Expression Of Glycogen Synthase Kinase-3β Research Articles

Ampelopsin attenuates 6-OHDA-induced neurotoxicity by regulating GSK-3β/NRF2/ARE signalling

Increasing evidence has demonstrated that oxidative stress is involved in the pathogenesis of Parkinson's disease (PD), suggesting that pharmacological targeting of the antioxidant machinery may have therapeutic potential. Ampelopsin, a natural flavonoid compound, has been reported to possess various pharmacological functions, including anti-inflammatory, anti-oxidative and anti-cancer effects. However, its neuroprotective effect and neurotoxicity-attenuating role as well as underlying mechanisms are still unclear. In the present study, we investigated the potential role of ampelopsin in neuroprotective function and neurotoxicity attenuation, and explored its corresponding mechanisms against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in the cell model of PD. The pretreatment with ampelopsin for 1 h significantly improved cell viability, reduced the release of lactate dehydrogenase (LDH) and inhibited the accumulation of reactive oxygen species (ROS) in 6-OHDA-stimulated PC12 cells. In addition, ampelopsin treatment attenuated 6-OHDA-induced apoptosis of PC12 cells by inhibiting the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Moreover, ampelopsin treatment significantly increased the localization of nuclear factor-erythroid 2-related factor 2 (Nrf2) and subsequent activation of haem oxygenase-1 (HO-1) gene associated with antioxidant response element (ARE) pathway in a dose-dependent manner. Further study demonstrated that ampelopsin treatment suppressed the expression of glycogen synthase kinase-3β (GSK-3β). The present results suggest that the neuroprotective role of ampelopsin is due to the increased expression of Nrf2 through suppressing GSK-3β signalling, thereby correspondingly inhibiting ROS/p-p38/p-JNK MAPK signalling pathway. Therefore, ampelopsin has promising therapeutic potential for PD.

Wnt/β-catenin signaling participates in the regulation of lipogenesis in the liver of juvenile turbot (Scophthalmus maximus L.).

In this study, the mechanism that Wnt/β-catenin signaling inhibits lipogenesis was investigated in the liver of juvenile turbot (Scophthalmus maximus L.) by LiCl or XAV939 treatment. Wnt/β-catenin signaling was activated by LiCl treatment or inhibited by XAV939 treatment through regulating the expression of glycogen synthase kinase-3β (GSK-3β) and Wnt10b. In addition, the expression of lipoprotein lipase (LPL), fatty acid synthetase (FAS), peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT/enhancer binding protein α (C/EBPα) was inhibited by LiCl treatment, but induced by XAV939 treatment. In the plasma of juvenile turbot, the level of nonesterified fatty acid (NEFA), glycerol, triglyceride (TG), total cholesterol (TC), and low density lipoprotein cholesterol (LDL-C) was decreased by LiCl treatment, which was related to the decrease of the activity of LPL and FAS. Thus the inhibitory effect of Wnt/β-catenin signaling on lipogenesis was associated with the expression of key enzymes and transcriptional factors. Wnt/β-catenin signaling may participate in inhibiting lipogenesis by inhibiting the expression of PPARγ and C/EBPα in the liver of juvenile turbot.

Diabetes in Pregnancy Adversely Affects the Expression of Glycogen Synthase Kinase-3β in the Hippocampus of Rat Neonates.

Diabetes during pregnancy causes a wide range of neurodevelopmental and neurocognitive abnormalities in offspring. Glycogen synthase kinase-3 (GSK-3) is widely expressed during brain development and regulates multiple cellular processes, and its dysregulation is implicated in the pathogenesis of diverse neurodegenerative and psychological diseases. This study was designed to examine the effects of maternal diabetes on GSK-3β messenger RNA (mRNA) expression and phosphorylation in the developing rat hippocampus. Female rats were maintained diabetic from a week before pregnancy through parturition, and male offspring was killed immediately after birth. We found a significant bilateral upregulation of GSK-3β mRNA expression in the hippocampus of pups born to diabetic mothers at P0, compared to controls. Moreover, at the same time point, there was a marked bilateral increase in the phosphorylation level of GSK-3β in the diabetic group. Unlike phosphorylation levels, there was a significant upregulation in hippocampal GSK-3β mRNA expression in the insulin-treated group, when compared to controls. The present study revealed that diabetes during pregnancy strongly influences the regulation of GSK-3β in the right/left developing hippocampi. These dysregulations may be part of the cascade of events through which diabetes during pregnancy affects the newborn's hippocampal structure and function.

