Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss)

Comparative Biochemistry and Physiology, Part B 150 (2008) 377–384

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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss) Koji Murashita a,b, Susumu Uji a, Takeshi Yamamoto c, Ivar Rønnestad b, Tadahide Kurokawa a,⁎ a b c

Nansei Station, National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, Minami-ise, Mie 516-0193, Japan Department of Biology, University of Bergen, Pb 7800, N-5020 Bergen, Norway Tamaki Station, National Research Institute of Aquaculture, Fisheries Research Agency, 224-1, Hiruta, Tamaki, Mie 519-0423, Japan

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Article history: Received 9 March 2008 Received in revised form 15 April 2008 Accepted 16 April 2008 Available online 24 April 2008 Keywords: Fish Leptin Obese gene Recombinant protein NPY POMC Feed ingestion Anorexic effect

A B S T R A C T Leptin is a key factor for the regulation of food intake and energy homeostasis in mammals, but information regarding its role in teleosts is still limited. There are large differences between mammalian and teleost leptin at both gene and protein levels, and in order to characterize the function of leptin in fish, preparation of species-specific leptin is therefore a key step. In this study, full-length cDNA coding for rainbow trout leptin was identified. In spite of low amino acid sequence similarity with other animals, leptin is highly conserved between trout and salmon (98.7%). Based on the cDNA, we produced pure recombinant trout leptin (rtleptin) in E. coli, with a final yield of 20 mg/L culture medium. We then examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs (POMC A1, A2 and B) in a short-term (8 h) experiment. The rt-leptin suppressed food intake and led to transient reduction of NPY mRNA levels, while the expression of POMCs A1 and A2, was elevated compared with vehicle-injected controls. These results for rainbow trout are the first that describe a physiological role of leptin using a species-specific orthologue in teleosts, and they suggest that leptin suppresses food intake mediated by hypothalamic regulation. This anorexic effect is similar to that observed in mammals and frogs and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution. © 2008 Elsevier Inc. All rights reserved.

1. Introduction The hormonal product of the obese -ob– gene, leptin, is a member of the class-1 alpha helical cytokines that in mammals is produced primarily by adipose tissue (Zhang et al., 1994). It has been shown to play a key role in food intake and regulation of energy balance (Schwartz et al., 2000; Margetic et al., 2002; Klok et al., 2007), and acute or chronic leptin treatment (intracerebroventricular injection, intraperitoneal injection or osmotic pump infusion) reduces food intake and body weight in mammals (Seeley et al., 1996; Sahu 1998; Sahu 2004; Wetzler et al., 2004). The regulatory role of leptin in energy homeostasis is mediated via the hypothalamus, and leptin influences the various hypothalamic orexigenic (such as neuropeptide Y, NPY and agouti-related protein, AgRP) and anorexigenic (such as pro-opiomelanocortin, POMC and cocaine- and amphetamine-regulated transcript, CART) neuropeptides in mammals (Schwartz et al., 2000). Since the discovery of leptin in mouse by Zhang et al. (1994), a good deal of research has been done on leptin orthologues and their biological roles in non-mammalian species. Although a leptin homologue has been isolated from chicken (Taouis et al., 1998), and ⁎ Corresponding author. Tel.: +81 599 66 1830; fax: +81 599 66 1962. E-mail address: [email protected] (T. Kurokawa). 1096-4959/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2008.04.007

recombinant chicken leptin injection reduced food intake (Dridi et al., 2000), there has been some doubt concerning this discovery (Huising et al., 2006b). To the best of our knowledge, only two studies have so far used species-specific leptin to explore its biological functions in ectothermic vertebrates. The first was performed on the African clawed frog (Xenopus laevis), where intracerebroventricular injection of recombinant frog leptin was shown to be potently anorexigenic (Crespi and Denver, 2006). The other study reported successful production of recombinant pufferfish leptin, but no physiological effects were characterized (Yacobovitz et al., 2008). Many other studies have examined the relationship between leptin and appetite in fish, but these have all been based on the mammalian orthologue. For many fish, including coho salmon, Oncorhynchus kisutch (Baker et al., 2000), catfish, Ictalurus punctatus (Silverstein and Plisetskaya, 2000) and green sunfish, Lepomis cyanellus (Londraville and Duball, 2002), administration of mammalian leptin did not affect food intake or body weight. On the other hand, Volkoff et al. (Volkoff et al. (2003) and de Pedro et al. (2006) reported that administration of mammalian leptin to goldfish (Carassius auratus) reduced both food intake and body weight. However, since these studies used mammalian leptin, and taking into account the very low sequence similarity between mammalian and fish genes (Kurokawa et al., 2005; Crespi and Denver, 2006; Huising et al., 2006a), the physiological roles of leptin in teleosts are in our opinion still obscure. In order to explore the

