罗格列酮上调高脂饮食老年大鼠骨骼肌GLUT4和PKB的表达

目的 观察高脂饮食对老年大鼠骨骼肌葡萄糖转运蛋白4(GLUT4)和蛋白激酶B(PKB)的表达变化及罗格列酮的干预效果.方法 老年大鼠随机分为对照组、高脂组(HF)、高脂+罗格列酮干预组(RSG),每组20只.应用清醒状态下正常葡萄糖高胰岛素钳夹技术的葡萄糖输注率评价胰岛素抵抗,用荧光定量PCR法和Western印迹技术检测骨骼肌GLUT4和PKB的表达.结果 高脂组骨骼肌长链脂酰辅酶A(LCACoA)升高而葡萄糖输注率明显下降(P＜0.01),骨骼肌GLUT4和PKB的表达明显降低(P＜0.05,P＜0.01),罗格列酮干预组显著缓解高脂组上述变化(P＜0.05,P＜0.01).结论 罗格列酮上调高脂饮食老年大鼠骨骼肌GLUT4和PKB的表达,是改善老年胰岛素抵抗的机制之一.     

Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells

Proliferation of activated hepatic stellate cells (HSC) is an important event in the development of hepatic fibrosis. Insulin-like growth factor-1 (IGF-1) has been shown to be mitogenic for HSC, but the intracellular signaling pathways involved have not been fully characterized. Thus, the aims of the current study were to examine the roles of the extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3-K) and p70-S6 kinase (p70-S6-K) signaling pathways in IGF-1- and platelet-derived growth factor (PDGF)-induced mitogenic signaling of HSC and to examine the potential crosstalk between these pathways. Both IGF-1 and PDGF increased ERK, PI3-K and p70-S6-K activity. When evaluating potential crosstalk between these signaling pathways, we observed that PI3-K is required for p70-S6-K activation by IGF-1 and PDGF, and is partially responsible for PDGF-induced ERK activation. PDGF and IGF-1 also increased the levels of cyclin D1 and phospho-glycogen synthase kinase-3beta. Coordinate activation of ERK, PI3-K and p70-S6-K is important for perpetuating the activated state of HSC during fibrogenesis.     

erbB-2/neu transformed rat cholangiocytes recapitulate key cellular and molecular features of human bile duct cancer

BACKGROUND & AIMS: Cholangiocarcinomas appear to arise from the malignant transformation of cholangiocytes lining the biliary tract. Because the development of an in vitro model of malignant transformation can provide a powerful new tool for establishing critical events governing the molecular pathogenesis of cholangiocarcinoma, we investigated the potential of achieving malignant transformation of cultured rat cholangiocytes in relation to aberrant overexpression of mutationally activated erbB-2/neu. METHODS: Malignant neoplastic transformation was achieved after infection of the rat cholangiocyte cell line, designated BDE1, with the retrovirus Glu664-neu, containing the transforming rat erbB-2/neu oncogene. RESULTS: Compared with untransformed control cells, malignant transformants carrying the activating erbB-2/neu mutation prominently overexpressed p185neu receptor protein, which was phosphorylated strongly at its major autophosphorylation site at tyrosine 1248. Moreover, erbB-2/neu transformation of BDE1 cells resulted in increased telomerase activity, up-regulation of cyclooxygenase-2 with overproduction of prostaglandin E(2), enhanced phosphorylation of mitogen-activated protein kinase and of serine/threonine kinase Akt/PKB, overexpression of vascular endothelial growth factor, and increased mucin 1 messenger RNA expression. Only erbB-2/neu transformants were tumorigenic when transplanted into isogeneic rats, yielding a 100% incidence of tumors closely resembling human desmoplastic ductal cholangiocarcinomas in their morphology. Malignant cholangiocytes in the tumors were strongly immunoreactive for biliary cytokeratin 19, p185neu, and cyclooxygenase-2. CONCLUSIONS: This unique malignant transformation model recapitulates key molecular features of the human disease and appears to be well suited for testing novel molecular therapeutic strategies against cholangiocarcinoma.     

p38 and JNK have distinct regulatory functions on the development of apoptosis during simulated ischaemia and reperfusion in neonatal cardiomyocytes

