鱼类leptin的生物学特性及功能

卢荣华孙君君梁旭方聂国兴杨 峰

(1. 河南师范大学水产学院, 新乡 453007; 2. 华中农业大学水产学院, 武汉 430070)

鱼类leptin的生物学特性及功能

卢荣华1孙君君1梁旭方2聂国兴1杨 峰1

(1. 河南师范大学水产学院, 新乡 453007; 2. 华中农业大学水产学院, 武汉 430070)

瘦素(Leptin)是肥胖基因(Obese gene)的产物, 属于I型细胞因子。在哺乳动物中, leptin主要由脂肪细胞合成与分泌, 是调控摄食、能量代谢、骨骼发育、甲状腺功能以及繁殖等生理过程的重要激素。目前, 多种硬骨鱼类的leptin基因已被克隆, 其功能也已得到初步研究。研究认为, 鱼类leptin的主要合成部位在肝脏, 其在氨基酸序列上与哺乳动物存在很大差异, 但蛋白质结构高度保守; 功能方面, leptin可调节鱼类的摄食、葡萄糖和脂肪代谢以及繁殖等生命活动过程。本文就鱼类leptin及其受体的特征结构、组织分布、表达调控及功能研究进展进行简要综述。

Leptin; 鱼类; 生物学特性; 食欲调节; 能量代谢

1994年, Zhang等[1]首次分离出哺乳动物的肥胖基因(Obese gene), 瘦素(Leptin)即是肥胖基因的产物, 该名字起源于希腊语leptos意为“瘦的”, 其得名是由于研究发现缺失leptin会造成哺乳动物的肥胖。哺乳动物leptin由脂肪组织分泌, 已证明其在糖脂代谢[2,3]、生殖[4,5]、免疫[6]、能量消耗[7]以及摄食中发挥重要作用, 被认为是机体的一种饱食因子[8—10]。与对陆生动物leptin的深入研究相比, 鱼类leptin虽然也已引起学者们的关注, 但相关功能研究尚开展的不多。

1 Leptin及Leptin受体的结构与同源性分析

1.1 Leptin的结构, 拷贝数及同源性分析

Johnson等[11]首次通过鼠leptin抗体在蓝绿鳞鳃太阳鱼(Lepomis macrochirus)、虹鳟(Oncorhynchus mykiss)、大口黑鲈(Pomonix annularis)和斑点叉尾鲴(Ictalurus punctatus)中检测到其血液、脑、心、肝中均存在leptin。日本学者Kurokawa等[12]通过基因组同线性方法从红鳍东方鲀(Takifugu rubripes)等变温动物中首次鉴定并分离出肥胖基因。随后, 包括日本青 鳉(Oryzias latipes)[12,13]、鲤(Cyprinus carpio)[14]、斑马鱼(Danio rerio)[15]、大麻哈鱼(Oncorhynchus keta)[16]、虹鳟[16]、草鱼(Ctenopharyngodon idella)[17]、北极鲑(Salvelinus alpinus)[18]、大西洋鲑(Salmo salar)[19]、 鲈 (Morone saxatilis)[20]、 黄 颡 鱼(Pelteobagrus fulvidraco)[21]和大黄鱼[22]等鱼类以及非洲爪蟾(Xenopus laevis)[23]、热带爪蟾(Xenopus tropicalis)[12,23]、宽虎纹钝口螈(Ambystoma tigrinum)[24]等两栖动物的leptin基因也被先后分离, 并在鲤[14]、草鱼[17]、虹鳟[16,25]和 青 鳉(Oryzias latipes)[26]等部分物种中进行了相对深入的研究。

在非洲爪蟾和目前已知的多数鱼类中, 肥胖基因由3个外显子和2个内含子组成, 与人类的肥胖基因结构相似[1], 且内含子的相对位置高度保守[12,16,17,23,27]。然而, 宽虎纹钝口螈[24]、鲤[14]和鳜(Siniperca chuatsi)的肥胖基因只含有 2个外显子和 1个内含子(第 2个内含子), 它们的第1个内含子在进化过程中已丢失[13,15,23,24,27—29]。基因组同线性分析表明, 人、小鼠、热带爪蟾、河鲀 和斑马鱼等的leptin基因在基因组中的线性排列非常相似[12,14,16,17,23,24,27], 表明它们起源相同。