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physiological roles of leptin in fish, preparation of species-specific leptin is thus essential. The first aim of this study was to produce recombinant leptin in rainbow trout (Oncorhynchus mykiss). Trout leptin cDNA was therefore cloned and recombinant trout leptin (rt-leptin) was successfully expressed in E. coli and purified. We also examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs as a first step towards understanding the biological roles of leptin in a teleost model. 2. Materials and methods 2.1. Animals and samples

Fig. 1. Schematic diagram of plasmid for expression of rt-leptin. T7 P, T7 promoter; RBS, ribosome binding site; GS T, glutathione S-transferase; PreScission site, PreScission protease recognition site; tLep, trout leptin mature region; T7 T, T7 transcription termination region; pUC ori, bacterial replication origin; Amp, ampicillin-resistant gene; Kan, kanamycin-resistant gene; f1 ori, filamentous phage replication origin.

RNA for cloning the leptin gene originated from rainbow trout (O. mykiss, Salmonidae) (less than one year old) reared at the Inland Station of the National Research Institute of Aquaculture (Mie, Japan) in indoor tanks supplied with a continuous flow of groundwater at 16 °C. The fish received a commercial pellet diet (Nippon Formula Feed, Japan) fed ad libitum using a self-feeder system (Yamamoto et al., 2002). The fish were killed with 2-phenoxyethanol (0.3 mL/L), and tissues were collected and stored with RNAlater RNA Stabilization Reagent (Qiagen, Hilden, Germany) at − 20 °C until further analysis.

Fig. 2. Molecular characterization of trout leptin. (A) Alignment of the amino acid sequences of leptins. Conserved cysteine residues involved in the formation of disulfide bridges are shaded. The asterisks indicate amino acids conserved in all sequences, whereas colons and dots reflect decreasing degrees of similarity. The α-helices, inferred from human leptin, are boxed. Accession numbers are as follows: trout, AB354909; salmon, BI468126; carp1, AJ830745; pufferfish, AB193547; Xenopus, AY884210; salamander, CN054256 and DQ064637; human, P41159. The cleavage sites were estimated by the SignalP program (http://www.cbs.dtu.dk/services/SignalP/). (B) Ribbon diagrams showing the tertiary structure of human and trout leptin. Secondary and tertiary protein structures were modeled using the ProMod II program at the SWISS-MODEL automated protein modeling server based on human leptin Protein Data Bank structure file 1AX8.pdb.

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2.2. Cloning of rainbow trout leptin cDNA Total liver RNA was isolated using RLT buffer (Qiagen) and Sepasol II Super (Nakalai Tesque, Tokyo, Japan) according to the manufacturer's instructions. The isolated total RNA (1 μg) was incubated with 10 U DNase I (Takara, Kyoto, Japan) at 37 °C for 30 min in order to digest genomic DNA. First-strand cDNA of liver with 3′ and 5′ adaptors added was synthesized using SMART cDNA Library Construction Kit (BD Biosciences Clontech, Palo Alto, CA, USA) for rapid amplification of cDNA ends (RACE) PCR. In order to obtain the full-length rainbow trout leptin sequences, 3′ and 5′ RACE PCR were performed according to Kurokawa et al. (Kurokawa et al. (2003). Primers were designed on the partial sequence of rainbow trout leptin available from GenBank accession no. AM042713 (3′ RACE outer, 5′-ATG GTG ATT AGG ATC AAA AAG CTG GAT A-3′; 3′ RACE inner, 5′-TAT CTC CTA ACC TGA TCG AGG GCA TGG A-3′, 5′ RACE outer, 5′-GTT CAG TAG TAA CCG ACC AAG GTA GCC C-3′; 5′ RACE inner, 5′-TTG AGT CTG TTT AGC GCA ACT GGA CCC A-3′). The RACE products were purified from agarose gel using GFX PCR DNA and Gel Band Purification Kit (GE Healthcare BioSciences, Piscataway, NJ, USA) and cloned into the pDrive vector (Qiagen). Shimadzu Biotech (Kyoto, Japan) sequenced the insert. 2.3. Phylogenetic and 3D structural analysis A phylogenetic tree based on the amino acid sequences of mature peptides was constructed by the neighbor-joining method of the Clustal W (http://www.ddbj.nig.ac.jp/search/clustalw-e.html)