Abstract. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (SI/R) were used as an in vitro model to delineate the role(s) of extracellular signal–regulated kinase (ERK), p38 and c–Jun NH₂–terminal protein kinase (JNK), as well as PKB in apoptosis. Exposure of the myocytes to SI (simulated ischaemia – energy depletion induced by KCN and 2-deoxy-D–glucose) reduced cell viability, as measured by the 3–[4,5–dimethylthiazol–2–yl]–2,5–diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis as evidenced by caspase–3 activation and poly(ADP–ribose) polymerase (PARP) cleavage. However, morphological evidence of increased apoptosis, detected by staining with Hoechst 33342, was only seen in response to reperfusion. This suggests that although ischaemic conditions are sufficient to induce cellular markers of apoptosis (PARP cleavage and caspase–3 activation), reperfusion is required to complete the apoptotic pathway in these cells. Furthermore, SI resulted in a rapid, strong, biphasic activation of p38 concomitant with a weak and transient activation of the two ERK isoenzymes, p42/p44–MAPK. Reperfusion for 5 minutes resulted in a strong phosphorylation of p42/p44–MAPK, while no additional p38 activation was seen at this stage. On the other hand, p46/p54–MAPK (JNK) was phosphorylated in response to 5 minutes of reperfusion only and not during SI alone. A peak of PKB/Akt (Ser⁴⁷³) activity was seen within 5 minutes of exposure to SI, whereas PKB/Akt (Thr³⁰⁸) phosphorylation remained at the baseline level. Both PKB/Akt phosphorylation sites (Ser⁴⁷³ and Thr³⁰⁸) were phosphorylated after 5 minutes of reperfusion. Inhibition of PI–3–kinase activity, using wortmannin, decreased phosphorylation on both sites during SI. However, only SI/R-induced PKB/Akt phosphorylation on Thr³⁰⁸ was reduced by wortmannin. Myocytes pre–treated with SB203580, a p38–inhibitor, displayed a significant increase in cell viability [63.67 ± 1.85 to 84.33 ± 4.8% (p < 0.05)] and attenuation of the apoptotic index during SI/R [22.6 ± 2.94% to 9 ± 0.43% (p < 0.001)], while SP600125, a specific JNK inhibitor, caused a significant increase in caspase–3 activation [1.66 ± 0.03 fold to 2.56 ± 0.27 fold (p < 0.001)] and apoptotic index [22.6 ± 2.94% to 32.75 ± 6.13% (p < 0.05)]. However, PD98059, an ERK inhibitor, failed to affect apoptosis during SI/R. Inhibition of PI–3–kinase prevented the increase in mitochondrial viability usually observed during reperfusion. Interestingly, wortmannin caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase–3 activation or the apoptotic index. Our results suggest that p38 has a pro–apoptotic role while JNK phosphorylation is protective in our cell model and that these kinases act via caspase–3 to prevent or promote cell survival in response to SI/R–induced injury.     

Nuclear Akt interacts with B23/NPM and protects it from proteolytic cleavage, enhancing cell survival

B23/NPM is a major nucleolar phosphoprotein that has a critical role in cell proliferation and cell death. Here, we show that it forms a complex with Akt on growth factor (GF) stimulation in both the cytoplasm and the nucleus, for which Akt activation is indispensable. The C terminus of B23 (239-294 residues) potently binds pleckstrin homology (PH) domain of Akt. Akt binding to B23 protects it from proteolytic degradation by caspase-3, leading to the up-regulation of cell survival. Interestingly, unsumoylated B23 K263R, but not wild-type B23, strongly interacts with Akt in the nucleoplasm in the absence of GFs. Furthermore, we show that Akt2, but not other isoforms, specifically regulates B23 sumoylation and protein stability. Also, nuclear Akt regulates the cell cycle progression activity of B23. Therefore, our findings support that nuclear Akt binds and stabilizes B23 in the nucleoplasm, and regulates its activities in cell survival and cell cycle.     

Xanthine oxidase interaction with vascular endothelial growth factor in human endothelial cell angiogenesis

OBJECTIVES: Reduced capillary density occurs early in cardiovascular diseases. Oxidant stress is implicated in endothelial apoptosis. We investigated the effects of xanthine oxidase (XO) on endothelial survival signaling: protein kinase B/Akt, its cross-talk with p38 MAPK and apoptosis pathways, and its effect on vascular tube formation in vascular endothelial growth factor (VEGF)-simulated human umbilical vein cells. METHODS: We studied primary cultured human endothelial cells from the umbilical cord. Reactive oxygen species (ROS) production was detected by dihydroethidium staining, cell-signaling pathways by western blots, cell survival by western blots, and nuclear chromatin and angiogenesis response by MTT proliferation assay and three-dimensional Matrigel cultures. RESULTS: Exogenous XO increased cellular ROS production and caused superoxide-dependent inhibition of Akt phosphorylation and enhancement of p38 MAPK phosphorylation in a time-and dose-dependent manner. In contrast, application of the XO inhibitor oxypurinol or allopurinol inhibited VEGF-stimulated Akt phosphorylation, indicating that endogenous XO promotes VEGF-induced endothelial cell (EC) survival signaling. Exogenous XO induced activation of caspase-3 and reduced expression of the anti-apoptosis protein Bcl-2. Exogenous XO also reduced EC viability, proliferation, and vascular tube formation by p38 MAPK-dependent, phosphoinositide 3-kinase (PI3-K) reversible mechanisms; whereas VEGF promoted EC survival by PI3-K-dependent, p38 MAPK-independent effects. CONCLUSIONS: Exogenous XO activity is an important contributor to endothelial mechanisms for microvascular rarefaction, by modulation of cell survival signaling pathways; however, endogenous XO is necessary for maintaining EC survival.     

