根皮苷对db/db小鼠糖尿病心肌病变保护机制的定量蛋白质组学研究
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摘要
第一部分根皮苷对db/db小鼠糖尿病心肌病变保护机制的定量蛋白质组学研究
研究背景
糖尿病(diabetes mellitus, DM)是一种由于胰岛素绝对或相对分泌不足而导致的以慢性血葡萄糖水平增高为特征的一种常见内分泌代谢性疾病。近年来,DM的发病率和患病率在全球范围内呈现逐年升高的趋势,已成为严重威胁人类健康与生活质量的世界性公告卫生问题。DM不仅仅表现为长期慢性的血糖升高,还可伴有多种并发症。其中心血管并发症已经成为DM并发症中致残率和致死率最高、危害最大的并发症。
糖尿病性心脏病是糖尿病患者致死的主要原因之一,尤其是在2型糖尿病患者中。广义的糖尿病心脏病包括冠状动脉粥样硬化性心脏病(冠心病),糖尿病心肌病变和糖尿病心脏自主神经病变等。糖尿病心脏病与非糖尿病患者相比常起病比较早,糖尿病患者伴冠心病常表现为无痛性心肌梗死,梗死面积比较大,穿壁梗死多,病情多比较严重,预后比较差,病死率较高；如冠状动脉造影和临床排除冠状动脉病变,糖尿病患者出现严重的心律失常心脏肥大肺淤血和充血性心力衰竭,尤其是难治性心力衰竭临床可考虑糖尿病心肌病(Diabetic Cardimyopathy, DCM)。DCM是指发生在DM中,不能用高血压性心脏病、冠心病、心脏瓣膜病及其他心脏病来解释的心肌疾病。这一疾病在代谢紊乱及微血管病变的基础上引发心肌广泛灶性坏死,出现亚临床心功能异常,最终进展为心律失常、心力衰竭及心源性休克。迄今为止,针对糖尿病引起的心肌损伤目前尚无特效疗法,临床治疗首先是控制DM,然后主要针对心肌损害、心律失常、心力衰竭和抗血栓进行治疗。而且由于部分患者早期并无明显症状,而上述治疗方法均存在一定局限性,并不能有效及时的阻止它的的发展。因此,在临床实践中迫切需要寻找能有效防治糖尿病心肌病变的新型药物与途径,进一步完善与充实糖尿病心肌病变的治疗策略。
根皮苷(phlorizin, PHL)是根皮素(phloretin)的2’-β-D-葡萄糖苷,是从苹果,苹果树皮及叶中提取的一种二氢查尔酮苷。大量研究表明,PHL具有多种生物活性与药理作用,如调节血糖、抗炎、抗肿瘤、改善认知以及抗氧化等等,目前已被应用于医药、化妆品、食品等领域。近年来,PHL作为新型药物在防治DM及其并发症中的作用获得广泛关注。动物实验发现,长期口服PHL对DM并发症的靶器官如肾脏、心脏、大动脉和视网膜的病变都起到显著地保护和改善作用。虽然临床研究已经证实了PHL对于DM患者心脏的保护作用,但是PHL对糖尿病心肌病变的改善及其保护作用的可能机制,目前国内外鲜见报道；PHL治疗糖尿病心肌病变的作用分子靶点也尚未明确。
同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation, iTRAQ)技术是近年来最新开发的一种新的蛋白质组学定量研究技术,具有良好的定量效果、较高的重复性,并可对多达四种不同样本同时进行定量分析。目前,iTRAQ技术已经在蛋白质组学的定量研究中得到了极其广泛的应用。但是,国内外尚无报道采用iTRAQ标记及相关分析技术来研究根皮苷对2型糖尿病心肌损伤的保护机制。
因此,在本研究中,我们应用了目前较为普遍认可的db/db小鼠的2型DM动物模型,通过10周的PHL灌胃,观察PHL对于db/db小鼠体重、血糖、血脂、血胆固醇及血中AGEs水平的影响,并对db/db小鼠的心脏进行了组织病理学和超微结构的形态学观察,旨在观察天然药物PHL治疗对于db/db小鼠体重、DM有关代谢指标的影响及其对心脏的保护作用,为临床治疗糖尿病性心脏病提供新的治疗思路与途径。另外,我们还应用了iTRAQ蛋白质组学定量技术,Turbo SEQUEST软件和国际蛋白质索引(international protein index, IPI)数据库检索等生物信息学方法,分离与鉴定正常对照组(CC组),db/db小鼠组(DM组)与db/db小鼠PHL治疗组(DMT组)等小鼠心肌组织的差异表达蛋白,旨在揭示PHL对于db/db小鼠糖尿病心肌病变变的分子保护机制,为开展药物研发工作寻找新的药物靶标开辟新途径,为临治疗提供新的思路。
研究目的
1.研究db/db小鼠在糖尿病进展中体重及有关代谢指标的变化,通过对db/db小鼠的心脏组织病理学和超微结构的形态学观察,评价糖尿病心肌病变的特征与程度。初步探讨db/db小鼠糖尿病心肌病变的差异表达蛋白的特征,进一步了解糖尿病心肌病变的分子机制。
2.研究PHL对db/db小鼠体重及糖尿病有关代谢指标的变化的影响,通过对db/db小鼠的心脏组织病理学和超微结构的形态学观察,评价PHL对糖尿病心肌病变的保护作用。探讨db/db小鼠经PHL治疗后心肌组织的差异表达蛋白,根据其所参与的生物过程和代谢通路筛选出药物作用的关键蛋白与候选靶标,进一步揭示PHL对于糖尿病心肌病变的保护机制,为临床治疗提供新的理论基础。
研究方法
7周龄的雄性C57BLKS/J db/db小鼠16只,以及7周龄雄性C57BLKS/J db/m小鼠8只。小鼠均予观察1周后开始正式实验。用C57BLKS/J db/m小鼠8只作为正常对照组(CC),C57BLKS/J db/db小鼠分为两组：一组随机8只小鼠为DM模型组(DM),每日用生理盐水灌胃；另一组随机8只为PHL干预组(DMT),每日用体积相同生理盐水的20mg/kg/d的PHL溶液灌胃,干预时间为10周。实验期间每周定期测量每只小鼠的体重并记录。实验结束时,所有小鼠空腹过夜并处死,采血检测空腹血糖(FBG),血甘油三酯(TG),血总胆固醇(TC)及血清糖基化终末代谢产物(AGES)等指标；并迅速分离心脏组织,按照病理取材常规制作心肌石蜡切片。剩余心脏组织分离后立即存于液氮-80℃保存留待进步蛋白质组学实验用。
从正常对照组(CC组),db/db小鼠组(DM组)与db/db小鼠PHL治疗组(DMT组)三组小鼠中各选取4只,分离心脏。每只小鼠取50mg心脏组织,进行心肌组织研磨、切碎、超声破碎、裂解来提取心肌总蛋白。各组取60ug肽段消化后用iTRAQ染料进行标记,正常对照组用114标记,PHL干预组用116标记,db/db糖尿病组用117则标记。将各组已标记的肽段混合,用强阳离子交换柱(strong cation exchange, SCX)进行分离分级,收集穿流以及洗脱部分合并成10组。接下来用Thermo Finnigan LTQ Velos质谱仪进行液相质谱-串联质谱分析(LC-ESI-MS/MS)。最后,使用相关软件将定量及检定结果进行合并处理,得到定量和鉴定结果。搜索使用的数据库为ipi.MOUSE.v3.72.REVERSED.fasta蛋白库(Sequest结果过滤参数为：Protein FDR<0.01; Peptide FDR<0.01),应用EXPASY蛋白质组学工具来分析等电点、分子量等。然后采用Ingenuity Pathway Analysis软件(www.ingenuity.com)对经鉴定的心肌蛋白质进行蛋白功能和通路的分析。最后将筛选出的部分差异蛋白用蛋白质免疫印迹方法(Western blot)进
一步验证其在心肌组织中的表达。
研究结果
1.一般观察
CC组小鼠生长良好,精神状况佳,毛发光亮顺泽,活跃。DM组小鼠在实验进程中,逐渐表现为毛发污秽无光泽,被毛蓬松,少动,出现明显的多饮、多食、多尿,体重迅速增加。而DMT组小鼠的上述表现较DM组有所减轻。
2.PHL对db/d小鼠体重、FBG、TG、TC与AGEs的影响
随着观察时间的延长,DM组与DMT组小鼠的体重较对照组呈显著增加。从实验第2周开始,DM组小鼠体重逐渐增加,一直持续到实验结束的第10周(P<0.05)。然而,DMT组小鼠与DM组相比,PHL灌胃显著改善了DM小鼠体重的快速增加(P<0.05)。
实验开始时,各组小鼠的血FBG、TG、TC及AGES水平无明显差异。至实验结束时DM组小鼠的FBG、TG、TC及AGES水平与CC组比较,均明显升高(P<0.05)。给予PHL干预的DMT组小鼠的FBG、TG、TC及与AGES水平与DM组比较则明显下降(P<0.05)。
3.PHL对db/db小鼠心肌组织病理学变化的影响
实验结束后,将db/db小鼠心肌组织切片进行苏木素-伊红(HE)染色进行观察。DM组的小鼠的心肌组织较CC组呈现出明显的心肌细胞肥大和心肌细胞排列不规则,并伴有细胞核损伤。然而,DMT组经过PHL的治疗大大减少了心肌组织肥大以及细胞核损伤的情况。
4.PHL对db/db小鼠心肌组织超微结构变化的影响
通过电子显微镜下对db/db小鼠心肌组织超微结构的观察,发现CC组小鼠心肌细胞肌原纤维完整,排列整齐,线粒体结构正常,呈线性排列；细胞核形态良好,核膜完整。而DM组心肌细胞中可以观察到线粒体肿胀,嵴溶解,有很大区域的肌原纤维及线粒体排列混乱不规则；细胞核的形态有所改变,核膜不完整。然而,在DMT组,PHL的治疗明显的降低了心肌细胞中损伤的线粒体的数量,并且减轻了肌原纤维的不规则排列情况。
5.iTRAQ鉴定结果
经LS-ESI-MS/MS鉴定并予软件分析、数据库检索后,共鉴定出蛋白1627种,符合鉴定条件者可信蛋白1591中,具有唯一肽段7836个。其中鉴定在DM组与正常对照组心肌蛋白相比表达有变化但经PHL治疗后回调的差异表达蛋白共113个,其中在DM组表达上调的但经PHL治疗后回调的有29个,在DM组表达下调的但经PHL治疗后回调的有84个
6.PHL干预后重要差异蛋白质特征
在这些经PHL治疗后回调的差异表达蛋白中,我们发现一些涉及到糖尿病心肌病变发生发展的重要蛋白,并把它们按照功能和所参与的代谢过程分为：与心肌脂代谢有关的蛋白、与心肌线粒体有关的蛋白和与心肌病发生发展有关的蛋白,进一步明确这些重要蛋白在糖尿病心肌病变中的可能起的作用。
7.PHL干预后差异蛋白的生物信息学分析
对于PHL干预后的113个差异蛋白点,采用IPA软件对有显著改变的蛋白所参与的重要生物过程和疾病进行分析。结果发现,这些蛋白所参与的生物过程和疾病包括心血管疾病、脂代谢、心血管系统的发育和功能、内分泌系统疾病、自由基的修复和能量的产生等等,都是与糖尿病心肌病变的发展密切相关的生物过程。
对于PHL干预后的113个差异蛋白点,采用IPA软件绘制差异蛋白功能网络来说明这些蛋白之间的相互关系。网络中共出现35个蛋白,其中有24个蛋白是我们筛选出的重要的差异蛋白,比如Dapk3, Titin, Prkaa, Des, ILK, Nampt等。此蛋白相互作用网络为我们进一步研究脂质代谢、线粒体功能和心肌病的发生和发展之间的关系提供了新的思路。
8.筛选的差异蛋白在心肌组织的表达改变
为了验证蛋白质组学鉴定出的部分差异蛋白在心肌组织的表达,应用Western blot方法检测其中差异蛋白如钙联蛋白(Calnexin)与整合素连接激酶(integrin-linked protein kinase,ILK)在各组小鼠心肌组织中的表达。结果显示,与在正常对照组小鼠心肌中的表达比较,Calnexin在db/db小鼠中表达明显增高,然而PHL治疗后,表达增高的Calnexin有所下调。另外,ILK在db/db小鼠中表达中与正常对照组比较表达后明显降低,经过PHL干预后该蛋白表达回调。经过VisionWorks LS image acquisition and analysis software软件分析亦显示该蛋白质印迹实验结果与iTRAQ鉴定结果是一致的。
结论
1.与正常对照组比较,db/db小鼠随年龄增长体重显著增加,血FBG、TG、TC及AGEs水平显著升高；PHL灌胃干预可以明显抑制db/db小鼠的肥胖趋势,并且能够显著降低其FBG、TG、TC及与AGEs水平。说明PHL可能通过改善db/db小鼠的糖尿病整体代谢紊乱状态来预防DM的进展和并发症的发生
2.心肌组织的形态学观察显示,PHL治疗可以明显改善db/db小鼠心肌组织肥大以及细胞核损伤的情况；而且,PHL治疗明显减轻db/db小鼠心肌细胞中肌原纤维的不规则排列情况,降低损伤的线粒体的数量。说明PHL对db/db小鼠的糖尿病心肌病变具有保护作用。
3.作为定量蛋白质组学的新技术,iTRAQ具有高灵敏性、高通量,良好的定量效果、较高的重复性,能够对于多组样本同时进行比较分析以便获得更好的蛋白质组覆盖率。所以,iTRAQ能够用于研究PHL对db/db小鼠糖尿病心肌病变保护作用的分子机制,帮助发现新的药物靶标。
