鱼类神经肽Y基因及其食欲调控机理研究

下丘脑的神经肽Y(Neuropeptide Y，NPY)是1982年首次从猪脑中分离出来的一种含36个氨基酸的单链多肽，属胰多肽家族。NPY是促进脊椎动物采食量的一个最强有力的诱导因子，所研究过的脊椎动物各物种的NPY存在高度同源性。与哺乳动物相似，鱼类NPY的结合区域也主要位于食欲调节区域。NPY在鱼类摄食活动中也被证实发挥了重要的食欲促进作用。3种鱼类NPY受体在斑马鱼中被成功克隆，为鱼类NPY受体功能及其作用机理研究奠定了基础。     

鱼类肽YY与胰肽Y的研究进展

酪酪肽与胰肽Y同属于胰多肽家族,是广泛存在的多功能调节肽;胰肽Y是酪酪肽基因组复制产生的鱼类中特有的神经肽。目前已在多种鱼类中分离得到酪酪肽与胰肽Y的基因,与特定受体发挥生理功能,包括使机体产生饱腹感而食欲下降,减少摄食,降低体质量,在调控养殖鱼类摄食,提高饲料转化率,促进鱼类健康养殖以及消化道、肝胰腺、肥胖等治疗方面发挥重要作用。本文通过概述鱼类中酪酪肽与胰肽Y的结构特征、体内分布、作用的形式及受体、影响因素等生物学功能,为鱼类肽YY及胰肽Y的研究提供参考。     

Apelin与鱼类摄食

鱼类通过摄食活动获得能量和营养物质，而食欲是影响鱼类摄食的重要因素之一。食欲调控是一个复杂的网络，大脑整合机体能量和摄食信号，通过食欲调节因子（包括促进食欲因子和抑制食欲因子）调控鱼类摄食行为。Apelin作为一种新型内分泌因子，在体内发挥广泛的生物学作用。其中，apelin在摄食调节方面的作用正在引起学者的关注。本文以哺乳动物为参照，综述了apelin的组织分布、饥饿和营养物质对apelin表达的影响、apelin处理对鱼类摄食的影响及其可能的机制和信号通路，旨在为促进鱼类摄食和水产养殖提供参考。     

团头鲂AGRP和NPY基因cDNA克隆及其在饥饿与再摄食状态下的表达分析

为研究刺鼠相关蛋白（AGRP）和神经肽Y（NPY）基因在团头鲂幼鱼应答饥饿与再摄食过程中所起的调控作用，本研究采用RACE PCR技术首次克隆了团头鲂AGRP和NPY基因全长cDNA序列，通过设置正常对照组（control，体质量3%持续投喂4周）、饥饿再投喂组（F/refeeding，饥饿2周+3%再投喂2周）、饥饿过量投喂组（F/excessive refeeding，饥饿2周+8%再投喂2周）和饥饿组（fasted，持续饥饿4周）4个处理组，采用qRT-PCR技术分析团头鲂在脑和肠道组织中这两种基因在饥饿再投喂过程中的表达变化特征。结果显示，（1）组织学分析表明，持续饥饿组肠道黏膜下层出现明显的白细胞浸润现象。（2）团头鲂AGRP和NPY基因cDNA序列全长分别为770 和557 bp，其中有5’非编码区77和101 bp以及3’非编码区315和120 bp，开放阅读框为378和336 bp，共编码了125个氨基酸和111个氨基酸。系统进化树分析表明，团头鲂的AGRP和NPY基因分别与其他鲤科鱼类聚类一支，具有最近的亲缘关系。（3）qRT-PCR分析显示，AGRP和NPY基因在所有检测组织中均有表达，二者均在脑组织中表达量最高，其次是肝脏和肠道。（4）饥饿再投喂处理对团头鲂脑和肠道中AGRP和NPY基因表达影响显著，饥饿组团头鲂脑和肠道组织AGRP基因表达量均呈先升后降趋势，第28天，饥饿再投喂组能够显著上调团头鲂AGRP基因在脑组织中的表达，但饥饿过量投喂组能够显著上调AGRP基因在肠道中表达量。持续饥饿组团头鲂脑和肠道组织NPY基因表达呈先上升后下降趋势，且饥饿组脑和肠道组织中NPY表达量在28 d均显著高于其余3组。上述结果表明，AGRP和NPY基因在团头鲂摄食调控中发挥重要作用，且在脑和肠道组织具有不同表达模式。     

