Total Glycogen Synthase Kinase-3 Research Articles

Clinical significance of glycogen synthase kinase 3 (GSK-3) expression and tumor budding grade in colorectal cancer: Implications for targeted therapy

IntroductionGlycogen synthase kinase 3 (GSK-3) has been proposed as a novel cancer target due to its regulating role in both tumor and immune cells. However, the connection between GSK-3 and immunoevasive contexture, including tumor budding (TB) has not been previously examined. Methods: we investigated the expression levels of total GSK-3 as well as its isoforms (GSK-3β and GSK-3α) and examined their potential correlation with TB grade and the programmed cell death-ligand 1 (PD-L1) in colorectal cancer (CRC) tumor samples. Additionally, we compared the efficacy of GSK-3-inhibition with PD-1/PD-L1 blockade in humanized patient-derived (PDXs) xenografts models of high-grade TB CRC. Resultswe show that high-grade (BD3) TB CRC is associated with elevated expression levels of total GSK-3, specifically the GSK-3β isoform, along with increased expression of PD-L1 in tumor cells. Moreover, we define an improved risk stratification of CRC patients based on the presence of GSK-3+/PD-L1+/BD3 tumors, which are associated with a worse prognosis. Significantly, in contrast to the PD-L1/PD-1 blockade approach, the inhibition GSK-3 demonstrated a remarkable enhancement in the antitumor response. This was achieved through the reduction of tumor buds via necrosis and apoptosis pathways, along with a notable increase of activated tumor-infiltrating CD8+ T cells, NK cells, and CD4- CD8- T cells. Conclusionsour study provides compelling evidence for the clinical significance of GSK-3 expression and TB grade in risk stratification of CRC patients. Moreover, our findings strongly support GSK-3 inhibition as an effective therapy specifically targeting high-grade TB in CRC.

Neuroprotective Properties of Minocycline Against Methylphenidate-Induced Neurodegeneration: Possible Role of CREB/BDNF and Akt/GSK3 Signaling Pathways in Rat Hippocampus

Neurodegeneration is a side effect of methylphenidate (MPH), and minocycline possesses neuroprotective properties. This study aimed to investigate the neuroprotective effects of minocycline against methylphenidate-induced neurodegeneration mediated by signaling pathways of CREB/BDNF and Akt/GSK3. Seven groups of seventy male rats were randomly distributed in seven groups (n = 10). Group 1 received 0.7ml/rat of normal saline (i.p.), and group 2 was treated with MPH (10mg/kg, i.p.). Groups 3, 4, 5, and 6 were simultaneously administered MPH (10mg/kg) and minocycline (10, 20, 30, and 40mg/kg, i.p.) for 21days. Minocycline alone (40mg/kg, i.p.) was administrated to group 7. Open field test (OFT) (on day 22), forced swim test (FST) (on day 24), and elevated plus maze (on day 26) were conducted to analyze the mood-related behaviors; hippocampal oxidative stress, inflammatory, and apoptotic parameters, as well as the levels of protein kinase B (Akt-1), glycogen synthase kinase 3 (GSK3), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF), were also assessed. Furthermore, localization of total CREB, Akt, and GSK3 in the DG and CA1 areas of the hippocampus were measured using immunohistochemistry (IHC). Histological changes in the mentioned areas were also evaluated. Minocycline treatment inhibited MPH-induced mood disorders and decreased lipid peroxidation, oxidized form of glutathione (GSSG), interleukin 1 beta (IL-1β), alpha tumor necrosis factor (TNF-α), Bax, and GSK3 levels. In the contrary, it increased the levels of reduced form of glutathione (GSH), Bcl-2, CREB, BDNF, and Akt-1 and superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) activities in the experimental animals' hippocampus. IHC data showed that minocycline also improved the localization and expression of CREB and Akt positive cells and decreased the GSK3 positive cells in the DG and CA1 regions of the hippocampus of MPH-treated rats. Minocycline also inhibited MPH-induced changes of hippocampal cells' density and shape in both DG and CA1 areas of the hippocampus. According to obtained data, it can be concluded that minocycline probably via activation of the P-CREB/BDNF or Akt/GSK3 signaling pathway can confer its neuroprotective effects against MPH-induced neurodegeneration.

