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Aquaculture 272 (

Recombinant tilapia Neuropeptide Y promotes growth andantioxidant defenses in African catfish (Clarias gariepinus) fry

Yamila Carpio, Karen León, Jannel Acosta, Reynold Morales, Mario Pablo Estrada ⁎

Aquatic Biotechnology Department, Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology.P.O. Box 6162, Havana, 10 600, Cuba

Received 13 April 2007; received in revised form 6 August 2007; accepted 7 August 2007

Abstract

Neuropeptide Y (NPY) is a 36 amino acid peptide with a direct action on food intake and growth. This study assessed the effectof NPY administration by immersion on growth of Clarias gariepinus fry. The results of the experiment showed that the weightincrease in the NPY group was 87% and 64% greater than the weight in the control group after 18 and 30days, respectively. Thelength increase in the NPY group was 27% and 16% greater than in the control group at days 18 and 30. In recent years, it hasbecome increasingly apparent that the immune and neuroendocrine systems are close linked. Therefore, the actions of this peptideon antioxidant defenses (superoxide dismutase, catalase and reduced glutathione) and innate immunity (lysozyme, lectins and nitricoxide) have also been explored in this research. The results have showed that the administration of recombinant tilapia NPY byimmersion induced a specific increase in reduced glutathione concentration and superoxide dismutase activity.© 2007 Elsevier B.V. All rights reserved.

Keywords: Antioxidant defenses; Fish; Growth; Innate immunity; Neuropeptide Y

1. Introduction

Neuropeptide Y (NPY) is a 36 amino acid peptideabundant in the brain, peripheral sympathetic nerves andadrenal medulla. NPY acts as a co-transmitter, neuro-hormone and neuromodulator (Zukowska et al., 2003).Originally isolated and characterized from porcine brain(Tatemoto and Neuropeptide, 1982), it is now known tobe ubiquitous also outside of the nervous system, in theendothelium, immune cells, megakaryocytes and the gut(Zukowska et al., 2003). This molecule that belongs tothe Neuropeptide Y family of peptides shows remark-

⁎ Corresponding author. Animal Biotechnology Division, Center forGenetic Engineering and Biotechnology, P.O. Box: 6162, Havana10600, Cuba. Tel.: +53 7 2716022x5154; fax: +53 7 2731779.

E-mail address: mario.pablo@cigb.edu.cu (M.P. Estrada).

0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2007.08.024

able sequence homology from fish through mammals(Larhammar, 1996).

It is well known that the endocrine system is involvedin the control of food intake (Silverstein and Plisetskaya,2000) and thatNPYhas been implicated in these processesin many animals (Narnaware et al., 2000; Kuo et al.,2001). Recent findings have shown that intramuscularinjection and oral administration of NPY increases foodintake and growth in postlarvae of penaeid shrimps (Kiriset al., 2004). Similar results were found when the peptidewas administered intraperitoneally to tilapia fry (Carpioet al., 2006). These were the first studies integrating theeffect of NPYon stimulation of feeding, GH secretion andgrowth enhancement in aquatic organisms.

In recent years, it has become increasingly apparentthat the immune and neuroendocrine systems areintimately linked and that bi-directional communication

650 Y. Carpio et al. / Aquaculture 272 (2007) 649–655

between the two is essential for the maintenance ofhomeostatic function (Harris and Bird, 2000).

Increase lines of evidence indicate that NPY is animportant regulator of the immune functions. Manyimmune cells express NPY either constitutively, [e.g., inrat lymphocytes (Von Horsten et al., 1988) and in humanlymphoblasts (Schwarz et al., 1994)], or inducibly, [e.g., inhuman lymphocytes and monocytes (Schwarz et al.,1994)]. Studies of NPY receptors in immune cells aresparse, although the existing evidence indicates that similartypes of receptors to those expressed in the neural cellsexist in the immune system aswell (Zukowska et al., 2003).

The innate immune system is a fundamental defensemechanism of fish. The immune system in newlyhatched larvae is not fully developed. Their immuno-logical capacity is very limited at this stage and they areexposed to a potentially pathogenically hostile environ-ment (Ellis, 1988). Fish possess non-lymphoid cellulardefense mechanisms attributable to many factors such aslysozymes and lectins (Fletcher, 1982; Hanif et al.,2004). Other mechanism like nitric oxide (NO) responseseems to be involved in the innate defenses of small-sized fish against some diseases (Acosta et al., 2004).