黄条鰤 npy基因克隆及其对饥饿再投喂的应答特性

为探究神经肽Y (neuropeptide Y, NPY)在黄条鰤 (Seriola aureovittata)摄食调控中的作用及机制,本研究采用同源克隆的方法获得了黄条鰤 npy基因的开放阅读框(ORF)序列,并利用实时荧光定量PCR技术分析了npy基因的组织分布以及其对饥饿再投喂的应答特性。黄条鰤 npy基因ORF序列长度为300 bp,编码99个氨基酸的前体蛋白,其中包括28个氨基酸的信号肽、36个氨基酸的成熟肽。氨基酸序列同源性比对发现,黄条鰤 npy编码的氨基酸序列与斑马鱼(Danio rerio)等其他硬骨鱼高度保守(>90%);系统进化树分析表明,黄条鰤 npy与高体鰤 (Seriola dumerili)的关系最近。npy mRNA在所检测的12种组织中均有表达,其中,在脑组织表达量最高,在垂体和胃中表达量次之。在饥饿再投喂实验中,饥饿刺激了npy mRNA的表达,特别是饥饿21 d时,实验组垂体npy mRNA表达量显著高于对照组,再投喂7 d后恢复到对照组水平。上述结果表明,npy可能参与了黄条鰤的摄食调控,在饥饿代谢补偿机制中发挥了重要作用。     

鱼类下丘脑增食欲素(Orexin)研究进展

鱼体内的物质、能量代谢既与摄入食物、环境因素及运动强度有关,也与内分泌调控因子的参与密切相关。Orexin是新近发现的一种能够调节机体摄食和能量代谢的下丘脑神经多肽物质。目前在人类和其他哺乳动物上的研究越来越多,而在海洋鱼类上的研究则比较少见。本文首先综述了鱼类Orexin的基因、结构、组织分布与生理功能等方面的最新研究成果,初步阐明了Orexin在鱼类摄食、营养、生活节律等生理学基础领域中的作用,可为今后鱼类养殖领域开展该项目的应用性研究奠定基础。     

长牡蛎中I84蛋白酶抑制因子家族的扩张和功能分化

蛋白酶抑制因子是极其多样的蛋白质或多肽,能抑制蛋白酶的水解活性。研究发现蛋白酶抑制因子能通过抑制病原蛋白酶活性,从而抑制病原的入侵。I84蛋白酶抑制因子家族是MEROPS数据库中新增的一个家族,其部分成员在免疫防御过程中的作用得到了一定的揭示。为探究I84蛋白酶抑制因子家族在长牡蛎中的分布和功能状况,实验鉴定了长牡蛎中23个潜在的I84家族基因,根据系统进化树分析,挑选了5个同源基因进行时空表达和功能分析。首先,利用克隆技术验证了长牡蛎中5个I84家族同源基因CgSi3、CgSi5、CgSi6、CgSi16和CgSi19可表达性。结果显示,5个基因在外套膜、闭壳肌、性腺、血淋巴细胞、肝胰腺和鳃等6个组织中均表达,但肝胰腺中表达量显著高于其他组织。同时,5个基因在长牡蛎幼体不同发育阶段表达模式不同,其中各基因在受精卵时期均不表达,CgSi3表达量则在眼点幼虫期显著上升后下降,而CgSi6在壳顶幼虫期开始表达后,表达量随长牡蛎发育而持续增加。另外,对牡蛎进行病原相关模式分子(PAMPs)注射后,5个基因也表现出不同的表达模式,其中LPS和PGN注射后CgSi6表达量变化明显,而poly (...     

锯缘青蟹脑内5-HT和NPY的免疫细胞化学定位

为探讨5-羟色胺和神经肽Y在锯缘青蟹脑内的存在与否和分布状况，应用免疫细胞化学技术，在光学显微镜下观察5-羟色胺和神经肽Y阳性细胞和神经髓质的形态和分布。结果表明：在锯缘青蟹脑中共12个胞体群和11个神经髓质中，前脑和中脑中有4个细胞群和6个神经髓质检出5-HT免疫阳性反应；中脑和后脑中有7个细胞群和3个神经髓质具有NPY免疫阳性反应。5-羟色胺和神经肽Y在锯缘青蟹脑内的特异性分布，为其参与神经牛理活动提供了形态学证据。     