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(Thompson et al., 1994) and MEGA 3.1 program (http://www. megasoftware.net/index.html) (Kumar et al., 2004). Secondary and tertiary protein structures were estimated by the ProMod II program at the SWISS-MODEL automated protein modeling server (http://swissmodel.expasy.org//SWISS-MODEL.html) (Schwede et al., 2003) based on human leptin Protein Data Bank (PDB) structure file 1AX8.pdb to compare structural similarities of human and rainbow trout. 2.4. Expression and purification of the rt-leptin The fragment encoding the T7 promoter (T7 P), ribosome binding site (RBS), glutathione S-transferase (GST), PreScission protease recognition site, putative mature region of trout leptin and T7 transcription termination region (T7 T) was synthesized using the Translation System RTS E. coli Linear Template Generation set (Roche Diagnostics, Mannheim, Germany), and was cloned to the pCR II vector (Invitrogen). To delete the pCR II vector internal T7 P, the region of the vector without pCR II T7 P (5.24 kbp) was amplified using Ex Taq (Takara) with primer set of 5′-CCC GCG AAA TTA ATA CGA CTC ACT ATA GGG-3′ and 5′-ACT GGC CGT CGT TTT ACA ACG TCG-3′, and the PCR product was self-ligated (Fig. 1). The sequence between T7 P and T7 T of self-ligated plasmid was confirmed. The vector was transformed into the BL21-AI E. coli strain for protein expression. Transformed cells were grown in 500 mL of Luria-Broth (LB) medium containing 50 μg/mL ampicillin at 37 °C. When absorbance at 600 nm reached between 0.5 and 0.8, arabinose was added to the tube to a final concentration of

Fig. 3. Phylogenetic analysis of vertebrate leptin amino acid sequences. Scale bar indicates the substitution rate per residue. Numbers at nodes indicate the bootstrap value, as percentages, obtained for 1000 replicates. Growth hormone (GH) is included as outgroup. Accession numbers are as follows: cow leptin, P50595; pig leptin, Q29406; cat leptin, AB041360; dog leptin, O02720; human leptin, P41159; macaque leptin, Q28504; mouse leptin, P41160; rat leptin, P50596; dunnart leptin, AF159713; salamander leptin, CN054256 and DQ064637; Xenopus leptin, AY884210; trout leptin, AB354909; salmon leptin, BI468126; zebrafish leptin, BN000830; carp leptin-1, AJ830745; carp leptin-2, AJ830744; medaka leptin, AB193548; pufferfish leptin, AB193547; green puffer leptin, AB193549; human GH, P01241; trout GH-1, AAA49555.

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Fig. 4. Expression of trout leptin mRNA. The β-actin fragment was also amplified to confirm the steady-state level of expression of the housekeeping gene.