4.应用LS-ESI-MS/MS鉴定得到db/db小鼠组与正常对照组心肌蛋白相比表达有变化但经PHL治疗后回调的差异表达蛋白共113个,其中在DM组表达上调的但经PHL治疗后回调的有29个,在DM组下调的但经PHL治疗后回调的有84个。采用IPA分析发现这些蛋白参与的生物过程和疾病涉及到心血管疾病、脂代谢、心血管系统的发育和功能、内分泌系统疾病、自由基的修复和能量的产生等等,提示这些过程可能与糖尿病心肌病变的发生与发展密切相关。
第二部分质膜微囊的脂肪酸组成和小窝蛋白-1的脂肪酸化的研究
研究背景
质膜微囊(Caveolea),是一种特殊类型的脂筏,是哺乳动物细胞质膜上呈细颈烧瓶状的内陷结构,内含丰富的胆固醇、鞘磷脂和鞘糖脂。质膜微囊大量存在于内皮细胞、脂肪细胞、血管平滑肌细胞、纤维母细胞和肺上皮细胞,参与许多细胞生命活动,细胞内吞、胆固醇运输、细胞膜组装、信号传导和肿瘤生成。小窝蛋白(caveolin)是质膜微囊区别于其它脂筏结构的特征性蛋白分子,它与信号传导、物质转运、细胞增殖等密切相关,维持着质膜微囊的结构和功能。Caveolin-1能与多种信号分子如G蛋白α亚基、酪氨酸激酶受体、PKCs、Src家族酪氨酸激酶和eNOS相互作用。生物体内小窝蛋白异常可以引起多种疾病或异常。
除了富含胆固醇和鞘磷脂,caveolea内也含有多种脂肪酸。一般认为,大多数细胞的蛋白质脂肪酸化是以共价键形式与肉豆蔻酸(C14：0)和／或棕榈酸(C16：0)结合,并且蛋白质的脂肪酸化对其在细胞膜上的定位十分重要。从目前国内外的研究情况来看,虽然caveolea的结构成分已经较为清楚,但是对于其所含脂肪酸的种类组成与绝对定量的研究并不多见；此外,目前对于caveolea内的许多蛋白质(如eNOS)的脂肪酸化及功能研究报道诸多,但是作为caveolea的支架蛋白,有关caveolin-1的脂肪酸化及其在caveolea上定位的影响却报道甚少。在本研究中,我们采用了气相色谱-质谱技术(GC/MS)来定性和定量中国仓鼠卵巢(CHO)细胞中脂肪酸、caveolea内含有的脂肪酸和以共价键形式结合在caveolin-1上的脂肪酸,并用同位素标记的脂肪酸验证了caveolin-1的脂肪酸化,以及观察了caveolin-1的脂肪酸化对其在caveolae上的定位是否有影响,旨在明确特定脂肪酸与caveolea、caveolin-1的关系,为日后研究某种特定脂肪酸对蛋白质的酸化作用奠定理论基础。
研究目的
1.采用气相色谱-质谱技术(GC/MS)对CHO细胞中脂肪酸、caveolea内含有的脂肪酸和以共价键形式结合在caveolin-1上的脂肪酸来进行定性和定量,旨在明确其相关脂肪酸组成及含量。
2.采用同位素标记的脂肪酸,通过观察与非标记脂肪酸竞争性结合caveolin-1,来验证caveolin-1的脂肪酸化的种类。
3.通过将CHO细胞培养在含有或不含有特定脂肪酸的环境中,观察caveolin-1的脂肪酸化对其在caveolae上的定位是否有影响。
研究方法
中国仓鼠卵巢(CHO)细胞在10cm的培养盘中使用Ham's F-12培养基(含5%的FBS,2mmol/L的L-谷氨酰胺,100U/mL的青霉素和100μg/ml的链霉素)培养至90%融合,用冷的PBS洗5遍后溶解于1mlMBST/OG(含25mM的MES,150mM NaCl,1%Triton-100,60mM心肌糖苷,PH=6.7),在冰上放置30分钟。使用Op ti-prep的方法将质膜微囊(caveolea)、细胞质(cytosol)、质膜(plasma membrane)、内膜(internal membrane)和去核后上清液(post nuclear supematan, PNS)分离。为了提取用于脂肪酸定量的caveolin-1,CHO细胞的裂解液中加入抗caveolin-1IgG在4。C孵育18小时,然后用蛋白A磁珠孵育另外2小时。离心,收集珠子,然后使用高盐缓冲液(500mM NaCl)洗5次。用于定量分析的脂肪酸珠子用1M的NaOH孵育过夜,然后用1M HC1中和,再用Folch试剂(氯仿：甲醇=2：1)提取脂质,并用GC/MS分析。非免疫兔IgG被用作阴性对照。用GC/MS对脂肪酸进行定量方法是：用Folch/BHT试剂来提取样本中的脂质,50μl的二十三烷酸(23：0)(5mg/ml的氯仿)加入提取液中作为内参。然后样本中全部的脂质用BF3/甲醛(10%)进行甲基脂化。用装有omegawax250毛细管柱的气相色谱系统(Agilent6890GC G2579A)来分析脂肪酸甲基脂。大型选择性探头(MSD, Agilent5973)用来识别目标峰,氢火焰离子化检测器(FID)用于定量脂肪酸。蛋白质印迹方法原理与步骤同第一部分。一抗为抗caveolin-1IgG多克隆抗体(滴度1：200,Sigma. USA)。为了测定Caveolin-1的脂肪酸化,CHO细胞在10cm培养皿中使用Ham's F-12培养基中培养至80%融合(含5%的FBS,2mmol/L的L-谷氨酰胺,100U/mL的青霉索和100μg/ml的链霉素)。使用含1%BSA的培养基饥饿细胞十八小时后,用2.5mCi的3H棕榈酸(3H-C16：0)或者25μCi的14C硬脂酸(14C-C18：0)在有／无30倍浓度的非标记棕榈酸和硬质酸的培养基中室温标记3小时。将细胞溶解于MBST/OG缓冲液,用抗caveolin-1蛋白A免疫沉淀。非免疫兔IgG被用作阴性对照。免疫沉淀下来的caveolin-1用SDS-PAGE分离,转移到PVDF膜上,caveolin-1的脂肪酸化用自动放射性监测仪检测在-80℃存放6周,用Kodak MS胶卷显影。
研究结果
1.CHO细胞中FA的含量及组成
通过用GC/MS检测CHO全细胞裂解液中的脂肪酸的种类及含量,我们发现CHO细胞中含有多种脂肪酸,包括饱和与不饱和脂肪。在这些脂肪酸中油酸占了高达59.5%的比例(C18:1,307±18.4μg/5×107cells),其次是棕榈酸(C16:0,91.8±9.5μg/5x107cells;17.8%)),硬脂酸(C18:0,51.8±10.2μg/5×107cells;10%)),鳕油酸(C20:1,22.5±1.4μg/5×107cells;4.4%),棕榈油酸(C16:1,18.7±1.1μg/5x107cells；3.6%),花生四烯酸(C20:4,14.8±1.8μg/5×107cells;2.9%)和肉豆蔻酸(C14:0,9.0±1.4μg/5×107cells;1.7%).
2Caveolae中FA的含量及组成
通过用GC/MS检测caveolea中脂肪酸的含量与组成,我们发现caveolea中含有有限的几种脂肪酸,主要为棕榈酸(C16:0,0.48±0.06μg/5×107cells;24%),硬脂酸(C18:0,0.61±0.07μg/5×107cells;30%)和油酸(C18:1,0.83±0.21μg/5x107cells;40%)。
3.与Caveolin-1结合的FA的含量及组成
一般认为,质膜蛋白会容易被肉豆蔻酸(C14：0)和棕榈酸(C16：0)所脂肪酸化。然而通过用GC/MS检测与caveolin-1结合的脂肪酸含量与组成,我们发现约60%的与caveolin-1结合的脂肪酸为硬脂酸(C18：0),40%的为棕榈酸(C16：0)。我们并没有没有检测到任何的肉豆蔻酸(C14：0)与CHO细胞的caveolin-1相结合。
4.测定Caveolin-1的脂肪酸化
为了进一步验证结合于caveolin-1上的脂肪酸种类及相互作用,我们采用了同位素标记的脂肪酸来检测Caveolin-1脂肪酸化。结果显示,在用3H-棕榈酸(3H-C16：0)标记的细胞中,我们检测到了结合到caveolin-1上的大量3H-棕榈酸(3H-C16：0),说明棕榈酸可以直接与caveolin-1相结合。当有过量的非标记棕榈酸(C16：0)和硬脂酸(C18：0)存在时,非标记棕榈酸(C16：0)和硬脂酸(C18：0)有效的阻滞了3H-棕榈酸(3H-C16：0)与caveolin-1的结合。但是过量的非标记油酸(C18：1)只是轻度的阻滞了3H-棕榈酸(3H-C16：0)和caveolin-1的结合。
同样的,在用14-硬脂酸(14C-C18:0)标记的细胞中,我们检测到了与caveolin-1结合的大量14-硬脂酸,说明硬脂酸可以直接与caveolin-1相结合。当有过量的非标记硬脂酸(C18：0)存在时,非标记硬脂酸(C18：0)能够有效地抑制14C-硬脂酸(14C-C18：0)与caveolin-1的结合。过量的非标记油酸(C18：1)和棕榈酸(C16：0)虽然也阻滞了14C-硬脂酸(14C-C18：0)与caveolin-1的结合,但是阻滞的效果较之非标记硬脂酸(C18：0)不明显。
这些数据说明棕榈酸(C16：0)和硬脂酸(C18：0)可以直接结合于caveolin-1上,并且是其结合的主要脂肪酸。5.脂肪酸化对Caveolin-1亚细胞定位的影响
蛋白质的脂肪酸化可以影响蛋白的亚细胞定位。为了检测caveolin-1的脂肪酸化是否影响了caveolin-1的亚细胞定位,我们把CHO细胞分别培养在含有20%FBS,1%BSA,1%BSA+棕榈酸(过量)和1%BSA+硬脂酸(过量)培养基中。结果显示,caveolin-1在caveolea上的定位在20%FBS与1%BSA培养基中没有明显差别,在加入和不加入过量棕榈酸或硬脂酸培养基也未见明显差别。说明棕榈酸和硬脂酸对于caveolin-1在caveolea上的定位没有明显影响。
结论
Caveolea含有有限种类的部分脂肪酸,其中饱和脂肪酸的含量较高,这与CHO全细胞中脂肪酸的组成有所不同。与caveolin-1结合的最主要脂肪酸是硬脂酸,而不是之前推测的肉豆蔻酸。Caveolea独特的脂肪酸结构和caveolin-1的脂肪酸化可能对于caveolea的形成和维持其功能非常重要。对这些相关脂肪酸的定性和定量研究将会对进一步揭示Caveolea的功能和caveolin-1的脂肪酸化对于细胞膜信号传导所起的作用有重要的意义。
Investigation of the Protective Effects of Phlorizin on Cardiac Damage in db/db Mice by Quantitative Proteomics
Background
Diabetes mellitus (diabetes mellitus, DM) is a common endocrine and metabolic disease caused by absolute or relative insulin secretion defect and characterized by chronic increased blood glucose levels. In recent years, with the improvement of the living standard, modern life styles and aging, the prevalence of diabetes mellitus is rapidly increasing worldwide. It has become a worldwide public health problem and a serious threat to human health and quality of life. DM not only develops sustained elevated circulating glucose, but can be accompanied by a variety of complications attacking both macrovascular and microvascular lesion leading to a variety of complications. Among these, cardiovascular complication has become one of the most harmful complications with the highest morbidity and mortality.
