第 41 卷 第 6 期 水 生 生 物 学 报 Vol. 41, No. 6

2017 年 11 月 ACTA HYDROBIOLOGICA SINICA Nov., 2017 doi: 10.7541/2017.162

EFFECTS OF γ-AMINOBUTYRIC ACID ON FOOD INTAKE AND APPETITE IN MANDARIN FISH SINIPERCA CHUATSI

HUANG 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- way CLC number: S965.1 Document code: A Article ID: 1000-3207(2017)06-1311-07

γ-Aminobutyric acid (GABA), a non-protein were observed following injections of the agonist or amino acid, distributes widely in nature and serves as antagonists of GABA into the hypothalamus[2, 11—15]. a major inhibitory neurotransmitter in the central Likewise, GABA is also involved in the regulation of nervous system[1, 2]. It has been implicated in several a number of physiological functions in fish. It was centrally mediated physiological functions such as found that injecting GABA intraperitoneally caused neurotransmission[3], blood pressure regulator[4], hor- an increase of serum GTH levels in regressed or early monal regulator[5], relaxation regulator[6] and sleep- maturing fish, rather in late maturing animals[16]. An lessness and depression regulator[7]. Moreover, it has ICV injection of caused an acute and dose been established as an important regulator of food in- dependent increase in locomotor activity of juvenile take in mammals[8—10]. Many previous studies were spring chinook salmon[17]. Interactions in goldfish carried out by using commercial GABA, GABA recep- were found between feeding behaviors and ORXR-β- [18] tor agonist or GABA in mamma- GABAAR subunit . However, effects of GABA on lian species. It was reported that injection of GABA into food intake of teleost remain largely unknown. There- the ventromedial hypothalamus of satiated rats in- fore, further studies are needed to elucidate the poten- duced further consumption of food. Similar effects tial role of GABA on food intake of fish.

Received date: 2016-11-24; Accepted date: 2017-04-27 Foundation 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: [email protected] Corresponding author: Liang Xu-Fang, E-mail: [email protected] 1312 水 生 生 物 学 报 41 卷