0.2% to induce the expression of GST-rt-leptin protein. Cells were grown for an additional 4 h and collected by centrifugation at 3700 g for 20 min at 4 °C. The cell pellet was resuspended in 10 mL of CelLytic B (Sigma-Aldrich, St. Louis, MO, USA) and broken up by sonication. Phenylmethylsulfonyl fluoride (PMSF) was then added to a final concentration of 1 mM. The soluble (supernatant) fraction was collected after centrifugation at 15,000 g for 15 min at 4 °C. The GST-rtleptin fusion protein was purified from the soluble fraction using Glutathione Sepharose 4B (GE Healthcare Bio-Sciences) and the rtleptin was cut off from the fusion protein using PreScission Protease (GE Healthcare Bio-Sciences) by a bach method according to the manufacturer's instructions. The purified rt-leptin was dialyzed against 10 mM ammonium bicarbonate, freeze-dried and stored at −20 °C until use. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA) using BSA as a standard, and 10 mg of protein was obtained from 500 mL of bacterial culture. A single band was detected by SDS-PAGE and Western blot analysis using anti-trout leptin peptide antibody under reduction conditions. The anti-trout leptin peptide antibody was prepared in rabbit using two mixtures of synthetic peptides (predicted part for antigenicity: CPA RQQ KQT GEG GLS and C+G PVA LNR LKG YLG R) conjugated with BSA as an antigen by Operon Biotechnologies (Tokyo, Japan). 2.5. Tissue expression analysis for trout leptin Rainbow trout total RNA of the brain, intraperitoneal adipose tissue, eye, heart, kidney, liver, muscle, stomach, intestine, pancreas, dorsal skin and ventral skin was prepared as described above (Section 2.2), and fist-strand cDNA was synthesized using oligo (dT) primer with reverse transcription kit (Invitrogen, Carlsbad, CA, USA). The tissue distributions of the mRNA of trout leptin were analyzed by RT-PCR using following primer set: 5′-TTG CAT GTA GTG AGG ACC AAG GTC-3′ and 5′-CAG TAG TAA CCG ACC AAG GTA GCC C-3′. The PCR parameters were 40 cycles at 95 °C for 30 s, 61 °C for 30 s and 72 °C for 30 s. Rainbow trout β-actin (GenBank accession no. AJ438158) was amplified to confirm the steady-state level of expression of the housekeeping gene.

expressed as cumulative food intake and feeding activity (food intake per hour). The food intake in mg/g BW was calculated as the total feeding amount (mg) / the total BW (g) of each tank. 2.7. Effect of rt-leptin on gene expression of hypothalamic NPY and POMC In order to assess the effect of rt-leptin on gene expression in hypothalamus a replicate experiment was performed using the same stock of rainbow trout (mean BW: 58.3 g). Similar to the previous experiment rtleptin was administered to the fish by IP injection, and the fish were then sampled at 0, 1, 2 and 4 h after injection (n = 5 fish/sampling time). In rainbow trout, plural POMC genes have been reported by Leder and Silverstein (2006). The hypothalamus, including the preoptic area (POA), was sampled from each fish and NPY and three POMCs (POMC A1, POMC A2 and POMC B) mRNA levels were analyzed by real-time quantitative RT-PCR (qPCR) using SYBR Green assays (iCycler, Bio-Rad Laboratories, CA, USA) according to the manufacturer's instructions. Primer set for the qPCR of NPY was designed in the nucleotide sequence of trout NPY available from GenBank (GenBank accession no: AF203902) (5′-CCC TTG CGG AAG GCT ACC CGG TCA-3′ and 5′-GAC TGT GGG AGC GTG TCT G-3′). Primer sets for the qPCR of POMCs were designed in the same manner as described by Leder and Silverstein (2006) (POMC A1, 5′-CTC GCT GTC AAG ACC TCA ACT CT-3′ and 5′-GAG TTG GGT TGG AGA TGG ACC TC-3′; POMC A2, 5′-TCC CCG TCA AGG TGC ATC T-3′ and 5′-TCA GTG CCC AGC TCT GGT CT-3′; POMC B, 5′-CCA GAA CCC TCA CTG TGA CGG-3′ and 5′CCT GCT GCC CTC CTC TAC TGC-3′). The PCR parameters were 40 cycles

2.6. Effect of rt-leptin on food intake Rainbow trout (mean body weight, BW: 58.8 g) were used for the food intake experiment. The fish were allocated to six tanks (60 L tank, n = 23–24 fish for each tank) and food intake did not differ among the tanks for four days prior to the experiment. Food was withheld for 24 h prior to the start of experimental treatment. The injection dose of rt-leptin was based on reports on mice (Campfield et al., 1995; Pelleymounter et al., 1995; Rentsch et al., 1995) and goldfish (Volkoff et al., 2003; Matsuda et al., 2005; de Pedro et al., 2006). The freezedried rt-leptin was dissolved in 10 mM PBS (pH 7.4) at 120 ng/μL and 6 μL/g BW (total 720 ng/g BW) was injected IP into fish in three tanks. Fish in the remaining three tanks were given injections of the same volume of PBS (control group). The amount of food ingested during the first 8 h after treatment was counted every hour using a selffeeder system on each tank (Yamamoto et al., 2002). The results were

Fig. 5. Effect of rt-leptin on food intake. (A) Feeding activities are shown as food intake during each hour (mg per g body weight per hour). The results are expressed as mean± SEM. (B) Cumulative food intake (mg per g body weight). The results are expressed as mean ± SEM. Values shown are for three replicate experiments with 23–24 fish per group (n = 3). Significance was compared with PBS-injected fish at each time point (⁎p b 0.05, ⁎⁎p b 0.01).

