PI3K/Akt 及其靶蛋白FOXO1 与非酒精性脂肪性肝病

FOXO转录因子是Forkhead蛋白大家族的一个亚群,在人类的4个同源基因中包括FoxO1、FoxO2、FoxO3a和FoxO4。FoxO蛋白质通过丝氨酸或苏氨酸以及赖氨酸残基的磷酸化和乙酰化等后转录修饰后而发挥作用。其中Foxo1是含有高度保守DNA结合位点的核转录蛋白,其主要功能是磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)的底物,在胰岛素信号转导中起负性调节作用,Foxo1通过介导胰岛素依赖性微粒体甘油三酯转运蛋白(MTP)的表达,影响肝脏装配和分泌极低密度脂蛋白(VLDL),维持脂代谢稳定。在胰岛素抵抗和脂肪肝状态下,肝细胞核内Foxo1表达明显升高,引起高甘油三酯血症和脂肪肝。有针对性的干预PI3K/Akt及Foxo1的表达,可能从分子机制上为非酒精性脂肪肝的防治提供广阔前景。     

FoxO1 对肝脏脂代谢的影响

Fox O1是叉头转录家族O亚族的一员,因其对胰岛素作用起重要的调控作用而被人所熟知,越来越多的研究表明,Fox O1对于肝脏脂质代谢也起重要的调控作用,其作用机制可能是通过上调微粒体甘油三酯转运蛋白(MTP)、载脂蛋白B(Apo B)的表达,从而促进极低密度脂蛋白(VLDL)在肝脏中的合成,增加循环中VLDL含量;Fox O1还可通过促进载脂蛋白CⅢ(Apo CⅢ)的表达,进而抑制脂蛋白酯酶(LPL)活性,减少循环中甘油三酯(TG)分解,导致高甘油三脂血症的发生;同时,Fox O1还能抑制固醇调节元件结合蛋白SREBP-1c表达,抑制脂肪合成。本文主要通过以上几个方面概述了Fox O1与肝脏脂代谢的影响。     

多不饱和脂肪酸调控基因表达的分子机制的研究进展

多不饱和脂肪酸（PUFA）,尤其是n-3系的PUFA,可以降低脂肪酸以甘油三酯形式的沉积,同时促进脂肪酸氧化和葡萄糖合成糖原。其具体机制是PUFA通过激活过氧化物酶体活化增生因子受体α（PPARα）来控制氧化途径过程中的基因表达,而其对脂肪合成途径中有关基因的抑制则是通过降低能传递胰岛素和碳水化合物信息的转录因子与DNA的亲和力和转录因子的核内丰度。尤其是PUFA抑制了类固醇空单元结合蛋白－1（SREBP－1）的核内丰度和表达,降低了核因子Y(NF-Y)、Sp1和肝核因子－4（MNF－4）与DNA的亲和力。     

固醇调节元件结合蛋白-1信号通路研究进展

固醇调节元件结合蛋白-1(sterol regulatory element-binding protein-1,SREBP-1)是脂质代谢重要的核转录因子之一,主要调控脂肪酸、甘油三酯和胆固醇的生物合成。SREBP-1及其靶基因的异常表达能够引起胰岛素抵抗、糖尿病和脂肪肝等一系列代谢性疾病。因此,认识SREBP-1信号通路上下游各因素的表达调控作用就显得非常重要。该文总结了受SREBP-1调控表达的靶基因的特点,着重介绍了胰岛素等上游因子在SREBP-1调控过程中的作用,为指导治疗各类代谢性疾病提供新的思路。     

胰岛素受体底物家族与Ⅱ型糖尿病关系性的研究进展

胰岛素受体底物分子(IRS)是调节胰岛素信号通路的关键物质,在维持细胞生长,分裂和代谢中起着重要作用。目前已发现的家族成员有四个(IRS-1、IRS-2、IRS-3、IRS-4)。目前研究表明,糖尿病的发生与之密切相关:胰岛素信号通路与其他信号通路发生交叉发生干扰,从而导致胰岛素抵抗,引发Ⅱ型糖尿病;IRS蛋白的结构、表达水平异常导致胰岛素信号的中断或减弱,并表现为胰岛素抵抗;四种IRS分子表达的不平衡,致使胰岛素分泌调节的稳态被破坏也可能是糖尿病发病的原因之一。Fox蛋白家族是动物细胞内的一类转录因子,与细胞代谢密切相关。Fox蛋白靶点有可能作为研究治疗糖尿病方法的一种新思路。     

