摄食可维持动物机体的稳定，促进动物生长[1]。摄食主要通过中枢摄食系统和外周摄食系统的增食欲及厌食欲因子(神经肽、单胺类、胃肠肽和激素等)互作进行调控[2]。胃饥饿素(Ghrelin)，又称作生长激素释放肽(growth-hormone-releasing peptide)，是机体内重要的增食欲因子之一[3]。1999年，日本学者Kojima等[3]在大鼠上鉴定出含28个氨基酸的多肽，能刺激生长激素(GH)的释放，故将此多肽命名为生长激素释放肽，进一步通过组织表达分析发现其在胃和下丘脑中存在。随后，2000年Wajnrajch等[4]在人的胃和下丘脑处检测到Ghrelin的表达，因此认为Ghrelin是一种脑肠肽[3]。除大鼠和人外，Ghrelin在其他哺乳动物[5-7]中也已鉴定存在。Ghrelin作为增食欲因子，在鱼类摄食相关领域中已成为研究热点之一。为此，本文着重阐述Ghrelin对鱼类摄食的调控及其机制，以期为鱼类摄食调控及生长的研究和生产实践提供理论依据。
1 Ghrelin的结构 1.1 Ghrelin的基因结构目前，学者们已经在多个物种上成功鉴定了Ghrelin基因。在哺乳动物上，Ghrelin基因一般含有5个外显子和4个内含子(图 1-A)。对人[4]、大鼠[3]和小鼠[5]基因序列比对发现，19 bp的第1外显子区域和5′端启动子区域的TATA盒样序列高度同源性，暗示Ghrelin基因在哺乳动物中结构的保守性。在鱼类上，最早的报道是2002年，Unniappan等[8]通过cDNA末端快速扩增(RACE)技术在金鱼(Carassius auratus)上成功克隆了Ghrelin基因，含4个外显子和3个内含子(图 1-B)。类似的如莫桑比克罗非鱼(Oreochromis mossambicus)[9]、尼罗罗非鱼(Oreochromis niloticus)[10]、黑鲷(Acanthopagrus schlegeli)[11]和斑马鱼(Barchydanio rerio)[12]。而有些鱼类如虹鳟(Oncorhynchus mykiss)[13]、斑点叉尾(Ietalurus Punetaus)[14]、大西洋鲑(Salmo salar)[15]的Ghrelin基因存在亚型(Ghrelin1和Ghrelin2)，包括5个外显子和4个内含子，结构与哺乳动物Ghrelin基因结构相似。这些差异是否与不同鱼类的生活习性及发挥某些生物学功能有关待进一步研究。
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A：哺乳动物以及虹鳟、斑点叉尾和大西洋鲑等鱼类Ghrelin基因结构（5个外显子和4个内含子）；B：金鱼、莫桑比克罗非鱼、尼罗罗非鱼、黑鲷和斑马鱼等鱼类Ghrelin基因结构（4个外显子和3个内含子）。 A:the structure of Ghrelin gene in mammals and a part of fish species including Oncorhynchus mykiss, Ietalurus punetaus and Salmo salar(5 exons and 4 introns); B:the structure of Ghrelin gene in a part of fish species including Carassius auratus, Oreochromis mossambicus, Oreochromis niloticus, Acanthopagrus schlegeli and Barchydanio rerio(4 exons and 3 introns). 图 1 Ghrelin基因结构 Figure 1 The structure of Ghrelin gene |