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at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. After standardization by β-actin gene, the NPY and POMCs mRNA levels were set relative to the mean of 0-time fish mRNA levels = 1.

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2.8. Statistical analysis Results were considered statistically significant at p b 0.05. Cumulative food intake was analyzed by repeated measures ANOVA with Bonferroni's test. Feeding activity and gene expression analysis were analyzed by means of Student's t-test. 3. Results 3.1. Phylogenetic analysis of trout leptin protein Full-length cDNA sequences of trout leptin were obtained by 3′ and 5′ RACE PCR (966 bp, GenBank accession no. AB354909). The trout leptin nucleotide sequence encoded 171 amino acid residues, including the 21residue putative signal peptide and 150-residue putative mature peptide (Fig. 2A). The putative mature region of trout leptin indicated extremely high degree of identity with salmon (Atlantic salmon, Salmo salar) leptin (98.7%, from EST database), though there are low identities with the other species (20.8–26.8%, Fig. 2A). Trout leptin has two cysteine residues for the disulphide bonds of α-helices C and D (Zhang et al., 1997). The predicted tertiary structures of trout and human leptin are highly conserved, in spite of considerable differences in the primary structures (Fig. 2B). The phylogenetic analysis of vertebrate leptin separated fish leptin from tetrapod leptin, and also separated salmonid leptin from the leptin of other fish species (Fig. 3). 3.2. Tissue expression of trout leptin In order to better characterize the rainbow trout leptin, tissue expression was analyzed by RT-PCR. A strong band for leptin was detected in the liver, and weaker bands were observed in the eye, heart, muscle and ventral skin (Fig. 4). 3.3. Effect of rt-leptin on food intake IP injection of rt-leptin had a strong anorexic effect on the feeding behavior of rainbow trout (Fig. 5). After IP administration of 720 ng/g BW of rt-leptin, feeding activity assessed as ingested feed pr hr (Fig. 5A) was significantly suppressed at 2 h after rt-leptin injection (20% of the PBS group). Food intake was significantly suppressed at 2, 5, 6, 7 and 8 h (21, 63, 63, 69 and 71% of that of the PBS group, respectively, Fig. 5B). The data suggested that the anorexic effect was present already 1 h after injection, but this was not significant. There was no compensation for the decline in feed intake in the rt-leptin-injected group during the duration of the experiment. 3.4. Effect of rt-leptin on gene expression of NPY and POMCs IP injection of rt-leptin had different effects on gene expression of hypothalamic NPY and POMCs (Fig. 6). Levels of NPY mRNA at 2 h were significantly lower in rt-leptin-injected fish than in PBS-injected fish (0.36-fold, Fig. 6A). On the other hand, POMC A1 and A2 mRNA levels were significantly higher in rt-leptin-injected fish than in PBS-injected fish at 2 h (1.93 and 7.31-fold, respectively, Fig. 6B and C). The expression of POMC B was not affected by rt-leptin injection (Fig. 6D). 4. Discussion

Fig. 6. Effect of rt-leptin on gene expression of NPY and POMCs in hypothalamus. (A), NPY; (B), POMC A1; (C), POMC A2; (D), POMC B. The results are expressed as mean ± SEM (n = 5 fish). Significance was compared with PBS-injected fish at each sampling point (⁎p b 0.05, ⁎⁎p b 0.01).

