Expression of girdersofactin filaments (Girdin) in pituitary adenomasand its role in promoting cell proliferation / 第二军医大学学报

Objective To investigate the expression of girders of actin filaments (Girdin) in pituitary adenomas, and its role in promoting cell proliferation and the related molecular mechanism. Methods Two prolactinoma, growth hormone adenoma and non-functioning pituitary adenoma tissues, and one normal pituitary gland tissue were collected. The protein expression of Girdin in the different tissues was detected by Western blotting, and then the expression of Girdin was further confirmed by immunofluorescence. Rat pituitary tumor cell lines GH3 cell model with Girdin knockdown and overexpression was established by RNA interference and overexpression of Girdin, respectively. The protein expression of Girdin and Akt and phosphorylation level of Akt in the GH3 cell models were detected by Western blotting. The function and biological behavior of Girdin in pituitary adenomas tissues were studied by cell proliferation assay and cell apoptosis assay. Results The expression of Girdin in the non-functioning pituitary adenomas was the highest, followed by growth hormone pituitary adenomas. The high expression of Girdin in the non-functioning pituitary adenomas was also verified by immunofluorescence assay. RNA interference and overexpression of Girdin effectively knocked down and increased the expression of Girdin, respectively, accompanied by the simultaneous changes of Akt phosphorylation. In addition, overexpression of Girdin promoted the proliferation of GH3 cells. Conclusion Girdin is highly expressed in non-functioning pituitary adenomas and can promote the proliferation of pituitary adenoma cell by regulating the Akt phosphorylation.

Effects of PTEN and its mutants on AKT phosphorylation in gastric cancer cells / 实用医学杂志

Objective To study the effects of PTEN and missense mutations in PTEN phosphotase domain on AKT phosphorylation in AGS and BGC-823 cells. Methods The plasmids of wtPTEN,PTEN-C124S which PTEN mutant is in both lipid and protein phosphotase domain and PTEN-G129E which PTEN mutant is only in lipid phosphotase domain were respectively transfected into AGS and BGC-823 cells. The cells were stimulated with insulin or rhEGF after serum starvation overnight. The levels of AKT phosphorylation were detected by Western blot. Results Both insulin and rhEGF can activate AKT phosphorylation in gastric cancer cells. Overexpressed PTEN inhibitedAKT phosphorylation induced by insulin or rhEGF(P 0.05). Conclusions PTEN can inhibit AKT phosphorylation induced by insulin or rhEGF in gastric cancer cells. Missense mutations in the 124th or 129th amino acid of PTEN phospho-tase domain do not exert inhibitive function.

Different Effects of Orbital Shear Stress on Vascular Endothelial Cells: Comparison with the Results of In Vivo Study with Rats

PURPOSE: An attempt was made to characterize the orbital shear stress by comparing the effects of orbital shear stress on vascular endothelial cells (ECs) with the results of animal experiments. MATERIALS AND METHODS: In the laboratory study, cultured ECs of well were distinguished by center and periphery then exposed to orbital shear stress using an orbital shaker. In the animal study, arteriovenous (AV) fistulas were made at the right femoral arteries of Sprague-Dawley rats to increase the effect of the laminar flow. The condition of the stenosis was given on the left femoral arteries. The protein expression of inducible nitric oxide synthase (iNOS) and Akt phosphorylation were observed and compared. RESULTS: Under orbital shear stress, ECs showed an increase in iNOS protein expression and phosphorylation of Akt but most of the protein expressions derived from the periphery. When compared to the animal study, the increased expression of iNOS protein and phosphorylation of Akt were observed in the sample of AV fistula conditions and the iNOS protein expression was decreased in the stenosis conditions. CONCLUSION: Orbital shear stress did not show the characteristics of a pure turbulent shear force. By comparing the observation with the morphological changes of vascular ECs and site-specific protein expression on the results of animal experiments, uniform directional lamina shear stress forces were expressed at the periphery.

Beneficial effects of Ginkgo biloba extract on insulin signaling cascade, dyslipidemia, and body adiposity of diet-induced obese rats