Diabetic heart disease is the main cause for death of diabetic patients, especially in type2diabetes. Broadly-defined concept of diabetic heart disease includes coronary heart disease, diabetic cardiomyopathy and diabetic cardiovascular autonomic neuopathy. Patients with diabetes often develop hypertension and atherosclerosis leading to cardiovascular complications. However, some diabetic patients develop heart failure without hypertension and coronary artery disease. This phenomenon was first described by Rubler et al. and was termed "diabetic cardiomyopathy"(DCM). Diabetic cardiomyopathy is characterized by structural and functional changes in the heart, such as elevated left ventricular (LV) mass, myocardial fibrosis, and abnormal diastolic function, eventually progress to arrhythmia, heart failure and cardiogenic shock. However, the mechanistic details of diabetic cardiac damage remain unclear, and this disease has not yet been sufficiently studied. The clinical treatment of diabetic cardiac damage focuses on controlling DM, preventing myocardial damage, arrhythmia, heart failure and antithrombotic therapy. So far, current therapeutic options for the treatment of diabetic heart disease, as described above, have certain limitations and are far from satisfactory. Therefore, there is an urgent need to find the more effective approaches for the intervention of diabetic heart disease.
Phlorizin (phloretin-2'-O-glucoside, PHL), a dihydrochalcone derived from apple peels, bark and leaves, is a known antioxidant. Previews studies have shown that PHL has many biological activities and pharmacological actions, such as blood glucose regulation, anti-inflammation, anti-tumor, cognition improvement, antioxidative action and so on. It has been used in medicine, cosmetics, food and other fields. The main pharmacological property of phlorizin is to produce renal glycosuria and block intestinal glucose absorption through inhibition of sodium/glucose cotransporters in the kidney and intestine. In addition, animal experiments showed that long-term oral PHL administration has protective effect on DM complications related to targeting organs such as the kidney, heart, aorta and retina. Although cardioprotective benefits of phlorizin have been reported, little is known about the effect of phlorizin on cardiac damage in type2diabetes mellitus (T2DM).
Isotope labeling relative and absolute quantitative (isobaric tags for relative and absolute quantitation, iTRAQ) technology is a new proteomics quantitative research technique in recent years, which has good quantitative effect, high repeatability, and can be used for separate up to four different samples at the same time. This technology can help researchers find differentially expressed proteins, analyze the protein function, and conduct accurate identification and quantitation of all the proteins present in a genome or a complicated hybrid system.
In this study, we used phlorizin to treat T2DM in db/db mice, a common used diabetic animal model, for10weeks to observe the effects of PHL on their body weight, blood glucose, blood triglycerides, blood cholesterol and blood AGE levels.We also observed the morphological changes in histopathology and ultrastructure of db/db mouse heart. Additionally, we applied proteomics relative quantitative techniques iTRAQ, bioinformatics methods like Turbo SEQUEST software and international protein index (international protein index, IPI) database retrieval to separate and identify differentially expressed proteins in myocardial tissue of mice in normal control group (CC group), untreated db/db diabetic mice group (DM group) and PHL treatment db/db mice group (DMT group), which aims at revealing mechanism of protective effect of PHL for diabetic cardiac damage in db/db mice. It provides a novel way to find new drug targets for drug research and development and helps more rational and efficient treatment for patients with diabetic heart disease.
Objectives
1. The aim of the study was to examine the effects of phlorizin on body weight, metabolic parameters and serum AGEs levels, and the pathological changes of myocardium via histology and ultrastructure in diabetic db/db mice.
2. The purpose of the study was to identify and quantify the differently expressed proteins in normal control group (CC group), untreated db/db diabetic mice group (DM group) and PHL treatment db/db mice group (DMT group), hoping to clarify the mechaniam underlining the protection of phlorizin against diabetic cardiac damage..
Methods
Male C57BLKS/J db/db (n=16,7weeks old) and db/m mice (n=8,7weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China). The mice were kept under observation for one week before the experiments started. C57BLKS/J db/m mice were selected as control group (CC, n=8). The db/db mice were divided into2groups:untreated diabetic group (DM, n=8) administrated with normal saline solution by intragastric gavage and diabetic group treated with phlorizin at a dosage of20mg/kg/d (DMT, n=8) for10weeks. All mice were weighed every week. At the end of the experiments, all mice were fasted overnight and sacrificed. Fasting blood was collected. Fasting blood glucose (FBG), blood triglycerides (TC), blood cholesterol and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured. Whole heart from the controls, untreated db/db mice and phlorizin treated db/db mice were immediately enucleated and were fixed in4%paraformaldehyde and then embedded in paraffin. Some parts of hearts were dissected for histological procedures. The rest parts of hearts tissue were kept at-80℃for further proteomic studies.
Heart tissue (50mg) from each of four mice per group was prepared and digested with trypsin. A total of60ug of peptides from each group were labeled with iTRAQ reagents following the manufacturer's instructions. The control group peptides were labeled with Reagent114; the DMT group, Reagent116; and the DM group, Reagent117. The labeled samples were then separated into10fractions using PolySulfoethyl A strong cation-exchange (SCX) columns. Mass spectrometric analysis was performed using a liquid chromatography system and an LTQ-Velos ion trap mass spectrometer. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis.The data packet containing the differentially expressed proteins identified in the iTRAQ experiment was converted by IPA to "fold change" and uploaded into IPA. Each identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base.
Results
1. General observation
Mice in CC group grew in good condition with smooth furs and active state duing experiments. The db/db mice exhibited sedentary, polydipsia, polyuris and polyphagia with shaggy furs and a rapidly increased body size. Phlorizin treated db/db mice also exhibited abnormal condition, but much better than untreated db/db mice.
2. The effect of PHL diabetes on body weight, FBG, TG, TC and AGES in db/db mice
During the observation period, the DM and DMT groups gained substantially more weight than the control group (P<0.05). Nevertheless, phlorizin treatment significantly reduced body weight gain in db/db mice at the second week after phlorizin administration (P<0.05). After10weeks, serum FBG, TG, and TC levels in the DM group were significantly higher than those in the control group (P<0.05). However, phlorizin treatment dramatically reduced these values in the DMT group compared with the DM group (P<0.05). In addition, db/db mice had significantly elevated serum AGE levels. After phlorizin treatment, AGE levels in db/db mice were reduced.(P<0.05)
3. The effect of PHL on histological observation of in db/db mice
On H&E-stained sections, the myocardium in DM group exhibited significant myocardial hypertrophy and myofiber disarray accompanied by damaged nuclei and increased degeneration. However, phlorizin treatment attenuated this cardiomyocyte hypertrophy to a similar level to the control group.
4. The effect of PHL on ultrastructural observation of myocardium in db/db mice
Myocardial ultrastructure could be visualized by electron microscopy. In the control group, the myofibrils were arranged in a striated pattern, and the mitochondria were positioned in rows along the myofibrils. Although the sarcomere was of the same length, some cardiomyocyte mitochondria in the DM group showed cristae loss. Large areas of the myocardium exhibited a complete disruption of myofibril and mitochondrial arrangements. The shape of the nuclei was altered, and the nuclear membrane was disrupted. However, due to the protective effect of phlorizin in the DMT group, the number of degenerated mitochondria was significantly decreased, and the myofibril disorder was markedly attenuated.
5. Differentialy expressed proteins identified by Mass spectrometry
Using the iTRAQ approach followed by LC-MS/MS identification and IPI database searching, we analyzed the effect of phlorizin on the myocardial protein profile of db/db mice. Of the113differentially expressed proteins,29were elevated in the DM group compared with the control group but were still decreased by phlorizin treatment. An additional84proteins were decreased in the DM group compared with the control group, but these were restored by the phlorizin treatment.
6. Features of differentialy expressed proteins after PHL intervention
According to the features of differentialy expressed proteins, we made functional classification of proteins involved in metabolic disorders in db/db mice into altered proteins in cardiac lipid metabolism, altered proteins related to myocardial mitochondria and altered proteins involved in cardiomyopathy.
7. Bioinformatics analysis of differentially expressed proteins
The top-ranked biological process and involved disease include cardiovascular disease,lipid metabolism, cardiovascular system development and function, endocrine system disorder, free radical scavenging and energy production, which may play important role during diabetic cardiac damage.
The top protein network was generated by pathway analysis of differentially expressed proteins. There was a cluster of35proteins in the network, of which24are included on our list such as Dapk3, Titin, Prkaa, Des, ILK, Nampt and so on. This network may provide important information of interaction between proteins involved in lipid metabolism, mitochondrial function, and cardiomyopathy.
8. Validation of iTRAQ data for selected candidate proteins
We selected two proteins for Western blot analysis to validate the iTRAQ data. Calnexin was found to be decreased, whereas integrin-linked protein kinase (ILK) was increased in the DMT group compared with the DM group. Quantification of band intensity showed that the results from density of bands are almost consistent with the iTRAQ data. This indicates that the iTRAQ data are reliable.
Conclusion
1. PHL treatment can significantly prevent the obesity trend of db/db mice and reduce the FBG. TG, TC and serum AGEs level of db/db mice.
2. PHL treatment can significantly improve the hypertrophy of cardiomyocytes and damage of the nucleus in myocardium of db/db mice. It can also reduce the number of injury mitochondria in cardiomyocyte, and attenuate irregular arrangement of myofibrils.
3. As a novel technology of quantitative proteomics, iTRAQ has been widely used to tag peptides for multiplexed protein quantification and provides increased experimental throughput and lower variability. Thus, using iTRAQ is available to study the mechanism of the protection of phlorizin against diabetic cardiac damage in this study.
4. A total of113proteins differentially changed with phlorizin treatment in myocardium of db/db mice were identified using LS-ESI-MS/MS methods. Of these,29were elevated in the DM group compared with the control group but were still decreased by phlorizin treatment. An additional84proteins were decreased in the DM group compared with the control group, but these were restored by the phlorizin treatment. The top-ranked biological processes and top protein network
was generated by pathway analysis suggested that the altered proteins are mainly related to cardiac lipid metabolism, mitochondrial energy production and development of cardiomyopathy.