Ghrelin的蛋白质结构包括信号肽、成熟肽和C-末端肽区域，成熟肽含17~28个氨基酸不等。不同物种间Ghrelin蛋白质结构成熟肽保守型较高，且成熟肽第3位氨基酸均为丝氨酸(Ser)，可发生酰化修饰，而成熟肽之后具有甘氨酸(Gly)-精氨酸(Arg)-Arg或Gly-Arg酰胺化信号结构。在哺乳动物上，人[4]和大鼠[3]Ghrelin蛋白质结构均已鉴定。在鱼类上，最早发现金鱼Ghrelin前体蛋白有103个氨基酸，含22个成熟肽，酰胺化信号在成熟肽第19个氨基酸之后[8]。罗非鱼[9-10]、黑鲷[11]、斑马鱼[12]均含有一种Ghrelin蛋白质结构。虹鳟、大西洋鲑因具有2种Ghrelin基因亚型，存在2种Ghrelin蛋白质结构，在虹鳟上其成熟肽分别含24和21个氨基酸[13]，在大西洋鲑上其成熟肽分别含23和20个氨基酸[15]；虽然也具有2种Ghrelin基因亚型，但在斑点叉尾上仅发现1种Ghrelin蛋白质结构，其成熟肽含22个氨基酸[14]。各物种Ghrelin成熟肽的前7位氨基酸的保守性高，鱼类Ghrelin成熟肽区域保守性较高。这种保守性可能与Ghrelin发挥生物学功能有关，还需进一步研究。
2 Ghrelin的组织分布Ghrelin广泛分布于各物种的中枢和外周系统中。在哺乳动物中，Ghrelin在中枢系统中的脑组织表达量最高，在外周系统中的胃和肠道广泛表达[3, 16-17]。Ghrelin的组织分布在金鱼[8]、日本鳗鲡(Anguilla japonica)[18]、黑鲷[11]、斑马鱼[12]、雅鱼[19-20]和红腹食人鱼(Pygocentrus nattereri)[21]等鱼类上已有报道。Unniappan等[8]通过Northern blot检测出金鱼的肠道组织中有Ghrelin mRNA的表达，进一步用反转录PCR(RT-PCR)分析出脾脏中Ghrelin mRNA表达量最高，其次是肠道，但是在中脑、后脑和垂体以及其他外周组织中未检测出Ghrelin mRNA。Kaiya等[18]通过RT-PCR发现日本鳗鲡Ghrelin在脑、心脏、胃、肠道、体肾和头肾处有表达，胃和前肠的表达量最高。Yeung等[11]发现黑鲷Ghrelin仅在胃部大量表达。而Amole等[12]发现，除胃部外，斑马鱼脑和肝胰脏等也有Ghrelin分布。此外，报道了齐口裂腹鱼(Schizothorax prenanti)Ghrelin在脑和肠道中表达量较高[20]，而重口裂腹鱼(Schizothorax davidi)Ghrelin在肠道中丰富表达[19]。Volkoff[21]通过实时定量PCP(qRT-PCR)发现在红腹食人鱼脑、消化道、肝脏和脾脏等组织中Ghrelin表达量较为丰富。
Ghrelin在不同物种各组织中的广泛表达模式暗示其具有多种生物学功能，在脑组织中的丰富表达可能暗示其参与多种生命活动的中枢调控，在消化道中的高表达可能暗示其与动物的摄食、消化和吸收等功能有关。
3 Ghrelin调节鱼类摄食Ghrelin对动物摄食功能的调节是其生物学功能研究的热点之一，在哺乳动物上的研究较多，鱼类上也开展了一些研究，研究主要集中在：1)喂养策略对Ghrelin表达量的影响；2)注射Ghrelin对鱼类摄食量的影响。
3.1 喂养策略对Ghrelin表达量的影响摄食前后和禁食后复投喂等喂养策略可引起Ghrelin表达水平改变。学者们发现增食欲因子在摄食前或禁食后表达量上升，在摄食后表达量下降，而厌食欲因子的表达模式则相反[22]。在哺乳动物上的报道显示，中枢神经系统和外周组织中的Ghrelin表达模式为摄食后表达量下降、禁食(短期或长期)后表达量上升，长期禁食后复投喂表达量下降[23-25]。在鱼类上，Wei等[20]发现齐口裂腹鱼摄食后1.5和9.0 h脑中Ghrelin表达量显著下降，摄食后6 h肠道中Ghrelin表达量也显著下降。类似的，异育银鲫摄食后1和3 h Ghrelin表达量显著降低，禁食7 d后Ghrelin表达量显著升高[26]。Amole等[12]报道斑马鱼禁食3、5和7 d后脑和肠道中Ghrelin mRNA表达量均显著升高，复投喂后Ghrelin mRNA表达量恢复至正常投喂组水平。此外，在重口裂腹鱼[27]、南亚野鲮(Labeo rohita)[28]和草鱼(Ctenopharyngodon idellus)[29]等上的研究也发现长期禁食显著提高Ghrelin表达量，复投喂后恢复至正常水平。这些研究表明Ghrelin作为促食欲因子参与鱼类摄食调控。
与上述研究结果不同的是，有关尼罗罗非鱼[10]、虹鳟[30]及斑点叉尾[31]的报道显示喂养策略不能影响Ghrelin表达量的改变。鱼类种类繁多，分类地位不同，摄食规律多样，Ghrelin是否在不同鱼类上均发挥摄食调控的功能还有待进一步研究。