The Effects of Early Life Lead Exposure on the Expression of Glycogen Synthase Kinase-3β and Insulin-like Growth Factor 1 Receptor in the Hippocampus of Mouse Pups.

The present study was undertaken to investigate the effects of maternal lead exposure on expression of GSK-3β and IGF1R in the hippocampus of mice offspring. Lead exposure initiated from beginning of gestation to weaning. Lead acetate administered in drinking solutions was dissolved in distilled deionized water at the concentrations of 0.1, 0.5, and 1%, respectively. On the 21st postnatal day, the Pb levels were determined by graphite furnace atomic absorption spectrometry. The expression of GSK-3β and IGF1R in hippocampus was examined by immunohistochemistry and Western blotting. The lead levels in blood and hippocampus of all lead exposure groups were significantly higher than those of the control group (P < 0.05). Compared with the control group, the expression of GSK-3β was increased in lead-exposed groups (P < 0.05), but the expression of IGF1R was decreased (P < 0.05). The high expression of GSK-3β and low expression of IGF1R in the hippocampus of pups may contribute to the neurotoxicity associated with maternal Pb exposure.

Treadmill exercise decreases incidence of Alzheimer's disease by suppressing glycogen synthase kinase-3β expression in streptozotocin-induced diabetic rats.

Diabetes is a metabolic disorder, and it is considered as a major risk factor for Alzheimer’s disease (AD). In the present study, we evaluated whether treadmill exercise ameliorates progression of AD in relation with glycogen synthase kinase-3β (GSK-3β) activity using streptozotocin (STZ)-induced diabetic rats. For this study, step-down avoidance task, immunohistochemistry for glycogen synthase kinase-3β (GSK-3β) and tau, and western blot for phosphor-phosphoinositide 3 kinase (p-PI3K)/PI3K and phosphor-Akt (p-Akt)/Akt were performed. Diabetes mellitus was induced by intraperitoneal injection of STZ. The rats in the exercise groups were made to run on the treadmill for 30 min per one day, five times a week, during 12 weeks. The present results showed that short-term and long-term latencies in the step-down avoidance task were decreased by induction of diabetes, and treadmill exercise inhibited these latencies in the diabetic rats. Induction of diabetes suppressed the ratio of p-PI3K to PI3K and the ratio of p-Akt to Akt, and treadmill exercise increased these ratios in the diabetic rats. The numbers of GSK-3β-positive and tau-positive cells in the hippocampal dentate gyrus was higher in the diabetes-induction group than that in the control group, and treadmill exercise inhibited these numbers in the diabetic rats. In the present study, treadmill exercise suppressed hyperphosphorylation of tau in the hippocampus by decreased GSK-3β activity through PI3K/Akt pathway activation in the diabetic rats. Based on the present results, treadmill exercise may helpful to prevent diabetes-associated AD occurrence.

Expression of glycogen synthase kinase-3β in pancreatic cancer and its correlation with epithelialmesenchymal transition

Objective To explore the glycogen synthase kinase-3β (GSK-3β) expression in pancreatic carcinoma and its correlation with epithelial-mesenchymal transition.Methods Sixty cases of pancreatic adenocarcinoma specimens dated from 2006-2010 were adopted in this study,35 of which had paired adjacent normal tissues.Immunohistochemical staining and Western blotting were used to detect the expression of GSK-3β,Ser-9,Tyr-216,Sanil and E-cadherin.The correlation between GSK-3 β and pancreatic cancer clinicopathological parameters,or epithelial-mesenchymal transition was analyzed.Results The positive expression rate of GSK-3 β in pancreatic carcinoma was 65.0％,which was significantly higher than in paired adjacent normal tissues (22.8％),and GSK-3β expression was positive correlated with pancreatic cancer clinical stage classification,degree of differentiation and lymph node metastasis (P ＜ 0.05),but not with patient's age,gender and tumor size (P ＞ 0.05).Phosphorylation of GSK-3 β (Ser-9) in the paired adjacent normal tissues was significantly higher than in pancreatic cancer,and the expression of Tyr-216 and Snail was quite the opposite.E-cadherin expression was lower in both cancer and paired adjacent normal tissues.Pearson correlation analysis revealed that GSK-3β was correlated with other abovementioned markers.Conclusion GSK-3β expression was significantly higher in pancreatic cancer than in the paird adjacent normal tissues,and had a positive correlation with clinical stage,TNM stage,tumor differentiation and lymph node metastasis.In addition,GSK-3β had significant relationship with epithelialmesenchymal transition. Key words: Pancreatic neoplasms ; Glycogen synthase kinase-3 β ; Epithelial mesenchymal transition ; Tumor invasion