Part Two
Caveolar Fatty Acids and Acylation of Caveolin-1
Backgroud
Caveolae are cholesterol and sphingomyelin-rich plasma membrane microdomains presented in most types of mammalian cells and tissues. Caveolae were originally identified as50-100nm flask-shaped, non-clathrin-coated invagination of the plasma membrane and found to be involved in endocytosis and potocytosis. However, later studies revealed that these microdomains concentrate a variety of signaling molecules which provide a platform for signal transduction.Thus, changes in the components of caveolae may have a profound effect on cellular functions. Caveolin-1, a22-kDa protein, is the principal structural component of caveolae and a determinant for caveolae formation.A deficiency of caveolin-1in mice eliminated caveolae, which subsequently impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone.
In addition to cholesterol and sphingomyelin, caveolae also contain a variety of fatty acids. While it is generally believed that covalent attachment of myristic and/or palmitic acid occurs on a wide variety of cellular proteins and the acylation of protein is critical for membrane targeting, the fatty acid compositions of caveolae, the fatty acids bound to caveolin-1, and the effect of acylation of caveolin-1on caveolin-1targeting to caveolae remain poorly understood. Our results revealed that caveolae contain a limited subset of fatty acids, highly enriched with saturated fatty acids, which is quite different from the fatty acid compositions in whole cells. We further demonstrated that the primary fatty acid associated with caveolin-1is stearic acid, not myristic acid as previous speculated.
Objectives
The aim of the study is to use gas chromatography/mass spectrometry (GC/MS) to identify and quantify the fatty acid compositions of caveolae and fatty acids covalently bound to caveolin-1in Chinese hamster ovary (CHO) cells, a cell system with high caveolin-1expression, and then characterize the fatty acid esterification of caveolin-1with isotope-labeled fatty acids. Another purpose of the study is to observe weather the fatty acid esterification of caveolin-1can affect its targeting to caveolae.
Methods
Caveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1igG and analyzed with GC/MS. For characterization of fatty acid esterification of caveolin-1, CHO cells were cultured in Ham's F-12medium containing5%FBS,2mmol/L L-glutamine,100U/ml penicillin and100μg/ml streptomycin to80%confluency in10cm culture dish. The cells were starved18h in Ham's F-12medium containing1%BSA and then labeled with2.5mCi of3H-palmitic acid or25μCi of14C-stearic acid for3h at37o C in the presence/absence of30times of non-labeled palmitic acid (16:0), stearic acid (18:0) or oleic acid (18:1). The cells were dissolved in MBST/OG buffer and immunoprecipitated with anti-caveolin-1/protein A. Non-immune rabbit IgG was used as negative control. The immunoprecipitated caveolin-1was separated by SDS-PAGE and transferred to PVDF membrane. The fatty acid esterification of caveolin-1was detected by autoradiogram at-80°C for6weeks using a Kodak MS film.
Results
In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids,0.48μg palmitic acid,0.61μg stearic acid and0.83μ g oleic acid/caveolae preparation/5x107cells. Unexpectedly, GC/MS analysis indicated that caveolin-1was not acylated by myristic acid; instead, it was acylated by stearic acid (0.23μg) and palmitic acid (0.15μg). In addition, the targeting of caveolin-1to caveolae was not affected by the presence of palmitic acid or stearic acid.
Conclusion
Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1may be important for caveolae formation and for maintaining the function of caveolae.
研究背景
糖尿病(diabetes mellitus, DM)是一种由于胰岛素绝对或相对分泌不足而导致的以慢性血葡萄糖水平增高为特征的一种常见内分泌代谢性疾病。近年来,DM的发病率和患病率在全球范围内呈现逐年升高的趋势,已成为严重威胁人类健康与生活质量的世界性公告卫生问题。DM不仅仅表现为长期慢性的血糖升高,还可伴有多种并发症。其中心血管并发症已经成为DM并发症中致残率和致死率最高、危害最大的并发症。
糖尿病性心脏病是糖尿病患者致死的主要原因之一,尤其是在2型糖尿病患者中。广义的糖尿病心脏病包括冠状动脉粥样硬化性心脏病(冠心病),糖尿病心肌病变和糖尿病心脏自主神经病变等。糖尿病心脏病与非糖尿病患者相比常起病比较早,糖尿病患者伴冠心病常表现为无痛性心肌梗死,梗死面积比较大,穿壁梗死多,病情多比较严重,预后比较差,病死率较高；如冠状动脉造影和临床排除冠状动脉病变,糖尿病患者出现严重的心律失常心脏肥大肺淤血和充血性心力衰竭,尤其是难治性心力衰竭临床可考虑糖尿病心肌病(Diabetic Cardimyopathy, DCM)。DCM是指发生在DM中,不能用高血压性心脏病、冠心病、心脏瓣膜病及其他心脏病来解释的心肌疾病。这一疾病在代谢紊乱及微血管病变的基础上引发心肌广泛灶性坏死,出现亚临床心功能异常,最终进展为心律失常、心力衰竭及心源性休克。迄今为止,针对糖尿病引起的心肌损伤目前尚无特效疗法,临床治疗首先是控制DM,然后主要针对心肌损害、心律失常、心力衰竭和抗血栓进行治疗。而且由于部分患者早期并无明显症状,而上述治疗方法均存在一定局限性,并不能有效及时的阻止它的的发展。因此,在临床实践中迫切需要寻找能有效防治糖尿病心肌病变的新型药物与途径,进一步完善与充实糖尿病心肌病变的治疗策略。
根皮苷(phlorizin, PHL)是根皮素(phloretin)的2’-β-D-葡萄糖苷,是从苹果,苹果树皮及叶中提取的一种二氢查尔酮苷。大量研究表明,PHL具有多种生物活性与药理作用,如调节血糖、抗炎、抗肿瘤、改善认知以及抗氧化等等,目前已被应用于医药、化妆品、食品等领域。近年来,PHL作为新型药物在防治DM及其并发症中的作用获得广泛关注。动物实验发现,长期口服PHL对DM并发症的靶器官如肾脏、心脏、大动脉和视网膜的病变都起到显著地保护和改善作用。虽然临床研究已经证实了PHL对于DM患者心脏的保护作用,但是PHL对糖尿病心肌病变的改善及其保护作用的可能机制,目前国内外鲜见报道；PHL治疗糖尿病心肌病变的作用分子靶点也尚未明确。
同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation, iTRAQ)技术是近年来最新开发的一种新的蛋白质组学定量研究技术,具有良好的定量效果、较高的重复性,并可对多达四种不同样本同时进行定量分析。目前,iTRAQ技术已经在蛋白质组学的定量研究中得到了极其广泛的应用。但是,国内外尚无报道采用iTRAQ标记及相关分析技术来研究根皮苷对2型糖尿病心肌损伤的保护机制。
因此,在本研究中,我们应用了目前较为普遍认可的db/db小鼠的2型DM动物模型,通过10周的PHL灌胃,观察PHL对于db/db小鼠体重、血糖、血脂、血胆固醇及血中AGEs水平的影响,并对db/db小鼠的心脏进行了组织病理学和超微结构的形态学观察,旨在观察天然药物PHL治疗对于db/db小鼠体重、DM有关代谢指标的影响及其对心脏的保护作用,为临床治疗糖尿病性心脏病提供新的治疗思路与途径。另外,我们还应用了iTRAQ蛋白质组学定量技术,Turbo SEQUEST软件和国际蛋白质索引(international protein index, IPI)数据库检索等生物信息学方法,分离与鉴定正常对照组(CC组),db/db小鼠组(DM组)与db/db小鼠PHL治疗组(DMT组)等小鼠心肌组织的差异表达蛋白,旨在揭示PHL对于db/db小鼠糖尿病心肌病变变的分子保护机制,为开展药物研发工作寻找新的药物靶标开辟新途径,为临治疗提供新的思路。
研究目的
1.研究db/db小鼠在糖尿病进展中体重及有关代谢指标的变化,通过对db/db小鼠的心脏组织病理学和超微结构的形态学观察,评价糖尿病心肌病变的特征与程度。初步探讨db/db小鼠糖尿病心肌病变的差异表达蛋白的特征,进一步了解糖尿病心肌病变的分子机制。
2.研究PHL对db/db小鼠体重及糖尿病有关代谢指标的变化的影响,通过对db/db小鼠的心脏组织病理学和超微结构的形态学观察,评价PHL对糖尿病心肌病变的保护作用。探讨db/db小鼠经PHL治疗后心肌组织的差异表达蛋白,根据其所参与的生物过程和代谢通路筛选出药物作用的关键蛋白与候选靶标,进一步揭示PHL对于糖尿病心肌病变的保护机制,为临床治疗提供新的理论基础。
研究方法