鱼类生长抑素调控垂体生长激素分泌的作用机制

生长抑素是一个多基因、多功能的家族。通过其受体的介导参与机体的生长、发育、代谢、生殖以及免疫等生理过程。本文简要概括鱼类生长抑素及其受体的研究进展,重点对生长抑素调控垂体生长激素分泌的信号转导机制进行概括讨论,旨在加深对鱼类生长抑素作用机制的认识和了解。迄今,在鱼类中已经鉴定出六种生长抑素基因和四种生长抑素受体。由于存在多种生长抑素基因以及不同的加工过程,一种鱼可能产生多种形式的生长抑素多肽。鱼类进化过程中存在基因组复制,导致一种受体又有多种亚型。鱼类生长抑素调控垂体生长激素分泌的作用机制主要源自金鱼中的研究,结果表明cAMP通路、钙离子通道以及PKC通路可能参与了金鱼生长抑素抑制垂体生长激素分泌的过程。生长抑素调控垂体生长激素分泌的作用机制是一个复杂的网络结构,多种信号通路参与其中;不同的物种间其作用机制不尽相同。鱼类生长抑素基因、受体及其调控垂体生长激素分泌的作用机制仍有待进一步研究。     

卵形鲳鲹Kiss1基因组结构特征及饵料类型对其表达的影响

Kiss基因编码的kisspeptin多肽是脊椎动物重要的神经激素,在调控机体生长发育、能量代谢和生殖活动中发挥重要作用。为了明确卵形鲳鲹Kiss1 (ToKiss1)基因序列及饵料类型对其表达的影响,实验利用RACE方法克隆分析了ToKiss1基因结构特征,荧光定量PCR (qRT-PCR)方法研究了饵料类型对ToKiss1 mRNA表达调控的影响。结果显示,ToKiss1基因全长2 768 bp,由3个外显子和2个内含子组成。ToKiss1基因cDNA全长505 bp,开放阅读框312 bp,编码104个氨基酸,包括信号肽序列和典型的kisspeptin-10结构域"YNLNSFGLRY"。卵形鲳鲹ToKiss1蛋白三级结构由2个α螺旋和无规则卷曲构成。qRTPCR表达分析结果显示,ToKiss1 mRNA在卵形鲳鲹脑、肠道、胃、脾脏、肌肉和心脏中均有表达,在脑中的表达量最高。饵料类型显著影响ToKiss1 mRNA在卵形鲳鲹肠道组织中的表达,颗粒饲料组表达量最高,其次为冰杂鱼组,冰鱿鱼组表达量最低,但饵料类型对肝脏组织中ToKiss1 mRNA的表达无显著影响,表明ToKiss1在卵形鲳鲹消化吸收过程中发挥了重要调控作用。该研究首次探讨了饵料类型对硬骨鱼肝脏和肠道组织中Kiss1 mRNA表达的影响,为深入研究硬骨鱼类Kiss1基因摄食调控生理机制奠定了基础。     

鱼类促性腺激素抑制激素及其受体的研究进展

促性腺激素抑制激素是2000年由日本学者首次从鹌鹑脑中分离出的一种新型下丘脑神经肽,通过其受体介导参与机体的生长、生殖以及摄食等生理过程。迄今,只在金鱼、斑马鱼、星点东方鲀、罗非鱼以及斜带石斑鱼等几种鱼中鉴定出促性腺激素抑制激素。目前,鱼类促性腺激素抑制激素的生理学功能研究相对较少,且存在争议。鱼类促性腺激素抑制激素及其受体的表达调控以及其他生理学功能仍有待进一步研究。本研究简要总结鱼类促性腺激素抑制激素及其受体的研究进展,并对促性腺激素抑制激素的生理学功能进行概括讨论,旨在加深对鱼类促性腺激素抑制激素的认识和了解,为进一步研究做铺垫。     

Identification,characterization and feeding response of peptide YYb (PYYb) gene in grass carp (Ctenopharyngodon idellus)

The peptide YYb (PYYb) is a fish-specific peptide belonging to the neuropeptide Y (NPY) family. In the present study, the full-length cDNA sequence and genomic structure of PYYb (gcPYYb) from Ctenopharyngodon idellus have been isolated and characterized. The gcPYYb gene consists of three exons interspaced by two introns, opposing to the typical architecture of most NPY-family genes as well as its paralogs. Alignment of deduced amino acid sequence indicates that the fish PYYb is more variable compared to NPY and PYY as shown by more residue changes in teleosts lineage, suggesting mild selective pressure imposed on the peptide. Real-time quantitative PCR analysis shows the gcPYYb mRNA in developing larvae is increased during the mixed endo- and exogenous-feeding period and is widely distributed throughout the intestine of fully grown individuals. Following a single meal, the gcPYYb mRNA in foregut is increased at 3 h post-feeding and subsequently decreased before the foregut contents are cleared. These results suggest that the gcPYYb has an important role in the early life stages of grass carp and is involved in food intake by transmitting feeding-related signals.     

