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Endocrinology Vol. 144, No. 12 5215-5226
Copyright © 2003 by The Endocrine Society

Peptide Purification, Complementary Deoxyribonucleic Acid (DNA) and Genomic DNA Cloning, and Functional Characterization of Ghrelin in Rainbow Trout

Hiroyuki Kaiya, Masayasu Kojima, Hiroshi Hosoda, Shunsuke Moriyama, Akiyoshi Takahashi, Hiroshi Kawauchi and Kenji Kangawa

Department of Biochemistry (H.Kai., H.H., K.K.), National Cardiovascular Center Research Institute, Osaka 565-8565, Japan; Division of Molecular Genetics (M.K.), Institute of Life Science, Kurume University, Fukuoka 839-0861, Japan; and School of Fisheries Sciences (S.M., A.T., H.Kaw.), Kitasato University, Iwate 022-0101, Japan

Address all correspondence and requests for reprints to: Hiroyuki Kaiya, Ph.D., Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: kaiya{at}ri.ncvc.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have identified ghrelin from the stomach of rainbow trout. Four isoforms of ghrelin peptide were isolated: the C-terminal amidated type of rainbow trout ghrelin (rt ghrelin) composed of 24 amino acids (GSSFLSPSQKPQVRQGKGKPPRV-amide) is a basic form; des-VRQ-rt ghrelin, which deleted three amino acids (V13R14Q15) from rt ghrelin; and further two types of rt ghrelin that retained the glycine residue at the C terminus, rt ghrelin-Gly, and des-VRQ-rt ghrelin-Gly. The third serine residue was modified by octanoic acid, decanoic acid, or the unsaturated form of those fatty acids. In agreement with the isolated peptides, two cDNAs of different lengths were isolated. The rt ghrelin gene has five exons and four introns, and two different mRNA molecules are predicted to be produced by alternative splicing of the gene. A high level of ghrelin mRNA expression was detected in the stomach, and moderate levels were detected in the brain, hypothalamus, and intestinal tracts. Des-VRQ-rt ghrelin stimulated the release of GH in the rat in vivo. Furthermore, des-VRQ-rt ghrelin stimulated the release of GH, but not the release of prolactin and somatolactin in rainbow trout in vivo and in vitro. These results indicate that ghrelin is a novel GH secretagogue in rainbow trout that may affect somatic growth or osmoregulation through GH. Because ghrelin is expressed in various tissues other than stomach, it may play important role(s) in cellular function as a local regulator.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN IS A 28-amino acid peptide identified in rat and human stomach (1) as an endogenous ligand for the GH secretagogue (GHS) receptor (GHS-R) (2, 3). The third serine residue is uniquely modified by octanoylation, and the acylation of the peptide is essential for both receptor binding (4) and eliciting biological activity (1). When ghrelin is injected iv or ip in rats, plasma GH levels increase in anesthetized or free-moving rats (1, 5, 6, 7). Release of GH is also observed after intracerebroventricular injection of ghrelin (5, 8). Ghrelin has also been shown to increase plasma GH levels in humans (9, 10). The mechanism(s) by which ghrelin stimulates GH release has been investigated. Date et al. (11) demonstrated that the gastric vagal afferent is the major pathway conveying ghrelin signals for GH secretion. Tamura et al. (12) indicated that the arcuate nuclei (Arc) are involved in the regulation of GH secretion into the systemic circulation through the use of monosodium glutamate-treated Arc-ablated rats. Shuto et al. (13) further demonstrated that the GHS-R in the Arc is involved in GH secretion using transgenic rats expressing antisense GHS-R mRNA. In addition, ghrelin can stimulate the release of GH by directly acting on the pituitary (1). Therefore, it is likely that, in mammals, the regulation of GH secretion by ghrelin is controlled by both direct and indirect pathways that converge on the pituitary.