2-Cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against beta-amyloid-induced activation of the apoptotic cascade in cultured cortical neurons.

Aggregated β-amyloid, implicated in the pathogenesis of Alzheimer's disease (AD), induces neurotoxicity by evoking a cascade of oxidative damage-dependent apoptosis in neurons. We investigated the molecular mechanisms underlying the protective effect of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride (KHG26377) against the beta-amyloid (Aβ25-35)-induced primary cortical neuronal cell neurotoxicity. Treatment with KHG26377 attenuated the Aβ25-35-induced apoptosis by decreasing the Bax/Bcl-2 ratio and suppressing the activation of caspase-3. A marked increase in calcium influx and in the level of reactive oxygen species together with a decrease in glutathione levels was found after Aβ25-35 exposure; however, KHG26377 treatment reversed these changes in a concentration-dependent manner. In addition, KHG26377 significantly suppressed Aβ25-35-induced toxicity concomitant with a reduction in the activation of extracellular signal-regulated kinases 1 and 2 and nuclear factor kappa B. The KHG26377-induced protection of neuronal cells against Aβ toxicity was also mediated by suppressing the expression of glycogen synthase kinase-3β, increasing the levels of β-catenin, and reducing the levels of phosphorylated tau. Our findings suggest that KHG26377 may modulate the neurotoxic effects of β-amyloid and provide a rationale for treatment of AD.

Glycogen Synthase Kinase-3β Expression and Phosphorylation in Peripheral Blood Mononuclear Cells of Patients with Amyotrophic Lateral Sclerosis

Aims: To quantify total glycogen synthase kinase (GSK)-3β and GSK-3β phosphorylated at serine 9 in the peripheral blood mononuclear cells from Amyotrophic Lateral Sclerosis (ALS) patients and to assess if GSK-3β could be a biomarker for ALS. Study Design: Cross-sectional observational study. Place and Duration of Study: Department of Immunology and Amyotrophic Lateral Sclerosis Unit, Hospital Carlos III, Madrid, Spain, between February 2011 and August 2012. Methodology: Blood samples were drawn from 44 ALS patients and 41 healthy controls. Peripheral blood mononuclear cells were isolated and cellular extracts were obtained to assess GSK-3β and serine 9 phosphorylated GSK-3β concentrations. Enzymes were measured by a quantitative enzyme-linked immunoassay in the peripheral blood Short Communication British Journal of Medicine & Medical Research, 4(1): 263-271, 2014 264 mononuclear cell extracts. Patients were divided into two groups according to the Amyotrophic Lateral Sclerosis Functional Rating Scale-revised (ALSFRS-R) median value for the comparative analysis. Results: Patients (n=22) showing a high functional impairment (ALSFRS-R ≤ 32) had GSK-3β levels (11.2±3.6 pg/μg protein) higher than healthy controls (8.7±4.7 pg/μg protein; P=0.04) and than those patients (n=22) with ALSFRS-R > 32 (6.9±4.4 pg/μg protein; P<0.01). A negative correlation between GSK-3β concentration and ALSFRS-R values (r = −0.39; P=0.006) was also observed. Conclusion: Our results show that GSK-3β expression is altered in non-neural cells of ALS patients and suggest that its overexpression may play a role in the pathogenesis of ALS. The quantification of GSK-3β in peripheral blood mononuclear cells may be used as a potential biomarker of ALS progression.

