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doi: 10.7541/2017.162EFFECTS OF γ-AMINOBUTYRIC ACID ON FOOD INTAKE AND APPETITEIN MANDARIN FISH SINIPERCA CHUATSIHUANG Dong1, 2, LIANG Xu-Fang1, 2, YUAN Xiao-Chen1, 2, CAI Wen-Jing1, 2, LI Ai-Xuan1, 2,HE Shan1, 2 and XUE Min3(1. College of Fisheries, Chinese Perch Research Center, Huazhong Agricultural University, Wuhan 430070, China; 2. Freshwa-ter Aquaculture, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Wuhan 430070, China; 3. National Aquafeed Safety Assessment Station, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China)Abstract: γ-Aminobutyric acid (GABA) has been established as an important regulator of food intake in mammals, but its role in fish remains scant. The aim of this study was to investigate the effects of GABA on food intake in mandarin fish (Siniperca chuatsi). Fish received intracerebroventricular (ICV) injection of sa-line and different doses of GABA (50, 125, 500 and 2000 μg). Food intake increased significantly in 125 μg GABA group within 2h. The RT-qPCR data demonstrated that up-regulation of NPY and AgRP, and down-regulation of CART and POMC were consistent with higher food intake of mandarin fish. The mRNA expres-sion of Leptin-R decreased significantly at all the four doses of GABA at 0.5h and 2h compared to the saline injected fish. These results indicated that GABA could affect the appetite by regulating leptin signaling path-way and thus resulting in alteration of food intake. Our findings could provide theory basis for GABA applica-tion in aquaculture feeds.Key words: γ-Aminobutyric acid; Mandarin fish; Food intake; Gene expression; Leptin signaling path-wayCLC number: S965.1 Document code: A Article ID: 1000-3207(2017)06-1311-07γ-Aminobutyric acid (GABA), a non-protein amino acid, distributes widely in nature and serves as a major inhibitory neurotransmitter in the central nervous system[1, 2]. It has been implicated in several centrally mediated physiological functions such as neurotransmission[3], blood pressure regulator[4], hor-monal regulator[5], relaxation regulator[6] and sleep-lessness and depression regulator[7]. Moreover, it has been established as an important regulator of food in-take in mammals[8—10]. Many previous studies were carried out by using commercial GABA, GABA recep-tor agonist or GABA receptor antagonist in mamma-lian species. It was reported that injection of GABA into the ventromedial hypothalamus of satiated rats in-duced further consumption of food. Similar effects were observed following injections of the agonist or antagonists of GABA into the hypothalamus[2, 11—15]. Likewise, GABA is also involved in the regulation of a number of physiological functions in fish. It was found that injecting GABA intraperitoneally caused an increase of serum GTH levels in regressed or early maturing fish, rather in late maturing animals[16]. An ICV injection of muscimol caused an acute and dose dependent increase in locomotor activity of juvenile spring chinook salmon[17]. Interactions in goldfish were found between feeding behaviors and ORXR-β-GABA A R subunit[18]. However, effects of GABA on food intake of teleost remain largely unknown. There-fore, further studies are needed to elucidate the poten-tial role of GABA on food intake of fish.第 41 卷第 6 期水生生物学报Vol.41, No.6 2017 年 11 月ACTA HYDROBIOLOGICA SINICA N o v.,2017Received date: 2016-11-24; Accepted date: 2017-04-27Foundation item: Supported by the National Basic Research Program of China (2014CB138601); the Fundamental Research Funds for the Central Universities (2015PY041)Brief introduction of author: Huang Dong (1991—), Male, born in Shanxi; Master student; major in molecular nutrition of fish. E-mail: 573009018@Corresponding author: Liang Xu-Fang, E-mail: xufang_liang@As is known to us, the regulation of appetite which controls food intake is a complex phenomenon involving interactions between the brain and periphe-ral signals. A few peptides are now recognized to play an important role in the regulation