Most living systems adapt to oxidative stress byincreasing their antioxidant potential (Hermes-Lima,2004). Apparently, antioxidant enzymes are the mostimportant molecules providing effective protectionagainst oxidative stress. They directly detoxify harmfulreactive oxygen species (ROS) or other compoundsinvolved in ROS generation and oxidative damage tocellular components.

A better understanding of the relationship betweenhormones and immunity opens new possibilities toimprove growth and disease resistance in fish withdesirable features for aquaculture. Surprisingly, despiteNPY is an attractive molecule due to its potent effect overGH secretion, studies on its immunomodulatory effects infish are missing. Accordingly, the present study assessedthe effect of NPYadministration by immersion on growthofClarias gariepinus larvae and the actions of this peptideon important parameters of innate immunity (lysozyme,lectins and NO response) and antioxidant defenses[superoxide dismutase (SOD), catalase and reducedglutathione (GSH)].

2. Materials and methods

2.1. Expression of recombinant tilapia NPY

The pTYB1-tilapia NPY recombinant plasmid, obtainedin the laboratory (Carpio et al., 2006), was transformed intoelectro competent E. coli strain BL21 harboring the lambdaDE3 lysogen that carries the T7 RNA polymerase under the

control of the lac UV5 promoter. One colony was used toinoculate 5mL of Luria Bertani (LB) medium containing50μg/mL of ampicillin for overnight growth at 37°C withvigorous shaking. This culture was used to inoculate 1L ofLB medium containing 50μg/mL of ampicillin. The expres-sion of the recombinant NPY was induced at OD600 of 0.5 byadding isopropyl-b-D-thiogalactopyranoside (IPTG) to0.5mM and grown for 6h at 28°C. After induction, cellswere harvested by centrifugation at 5000g for 20min andstore frozen for later use. The frozen cell pellet was thawedand resuspended in 50mL of lysis buffer (20mM Tris–HCl,pH 8, 500mM NaCl, 1mM ácido etilendiaminotetraacético[EDTA], 20mM phenylmethylsulphonyl fluoride [PMSF]).The expression of the fusion protein was tested on 10%Sodium dodecyl sulphate-polyacrilamide gel electrophoresis(SDS-PAGE) after sonication. Once the protein expressionhad been checked, the supernatant obtained after centrifuga-tion of sonicated cells was incubated at 4°C overnight withcysteine to induce the cleavage of NPY from the intein fusionprotein. The result was checked by Tricine-SDS-PAGE. Thetotal protein concentration was measured with BCA ProteinAssay Kit (Pierce, USA) according to manufacturer'sinstructions. The percent of cleavage peptide was measuredby comparing it with an epidermal growth factor (CIGB,Cuba) standard curve.

2.2. Growth-promoting effects by immersion

Two hundred C. gariepinus larvae of 0.0072±0.0001gweight and 9.3±0.2mm length in each group were acclimatedin 80L tanks with fresh running water for 1week prior to theexperiment. The fish were maintained at 28°C under a 12hlight/12h dark regime and fed with commercial pelleted feed(Center for the Production of Laboratory Animals, CENPA-LAB) to satiation twice a day. Prior to treatment, the tankswere cleaned and the level of the water was down to 2L. Then,10mL of the supernatant of lysed cells containing NPY wasadded. The treatment was carried out for 90min without waterrecirculation. Final concentration of NPY in the immersionmedia was 200μg per liter (200μg/L). This dose was selectedbased on previous dose–response experiments (unpublishedresults). The supernatant of broken cells of pTYB1-trans-formed E. coli treated with cysteine equivalent to the amountreceived by NPY-treated group was added to negative controlfish. The experiment was repeated three times in a week for4weeks. Sampling was done at 15 and 30 days from thebeginning of the experiment. The growth promoting effectswere evaluated by body weight and length increase. Data wereexpressed as mean±SE.

2.3. Homogenates of larvae

Larvae were washed three times with sterile bufferphosphate saline (PBS 1X: (137mM NaCl, 2.7mM KCl,4.3mM Na2HPO47H2O, pH 7.3), homogenized with 10mL ofPBS 1× per gram of tissue and centrifuged at 5000g for 15minat 4°C. The supernatants were collected and stored at − 70°C.