As is known to us, the regulation of appetite mrigola (initial body weight: 0.16±0.02 g) (Wuhan, which controls food intake is a complex phenomenon China) at 8:00 am and 17:00 pm every day. Animals involving interactions between the brain and periphe- were acclimated to these conditions for at least 2 weeks ral signals. A few peptides are now recognized to play before the experiment. an important role in the regulation of appetite, which 1.3 and treatment exist in the same neuron or may have interactions The GABA was purchased from Sigma Bio- with GABA. Neuropeptide Y (NPY) neurons coex- chemicals, Dorset, UK. The drug was dissolved in pressing GABA and agouti-related protein (AgRP) teleost saline (20 mg Na2CO3 per 100 mL of 0.6% [19] stimulate appetite . In addition, NPY, AgRP, and NaCl). Doses of drug were selected based on previ- GABA released in the ARC suppress the restraint of ous studies[24—26], which were chosen for the follow- proopiomelanocortin (POMC) neurons coexpressing ing experiments. The ICV injections were carried out α-melanocyte stimulating hormone (α-MSH) and co- using a 0.5 mm Microlance needle connected to a 5 μL caine and amphetamine regulated transcript (CART) [19] Hamilton microsyringe. The needle was performed on appetite . Leptin, a product of the obese (ob) freehand through the central junction between the gene, bindings with leptin receptor (Leptin-R) and frontal and parietal bones. The procedure of ICV in- acts directly on the hypothalamus to inhibit nore- jection and accuracy of injection placement into the pinephrine efflux (NE) efflux through GABA[20]. Al- ventricular regions of the fish brain followed the though information about the feeding regulation [27] method of De Pedro et al . After injection, the mechanism of these peptides is increasing, the intera- wounds were covered immediately with vaseline. ctions of GABA with them on food intake of fish are 1.4 Effects of ICV administration of GABA on food still limited. intake Mandarin fish (Siniperca chuatsi), a demersal After the acclimation period, 30 food-deprived piscivore, is one of the most valuable food fish native (24h) fish were anesthetized with MS-222 (1鲶10000) to the freshwaters of China. It is one of the chief eco- and weighted when they lost equilibrium, which were nomic edible fish in China and in other countries due [21, 22] divided into five groups that received ICV injection, to its large size, fast growth and delicious flesh . respectively. GABA at 50 μg, 125 μg, 500 μg and In addition, it has distinct food preference. In the 2000 μg, and one control group, which received ICV wild, it feed solely on live fry of other fish species once the fry start feeding[23]. injection of the same volume of teleost saline (5 μL). In the present study, we investigated food intake The fish were habituated in anaesthetic-free water and mRNA expression profiles of five representative within 1—2min to help them recovered equilibrium appetite-related genes in the brain of mandarin fish and normal swimming activity after injection. Every after GABA administration. This study will provide a mandarin fish was then transferred to a aquaria which better understanding of GABA in mediating food in- contained enough pre-weighted prey fish (average take of fish. body weight: 0.16 g) immediately. The food intake was measured by directly observing and recording the 1 Materials and methods number of prey fish eaten by individual mandarin fish 1.1 Ethics statement after 0.5h, 2h and 4h. The formula is as follows: This study was carried out in strict accordance FI=Wi–(Wf×F) where Wi=initial food weight, Wf=re- with the recommendations in the Guide for the Care maining food weight and F=correction factor[27]. and Use of Laboratory Animals of the National Vete- 1.5 Effects of ICV administration of GABA on gene rinary and Quarantine Service. The protocol was ap- expression proved by the Committee on the Ethics of Animal Ex- In order to examine effects of GABA on brain periments of the Huazhong Agricultural University. NPY, AgRP, CART, POMC and Leptin-R mRNA ex- 1.2 Animals pressions, another experiment was performed using The mandarin fish were obtained from Wuhan the uninjectd stock of mandarin fish, which received Freshwater Fish Farm (Wuhan, China). Prior to the the same treatment without giving prey fish. Fish initiation of the experiment, 100 mandarin fish were were then sampled at 0.5h and 2h after injection carried out in 2 cylindrical plastic tanks (350 L) and without feeding. Brains of both groups were dissec- were kept under controlled light-dark conditions (12 ted and immediately snap-frozen in liquid nitrogen Light /12 Dark) with a constant flow of filtered water and stored at –80℃ for RNA isolation and sub- and the water temperature was regulated to 23—25℃. sequent analyses. The fish were fed with (2% of bodyweight) Cirrhina Total RNA was extracted using Trizol reagent 6 期 黄 东等: γ-氨基丁酸对鳜摄食和食欲的影响 1313

(TaKaRa, Japan), treated with RQI RNase-Free DNase value method[29]. All amplifications were performed (TaKaRa, Japan) to remove DNA contaminant, and in triplicate for each RNA sample. then spectrophotometrically quantified. For each re- 1.6 Statistical analyses verse transcription reaction, RNA was subjected to All data were presented as mean±SEM (standard cDNA synthesis by SuperScriptTM II RT reverse tran- error of the mean). Statistical analysis was performed scriptase (TaKaRa, Japan) according to reagent’s in- by a one-way analysis of variance (one-way AN- structions. The selected gene coding sequence were OVA) using SPSS 19.0 with Duncan’s multiple com- based on the results of transcriptome sequencing of parisons test. Statistical significance was determined mandarin fish which were mapped to the mandarin at the 5% level. fish genome by the Short Oligonucleotide Analysis 2 Results [28] Package SOAP aligner/soap2 . Primers of the selec- 2.1 Effects of ICV injection of GABA on food in- ted genes for the real-time PCR (RT-qPCR) were de- take in mandarin fish signed in Tab. 1. The significant increases in food intake were ob- RT-qPCR was performed by a quantitative served at 0.5h and 2h post-injection at the dose of thermal cycler (BIO-RAD CFX96, BIO-RAD, USA) 125 μg GABA compared to the control group (P< using RPL13A, a housekeeping gene, as an endoge- 0.05). The food intake at 4h post-injection showed no nous reference to normalize the template amount. significant differences among tested dose points Each real-time PCR (in 20 μL) reaction contained 10 μL (P>0.05) (Fig. 1). SYBR Green Realtime PCR Master Mixture (Toyobo, Japan), 0.4 μmol/L primers and 1 μL normalized tem- 0.25 Control plate cDNA. The PCR parameters were 40 cycles at GABA ICV-50 μg ℃ ℃ ℃ 0.20 GABA ICV-125 μg 95 for 15s, 60 for 15s, and 72 for 45s, with an a GABA ICV-500 μg b additional initial 1-min denaturation at 95℃. The spe- a cificity of PCR products were confirmed by melting 0.15 GABA ICV-2000 μg a b a a curve analysis. Pooled cDNA samples of the respec- a 0.10 a tive brain tissues were used to generate the calibra- a ab a a tion curves. The amplification efficiencies of control (g/gBW) Food intake 0.05 a and target genes were approximately equal and a ranged from 94.2% to 101.7%. Gene expression 0 levels were quantified relative to the expression of 0 0.5 2 4 –ΔΔCt RPL13A using the optimized comparative Ct (2 ) Time after injection (h)