of appetite, which exist in the same neuron or may have interactions with GABA. Neuropeptide Y (NPY) neurons coex-pressing GABA and agouti-related protein (AgRP) stimulate appetite[19]. In addition, NPY, AgRP, and GABA released in the ARC suppress the restraint of proopiomelanocortin (POMC) neurons coexpressing α-melanocyte stimulating hormone (α-MSH) and co-caine and amphetamine regulated transcript (CART) on appetite[19]. Leptin, a product of the obese (ob) gene, bindings with leptin receptor (Leptin-R) and acts directly on the hypothalamus to inhibit nore-pinephrine efflux (NE) efflux through GABA[20]. Al-though information about the feeding regulation mechanism of these peptides is increasing, the intera-ctions of GABA with them on food intake of fish are still limited.Mandarin fish (Siniperca chuatsi), a demersal piscivore, is one of the most valuable food fish native to the freshwaters of China. It is one of the chief eco-nomic edible fish in China and in other countries due to its large size, fast growth and delicious flesh[21, 22]. In addition, it has distinct food preference. In the wild, it feed solely on live fry of other fish species once the fry start feeding[23].In the present study, we investigated food intake and mRNA expression profiles of five representative appetite-related genes in the brain of mandarin fish after GABA administration. This study will provide a better understanding of GABA in mediating food in-take of fish.1 Materials and methods1.1 Ethics statementThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Vete-rinary and Quarantine Service. The protocol was ap-proved by the Committee on the Ethics of Animal Ex-periments of the Huazhong Agricultural University.1.2 AnimalsThe mandarin fish were obtained from Wuhan Freshwater Fish Farm (Wuhan, China). Prior to the initiation of the experiment, 100 mandarin fish were carried out in 2 cylindrical plastic tanks (350 L) and were kept under controlled light-dark conditions (12 Light /12 Dark) with a constant flow of filtered water and the water temperature was regulated to 23—25℃. The fish were fed with (2% of bodyweight) Cirrhina mrigola (initial body weight: 0.16±0.02 g) (Wuhan, China) at 8:00 am and 17:00 pm every day. Animals were acclimated to these conditions for at least 2 weeks before the experiment.1.3 Drug and treatmentThe GABA was purchased from Sigma Bio-chemicals, Dorset, UK. The drug was dissolved in teleost saline (20 mg Na2CO3 per 100 mL of 0.6% NaCl). Doses of drug were selected based on previ-ous studies[24—26], which were chosen for the follow-ing experiments. The ICV injections were carried out using a 0.5 mm Microlance needle connected to a 5 μL Hamilton microsyringe. The needle was performed freehand through the central junction between the frontal and parietal bones. The procedure of ICV in-jection and accuracy of injection placement into the ventricular regions of the fish brain followed the method of De Pedro et al[27]. After injection, the wounds were covered immediately with vaseline.1.4 Effects of ICV administration of GABA on food intakeAfter the acclimation period, 30 food-deprived (24h) fish were anesthetized with MS-222 (1鲶10000) and weighted when they lost equilibrium, which were divided into five groups that received ICV injection, respectively. GABA at 50 μg, 125 μg, 500 μg and 2000 μg, and one control group, which received ICV injection of the same volume of teleost saline (5 μL). The fish were habituated in anaesthetic-free water within 1—2min to help them recovered equilibrium and normal swimming activity after injection. Every mandarin fish was then transferred to a aquaria which contained enough pre-weighted prey fish (average body weight: 0.16 g) immediately. The food intake was measured by directly observing and recording the number of prey fish eaten by individual mandarin fish after 0.5h, 2h and 4h. The formula is as follows: F I=W i–(W f×F) where W i=initial food weight, W f=re-maining food weight and F=correction factor[27].1.5 Effects of ICV administration