In teleost fish, several factors have been implicated in regulating GH release. Species-related or age-related differences, however, have been observed (14). For example, GHRH, a primary regulator of GH release in mammals, stimulates the release of GH in the goldfish, rainbow trout, and tilapia but has no effect on GH secretion in the eel (15). Shepherd et al. (16) demonstrated that ip injection of a ghrelin mimic, peptidyl-GHS secretagogue, KP-102, increases plasma GH levels but not plasma prolactin (PRL) levels in the tilapia, suggesting the presence of a ghrelin-GHS system in fish. Unniappan et al. (17) have recently reported the cloning of a cDNA encoding preproghrelin from the brain and intestine of goldfish. Kaiya et al. (18, 19) isolated ghrelin peptide from the stomach of the Japanese eel and Mozambique tilapia (Oreochromis mossambicus). Interestingly, the carboxyl termini of these peptides possess an amide structure, which has not been observed in tetrapod ghrelins including mammals, chicken, and bullfrog (20, 21). Eel and tilapia ghrelin potently stimulated the release of GH and PRL from organ-cultured tilapia pituitary (18, 19). Rat ghrelin, however, led to similar levels of GH and PRL release (22); thus, these were not species-specific effects. Taken together, these data support a model wherein ghrelin stimulates the release of GH and PRL at least in the tilapia, but the effect of ghrelin seems to differ in vivo or at the pituitary. In the present study, we report the isolation of ghrelin from the stomach of rainbow trout, a common model of fish physiology, and examined its effects on the release of GH, PRL, and somatolactin (SL), another pituitary hormone, both in vivo and in explanted rainbow trout pituitary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of rainbow trout ghrelin (rt ghrelin) from stomach
Ghrelin activity during the purification process was followed by measuring changes in intracellular calcium concentration ([Ca2+]i) in a cell line stably expressing rat GHS-R [Chinese hamster ovary (CHO)-GHSR62] as described previously (1, 20). Rainbow trout stomach was collected in Takatsuki-city, Osaka, Japan, in January. Frozen stomachs, approximately 17.5 g from 10 specimens, were used as the starting materials. The basic peptide-enriched SP-III fraction was prepared as described previously (20, 21). The SP-III fraction was subjected to ion exchange HPLC (TSKgel CM-2SW, 4.6 x 250 mm, Tosoh, Tokyo, Japan). Active fractions were purified by an antirat ghrelin[1–11] IgG immuno-affinity column. Adsorbed substances were separated by reverse-phase (RP)-HPLC using a µBondasphare C18 column (3.9 x 150 mm, Waters, Milford, MA) at a flow rate of 1 ml/min under a linear gradient from 10 to 60% acetonitrile/0.1% trifluoroacetic acid for 40 min. The active fractions were further purified by a diphenyl column (2.1 x 150 mm, 219TP5125, Vydac, Hesperia, CA) at a flow rate of 0.2 ml/min under a linear gradient from 10 to 60% acetonitrile/0.1% trifluoroacetic acid for 40 min. Fractions corresponding to each absorbance peak were collected. To analyze the peptide sequence, 5 pmol of the purified peptide were subjected to protein sequencing (model 494, PE Applied Biosystems, Foster City, CA). The molecular weight of the purified peptide was determined using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Voyage-DE PRO, PE Applied Biosystems). Acylation patterns were determined by the difference between detected molecular mass and theoretical molecular mass that was calculated from amino acid sequence of peptide (Table 1Go).