Type I collagen and divalent cation shifts disrupt cell-cell adhesion, increase migration, and decrease PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells

Background: We have shown in FG pancreatic cancer cells that α₂β₁ integrin-mediated type I collagen adhesion decreases parathyroid hormone-related protein (PTHrP), inerleukin-6 (IL-6), and interleukin-8 (IL-8) expression, decreases the localization of E-cadherin and β-catenin in cell-cell contacts, increases cell migration, and increases glycogen synthase kinase 3 (GSK3) and protein kinase B (PKB/Akt) phosphorylation states relative to α₅β₁ integrin-mediated fibronectin (Fn) adhesion. Aim of the Study: To extend our observations in FG cells to other pancreatic cancer cell lines, and to determine whether E-cadherin-mediated cell-cell adhesion and its downstream effectors were functionally involved in the ECM-mediated regulation of PTHrP, IL-6, and IL-8. Methods: We used standard biochemical techniques to determine ECM-specific differences in E-cadherin and β-catenin localization, GSK3 and PKB/Akt phosphorylation, haptokinetic cell migration, and cytokine expression in pancreatic cancer cells. We also conducted functional studies using pharmacological inhibitors for GSK3 and PKB/Akt, as well as elevated Mg²⁺/Ca²⁺ ratios similar to pancreatic juice, and examined their effects on cytokine expression. Results: Differences in E-cadherin and β-catenin localization along with GSK3 and PKB/Akt phosphorylation occur in multiple pancreatic cancer cell lines, resulting in differences in ECM-mediated haptokinesis and cytokine expression that are generally consistent with previous observations in FG cells. Our functional studies also suggest that E-cadherin-mediated cell-cell adhesion and downstream effectors are involved in PTHrP, IL-6, and IL-8 expression. Conclusions: These data indicate that α₂β₁ integrin-mediated type I collagen adhesion disrupts cell-cell adhesion architecture, resulting in increased migration and decreased PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.     

Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor

Aims/hypothesis Treatment with glucocorticoids, especially at high doses, induces insulin resistance. The aims of the present study were to identify the potential defects in insulin signalling that contribute to dexamethasone-induced insulin resistance in skeletal muscles, and to investigate whether the glycogen synthase-3 (GSK-3) inhibitor CHIR-637 could restore insulin-stimulated glucose metabolism.Materials and methods Skeletal muscles were made insulin-resistant by treating male Wistar rats with dexamethasone, a glucocorticoid analogue, for 12 days. Insulin-stimulated glucose uptake, glycogen synthesis and insulin signalling were studied in skeletal muscles in vitro.Results Dexamethasone treatment decreased the ability of insulin to stimulate glucose uptake, glycogen synthesis and glycogen synthase fractional activity. In addition, the dephosphorylation of glycogen synthase by insulin was blocked. These defects were paralleled by reduced insulin-stimulated protein kinase B (PKB) and GSK-3 phosphorylation. While expression of PKB, GSK-3 and glycogen synthase was not reduced by dexamethasone treatment, expression of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) was increased. Inhibition of GSK-3 by CHIR-637 increased glycogen synthase fractional activity in soleus muscle from normal and dexamethasone-treated rats, although the effect was more pronounced in control rats. CHIR-637 did not improve insulin-stimulated glucose uptake in muscles from dexamethasone-treated rats.Conclusions/interpretation We demonstrated that chronic dexamethasone treatment impairs insulin-stimulated PKB and GSK-3 phosphorylation, which may contribute to insulin resistance in skeletal muscles. Acute pharmacological inhibition of GSK-3 activated glycogen synthase in muscles from dexamethasone-treated rats, but GSK-3 inhibition did not restore insulin-stimulated glucose uptake.     

Bmi-1基因对胃癌细胞增殖的影响及机制

目的:探索干扰Bmi-1基因后对其可能的下游基因Akt/PKB活性和P16INK4a基因表达的影响及对肿瘤细胞增殖和细胞衰老的作用.方法:用s iRNA技术干扰Bmi-1表达后,运用Western blot检测Bmi-1蛋白及相关蛋白pAkt、Akt和P16INK4a的表达,同时进行SA-β-Gal染色检测细胞衰老,软琼脂克隆形成实验检测细胞的增殖能力.结果:转染Bmi-1 i质粒组平均细胞衰老率28%±3.5%,而对照Ctrl i组为16%±2.7%,有明显统计学差异( P<0.01).转染Bmi-1 i质粒组细胞平均克隆形成数为3.4±1.4个,而对照Ctrl i组为11±2.3个,两组比较有明显的统计学差异( P<0.01).Bmi-1 i 组较Ctrl i 组Bmi-1和pAkt蛋白表达明显下降,而P16INK4a蛋白表达升高.结论:干扰Bmi-1可以通过降低Akt/PKB活性和上调P16INK4a蛋白表达,促进肿瘤细胞衰老并减弱肿瘤细胞的增殖能力.