Protective effect of molecular hydrogen against H 2 O 2 -induced oxidative injury in cultured rat spinal cord neurons

Objective To investigate the protective effect of molecular hydrogen against hydrogen peroxide(H2O2)-induced oxidative injury in primary cultures of rat spinal cord neurons and the related mechanism.Methods Rat spinal cord neurons were cultured for 7days and were randomly assigned to four groups with different treatments-(1)Normal medium(NM)control:treated with NM;(2)Hydrogen-rich medium(HM):treated with HM;(3)NM+H2O2:treated with 100 μmol/L H2O2and 15μmol/L ferrous chloride(FeCl2)after pretreatment with NM for 2h;and(4)HM+H2O2:treated with 100μmol/L H2O2and 15μmol/L FeCl2after pretreatment with HM for 2h.The respective media were changed every 6hin each group,and 12hlater the cells were collected for assays of reactive oxygen species(ROS),hydroxyl radical(HO·), apoptosis,and the expression of glycogen synthase kinase-3β(GSK-3β)and p-GSK-3β.Results Compared with NM,HM significantly reduced the H2O2-induced intracellular production of ROS and HO·in purified neurons(P0.01),significantly decreased the number of apoptotic neurons(P0.01)and expression of caspase-3(P0.01),and significantly promoted phosphorylation of GSK-3β(P0.01).Conclusion Molecular hydrogen has protective effect against H2O2-induced oxidative injury in primary cultured neurons.The mechanisms might be related to reduction of intracellular production of ROS and HO·,inhibition of apoptosis in neurons and promotion of GSK-3βphosphorylation.

Silencing of p53 RNA through transarterial delivery ameliorates renal tubular injury and downregulates GSK-3β expression after ischemia-reperfusion injury

p53, a pivotal protein in the apoptotic pathway, has been identified as a mediator of transcriptional responses to ischemia-reperfusion (IR) injury. The characteristics and functional significance of the p53 response in vivo are largely unknown in IR-induced kidney injury. Therapeutic opportunities of delivering small interfering RNA (siRNA) via venous injection have gained recognition; however, systemic adverse effects of siRNA therapy should be considered. To prevent IR-induced kidney injury, we tested the efficacy of transarterial administration of siRNA targeting p53 (p53 siRNA). Female C57BL/6 mice underwent unilateral renal artery ischemia for 30 min, followed by reperfusion. siRNA experiments utilized short hairpin (sh) RNA plasmid-based approaches. Transfection of shRNA was performed using cationic polymer transfection reagent. Injection of synthetic p53 shRNA into the left renal artery just after ischemia improved tubular injury, apoptosis, and the swelling of mitochondria in cells of the thick ascending limb of Henle (mTALH) at the outer medullary regions. Staining of upregulated p53 was colocalized with the inducible expression of glycogen synthase kinase-3β (GSK-3β) at mTALH after IR injury. p53 shRNA inhibited GSK-3β expression and restored β-catenin expression at mTALH. For IR-induced kidney injury, transarterial delivery of p53 siRNA is an effective pharmacological intervention. Targeting siRNA to p53 leads to an attenuation of apoptosis and mitochondrial damage through the downregulation of GSK-3β expression and upregulation of β-catenin. Local delivery of vectors such as p53 siRNA through a transaortic catheter is clinically useful in reducing the adverse effect of siRNA-related therapy.

Polysaccharides from Liriopes Radix ameliorates hyperglycemia via various potential mechanisms in diabetic rats

Liriopes Radix, which is regarded as both drug and healthy diet, is drunk as tea and used in traditional Chinese medicine to treat diabetes. Based on our previous studies, investigated the hypoglycemic effects and explored the mechanisms of total polysaccharides from Liriope spicata var. prolifera (Liriopes Radix) in a diabetic rat model. TLSP reduced hyperglycemia in diabetic rats. The oral glucose tolerance test showed that TLSP could improve the glucose tolerance of diabetic rats. Damage to liver and pancreas tissue was inhibited after treatment with TLSP. Moreover, TLSP increased glycogen content, glucokinase (GK) and glycogen synthetase (GS) activities, and suppressed the elevation of glucose-6-phosphatase (G6Pase) and glycogen phosphorylase (GP) activities in liver. Compared with the diabetic control group, GK and GS mRNA expression were significantly elevated, while G6Pase and GP mRNA expression were decreased in TLSP groups. In addition, TLSP could inhibit glycogen synthase kinase-3β expression and increase insulin receptor, insulin receptor substrate-1, phosphoinositide 3-kinase, protein kinase B and glucose transport protein-4 expression in liver. TLSP showed hypoglycemic function. Improvement of glucose metabolism and insulin-signaling transduction were possible mechanisms.