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  		TABLE 1. Expected molecular forms of isolated ghrelins from rainbow trout stomach

 
cDNA cloning
Rapid amplification of cDNA ends (RACE) PCR method was used for cDNA cloning. Total RNA was extracted from a stomach using TRIzol reagent (Invitrogen, Carlsbad, CA). Poly(A)+ RNA was isolated using a mRNA purification kit (Takara Bio Inc., Kyoto, Japan). For 3'-RACE PCR, first-strand cDNAs were synthesized from 100 ng of poly(A)+ RNA using an adaptor primer supplied by the 3'-RACE system (Invitrogen) and the SuperScript II reverse transcriptase (RT) (Invitrogen). One tenth of the cDNA served as a template. Primary PCR was performed as described previously (20, 21) using four degenerated primers, based on the N-terminal seven amino acid sequence of mammalian ghrelin (GSSFLSP): GRL-s7, 5'-GGGTCGAGYTTCTTRTCNCC-3'; GRL-s8, 5'-GGGTCGAGYTTCTTRAGYCC-3'; GRL-s9, 5'-GGGTCGAGYTTCCTNTCNCC-3'; and GRL-s10, 5'-GGGTCGAGYTTCCTNAGYCC-3'. Amplification was performed as follows: 94 C for 1 min, 35 cycles at 94 C for 30 sec, 58 C for 30 sec and 72 C for 1 min, and a final extension for 3 min at 72 C. Amplified products were purified using Wizard PCR preps DNA purification system (Promega, Madison, WI). For second-round nested PCR, a nested sense primer for eel ghrelin[7–13] (5'-CCCTCACAGAGACCGCAGGG-3') was used. The amplification reaction was 94 C for 1 min, 35 cycles at 94 C for 30 sec, 52 C for 30 sec and 72 C for 1 min, and a final extension for 3 min at 72 C. The candidate ghrelin cDNA fragment was subcloned using a TOPO TA cloning kit (pCR II-TOPO vector, Invitrogen), and sequence of inserted DNA was determined.

For 5'-RACE PCR, first-strand cDNAs were synthesized from 200 ng of poly(A)+ RNA with oligo-dT12–18 primer or random primer and the SuperScript II RT or Thermoscript RT (Invitrogen). RT reaction was conducted at 42 C for 1 h by the SuperScript II RT and at 42 C for 10 min followed at 60 C for 50 min by the Thermoscript RT. One fifth of the purified cDNA was subjected to a TdT-tailing reaction of the 5'-ends of the first-strand cDNA with deoxy CTP according to the manufacturer’s protocol (Invitrogen). The resultant dC-tailed cDNAs served as a template. A gene-specific primer (GSP) was designed based on the sequence of the rt ghrelin cDNA as determined by 3'-RACE PCR: GSP-1, 5'-TTGGAGAACAGGGAATGGAGG-3'. Primary PCR was performed using GSP-1, an abridged anchor primer supplied with the 5'-RACE kit, and Ex Taq DNA polymerase with the following reaction conditions: 94 C for 1 min, 35 cycles at 94 C for 30 sec, 60 C for 30 sec and 72 C for 1 min, and a final extension for 3 min at 72 C. The resultant product was purified by PCR preps, and the second-round nested PCR was performed with the same GSP-1 and abridged universal amplification primers. The amplification reaction was 94 C for 1 min, 30 cycles at 94 C for 30 sec, 55 C for 30 sec and 72 C for 1 min, and a final extension for 3 min at 72 C. The candidate PCR product was subcloned into the pCR-II TOPO vector, and sequence of inserted DNA was determined. The nucleotide sequence of the isolated cDNA fragment was determined by automated sequencing (DNA sequencer: model 373 or 3100, PE Applied Biosystems) according to protocol for the Thermosequence II dye terminator cycle sequencing kit (Amersham Bioscience KK, Tokyo, Japan) or the BigDye terminator cycle sequencing kit (PE Applied Biosystems). Approximately 430 and 480 bp of cDNA were determined by 3'- and 5'-RACE PCR, respectively.