7周龄的雄性C57BLKS/J db/db小鼠16只,以及7周龄雄性C57BLKS/J db/m小鼠8只。小鼠均予观察1周后开始正式实验。用C57BLKS/J db/m小鼠8只作为正常对照组(CC),C57BLKS/J db/db小鼠分为两组：一组随机8只小鼠为DM模型组(DM),每日用生理盐水灌胃；另一组随机8只为PHL干预组(DMT),每日用体积相同生理盐水的20mg/kg/d的PHL溶液灌胃,干预时间为10周。实验期间每周定期测量每只小鼠的体重并记录。实验结束时,所有小鼠空腹过夜并处死,采血检测空腹血糖(FBG),血甘油三酯(TG),血总胆固醇(TC)及血清糖基化终末代谢产物(AGES)等指标；并迅速分离心脏组织,按照病理取材常规制作心肌石蜡切片。剩余心脏组织分离后立即存于液氮-80℃保存留待进步蛋白质组学实验用。
从正常对照组(CC组),db/db小鼠组(DM组)与db/db小鼠PHL治疗组(DMT组)三组小鼠中各选取4只,分离心脏。每只小鼠取50mg心脏组织,进行心肌组织研磨、切碎、超声破碎、裂解来提取心肌总蛋白。各组取60ug肽段消化后用iTRAQ染料进行标记,正常对照组用114标记,PHL干预组用116标记,db/db糖尿病组用117则标记。将各组已标记的肽段混合,用强阳离子交换柱(strong cation exchange, SCX)进行分离分级,收集穿流以及洗脱部分合并成10组。接下来用Thermo Finnigan LTQ Velos质谱仪进行液相质谱-串联质谱分析(LC-ESI-MS/MS)。最后,使用相关软件将定量及检定结果进行合并处理,得到定量和鉴定结果。搜索使用的数据库为ipi.MOUSE.v3.72.REVERSED.fasta蛋白库(Sequest结果过滤参数为：Protein FDR<0.01; Peptide FDR<0.01),应用EXPASY蛋白质组学工具来分析等电点、分子量等。然后采用Ingenuity Pathway Analysis软件(www.ingenuity.com)对经鉴定的心肌蛋白质进行蛋白功能和通路的分析。最后将筛选出的部分差异蛋白用蛋白质免疫印迹方法(Western blot)进
一步验证其在心肌组织中的表达。
研究结果
1.一般观察
CC组小鼠生长良好,精神状况佳,毛发光亮顺泽,活跃。DM组小鼠在实验进程中,逐渐表现为毛发污秽无光泽,被毛蓬松,少动,出现明显的多饮、多食、多尿,体重迅速增加。而DMT组小鼠的上述表现较DM组有所减轻。
2.PHL对db/d小鼠体重、FBG、TG、TC与AGEs的影响
随着观察时间的延长,DM组与DMT组小鼠的体重较对照组呈显著增加。从实验第2周开始,DM组小鼠体重逐渐增加,一直持续到实验结束的第10周(P<0.05)。然而,DMT组小鼠与DM组相比,PHL灌胃显著改善了DM小鼠体重的快速增加(P<0.05)。
实验开始时,各组小鼠的血FBG、TG、TC及AGES水平无明显差异。至实验结束时DM组小鼠的FBG、TG、TC及AGES水平与CC组比较,均明显升高(P<0.05)。给予PHL干预的DMT组小鼠的FBG、TG、TC及与AGES水平与DM组比较则明显下降(P<0.05)。
3.PHL对db/db小鼠心肌组织病理学变化的影响
实验结束后,将db/db小鼠心肌组织切片进行苏木素-伊红(HE)染色进行观察。DM组的小鼠的心肌组织较CC组呈现出明显的心肌细胞肥大和心肌细胞排列不规则,并伴有细胞核损伤。然而,DMT组经过PHL的治疗大大减少了心肌组织肥大以及细胞核损伤的情况。
4.PHL对db/db小鼠心肌组织超微结构变化的影响
通过电子显微镜下对db/db小鼠心肌组织超微结构的观察,发现CC组小鼠心肌细胞肌原纤维完整,排列整齐,线粒体结构正常,呈线性排列；细胞核形态良好,核膜完整。而DM组心肌细胞中可以观察到线粒体肿胀,嵴溶解,有很大区域的肌原纤维及线粒体排列混乱不规则；细胞核的形态有所改变,核膜不完整。然而,在DMT组,PHL的治疗明显的降低了心肌细胞中损伤的线粒体的数量,并且减轻了肌原纤维的不规则排列情况。
5.iTRAQ鉴定结果
经LS-ESI-MS/MS鉴定并予软件分析、数据库检索后,共鉴定出蛋白1627种,符合鉴定条件者可信蛋白1591中,具有唯一肽段7836个。其中鉴定在DM组与正常对照组心肌蛋白相比表达有变化但经PHL治疗后回调的差异表达蛋白共113个,其中在DM组表达上调的但经PHL治疗后回调的有29个,在DM组表达下调的但经PHL治疗后回调的有84个
6.PHL干预后重要差异蛋白质特征
在这些经PHL治疗后回调的差异表达蛋白中,我们发现一些涉及到糖尿病心肌病变发生发展的重要蛋白,并把它们按照功能和所参与的代谢过程分为：与心肌脂代谢有关的蛋白、与心肌线粒体有关的蛋白和与心肌病发生发展有关的蛋白,进一步明确这些重要蛋白在糖尿病心肌病变中的可能起的作用。
7.PHL干预后差异蛋白的生物信息学分析
对于PHL干预后的113个差异蛋白点,采用IPA软件对有显著改变的蛋白所参与的重要生物过程和疾病进行分析。结果发现,这些蛋白所参与的生物过程和疾病包括心血管疾病、脂代谢、心血管系统的发育和功能、内分泌系统疾病、自由基的修复和能量的产生等等,都是与糖尿病心肌病变的发展密切相关的生物过程。
对于PHL干预后的113个差异蛋白点,采用IPA软件绘制差异蛋白功能网络来说明这些蛋白之间的相互关系。网络中共出现35个蛋白,其中有24个蛋白是我们筛选出的重要的差异蛋白,比如Dapk3, Titin, Prkaa, Des, ILK, Nampt等。此蛋白相互作用网络为我们进一步研究脂质代谢、线粒体功能和心肌病的发生和发展之间的关系提供了新的思路。
8.筛选的差异蛋白在心肌组织的表达改变
为了验证蛋白质组学鉴定出的部分差异蛋白在心肌组织的表达,应用Western blot方法检测其中差异蛋白如钙联蛋白(Calnexin)与整合素连接激酶(integrin-linked protein kinase,ILK)在各组小鼠心肌组织中的表达。结果显示,与在正常对照组小鼠心肌中的表达比较,Calnexin在db/db小鼠中表达明显增高,然而PHL治疗后,表达增高的Calnexin有所下调。另外,ILK在db/db小鼠中表达中与正常对照组比较表达后明显降低,经过PHL干预后该蛋白表达回调。经过VisionWorks LS image acquisition and analysis software软件分析亦显示该蛋白质印迹实验结果与iTRAQ鉴定结果是一致的。
结论
1.与正常对照组比较,db/db小鼠随年龄增长体重显著增加,血FBG、TG、TC及AGEs水平显著升高；PHL灌胃干预可以明显抑制db/db小鼠的肥胖趋势,并且能够显著降低其FBG、TG、TC及与AGEs水平。说明PHL可能通过改善db/db小鼠的糖尿病整体代谢紊乱状态来预防DM的进展和并发症的发生
2.心肌组织的形态学观察显示,PHL治疗可以明显改善db/db小鼠心肌组织肥大以及细胞核损伤的情况；而且,PHL治疗明显减轻db/db小鼠心肌细胞中肌原纤维的不规则排列情况,降低损伤的线粒体的数量。说明PHL对db/db小鼠的糖尿病心肌病变具有保护作用。
3.作为定量蛋白质组学的新技术,iTRAQ具有高灵敏性、高通量,良好的定量效果、较高的重复性,能够对于多组样本同时进行比较分析以便获得更好的蛋白质组覆盖率。所以,iTRAQ能够用于研究PHL对db/db小鼠糖尿病心肌病变保护作用的分子机制,帮助发现新的药物靶标。
4.应用LS-ESI-MS/MS鉴定得到db/db小鼠组与正常对照组心肌蛋白相比表达有变化但经PHL治疗后回调的差异表达蛋白共113个,其中在DM组表达上调的但经PHL治疗后回调的有29个,在DM组下调的但经PHL治疗后回调的有84个。采用IPA分析发现这些蛋白参与的生物过程和疾病涉及到心血管疾病、脂代谢、心血管系统的发育和功能、内分泌系统疾病、自由基的修复和能量的产生等等,提示这些过程可能与糖尿病心肌病变的发生与发展密切相关。
第二部分质膜微囊的脂肪酸组成和小窝蛋白-1的脂肪酸化的研究
研究背景
质膜微囊(Caveolea),是一种特殊类型的脂筏,是哺乳动物细胞质膜上呈细颈烧瓶状的内陷结构,内含丰富的胆固醇、鞘磷脂和鞘糖脂。质膜微囊大量存在于内皮细胞、脂肪细胞、血管平滑肌细胞、纤维母细胞和肺上皮细胞,参与许多细胞生命活动,细胞内吞、胆固醇运输、细胞膜组装、信号传导和肿瘤生成。小窝蛋白(caveolin)是质膜微囊区别于其它脂筏结构的特征性蛋白分子,它与信号传导、物质转运、细胞增殖等密切相关,维持着质膜微囊的结构和功能。Caveolin-1能与多种信号分子如G蛋白α亚基、酪氨酸激酶受体、PKCs、Src家族酪氨酸激酶和eNOS相互作用。生物体内小窝蛋白异常可以引起多种疾病或异常。
除了富含胆固醇和鞘磷脂,caveolea内也含有多种脂肪酸。一般认为,大多数细胞的蛋白质脂肪酸化是以共价键形式与肉豆蔻酸(C14：0)和／或棕榈酸(C16：0)结合,并且蛋白质的脂肪酸化对其在细胞膜上的定位十分重要。从目前国内外的研究情况来看,虽然caveolea的结构成分已经较为清楚,但是对于其所含脂肪酸的种类组成与绝对定量的研究并不多见；此外,目前对于caveolea内的许多蛋白质(如eNOS)的脂肪酸化及功能研究报道诸多,但是作为caveolea的支架蛋白,有关caveolin-1的脂肪酸化及其在caveolea上定位的影响却报道甚少。在本研究中,我们采用了气相色谱-质谱技术(GC/MS)来定性和定量中国仓鼠卵巢(CHO)细胞中脂肪酸、caveolea内含有的脂肪酸和以共价键形式结合在caveolin-1上的脂肪酸,并用同位素标记的脂肪酸验证了caveolin-1的脂肪酸化,以及观察了caveolin-1的脂肪酸化对其在caveolae上的定位是否有影响,旨在明确特定脂肪酸与caveolea、caveolin-1的关系,为日后研究某种特定脂肪酸对蛋白质的酸化作用奠定理论基础。
研究目的
1.采用气相色谱-质谱技术(GC/MS)对CHO细胞中脂肪酸、caveolea内含有的脂肪酸和以共价键形式结合在caveolin-1上的脂肪酸来进行定性和定量,旨在明确其相关脂肪酸组成及含量。
2.采用同位素标记的脂肪酸,通过观察与非标记脂肪酸竞争性结合caveolin-1,来验证caveolin-1的脂肪酸化的种类。
3.通过将CHO细胞培养在含有或不含有特定脂肪酸的环境中,观察caveolin-1的脂肪酸化对其在caveolae上的定位是否有影响。
研究方法
中国仓鼠卵巢(CHO)细胞在10cm的培养盘中使用Ham's F-12培养基(含5%的FBS,2mmol/L的L-谷氨酰胺,100U/mL的青霉素和100μg/ml的链霉素)培养至90%融合,用冷的PBS洗5遍后溶解于1mlMBST/OG(含25mM的MES,150mM NaCl,1%Triton-100,60mM心肌糖苷,PH=6.7),在冰上放置30分钟。使用Op ti-prep的方法将质膜微囊(caveolea)、细胞质(cytosol)、质膜(plasma membrane)、内膜(internal membrane)和去核后上清液(post nuclear supematan, PNS)分离。为了提取用于脂肪酸定量的caveolin-1,CHO细胞的裂解液中加入抗caveolin-1IgG在4。C孵育18小时,然后用蛋白A磁珠孵育另外2小时。离心,收集珠子,然后使用高盐缓冲液(500mM NaCl)洗5次。用于定量分析的脂肪酸珠子用1M的NaOH孵育过夜,然后用1M HC1中和,再用Folch试剂(氯仿：甲醇=2：1)提取脂质,并用GC/MS分析。非免疫兔IgG被用作阴性对照。用GC/MS对脂肪酸进行定量方法是：用Folch/BHT试剂来提取样本中的脂质,50μl的二十三烷酸(23：0)(5mg/ml的氯仿)加入提取液中作为内参。然后样本中全部的脂质用BF3/甲醛(10%)进行甲基脂化。用装有omegawax250毛细管柱的气相色谱系统(Agilent6890GC G2579A)来分析脂肪酸甲基脂。大型选择性探头(MSD, Agilent5973)用来识别目标峰,氢火焰离子化检测器(FID)用于定量脂肪酸。蛋白质印迹方法原理与步骤同第一部分。一抗为抗caveolin-1IgG多克隆抗体(滴度1：200,Sigma. USA)。为了测定Caveolin-1的脂肪酸化,CHO细胞在10cm培养皿中使用Ham's F-12培养基中培养至80%融合(含5%的FBS,2mmol/L的L-谷氨酰胺,100U/mL的青霉索和100μg/ml的链霉素)。使用含1%BSA的培养基饥饿细胞十八小时后,用2.5mCi的3H棕榈酸(3H-C16：0)或者25μCi的14C硬脂酸(14C-C18：0)在有／无30倍浓度的非标记棕榈酸和硬质酸的培养基中室温标记3小时。将细胞溶解于MBST/OG缓冲液,用抗caveolin-1蛋白A免疫沉淀。非免疫兔IgG被用作阴性对照。免疫沉淀下来的caveolin-1用SDS-PAGE分离,转移到PVDF膜上,caveolin-1的脂肪酸化用自动放射性监测仪检测在-80℃存放6周,用Kodak MS胶卷显影。
研究结果
1.CHO细胞中FA的含量及组成
通过用GC/MS检测CHO全细胞裂解液中的脂肪酸的种类及含量,我们发现CHO细胞中含有多种脂肪酸,包括饱和与不饱和脂肪。在这些脂肪酸中油酸占了高达59.5%的比例(C18:1,307±18.4μg/5×107cells),其次是棕榈酸(C16:0,91.8±9.5μg/5x107cells;17.8%)),硬脂酸(C18:0,51.8±10.2μg/5×107cells;10%)),鳕油酸(C20:1,22.5±1.4μg/5×107cells;4.4%),棕榈油酸(C16:1,18.7±1.1μg/5x107cells；3.6%),花生四烯酸(C20:4,14.8±1.8μg/5×107cells;2.9%)和肉豆蔻酸(C14:0,9.0±1.4μg/5×107cells;1.7%).