3.2 注射Ghrelin对鱼类摄食量的影响通过中枢和外周注射Ghrelin可进一步探究Ghrelin对动物摄食的调控功能。在哺乳动物上的研究发现中枢或外周注射Ghrelin均可促进摄食，并通过其受体生长激素释放激素受体(GHS-R)调节[32-36]。对鱼类的研究结果与哺乳动物基本类似。Unniappan等[8]报道，金鱼脑室注射Ghrelin后显著增加1 h内摄食量。Matsuda等[37]给金鱼脑室和腹腔注射酰化Ghrelin，1 h内摄食量均极显著增加。Shepherd等[38]给虹鳟幼鱼静脉注射Ghrelin，摄食量显著增加。Miura等[39]发现金鱼腹腔或脑室注射酰化Ghrelin均显著增加摄食量，而注射非酰化Ghrelin则摄食量无显著变化。Tinoco等[40]报道，给虹鳟幼鱼腹腔注射Ghrelin 7 d后摄食量显著提高。此外，Velasco等[41]报道，给虹鳟注射Ghrelin 24 h后摄食量显著增加。在塞内加尔鳎上的研究也显示Ghrelin能够促进其摄食[42]。不同的是，Saito等[43-44]给初生小鸡脑室注射不同剂量Ghrelin，结果发现2 h内摄食量呈现剂量依赖性的显著下降；JÖnsson等[30]报道虹鳟腹腔注射Ghrelin 12 h内摄食量无显著改变；JÖnsson等[45]给虹鳟幼鱼长期(14 d)腹腔注射Ghrelin，其摄食量显著下降。综上，在鱼类上Ghrelin可发挥促进摄食的功能，但在不同鱼类上存在差异，这可能与药物来源、给药方式及注射剂量和时间有关。
4 Ghrelin调节鱼类摄食机制Ghrelin对动物摄食调控的作用机制目前还不十分清楚。作者根据已有哺乳动物和鱼类研究，从以下3点分析Ghrelin调节鱼类摄食的作用机制：1)Ghrelin调节鱼类的胃肠运动；2)Ghrelin与胃中消化因子的关系；3)Ghrelin与其他食欲调节因子的关系。
4.1 Ghrelin调节鱼类的胃肠运动组织分布的研究发现Ghrelin在机体胃肠道中丰富表达，生物学功能的研究发现其参与动物的摄食调控。此外，Ghrelin与胃动素蛋白结构相似性高[34]，有学者提出Ghrelin通过影响动物的胃肠运动来调节摄食。在小鼠上的研究显示，GHS-R敲除后胃排空下降，而中枢和外周注射Ghrelin可通过GHS-R促进机体胃排空，增加胃肠动力。在鱼类上关于Ghrelin对胃肠运动影响的研究较少，还未见Ghrelin与胃排空关系的报道。Olsson等[46]以斑马鱼为研究对象，通过力量位移传感器记录了随Ghrelin浓度的增加升高了肠道紧张性收缩频率。而Kitazawa等[47]发现，在虹鳟上Ghrelin不能引起胃和肠道明显收缩，在金鱼上Ghrelin可引起肠道的小幅度收缩，但效果不明显。Ghrelin对鱼类胃肠运动的调节还有待进一步探究。
4.2 Ghrelin与胃中消化因子的关系Ghrelin主要在胃肠道分泌，胃肠道是动物体消化的主要场所。因此，Ghrelin与动物胃中消化因子(胃酸和胃消化酶等)可能存在联系。Masuda等[48]发现给大鼠静脉注射Ghrelin可增加胃酸的分泌。类似的，Date等[49]报道给大鼠脑室注射Ghrelin也呈现剂量依赖性的增加胃酸的分泌。不同的是，de la Cour等[50]发现酰化Ghrelin或去酰化Ghrelin均不能改变大鼠胃G细胞对胃酸分泌。Ghrelin对动物胃酸分泌的调节作用还有待进一步研究。此外在，哺乳动物中，适量的Ghrelin能够提高胃蛋白酶及肝脂肪酶活性。Du等[51]报道，用Ghrelin处理胃黏膜细胞4 h，胃蛋白酶活性无显著性变化。而杜改梅等[52-53]用1×10-3 μmol/L的Ghrelin处理大鼠胃黏膜细胞时显著增加了胃蛋白酶活性，进一步对大鼠左侧腿部肌肉注射重组Ghrelin，发现胃蛋白酶的活性显著提高。Nieminen等[54]对田鼠腹腔注射Ghrelin，持续注射4 d后肝脏中脂肪酶活性极显著提高。目前未见Ghrelin对动物淀粉酶活性影响的研究，在鱼类上关于Ghrelin与消化酶关系的研究也尚属空白。