Cloning of the rt ghrelin gene
Genomic DNA was extracted using the GenomicPrep Cells and Tissue DNA isolation Kit (Bioscience KK) from rainbow trout stomach. PCR for full-length genomic DNA was performed using 500 ng genomic DNA as a template and ExTaq DNA polymerase. GSPs that were used are as follows: cdna-s, 5'-GATATCAATGTCCAAGGTATATCTG-3'; and cdna-as, 5'-AAGGAAGCAGTGTTATTTTATTCA-3'. The amplification conditions were 94 C for 2 min, and 30 cycles of 94 C for 1 min, 53 C for 30 sec, and 72 C for 6 min, and a final extension for 3 min at 72 C. A product of approximately 3.2 kbp was amplified and subcloned using the TOPO XL PCR Cloning Kit (pCR-XL-TOPO vector, Invitrogen). The nucleotide sequence was determined as described above.

Gene expression analysis by quantitative RT-PCR
Total RNA of brain, hypothalamus, heart, stomach, pyloric appendage, intestinal tracts, liver, spleen, kidney, head kidney, and gill was extracted separately from four individuals to examine the variation in mRNA levels. First-strand DNA was synthesized from 5 µg of DNase I (Invitrogen)-treated total RNA using the Superscript II RT (Invitrogen) and random primer at 25 C for 10 min followed by 42 C for 1 h. Synthesized cDNA was cleaned up with phenol-chloroform treatment followed by ethanol precipitation. PCR was performed using the HotStarTaq Master Mix Kit (QIAGEN GmbH, Hilden, Germany) with a sense primer (5'-ACTGATGCTGTGTACTCTGG-3') and an antisense primer (5'-CACCATACTCCTGGAACTCC-3'). The amplification reaction was performed with 95 C for 15 min, subsequent 35 cycles at 95 C for 30 sec, 57 C for 30 sec, and 72 C for 1 min, and a final extension for 3 min at 72 C. Amplicon of 248 bp was expected. As an internal control, a 369 bp of 18S ribosomal RNA fragment (DDBJ/EMBL/GenBank accession no. AF243428) was amplified with a sense primer (5'-TTAGTTGGTGGAGCGATTTGT-3') and an antisense primer (5'-AGTGGCGACGGGCGGTGTGTA-3') following reaction conditions: 95 C for 15 min, subsequent 16 cycles at 95 C for 30 sec, 57 C for 30 sec, and 72 C for 1 min, and a final extension for 3 min at 72 C. Amplified products were electrophoresed on a 2% (wt/vol) agarose, and incorporated ethidium bromide was visualized with a FLA2000 (Fuji Photo Film Co. Ltd., Kanagawa, Japan). In addition, quantitative, real-time RT-PCR analysis was performed by the LightCycler system (Roche Applied Science, Mannheim, Germany) using a QutantiTect SYBR Green PCR kit (QIAGEN GmbH) and above primer sets for each ghrelin or 18S ribosomal RNA.

GH-releasing activity of ghrelin in rats
Octanoylated rt ghrelin and des-VRQ-rt ghrelin were synthesized at the Daiichi Suntory Pharma Co., Ltd., Institute for Medicinal Research and Development, as described previously (23), and were used for some biological experiments described below.

Male Sprague Dawley rats, weighing 250–280 g, were cannulated in the femoral artery and vein under pentobarbital sodium anesthesia. After sampling untreated blood (time 0), 2 nmol/250 g body weight of either des-VRQ-rt ghrelin or rat ghrelin was injected into the femoral vein. Blood (150 µl) was collected from the femoral artery in a syringe containing EDTA (1 mg/ml blood) 5, 10, 15, 20, 30, and 60 min after injection. GH concentration in plasma was measured using a rat GH enzyme-immunoassay kit (Amersham Bioscience KK). Data were analyzed by two-way ANOVA to evaluate effects of time or time vs. ghrelin species.

