Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.     

Tissue-specific analysis of glycogen synthase kinase-3α (GSK-3α) in glucose metabolism: effect of strain variation

Background

Over-activity and elevated expression of glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology of insulin resistance and Type 2 diabetes. Administration of specific GSK-3 inhibitors to diabetic or obese rodent models improves glycaemic control and insulin sensitivity. However, due to the indiscriminatory nature of these inhibitors, the relative contribution of the two isoforms of GSK-3 (GSK-3α and GSK-3β) is not known. Recently, we demonstrated that an out-bred strain of mice (ICR) lacking expression of GSK-3α in all tissues displayed improved insulin sensitivity and enhanced hepatic glucose metabolism. We also found that muscle (but not liver) inactivation of GSK-3β conferred insulin and glucose sensitization in an in-bred strain of mice (C57BL/6).

Methodology/Principal Findings

Here, we have employed tissue-specific deletion of GSK-3α, to examine the relative contribution of two insulin-sensitive tissues, muscle and liver, towards the insulin sensitization phenotype originally observed in the global GSK-3α KO animals. We found that mice in which GSK-3α has been inactivated in either skeletal-muscle or liver displayed no differences in glucose tolerance or insulin sensitivity compared to wild type littermates. Given the strain differences in our original analyses, we examined the insulin and glucose sensitivity of global GSK-3α KO animals bred onto a C57BL/6 background. These animals also revealed no significant differences in glucose metabolism/insulin sensitivity compared to their wild type littermates. Furthermore, deletion of hepatic GSK-3α on the out-bred, ICR background failed to reproduce the insulin sensitivity manifested by the global deletion of this isoform.

Conclusions/Significance

From these data we conclude that the improved insulin sensitivity and hepatic glucose homeostasis phenotype observed upon global inactivation of GSK-3α is strain-specific. We surmise that the insulin-sensitization observed in the out-bred strain of mice lacking GSK-3α is mediated by indirect means that do not require intrinsic function of GSK-3α in skeletal muscle and liver tissues.     

Suppressive Regulatory T Cell Activity Is Potentiated by Glycogen Synthase Kinase 3β Inhibition

The mechanism by which regulatory T (Treg) cells suppress the immune response is not well defined. A recent study has shown that β-catenin prolongs Treg cell survival. Because β-catenin is regulated by glycogen synthase kinase 3β (GSK-3β)-directed phosphorylation, we focused on GSK-3β and the role it plays in Treg cell function. Inhibition of GSK-3β led to increased suppression activity by Treg cells. Inhibitor-treated Treg cells exhibited prolonged FoxP3 expression and increased levels of β-catenin and of the antiapoptotic protein Bcl-xL. Systemic administration of GSK-3β inhibitor resulted in prolonged islet survival in an allotransplant mouse model. Our data suggest that GSK-3β could be a useful target in developing strategies designed to increase the stability and function of Treg cells for inducing allotransplant tolerance or treating autoimmune conditions.     

Glycogen synthase kinase 3β inhibition enhanced proliferation,migration and functional re-endothelialization of endothelial progenitor cells in hypercholesterolemia microenvironment