Tab. 1 Primers used in this study Fig. 1 Effects of ICV administration of GABA at the dose of 0, Amplicon 50, 125, 500 and 2000 μg on food intake before injection and 0.5h, Gene name Primer name Sequence (5′—3′) length (bp) 2h and 4h after injection TATCCCCCCACCCTAT RPL13A RPL13A-F 245 Data represent means±SE (n=6). Significant level is marked with GACA ACGCCCAAGGAGAGC different letters (P<0.05), compared with the values from saline in- RPL13A-R GAACT jected mandarin fish at each dose GAGCCAAGCGAAGAC AgRP AgRP-F 151 CAGA 2.2 Effects of ICV injection of GABA on gene ex- GCAGCACGGCAAATG AgRP-R AGAG pression GTTGAAGGAAAGCAC NPY NPY-F 191 The expressions of selected genes representing AGACA GCTCATAGAGGTAAA feeding regulation were measured by RT-qPCR me- NPY-R AGGGG thod after GABA treatment. The mRNA levels of CTGCTGTCCGTCATTT CART CART-F 171 GTCAC NPY in brain increased significantly at the dose of TGGGATGCTTCCTCTT CART-R 125 μg at 0.5h and 2h after GABA injection com- TTCTC GTGTCATCCTCGTTAC pared to those of the control group (P<0.05). Injec- POMC POMC-F 162 TGC tion of GABA significantly promoted AgRP mRNA GCGACGCTCCTATTC POMC-R AAT expressions at the dose of 125 μg at 0.5h, while the CTCTGAGAACATTTCC Leptin-R LeptinR-F 100 mRNA levels decreased significantly at all the four AAAGTCG GGCTGGGGCGGGAGT doses at 2h compared to the control group (P<0.05) LeptinR-R TAG (Fig. 2). The mRNA levels of the anorexigenic genes 1314 水 生 生 物 学 报 41 卷

CART and POMC were also assessed. The results of POMC compared to the control group at 0.5h showed a marked decrease of CART mRNA levels at (P>0.05) (Fig. 2). 0.5h and 2h by GABA administration (P<0.05), while The mRNA abundance of Leptin-R in the brain the mRNA levels of POMC were decreased at 2h decreased significantly at 0.5h and 2h after GABA compared to those in the control group (P<0.05). ICV injection compared to the control group (P<0.05) injection of GABA did not affect the expression level (Fig. 2).

1.5 b 2.5 b 1.5 A 0.5h B 0.5h C 0.5h b 2h 2.0 2h b 2h a a a b 1.0 a b a 1.0 1.5 a a a a a a a a a a a a a a 1.0 a 0.5 a a 0.5 a a 0.5 Relative mRNA expression mRNA Relative expression mRNA Relative expression mRNA Relative 0 0 0 0 501255002000 0 50 1255002000 0 50125 500 2000 0 50 125 500 2000 0 50125 500 2000 0 50125 500 2000 ICV GABA (μg) ICV GABA (μg) ICV GABA (μg)