In vivo effect of trout ghrelin on the secretion of pituitary hormones in rainbow trout
Immature rainbow trout, weighing about 100 g, were obtained from a commercial supplier in Iwate Prefecture (Kamaishi-city, Japan) fisheries experimental station. Trout were maintained in an outdoor tank with running fresh water at 10–16 C under natural photoperiod for more than 2 wk until use without feeding. This experiment was conducted between 1000 and 1600 h. Intraperitoneal injection of rt ghrelin or des-VRQ-rt ghrelin was conducted according to the method of Moriyama et al. (24). Fish were lightly anesthetized with 0.01% 2-phenoxyethanol, and the ghrelins were ip injected with either 25 or 250 ng/g body weight. Control fish received 0.9% NaCl solution only (1 µl/g body weight). Blood samples were collected from the caudal vessels at 0.5, 1, and 3 h after injection or at 1, 3, and 6 h after injection as indicated (n = 5 at each time point). Body weights and sex of fish used were: short-term experiments, 74.4 ± 4.8 g (mean ± SD), 27 males and 23 females; moderate-term experiments, 89.1 ± 5.4 g, 25 males and 25 females.

Effects of trout ghrelin on the secretion of pituitary hormones in trout pituitary gland
Immature rainbow trout, weighing about 100 g, were used in this study. After anesthesia with 0.1% 2-phenoxyethanol, trout were decapitated, and the pituitary gland was dissected out. Pituitaries were rinsed once with basic culture medium (BCM; Eagle’s MEM with Earle’s salts (Life Technologies, Inc., Gaithersburg, MD) with kanamycin [60 mg/ml (pH 7.1–7.4)] with sodium bicarbonate (2.2 g /liter) of double concentration of kanamycin (120 mg/ml) for disinfection, then rinsed once with BCM, and placed in a 96-well plate with one pituitary per well containing 200 µl/well of BCM. The pituitary glands were precultured for 48 h at 11 C. After preincubation, media were removed, and 200 µl/well of rt ghrelin or des-VRQ-rt ghrelin diluted with fresh BCM were replaced at a concentration from 0.1 to 100 nM (n = 5 in each dose). After a 24-h incubation, cultured media were collected and stored at -30 C until RIA.

Hormone measurements
Concentrations of GH, PRL, and SL in plasma were measured by homologous RIAs according to the methods of Bolton et al. (25), Hirano et al. (26), and Kakizawa et al. (27) with some modifications. Antibody-bound hormone complexes were precipitated with 100 µl of 0.25% PANSORBIN Cells (Carbiochem, San Diego, CA) suspended in the RIA buffer, and the radioactivity of precipitate was counted by a {gamma} counter (Packard Instrument Co., COBRA Quantum, Meriden, CT). The lowest detectable level of GH was 0.78 ng/ml, that of PRL was 0.37 ng/ml, and that of SL was 0.64 ng/ml. Intraassay coefficient of variation was less than 4%.

Statistics
All data are expressed as the mean ± SE. Group comparisons were performed using one-way ANOVA, followed by Fisher’s least significant difference test. Differences of P < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of rt ghrelin
Seven groups of ghrelin activity were identified by ion exchange HPLC (pH 4.8) for the SP-III fraction (Fig. 1AGo). Each active group was purified by passage over an antirat ghrelin[1–11] IgG immuno-affinity column, and followed by two different rounds of RP-HPLC. Figure 1BGo shows the final isolation of the major rt ghrelin from group D in Fig. 1AGo. Indeed, two active peaks were isolated in this group. We were able to isolate 14 active peptides from seven groups in carboxymethyl (CM)-HPLC (Table 1Go). Peptide sequence analysis revealed that peptides of two different lengths were isolated; one sequence was GSXFLSPSQKPQVRQGKGKPP, and the other one was GSXFLSPSQKPQGKGKPP (X was unidentified by the sequencer because of acyl modification). The latter form has a three amino acid deletion (VRQ) at positions 13 to 15 of the former. From the sequence homology compared with other ghrelins, we determined these isolated peptides to be rt ghrelins.



