant changes compared to levels for the FL/FLβ control mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 7.

Analysis of insulin signaling in MβKO mice. (A and C) Insulin signaling in muscle (A and C, top) and liver (C, bottom). Six-week-old male FL/FLβ control and MβKO mice were fasted overnight and injected i.p. with 100 mU/g insulin or with 1 mg/g glucose for 20 min or for the times indicated. Following treatment, different SM types (top panels) and liver tissue (bottom left) were extracted, lysed as described in Materials and Methods, and immunoblotted with the indicated antibodies. A representative blot is shown (left), with the quantifications of results from at least four independent experiments displayed (right). *, P < 0.05; NS, not significant. (B) GS regulation. (Left) Signal intensities of the insulin-stimulated dephosphorylation of GS from muscle (gastrocnemius) tissues of the FL/FLβ control and MβKO mice were quantified, and the data are presented as a bar graph showing signal intensities as percentages of the WT level (100%). Values are the means ± SEMs from at least four independent experiments. *, P < 0.05 (FL/Flβ versus MβKO, insulin treated). (Right) Muscle tissues were extracted from 6-week-old male FL/FLβ control and MβKO mice and homogenized, and GS activity was measured in the presence of 0.1 mM (low) or 10 mM (high) G6P as described in Materials and Methods. Values are the means ± SEMs of results from six different muscle samples, with each assayed in duplicate. *, P < 0.05 (FL/Flβ versus MβKO, insulin treated). (D) GSK-3 kinase assays. Muscle (gastrocnemius) tissue was extracted from 6-week-old male FL/FLβ control and MβKO mice and partially purified with CM-Sepharose chromatography as described in Materials and Methods. GSK-3 kinase activity was determined using a quantitative peptide phosphorylation assay. The inset shows an immunoblot of GSK-3 that was used in the kinase assay reaction. GSK-3 kinase activities are expressed relative to that for the FL/FLβ control (which is set at 100%) and are the means ± SEMs of results from five different muscle samples, with each assayed in triplicate.

FIG. 8.

Analysis of glycogen deposition and glucose transport in MβKO mice. (A) Glycogen content in muscle. Muscle tissue (gastrocnemius) was extracted from fasted (overnight, 16 h) or fed (ad libitum) 6- or 12-week-old male FL/FLβ control and MβKO mice, and glycogen content was determined by acid hydrolysis and expressed as μmol of glucose units per gram of muscle tissue. The data are presented as means ± SEMs, with each muscle sample assayed in triplicate, and n indicates the number of mice in each group. *, P < 0.05; NS, not significant. (B) GLUT4 expression analysis. Muscle tissue (gastrocnemius) was extracted from 6-week-old male FL/FLβ control and MβKO mice and total cell membrane isolated as described in Materials and Methods and immunoblotted with the indicated antibodies. Equal loading of the lanes was assessed using an anticaveolin antibody, and the blots shown are representative of at least four independent experiments. (C) In vitro glucose transport measurement. Intact soleus and EDL muscle fibers were isolated from deeply anesthetized FL/FLβ control and MβKO mice and were incubated with or without 2 mU of insulin per ml for 30 min. 2-Deoxy-d-glucose uptake was measured over a 20-min period as described in Materials and Methods. The data are presented as means ± SEMs, with the number (n) of mice in each group indicated. NS, not significant. (D) In vivo glucose uptake. Six-week-old male FL/FLβ control and MβKO mice were fasted overnight and injected i.p. with a mixture of unlabeled dextrose and [³H]2DG, and the accumulation of the 2DG in muscle tissue (gastrocnemius) was determined as described in Materials and Methods. Data are presented as means ± SEMs, with the number (n) of mice in each group indicated. (E) Glycogen phosphorylase regulation. (Left) Muscle tissue (gastrocnemius) was extracted from 6-week-old male FL/FLβ control and MβKO mice and immunoblotted with the indicated antibodies. A representative blot is shown (below), with quantification of results from three independent experiments displayed as a bar graph (above). Total glycogen phosphorylase expression was normalized to GAPDH levels and expressed as signal intensities, while the signal intensities from the phosphorylated form of the protein are expressed relative to the WT level (100%). (Right) Muscle tissues were extracted from male FL/FLβ control and MβKO mice and homogenized, and glycogen phosphorylase activity was measured in the absence or presence of AMP as described in Materials and Methods.