Tideglusib inhibition of GSK3 promotes the oxidative muscle phenotype and reduces serum creatine kinase in D2 mdx mice

Introduction Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by an absence of dystrophin that compromises membrane integrity, ultimately resulting in muscle weakness, wasting, and premature death. The fast glycolytic muscle fibres are known to be most susceptible to dystrophic pathology, while slow oxidative fibres are less affected. Thus, promoting the slow oxidative phenotype has become a viable therapeutic strategy. Recent work from our lab has shown that inhibiting the enzyme glycogen synthase kinase 3 (GSK3) can promote the slow oxidative phenotype leading to enhancements in fatigue resistance. Furthermore, we have shown that inhibiting GSK3 augments muscle specific force production and myoblast fusion. Therefore, inhibiting GSK3 may aid in alleviating dystrophic pathology. Tideglusib is a potent GSK3 inhibitor currently undergoing clinical trials for myotonic dystrophy, another form of muscular dystrophy. Here, we tested whether treating the DMD preclinical mdx mouse with tideglusib would alter muscle oxidative phenotype, improve muscle function, and reduce serum creatine kinase (CK) levels, a marker of cellular damage. Methods Male DBA/2J wild type (WT) and mdx mice were ordered from Jackson Laboratories at 5-6 weeks of age. Three groups were included in this study; 1) WT healthy control, 2) mdx tideglusib (10 mg/kg/day, via oral gavage), and 3) mdx vehicle (26% peg400, 15% Chremaphor EL and water). The mdx-tideglusib and vehicle mice underwent their respective treatments for 2 weeks with ad libitum access to food and water. Subsequently, all mice were subjected to a hangwire test to assess muscle function. Mice were then euthanized and their serum extracted for CK activity analyses using a commercially available kit that was fitted onto a 96-well plate. Extensor digitorum longus (EDL) muscles were collected and homogenized for Western blotting to investigate phosphorylated (serine 9) and total GSK3b content along with myosin heavy chain (MHC) I and IIa levels. Results The hangwire test results showed that mdx mice treated with tideglusib were able to sustain hanging on the wire for a significantly longer period of time compared with the mdx vehicle group (p = 0.03). Serum CK analyses revealed that while the mdx vehicle group had significantly higher levels of activity compared with WT (p = 0.009), there was a 30% reduction in the mdx tideglusib mice that was no longer significantly different from WT. Western blotting revealed that though phosphorylated GSK3b levels were unaltered, tideglusib treatment led to a significant reduction in GSK3b content compared with vehicle (p = 0.04). Additionally, there was a significant increase in the oxidative MHC isoforms (I and IIa, p = 0.02) in the mdx tideglusib group compared with vehicle. Conclusions Our results demonstrate the potential use of tideglusib for DMD. We show that tideglusib treatment inhibited GSK3 in mdx mice through a reduction in total GSK3 content. In turn, we also found a significant increase in oxidative MHC isoforms (I and IIa), which was associated with enhanced muscle performance and reduced muscle damage.

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Abstract 52: Glycogen synthase kinase inhibition associated with Notch-1 reduction in cholangiocarcinoma