Nuclear expression of Glycogen synthase kinase‐3β and lack of membranous β‐catenin is correlated with poor survival in colon cancer

Dysregulation of Wnt/β-catenin signaling is a hallmark of colon cancer. Glycogen synthase kinase-3β (GSK-3β) can be a positive regulator of survival and proliferation of cultured colon cancer cell but its role in clinical colon cancer is unknown. Our objectives were to evaluate the role of GSK-3β in colon cancer. A tumor tissue microarray of primary colon cancers and metastases was used to evaluate expression and subcellular localization of GSK-3β and β-catenin. In total, 85 primary colon cancer samples were evaluated by immunohistochemistry. Immunoreactivity was correlated to known markers of adverse prognosis. Overall survival was the primary end-point. We found nuclear accumulation of GSK-3β in 39% (33/85) of evaluated tumors. Nuclear GSK-3β was significantly associated with shorter overall survival (p = 0.008), larger tumor size (p = 0.015), distant metastasis (p = 0.029) and loss of membranous β-catenin (p = 0.007). Loss of membranous β-catenin occurred in 37% (30/82) of the tumors and was associated with poor survival (p = 0.016). The combination of nuclear GSK-3β and lack of membrane β-catenin occurred in a total of 26% of the studied tumors (21/61) and was significantly and independently associated with poor prognosis. Our results suggest that nuclear expression of GSK-3β and loss of membrane β-catenin identify a subset of colon carcinomas with worse prognosis.

Involvement of the Wnt signaling pathway and cell apoptosis in the rat hippocampus following cerebral ischemia/reperfusion injury.

We investigated the role of the Wnt signaling pathway in cerebral ischemia/reperfusion injury by examining β-catenin and glycogen synthase kinase-3β protein expression in the rat hippocampal CA1 region following acute cerebral ischemia/reperfusion. Our results demonstrate that cell apoptosis increases in the CA1 region following ischemia/reperfusion. In addition, β-catenin and glycogen synthase kinase-3β protein expression gradually increases, peaking at 48 hours following reperfusion. Dickkopf-1 administration, after cerebral ischemia/reperfusion injury, results in decreased cell apoptosis, and β-catenin and glycogen synthase kinase-3β expression, in the CA1 region. This suggests that β-catenin and glycogen synthase kinase-3β, both components of the Wnt signaling pathway, participate in cell apoptosis following cerebral ischemia/reperfusion injury.

Pomegranate Bioactive Constituents Suppress Cell Proliferation and Induce Apoptosis in an Experimental Model of Hepatocellular Carcinoma: Role of Wnt/β-Catenin Signaling Pathway

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and chemoprevention represents a viable approach in lowering the mortality of this disease. Pomegranate fruit, an abundant source of anti-inflammatory phytochemicals, is gaining tremendous attention for its wide-spectrum health benefits. We previously reported that a characterized pomegranate emulsion (PE) prevents diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis though inhibition of nuclear factor-kappaB (NF-κB). Since NF-κB concurrently induces Wnt/β-catenin signaling implicated in cell proliferation, cell survival, and apoptosis evasion, we examined antiproliferative, apoptosis-inducing and Wnt/β-catenin signaling-modulatory mechanisms of PE during DENA rat hepatocarcinogenesis. PE (1 or 10 g/kg) was administered 4 weeks before and 18 weeks following DENA exposure. There was a significant increase in hepatic proliferation (proliferating cell nuclear antigen) and alteration in cell cycle progression (cyclin D1) due to DENA treatment, and PE dose dependently reversed these effects. PE substantially induced apoptosis by upregulating proapoptotic protein Bax and downregulating antiapoptotic protein Bcl-2. PE dose dependently reduced hepatic β-catenin and augmented glycogen synthase kinase-3β expression. Our study provides evidence that pomegranate phytochemicals exert chemoprevention of hepatic cancer through antiproliferative and proapoptotic mechanisms by modulating Wnt/β-catenin signaling. PE, thus, targets two interconnected molecular circuits (canonical NF-κB and Wnt/β-catenin pathways) to exert chemoprevention of HCC.