This is the first study to successfully express recombinant fish leptin and to demonstrate a biological activity of a homologous leptin in vivo in a teleost species. These results for rainbow trout suggest that trout leptin has an anorexic effect that suppresses food intake mediated by hypothalamic regulation. This anorexic effect compares well with that observed in mammals, where leptin is one of the key hormones regulating food intake (Stanley et al., 2005; Wynne et al., 2005). It is interesting to note that the intraperitoneal (IP) injection of rt-leptin

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resulted in rapid suppression of food intake, with most of the effect occurring during the first 2 h, and also that the reduced initial feed intake was not corrected for during the 8 h that the experiment lasted. This suggests a strong anorexic short-term effect although limitations of the administration technique (IP injection) prevent too firm conclusions from being drawn with regard to effects on feeding frequency and meal size in long term-experiments. It is also clear that the currently available data for leptin in fish fail to support all aspects of the mammalian model for the physiological effects of leptin. Huising et al. (2006a) reported a short-term postprandial increase in leptin mRNA expression in carp liver, while both short-term and long-term food deprivation failed to alter gene expression. A long-term experiment using leptin infusion techniques should be performed in order to determine whether the observed anorexic effects in the present study are sustained up to a level at which growth and energy homeostasis are negatively affected. Further, the anorexigenic properties of the rtleptin should be investigated with dosed delivery. This study suggests that the rapid suppression of food intake by leptin injection is mediated by hypothalamic regulation due to reduced mRNA levels of NPY and increased expression of POMC A1 and A2. It is important to emphasize that the presently used samples from the hypothalamus included the preoptic area (POA). This area has by far the highest level of NPY gene expression in the brain of rainbow trout (Doyon et al., 2003), yet this region is typically not included with the hypothalamus during dissections. The changes observed in rainbow trout appear similar to that observed in the mammalian model, where leptin suppresses food intake mediated by NPY/AgRP and POMC/CART neurons in the arcuate nucleus in the hypothalamus (Schwartz et al., 2000). NPY has been regarded as one of the most potent orexigenic peptides in mammals (Stanley et al., 1986; Woods et al., 1998; Gehlert 1999; Hillebrand et al., 2002; Konturek et al., 2004), and it is known that hypothalamic NPY gene expression is inhibited by leptin (Stephens et al., 1995; Schwartz et al., 1996; Morishita et al., 1998; Dryden et al., 1999). In fish, NPY also plays an orexigenic role; administration of mammalian or fish NPY causes a dose-dependent increase in food intake or body weight in goldfish (Lopez-Patino et al., 1999; de Pedro et al., 2000; Narnaware et al., 2000), tilapia (Oreochromis sp.) (Carpio et al., 2006; Kiris et al., 2007), and catfish (Silverstein and Plisetskaya, 2000). On the other hand, POMC plays an anorexigenic role through the action of the peptide α-MSH (one of the cleavage forms of POMC) (Nahon 2006). POMC neurons in the hypothalamus contain the long-signaling form of the leptin receptor (Cheung et al., 1997), and leptin raises mRNA levels of POMC (Schwartz et al., 1997; Thornton et al., 1997; Elias et al., 1998; Mizuno et al.,1998). In fish, including rainbow trout, several POMC genes have been identified (de Souza et al., 2005; Takahashi et al., 2005; Leder and Silverstein, 2006). In this experiment, NPY and POMC (A1 and A2) mRNA levels showed opposite patterns 2 h after rt-leptin injection. Moreover, this latency was also synchronized with feeding activity, which was suppressed 2 h after injection. These data strongly suggest that leptin has an anorexigenic function that is mediated by hypothalamic regulation in rainbow trout. Moreover, since recombinant frog leptin also suppress food intake in frogs (Crespi and Denver, 2006) the anorexigenic activity of leptin may be conserved from fish to frog to mammal and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution. In this experiment, we did not test the effects of the injection itself on feeding behavior. But after the injection, the mRNA levels of POMCs were decreased in both of PBS and rt-leptin group after 1 h. There was possibility that the injection itself induced stress and perhaps changes in feeding behavior. An important prerequisite for running experiments of this type that aim to explore the physiological roles played by leptin in teleosts is the use of species-specific leptin. Since its discovery in mouse in 1994 (Zhang et al., 1994) much effort has been put into to identifying the leptin gene in fish. When leptin was finally identified in teleosts in 2005, using a synteny cloning approach on pufferfish, Takifugu

