大鼠脊髓腰节段神经元存活信号转导通路及凋亡调控因子的增龄变化

目的 观察大鼠脊髓腰节段神经元存活信号转导通路中重要因子和调控凋亡相关因子的增龄变化及与老化的关系.方法 按月龄将雄性SD大鼠分为1、3、6、12、18、24月龄6组.应用免疫组织化学染色和Western印迹法观察增龄大鼠脊髓腰节段神经元存活信号转导通路中的转导因子PI3K、p-p70S6K、磷酯化环腺苷酸应答元件结合蛋白(p-CREB)及凋亡调控因子Bax和Bcl-2的表达情况.结果 大鼠脊髓腰节段神经元p-p70S6K和p-CREB随增龄变化明显,1月龄时表达量较低,3月龄时有所增高,至6月龄、12月龄达高峰,24月龄时明显减低;而PI3K的变化不大.大鼠脊髓腰节段Bax表达随年龄增高逐渐增加,至24月龄表达量达高峰;Bcl-2表达随年龄增加而逐渐降低,至24月龄时降至最低点.与6月龄(青年期)大鼠相比,24月龄(老年期)大鼠脊髓腰节段神经元p-p70S6K、p-CREB和Bcl-2表达明显降低,Bax表达显著增高.结论 老年大鼠脊髓腰节段p-p70S6K、p-CREB和Bcl-2的表达明显降低, Bax表达显著增多,提示脊髓老化与神经元存活信号转导通路和凋亡调控因子的变化有关.     

β-Arrestins在胰岛素/胰岛素样生长因子-1信号转导中的作用

β-Arrestins是G蛋白耦联受体信号转导通路的负调节因子,越来越多的证据表明,β-arrestins也能作用于细胞内的多种信号分子,调节胰岛素/胰岛素样生长因子-1(IGF-1)信号转导通路.在胰岛素的刺激下,β-arrestin 2能够募集蛋白激酶B(Akt)和酪氨酸激酶Src到胰岛素受体,从而调节胰岛素介导的糖代谢效应;而β-arrestin 1则与胰岛素受体底物-1(IRS-1)竞争性结合泛素连接酶Mdm2,从而减少IRS-1的泛素化和降解,促进磷脂酰肌醇3激酶(PI3K)通路的信号转导.在IGF-1介导的信号转导通路中,β-arrestin 1结合并介导了IGF-1受体(IGF-1R)的内吞,促进胞外信号调节激酶活化,正性调节丝裂原活化蛋白激酶通路.此外,β-arrestin 1与IGF-1R相耦联后,能越过信号分子IRS-1而激活PI3K,进而活化Akt,表现出对P13K途径的正性调控作用.     

大黄素对糖尿病KKAy小鼠PI3-K信号转导通路的影响

目的 观察大黄素对2型糖尿病模型KKAy小鼠血糖、胰岛素水平及磷脂酰肌醇3-激酶(PI3-K)信号转导通路的影响.方法 随机血糖均≥13.9 mmol/L的SPF级KKAy小鼠16只,随机分为模型组和治疗组各8只,另选8只C57BL/6J小鼠为正常组.正常组和模型组灌服20 mL/(d·kg)无菌水,治疗组予50 mg/kg大黄素灌胃.8周后测定各组小鼠空腹血糖(FPG)、空腹胰岛素(HNS)并计算胰岛素敏感指数(ISI);用Western blot法测定三组小鼠骨骼肌、脂肪组织中胰岛素受体底物-1( IRS-1)、PI3-K水平及Akt丝氨酸(Ser)473磷酸化水平.结果 模型组小鼠较正常组FPG、FINS明显升高,ISI明显降低,而治疗组小鼠的FPG、FINS较模型组明显降低,ISI明显升高(P均＜0.05).模型组IRS-1、PL3 -K表达水平及胰岛素刺激后Akt Ser473磷酸化升高倍数低于正常组,治疗组IRS-1、PI3-K表达水平及Akt Ser473磷酸化升高倍数高于模型组(P均＜0.05).结论 大黄素灌胃可降低2型糖尿病模型KKAy小鼠血糖、胰岛素水平,并增强胰岛素敏感度,提高小鼠骨骼肌及脂肪组织中的IRS-1、PI3 -K水平及Akt Ser473磷酸化水平.     