The peripheral messenger RNA expression of glycogen synthase kinase‐3β genes in Alzheimer's disease patients: a preliminary study

To explore the peripheral leucocytic messenger RNA (mRNA) expression of glycogen synthase kinase-3β (GSK-3β) gene in Alzheimer's disease (AD) patients. Using TaqMan relative quantitative real-time polymerase chain reaction, we analyzed leucocytic gene expression of GSK-3β in 48 AD patients and 49 healthy controls. Clinical data of AD patients were also collected. The mRNA expression level of the GSK-3β gene was significantly higher in the AD group (3.13±0.62) than in the normal group (2.77±0.77). Correlational analyses showed that the mRNA expression level of GSK-3β gene in AD patients was associated with the age of onset (P=0.047), age (P=0.055), and Behavioral Pathology in Alzheimer's Disease Rating Scale total score (P=0.062) and subscores: aggressiveness score (P=0.073) and anxieties and phobias score (P=0.067). Through multivariate regression model, older age, higher anxieties and phobias score and aggressiveness score were associated with higher mRNA expression level of GSK-3β gene. In AD patients, the mRNA expression level of the GSK-3β gene is increased and may be related to age and behavioural pathology in AD.

Pro-inflammatory interleukin-18 increases Alzheimer’s disease-associated amyloid-β production in human neuron-like cells

BackgroundAlzheimer’s disease (AD) involves increased accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles as well as neuronal loss in various regions of the neocortex. Neuroinflammation is also present, but its role in AD is not fully understood. We previously showed increased levels of pro-inflammatory cytokine interleukin-18 (IL-18) in different regions of AD brains, where it co-localized with Aβ-plaques, as well as the ability of IL-18 to increase expression of glycogen synthase kinase-3β (GSK-3β) and cyclin dependent kinase 5, involved in hyperphosphorylation of tau-protein. Elevated IL-18 has been detected in several risk conditions for AD, including obesity, type-II diabetes, and cardiovascular diseases as well as in stress.MethodsWe differentiated SH-SY5Y neuroblastoma cells as neuron-like and exposed them to IL-18 for various times. We examined the protein levels of amyloid-β precursor protein (APP) and its processing products, its cleaving enzymes, involved in amyloidogenic processing of APP, and markers of apoptosis.ResultsIL-18 increased protein levels of the β-site APP-cleaving enzyme BACE-1, the N-terminal fragment of presenilin-1 and slightly presenilin enhancer 2, both of which are members of the γ-secretase complex, as well as Fe65, which is a binding protein of the C-terminus of APP and one regulator for GSK-3β. IL-18 also increased APP expression and phosphorylation, which preceded increased BACE-1 levels. Further, IL-18 altered APP processing, increasing Aβ40 production in particular, which was inhibited by IL-18 binding protein. Increased levels of soluble APPβ were detected in culture medium after the IL-18 exposure. IL-18 also increased anti-apoptotic bcl-xL levels, which likely counteracted the minor increase of the pro-apoptotic caspase-3. Lactate dehydrogenase activity in culture medium was unaffected.ConclusionsThe IL-18 induction of BACE-1, APP processing, and Aβ is likely to be linked to stress-associated adaptations in neurons during the course of normal functioning and development. However, in the course of wider changes in the aging brain, and particularly in AD, the effects of heightened or prolonged levels of IL-18 may contribute to the process of AD, including via increased Aβ.