在人和小鼠等哺乳动物以及非洲爪蟾的基因组中, 肥胖基因仅有一个拷贝, 编码一种蛋白产物。然而最近的研究发现, 在斑马鱼、日本青 鳉和石斑鱼(Epinephelus coioides)的基因组中存在2个肥胖基因拷贝, 编码两种不同的产物(分别命名为leptin-A型和leptin-B型)[13,15,30]。斑马鱼的leptin-A和leptin-B仅含有24%的氨基酸同源性, 表明它们属于两种不同的leptin亚型。因此鱼类可能普遍存在两种leptin亚型[13,15]。尽管先前的研究发现鲤中也存在两种leptin转录本, 然而它们之间的氨基酸同源性高达84%,系统树分析发现它们同聚于leptin-A进化分枝, 表明它们很可能同属于leptin-A型, 这可能是由鲤(四倍体)在～16Mya发生的基因组加倍引起。系统树分析和氨基酸同源性分析显示, 目前分离得到的鱼类leptin绝大部分属于leptin-A型, 包括绿河豚(Tetraodon nigroviridis)[12]、虹鳟[16]、北极红点鲑(Salvelinus alpinus)[18]、鲢(Hypophthalmichthys molitrix)和草鱼[17]等, 在鲤及其他大部分鱼类中很可能还存在leptin-B型[15]。两种leptin亚型在鱼类中的出现以及它们组织表达的差异, 可能赋予leptin新的功能。最新的研究发现, 某些种类如大西洋鲑的leptin甚至出现了多达四个亚型同源物(LepA1, LepA2, LepB1/B2)[31]。

Leptin的蛋白序列在哺乳动物中高度保守, 鱼类及其他变温动物leptin与哺乳动物leptin的氨基酸同源性却很低(如河 鲀 、斑马鱼、虹鳟和非洲爪蟾 leptin等与人leptin的氨基酸同源性分别为13.2%、22%、21.4%和35.0%)[12,14,16,17,23,24,27], 此外, 不同种属鱼类leptin之间的氨基酸同源性同样很低(如 河鲀 与斑马鱼、虹鳟leptin的氨基酸同源性为19%和20.9%, 鲤和虹鳟leptin的氨基酸同源性为26.8%)[12—17,23,24,27]。分析发现, 它们用于形成二硫键的半胱氨酸却高度保守, 蛋白二级结构(4个α螺旋)也同样高度保守, 而且三级结构预测表明它们之间的空间结构十分相似[12—17,23,24,27]。

1.2 Leptin受体的基因结构

人等哺乳动物的leptin受体(Leptin receptor, leptin-R)为单一拷贝, 但其可通过不同的剪接方式产生多达6种长度不同的转录本[32]。其中, 长型leptin-R在介导leptin的调控功能中起重要作用[32]。人的leptin-R含有20个外显子, 非洲爪蟾的外显子则多达26个[23]。目前, 已在海洋青鳉鱼(Oryzias melastigma)[33]、 日本青 鳉[13]、 河 鲀[34]和草鱼[35]等鱼类中分离了leptin-R基因, 日本青鳉leptin-R基因含有20个外显子[13], 河 鲀leptin-R基因则含有21个外显子[34]。目前在非洲爪蟾中仅发现长型leptin-R一种类型,其他在哺乳动物中出现的5种类型至今未见报道。

2 Leptin及leptin受体的组织差异表达

2.1 Leptin的组织分布及分泌浓度

Leptin在多种组织中均有表达, 在人等哺乳动物中, 脂肪组织是leptin的主要合成位点[1,36]。此外, 其在胎盘等多种组织中也可以合成[36]。与哺乳动物不同, 非洲爪蟾leptin在脑和心脏中大量表达[23], 宽虎纹钝口螈 leptin主要在皮肤和卵巢中表达[24], 而鱼类的leptin虽然在包括肠道、脂肪、大脑等不同组织中都有少量表达, 但主要表达部位是肝脏[11—17,19,27,34]。此外, 不同亚型leptin的组织表达情况也有差异。在日本青 鳉中, leptin-A主要表达在鳃、肝脏、脑垂腺、脾、肠、心脏等组织中, leptin-B在卵巢、脑垂腺、鳃和心脏中表达水平较高。在斑马鱼中, leptin-A 主要在肝脏中表达, 而leptin-B在脑和眼中大量表达[15]。leptin亚型组织分布的不同, 提示其可能有各自独特的功能。如leptin-B在卵巢的大量表达暗示其可能在调控生殖功能中起重要作用[13]。