Role of NPY receptor 8 in regulating of food intake in Chinese perch (Siniperca chuatsi)

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor, whose role in feeding regulation remains poorly understood. In the present study, two subtypes of NPY8R (NPY8Ra and NPY8Rb) were identified in Chinese perch, and their amino acid sequences were highly conserved in fish. The predicted tertiary protein structures of human NPY1R, Chinese perch NPY8Ra, and NPY8Rb tended to be conserved as a whole. However, the predicted tertiary protein structures of sc-NPY8Ra and sc-NPY8Rb were different at several sites. Chinese perch NPY8Ra and NPY8Rb were mainly expressed in brain. Intracerebroventricular administration of siRNA-NPY8Rb significantly decreased the mRNA expression levels of NPY8Rb and POMC (appetite-suppressive gene) and significantly increased the food intake of Chinese perch at 2 h and 8 h after injection. Similar result has not been observed in the siRNA-NPY8Ra administration group. The above results suggested that NPY8Ra might not play a role in appetite regulation and NPY8Rb might be an effector in the negative feedback regulation of feeding in Chinese perch.

     

Characterization,cerebral distribution and gonadotropin release activity of neuropeptide Y (NPY) in the goldfish

The presence of a peptide closely related to porcine NPY has been demonstrated in the goldfish brain and pituitary by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). The RIA data demonstrate that displacement curves of brain extracts are parallel to a porcine NPY standard and that in HPLC a compound present in brain extracts is co-eluted with porcine NPY. The distribution of this NPY-like factor within the central nervous system was studied by radioimmunoassay and immunohistochemistry. The results indicated that NPY has a widespread distribution with the highest concentrations being found in the telencephalon and diencephalon. In the pituitary gland, NPY immunoreactive terminals characterized at the electron microscope level were found in the different lobes and, in particular, in close association with the gonadotrophin (GTH) secreting cells. Using anin vitro perifusion system, it was shown that NPY causes a dose dependent increase of GTH release from anterior lobe fragments. These data indicate for the first time in teleosts that NPY is present and widely distributed in the brain and pituitary, and that among other putative functions, could be implicated in the multihormonal release of GTH from the pituitary.     

Cloning,expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper,Epinephelus coioides

As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp^6.59 might be a common NPY-binding site, while Asp^2.68 of the Y8b receptor and Glu^5.24 of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.     

Endogenous opioids

The discovery of the opioid receptors and their corresponding ligands, the opioid peptides, is of fundamental importance as regards our understanding of a variety of functional mechanisms in the central and peripheral nervous system. This review considers the distribution of the multiple opioid peptides within the organism as well as their tissue-specific enzymatic processing. These parameters differ considerably between species. The multiple opioid peptides are paralleled by multiple opioid receptors. These opioid systems affect a broad spectrum of functions, such as behaviour, pain perception, the cardiovascular system, respiration, appetite, gut motility and secretion, water- and electrolyte balance and the complex field of endocrine mechanisms. The task of future pharmacological research is to study these functions by means of more selective opioid agonists and antagonists. An awareness of our present knowledge and of probable findings to come suggests novel therapeutic possibilities in the field of veterinary science.     

Role of neuropeptide Y (NPY) in the regulation of reproduction: study based on catfish model

Significance of NPY in the regulation of GnRH–LH axis was evaluated. Considerable NPY immunoreactivity was seen in the components like olfactory system, basal telencephalon, preoptic and tuberal areas, and the pituitary gland that serve as neuroanatomical substrates for processing reproductive information. Close anatomical association as well as colocalizations of NPY and GnRH were seen in the olfactory receptor neurons, olfactory nerve fibers and their terminals in the glomeruli, ganglion cells of nervus terminalis, medial olfactory tracts, fibers in the ventral telencephalon and pituitary. In the pituitary, NPY fibers seem to innervate the GnRH as well as LH cells. Intracranial administration of NPY resulted in significant increase in the GnRH immunoreactivity in all the components of the olfactory system. In the pituitary, NPY augmented the population of GnRH fibers and LH cells. HPLC analysis showed that salmon GnRH content in the olfactory organ, bulb, preoptic area+telencephalon and pituitary was also significantly increased following NPY treatment. NPY may play a role in positive regulation of GnRH throughout the neuraxis and also up-regulate the LH cells in the pituitary.