Hypercholesterolemia impairs the quantity and function of endothelial progenitor cell. We hypothesized that glycogen synthase kinase 3β activity is involved in regulating biological function of endothelial progenitor cells in hypercholesterolemia microenvironment. For study, endothelial progenitor cells derived from apolipoprotein E-deficient mice fed with high-fat diet were used. Glycogen synthase kinase 3β activity was interfered with glycogen synthase kinase 3β inhibitor lithium chloride or transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β (GSK3β-KM). Functions of endothelial progenitor cells, proliferation, migration, secretion and network formation of endothelial progenitor cells were assessed in vitro. The expression of phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 in endothelial progenitor cells was detected by Western blot. The in vivo function re-endothelialization and vasodilation were also analyzed by artery injury model transplanted with glycogen synthase kinase 3β-inhibited endothelial progenitor cells. We demonstrated that while the proliferation, migration, network formation as well as VEGF and NO secretion were impaired in apolipoprotein E-deficient endothelial progenitor cells, glycogen synthase kinase 3β inhibition significantly improved all these functions. Apolipoprotein E-deficient endothelial progenitor cells showed decreased phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 expression, whereas these signals were enhanced by glycogen synthase kinase 3β inhibition and accompanied with β-catenin nuclear translocation. Our in vivo model showed that glycogen synthase kinase 3β inhibition remarkably increased re-endothelial and vasodilation. Taken together, our data suggest that inhibition of glycogen synthase kinase 3β is associated with endothelial progenitor cell biological functions both in vitro and in vivo. It might be an important interference target in hypercholesterolemia microenvironment.     

Inhibitory Phosphorylation of GSK-3 by CaMKII Couples Depolarization to Neuronal Survival

Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3α and GSK-3β, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3α/β. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3α/β mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3β protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90^RSK. CaMKII associated with and phosphorylated GSK-3α/β. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca²⁺/calmodulin/CaMKII/GSK-3 pathway that couples depolarization to neuronal survival.     

Axil, a Member of the Axin Family, Interacts with Both Glycogen Synthase Kinase 3β and β-Catenin and Inhibits Axis Formation of Xenopus Embryos

Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3β (GSK-3β). This protein had 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway.We designated this protein Axil for Axin like. Like Axin, Axil ventralized Xenopus embryos and inhibited Xwnt8-induced Xenopus axis duplication. Axil was phosphorylated by GSK-3β. Axil bound not only to GSK-3β but also to β-catenin, and the GSK-3β-binding site of Axil was distinct from the β-catenin-binding site. Furthermore, Axil enhanced GSK-3β-dependent phosphorylation of β-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3β-dependent phosphorylation of β-catenin, thereby inhibiting axis formation.     

Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.     

Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β) gene with differential expression patterns and regulatory functions

Background

Glycogen synthase kinase 3 (GSK3α and GSK3β) are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer’s disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment.

Methodology

Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms.

Conclusions

We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.     

PKCβ Positively Regulates RANKL-Induced Osteoclastogenesis by Inactivating GSK-3β

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-κB ligand (RANKL) signaling has remained elusive. We now demonstrate that PKCβ acts as a positive regulator which inactivates glycogen synthase kinase-3β (GSK-3β) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, PKCβ expression is increased by RANKL. Pharmacological inhibition of PKCβ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-3β was decreased by PKCβ inhibition. Likewise, down-regulation of PKCβ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-3β phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the PKCβ pathway, leading to GSK-3β inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for PKCβ’s therapeutic targeting to treat inflammation-related bone diseases.     

Resveratrol regulates na?ve CD 8+ T-cell proliferation by upregulating IFN-γ-induced tryptophanyl-tRNA synthetase expression

We found that resveratrol enhances interferon (IFN)-γ-induced tryptophanyl-tRNA-synthetase (TTS) expression in bone marrow-derived dendritic cells (BMDCs). Resveratrol-induced TTS expression is associated with glycogen synthase kinase-3β (GSK-3β) activity. In addition, we found that resveratrol regulates naïve CD8⁺ T-cell polarization by modulating GSK-3β activity in IFN-γ-stimulated BMDCs, and that resveratol induces upregulation of TTS in CD8⁺ T-cells in the in vivo tumor environment. Taken together, resveratrol upregulates IFN-γ-induced TTS expression in a GSK-3β-dependent manner, and this TTS modulation is crucial for DC-mediated T-cell modulation. [BMB Reports 2015; 48(5): 283-288]     