2.0 1.5 D 0.5h E 0.5h a 2h b b 2h 1.5 a b 1.0 a a a a a a a 1.0 a a a a a 0.5 a 0.5 a a Relative mRNA expression mRNA Relative Relative mRNA expression mRNA Relative 0 0 0 50 12550020000 50 1255002000 0 501255002000 0 50 1255002000 ICV GABA (μg) ICV GABA (μg) 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 2h A. 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 point

3 Discussion tain toxicity[33], which may have negative impact on The present findings showed a clear effect of fish. Additionally, there was no significant change of exogenous GABA on food intake and appetite in food intake at 4h by GABA administration. This mandarin fish. The significant increases in food in- short-lived effect is similar to that observed in other take were observed at 0.5h and 2h post-injection at species studied previously. Injections of GABA the dose of 125 μg GABA compared to the control (0.97 nmol/L) into the ventromedial hypothalamus in- [34] group, indicating that GABA of 125 μg had orexige- creased food intake for only 15min in satiated rats . nic effects on fish. This result confirms the previous And injections of muscimol into the paraventricular findings that systemic administration of GABA has a hypothalamus and ventronaedial increased food in- [35, 36] hyperphagic effect on mammals and birds[30, 31]. To take during 1h or 2h post injection . The time of our knowledge, this is the first report showing that each experiment that the orexigenic effect lasted was ICV GABA can lead to a major increase in food con- not totally identical, maybe because of the different sumption in fish. However, ICV injection of GABA experimental species or the different GABA doses. at the dose of 500 and 2000 μg did not alter food in- Therefore, the effects of GABA may have a close re- take in 2h. One explanation is that high doses of lationship with dose and time. GABA may have sedating or muscle relaxant To examine how GABA exerted effects on food effects[32], which make the mandarin fish could not intake, we assessed the mRNA expressions of selec- catch the prey fish. The other plausible explanation ted genes involved in the regulation of food intake. may be high doses of GABA may give birth to cer- The results in the present study showed that the ex- 6 期 黄 东等: γ-氨基丁酸对鳜摄食和食欲的影响 1315 pression of NPY and AgRP were significantly in- reported that there were some interactions between creased at 0.5h post-injection at the dose of 125 μg GABA and Leptin-R. One study showed that mice GABA, indicating that GABA injection can regulate with disruption of GABA transporter in Leptin-R-ex- food intake through upregulating the expression of pressing neurons would develop mild obesity on NPY and AgRP genes, which may be responsible for chow diet and were sensitive to diet-induced [37] the ability of GABA to promote appetite . However, obesity[53]. Another study indicated that leptin’s ef- the expression of AgRP decreased at 2h. The de- fects on norepinephrine efflux (NE) could be media- creased gene expression of AgRP might be due to an ted at least partially through GABA[54]. These results unclear interaction between GABA and AgRP in indicated that GABA might affect the appetite via [38] mandarin fish brain . Therefore, further study about Leptin signaling pathway. the regulation of AgRP expression mediated by In conclusion, these results indicate that GABA GABA in fish satiety signaling should be considered. could affect the appetite by regulating leptin signa- Besides, the down-regulated relative values of CART ling pathway and thus resulting in alteration of food and POMC were consistent with the ability of GABA intake. to promote appetite. CART acts as an anorexigenic factor in mammals[39], birds[39] and fish, including References: zebrafish, Danio rerio[40], goldfish, Carassius [1] Mccormick D A. 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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)。结果显示, 注射125 μg GABA组的鳜在2h内摄食量显著升高。实时荧 光定量PCR (RT-qPCR)结果显示, 注射125 μg GABA 0.5h后, 鳜鱼脑中AgRP和NPY mRNA表达量上调, CART和POMC mRNA表达量下调, 都和鳜摄食量增加相一致。相比于对照组, 注射GABA后Leptin-R的 mRNA表达量在0.5h和2h都有显著下降。这些结果表明GABA可能通过leptin的信号通路来影响食欲, 进而影 响摄食量。研究结果可以为GABA在水产饲料中的应用提供理论依据。

关键词: γ-氨基丁酸; 鳜; 摄食; 基因表达; leptin信号通路