2Caveolae中FA的含量及组成
通过用GC/MS检测caveolea中脂肪酸的含量与组成,我们发现caveolea中含有有限的几种脂肪酸,主要为棕榈酸(C16:0,0.48±0.06μg/5×107cells;24%),硬脂酸(C18:0,0.61±0.07μg/5×107cells;30%)和油酸(C18:1,0.83±0.21μg/5x107cells;40%)。
3.与Caveolin-1结合的FA的含量及组成
一般认为,质膜蛋白会容易被肉豆蔻酸(C14：0)和棕榈酸(C16：0)所脂肪酸化。然而通过用GC/MS检测与caveolin-1结合的脂肪酸含量与组成,我们发现约60%的与caveolin-1结合的脂肪酸为硬脂酸(C18：0),40%的为棕榈酸(C16：0)。我们并没有没有检测到任何的肉豆蔻酸(C14：0)与CHO细胞的caveolin-1相结合。
4.测定Caveolin-1的脂肪酸化
为了进一步验证结合于caveolin-1上的脂肪酸种类及相互作用,我们采用了同位素标记的脂肪酸来检测Caveolin-1脂肪酸化。结果显示,在用3H-棕榈酸(3H-C16：0)标记的细胞中,我们检测到了结合到caveolin-1上的大量3H-棕榈酸(3H-C16：0),说明棕榈酸可以直接与caveolin-1相结合。当有过量的非标记棕榈酸(C16：0)和硬脂酸(C18：0)存在时,非标记棕榈酸(C16：0)和硬脂酸(C18：0)有效的阻滞了3H-棕榈酸(3H-C16：0)与caveolin-1的结合。但是过量的非标记油酸(C18：1)只是轻度的阻滞了3H-棕榈酸(3H-C16：0)和caveolin-1的结合。
同样的,在用14-硬脂酸(14C-C18:0)标记的细胞中,我们检测到了与caveolin-1结合的大量14-硬脂酸,说明硬脂酸可以直接与caveolin-1相结合。当有过量的非标记硬脂酸(C18：0)存在时,非标记硬脂酸(C18：0)能够有效地抑制14C-硬脂酸(14C-C18：0)与caveolin-1的结合。过量的非标记油酸(C18：1)和棕榈酸(C16：0)虽然也阻滞了14C-硬脂酸(14C-C18：0)与caveolin-1的结合,但是阻滞的效果较之非标记硬脂酸(C18：0)不明显。
这些数据说明棕榈酸(C16：0)和硬脂酸(C18：0)可以直接结合于caveolin-1上,并且是其结合的主要脂肪酸。5.脂肪酸化对Caveolin-1亚细胞定位的影响
蛋白质的脂肪酸化可以影响蛋白的亚细胞定位。为了检测caveolin-1的脂肪酸化是否影响了caveolin-1的亚细胞定位,我们把CHO细胞分别培养在含有20%FBS,1%BSA,1%BSA+棕榈酸(过量)和1%BSA+硬脂酸(过量)培养基中。结果显示,caveolin-1在caveolea上的定位在20%FBS与1%BSA培养基中没有明显差别,在加入和不加入过量棕榈酸或硬脂酸培养基也未见明显差别。说明棕榈酸和硬脂酸对于caveolin-1在caveolea上的定位没有明显影响。
结论
Caveolea含有有限种类的部分脂肪酸,其中饱和脂肪酸的含量较高,这与CHO全细胞中脂肪酸的组成有所不同。与caveolin-1结合的最主要脂肪酸是硬脂酸,而不是之前推测的肉豆蔻酸。Caveolea独特的脂肪酸结构和caveolin-1的脂肪酸化可能对于caveolea的形成和维持其功能非常重要。对这些相关脂肪酸的定性和定量研究将会对进一步揭示Caveolea的功能和caveolin-1的脂肪酸化对于细胞膜信号传导所起的作用有重要的意义。
Investigation of the Protective Effects of Phlorizin on Cardiac Damage in db/db Mice by Quantitative Proteomics
Background
Diabetes mellitus (diabetes mellitus, DM) is a common endocrine and metabolic disease caused by absolute or relative insulin secretion defect and characterized by chronic increased blood glucose levels. In recent years, with the improvement of the living standard, modern life styles and aging, the prevalence of diabetes mellitus is rapidly increasing worldwide. It has become a worldwide public health problem and a serious threat to human health and quality of life. DM not only develops sustained elevated circulating glucose, but can be accompanied by a variety of complications attacking both macrovascular and microvascular lesion leading to a variety of complications. Among these, cardiovascular complication has become one of the most harmful complications with the highest morbidity and mortality.
Diabetic heart disease is the main cause for death of diabetic patients, especially in type2diabetes. Broadly-defined concept of diabetic heart disease includes coronary heart disease, diabetic cardiomyopathy and diabetic cardiovascular autonomic neuopathy. Patients with diabetes often develop hypertension and atherosclerosis leading to cardiovascular complications. However, some diabetic patients develop heart failure without hypertension and coronary artery disease. This phenomenon was first described by Rubler et al. and was termed "diabetic cardiomyopathy"(DCM). Diabetic cardiomyopathy is characterized by structural and functional changes in the heart, such as elevated left ventricular (LV) mass, myocardial fibrosis, and abnormal diastolic function, eventually progress to arrhythmia, heart failure and cardiogenic shock. However, the mechanistic details of diabetic cardiac damage remain unclear, and this disease has not yet been sufficiently studied. The clinical treatment of diabetic cardiac damage focuses on controlling DM, preventing myocardial damage, arrhythmia, heart failure and antithrombotic therapy. So far, current therapeutic options for the treatment of diabetic heart disease, as described above, have certain limitations and are far from satisfactory. Therefore, there is an urgent need to find the more effective approaches for the intervention of diabetic heart disease.
Phlorizin (phloretin-2'-O-glucoside, PHL), a dihydrochalcone derived from apple peels, bark and leaves, is a known antioxidant. Previews studies have shown that PHL has many biological activities and pharmacological actions, such as blood glucose regulation, anti-inflammation, anti-tumor, cognition improvement, antioxidative action and so on. It has been used in medicine, cosmetics, food and other fields. The main pharmacological property of phlorizin is to produce renal glycosuria and block intestinal glucose absorption through inhibition of sodium/glucose cotransporters in the kidney and intestine. In addition, animal experiments showed that long-term oral PHL administration has protective effect on DM complications related to targeting organs such as the kidney, heart, aorta and retina. Although cardioprotective benefits of phlorizin have been reported, little is known about the effect of phlorizin on cardiac damage in type2diabetes mellitus (T2DM).
Isotope labeling relative and absolute quantitative (isobaric tags for relative and absolute quantitation, iTRAQ) technology is a new proteomics quantitative research technique in recent years, which has good quantitative effect, high repeatability, and can be used for separate up to four different samples at the same time. This technology can help researchers find differentially expressed proteins, analyze the protein function, and conduct accurate identification and quantitation of all the proteins present in a genome or a complicated hybrid system.
In this study, we used phlorizin to treat T2DM in db/db mice, a common used diabetic animal model, for10weeks to observe the effects of PHL on their body weight, blood glucose, blood triglycerides, blood cholesterol and blood AGE levels.We also observed the morphological changes in histopathology and ultrastructure of db/db mouse heart. Additionally, we applied proteomics relative quantitative techniques iTRAQ, bioinformatics methods like Turbo SEQUEST software and international protein index (international protein index, IPI) database retrieval to separate and identify differentially expressed proteins in myocardial tissue of mice in normal control group (CC group), untreated db/db diabetic mice group (DM group) and PHL treatment db/db mice group (DMT group), which aims at revealing mechanism of protective effect of PHL for diabetic cardiac damage in db/db mice. It provides a novel way to find new drug targets for drug research and development and helps more rational and efficient treatment for patients with diabetic heart disease.
Objectives
1. The aim of the study was to examine the effects of phlorizin on body weight, metabolic parameters and serum AGEs levels, and the pathological changes of myocardium via histology and ultrastructure in diabetic db/db mice.
2. The purpose of the study was to identify and quantify the differently expressed proteins in normal control group (CC group), untreated db/db diabetic mice group (DM group) and PHL treatment db/db mice group (DMT group), hoping to clarify the mechaniam underlining the protection of phlorizin against diabetic cardiac damage..
Methods
Male C57BLKS/J db/db (n=16,7weeks old) and db/m mice (n=8,7weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China). The mice were kept under observation for one week before the experiments started. C57BLKS/J db/m mice were selected as control group (CC, n=8). The db/db mice were divided into2groups:untreated diabetic group (DM, n=8) administrated with normal saline solution by intragastric gavage and diabetic group treated with phlorizin at a dosage of20mg/kg/d (DMT, n=8) for10weeks. All mice were weighed every week. At the end of the experiments, all mice were fasted overnight and sacrificed. Fasting blood was collected. Fasting blood glucose (FBG), blood triglycerides (TC), blood cholesterol and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured. Whole heart from the controls, untreated db/db mice and phlorizin treated db/db mice were immediately enucleated and were fixed in4%paraformaldehyde and then embedded in paraffin. Some parts of hearts were dissected for histological procedures. The rest parts of hearts tissue were kept at-80℃for further proteomic studies.
Heart tissue (50mg) from each of four mice per group was prepared and digested with trypsin. A total of60ug of peptides from each group were labeled with iTRAQ reagents following the manufacturer's instructions. The control group peptides were labeled with Reagent114; the DMT group, Reagent116; and the DM group, Reagent117. The labeled samples were then separated into10fractions using PolySulfoethyl A strong cation-exchange (SCX) columns. Mass spectrometric analysis was performed using a liquid chromatography system and an LTQ-Velos ion trap mass spectrometer. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis.The data packet containing the differentially expressed proteins identified in the iTRAQ experiment was converted by IPA to "fold change" and uploaded into IPA. Each identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base.
Results
1. General observation
Mice in CC group grew in good condition with smooth furs and active state duing experiments. The db/db mice exhibited sedentary, polydipsia, polyuris and polyphagia with shaggy furs and a rapidly increased body size. Phlorizin treated db/db mice also exhibited abnormal condition, but much better than untreated db/db mice.
2. The effect of PHL diabetes on body weight, FBG, TG, TC and AGES in db/db mice
During the observation period, the DM and DMT groups gained substantially more weight than the control group (P<0.05). Nevertheless, phlorizin treatment significantly reduced body weight gain in db/db mice at the second week after phlorizin administration (P<0.05). After10weeks, serum FBG, TG, and TC levels in the DM group were significantly higher than those in the control group (P<0.05). However, phlorizin treatment dramatically reduced these values in the DMT group compared with the DM group (P<0.05). In addition, db/db mice had significantly elevated serum AGE levels. After phlorizin treatment, AGE levels in db/db mice were reduced.(P<0.05)
3. The effect of PHL on histological observation of in db/db mice
On H&E-stained sections, the myocardium in DM group exhibited significant myocardial hypertrophy and myofiber disarray accompanied by damaged nuclei and increased degeneration. However, phlorizin treatment attenuated this cardiomyocyte hypertrophy to a similar level to the control group.
4. The effect of PHL on ultrastructural observation of myocardium in db/db mice
Myocardial ultrastructure could be visualized by electron microscopy. In the control group, the myofibrils were arranged in a striated pattern, and the mitochondria were positioned in rows along the myofibrils. Although the sarcomere was of the same length, some cardiomyocyte mitochondria in the DM group showed cristae loss. Large areas of the myocardium exhibited a complete disruption of myofibril and mitochondrial arrangements. The shape of the nuclei was altered, and the nuclear membrane was disrupted. However, due to the protective effect of phlorizin in the DMT group, the number of degenerated mitochondria was significantly decreased, and the myofibril disorder was markedly attenuated.
5. Differentialy expressed proteins identified by Mass spectrometry
Using the iTRAQ approach followed by LC-MS/MS identification and IPI database searching, we analyzed the effect of phlorizin on the myocardial protein profile of db/db mice. Of the113differentially expressed proteins,29were elevated in the DM group compared with the control group but were still decreased by phlorizin treatment. An additional84proteins were decreased in the DM group compared with the control group, but these were restored by the phlorizin treatment.