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  		FIG. 1. Purification of rt ghrelin from stomach extract. Black bars indicate the fluorescence changes in [Ca2+]i in CHO-GHSR62 cells. A, CM-cation exchange HPLC (pH 4.8) of SP-III fraction of stomach extract. Each active fraction (A–G) was separately purified by an antirat ghrelin[1–11] IgG immuno-affinity chromatography. B, Final purification of active fraction D in CM-HPLC by reverse-phase RP-HPLC. Two active ghrelins (Table 1Go, peaks 7 and 8) were isolated from this fraction.

 
Cloning of rt ghrelin cDNA
cDNA encoding preproghrelin was isolated from stomach mRNA. cDNAs of two different lengths were isolated; both were identical in 5'-untranslated region and 3'-untranslated region length (57 bp and 142 bp, respectively), but differed in length of coding region between 336 bp (DDBJ/EMBL/GenBank accession no. AB096919) and 327 bp (DDBJ/EMBL/GenBank accession no. AB101443). Long-type cDNA was obtained only in the case that RT reaction was conducted using a random primer and Thermoscript RT enzyme. The deduced amino acid sequence of the coding regions of the two cDNAs indicated that the preproghrelin-1 and preproghrelin-2 are composed of 111 and 108 amino acids, respectively (Fig. 2Go). Figure 3Go shows alignments of ghrelin precursors. The amino acid sequence of preproghrelin-1 in rainbow trout was 49, 57, and 47% identical to that of tilapia, eel, and goldfish, respectively (Fig. 3AGo). The identity to that of bullfrog, chicken, and human was 25, 40, and 33%, respectively (Fig. 3BGo). From this cDNA analysis, the unidentified third amino acid was determined to be serine, as seen in other species except bullfrog, in which this residue is threonine (20). An amidation signal Gly-Arg-Arg present in eel and goldfish ghrelin precursors followed at the C-terminal end of the mature ghrelin. It is predicted that posttranslational processing of the two isolated mRNAs will yield 23- or 20-amino acid peptides with an amidated C-terminal. Because a C-terminal amidated peptide is hard to read in a peptide sequencer, these results are in agreement with the results of peptide purification.



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  		FIG. 2. Nucleotide sequence and deduced amino acid sequence of the rt ghrelin cDNA. The rt ghrelin cDNA was 535 bp (preproghrelin-1) and 526 bp (preproghrelin-2) in length. The mature sequence of rt ghrelin is underlined. The C-terminal amidation signal is shown with a dashed line. A typical dibasic processing sequence is boxed. Doubled underline indicates the polyadenylation signal (AATAAA). The nucleotide sequence for the rt ghrelin precursor has been deposited in the DDBJ/EMBL/GenBank databases with the accession no. AB096919 (preproghrelin-1) and AB101443 (preproghrelin-2).

 


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  		FIG. 3. Comparison of amino acid sequence of preproghrelin. Mature ghrelin was boxed. Asterisks indicate identical amino acids across all species. Alignment in fish species (A) and in bullfrog, chicken, and human (B). Amino acid sequences are available from the DDBJ/EMBL/GenBank databases (accession no. AB062427, eel; AF454389, goldfish; AB077764, tilapia; AB058510, bullfrog; AB075215, chicken; and AB029434, human).

 
Structural determination
To determine the molecular weights of the rt ghrelins, isolated peptides were analyzed by MOLDI-TOF mass spectrometry. Table 1Go shows the actual measured molecular mass of the isolated peptides, the expected molecular forms, and the isolated yields. From the molecular masses in addition to the analyses of peptide sequence, four types of amino acid sequence (20, 21, 23, and 24 amino acids) and two types of the C-terminal end (amidated and nonamidated forms) were predicted. Both 21- and 24-amino acid ghrelins are produced by glycine retention before dibasic processing signal without C-terminal amidation. We concluded that all isolated peptides were rt ghrelin and its isoforms. The major rt ghrelin, with isolated yields of 54 pmol, was a peptide isolated from group D, peak 8. The expected peptide sequence was GSSFLSPSQKPQGKGKPPRV-amide with unsaturated decanoic acid (C10:2). Octanoylated and decanoylated ghrelins, which have primarily been identified in other animal species, were also identified, but those yields were relatively low (Table 1Go, peaks 5, 7, 9, and 12). We designated the 23-amino acid peptide (GSSFLSPSQKPQVRQGKGKPPRV-amide) as a basic form; rt ghrelin and rt ghrelin that deleted three amino acids of V13R14Q15 were designated as des-VRQ-rt ghrelin (GSSFLSPSQKPQGKGKPPRV-amide). Furthermore, ghrelins composed of 24 amino acids (GSSFLSPSQKPQVRQGKGKPPRVG-OH) and 21 amino acids (GSSFLSPSQKPQGKGKPPRVG-OH) were designated as rt ghrelin-Gly and des-VRQ-rt ghrelin-Gly, respectively.

