Effects Of Glycogen Synthase Kinase-3 Inhibition Research Articles

Activation of Wnt/β‐catenin signalling and HIF1α stabilisation alters pluripotency and differentiation/proliferation properties of human‐induced pluripotent stem cells

Wnt/β-catenin signalling, in the microenvironment of pluripotent stem cells (PSCs), plays a critical role in their differentiation and proliferation. Contradictory reports on the role of Wnt/β-catenin signalling in PSCs self-renewal and differentiation, however, render these mechanisms largely unclear. Wnt/β-catenin signalling pathway in human-induced pluripotent stem cells (hiPSCs) was activated by inhibiting glycogen synthase kinase 3 (GSK3), driving the cellsinto a mesodermal/mesenchymal state, exhibiting proliferative, invasive and anchorage-independent growth properties, where over 70% of cell population became CD 44 (+)/CD133 (+). Wnt/β-catenin signalling activation also altered the metabolic state of hiPSCs from aerobic glycolysis to oxidative metabolismand changed their drug and oxidative stress sensitivities.These effects of GSK3inhibition were suppressedinHIF1α-stabilised cells. Persistentactivation of Wnt/β-catenin signalling endows hiPSCs with proliferative/invasive 'teratoma-like'states, shifting their metabolic dependence and allowing HIF1α-stabilisation to inhibit their proliferative/invasive properties. The hiPSC potential to differentiate into 'teratoma-like' cells suggest that stem cells may exist in two states with differential metabolic and drug dependency.

Activation of Neurogenesis in Multipotent Stem Cells Cultured In Vitro and in the Spinal Cord Tissue After Severe Injury by Inhibition of Glycogen Synthase Kinase-3

The inhibition of glycogen synthase kinase-3 (GSK-3) can induce neurogenesis, and the associated activation of Wnt/β-catenin signaling via GSK-3 inhibition may represent a means to promote motor function recovery following spinal cord injury (SCI) via increased astrocyte migration, reduced astrocyte apoptosis, and enhanced axonal growth. Herein, we assessed the effects of GSK-3 inhibition in vitro on the neurogenesis of ependymal stem/progenitor cells (epSPCs) resident in the mouse spinal cord and of human embryonic stem cell–derived neural progenitors (hESC-NPs) and human-induced pluripotent stem cell–derived neural progenitors (hiPSC-NPs) and in vivo on spinal cord tissue regeneration and motor activity after SCI. We report that the treatment of epSPCs and human pluripotent stem cell–derived neural progenitors (hPSC-NPs) with the GSK-3 inhibitor Ro3303544 activates β-catenin signaling and increases the expression of the bIII-tubulin neuronal marker; furthermore, the differentiation of Ro3303544-treated cells prompted an increase in the number of terminally differentiated neurons. Administration of a water-soluble, bioavailable form of this GSK-3 inhibitor (Ro3303544-Cl) in a severe SCI mouse model revealed the increased expression of bIII-tubulin in the injury epicenter. Treatment with Ro3303544-Cl increased survival of mature neuron types from the propriospinal tract (vGlut1, Parv) and raphe tract (5-HT), protein kinase C gamma–positive neurons, and GABAergic interneurons (GAD65/67) above the injury epicenter. Moreover, we observed higher numbers of newly born BrdU/DCX-positive neurons in Ro3303544-Cl–treated animal tissues, a reduced area delimited by astrocyte scar borders, and improved motor function. Based on this study, we believe that treating animals with epSPCs or hPSC-NPs in combination with Ro3303544-Cl deserves further investigation towards the development of a possible therapeutic strategy for SCI.

GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery

SUMMARYCanonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the β-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, β-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.

Glycogen synthase kinase 3 is part of the molecular machinery regulating the adaptive response to LPS stimulation in microglial cells