rubripes (Kurokawa et al., 2005) both the sequence of the orthologue gene and the protein were shown to be very different from those of mammals. Subsequent identifications of additional leptin in more teleosts species including carp, Cyprinus carpio, zebrafish, Danio rerio (Huising et al., 2006a), medaka, Oryzias latipes, green puffer, Tetraodon nigroviridis (Kurokawa et al., 2005) have continued to confirm the unusually large differences within the teleosts. The amino acid identity between identified teleost and human leptin is as low as 16–25%, while the identity within the teleost group is also low (between cyprinid fish species and other fish species identity is 19–28%; Huising et al., 2006a). At the same time, the rainbow trout leptin sequence identified in the present paper showed a very high degree of similarity in the mature region with Atlantic salmon leptin (from EST database, 98.7%). These could be explained from the evolutionary distance; teleost and tetrapod diverged from a common ancestor about 450 million years ago (MYA) (Kumar and Hedges, 1998) and only 150 million years after that (300 MYA), cyprinid fish diverged from teleost ancestor (Taylor et al., 2003). Moreover, Atlantic salmon diverged from the Pacific salmon (Oncorhynchus sp. including rainbow trout) just recently about 20 MYA (McKay et al., 1996). This also suggests that leptin is highly conserved among salmonids, but it is still unknown if and to what extent experimental and analytical methods based on species-specific leptin may be interchangeable between species. It is interesting to speculate why mammalian leptin have effects in some fish species but not in others. Despite the low amino acid sequence similarity between the hitherto identified leptin (Fig. 3) the various orthologues seem to have a highly conserved tertiary structure that involves a four-helix structure and a disulfide bridge. Crespi and Denver (2006) suggested that a conserved tertiary structure has been maintained by natural selection and presumably has been constrained by the structure of the receptor-binding pocket. These authors also demonstrated that recombinant frog leptin activated both the mouse and the frog leptin receptors in vitro (Crespi and Denver, 2006). Based on alignment they demonstrated a similarity of residues in the AB loop (GLDFIP; Fig. 2) among mammalian and frog leptin and suggested that this sequence may be important for receptor binding. The teleosts analyzed to date do not support that this is a conserved sequence in leptin orthologues (Fig. 2) and it remains to be shown which amino acids that are critical to ensure a specific leptin receptor binding affinity. The injected doses of rt-leptin were determined on the basis of a combination of data from the literature on other animal studies (Campfield et al., 1995; Pelleymounter et al., 1995; Rentsch et al., 1995; Volkoff et al., 2003; Matsuda et al., 2005; de Pedro et al., 2006) in combination with a preliminary titration assay on trout (data not shown). In rodents, endogenous blood leptin levels lie between 1.5 and 5 ng/mL (Ahima et al., 1996; Chehab et al., 1997; Halaas et al., 1997). The reported efficacy of leptin is somewhat variable in mammals, but effective doses are usually between 0.1 and 10 μg/g BW (Campfield et al., 1995; Pelleymounter et al., 1995; Rentsch et al., 1995). We used 720 ng/g BW doses for IP injection. To date, however, blood leptin levels have never been established for any fish species, and appropriate physiological doses remain to be determined. Adipose tissue is the main site of leptin synthesis in mammals (Zhang et al., 1994; Montague et al., 1997). In contrast to mammalians, rainbow trout leptin was mainly expressed in liver as reported in pufferfish (Kurokawa et al., 2005) and carp (Huising et al., 2006a), which suggests that liver is the major site for leptin expression in fish. The first successful production of recombinant fish leptin was in pufferfish (Yacobovitz et al., 2008), but the physiological function of leptin was not characterized in that study. In this study, we first cloned trout leptin cDNA from liver to produce recombinant trout leptin. A high production yield (of the order of g/L of bacterial culture) of recombinant human leptin has previously been reported, based on a commercially available expression vector in an E. coli system (Varnerin et al., 1998). However, we failed to establish a production system for rt-leptin after testing several commercially

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available expression vectors. This was possibly caused by protein instability or enzymatic degradation of the rt-leptin by bacterial proteases. There was also apparent toxicity of the rt-leptin itself on the producing bacterial cells (data not shown). An expression vector produced in our own laboratory enabled us to produce pure rt-leptin with a yield of 20 mg/L of bacterial culture. With this efficiency, sufficient amounts of rtleptin for an experiment can be produced rapidly and at low cost. In conclusion, we have cloned trout leptin cDNA and successfully produced rt-leptin in E. coli. Furthermore, the results of the rt-leptin injection test suggest that fish leptin has an anorexigenic function. This rt-leptin will be a useful tool for identifying the function of leptin in salmonids, since leptin seems to be highly conserved in this family. The rt-leptin will also enable production of an assay for the analysis of plasma leptin levels. 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