Abstract Introduction: Cholangiocarcinoma (CCA) is the most common biliary cancer and the second most common hepatic malignancy after hepatocellular carcinoma with incidence in the United States steadily increasing. It is a highly malignant adenocarcinoma characterized by poor prognosis and chemotherapeutic inefficacy with 5-year survival rates remaining at less than 10%. Currently, surgical resection is the only effective strategy; however, the majority of patients (90%) are not candidates due to metastasis and concomitant liver disease. Furthermore, local and distant recurrence and high morbidity rates are common following resection. Therefore, new targeted molecular strategies against CCA are imperative. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target as it is overexpressed in various cancer types including CCA acting as an oncogene in these cancers. However, the role of the GSK-3 isoform and their interaction with Notch-1 remains unclear in CCA. Our findings and other reports demonstrate that the inhibition of Notch-1 in CCA leads to growth suppression. We hypothesize that GSK-3 stabilizes Notch-1 and thereby promoting cellular proliferation in CCA. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 (AR) on the levels of GSK-3 isoforms and further delineated the effect of specific GSK-3 inhibition on Notch-1 signaling and growth in CCA. Methods: The effects of AR on CCA cell lines (CCLP-1 and SG-231) were assessed by MTT assay and live cell imaging. Cell lysates were analyzed via Western blotting for active and total GSK-3, β-catenin, Notch-1, pro-apoptotic and anti-apoptotic proteins. Transient knockout against Notch-1 was used to determine the interaction of Notch-1 in CCA whereas knockout of GSK-3α or β was used to analyze the interaction with Notch-1 and delineate isoform specificity in CCA. Results: Increasing AR treatment (0μM - 20μM) had a significant dose-dependent growth reduction (p<0.001); starting at 5μM in CCA cells, compared to control. Upon Western analysis, growth suppression due to apoptosis was evidenced by increased expression of pro-apoptotic and reduced anti-apoptotic proteins. Inhibition of GSK-3 by AR reduced the levels of active GSK-3 expression with concomitant reduction in Notch-1 expression in a dose-dependent manner. Importantly, specific knock-down of GSK-3α resulted in Notch-1 reduction compared to GSK-3β knock-down. However, shRNA against Notch-1 had no effect on the expression of either GSK-3 isoform. Conclusion: AR-A014418, a GSK-3 inhibitor, effectively inhibits CCA growth in cell culture through both GSK-3 and Notch-1 pathways. Transient knockout of GSK-3 isoforms demonstrated specificity toward GSK-3α in reducing Notch-1 suggesting GSK-3α may be required for Notch-1 stability. This is the first study to effectively correlate GSK-3α with Notch-1 in cholangiocarcinoma. Citation Format: Kevin M. Sokolowski, Selvi Kunnimalaiyaan, T Clark Gamblin, Muthusamy Kunnimalaiyaan. Glycogen synthase kinase inhibition associated with Notch-1 reduction in cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 52. doi:10.1158/1538-7445.AM2015-52

The role of activation of PERK in activating glycogen synthase kinase 3(GSK-3) by oleic acid(OA) in type 2 diabetes

ER-stress induced apoptosis of beta cells is an important mechanism of type 2 diabetes. PERK can be activated by the overactivation of ER-stress which will induce beta cells apoptosis. This study is to reveal the role of ER-stress, GSK-3 and the potential signal pathway during beta cells apoptosis. The alterations of ER-stress related signal factors and kinases induced by OA are assessed by western blot, and the changes of PERK and AMPK are analyzed by ELISA meanwhile. The phosphorylated GSK-3β and total GSK-3 are detected by western blot, while PERK are inhibited by the transfection of P58IPK plasmid and AMPK are inhibited by the inhibitor Compound C. Finally, the interaction between PERK and GSK-3 are identified by co- immunoprecipitation and direct immuno-fluorescence. The study shows 1. The expression of GRP78, ATF6, XBP1, PERK and AMPK significant increased in the presence of 0.4mM OA(P 0.05). 4. Co-immunoprecipitation of PERK and GSK-3 verified the association of PERK and GSK-3. 5. Obvious co-localization was observed after merging FITC-conjugated PERK and Rhodamine-conjugated GSK-3β. These results suggest activation of GSK-3 can induce beta cells apoptosis by ER-stress which is caused after long-time exposure to free fatty acids. PERK will activate GSK-3 directly. Collectively, PERK-GSK-3 signal pathway will play an important role during beta cell apoptosis in type 2 DM.