6. Features of differentialy expressed proteins after PHL intervention
According to the features of differentialy expressed proteins, we made functional classification of proteins involved in metabolic disorders in db/db mice into altered proteins in cardiac lipid metabolism, altered proteins related to myocardial mitochondria and altered proteins involved in cardiomyopathy.
7. Bioinformatics analysis of differentially expressed proteins
The top-ranked biological process and involved disease include cardiovascular disease,lipid metabolism, cardiovascular system development and function, endocrine system disorder, free radical scavenging and energy production, which may play important role during diabetic cardiac damage.
The top protein network was generated by pathway analysis of differentially expressed proteins. There was a cluster of35proteins in the network, of which24are included on our list such as Dapk3, Titin, Prkaa, Des, ILK, Nampt and so on. This network may provide important information of interaction between proteins involved in lipid metabolism, mitochondrial function, and cardiomyopathy.
8. Validation of iTRAQ data for selected candidate proteins
We selected two proteins for Western blot analysis to validate the iTRAQ data. Calnexin was found to be decreased, whereas integrin-linked protein kinase (ILK) was increased in the DMT group compared with the DM group. Quantification of band intensity showed that the results from density of bands are almost consistent with the iTRAQ data. This indicates that the iTRAQ data are reliable.
Conclusion
1. PHL treatment can significantly prevent the obesity trend of db/db mice and reduce the FBG. TG, TC and serum AGEs level of db/db mice.
2. PHL treatment can significantly improve the hypertrophy of cardiomyocytes and damage of the nucleus in myocardium of db/db mice. It can also reduce the number of injury mitochondria in cardiomyocyte, and attenuate irregular arrangement of myofibrils.
3. As a novel technology of quantitative proteomics, iTRAQ has been widely used to tag peptides for multiplexed protein quantification and provides increased experimental throughput and lower variability. Thus, using iTRAQ is available to study the mechanism of the protection of phlorizin against diabetic cardiac damage in this study.
4. A total of113proteins differentially changed with phlorizin treatment in myocardium of db/db mice were identified using LS-ESI-MS/MS methods. Of these,29were elevated in the DM group compared with the control group but were still decreased by phlorizin treatment. An additional84proteins were decreased in the DM group compared with the control group, but these were restored by the phlorizin treatment. The top-ranked biological processes and top protein network
was generated by pathway analysis suggested that the altered proteins are mainly related to cardiac lipid metabolism, mitochondrial energy production and development of cardiomyopathy.
Part Two
Caveolar Fatty Acids and Acylation of Caveolin-1
Backgroud
Caveolae are cholesterol and sphingomyelin-rich plasma membrane microdomains presented in most types of mammalian cells and tissues. Caveolae were originally identified as50-100nm flask-shaped, non-clathrin-coated invagination of the plasma membrane and found to be involved in endocytosis and potocytosis. However, later studies revealed that these microdomains concentrate a variety of signaling molecules which provide a platform for signal transduction.Thus, changes in the components of caveolae may have a profound effect on cellular functions. Caveolin-1, a22-kDa protein, is the principal structural component of caveolae and a determinant for caveolae formation.A deficiency of caveolin-1in mice eliminated caveolae, which subsequently impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone.
In addition to cholesterol and sphingomyelin, caveolae also contain a variety of fatty acids. While it is generally believed that covalent attachment of myristic and/or palmitic acid occurs on a wide variety of cellular proteins and the acylation of protein is critical for membrane targeting, the fatty acid compositions of caveolae, the fatty acids bound to caveolin-1, and the effect of acylation of caveolin-1on caveolin-1targeting to caveolae remain poorly understood. Our results revealed that caveolae contain a limited subset of fatty acids, highly enriched with saturated fatty acids, which is quite different from the fatty acid compositions in whole cells. We further demonstrated that the primary fatty acid associated with caveolin-1is stearic acid, not myristic acid as previous speculated.
Objectives
The aim of the study is to use gas chromatography/mass spectrometry (GC/MS) to identify and quantify the fatty acid compositions of caveolae and fatty acids covalently bound to caveolin-1in Chinese hamster ovary (CHO) cells, a cell system with high caveolin-1expression, and then characterize the fatty acid esterification of caveolin-1with isotope-labeled fatty acids. Another purpose of the study is to observe weather the fatty acid esterification of caveolin-1can affect its targeting to caveolae.
Methods
Caveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1igG and analyzed with GC/MS. For characterization of fatty acid esterification of caveolin-1, CHO cells were cultured in Ham's F-12medium containing5%FBS,2mmol/L L-glutamine,100U/ml penicillin and100μg/ml streptomycin to80%confluency in10cm culture dish. The cells were starved18h in Ham's F-12medium containing1%BSA and then labeled with2.5mCi of3H-palmitic acid or25μCi of14C-stearic acid for3h at37o C in the presence/absence of30times of non-labeled palmitic acid (16:0), stearic acid (18:0) or oleic acid (18:1). The cells were dissolved in MBST/OG buffer and immunoprecipitated with anti-caveolin-1/protein A. Non-immune rabbit IgG was used as negative control. The immunoprecipitated caveolin-1was separated by SDS-PAGE and transferred to PVDF membrane. The fatty acid esterification of caveolin-1was detected by autoradiogram at-80°C for6weeks using a Kodak MS film.
Results
In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids,0.48μg palmitic acid,0.61μg stearic acid and0.83μ g oleic acid/caveolae preparation/5x107cells. Unexpectedly, GC/MS analysis indicated that caveolin-1was not acylated by myristic acid; instead, it was acylated by stearic acid (0.23μg) and palmitic acid (0.15μg). In addition, the targeting of caveolin-1to caveolae was not affected by the presence of palmitic acid or stearic acid.
Conclusion
Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1may be important for caveolae formation and for maintaining the function of caveolae.
引文
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24. Tyagi NK, Kumar A, Goyal P, Pandey D, Siess W, et al. (2007) D-Glucose-recognition and phlorizin-binding sites in human sodium/D-glucose cotransporter 1 (hSGLTl):a tryptophan scanning study. Biochemistry 46: 13616-13628.
25. Osorio H, Coronel I, Arellano A, Pacheco U, Bautista R, et al. (2012) Sodium-glucose cotransporter inhibition prevents oxidative stress in the kidney of diabetic rats. Oxid Med Cell Longev 2012:542042.
26. Chen ZC, Cheng YZ, Chen L.T, Cheng KC, Li Y, et al. (2012) Increase of ATP-sensitive potassium (K(ATP)) channels in the heart of type-1 diabetic rats. Cardiovasc Diabetol 11:8.
27. Shen L, You BA, Gao HQ, Li BY, Yu F, et al. (2012) Effects of phlorizin on vascular complications in diabetes db/db mice. Chin Med J (Engl) 125:3692-3696.
28. Fort PE, Losiewicz MK, Reiter CE, Singh RS, Nakamura M, et al. (2011) Differential roles of hyperglycemia and hypoinsulinemia in diabetes induced retinal cell death:evidence for retinal insulin resistance. PLoS One 6:e26498.
29. Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, et al. (1996) Progress with proteome projects:why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 13:19-50.
30. Abbott A (2001) And now for the proteome. Nature 409:747.
31. Fields S (2001) Proteomics. Proteomics in genomeland. Science 291:1221-1224.
32.高海青,邱洁.心血管蛋白质组学[M].第1版.北京：人民卫生出版社,2010.
33. Hu J, Qian J, Borisov O, Pan S, Li Y, et al. (2006) Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags. Proteomics 6:4321-4334.
34. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, et al. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154-1169.
35. Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, Jerums G (1991) Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes 40:1328-1334.
36. American Diabetes A (2011) Executive summary:standards of medical care in diabetes-2011. Diabetes Care 34 Suppl 1:S4-10.
37.高海青,李保应.葡萄多酚：基础与临床[M].第1版.北京：人民卫生出版社,2012.
38. Futterman LG, Lemberg L (2000) The Framingham Heart Study:a pivotal legacy of the last millennium. Am J Crit Care 9:147-151.
39. Brownlee M (2005) The pathobiology of diabetic complications:a unifying mechanism. Diabetes 54:1615-1625.
40. An D, Rodrigues B (2006) Role of changes in cardiac metabolism in development of diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 291:HI489-1506.
41. Lu WD, Li BY, Yu F, Cai Q, Zhang Z, et al. (2012) Quantitative proteomics study on the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice. Mol Med Report 5:1285-1294.
42. Hummel KP, Dickie MM, Coleman DL (1966) Diabetes, a new mutation in the mouse. Science 153:1127-1128.
43. Herberg L, Coleman DL (1977) Laboratory animals exhibiting obesity and diabetes syndromes. Metabolism 26:59-99.
44. Berglund O, Frankel BJ, Hellman B (1978) Development of the insulin secretory defect in genetically diabetic (db/db) mouse. Acta Endocrinol (Copenh) 87: 543-551.
45. Lee SM, Bressler R (1981) Prevention of diabetic nephropathy by diet control in the db/db mouse. Diabetes 30:106-111.
46. Wende AR, Abel ED (2010) Lipotoxicity in the heart. Biochim Biophys Acta 1801: 311-319.
47. Vasantha Rupasinghe HP, Yasmin A (2010) Inhibition of oxidation of aqueous emulsions of omega-3 fatty acids and fish oil by phloretin and phloridzin. Molecules 15:251-257.
48. Bartels ED. Nielsen JM, Hellgren LI, Ploug T. Nielsen LB (2009) Cardiac expression of microsomal triglyceride transfer protein is increased in obesity and serves to attenuate cardiac triglyceride accumulation. PLoS One 4:e5300.
49. Dougan SK, Rava P, Hussain MM, Blumberg RS (2007) MTP regulated by an alternate promoter is essential for NKT cell development. J Exp Med 204: 533-545.
50. Mohler PJ, Zhu MY, Blade AM, Ham AJ, Shelness GS, et al. (2007) Identification of a novel isoform of microsomal triglyceride transfer protein. J Biol Chem 282: 26981-26988.
51. Chang YH, Chang DM, Lin KC, Shin SJ, Lee YJ (2011) Visfatin in overweight/obesity, type 2 diabetes mellitus, insulin resistance, metabolic syndrome and cardiovascular diseases:a meta-analysis and systemic review. Diabetes Metab Res Rev 27:515-527.
52. Peiro C, Romacho T, Carraro R, Sanchez-Ferrer CF (2010) Visfatin/PBEF/Nampt: A New Cardiovascular Target? Front Pharmacol 1:135.
53. Neely JR, Rovetto MJ, Oram JF (1972) Myocardial utilization of carbohydrate and lipids. Prog Cardiovasc Dis 15:289-329.
54. Saddik M, Lopaschuk GD (1991) Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts. J Biol Chem266:8162-8170.
55. Saito F, Kawaguchi M, Izumida J, Asakura T, Maehara K, et al. (2003) Alteration in haemodynamics and pathological changes in the cardiovascular system during the development of Type 2 diabetes mellitus in OLETF rats. Diabetologia 46: 1161-1169.
56. Anguera I, Magrina J, Setoain FJ, Esmatges E, Pare C, et al. (1998) [Anatomopathological bases of latent ventricular dysfunction in insulin-dependent diabetics]. Rev Esp Cardiol 51:43-50.
57. Rodrigues B, Cam MC, McNeill JH (1995) Myocardial substrate metabolism: implications for diabetic cardiomyopathy. J Mol Cell Cardiol 27:169-179.
58. Borbely A, van Heerebeek L, Paulus WJ (2009) Transcriptional and posttranslational modifications of titin:implications for diastole. Circ Res 104: 12-14.
59. Satoh M, Takahashi M, Sakamoto T, Hiroe M, Marumo F, et al. (1999) Structural analysis of the titin gene in hypertrophic cardiomyopathy:identification of a novel disease gene. Biochem Biophys Res Commun 262:411-417.