Fig. 1. Effect of NPYadministration on A) body weight and B) length ofClarias gariepinus larvae at 0 (n=20), 15 (n=20) and 30 days (n=80) ofthe beginning of the experiment. The peptide was administered byimmersion. NPY group: Larvae treated with genetically engineered E.coli broke cell supernatants expressing NPY; BL21 group: larvae treatedwith E. coli transformed with pTYB1 broke cell supernatants. Data areexpressed as arithmetic mean±SE. ⁎pb0.0001. The weight in gramsand the length in mmwere used in statistical tests. AMann–Whitney testwas used for body weight comparison between groups. In the case oflength, a Student's t test was used with the data of day 15 and a Mann–Whitney test was used with the data of day 30.

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2.4. Protein concentration

Larval homogenates protein concentration was measuredwith BCA Protein Assay Kit (Pierce, USA) according tomanufacturer's instructions.

Table 1Protein concentration and innate immunity parameters (lysozyme and lectins)of the immersion experiment

Parameters analyzed Number of larvae Number ofreplicates

Statistical analysis

Protein concentration(mg/g of tissue) a

20 2 pN0.05; Student's t

Lysozyme(U/g of tissue) a

20 2 pN0.05; Mann–Wh

Lectins (titre) b 4 pools of 5 larvae 1 pN0.05; Mann–Wha Data represent arithmetic mean±standard error.b Data represent geometric mean±confidence interval.

2.5. Antioxidant defenses

SOD activity was determined based on the ability of theenzyme to inhibit the auto-oxidation of pyrogallol. Theinhibition by SOD was measured at 420nm. The rate of theauto-oxidation of pyrogallol in the absence of tissue was used asthe reference rate. One unit of SODwas defined as the amount ofprotein that inhibits in 50% the auto-oxidation rate of pyrogallolat 25°C and pH 8.2. The SOD activity was expressed in units pergram of tissue per minute (U/g min) (Ecobichon, 1984).

Catalase activity was evaluated by measuring the decreasein hydrogen peroxide (H2O2) concentration at 240nm. Briefly,the assay mixture consisted of 1.5mL buffer phosphate(50mM, pH 7.0), 500μL H2O2 (53mM) and 100μL of sample.Change in absorbance was recorded at 240nm during 1min at20°C. Enzyme activity was expressed in units of CAT activityper gram of tissue per min (U/g min) (Buege and Aust, 1978).

Reduced GSH was measured by 5, 5′-dithiobis(2-nitro-benzoate) (DNTB) assay (Boehringer-Mannheim, 1987). Thelipids were removed from the samples by adding to 400μL oflarval homogenates, 120μL of chloroform and 200μL ofmethanol, mix for 20s and centrifuge at 5000g for 10min. Thesupernatants were used for the assay. An aliquot from thesample (50μL) was added to 2.9mL of phosphate buffer 95mMpH 8 and 50μL of DTNB 50mM. Optical density (OD) wasrecorded at 412nm using buffer–DTNB without sample as ablank. The amount of GSH present in the samples wascalculated from a standard curve where purified GSH was usedinstead of samples. The values were expressed as microgramper gram of tissue (μg/g).

2.6. Lysozyme activity

The lysozyme activity of samples (larval homogenates) wasmeasured using a method based on the ability of lysozyme tolyse the bacteriumMicrococcus lysodeikticus (Ellis, 1990). In a96-well microtray, 100μL of samples in four twofold serialdilutions in phosphate buffer (0.05M, pH 6.2) was mixed with100μL of a 0.4mg/mL suspension ofM. lysodeikticus (Sigma) inphosphate buffer. The microtray was incubated at 22°C and theOD was read at 450nm at 0, 15, 30 and 60 min. For a positivecontrol, larval homogenates was replaced by hen egg white

in control and NPY-treated groups at 15 and 30 days from the beginning

15 days 30 days

BL21 (control) NPY BL21 (control) NPY

test 61.80±5.32 67.39±5.87 56.93±5.40 46.27±3.68

itney test 2660±300 2040±100 1570±130 1660±50

itney test 646±3 406±3 215±2 256±1

Fig. 2. Antioxidant defenses in control and NPY-treated groups at30 days from the beginning of the immersion experiment. A: Catalase;B: superoxide dismutase (SOD) and C: reduced glutathione (GSH).Data represents the arithmetic mean of 4 pools of 5 larvae±SE.Differences between NPYand BL21 control group were analyzed by aMann–Whitney test. ⁎pb0.05.