Benfotiamine prevents increased β-amyloid production in HEK cells induced by high glucose

To determine whether high glucose enhances β-amyloid (Aβ) production in HEK293 Swedish mutant (APPsw) cells with Aβ precursor protein (APP) overexpression, and whether under this condition benfotiamine reduces the increased Aβ production. HEK293 APPsw cells were cultured with different concentrations of glucose for different times. The Aβ content in the supernatant was determined by ELISA. To investigate the mechanism by which benfotiamine reduced Aβ production, glycogen synthase kinase-3 (GSK-3) activity and expression were measured after the cells were cultured with 5.5 g/L glucose for 12 h. With 1.0, 3.0, 4.5, 5.5, 6.5, 7.5, 8.5, or 10.5 g/L glucose, Aβ production by HEK293 APPsw cells was highest in the presence of 5.5 g/L glucose for 6 and 12 h. The difference in Aβ content between 5.5 and 1.0 g/L was most marked after incubation for 12 h. Benfotiamine at 20 and 40 μg/mL significantly reduced Aβ production in cells incubated with 5.5 g/L glucose for 12 h. Moreover, 40 μg/mL benfotiamine significantly enhanced the ratio of phosphorylated GSK-3 to total GSK-3, together with consistent down-regulation of GSK-3 activity. High glucose increases Aβ production by HEK293 APPsw cells while benfotiamine prevents this increase. This is correlated with the modulation of GSK-3 activity.

Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog

The terrestrial anuran Rana sylvatica tolerates extended periods of whole-body freezing during the winter. Freezing survival is facilitated by extensive glycogen hydrolysis and distribution of high concentrations of the cryoprotectant glucose into blood and all tissues. As glycogenesis is both an energy-expensive process and counter-productive to maintaining sustained high cryoprotectant levels, we proposed that glycogen synthase kinase-3 (GSK-3) would be activated when wood frogs froze and would phosphorylate its downstream substrates to inactivate glycogen synthesis. Western blot analysis determined that the amount of phosphorylated (inactive) GSK-3 decreased in all five tissues tested in 24 h frozen frogs compared with unfrozen controls. Total GSK-3 protein levels did not change, with the exception of heart GSK-3, indicating that post-translational modification was the primary regulatory mechanism for this kinase. Kinetic properties of skeletal muscle GSK-3 from control and frozen frogs displayed differential responses to a temperature change (22 versus 4°C) and high glucose. For example, when assayed at 4°C, the K(m) for the GSK-3 substrate peptide was ∼44% lower for frozen frogs than the corresponding value in control frogs, indicating greater GSK-3 affinity for its substrates in the frozen state. This indicates that at temperatures similar to the environment encountered by frogs, GSK-3 in frozen frogs will phosphorylate its downstream targets more readily than in unfrozen controls. GSK-3 from skeletal muscle of control frogs was also allosterically regulated. AMP and phosphoenolpyruvate activated GSK-3 whereas inhibitors included glucose, glucose 6-phosphate, pyruvate, ATP, glutamate, glutamine, glycerol, NH(4)Cl, NaCl and KCl. The combination of phosphorylation and allosteric control argues for a regulatory role of GSK-3 in inactivating glycogenesis to preserve high glucose cryoprotectant levels throughout each freezing bout.

Insulin Receptor Expression and Activity in the Brains of Nondiabetic Sporadic Alzheimer’s Disease Cases

We investigated the contents of the insulin receptor-beta subunit (IRβ) and [Tyr1162/1163]-phosphorylated IRβ as surrogate indices of total IR content and IR activation in postmortem hippocampal formation brain specimens from nondiabetic sporadic Alzheimer's disease (AD) cases. We found no significant changes in the brain contents of total IRβ or [Tyr1162/1163]-phosphorylated IRβ, suggesting normal IR content and activation in the brains of nondiabetic sporadic AD cases. Moreover, total IRβ and [Tyr1162/1163]-phosphorylated IRβ levels in the hippocampal formation are not correlated with the severity of amyloid or tau-neuropathology. Exploring the regulation of glycogen synthase kinase 3 (GSK3) α/β, key IR-signaling components, we observed significantly lower levels of total GSK3 α/β in brain specimens from nondiabetic AD cases, suggesting that impaired IR signaling mechanisms might contribute to the onset and/or progression of AD dementia. Outcomes from our study support the development of insulin-sensitizing therapeutic strategies to stimulate downstream IR signaling in nondiabetic AD cases.