Ginkgo biloba extract (GbE) has been indicated as an efficient medicine for the treatment of diabetes mellitus type 2. It remains unclear if its effects are due to an improvement of the insulin signaling cascade, especially in obese subjects. The aim of the present study was to evaluate the effect of GbE on insulin tolerance, food intake, body adiposity, lipid profile, fasting insulin, and muscle levels of insulin receptor substrate 1 (IRS-1), protein tyrosine phosphatase 1B (PTP-1B), and protein kinase B (Akt), as well as Akt phosphorylation, in diet-induced obese rats. Rats were fed with a high-fat diet (HFD) or a normal fat diet (NFD) for 8 weeks. After that, the HFD group was divided into two groups: rats gavaged with a saline vehicle (HFD+V), and rats gavaged with 500 mg/kg of GbE diluted in the saline vehicle (HFD+Gb). NFD rats were gavaged with the saline vehicle only. At the end of the treatment, the rats were anesthetized, insulin was injected into the portal vein, and after 90s, the gastrocnemius muscle was removed. The quantification of IRS-1, Akt, and Akt phosphorylation was performed using Western blotting. Serum levels of fasting insulin and glucose, triacylglycerols and total cholesterol, and LDL and HDL fractions were measured. An insulin tolerance test was also performed. Ingestion of a hyperlipidic diet promoted loss of insulin sensitivity and also resulted in a significant increase in body adiposity, plasma triacylglycerol, and glucose levels. In addition, GbE treatment significantly reduced food intake and body adiposity while it protected against hyperglycemia and dyslipidemia in diet-induced obesity rats. It also enhanced insulin sensitivity in comparison to HFD+V rats, while it restored insulin-induced Akt phosphorylation, increased IRS-1, and reduced PTP-1B levels in gastrocnemius muscle. The present findings suggest that G. biloba might be efficient in preventing and treating obesity-induced insulin signaling impairment.

Effects of resveratrol on the insulin signaling pathway of obese mice

The present study was conducted to investigate the effects of resveratrol on the insulin signaling pathway in the liver of obese mice. To accomplish this, we administered resveratrol to high fat diet-induced obese mice and examined the levels of protein phosphorylation in the liver using an antibody array. The phosphorylation levels of 10 proteins were decreased in the high fat diet and resveratrol (HFR) fed group relative to the levels in the high fat diet (HF) fed group. In contrast, the phosphorylation levels of more than 20 proteins were increased in the HFR group when compared with the levels of proteins in the HF group. Specifically, the phosphorylation levels of Akt (The308, Tyr326, Ser473) were restored to normal by resveratrol when compared with the levels in the HF group. In addition, the phosphorylation levels of IRS-1 (Ser636/Ser639), PI-3K p85-subunit alpha/gamma(Tyr467/Tyr199), PDK1 (Ser241), GSK-3alpha (S21) and GSK-3 (Ser9), which are involved in the insulin signaling pathway, were decreased in the HF group, whereas the levels were restored to normal in the HFR group. Overall, the results show that resveratrol restores the phosphorylation levels of proteins involved in the insulin signaling pathway, which were decreased by a high fat diet.

Anti-diabetic effects of benfotiamine on an animal model of type 2 diabetes mellitus / 대한수의학회지

Although benfotiamine has various beneficial anti-diabetic effects, the detailed mechanisms underlying the impact of this compound on the insulin signaling pathway are still unclear. In the present study, we evaluated the effects of benfotiamine on the hepatic insulin signaling pathway in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which are a type 2 diabetes mellitus model. OLETF rats treated with benfotiamine showed decreased body weight gain and reduced adipose tissue weight. In addition, blood glucose levels were lower in OLETF rats treated with benfotiamine. Following treatment with benfotiamine, the levels of Akt phosphorylation (S473/T308) in the OLETF groups increased significantly compared to the OLETF control group so that they were almost identical to the levels observed in the control group. Moreover, benfotiamine restored the phosphorylation levels of both glycogen synthase kinase (GSK)-3alpha/beta (S21, S9) and glycogen synthase (GS; S641) in OLETF rats to nearly the same levels observed in the control group. Overall, these results suggest that benfotiamine can potentially attenuate type 2 diabetes mellitus in OLETF rats by restoring insulin sensitivity through upregulation of Akt phosphorylation and activation of two downstream signaling molecules, GSK-3alpha/beta and GS, thereby reducing blood glucose levels through glycogen synthesis.

Midazolam protects B35 neuroblastoma cells through Akt-phosphorylation in reactive oxygen species derived cellular injury / 대한마취과학회지