限制热量对NIT-1细胞胰岛素／胰岛素样生长因子通路及胰岛素分泌功能的影响

目的 研究限制热量(CR)对胰岛素β瘤细胞(NIT-1)细胞胰岛素/胰岛素样生长因子(IGF-1)信号通路及胰岛素分泌功能的影响,为2型糖尿病(T2DM)的发病机制及防治提供实验依据和理论基础.方法 NIT-1细胞培养至指数生长期,分别用PI3-K的阻断剂LY294002(LY)进行干扰,实验分成4组:对照组;CR组;LY+CR组;LY组.用RT-PCR、免疫细胞化学等方法检测Foxo1及其下游基因的转录及表达,用放射免疫及免疫细胞化学法检测胰岛素的分泌及表达,用细胞计数法检测细胞倍增周期.结果 ①CR可使NIT-1细胞倍增周期延长;②Foxo1主要定位于胞浆,阻断PI3-K后Foxo1从胞浆移入胞核,mRNA转录减少;③CR在正常状态及PI3-K通路障碍的细胞均可使Foxo1mRNA转录增加,并影响其下游基因p27、Bim的转录;④PI3-K通路障碍可使NIT-1细胞胰岛素分泌增加,CR则可使PI3-K通路障碍的NIT-1细胞胰岛素分泌减少.结论 CR不需通过上游PI3-K/Akt,可直接或间接作用于Foxo1参与对胰岛素/IGF-1信号通路的调控,延长细胞寿命,增强NIT-1细胞的糖耐量,改善胰岛素信号的传导及NIT-1的胰岛素分泌功能;可能参与了T2DM的发生发展.     

IGF-1对Rh1肉瘤细胞PI3K/Akt/mTOR信号通路的影响

目的 探讨胰岛素样生长因子(IGF)1对Rh1肉瘤细胞生长活性和PI3 K/Akt/mTOR信号通路的背景变化.方法 常规细胞培养,用无血清培养基消除内源性因子影响27h,再用IGF-1(终浓度为10 ng/ml)刺激72 h,流式细胞仪检测细胞生长活性;另外Western印迹方法观察IGF-1刺激细胞5、10、20、30和60min后Akt(s473)、S6的动态变化.结果 与对照组相比,IGF-1可促进Rh1细胞存活.IGF-1刺激不同时间后S6磷酸化则随着时间的延长逐渐增强;IGF-1亦导致Akt(s473)位点的磷酸化,随时间的延长,磷酸化Akt在5min时达高峰,此后逐渐减弱.结论 Akt、S6等是PI3K/Akt/mTOR信号通路中的重要信号分子,对Rhl细胞而言,在IGF-1刺激下S6有逐渐增强的变化,Akt (s473)位点磷酸化则有减弱的动态变化.     

Apelin抑制人成骨细胞的凋亡

目的 研究apelin对人成骨细胞凋亡的作用.方法 用茜素红染色观察原代培养的人成骨细胞矿化结节的形成.碱性磷酸酶(ALP)用ELISA检测;骨钙素(OC)用放射免疫测定法测定;Ⅰ型胶原用ELISA法检测.用ELISA法检测细胞凋亡.Bcl-2,Bax,caspase-3,cytochmme C,P-Akt,Akt用Western blot检测.PI3-K p85α用免疫沉淀法检测.用小分子RNA干扰技术(siRNAs)抑制APJ表达,联合PI3-K信号转导阻断剂LY294002及Akt信号转导阻断剂HIMO干预,以观察Apelin对成骨细胞凋亡的作用及信号转导.结果Apelin能抑制无血清诱导的人成骨细胞凋亡,siRNAs转染阻断APJ表达可消除此效应.Apelin干预可诱导入成骨细胞Bcl-2蛋白质表达,抑制Bax蛋白质表达和Cytochrome C的分泌及caspase-3的裂解活化.Apelin可诱导人成骨细胞PI3-K及Akt磷酸化;siRNAs转染沉默APJ可消除此效应;PI3-K信号转导阻断剂LY294002或Akt阻断剂HIMO能消除Apelin对人成骨细胞Akt的活化作用;LY294002或HIMO也能阻断Apelin对人成骨细胞抑制凋亡作用.结论 Apelin可抑制人成骨细胞的凋亡;Apelin通过APJ/PI3-K/Akt信号通路介导参与对成骨细胞凋亡的调节.     