Glucose Uptake in Rat Extraocular Muscles: Effect of Insulin and Contractile Activity

Extraocular muscles show specific adaptations to fulfill the metabolic demands imposed by their constant activity. One aspect that has not been explored is the availability of substrate for energy pathways in extraocular muscles. In limb muscles, glucose enters by way of GLUT1 and GLUT4 transporters in a process regulated by insulin and contractile activity to match metabolic supply to demand. This mechanism may not apply to extraocular muscles because their constant activity may require high basal (insulin- and activity-independent) glucose uptake. The authors tested the hypothesis that glucose uptake by extraocular muscles is not regulated by insulin or contractile activity. Extraocular muscles from adult male Sprague-Dawley rats were incubated with 100 nM insulin or were electrically stimulated to contract (activity); glucose uptake was measured with 2-deoxy-d[1,2-(3)H]glucose. The contents of GLUT1, GLUT4, total and phosphorylated protein kinase B (Akt), phosphorylated AMP-activated protein kinase (AMPK), and glycogen synthase kinase 3 (GSK3) underwent Western blot analysis. Insulin and activity increased glucose uptake over the basal rate to 108% and 78%, respectively. GLUT1 and GLUT4 were detectable in extraocular muscles. Phosphorylated AKT/total AKT increased by twofold after insulin stimulation, but there was no change with activity. AMPK phosphorylation increased 35% with activity. Phosphorylated-GSK3/total GSK3 did not change with insulin or activity. Glucose uptake in extraocular muscles is regulated by insulin and contractile activity. There is evidence of differences in the insulin signaling pathway that may explain the low glycogen content in these muscles.

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.

Diet-Induced Hyperhomocysteinemia Increases Amyloid-β Formation and Deposition in a Mouse Model of Alzheimers Disease

Hyperhomocysteinemia (HHcy) has been recognized as a risk factor for developing Alzheimer's disease (AD). However, its underlying molecular mechanisms are still elusive. Here we show that HHcy induces an elevation of amyloid beta (Abeta) levels and deposition, as well as behavioral impairments, in a mouse model of AD-like amyloidosis, the Tg2576 mice. This elevation is not associated with significant change of the steady state levels of the Abeta precursor protein (APP), beta- or alpha-secretase pathways, nor with the Abeta catabolic pathways. By contrast, HHcy significantly reduces glycogen synthase kinase 3 (GSK3) Ser21/9 phosphorylation, but not total GSK3 protein levels. Similar results are obtained in brains homogenates from a genetic mouse model of HHcy. In vitro studies show that homocysteine increases Abeta formation, reduces phosphorylated GSK3 levels, without changes in total APP and its metabolism, and these effects are prevented by selective GSK3 inhibition. Overall, these data support a potential link between GSK3 and the pro-amyloidotic effect of HHcy in vivo and in vitro.

Cocaine regulates protein kinase B and glycogen synthase kinase‐3 activity in selective regions of rat brain

Protein kinase B (also known as Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase 3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of three daily injections of 15 mg/kg cocaine or 1 mL/kg saline spaced 1 h apart for 1, 3, or 14 days. Amygdala, nucleus accumbens, caudate putamen, and hippocampus tissues were dissected 30 min following the last injection and analyzed for phosphorylated and total Akt and GSK3(alpha and beta) protein levels using western blot analysis. Phosphorylation of Akt on the threonine-308 (Thr308) residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 was significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure.

Hepatocyte CYP2E1 Overexpression and Steatohepatitis Lead to Impaired Hepatic Insulin Signaling

Insulin resistance and increased cytochrome P450 2E1 (CYP2E1) expression are both associated with and mechanistically implicated in the development of nonalcoholic fatty liver disease. Although currently viewed as distinct factors, insulin resistance and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signaling. To test this possibility, the effects of in vitro and in vivo CYP2E1 overexpression on hepatocyte insulin signaling were examined. CYP2E1 overexpression in a hepatocyte cell line decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in response to insulin. CYP2E1 overexpression was also associated with increased inhibitory serine 307 and 636/639 IRS-1 phosphorylation. In parallel, the effects of insulin on Akt activation, glycogen synthase kinase 3, and FoxO1a phosphorylation, and glucose secretion were all significantly decreased in CYP2E1 overexpressing cells. This inhibition of insulin signaling by CYP2E1 overexpression was partially c-Jun N-terminal kinase dependent. In the methionine- and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression, insulin-induced IRS-1, IRS-2, and Akt phosphorylation were similarly decreased. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signaling, potentially contributing to the insulin resistance associated with nonalcoholic fatty liver disease.