Repeated stimulation of TLR4 signaling by lipopolysaccharide (LPS) in microglia induces a state of tolerance/sensitization consisting in the reprogramming of the expression of pro-inflammatory genes in favor of anti-inflammatory ones. The molecular mechanisms underlying this adaptive response are far to be elucidated. Glycogen synthase kinase 3 (GSK3) has emerged as crucial regulator of TLR signaling, mediating the balance between pro- and anti-inflammatory functions in both periphery and central nervous system. The present study extends this notion identifying GSK3 as part of the molecular machinery regulating the LPS-adaptive response in microglial cells, by using primary microglial cultures and organotypic hippocampal slices (OHSCs). We found that lithium chloride (LiCl), a widely used GSK3 inhibitor and the mainstay treatment for bipolar disorder, reinforced the LPS adaptive response by enhancing both downregulation of pro-inflammatory genes (inducible nitric oxide synthase, interleukin 1β, interleukin 6, tumor necrosis factor α), and upregulation of genes typically associated to anti-inflammatory functions (interleukin 10 and MRC1). The effects of GSK3 inhibition were mimicked by Wnt3a, added exogenously, and reversed by Inhibitor of Wnt-Response-1-endo, a pharmacological disruptor of the canonical Wnt/β-catenin pathway, and GW9662, a selective peroxisome proliferator activated receptor γ antagonist, suggesting that these two pathways are involved in the regulation of LPS-tolerance/sensitization by GSK. Finally, LiCl treatment of OHSCs enhanced the protective functional consequences of the microglial adaptive response to LPS on oligodendrocyte maturation, as indicated by MBP mRNA upregulation. These results further indicate GSK3 as key component in the orchestration of neuroinflammation and target for neuroprotective strategies.

Abstract B167: Targeting glioblastoma invasion with GSK-3 inhibitors: Rapid effects on vimentin dynamics.

Abstract Tumor cell invasion of surrounding normal brain remains a formidable obstacle to the effective treatment of the lethal brain tumor glioblastoma. Moreover, invasion may be enhanced by radiation and bevacizumab, which are important clinical agents in glioblastoma treatment. At present there are no agents available clinically that specifically target the invading tumor cell population. We previously identified that small molecule inhibitors of the protein kinase GSK-3 (glycogen synthase kinase-3), are able to specifically block glioblastoma invasion in vitro and in vivo, where they also prolong survival in animal models bearing intracranial invasive orthotopic glioblastoma xenografts. GSK-3 is a ubiquitous multifunctional serine/threonine protein kinase involved in many signaling pathways. To better understand the functions of GSK-3 in glioblastoma we used proteomics and gene/microRNA expression profiling to determine the molecular effects of GSK-3 inhibition in glioblastoma cells with the inhibitors Lithium chloride and 6-bromoindirubin-3’-oxime (BIO). This revealed distinct and overlapping effects of each compound, including the expected upregulation of β-catenin targets such as Axin2. Proteomics revealed major changes in the cytoskeleton, with downregulation of the EMT marker vimentin as the most significant alteration. Vimentin is an intermediate filament protein that functions as an organizer of a number of critical proteins involved in attachment, migration, and cell signaling. The downregulation of vimentin was rapid and due to alterations in its dynamics in response to GSK-3 inhibition. GSK-3 and vimentin were shown to associate with each other in glioblastoma cells, and reduction in phosphorylation of vimentin serine 38 was observed in response to treatment with inhibitors. We showed that vimentin is highly expressed in patient glioblastoma samples and higher levels of vimentin are associated with poorer prognosis in glioblastoma patients. Vimentin knockdown also reduced glioblastoma cell migration. The mechanism of action of GSK-3 inhibition in the context of glioblastoma invasion and the potential of developing a therapeutic strategy based on these observations will be discussed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B167. Citation Format: Sean E. Lawler, Michal O. Nowicki, E. Antonio Chiocca. Targeting glioblastoma invasion with GSK-3 inhibitors: Rapid effects on vimentin dynamics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B167.

Glycogen synthase kinase-3 (GSK-3) regulates TGF-β₁-induced differentiation of pulmonary fibroblasts.

Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β₁-induced myofibroblast differentiation is currently largely unknown. To determine the contribution of GSK-3 signalling in TGF-β₁-induced myofibroblast differentiation. We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively. Stimulation of MRC5 and primary human lung fibroblasts with TGF-β₁ resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β₁-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β₁-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling. We demonstrate that GSK-3 signalling regulates TGF-β₁-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.

Effect of GSK-3 inhibition in vitro and in vivo on antitumor effect of sorafenib in renal cell carcinoma (RCC).