Zeste同源2与年龄相关性胰岛细胞功能关系的研究

目的 探讨增强Zeste同源2（EZH2）与年龄相关性胰岛细胞功能的关系. 方法 健康SD大鼠分为1、6、12及24月龄组,每组各6只.取大鼠胰腺,行免疫组化染色.Western blot检测胰岛内EZH2及胰岛素水平.行Ki67染色及TUNEL染色分别检测胰岛细胞增殖及凋亡. 结果 随年龄增加,胰岛内EZH2表达逐渐减少：1月龄组（0.22516±0.03091）;6月龄组（0.18427±0.02315）;12月龄组（0.03165±0.01675）;24月龄组0（P＜0.01）.胰岛细胞增殖逐渐减少：6月龄组（0.36±0.03）％;12月龄组（0.61±0.02）％;24月龄组（0.12±0.02）％（P＜0.01）.胰岛细胞凋亡逐渐增加：12月龄组（0.02±0.03）％;24月龄组（0.09±0.04）％（P＜0.01）.胰岛内胰岛素水平：1月龄组（206.5±27.8） mmol/L;6月龄组（267.4±25.3）mmol/L; 12月龄组（376.2±31.9）mmol/L; 24月龄组（187.8±25.1）mmol/L （P＜0.01）. 结论 随年龄增加,胰岛细胞逐渐衰老,胰岛细胞内EZH2表达逐渐减少,伴随着胰岛细胞增殖能力下降,凋亡增加,胰岛素水平逐渐减低.     

花生四烯酸改善饱和脂肪酸诱导肝细胞胰岛素抵抗的作用机制

目的探讨花生四烯酸（AA）改善软脂酸（PA）诱导的肝细胞胰岛素抵抗（IR）的作用机制。方法分别用0.25 mmol/L PA和PA＋50μmol/L AA与HepG2细胞共同培养24 h,并设正常对照组。测定每组在有无胰岛素刺激时及胰岛素刺激情况下加和不加入PI3K抑制剂WT或PKC抑制剂CC时细胞内糖异生限速酶磷酸烯醇式丙酮酸羧激酶（PEPCK）活性和胰岛素受体底物2（IRS-2）蛋白水平。结果PA组基础及胰岛素刺激的PEPCK活性比对照组和PA＋AA组明显升高,IRS-2蛋白下降（P〈0.05）。加入WT与否,对照组和PA＋AA组PEPCK活性、IRS-2蛋白水平有明显差异（P〈0.05）;加入CC与否,对照组和PA＋AA组IRS-2蛋白水平有明显差异（P〈0.05）;PA组均无变化。结论肝细胞IR时,胰岛素信号转导通路的PI3K途径和PKC通路可能存在严重障碍。AA通过纠正异常的糖代谢关键酶活性和胰岛素信号蛋白量,改善胰岛素信号转导通路缺陷,缓解PA引起的IR。     

细胞因子在干细胞移植存活与分化中的作用

由于心肌坏死后局部组织缺血、缺氧,引起氧化应激和炎性反应,致使移植干细胞的生存和分化能力受到一定影响.胰岛素样生长因子-1、肝细胞生长因子、骨形态发生蛋白-2等细胞因子可通过与其受体结合,激活PI3-K/Akt信号转导途径,从而介导干细胞在移植微环境中的存活,有利于移植干细胞向心肌分化.     

苦酸通调方对HepG2细胞胰岛素抵抗模型中IRS-1、PI3K、Akt蛋白表达的影响

目的观察苦酸通调方对HepG2细胞胰岛素抵抗(IR)模型中内胰岛素受体底物(IRS)-1、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)信号分子的影响并探讨相关机制。方法通过0.25 mmol/L棕榈酸联合30 mmol/L高糖孵育24 h诱导HepG2 IR细胞模型,予以不同浓度的苦酸通调方(50,100,200μg/ml)。葡萄糖试剂盒检测细胞培养液上清葡萄糖含量,肝糖原试剂盒测定HepG2细胞内肝糖原含量,蛋白免疫印迹法(Western印迹)检测细胞内IRS-1、PI3K、Akt的蛋白表达。结果与模型组相比,苦酸通调方干预后,呈剂量依赖性增加IR HepG2细胞的葡萄糖消耗量及细胞内肝糖原含量(P<0.05),上调IRS-1、PI3K、Akt蛋白磷酸活化水平(P<0.05)。结论苦酸通调方改善2型糖尿病IR作用可能与影响IRS-1、PI3K、Akt蛋白表达相关。     