of GABA on gene expressionIn order to examine effects of GABA on brain NPY, AgRP, CART, POMC and Leptin-R mRNA ex-pressions, another experiment was performed using the uninjectd stock of mandarin fish, which received the same treatment without giving prey fish. Fish were then sampled at 0.5h and 2h after injection without feeding. Brains of both groups were dissec-ted and immediately snap-frozen in liquid nitrogen and stored at –80℃ for RNA isolation and sub-sequent analyses.Total RNA was extracted using Trizol reagent1312水生生物学报41 卷(TaKaRa, Japan), treated with RQI RNase-Free DNase (TaKaRa, Japan) to remove DNA contaminant, and then spectrophotometrically quantified. For each re-verse transcription reaction, RNA was subjected to cDNA synthesis by SuperScript TM II RT reverse tran-scriptase (TaKaRa, Japan) according to reagent’s in-structions. The selected gene coding sequence were based on the results of transcriptome sequencing of mandarin fish which were mapped to the mandarin fish genome by the Short Oligonucleotide Analysis Package SOAP aligner/soap2[28]. Primers of the selec-ted genes for the real-time PCR (RT-qPCR) were de-signed in Tab. 1.RT-qPCR was performed by a quantitative thermal cycler (BIO-RAD CFX96, BIO-RAD, USA) using RPL13A, a housekeeping gene, as an endoge-nous reference to normalize the template amount. Each real-time PCR (in 20 μL) reaction contained 10 μL SYBR Green Realtime PCR Master Mixture (Toyobo, Japan), 0.4 μmol/L primers and 1 μL normalized tem-plate cDNA. The PCR parameters were 40 cycles at 95℃ for 15s, 60℃ for 15s, and 72℃ for 45s, with an additional initial 1-min denaturation at 95℃. The spe-cificity of PCR products were confirmed by melting curve analysis. Pooled cDNA samples of the respec-tive brain tissues were used to generate the calibra-tion curves. The amplification efficiencies of control and target genes were approximately equal and ranged from 94.2% to 101.7%. Gene expression levels were quantified relative to the expression of RPL13A using the optimized comparative Ct (2–ΔΔCt)value method[29]. All amplifications were performed in triplicate for each RNA sample.1.6 Statistical analysesAll data were presented as mean±SEM (standard error of the mean). Statistical analysis was performed by a one-way analysis of variance (one-way AN-OVA) using SPSS 19.0 with Duncan’s multiple com-parisons test. Statistical significance was determined at the 5% level.2 Results2.1 Effects of ICV injection of GABA on food in-take in mandarin fishThe significant increases in food intake were ob-served at 0.5h and 2h post-injection at the dose of 125 μg GABA compared to the control group (P< 0.05). The food intake at 4h post-injection showed no significant differences among tested dose points (P>0.05) (Fig. 1).2.2 Effects of ICV injection of GABA on gene ex-pressionThe expressions of selected genes representing feeding regulation were measured by RT-qPCR me-thod after GABA treatment. The mRNA levels of NPY in brain increased significantly at the dose of 125 μg at 0.5h and 2h after GABA injection com-pared to those of the control group (P<0.05). Injec-tion of GABA significantly promoted AgRP mRNA expressions at the dose of 125 μg at 0.5h, while the mRNA levels decreased significantly at all the four doses at 2h compared to the control group (P<0.05) (Fig. 2). The mRNA levels of the anorexigenic genesTab. 1 Primers used in this studyGene name Primer name Sequence (5′—3′)Amplicon length (bp)RPL13A RPL13A-F TATCCCCCCACCCTATGACA245RPL13A-R ACGCCCAAGGAGAGC GAACTAgRP AgRP-F GAGCCAAGCGAAGACCAGA151AgRP-R GCAGCACGGCAAATG AGAGNPY NPY-F GTTGAAGGAAAGCAC AGACA191NPY-R GCTCATAGAGGTAAA AGGGGCART CART-F CTGCTGTCCGTCATTT GTCAC171CART-R TGGGATGCTTCCTCTT TTCTCPOMC POMC-F GTGTCATCCTCGTTACTGC162POMC-R GCGACGCTCCTATTC AATLeptin-R LeptinR-F CTCTGAGAACATTTCC AAAGTCG100LeptinR-R GGCTGGGGCGGGAGTTAGFig. 