Glycogen synthase kinase-3 is increased in white cells early in Alzheimer's disease

Alzheimer's disease (AD) is a disorder without a molecular marker in peripheral tissues or a disease modifying treatment. As increasing evidence has suggested a role for glycogen synthase kinase-3 (GSK-3) in the pathogenesis of the condition we measured total GSK-3 protein (alpha and beta isoforms) and GSK-3 activity (serine 9 phosphorylation) in a group of healthy elderly people, in AD and in mild cognitive impairment (MCI). Total GSK-3 protein was increased in both AD and in MCI without a compensatory decrease in activity. These data suggest that GSK-3 assays might be a useful diagnostic marker in a readily available tissue and moreover that GSK-3 activity is increased in the prodromal phase of the disorder suggesting that inhibition of GSK-3 might be a useful therapeutic strategy.

Increased total and phosphorylated glycogen synthase kinase-3 (GSK-3) in the adipocytes of patients with the polycystic ovary syndrome (PCOS)

Increased total and phosphorylated glycogen synthase kinase-3 (GSK-3) in the adipocytes of patients with the polycystic ovary syndrome (PCOS)

GSK-3 parameters in postmortem frontal cortex and hippocampus of schizophrenic patients

The protein kinase glycogen synthase kinase-3 (GSK-3) is highly abundant in brain and involved in signal transduction cascades, particularly during neurodevelopment. We have previously found reduced GSK-3β mRNA levels, protein levels and GSK-3 total (α+β isoforms) activity in postmortem frontal cortex of schizophrenic patients in the Stanley Medical Research Institute's Brain Collection. To verify and extend these findings, GSK-3 parameters were now measured in the frontal cortex (BA9) and hippocampus obtained from the Rebecca L. Cooper Research Laboratories postmortem brain collection. Fifteen pairs of schizophrenic patients and matched control subjects have been studied. No significant differences in GSK-3α and GSK-3β mRNA levels, GSK-3β protein levels or total GSK-3 (α+β) activity were found in the frontal cortex of the two diagnostic groups. Hippocampal GSK-3α and GSK-3β mRNA levels were significantly lower (22% and 28%, respectively) in the tissue from the schizophrenic patients compared with the normal controls. Hippocampal GSK-3β protein levels in the schizophrenic patients were 24% significantly lower than control values only after omission of three outlier subjects. Hippocampal total GSK-3 (α+β) activity in the patients was 31% lower in the schizophrenic patients vs. control subjects. This difference was marginally significant. While our previous data on GSK-3β in postmortem brain and the recent report that there is impaired AKT1-GSK-3β signaling in schizophrenia suggest that changes in pathways involving protein kinases such as AKT1 and GSK-3β in schizophrenia are complex, our present data do not provide strong evidence in support of the involvement of GSK-3β in schizophrenia. Therefore, further investigation in a greater number of brain samples is warranted to better clarify the possible role of this enzyme in the pathophysiology of schizophrenia.

GSK-3 parameters in lymphocytes of schizophrenic patients

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase that is involved in the signal transduction cascades of multiple cellular processes. GSK-3 has two isoforms, designated α and β. GSK-3β protein levels and GSK-3 enzyme activity have been reported to be reduced by over 40% in postmortem frontal cortex of schizophrenic patients. GSK-3 is also present in peripheral tissue such as lymphocytes. In this study we aimed to find whether the reduction in brain GSK-3β measures is reflected in peripheral tissue of schizophrenic patients. Fresh lymphocytes from schizophrenic patients showed no difference in GSK-3α and GSK-3β mRNA levels, GSK-3β protein levels, or total GSK-3 (α+β) enzyme activity compared with findings in control subjects. In addition, lymphocyte-derived cell lines from schizophrenic patients did not differ in their GSK-3β protein levels from levels in normal control subjects. The results rule out the use of lymphocyte GSK-3 as a marker for central GSK-3 abnormalities in schizophrenia.