大鼠空肠形态学及肠壁交感神经的增龄变化

目的探讨大鼠空肠形态学及肠壁交感神经的随龄变化。方法雄性SD大鼠104只,按不同发育阶段分为6组。光镜观察空肠形态学变化,增殖细胞核抗原(PCNA)、交感神经酪氨酸羟化酶(Tyrosine hydroxylase,TH)进行免疫组织化学染色,观察空肠增殖能力及交感神经的变化。结果自1月龄开始大鼠空肠绒毛高度和数量、肠壁肌层厚度逐渐增加,至6月龄达到高峰,而后逐渐降低,24月龄显著降低。不同月龄的大鼠空肠PCNA、交感神经TH也呈现相同变化趋势。结论老化状态下,大鼠空肠形态学发生明显变化,黏膜细胞增殖能力减弱,肌间神经丛交感神经减少,交感神经合成去甲肾上腺素能力下降。     

PI3-kinase/Akt/mTOR signaling: Impaired on/off switches in aging,cognitive decline and Alzheimer's disease

The normal on and off switching of the PI3-K (phosphoinositide 3-kinase)/Akt pathway, particularly by its major activators insulin and IGF-1 (insulin-like growth factor-1), is a powerful integrator of physiological responses rudimentary to successful aging. This is highlighted by extensive studies showing that reducing, but not obliterating, activation of the PI3-K/Akt/mTOR signal, at several levels, can extend healthy lifespan in organisms from yeast to mammals. Moreover, aberrant control of the PI3-K/Akt axis is emerging to be a primary causative node in all major diseases of aging: cancer, type 2 diabetes mellitus (T2DM), heart disease and neurodegeneration. Aging is the major risk factor for AD, the most common dementia disorder. The integrated coordination of neuronal responses through the PI3-K/Akt pathway has significant functional impact on key events that go awry in Alzheimer's disease (AD), including: synaptic plasticity, neuronal polarity, neurotransmission, proteostasis, use-dependent translation, metabolic control and stress responses including DNA repair. Investigation of the status of the PI3-K/Akt system in brains of individuals who have had AD shows aberrant and sustained activation of neuronal PI3-K/Akt/mTOR signaling to be an early feature of the disease. This is mechanistically linked to progressive desensitization of normal brain insulin and IGF-1 responses, aberrant proteostasis of Aβ and tau, synaptic loss and cognitive decline in the disease. Notably, concomitantly with feedback inhibition of insulin and IGF-1 responses, increased activation of the neuronal PI3-K/Akt/mTOR axis is a major candidate effector system for transmission of pathophysiological signals from Aβ to tau in the context of defects in synaptic transmission that lead to cognitive decline. Therapeutic approaches targeted at normalizing signaling through either the neuronal PI3-kinase/Akt/mTOR pathway or its activation by insulin and IGF-1 have been shown to be protective against the development of AD pathology and cognitive decline in animal models of AD and some of these therapies are entering clinical trials in patients with the disease.     

Regulation of insulin receptor substrate-2 tyrosine phosphorylation in animal models of insulin resistance

Insulin induces a wide variety of growth and metabolic responses in many cell types. These actions are initiated by insulin binding to its receptor and involve a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (insulin receptor substrates [IRSs]). We investigated IRS-1 and IRS-2 tyrosine phosphorylation; their association with phosphatidylinositol-3-OH kinase (PI3-K); and the phosphorylation of Akt, a serine-threonine kinase situated downstream of PI3-K, in liver and muscle of two animal models of insulin resistance: epinephrine- or dexamethasone-treated rats. We used in vivo insulin infusion followed by tissue extraction, immunoprecipitation, and immunoblotting. IRS-1 and IRS-2 protein expression did not change in liver and muscle of the epinephrine-treated rats, but in dexamethasone-treated rats IRS-1 presented an increase in liver and a decrease in muscle tissue. PI3-K and Akt protein expression did not change in liver or muscle of the two animal models of insulin resistance. There was a downregulation in insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation and association with PI3-K in both models of insulin resistance. In parallel, insulin-induced Akt phosphorylation was reduced in both tissues of epinephrine-treated rats, and in liver but not in muscle of dexamethasonetreated rats. The reduction in insulin-induced Akt phosphorylation may help to explain the insulin resistance in liver and muscle of epinephrine-treated rats and in the liver of dexamethasone-treated rats.     