60. Endo A, Surks HK, Mochizuki S, Mochizuki N, Mendelsohn ME (2004) Identification and characterization of zipper-interacting protein kinase as the unique vascular smooth muscle myosin phosphatase-associated kinase. J Biol Chem 279:42055-42061.
61. Kawai T, Matsumoto M, Takeda K, Sanjo H, Akira S (1998) ZIP kinase, a novel serine/threonine kinase which mediates apoptosis. Mol Cell Biol 18:1642-1651.
62. Ihara E, MacDonald JA (2007) The regulation of smooth muscle contractility by zipper-interacting protein kinase. Can J Physiol Pharmacol 85:79-87.
63. MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshome DJ, et al. (2001) Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci U S A 98:2419-2424.
64. Kamm KE, Stull JT (2011) Signaling to myosin regulatory light chain in sarcomeres. J Biol Chem 286:9941-9947.
65. Wang Y, Chiu JF, He QY (2006) Proteomics approach to illustrate drug action mechanisms. Curr Drug Discov Technol 3:199-209.
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2. Yamada E (1955) The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol 1:445-458.
3. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, et al. (1992) Caveolin, a protein component of caveolae membrane coats. Cell 68:673-682.
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5. Anderson RG, Kamen BA, Rothberg KG, Lacey SW (1992) Potocytosis: sequestration and transport of small molecules by caveolae. Science 255:410-411.
6. Allen JA, Halverson-Tamboli RA, Rasenick MM (2007) Lipid raft microdomains and neurotransmitter signalling. Nat Rev Neurosci 8:128-140.
7. Parton RG, Simons K (2007) The multiple faces of caveolae. Nat Rev Mol Cell Biol8:185-194.
8. Insel PA, Patel HH (2009) Membrane rafts and caveolae in cardiovascular signaling. Curr Opin Nephrol Hypertens 18:50-56.
9. Patel HH, Murray F, Insel PA (2008) Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annu Rev Pharmacol Toxicol 48:359-391.
10. Li XA, Everson WV, Smart EJ (2005) Caveolae, lipid rafts, and vascular disease. Trends Cardiovasc Med 15:92-96.
11. Jin Y, Lee SJ, Minshall RD, Choi AM (2011) Caveolin-1:a critical regulator of lung injury. Am J Physiol Lung Cell Mol Physiol 300:L151-160.
12. Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, et al. (2001) Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem 276:38121-38138.
13. Drab M, Verkade P, Elger M. Kasper M, Lohn M. et al. (2001) Loss of caveolae. vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293:2449-2452.
14. Resh MD (1999) Fatty acylation of proteins:new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta 1451: 1-16.
15. Michel JB, Feron O. Sacks D, Michel T (1997) Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J Biol Chem 272: 15583-15586.
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22. Ehrenkranz JR, Lewis NG, Kahn CR, Roth J (2005) Phlorizin:a review. Diabetes Metab Res Rev 21:31-38.
23. Pajor AM, Randolph KM, Kerner SA, Smith CD (2008) Inhibitor binding in the human renal low-and high-affinity Na+/glucose cotransporters. J Pharmacol Exp Ther 324:985-991.
24. Tyagi NK, Kumar A, Goyal P, Pandey D, Siess W, et al. (2007) D-Glucose-recognition and phlorizin-binding sites in human sodium/D-glucose cotransporter 1 (hSGLTl):a tryptophan scanning study. Biochemistry 46: 13616-13628.
25. Osorio H, Coronel I, Arellano A, Pacheco U, Bautista R, et al. (2012) Sodium-glucose cotransporter inhibition prevents oxidative stress in the kidney of diabetic rats. Oxid Med Cell Longev 2012:542042.
26. Chen ZC, Cheng YZ, Chen L.T, Cheng KC, Li Y, et al. (2012) Increase of ATP-sensitive potassium (K(ATP)) channels in the heart of type-1 diabetic rats. Cardiovasc Diabetol 11:8.
27. Shen L, You BA, Gao HQ, Li BY, Yu F, et al. (2012) Effects of phlorizin on vascular complications in diabetes db/db mice. Chin Med J (Engl) 125:3692-3696.
28. Fort PE, Losiewicz MK, Reiter CE, Singh RS, Nakamura M, et al. (2011) Differential roles of hyperglycemia and hypoinsulinemia in diabetes induced retinal cell death:evidence for retinal insulin resistance. PLoS One 6:e26498.
29. Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, et al. (1996) Progress with proteome projects:why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 13:19-50.
30. Abbott A (2001) And now for the proteome. Nature 409:747.
31. Fields S (2001) Proteomics. Proteomics in genomeland. Science 291:1221-1224.
32.高海青,邱洁.心血管蛋白质组学[M].第1版.北京：人民卫生出版社,2010.
33. Hu J, Qian J, Borisov O, Pan S, Li Y, et al. (2006) Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags. Proteomics 6:4321-4334.
34. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, et al. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154-1169.
35. Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, Jerums G (1991) Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes 40:1328-1334.
36. American Diabetes A (2011) Executive summary:standards of medical care in diabetes-2011. Diabetes Care 34 Suppl 1:S4-10.
37.高海青,李保应.葡萄多酚：基础与临床[M].第1版.北京：人民卫生出版社,2012.
38. Futterman LG, Lemberg L (2000) The Framingham Heart Study:a pivotal legacy of the last millennium. Am J Crit Care 9:147-151.
39. Brownlee M (2005) The pathobiology of diabetic complications:a unifying mechanism. Diabetes 54:1615-1625.
40. An D, Rodrigues B (2006) Role of changes in cardiac metabolism in development of diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 291:HI489-1506.
41. Lu WD, Li BY, Yu F, Cai Q, Zhang Z, et al. (2012) Quantitative proteomics study on the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice. Mol Med Report 5:1285-1294.
42. Hummel KP, Dickie MM, Coleman DL (1966) Diabetes, a new mutation in the mouse. Science 153:1127-1128.
43. Herberg L, Coleman DL (1977) Laboratory animals exhibiting obesity and diabetes syndromes. Metabolism 26:59-99.
44. Berglund O, Frankel BJ, Hellman B (1978) Development of the insulin secretory defect in genetically diabetic (db/db) mouse. Acta Endocrinol (Copenh) 87: 543-551.
45. Lee SM, Bressler R (1981) Prevention of diabetic nephropathy by diet control in the db/db mouse. Diabetes 30:106-111.
46. Wende AR, Abel ED (2010) Lipotoxicity in the heart. Biochim Biophys Acta 1801: 311-319.
47. Vasantha Rupasinghe HP, Yasmin A (2010) Inhibition of oxidation of aqueous emulsions of omega-3 fatty acids and fish oil by phloretin and phloridzin. Molecules 15:251-257.
48. Bartels ED. Nielsen JM, Hellgren LI, Ploug T. Nielsen LB (2009) Cardiac expression of microsomal triglyceride transfer protein is increased in obesity and serves to attenuate cardiac triglyceride accumulation. PLoS One 4:e5300.
49. Dougan SK, Rava P, Hussain MM, Blumberg RS (2007) MTP regulated by an alternate promoter is essential for NKT cell development. J Exp Med 204: 533-545.
50. Mohler PJ, Zhu MY, Blade AM, Ham AJ, Shelness GS, et al. (2007) Identification of a novel isoform of microsomal triglyceride transfer protein. J Biol Chem 282: 26981-26988.
51. Chang YH, Chang DM, Lin KC, Shin SJ, Lee YJ (2011) Visfatin in overweight/obesity, type 2 diabetes mellitus, insulin resistance, metabolic syndrome and cardiovascular diseases:a meta-analysis and systemic review. Diabetes Metab Res Rev 27:515-527.
52. Peiro C, Romacho T, Carraro R, Sanchez-Ferrer CF (2010) Visfatin/PBEF/Nampt: A New Cardiovascular Target? Front Pharmacol 1:135.
53. Neely JR, Rovetto MJ, Oram JF (1972) Myocardial utilization of carbohydrate and lipids. Prog Cardiovasc Dis 15:289-329.
54. Saddik M, Lopaschuk GD (1991) Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts. J Biol Chem266:8162-8170.
55. Saito F, Kawaguchi M, Izumida J, Asakura T, Maehara K, et al. (2003) Alteration in haemodynamics and pathological changes in the cardiovascular system during the development of Type 2 diabetes mellitus in OLETF rats. Diabetologia 46: 1161-1169.
56. Anguera I, Magrina J, Setoain FJ, Esmatges E, Pare C, et al. (1998) [Anatomopathological bases of latent ventricular dysfunction in insulin-dependent diabetics]. Rev Esp Cardiol 51:43-50.
57. Rodrigues B, Cam MC, McNeill JH (1995) Myocardial substrate metabolism: implications for diabetic cardiomyopathy. J Mol Cell Cardiol 27:169-179.
58. Borbely A, van Heerebeek L, Paulus WJ (2009) Transcriptional and posttranslational modifications of titin:implications for diastole. Circ Res 104: 12-14.
59. Satoh M, Takahashi M, Sakamoto T, Hiroe M, Marumo F, et al. (1999) Structural analysis of the titin gene in hypertrophic cardiomyopathy:identification of a novel disease gene. Biochem Biophys Res Commun 262:411-417.
60. Endo A, Surks HK, Mochizuki S, Mochizuki N, Mendelsohn ME (2004) Identification and characterization of zipper-interacting protein kinase as the unique vascular smooth muscle myosin phosphatase-associated kinase. J Biol Chem 279:42055-42061.
61. Kawai T, Matsumoto M, Takeda K, Sanjo H, Akira S (1998) ZIP kinase, a novel serine/threonine kinase which mediates apoptosis. Mol Cell Biol 18:1642-1651.
62. Ihara E, MacDonald JA (2007) The regulation of smooth muscle contractility by zipper-interacting protein kinase. Can J Physiol Pharmacol 85:79-87.
63. MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshome DJ, et al. (2001) Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci U S A 98:2419-2424.
64. Kamm KE, Stull JT (2011) Signaling to myosin regulatory light chain in sarcomeres. J Biol Chem 286:9941-9947.
65. Wang Y, Chiu JF, He QY (2006) Proteomics approach to illustrate drug action mechanisms. Curr Drug Discov Technol 3:199-209.
1. Sowa G (2012) Caveolae, caveolins, cavins, and endothelial cell function:new insights. Front Physiol 2:120.
2. Yamada E (1955) The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol 1:445-458.
3. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, et al. (1992) Caveolin, a protein component of caveolae membrane coats. Cell 68:673-682.
4. Schnitzer JE, Oh P, McIntosh DP (1996) Role of GTP hydrolysis in fission of caveolae directly from plasma membranes. Science 274:239-242.
5. Anderson RG, Kamen BA, Rothberg KG, Lacey SW (1992) Potocytosis: sequestration and transport of small molecules by caveolae. Science 255:410-411.
6. Allen JA, Halverson-Tamboli RA, Rasenick MM (2007) Lipid raft microdomains and neurotransmitter signalling. Nat Rev Neurosci 8:128-140.
7. Parton RG, Simons K (2007) The multiple faces of caveolae. Nat Rev Mol Cell Biol8:185-194.
8. Insel PA, Patel HH (2009) Membrane rafts and caveolae in cardiovascular signaling. Curr Opin Nephrol Hypertens 18:50-56.
9. Patel HH, Murray F, Insel PA (2008) Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annu Rev Pharmacol Toxicol 48:359-391.
10. Li XA, Everson WV, Smart EJ (2005) Caveolae, lipid rafts, and vascular disease. Trends Cardiovasc Med 15:92-96.
11. Jin Y, Lee SJ, Minshall RD, Choi AM (2011) Caveolin-1:a critical regulator of lung injury. Am J Physiol Lung Cell Mol Physiol 300:L151-160.
12. Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, et al. (2001) Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem 276:38121-38138.
13. Drab M, Verkade P, Elger M. Kasper M, Lohn M. et al. (2001) Loss of caveolae. vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293:2449-2452.
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