1 Effects of ICV administration of GABA at the dose of 0,50, 125, 500 and 2000 μg on food intake before injection and 0.5h,2h and 4h after injectionData represent means±SE (n=6). Significant level is marked withdifferent letters (P<0.05), compared with the values from saline in-jected mandarin fish at each dose6 期黄东等: γ-氨基丁酸对鳜摄食和食欲的影响1313CART and POMC were also assessed. The results showed a marked decrease of CART mRNA levels at 0.5h and 2h by GABA administration (P <0.05), while the mRNA levels of POMC were decreased at 2h compared to those in the control group (P <0.05). ICV injection of GABA did not affect the expression levelof POMC compared to the control group at 0.5h (P >0.05) (Fig. 2).The mRNA abundance of Leptin-R in the brain decreased significantly at 0.5h and 2h after GABA injection compared to the control group (P <0.05)(Fig. 2).3 DiscussionThe present findings showed a clear effect of exogenous GABA on food intake and appetite in mandarin fish. The significant increases in food in-take were observed at 0.5h and 2h post-injection at the dose of 125 μg GABA compared to the control group, indicating that GABA of 125 μg had orexige-nic effects on fish. This result confirms the previous findings that systemic administration of GABA has ahyperphagic effect on mammals and birds [30, 31]. To our knowledge, this is the first report showing that ICV GABA can lead to a major increase in food con-sumption in fish. However, ICV injection of GABA at the dose of 500 and 2000 μg did not alter food in-take in 2h. One explanation is that high doses of GABA may have sedating or muscle relaxanteffects [32], which make the mandarin fish could not catch the prey fish. The other plausible explanation may be high doses of GABA may give birth to cer-tain toxicity [33], which may have negative impact on fish. Additionally, there was no significant change of food intake at 4h by GABA administration. This short-lived effect is similar to that observed in other species studied previously. Injections of GABA (0.97 nmol/L) into the ventromedial hypothalamus in-creased food intake for only 15min in satiated rats [34].And injections of muscimol into the paraventricular hypothalamus and ventronaedial increased food in-take during 1h or 2h post injection [35, 36]. The time of each experiment that the orexigenic effect lasted was not totally identical, maybe because of the different experimental species or the different GABA doses.Therefore, the effects of GABA may have a close re-lationship with dose and time.To examine how GABA exerted effects on food intake, we assessed the mRNA expressions of selec-ted genes involved in the regulation of food intake.The results in the present study showed that the ex-Fig. 2 The expressions of neuropeptide Y (NPY ), agouti-related protein (AgRP ), cocaine and amphetamine regulated transcript (CART ),proopiomelanocortin (POMC ) and leptin receptor (Leptin-R ) genes in the brain after ICV of GABA at the dose of 0, 50, 125, 500 and 2000 μg at 0.5h and 2hA. NPY ;B. AgRP ;C. CART ;D. POMC ;E. Leptin-R . Data represent means±SE (n =6). Significant levels are marked with different letters (P <0.05), compared with the values from saline injected mandarin fish at each time point1314水 生 生 物 学 报41 卷pression of NPY and AgRP were significantly in-creased at 0.5h post-injection at the dose of 125 μg GABA, indicating that GABA injection can regulate food intake through upregulating the expression of NPY and AgRP genes, which may be responsible for the ability of GABA to promote appetite[37]. However, the expression of AgRP decreased at 2h. The de-creased gene expression of AgRP might be due to an unclear interaction between GABA and AgRP in mandarin fish brain[38]. Therefore, further study about the regulation of AgRP expression mediated by GABA in fish satiety signaling should be considered. Besides, the down-regulated relative values of CART and POMC were consistent with the ability of GABA to promote appetite. CART acts as an anorexigenic factor in mammals[39], birds[39] and fish, including zebrafish, Danio rerio[40], goldfish, Carassius auratus[41, 42], red-bellied piranha, Pygocentrus natte-reri[43], common carp, Cyprinus carpio[44], Atlantic