胰岛素对β细胞FoxO1胞质-胞核穿梭定位的影响

目的:通过激光扫描共聚焦显微镜对小鼠胰岛素瘤min6细胞免疫化学染色后观察外源性高胰岛素对Fox O1胞质-胞核穿梭定位的影响。方法:小鼠胰岛素瘤min6细胞用DMEM(low glucose)培养基(含有15%FBS、100 U/m L青霉素、100 U/m L链霉素)于25 m L培养瓶放置在37℃、5%CO2浓度的细胞孵箱中培养。细胞爬片后给予100μIU/m L浓度胰岛素分别刺激12、24和48小时。细胞免疫化学染色后激光扫描共聚焦显微镜观察Fox O1的表达位置变化,用Image pro plus软件对Fox O1荧光强度进行半定量分析。结果:与低糖孵育的对照组相比,胰岛素孵育12 h、24 h和48 h时胞质内Fox O1荧光强度逐渐增强,而细胞核Fox O1荧光强度减弱(P0.05)。结论:高浓度胰岛素孵育min6细胞使Fox O1出细胞核转位至细胞质,并且具有时间依赖性,提示Fox O1是高胰岛素血症对β细胞功能影响的机制之一。     

叉头框(Fox)转录因子家族的结构与功能

叉头框(forkheadbox,Fox)蛋白家族是一类DNA结合区具有翼状螺旋结构的转录因子,目前已有17个亚族。Fox蛋白不仅能作为典型的转录因子通过招募共激活因子等调节基因转录,有些还能直接同凝聚染色质结合参与其重构,协同其他转录因子参与转录调节。PI3K-Akt/PKB、TGFβ-Smad和MAPKinase等多条信号通路都可以影响Fox蛋白的磷酸化水平,从而调节其活性。Fox蛋白在胚胎发育、细胞周期调控、糖类和脂类代谢、生物老化和免疫调节等多种生物学过程中发挥作用。     

固醇调节元件结合蛋白2信号通路与非酒精性脂肪性肝病

非酒精性脂肪性肝病（non-alcoholic fatty liver disease,NAFLD）是以肝细胞内甘油三酯和胆固醇等脂毒性脂肪过度沉积为主要特征的一种临床获得性代谢综合征。最新研究表明,NAFLD向非酒精性脂肪肝炎(NASH)进展时,肝内胆固醇积累可能较甘油三酯更具有细胞毒性风险。固醇调节元件结合蛋白2（sterol regulatory element-binding protein 2,SREBP2）是脂质代谢重要的核转录因子之一,主要调控胆固醇的生物合成和体内平衡。SREBP2及其靶基因调控的胆固醇异常是引起非酒精性脂肪肝病发生发展的重要因素之一。因此,认识SREBP2信号通路中,上下游各因素的表达调控作用与NAFLD发病机制之间关系,就显得非常重要。本文总结了受SREBP2调控表达的靶基因的特点,着重介绍SREBP2调控胆固醇体内合成与平衡的信号通路与NAFLD发病机制之间关系,为研究和指导治疗NAFLD及其代谢性疾病提供新的思路。     

心肌细胞核钙调素Ⅰ介导的bcl-2转录调节在大鼠心肌肥厚中的作用

为探讨心肌细胞核钙调素Ⅰ（calmodulinⅠ,CaMⅠ）介导的bcl-2转录调节存人鼠心肌肥脬中的作用及其可能机制,实验随机分为对照组和心肌肥厚组,采用腹卡动脉缩窄法制备人鼠心肌肥厚模犁。模型复制成功后4周,以改良差速离心和密度梯度离心提取并纯化细胞核;蛋白印迹法测定心肌细胞核cAMP反应元件结合蛋白（cAMP response-element binding protein,CREB）及磷酸化CREB（phosphorylated cAMP response-element binding protein,pCREB）表达;免瘦组化法观察左审心肌组织CaMI蛋白表达及分布;延续转录分析法观察阻断CaMⅠ后心肌细胞核bcl-2 mRNA的变化。结果表明,心肌肥厚组pCREB蛋白表达较对照组明显增加（P〈0．05）,CREB蛋门表达无明显变化（P〉0．05）;CaMⅠ分布于细胞核及细胞浆,心肌肥厚组CaMⅠ蛋白表达较对照组明显增加（P〈0．05）;使用CaM抑制刺后心肌细胞核bcl-2 mRNA表达明显上调（P〈0．05）。结果提示,压力超负荷时心肌细胞核内CaMⅠ激活,抗凋亡基因bcl-2表达下调,核转录因子CREB磷酸化增加,但CREB在调节bcl-2基因转录过程中可能发挥次要作用。     