MDG-1, a polysaccharide from Ophiopogon japonicus exerts hypoglycemic effects through the PI3K/Akt pathway in a diabetic KKAy mouse model

Ethnopharmacological relevanceOphiopogon japonicus is a traditional Chinese medicine that might be helpful for the treatment of type 2 diabetes. Recent studies have confirmed its beneficial properties, but not the mechanism of action. Aim of studyIn this study, we examined the effects of a water-soluble β-d-fructan (MDG-1) from O. japonicus on type 2 diabetes through the PI3K/Akt pathway in a diabetic KKAy mouse model. Materials and methodsMDG-1 was extracted from the tube root of O. japonicus and purified as described previously (Xu et al., 2005). The KKAy mice were gavaged once daily with either distilled water, MDG-1or rosiglitazonefor 8 weeks. Blood glucose levels were tested regularly for the fed and fasted mice. In order to evaluate the effect of MDG-1 on disease progression, the proteins of InsR/IRS-1/PI3K/Akt/GSK-3/Glut-4 were detected by Western blotting and serum TG, TC, HDL-C, LDL-C were also dertermined. ResultsMDG-1 reduced the hyperglycemia, hyperinsulinemia and hyperlipidemia in the KKAy mice. The oral glucose tolerance test (OGTT) and the level of insulin in the serun showed that insulin resistance in KKAy mice was ameliorated after MDG-1 treated. After 8 weeks treatment with 300mg/kg MDG-1, the content of triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) the serum decreased significantly. Meanwhile high density lipoprotein cholesterol (HDL-C) content increased notably. MDG-1 did not have any effect on total cholesterol (TC) content in the serum, whereas rosiglitazone significantly decreased the TC content. In addition, MDG-1 upregulates the phosphoinositide 3-kinase p85 subunit, Akt, insulin receptor (InsR), insulin receptor substrate-1 (IRS-1) and Glut-4 expression, but downregulates glycogen synthase kinase 3β expression. ConclusionsThese data indicate that MDG-1 has remarkable anti-diabetic activity through the InsR/IRS-1/PI3K/Akt/GSK-3/Glut-4 signaling pathway. We believe that MDG-1 is a promising anti-diabetic compound that will be helpful for the treatment of T2DM.

Inhibitory effects of lupeal acetate of Cortex periplocae on N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis

Lupeal acetate of Cortex periplocae (CPLA), a triterpene compound extracted from a traditional Chinese herb, has been identified as an inhibitor of cancer cell growth. The objective of the present study was to evaluate the potential mechanisms through which CPLA inhibits N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. We treated F344 rats subcutaneously with the esophageal carcinogen NMBA (0.5 mg/kg body weight) and intramuscularly with CPLA (20 mg/kg), 3 times a week for 5 weeks. Rats were then sacrificed at weeks 9, 15 or 25, esophageal tissues were collected and tumor data were recorded. To investigate the mechanisms by which CPLA modulates tumorigenesis in esophagus, we evaluated the protein expression of glycogen synthase kinase-3β (GSK-3β) and β-catenin and the gene expression of c-myc. CPLA significantly (P<0.05) reduced the incidence of esophageal tumors observed at 25 weeks from 93.3% in NMBA-treated controls to 33.3% in the NMBA- and CPLA-treated rats. CPLA reduced β-catenin and c-myc expression, but increased GSK-3β expression, in preneoplastic lesions of the esophagus. These results suggest a novel tumor-suppressive role of CPLA through the activation of GSK-3β expression and the inhibition of β-catenin and c-myc expression. Therefore, CPLA is a potential therapeutic candidate for esophageal squamous cell carcinoma.

Effect of valsartan and fluvastatin on the expression of glycogen synthase kinase-3β in retina of diabetic rat