salmon, Salmo salar[45], cunner, Tautogolabrus ad-spersus[46] and channel catfish, Ictalurus punctatus[47]. POMC produces many biologically active peptides in the different hypothalamic and extra-hypothalamic sites involved with food intake, which could lead to subtle variations in the anorexic signal being transmit-ted[48]. Interestingly, there was no significant differ-ence among the expression of POMC at 0.5h. A plausible explanation of the expression of POMC at 0.5h could be that GABA could not directly inhibit the expression of POMC, but inhibit the pyrolysis product of POMC α-MSH[49] to promote appetite. It took time for POMC to get cracked to α-MSH to in-teract with GABA might be reason for the unchanged expression of POMC at 0.5h.On the other hand, ICV injection of GABA sig-nificantly decreased the expression the Leptin-R at all the four doses at 0.5h and 2h, indicating that GABA might have an effect on food intake by mediating the signaling pathway of leptin in fish. Recent studies have indicated that leptin has an anorexigenic role in fish and thus it is possibly involved in the regulation of growth and energy balance[50]. Indeed, the potent chemical signal leptin is shuttled across the blood brain barrier by a trunctuated form of the leptin re-ceptor[51] and leptin receptors have been found to co-express NPY/AGRP and POMC/CART neurons[52], which suggests that leptin affects food intake by simul-taneously inhibiting orexigenic factors and stimulat-ing anorexigenic factors. Our data showed that GABA may serve as a mediator of the leptin sup-pressive action on food intake. 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Endocrinology ,2012, 153(5): 2223—2233[53]Francis J, Mohankumar S M, Mohankumar P S. Leptin in-hibits norepinephrine efflux from the hypothalamus in vitro:role of gamma aminobutyric acid [J]. Brain Research , 2004,1021(2): 286—291[54]γ-氨基丁酸对鳜摄食和食欲的影响黄 东1, 2梁旭方1, 2袁小琛1, 2蔡文静1, 2李艾璇1, 2何 珊1, 2薛 敏3(1. 华中农业大学水产学院, 华中农业大学鳜鱼研究中心, 武汉 430070; 2. 淡水水产健康养殖湖北省协同创新中心, 农业部淡水生物繁育重点实验室, 武汉 430070; 3. 中国农业科学研究院饲料研究所, 国家水产饲料安全评价基地, 北京 100081)摘要: 为阐明γ-氨基丁酸(GABA)对鳜(Siniperca chuatsi )摄食和食欲的影响, 对鳜脑室注射生理盐水和不同剂量的GABA(50、125、500和2000 μg)。
（三）5G+云计算行业应用技能试题及答案1. 以消费者为导向的，以需定产的方式对立的是传统大规模量产的生产模式称为（） [单选题] *柔性制造(正确答案)定制制造批量制造工业制造2. 医养服务模式分为（）三种。
*养老机构康养服务(正确答案)医疗机构医疗服务(正确答案)居家照护指导及护理服务(正确答案)康养服务3. 外呼过程中，用户可以打断说话。
打断后也能快速回复问题。
[单选题] *正确(正确答案)错误4. 智慧交通解决方案是依托5G+（），搭建3张基础网络，研发1大核心平台，打造5个智慧交通核心场景 [单选题] *AICDE(正确答案)IDCICTDICT5. 全域旅游中AI客服可以提供（）在线服务。
[单选题] *5*24小时6*24小时7*24小时(正确答案)6. （）模式中边缘网络主要通过UPF下沉，构建贴近业务的边缘网络，降低传输时延，实现数据不出场。
[单选题] *共享优享专享(正确答案)尊享7. 5G港机远控业务对网络需求：单台设备上行（）以上，上行业务背景下ping时延均值（）以下。
[单选题] *20Mbps 18ms30Mbps 18ms(正确答案)30Mbps 25ms20Mbps 25ms8. 面向行业客户的5G专网的产品体系和商业模式中，（）是贯穿“优专尊”和“BAF”定制网络重要的一环。
[单选题] *DNNQosMEP切片(正确答案)9. 互联网手机进军营的功能不包括（） [单选题] *自带手机使用(正确答案)手机信息提取红包监测10. 智慧养猪管理系统-移小猪，帮助猪场实现养猪过程的（） [单选题] *可视化精准化(正确答案)全面化智能化11. 中国移动以5G、物联网、人工智能、大数据、（）为基础，打造“云”、“网”、“端”、“应用”结合的精准种植整体解决方案 [单选题] *3S技术(正确答案)4S技术5S技术2S技术12. 下列哪一个不属于智慧医院管理类-后勤管理应用功能？ [单选题] *耗材管理物流管理财务管理病人主索引(正确答案)13. 5G可以为教育行业用户解决的问题不包括（） [单选题] *教育均衡问题个性学习问题校园安全问题生源问题(正确答案)14. （）是端到端网络切片的纽带，用统一的ID标识贯通全程全网，实现一点订购、集中编排、统一管理与全网服务 [单选题] *Qos5QIRB网络切片标识(正确答案)15. 智能网联汽车，即ICV（全称IntelligentConnectedVehicle）,是指车联网与智能车的有机联合 [单选题] *正确(正确答案)错误16. 智慧城市政务一体化平台不包括以下哪些内容（） [单选题] *智慧政务监管/决策平台公共信息服务/办事平台5G机器人12345政务服务热线(正确答案)17. 以下不属于智慧航港落地停用的是（） [单选题] *智慧养护(正确答案)货运管理智慧闸口自动装卸18. 中国移动“1+4+N”高校产品体系中“4”是指4个智慧高校应用，其重点推广场景不包括（） [单选题] *5G教培实验室云实训智慧教室(正确答案)平安校园19. 重点聚焦局域类边缘业务，按需下沉边缘节点，满足垂直行业客户对（）的旺盛需求 [单选题] *下沉园区基站独享边缘计算(正确答案)共享UPF20. 在智慧医院-5G医疗云网融合中，中国移动在满足医疗业务、连接、计算、安全等可监管可控可感知的产品叫什么？ [单选题] *Onehealth移动边缘云移动态势感知(正确答案)移动医疗专网21. 利用边缘计算技术，可使用用户网元（）下沉到用户边缘，实现数据不出场安全隔离要求 [单选题] *AMFSMFUPF(正确答案)NEF22. 以下哪些属于交通“十四五”规划内容（） *加快推动发展数字产业化(正确答案)实现交通驾驶无人化推进现有产业的数据化转型(正确答案)构筑美好数字生化新图景(正确答案)23. 下面哪个客户是使用了移动云的电力客户 [单选题] *国家电投国家电网国投电力(正确答案)华电集团24. 视频点播服务的底层提供服务商是（） [单选题] *咪咕视频(正确答案)百度视频腾讯视频网易视频25. 交通行业场景包括（） *轨道交通(正确答案)铁路交通(正确答案)航空运输(正确答案)快递运输26. 校园物联装备管理现阶段存在的痛点有（） *设备利用率低(正确答案)维护成本高(正确答案)缺乏监管手段(正确答案)缺乏资金27. 端到端切片方案包括无线、传输、（）领域，需要跨领域协同。