叶酸缺乏致胎鼠宫内发育迟缓及肝脏胰岛素生长因子系统表达变化

目的：叶酸是一种水溶性B族维生素,在体内氨基酸与核苷酸代谢中起重要作用,是胎儿生长发育所必须的营养素。本文通过建立叶酸缺乏的孕鼠模型,探讨叶酸缺乏对胎鼠宫内发育的影响,并研究胎鼠肝脏组织中胰岛素生长因子（IGF）系统的表达变化。方法：雌性C57BL／6J小鼠叶酸缺乏组6只、正常对照组6只,分别饲以不舍叶酸和含2mg叶酸／kg的纯合饲料。四周后与雄鼠交配,于怀孕第13．5天（13．5dpc）对孕鼠剖腹取胎,观察和评价胎鼠发育指标,并对宫内发育迟缓（IUGR）比率进行统计。用Real-timePCR法检测胎鼠肝脏组织中胰岛素生长因子I（IGFI）、胰岛素生长因子I受体（IGFIR）、胰岛素生长因子II（IGFII）、胰岛素生长因子II受体（IGFIIR）、胰岛素生长因子结合蛋白1（IGFBP-1）和胰岛素生长因子结合蛋白3（IGFBP-3）mRNA的相对表达水平。结果：叶酸缺乏组雌鼠合笼前每日体重增长量降低,13．5dpc胎鼠吸收胎和死胎比率升高,胎重下降,IUGR比率显著升高,差异有统计学意义（P〈0．05）;叶酸缺乏组胎鼠肝脏组织中IGFII和IGFIIRmRNA的相对表达水平均低于正常对照组（P〈0．05）,IGFI、IGFIR、IGFBP-1和IGFBP-3mRNA的相对表达水平两组间没有差异（P〉0．05）。结论：叶酸缺乏会导致小鼠孕中期胎鼠IUGR比率升高及胎肝IGFII和IGFIIRmRNA的表达水平降低,提示叶酸缺乏对IGF系统基因的调控,可能与胎鼠I-UGR发生机制有关。     

Insulin regulates menin expression, cytoplasmic localization, and interaction with FOXO1

Menin is the ubiquitously expressed nuclear protein product of the MEN1 gene, which interacts with PKB/Akt in the cytoplasm to inhibit its activity. This study describes a novel insulin-dependent mechanism of menin regulation and interaction with other metabolic proteins. We show that insulin downregulated menin in a time-dependent manner via the human insulin receptor. Inhibition analysis indicated a critical role for the protein kinase Akt in regulation of menin expression and localization. Insulin-mediated decrease in menin expression was abrogated by the PI3K/Akt inhibitor LY-294002 at early time points, from 2 to 7 h. Furthermore, exposure to insulin resulted in the cytoplasmic localization of menin and increased interaction with FOXO1. Fasting followed by refeeding modulates serum insulin levels, which corresponded to an increase in menin interaction with FOXO1 in the liver. Liver-specific hemizygous deletion of menin resulted in increased expression of FOXO1 target genes, namely IGFBP-1, PGC-1α, insulin receptor, Akt, and G-6-Pase. This study provides evidence that menin expression and localization are regulated by insulin signaling and that this regulation triggers an increase in its interaction with FOXO1 via Akt with metabolic consequences.     

PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27Kip1

Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We previously demonstrated that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in primary MKs in a PI3K dependent fashion. UT-7/TPO, a megakaryocytic cell line, stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to reduce p27Kip1 expression after TPO stimulation, and fail to induce p27Kip1 expression following TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In addition, the number of MKs is significantly increased in bone marrow from Foxo3a-deficient mice. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings indicate that the PI3K/Akt/FOXO3a/p27Kip1 pathway contributes to normal TPO-induced MK proliferation.     

Activation of peroxisome proliferation–activated receptor-γ inhibits transforming growth factor-β1-induced airway smooth muscle cell proliferation by suppressing Smad–miR-21 signaling

The aims of the current study were to examine the signaling mechanisms for transforming growth factor-β1 (TGF-β1)-induced rat airway smooth muscle cell (ASMC) proliferation and to determine the effect of activation of peroxisome proliferation–activated receptor-γ (PPAR-γ) on TGF-β1-induced rat ASMC proliferation and its underlying mechanisms. TGF-β1 upregulated microRNA 21 (miR-21) expression by activating Smad2/3, and this in turn downregulated forkhead box O1 (FOXO1) mRNA expression. In addition, TGF-β1–Smad–miR-21 signaling also downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and thus de-repressed the PI3K–Akt pathway. Depletion of PTEN reduced the nuclear FOXO1 protein level without affecting its mRNA level. Inhibition of the PI3K–Akt pathway or proteasome function reversed PTEN knockdown-induced nuclear FOXO1 protein reduction. Our study further showed that loss of FOXO1 increased cyclin D1 expression, leading to rat ASMC proliferation. Preincubation of rat ASMCs with pioglitazone, a PPAR-γ activator, blocked TGF-β1-induced activation of Smad2/3 and its downstream targets changes of miR-21, PTEN, Akt, FOXO1, and cyclin D1, resulting in the inhibition of rat ASMC proliferation. Our study suggests that the activation of PPAR-γ inhibits rat ASMC proliferation by suppressing Smad–miR-21 signaling and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.