在正常生理状态下, 人的血浆leptin浓度为0.2—0.3 nmol/L[28,29], 鼠类的血浆leptin浓度为0.09—0.3 nmol/L[37,38]。而Kling等[39]测定了多种鱼类的血浆leptin浓度, 结果表明鱼类的血浆leptin浓度为1.1—5 nmol/L, 比哺乳动物的血浆leptin浓度高。哺乳动物能依据血浆中的leptin浓度传达体内脂肪含量信息给大脑, 进而指导摄食行为、代谢及生理内分泌, 使其与机体的营养状况相一致[40]。上述leptin组织表达及分泌浓度的差异, 暗示鱼类的leptin在功能及调节通路上可能与哺乳动物有较大差异。

2.2 Leptin受体的组织分布

不论在哺乳动物还是鱼类中, leptin均通过其受体发挥调控摄食及能量平衡等功能[41—47]。哺乳动物长型leptin-R主要在下丘脑中表达[1,32,36], 小鼠缺乏leptin-R会因食欲过强导致肥胖[48]。海洋青鳉的leptin-R主要在鳃、脾脏、肾脏和肌肉中表达, 且雌性海洋青鳉leptin-R的表达量要高于雄性的[33]。薛俊莲等[49]通过RT-PCR方法获得鲫鱼的leptin受体序列,并制备了鲫鱼2个瘦素受体的多克隆抗体; 检测了鲫鱼组织和血清中的受体, 初步确定了鲫鱼血清中存在可溶瘦素受体。

3 Leptin的功能

3.1 Leptin在调控摄食中的作用

用哺乳动物的 leptin在金鱼体内通过腹腔和侧脑室注射后显示其摄食减少、体重下降以及调控摄食和能量代谢相关基因表达水平发生改变[50,51]。在鲈腹腔内注射人leptin也可抑制其摄食[20]。然而, 以哺乳动物 leptin处理银大马哈鱼(Oncorhynchus kisutch)[52]、鲶(Ictalurus punctatus)[53]和绿海鲂(Lepomis cyanellus)[54], 却并不能改变它们的摄食行为或能量代谢。这可能与特异性的鱼类 leptin的功能有关。目前在绝大多数鱼类研究中发现 leptin具有抑制摄食的作用[16,17,25]。Murashita等[16]用重组虹鳟 leptin处理后发现可显著抑制虹鳟的摄食行为、刺激抑制食欲因子阿片促黑色素原 A1/A2 (proopiomelanocorein- A1/A2, POMC-A1/A2)基因表达并降低促食欲因子神经肽Y(neuropeptide Y, NPY)基因的表达水平。进一步研究发现重组鲑 leptin也可显著抑制大西洋鲑的生长, 分析原因认为 leptin可能通过显著提高 POMC-A1转录, 抑制食物摄入而导致生长减慢[55]。Li等[17]制备了草鱼leptin蛋白,通过短期(1 d)和长期(13 d)腹腔注射实验, 发现leptin短期处理可明显抑制草鱼的摄食、抑制 NPY等摄食相关基因的表达。在石斑鱼中, 饥饿和再投喂实验表明, Leptin-A在调节摄食和能量代谢中发挥重要作用[30]。