4617 Background: Sorafenib is a multikinase inhibitor which has been approved as the first-line treatment of advanced and/or metastaticrenal cell carcinoma (RCC). Sorafenib suppresses tumor growth directly by affecting tumor cells and indirectly by targeting tumor vasculature and angiogenesis. While the detailed molecular mechanisms of its action in RCC are still unclear, combining sorafenib with other signal transduction inhibitors may improve its activity and provide additional benefits to patients with RCC. Recently we showed that glycogen synthase kinase 3 (GSK-3) might be a potential new therapeutic target for RCC and targeting GSK-3 is an effective way to directly induce apoptosis in RCC, which is resistant to apoptosis-triggering treatment modalities (Br J Cancer. 101:2005-14, 2009). Methods: Here we examined effects of sorafenib on human RCC cell lines in vitro and in vivo by tumor xenograft mouse model as a single agent or in combination with a GSK-3 small molecule inhibitor AR-A014418. Protein expression and phosphorylation states were analyzed by Western blots. GSK-3 expression in cells was also confirmed by Q-RT-PCR (using TaqMan technology). Cell viability in vitro was examined by MTS assay. Morphological and biochemical features of apoptosis were detected with standard methods. Results: We detected that sorafenib induced growth inhibition of human RCC cell lines in vitro in a time (24-96 hours) and dose (2.5-10μ M) dependent manner as well as in vivo xenograft tumors in mice (30 mg/kg). Sorafenib treatment triggered apoptosis demonstrated by PARP cleavage which was more prominent with higher doses. However, in vitro sorafenib upregulated total GSK-3 expression and almost completely depleted inactive GSK3 pool as shown by diminished phosphorylation at Ser9. GSK-3 pharmacological inhibition with AR-A014418 (25 μ M, 30 mg/kg) potentiated sorafenib antitumor activity both in vitro and in vivo. Conclusions: Our results confirmed the direct antitumor effect of sorafenib in RCC andrevealed GSK-3 activation as one of potential consequence of sorafenib treatment. Targeting GSK-3 may synergize with sorafenib and this combination treatment might be a potential new approach for RCC. No significant financial relationships to disclose.

Abstract 5479: Glycogen synthase kinase-3 (GSK3) differently targets in MLL leukemia cells

Abstract Propose: We evaluated the anti-leukemic effect of GSK3 inhibitor for specific type of MLL-tranlocated cell line. Materials and methods: l GSK3 inhibitor: LiCl, SB216763, GSK3-IX l Cell line: MV4-11 [human lymphoblast, t(4:11)] MOLM-13 [human acute myeloid leukemia, ins(9:11)(q23;p22p23)] l Cell proliferation assay: XTT assay, Clonogenic assay l GSK3i activity & detection of histone acetylation and H3K4 methylation: Immunoblotting Summary: MLL gene encodes an enzyme which methylates histone H3 DNA-binding protein and positively regulates the expression of target genes in normal tissue. MLL rearrangement which is found in acute myeloid leukemia (AML), especially therapy-related AML, encodes fusion proteins that have lost H3 lys4-specific (H3K4) methyltransferase activity, and causes a transformation of hematopoietic cells into leukemia stem cells. GSK3, a serine/threonine kinase, plays a key role in glycogen metabolism and is constitutively expressed in resting cell. Although it has been thought that the inhibition of GSK3 causes apoptosis in neurons and other tissues, a paradoxical and unexpected cell death effect of GSK3 inhibition were reported recently in leukemic cell line with MLL translocation, especially t(4;11). Regarding the mechanism of the cytotoxic effect of GSK3 inhibitor, various molecules associated with the signaling pathway around MLL gene have been investigated. Acetylation of H3 peptides stimulates the activity of SET domain in MLL. MLL gene is cleaved at 2 conserved sites by taspase-1; N-terminal 320 kD fragment (N320) and a C-terminal 180 kD fragment (C180). RNA interference-mediated knockdown of taspase-1 resulted in an unprocessed MLL, and finally a loss of proper HOX gene expression. However, the exact mechanism has been still unknown and it remains to be uncertain whether GSK3 inhibitor shows cell death effect on various leukemic cell lines of MLL translocation with other partner gene. We intended to determine whether the inhibition of GSK3 induced cell death in different types of leukemia cell lines with MLL translocation. We examined the apoptotic effects of GSK3 inhibitors on MV4-11 and MOLM-13 cell line. LiCl and GSK3-IX induced cell death, H3K4 methylation and histone acetylation in dose-dependent manners on MV4-11 but did not induce on MOLM-13. However, SB216763 did not induced cell death both cell lines. Conclusion: Cell death effects with the inhibition of GSK3 may be different among leukemic cell line with MLL translocation according to the partner gene of MLL translocation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5479.