BACKGROUND: Soman, a potent irreversible acetylcholinesterase (AChE) inhibitor, induces delayed neuronal injury by reactive oxygen species (ROS). Midazolam is used in patients with pathologic effects of oxidative stresses such as infection, hemodynamic instability and hypoxia. We investigated whether midazolam protects the Central Nervous System (CNS) from soman intoxication. The present study was performed to determine whether midazolam protects B35 cells from ROS stress for the purpose of exploring an application of midazolam to soman intoxication. METHODS: Glucose oxidase (GOX) induced ROS stress was used in a B35 neuroblastoma cell model of ROS induced neuronal injury. To investigate the effect of midazolam on cell viability, LDH assays and fluorescence activated cell sorting (FACS) analysis was performed. Western blotting was used for evaluating whether Akt-phosphorylation is involved in cell-protective effects of midazolam. RESULTS: GOX derived ROS injury decreased cell viability about 1.6-2 times compared to control; midazolam treatment (5 and 10 microg/ml) dose-dependently increased cell viability during ROS injury. On western blots, Akt-phosphorylation was induced during pretreatment with midazolam; it was diminished during co-treatment with LY-294002, an inhibitor of Akt-phosphorylation. FACS analysis confirmed that the cell protective effect of midazolam is mediated by an anti-apoptotic effect. GOX-induced apoptosis was inhibited by midazolam and the finding was diminished by LY-294002. CONCLUSIONS: Midazolam protects neuronal cells from GOX-induced ROS injury; this effect is mediated by an anti-apoptotic effect through Akt-phosphorylation. This shows that midazolam may be useful in soman intoxication.

Inhibition of eNOS/sGC/PKG Pathway Decreases Akt Phosphorylation Induced by Kainic Acid in Mouse Hippocampus

The serine/threonine kinase Akt has been shown to play a role of multiple cellular signaling pathways and act as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI3K). It has been reported that phosphorylated Akt activates eNOS resulting in the production of NO and that NO stimulates soluble guanylate cyclase (sGC), which results in accumulation of cGMP and subsequent activation of the protein kinase G (PKG). It has been also reported that PKG activates PI3K/Akt signaling. Therefore, it is possible that PI3K, Akt, eNOS, sGC, and PKG form a loop to exert enhanced and sustained activation of Akt. However, the existence of this loop in eNOS-expressing cells, such as endothelial cells or astrocytes, has not been reported. Thus, we examined a possibility that Akt phosphorylation might be enhanced via eNOS/sGC/PKG/PI3K pathway in astrocytes in vivo and in vitro. Phosphorylation of Akt was detected in astrocytes after KA treatment and was maintained up to 72 h in mouse hippocampus. 2 weeks after KA treatment, astrocytic Akt phosphorylation was normalized to control. The inhibition of eNOS, sGC, and PKG significantly decreased Akt and eNOS phosphorylation induced by KA in astrocytes. In contrast, the decreased phosphorylation of Akt and eNOS by eNOS inhibition was significantly reversed with PKG activation. The above findings in mouse hippocampus were also observed in primary astrocytes. These data suggest that Akt/eNOS/sGC/PKG/PI3K pathway may constitute a loop, resulting in enhanced and sustained Akt activation in astrocytes.

Melatonin Induces Akt Phosphorylation through Melatonin Receptor- and PI3K-Dependent Pathways in Primary Astrocytes

Melatonin has been reported to protect neurons from a variety of neurotoxicity. However, the underlying mechanism by which melatonin exerts its neuroprotective property has not yet been clearly understood. We previously demonstrated that melatonin protected kainic acid-induced neuronal cell death in mouse hippocampus, accompanied by sustained activation of Akt, a critical mediator of neuronal survival. To further elucidate the neuroprotective action of melatonin, we examined in the present study the causal mechanism how Akt signaling pathway is regulated by melatonin in a rat primary astrocyte culture model. Melatonin resulted in increased astrocytic Akt phosphorylation, which was significantly decreased with wortmannin, a specific inhibitor of PI3K, suggesting that activation of Akt by melatonin is mediated through the PI3K-Akt signaling pathway. Furthermore, increased Akt activation was also significantly decreased with luzindole, a non-selective melatonin receptor antagonist. As downstream signaling pathway of Akt activation, increased levels of CREB phoshorylation and GDNF expression were observed, which were also attenuated with wortmannin and luzindole. These results strongly suggest that melatonin exerts its neuroprotective property in astrocytes through the activation of plasma membrane receptors and then PI3K-Akt signaling pathway.

The relation between poly(ADP-ribose) glycohydrolase,poly(ADP-ribose) polymerase and AKT phosphorylation in colorectal carcinoma cell / 重庆医科大学学报

Objective:Poly(ADP-ribose) glycohydrolase(PARG) plays an important role in the inflammation via regulating Poly(ADP-ribose) polymerase(PARP) and AKT phosphorylation,but it is not clear that how it in tumour.The current study was desiged to study the relationship between PARG,PARP,NF-?B and AKT phosphorylation in colorectal carcinoma cell lines LOVO.Methods:Western blot was used to test the expression of PARG,PARP,NF-?B,AKT and PI-AKT473.Gallotannin(GLTN) was served as PARG inhibitor.Results:The expression of PARG,PARP and NF-?B in gallotannin-treated LOVO was weaker than that in the control groups(gallotannin-untreated groups)(P=0.0068,P