Background Glycogen synthase kinase-3β (GSK-3β) plays an important role in glucose metabolism,and it may be affect the occurrence of diabetic retinopathy(DR). Objective The present study was to investigate the effect of valsartan and fluvastatin on the expression of GSK-3β in retina of diabetes rat model.Methods Diabetes mellitus models were induced by intrapenetoneal injection of streptozotocin(STZ) in 47 clean Sprague-Dawley(SD) rats and were then randomedly divided into 4 groups.Ten other normal rats were served as normal control group.Sodium carboxy methyl cellulose solution,valsartan,fluvastatin,valsartan+fluvastatin and sodium carboxy methyl cellulose solution was given by oral once per day for 12 weeks respectively in diabetes control group ( n =12),valsartan group ( n =12 ),fluvastatin group ( n =11 ),valsartau + fluvastatin group ( n =12 ) and normal control group.Twelve weeks after administration of drugs,blood glucose was measured and compared among various groups,and the expression of p-GSK-3β ( Ser-9 ) protein in retina was quantified and located by Western blot and immunohistochemistry,respectively. Results Twelve weeks after use of drugs,the level of blood glucose was(5.28±0.30),(26.08±3.33 ),(26.03 ±2.66 ),(25.90± 2.86 ),(25.99 ± 2.14 ) mmol/L in the normal control group,diabetes control group,valsartan,fluvastatin,valsartan + fluvastatin group,respectively,showing a significant difference among the 5 groups ( F =110.74,P＜0.01 ).Western blot showed that the grey value of p-GSK-3β ( Ser-9 ) /β-actin in retina in the diabetic control group was significant higher than the normal group(2.774±0.139 vs 1.927±0.111,q =15.79,P＜0.01 ),and that in valsartan,fluvastatin,valsartan+fluvastatin group was lower than the diabetic control group ( 1.895 ±0.090,2.051 ± 0.113,1.537 ± 0.071 vs 2.774 ± 0.139 ) ( q =1 3.69,13.48,23.06,P ＜ 0.01 ).The grey value of p-GSK-3β (Ser-9)/β-actin in the valsartan+fluvastatin group was declined in comparison with the valsartan group and fluvastatin group ( q =6.67,9.58,P＜0.01 ).Immunohistochemistry showed that the p-GSK-3β(Ser-9) protein was expressed all over the retinal layers and obviously in retinal ganglion cell layer(GCL) in normal control group.But the p-GSK-3β(Ser-9) protein was expressed significantly in diabetic control group.The expression of p-GSK-3β (Ser-9) protein was attenuated both in valsartan and fluvastatin groups and further attenuated in valsartan + fluvastatin group. Conclusions p-GSK-3β (Ser-9) protein is overexpressed in GCL of retina of diabetes rat.Both valsartan and fluvastatin can inhibit the expression of p-GSK-3β (Ser-9) and even getting stronger when they combined. Key words: Glycogen synthase kinase-3β; Diabetes mellitus/experimental; Retina

Rapid modulation of TRH and TRH-like peptide release in rat brain, pancreas, and testis by a GSK-3β inhibitor

Antidepressants have been shown to be neuroprotective and able to reverse damage to glia and neurons. Thyrotropin-releasing hormone (TRH) is an endogenous antidepressant-like neuropeptide that reduces the expression of glycogen synthase kinase-3β (GSK-3β), an enzyme that hyperphosphorylates tau and is implicated in bipolar disorder, diabetes and Alzheimer's disease. In order to understand the potential role of GSK-3β in the modulation of depression by TRH and TRH-like peptides and the therapeutic potential of GSK-3β inhibitors for neuropsychiatric and metabolic diseases, young adult male Sprague–Dawley (SD) rats were (a) injected ip with 1.8 mg/kg of GSK-3β inhibitor VIII (GSKI) and sacrificed 0, 2, 4, 6, and 8 h later or (b) injected with 0, 0.018, 0.18 or 1.8 mg/kg GSKI and bled 4 h later. Levels of TRH and TRH-like peptides were measured in various brain regions involved in mood regulation, pancreas and reproductive tissues. Large, 3–15-fold, increases of TRH and TRH-like peptide levels in cerebellum, for example, as well as other brain regions were noted at 2 and 4 h. In contrast, a nearly complete loss of TRH and TRH-like peptides from testis within 2 h and pancreas by 4 h following GSKI injection was observed. We have previously reported similar acute effects of corticosterone in brain and peripheral tissues. Incubation of a decapsulated rat testis with either GSKI or corticosterone accelerated release of TRH, and TRH-like peptides. Glucocorticoids, via inhibition of GSK3-β activity, may thus be involved in the inhibition of TRH and TRH-like peptide release in brain, thereby contributing to the depressogenic effect of this class of steroids. Corticosterone-induced acceleration of release of these peptides from testis may contribute to the decline in reproductive function and redirection of energy needed during life-threatening emergencies. These contrasting effects of glucocorticoid on peptide release appear to be mediated by GSK-3β.