3.2 Leptin在调控糖脂代谢中的作用

在哺乳动物中leptin可抑制脂肪的沉积, 促进脂肪的水解[56—58]等, 还可通过影响动物下丘脑和后脑的葡萄糖感受性神经元的活性, 抑制促食欲肽(NPY, AgRP, Orexin)和激活厌食欲肽(POMC, CART, CCK)的表达[59—61]进而来调控其摄食行为, 并促进能量消耗。因此, leptin调节摄食的作用也与对葡萄糖的代谢调控交织在一起; 除中枢神经系统外, leptin还可直接调节外周组织器官, 促进肝脏葡萄糖生成, 调控肝脏磷酸烯醇丙酮酸羧激酶(Phosphoenolpyruvate carboxykinase, PEPCK)基因表达和糖异生效率[62], 通过乳酸摄取增加直接刺激肝糖原产生。leptin对PEPCK及糖异生的影响限制了甘油三酯的合成, 因而哺乳类leptin主要通过调控糖和脂代谢与摄食的偶联来维持其体脂蓄积及机体能量代谢的稳定。

目前, 已有一些关于鱼类 leptin对脂类代谢的研究, 如 leptin能增加太阳鱼细胞内脂肪酸结合蛋白的量[54]; 重组草鱼 leptin蛋白通过腹腔注射后,可促进脂代谢基因或能量代谢基因解偶联蛋白2(Uncoupling protein 2, UCP2)、胆盐活化的胰脂肪酶(Bile salt-activated lipase, BSAL)和脂肪酸延长酶(Fatty acid elongase, ELO)的基因表达, 这与虹鳟实验中所得结果相似, 也与非洲爪蟾和哺乳动物的结果一致[17]; 笔者在草鱼营养性肝细胞脂变模型中的研究结果也表明, leptin剂量依赖性促进甘油的释放,并可影响9个脂肪代谢关键基因在转录和翻译水平的表达, 且其诱导脂肪分解的作用是由 JAK-STAT信号通路来介导的[3]。

迄今为止, 已有研究发现鱼类leptin和糖代谢(血糖变化)、饥饿及再投喂条件关系密切。在金鱼腹腔内注射leptin后, 可引起肝糖原、肌糖原以及循环激素水平的变化[51,52]; 在虹鳟脑室注射leptin后可提高糖合成和分解过程中相关酶的含量, GK活性以及与糖代谢反应有关基因的mRNA水平, 研究还发现leptin作用后会引起血糖浓度升高[25]。Huising等[14]以鲤为模型, 研究发现摄食后其肝脏leptin mRNA表达量上升, 其峰值出现在血糖含量的升高和血浆游离脂肪酸下降之后, 这可能是因为其leptin表达量由摄食后血糖含量变化所控制。Gorissen等[15]证实斑马鱼在禁食一周后, 其肝脏leptin-B的mRNA水平显著降低, 而leptin-A的mRNA水平则升高, 这与leptin的不同亚型有关。虹鳟在长期饥饿(3周)中, leptin的表达水平显著升高[39]。在鲈中, 饥饿3周其肝中leptin的mRNA水平显著减少, 再投喂3周后leptin mRNA水平则回升[20]; 上述这些研究表明了鱼类leptin可激活糖代谢系统, 这与高血糖条件下观察到的变化一致, 在鱼类脑中增加或降低血糖水平[63,64]伴随着GK的活性及其表达水平的变化、糖酵解能力以及葡萄糖和糖原水平的变化, 且这种变化与哺乳动物的糖代谢反应在某种程度上相类似[65]。

哺乳动物中的研究认为机体的血糖水平与摄食调节及能量平衡密切相关, 高血糖条件下食物的摄入被抑制[12]。而上述在绝大多数鱼类研究中的结果显示, 特异性的鱼类leptin具有抑制鱼类摄食的作用[16,17,25], 推测鱼类leptin对摄食的调节也与对糖脂代谢的作用相偶联, 即鱼类leptin通过调节糖(血糖)及脂类代谢进而调节鱼类的摄食及能量代谢的平衡。众所周知, 鱼类, 特别是肉食性鱼类, 对高糖具有不耐受性[66,67], 而鱼类的食性与leptin以及糖脂代谢是否存在更密切的关系, 尚需深入研究。