Pictures in Cell Biology: GSK-3 and regulation of kinesin function

Kinesins mediate trafficking of membrane-bounded organelles towards microtubule plus-ends. Different proteins and cargos are targeted for different locations, but, until recently, little was known about regulation of kinesin-based motility. Phosphorylation of kinesin light chains by glycogen synthase kinase 3 (GSK-3) [1xGlycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. Morfini, G et al. EMBO J. 2002; 23: 281–293Crossref | Scopus (261)See all References[1] has now been shown to provide one such regulatory mechanism. GSK-3 inhibits anterograde, but not retrograde, transport in squid axoplasm and reduces amounts of kinesin bound to membranes. Kinesin activities other than cargo binding are unaffected by GSK-3 phosphorylation. As predicted, active GSK-3 is localized preferentially in regions known to be sites of membrane delivery.In this set of images (Fig. 1Fig. 1), distributions for tubulin [green; (c)] and total GSK-3 [red; (a)] are compared in a rat hippocampal neuron and oligodendrocyte. Microtubule-containing regions in both neurons and glia co-label for GSK-3 [yellow (b)]. This distribution is similar to that seen for kinesin [1.xGlycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. Morfini, G et al. EMBO J. 2002; 23: 281–293Crossref | Scopus (261)See all References, 2.xMonoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. Pfister, K.K et al. J. Cell Biol. 1989; 108: 1453–1463Crossref | PubMedSee all References]. However, in locations where membrane proteins are delivered, such as tips of elongating processes (i.e. growth cones) or membrane sheets of oligodendrocytes, GSK-3 immunoreactivity is distinct from that of microtubules. Moreover, the GSK-3 in growth cones and similar regions is preferentially activated, consistent with a role for GSK-3 phosphorylation of kinesin in regulating delivery of some membrane proteins to specific subcellular domains.Fig. 1Localization of glycogen synthase kinase 3 [GSK-3; (a)] and tubulin (c) in a rat hippocampal neuron and oligodendrocyte. In the merged image (b), GSK-3 is shown in red and tubulin in green; colocalization is shown in yellow. Rat hippocampal cultures were a generous gift of Ann Marie Craig, Washington University, St Louis, MO, USA.View Large Image | Download PowerPoint Slide

Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes.

Glycogen synthase (GS) activity is reduced in skeletal muscle of type 2 diabetes, despite normal protein expression, consistent with altered GS regulation. Glycogen synthase kinase-3 (GSK-3) is involved in regulation (phosphorylation and deactivation) of GS. To access the potential role of GSK-3 in insulin resistance and reduced GS activity in type 2 diabetes, the expression and activity of GSK-3 were studied in biopsies of vastus lateralis from type 2 and nondiabetic subjects before and after 3-h hyperinsulinemic (300 mU x m(-2) x min(-1))-euglycemic clamps. The specific activity of GSK-3alpha did not differ between nondiabetic and diabetic muscle and was decreased similarly after 3-h insulin infusion. However, protein levels of both alpha and beta isoforms of GSK-3 were elevated (approximately 30%) in diabetic muscle compared with lean (P < 0.01) and weight-matched obese nondiabetic subjects (P < 0.05) and were unchanged by insulin infusion. Thus, both basal and insulin-stimulated total GSK-3 activities were elevated by approximately twofold in diabetic muscle. GSK-3 expression was related to in vivo insulin action, as GSK-3 protein was negatively correlated with maximal insulin-stimulated glucose disposal rates. In summary, GSK-3 protein levels and total activities are 1) elevated in type 2 diabetic muscle independent of obesity and 2) inversely correlated with both GS activity and maximally insulin-stimulated glucose disposal. We conclude that increased GSK-3 expression in diabetic muscle may contribute to the impaired GS activity and skeletal muscle insulin resistance present in type 2 diabetes.