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lysozyme (serial dilutions starting at 10μg/mL) and for anegative control, buffer replaced larval homogenates. A unit oflysozyme activity was defined as the amount of sample causing adecrease in the OD reading of 0.001min− 1.

2.7. Haemagglutination assay for lectins

Serial twofold dilutions of 100μL of larval extracts wereperformed using PBS pH 7.2 in U-bottom shaped (96 wells,Greiner, Microlon) microtitre wells to which an equal volume offreshly prepared 2% erythrocyte suspension (rabbit in PBS) wasadded (Jung et al., 2003). Wells were incubated for 1h at roomtemperature and the titre read visually and being equal to thedilution in the last well to show agglutination (as manifested by anevenly distributed layer of cells over the whole well bottom). Thehaemagglutinin activity of samples was examined and for each atitre valuewas obtained. The activitywas expressed as titre, i.e. thereciprocal of the highest dilution showing complete agglutination.

2.8. Determination of NOS metabolites in larval homogenates

Total NOS metabolites (nitrite + nitrate) were determinedby the nitrate reductase method (Schmidt et al., 1989). Briefly,larval homogenates samples (50μL) and serial dilutions ofsodium nitrite (1.56–100μM) were added to 96-well cultureplates (Costar) and incubated at 37°C for 1h with 50μL of afreshly prepared reaction mixture containing nitrate reductase(2U/mL) (Boehringer, 20U), reduced nicotinamide adeninedinucleotide phosphate (NADPH: 0.344mM) and flavineadenine dinucleotide (FAD: 0.044mM) in phosphate buffer.Total nitrite was determined adding 200μL of Griess reagent at540nm using a microplate reader.

2.9. Statistical analysis

Results were evaluated using GraphPad Prism version 4.0for Windows, GraphPad Software, San Diego, California,USA. Data with normal distribution and equal variances wereanalyzed using Student's t test. Mann–Whitney test was usedto evaluate differences between groups when pooled sampleswere used or data had unequal variances. Treatments wereconsidered to be significantly different if p b 0.05.

3. Results

The wet body weight and length of larvae were recorded inboth groups at days 15 and 30 from the beginning of theexperiment when differences between groups were evident.

The body weight was higher in the NPY-treated group at day15 (0.310±0.030g [p b 0.0001]) and day 30 (0.961±0.048g [p b0.0001]) from the beginning of the experiment compared tocontrol group (0.166±0.016g at day 15 and 0.587±0.024g atday 30) (Fig. 1A). Percentage of weight increase in the NPY-treated group was 87% and 64% greater than control group atdays 15 and 30, respectively.

The length was also higher in the NPY-treated group (33.85±1.16mm [p b 0.0001] at day 15 and 49.60±0.90mm [p b 0.0001]

at day 30) compared to negative control (26.65±0.92mmat day 15and 42.80±0.57mm at day 30) (Fig. 1B). Length increase in theNPY-treated groupwas 27%and16%greater than control group atdays 15 and 30, respectively.

No statistical differences in protein concentration, lyso-zyme activity and lectins were found at 15days from thebeginning of the immersion experiment (Table 1). It was notpossible to measure antioxidant defenses and nitric oxide atthis time point due to fish availability.

At day 30, increased SOD activity and GSH concentrationwere found in larval homogenates treated with NPY comparedwith control group (Fig. 2B, C). No differences were found inthe other parameters analyzed: protein concentration, lyso-zyme, lectins (Table 1) and catalase (Fig. 2A).

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There was no difference in NOSmetabolites at 30 days of theexperiment (Student's t test; n=20) between control (43±3 μM)and NPY-treated group (43±2 μM). Data represent arithmeticmean±standard error.

4. Discussion

Growth in fish is regulated by the brain neuroendocrinegrowth hormone (GH)-insulin like growth factor axis(Peter and Marchant, 1995). Thus, growth can bestimulated by manipulation of selected neuroendocrineregulators of GHwith an adequate food intake. The directaction of NPY on food intake and growth has beenexamined in recent studies in goldfish (Narnaware et al.,2000), tilapia (Carpio et al., 2006) and shrimp (Kiris et al.,2004). These effects were observed using several routessuch as central administration (Narnaware et al., 2000),intramuscular injection (Kiris et al., 2004), oral adminis-tration (Kiris et al., 2004) and intraperitoneal injection(Carpio et al., 2006).