3.3 Leptin的其他作用

已有研究证实 leptin与硬骨鱼类的繁殖活动密切相关。leptin及其受体在大西洋鲑和斑马鱼的脑(脑垂体)及性腺中表达很丰富[15,19,42]。Peyon等[68]报道重组鼠leptin对舌齿鲈(Dicentrarchus labrax)脑垂体中促黄体生成素(luteinizing hormone, LH)的产生有直接作用。高浓度的重组人 leptin可刺激虹鳟离体脑垂体分泌促性腺激素(gonadotropin, GtH)[69];在北极红点鲑性成熟季节, leptin mRNA在肝脏中保持较高的表达水平[18]。另有研究报道[70], leptin可影响鱼类的免疫系统, 采用重组虹鳟 leptin和虹鳟白细胞共孵育后可激活STAT3(Signal transducer and activator of transcription 3, STAT3), NF-κB(nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB), 以及三个主要的 MAPK(mitogen-activated protein kinases, MAPK)级联的信号通路(JNK, p38和 ERK); 并能减少虹鳟血液白细胞中超氧阴离子的量; 重组尼罗罗非鱼(Tilapia nilotica)leptin-A 可增强罗非鱼垂体催乳素的合成和释放, 而垂体催乳素反过来也可抑制肝 leptin蛋白的合成和分泌, 推测这种相互调节作用在急性高渗适应过程中对于动员能量储备是必要的[71]; Baltzegar等[72]在尼罗罗非鱼的研究也提示 leptin-A可能在其急性高渗应激中和皮质醇共同起重要的调节作用。魏 赟[73]对斜带石斑鱼(Epinephelus coioides)两种同源的瘦素基因(leptin-A, leptin-B)进行了多态性研究, 并与斜带石斑鱼12个生长性状进行关联分析, 筛选出了4个与生长性状紧密相关的分子标记。

4 展望

综上所述, leptin是一种具有多功能的激素, 在能量代谢、繁殖以及适应环境等方面发挥关键作用。虽然在鱼类中 leptin的研究已取得了一定的进展,但仍有很多重要的问题尚未解决, 如(1)鱼类 leptin受体是否同时作为 leptin-A和 leptin-B的受体？或是受体下游的作用机制决定 leptin类型？在鱼的全基因组中是否尚有未知的 leptin受体？(2)鱼类leptin是否具有像哺乳动物leptin在骨骼生成、甲状腺功能以及血管生成等方面的作用？进一步解析leptin在鱼类中的多种生理作用, 可为最终阐明影响脊椎动物能量稳态的 leptin系统的起源及进化奠定新的理论基础。

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BIOLOGICAL CHARACTERISTICS AND FUNCTIONS OF LEPTIN IN FISH

LU Rong-Hua1, SUN Jun-Jun1, LIANG Xu-Fang2, NIE Guo-Xing1and YANG Feng1
(1. College of Fisheries, Henan Normal University, Xinxiang 453007, China; 2. College of Fisheries, Huazhong Agricultural University, Shizishan Street 1, Wuhan 430070, China)

Leptin is the product of ob gene and a kind of Type I cytokine. It is primarily synthesized and secreted by adipocytes and plays an important role in the regulation of ingestion, energy metabolism, skeletal development, thyroid function, and reproduction in mammals. To date leptin gene has been cloned in various teleostean groups, and there have been studies of the physiological functions of leptin. Previous reports have suggested that fish leptin is mainly synthesized in liver, and the amino acid sequence is different from that in mammals; however, the protein structure is highly conservative. Because of its crucial roles in ingestion, glucose and lipid metabolism, and reproduction, Leptin is potentially a new drug target for improving the glucose utilization efficiency in fish in the future. Therefore in this review we discussed about fish leptin in terms of its structure, expression, functions, tissue-specific distribution and receptors.

Leptin; Fish; Biological characteristics; Appetite regulation; Energy metabolism

Q344+.1

A

1000-3207(2015)03-0583-07

10.7541/2015.76

2014-04-23;

2014-07-15

国家自然科学基金项目(31172420, 31402311); 河南省基础与前沿技术研究计划项目(142300410158)

卢荣华(1977—), 女, 河南虞城人; 博士; 主要研究方向为鱼类糖脂代谢调控机理研究。E-mail: laoaiyika@hotmail.com

梁旭方, 教授; E-mail: xfliang@mail.hzau.edu.cn