Novel MicroRNA Reporter Uncovers Repression of Let-7 by GSK-3β

Several members of the let-7 microRNA family are downregulated in ovarian and other cancers. They are thought to act as tumor suppressors by lowering growth-promoting and anti-apoptotic proteins. In order to measure cellular let-7 levels systematically, we have developed a highly sensitive let-7 reporter assay system based on the expression of a chimeric mRNA that contains the luciferase coding region and a 3′-untranslated region (UTR) bearing two let-7-binding sites. In cells expressing the reporter construct, termed pmirGLO-let7, luciferase activity was high when let-7 was absent, while luciferase activity was low when let-7 levels were elevated. The ovarian cancer cell lines BG-1 and UCI-101 were transfected with the let-7 reporter and surveyed with a library of kinase inhibitors in order to identify pathways affecting let-7 activity. Among the inhibitors causing changes in endogenous let-7 abundance, the lowering of glycogen synthase kinase 3 (GSK-3)β function specifically increased let-7 levels and lowered luciferase activity. Similarly, silencing GSK-3β increased both mature and primary-let-7 levels in BG-1 cells, and decreased BG-1 cell survival. Further studies identified p53 as a downstream effector of the GSK-3β-mediated repression of let-7 biosynthesis. Our studies highlight GSK-3β as a novel therapeutic target in ovarian tumorigenesis.     

Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.     

Novel Reporter Alleles of GSK-3α and GSK-3β

Glycogen Synthase Kinase 3 (GSK-3) is a key player in development, physiology and disease. Because of this, GSK-3 inhibitors are increasingly being explored for a variety of applications. In addition most analyses focus on GSK-3β and overlook the closely related protein GSK-3α. Here, we describe novel GSK-3α and GSK-3β mouse alleles that allow us to visualise expression of their respective mRNAs by tracking β-galactosidase activity. We used these new lacZ alleles to compare expression in the palate and cranial sutures and found that there was indeed differential expression. Furthermore, both are loss of function alleles and can be used to generate homozygous mutant mice; in addition, excision of the lacZ cassette from GSK-3α creates a Cre-dependent tissue-specific knockout. As expected, GSK3α mutants were viable, while GSK3β mutants died after birth with a complete cleft palate. We also assessed the GSK-3α mutants for cranial and sternal phenotypes and found that they were essentially normal. Finally, we observed gestational lethality in compound GSK-3β^−/−; GSK3α^+/− mutants, suggesting that GSK-3 dosage is critical during embryonic development.     

Resistin disrupts glycogen synthesis under high insulin and high glucose levels by down-regulating the hepatic levels of GSK3β

The effect of mouse resistin on hepatic insulin resistance in vivo and in vitro, and its possible molecular mechanism were examined. Focusing on liver glycogen metabolism and gluconeogenesis, which are important parts of glucose metabolism, in primary cultures of rat hepatocytes we found that glycogen content was significantly lower (P < 0.05) after treatment with recombinant murine resistin only in the presence of insulin plus glucose stimulation. Protein levels of factors in the insulin signaling pathway involved in glycogen synthesis were examined by Western blot analysis, with the only significant change observed being the level of phosphorylated (at Ser 9) glycogen synthase kinase-3β (GSK-3β) (P < 0.001). No differences in the protein levels for the insulin receptor β (IRβ), insulin receptor substrates (IRS1 and IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) or their phosphorylated forms were observed between control and resistin treated primary rat hepatocytes. In a mouse model with high liver-specific expression of resistin, fasting blood glucose levels and liver glycogen content changed. Fasting blood glucose levels were significantly higher (P < 0.001) in the model mice, compared to the control mice, while the glycogen content of the liver tissue was about 60% of that of the control mice (P < 0.05). The gluconeogenic response was not altered between the experimental and control mice. The level of phosphorylated GSK-3β in the liver tissue was also decreased (P < 0.05) in the model mice, consistent with the results from the primary rat hepatocytes. Our results suggest that resistin reduces the levels of GSK-3β phosphorylated at Ser 9 leading to impaired hepatic insulin action in primary rat hepatocytes and in a mouse model with high liver-specific expression of resistin.