IRS-3 inhibits IRS-2-mediated signaling in pancreatic beta-cells

IRS-2 plays an important role in the control of pancreatic beta-cell growth, however it is unclear if other IRS family members are also involved. Using recombinant adenoviruses, IRS-1, -2 and -3 expression was varied in the beta-cell line, INS-1. Increased IRS-1 expression had no appreciable effect on beta-cell growth. However, increased IRS-2 expression augmented glucose/IGF-1 induced beta-cell growth mitogenesis and decreased apoptosis due to glucose-deprivation. In contrast, increased IRS-3 expression significantly inhibited mitogenesis and increased apoptosis. IRS-3 was intransiently located to the beta-cell plasma membrane, and appeared to be inert in terms of IGF-1 induced signaling. However, increased IRS-3 expression blocked glucose/IGF-1 induced IRS-2 translocation from the cytosol to the plasma membrane, dampening IRS-2/IGF-1R interaction and subsequent activation of the PI3K/PKB/GSK3 signaling pathway. In contrast, glucose/IGF-1 induced Erk-1/-2 and p70S6K activation were unaffected by IRS-3. These data emphasize the importance of IRS-2/PI3K/PKB signal transduction for beta-cell growth and survival.     

Insulin-like growth factor-1 regulates platelet activation through PI3-Kalpha isoform

Platelets release insulin-like growth factor-1 (IGF-1) from alpha granules upon activation. We have investigated the regulation of IGF-1 in G(i)-dependent pathways leading to Akt activation and the role of IGF-1 in platelet activation. IGF-1 alone failed to induce platelet aggregation, but IGF-1 potentiated 2-MeSADP-induced platelet aggregation in a concentration-dependent manner. IGF-1 triggered platelet aggregation in combination with selective P2Y(1) receptor activation. IGF-1 also caused platelet aggregation without shape change when combined with selective G(z) stimulation by epinephrine, suggesting the role of IGF-1 in platelet aggregation by supplementing G(i) pathways. The potentiating effect of IGF-1 was not affected by intracellular calcium chelation. Importantly, IGF-1 was unable to potentiate platelet aggregation by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, suggesting a critical regulation by PI3-K. Moreover, the potentiating effect of IGF-1 was abolished by the presence of PI3-K p110alpha inhibitor PIK-75. Stimulation of platelets with IGF-1 resulted in phosphorylation of Akt, a downstream effector of PI3-K, which was completely inhibited by wortmannin. IGF-1-induced Akt phosphorylation was abolished by PIK-75 suggesting the contribution of PI3-K p110alpha for activation of Akt by IGF-1. These results demonstrate that IGF-1 plays a role in potentiating platelet aggregation by complementing G(i)- but not G(q)-signaling pathways via PI3-K p110alpha.     

Transgenic overexpression of insulin receptor substrate 1 in hepatocytes enhances hepatocellular proliferation in young mice only

Aim: The insulin receptor substrate-1 (IRS-1) is a multisite docking protein which plays a central role in the signal transduction of growth factors such as insulin and insulin-like growth factors (IGF-1 and IGF-2). It is found to be frequently overexpressed in human hepatocellular carcinoma (HCC). Methods: To study IRS-1 overexpression in hepatocytes in vivo, transgenic mice overexpressing IRS-1 exclusively in hepatocytes were created, showing enhanced hepatocyte proliferation in young animals. In the present study, the phenotype of IRS-1 transgenic animals was characterized over a period of two years. The livers of transgenic and control mice were analyzed for IRS-1 expression and phosphorylation, activation of the downstream mitogen-activated protein kinase (MAPK) cascade and phosphatidylinositol 3' kinase (PI3'K) and macroscopical and histological abnormalities. Results: The enhanced hepatocyte proliferation observed in young IRS-1 transgenic animals was no longer detectable in adult mice. Despite constitutive overexpression and phosphorylation of IRS-1, MAPK- and IRS-1-associated PI3'K activity were significantly reduced in older transgenic mice. Furthermore, no premalignant lesions or HCC were detected in IRS-1 transgenic animals up to the age of 24 months. Conclusions: Therefore, additional mechanisms such as enhanced growth factor expression or impaired negative feedback control mechanisms may augment IRS-1 overexpression in human hepatocarcinogenesis.