Although, the growth promoting effect of recombinanthormones by immersion has been demonstrated in severalfish species (Agellon et al., 1988; Moriyama andKawauchi, 1990), this study is the first report showingthat growth rate in fish is enhanced by immersion intoE. coli-derivedNPY. The results presented here confirmedthat NPY biological functions are well conserved in fish(Larhammar, 1996), since tilapia NPY stimulates growthin C. gariepinus larvae.

The absorption mechanism of proteins from ambientwater has not been demonstrated. Sherwood and Harvey(1986) showed that gonadotropin-releasing hormoneappeared quickly in the plasma of goldfish afterapplication to gills. Radiolabeled-BSA was observed onthe gill and epidermis of rainbow trout after immersion inthe solution suggesting the gill pillar cells as a possible siteof entry (Moriyama and Kawauchi, 1990). Thus, amechanismmust exist, probably in the gills, which allowsthe absorption of NPY in an efficient concentration topromote growth.

Contrary to the time-consuming and laborious work ofinjection (Kiris et al., 2004; Carpio et al., 2006), implan-tation (Cheng et al., 1998), and oral-incubation (Hertz et al.,1991), immersion seems to be a more efficient route forhormone administration in aquaculture. This approach notonly requires less complicated procedures for peptidepurification, but also causes minimum stress to fish whilebeing treated.

There is little information about the influence of NPYon protein synthesis. Previous observations showed thatintraperitoneal injection of NPY increase muscle proteincontent in tilapia fry (Carpio et al., 2006). In the present

study, no effect was found on larvae protein concentra-tion. We believe that factors like fish age and tissuecould influence the results. In fact, it has been shownthat the exact action of hormones on metabolism seemsto be dependent on the stage of the fish life (Sheridan,1986). Further experiments are needed to elucidate NPYrole over protein synthesis in relation to fish life stage.

Interactions between the endocrine and immunesystem through hormones and cytokines are importantto adjust defense mechanisms in both mammals and fish(Yada and Nakanishi, 2002).

Hormones have been described to modulate antioxi-dant enzymes activities in mammals (Bolzan et al., 1995;Hauck and Bartke, 2000). In the experiment described inthis paper, it was observed that NPY administrationincreased GSH concentration and SOD activity withoutany effect on catalase activity. These effects are probablylinked to the modified metabolic activity produced bygrowth acceleration. Modulation in antioxidant activitycaused bymetabolic variations has already been observedin fish (Wilhem Filho et al., 1993). The increase ofantioxidant defenses observed may neutralize deleteriousbyproducts of metabolism and counteract the oxidativestress associated with growth. Similar results wereobtained by Brown-Borg and Rakoczy (2003) in whichthey observed body weight increase and alteration ofmultiple components of the antioxidative defense systemafter growth hormone administration to dwarf mice.

The effect of hormones on innate immunity has beendemonstrated in various studies. For example, prolactinand GH increase lysozyme levels in fish (Marc et al.,1995; Yada et al., 2001, 2004). GH increases mannan-binding lectin serum levels in humans (Gravolt et al.,2004) and NO production in lymphoma cell lines(Robyn and Weigent, 2003). As NPY is a potent GHsecretagogue in pituitary; it is likely to function asmodulator of the GH gene expression in cells of fishimmune system and thus, an indirect modulator ofinnate immunity. However, in the present study no effectof NPY administration has been found on the innateimmunity parameters measured. Evidences in mammalssuggest the relationship between the age-dependentexpression of NPY and its receptors, and its function inthe immune system (Nair et al., 1993; Kitlinska et al.,2002). In fish, cloning of NPY receptors and studies ofthe anatomical distribution of its expression are recentlyinvestigated (Larhammar and Salaneck, 2004) but thereare no studies on the functional implication of thisdistribution. More experiments are needed to understandthe role of NPY over innate immunity in fish and,whether this role is age-dependent as in mammals ornot.

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As a conclusion, we showed that administration ofrecombinant tilapia NPYincreases growth inC. gariepinuslarvae confirming that NPY is a potent orexigenic factor infish. This peptide appears to modulate antioxidant defensemechanism through an induction of a specific increase inGSH concentration and SOD activity.

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