4.3 Ghrelin与其他食欲调节因子的互作Ghrelin主要在动物的胃肠道和中枢神经系统中下丘脑核团中表达量丰富。作为脑肠肽，Ghrelin可和下丘脑核团产生的多种食欲调节因子[神经肽Y(NPY)/刺鼠相关蛋白(AgRP)、阿黑皮素原(POMC)/可卡因-苯丙胺调节转录肽(CART)、促皮质激素释放激素(CRF)、增食欲素(Orexin)和哺乳动物雷帕霉素靶蛋白(mTOR)]以及外周或神经内分泌系统的肽类激素[瘦素(Leptin)、Nesfatin-1、GH和催乳素(PRL)]互作调节摄食(图 2)，其中NPY/AgRP、Orexin和GH促进动物摄食，POMC/CART、CRF、mTOR、Leptin、Nesfatin-1和PRL抑制动物摄食。
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Ghrelin：胃饥饿素或生长激素释放肽；GHS-R生长激素释放肽受体；POMC：阿黑皮素原；NPY：神经肽Y；CRF：促皮质激素释放激素；Orexin：增食欲素；mTOR：哺乳动物雷帕霉素靶蛋白；Leptin：瘦素；GH：生长激素；PRL：催乳素；Food itake：摄食量。下调箭头：抑制摄食；上调箭头：促进摄食；黑色字体：Ghrelin促进相关基因表达；白色字体：研究结果不一致或没有直接研究。 Ghrelin:growth-hormone-releasing peptide; GHS-R:growth hormone releasing peptide receptor; POMC:proopi-omelanocortin; NPY:neuropeptideY; CRF:corticotropin-releasing factor; mTOR:mammalian target of rapamycin; GH:growth hormone; PRL:prolactin.Down arrow:inhibiting food intake; up arrow:promoting food intake; blake font:Ghrelin promotes related gene expressions; white font:inconsistent research results or no direct study. 图 2 Ghrelin调控摄食机制 Figure 2 The mechanisms of Ghrelin regulation on feeding |
Ghrelin可能通过作用于NPY/AgRP和POMC/CART信号通路调节动物摄食。Asakawa等[34]在小鼠上的研究显示Ghrelin可通过作用于中枢系统的NPY及其受体Y1促进小鼠摄食。Miura等[55]以金鱼为研究对象，发现脑室或腹腔注射Ghrelin使摄食量显著增加，NPY mRNA表达量显著上升；当Ghrelin与Y1受体拮抗剂BIBP3226共注射后，摄食量显著下降。Gao等[56]研究显示，投喂含Ghrelin的饲粮8周，石斑鱼(Epinephelus coioides)脑NPY mRNA的表达量显著增加。Ariyasu等[57]发现，循环系统Ghrelin下降的小鼠，摄食量及下丘脑中神经肽(NPY/AgRP和POMC)的表达量未发生显著改变。Qi等[58]发现，长期弓状核Ghrelin过表达的小鼠前3周摄食量和体重显著增加，第4周开始摄食量无显著变化，但是体重仍显著增加，Ghrelin过表达6周后弓状核中NPY表达量无显著改变，而POMC表达量极显著升高，这可能是机体通过上调POMC的表达来补偿Ghrelin对摄食引起的刺激作用。Velasco等[59]研究发现，给虹鳟脑室注射Ghrelin显著降低下丘脑中POMC、CART表达量，显著增加下丘脑中NPY、AgRP表达量。Ghrelin通过NPY/AgRP和POMC/CART信号通路调节动物摄食的作用机制还需进一步探究。
4.3.2 Ghrelin与CRFGhrelin可能通过CRF信号通路调节动物的摄食。Asakawa等[60]给小鼠腹腔注射Ghrelin，下丘脑中CRF mRNA表达量显著增加。JÖnsson等[45]报道，给虹鳟幼鱼脑室注射Ghrelin，其摄食量显著降低，共注射Ghrelin与CRF受体拮抗剂ahCRF后，其摄食量得到恢复。然而，关于Ghrelin通过CRF信号通路调节动物摄食的研究资料很少，需要加大这方面的研究。