Deregulated GSK3 Sustains Gastrointestinal Cancer Cells Survival by Modulating Human Telomerase Reverse Transcriptase and Telomerase

Glycogen synthase kinase-3beta (GSK3beta) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3beta in gastrointestinal, pancreatic, and liver cancers. Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3beta expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3beta activity by in vitro kinase assay. The effects of small-molecule GSK3beta inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3beta inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Cancer cell lines and most cancer tissues showed increased GSK3beta expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3beta resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3beta inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. The results indicate that deregulated GSK3beta sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase.

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression.

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.

Short-term in vitro inhibition of glycogen synthase kinase 3 potentiates insulin signaling in type I skeletal muscle of Zucker Diabetic Fatty rats

Overactivity of glycogen synthase kinase 3 (GSK-3) is associated with insulin resistance of skeletal muscle glucose transport in prediabetic and type 2 diabetic rodent models. However, limited information is available concerning the potential molecular mechanisms underlying the role GSK-3 plays in the etiology of insulin resistance in the male Zucker Diabetic Fatty (ZDF) rat, a model of type 2 diabetes mellitus. Therefore, we assessed the functionality of proximal and distal insulin signaling elements in isolated type I (slow-twitch oxidative) soleus muscles of ZDF rats after in vitro exposure to a selective GSK-3 inhibitor (1 μmol/L CT98014, K i <10 nmol/L for GSK-3 α and GSK-3 β). Moreover, Ser307 phosphorylation of insulin receptor substrate 1 (IRS-1), which has been implicated in the development of insulin resistance, was also determined in the absence or presence of this GSK-3 inhibitor. Maximally insulin-stimulated (5 mU/mL) GSK-3 β serine phosphorylation was significantly less (35%, P < .05) in soleus muscle of ZDF rats compared with insulin-sensitive lean Zucker rats, indicating GSK-3 overactivity. In the absence of insulin, no effects of GSK-3 inhibition were detected. GSK-3 inhibition led to significant enhancement (28%) of insulin-stimulated glucose transport activity that was associated with significant up-regulation of tyrosine phosphorylation of IR (52%) and IRS-1 (50%), and with enhanced Akt Ser473 phosphorylation (48%) and GSK-3 β Ser9 phosphorylation (36%). Moreover, the selective GSK-3 inhibitor induced a significant reduction in the phosphorylation of IRS-1 Ser307 (26%) and c-jun N-terminal kinases 1 and 2 (31%), a mediator of IRS-1 Ser307 phosphorylation. These results indicate that selective inhibition of GSK-3 activity in type I skeletal muscle from overtly diabetic ZDF rats enhances IRS-1–dependent insulin signaling, possibly by a decrease in c-jun N-terminal kinase activation and a diminution of the deleterious effects of IRS-1 Ser307 phosphorylation.

Glycogen synthase kinase 3 controls endochondral bone development: Contribution of fibroblast growth factor 18

Glycogen synthase kinase 3 (GSK3) inhibits signaling pathways that are essential for bone development. To study the requirement for GSK activity during endochondral bone development, we inhibited GSK3 in cultured metatarsal bones with pharmacological antagonists. Interestingly, we find that inhibition of GSK3 strongly repressed chondrocyte and perichondrial osteoblast differentiation. Moreover, chondrocyte proliferation was inhibited, whereas perichondrial cell proliferation was stimulated. These results mirror the effects of fibroblast growth factor signaling (FGF), suggesting the FGF expression is induced. Indeed, we showed that (1) FGF18 expression is stimulated following inhibition of GSK3 and (2) GSK3 regulates FGF18 expression through the control of β-catenin levels. Stimulation of cultured metatarsal with FGF18 had similar effects on the differentiation and proliferation of chondrocytes and perichondrial cells as GSK3 repression. This suggests that the regulation of FGF18 expression is a major function of GSK3 during endochondral bone development. Consistent with this, we showed that the effect of GSK3 inhibition on chondrocyte proliferation is repressed in tissues lacking a receptor for FGF18, FGF receptor 3.

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 microM CT118637, Ki < 10 nM for GSK3alpha and GSK3beta). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced (P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR beta-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3beta serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.