4.3.3 Ghrelin与Orexin、mTORGhrelin与Orexin和mTOR信号通路互作调节动物摄食。Toshinai等[61]报道，通过Ghrelin诱导小鼠Orexin神经元的免疫活性，单独注射抗-Orexin引起摄食量显著下降，而与Ghrelin共注射后，摄食量显著低于Ghrelin单独注射组，但仍显著高于对照组，这说明抗-Orexin可以一定程度减弱Ghrelin引起的摄食量增加。Miura等[62]给金鱼脑室注射Orexin受体拮抗剂SB334867后摄食量下降，与Ghrelin共同注射后摄食量恢复到正常水平；此外，脑室注射Ghrelin后金鱼间脑中Orexin mRNA表达量显著增加。Penney等[63]以洞穴鱼为研究对象，发现腹腔注射Ghrelin 30 min内摄食量显著升高，全脑中mTOR和Orexin的表达量均显著升高。因此，中枢和外周注射Ghrelin均会提高Orexin和mTOR的表达量，但外周注射Ghrelin是否是通过反馈作用调节还需深入研究。
4.3.4 Ghrelin与Leptin、Nesfatin-1关于Ghrelin与外周组织中食欲调节因子相互关系的研究报道很少。Ghrelin受体和Leptin受体在小鼠弓状核中超过90%的神经元共表达[64]，在哺乳动物和鱼类上都发现Leptin系统与Ghrelin系统在调节摄食和能量代谢等生理作用上存在拮抗效应[65-68]。一些研究报道了Leptin可改变AgRP和NPY等食欲调节因子的表达量，与Ghrelin引起的变化模式相反[69-70]。此外，Ghrelin注射显著增加了Leptin缺失型小鼠的摄食量，而当Ghrelin与Leptin共注射后摄食量恢复到对照组水平[71]。Kohno等[72]的研究也显示注射Leptin能够抵抗Ghrelin引起的小鼠摄食量增加，二者共注射后摄食量与对照组无显著差异。Toshinai等[25]发现小鼠腹腔注射Leptin显著增加胃中Ghrelin mRNA的表达量。另外，Shimizu等[73]发现给小鼠腹腔注射Nesfatin-1显著抑制其摄食，而Stengel等[74]运用共聚焦显微镜研究发现Ghrelin与Nesfatin-1前体核酸结合蛋白2(NUCB2)共定位于大鼠的胃X/A样细胞中。因此，Ghrelin可能与Leptin、Nesfatin-1等外周食欲调节因子相互作用，但在鱼类上是否共同作用发挥调节摄食的功能及作用途径还不清楚。
4.3.5 Ghrelin与GH、PRL注射Ghrelin后可增加循环系统中与摄食相关的GH、PRL等激素的水平。Date等[75]报道大鼠脑室注射Ghrelin可增加血浆中GH水平。Kaiya等[18]在日本鳗鲡上的研究发现离体培养的垂体细胞用Ghrelin(0.1、1.0、10.0 nmol/L)处理，高剂量显著增加GH的释放量，并且各剂量均显著增加PRL的释放量。Riley等[76]21 d连续给莫桑比克罗非鱼腹腔注射Ghrelin，可显著增加垂体中GH mRNA的表达量。Shepherd等[38]也报道给虹鳟腹腔注射Ghrelin显著增加了血浆中GH的水平。因此，Ghrelin可能通过影响GH和PRL的分泌调节动物摄食。
综上，将Ghrelin与其他食欲因子的关系总结如下：1)Ghrelin可以作用于中枢神经系统中的NPY/AgRP和POMC/CART、CRF、Orexin和mTOR等信号通路调节动物摄食；2)Ghrelin可能与外周组织中的Leptin、Nesfatin-1等食欲因子互作；3)Ghrelin能够增加循环系统中GH、PRL等激素的水平。
5 小结Ghrelin作为一种脑肠肽，在动物中枢系统的脑组织及外周系统的胃中大量表达，是中枢和外周摄食调控系统中重要的增食欲因子。研究表明禁食后哺乳动物和鱼类Ghrelin表达量显著提高，中枢或外周注射Ghrelin可促进动物的摄食量。目前，有关Ghrelin摄食调控的探究主要集中在哺乳动物的人和大鼠上，在鱼类上主要集中在鲤科，其他鱼类的相关研究资料十分有限。鉴于Ghrelin作为在鱼类摄食相关领域中的研究热点，其摄食调控以及作用机制的研究不够深入，未来应在借鉴哺乳动物研究结果的基础上，深入探讨Ghrelin对不同鱼类的摄食调控机制，为鱼类摄食调控和生产应用提供理论依据。
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