Cloning of genomic DNA of trout ghrelin
It was observed that the position of the deleted amino acids (V13R14Q15) is similar to that seen in des-Gln14-rat ghrelin, suggesting that alternative splicing of the ghrelin gene also occurred in the rt ghrelin gene. We cloned the rt ghrelin gene containing full-length ghrelin cDNA. The rt ghrelin gene is 3270 bp in length and is comprised of five exons and four introns (Fig. 4AGo; DDBJ/EMBL/GenBank accession no. AB100839). The overall genomic organization is similar to that of the mouse and rat ghrelin genes. Figure 4BGo shows a portion of the nucleotide sequence of the rt ghrelin gene in exon 2, intron 2, and bounded exon 3. This exon-intron boundary is consistent with the AG-GT rule of the splicing, but two consensus sequences for 5' splice sites exist within 15 bases of the initial sequence of intron 2. These two AG nucleotides may be used as a splicing acceptor site at the 3'-end of intron 2. From this hypothesis, two lengths of ghrelin mRNA, 535 bp and 526 bp, would be produced.



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  		FIG. 4. Schematic representation of the rt ghrelin gene (A) and the model for splicing junction (B). A, Exons are shown by boxes. The sequence of the gene is available from the DDBJ/EMBL/GenBank databases (accession no. AB100839). B, The genomic sequence of the exon-intron boundaries of intron 2 of the ghrelin gene is shown. The splicing signals, GTs for the 5'-side and AG for the 3'-side of intron, are boxed.

 
Expression of ghrelin mRNA in rainbow trout tissues
Figure 5Go shows expression pattern of mRNA for ghrelin and 18S ribosomal RNA from four different individuals in a representative amplification condition. High levels of ghrelin mRNA were observed in the stomach in all fishes. A detailed mRNA expression analysis based on quantitative PCR also demonstrated highest mRNA levels in the stomach, followed by moderate expression levels in the central nervous system and intestinal tracts (Table 2Go).



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  		FIG. 5. Gene expression analysis of the rt ghrelin by RT-PCR. Gene expression pattern of ghrelin was examined in four different individuals. PCR product of each ghrelin and 18S ribosomal RNA was obtained with the amplification of 35 and 16 cycles, respectively. Detailed mRNA levels by quantitative PCR analysis are shown in Table 2Go.

 

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  		TABLE 2. Quantitative PCR analyses of ghrelin and 18S ribosomal RNA

 
Effects of rt ghrelin on rat GHS-R
We examined the ability of synthesized rt ghrelin and des-VRQ-rt ghrelin to stimulate the rat GHS-R using GHSR62 cells. Both octanoylated rt ghrelin and des-VRQ-rt ghrelin increased [Ca2+]i in a dose-dependent manner with similar potency to rat ghrelin, a full agonist for the GHS-R (Fig. 6AGo). No differences were seen among three ghrelins. Next, we examined the effect of ghrelin on the release of GH in the rat. Only des-VRQ-rt ghrelin was examined because the similar potency was shown between the two rt ghrelins in the rat GHS-R assay. Plasma GH levels were increased by a bolus iv injection of des-VRQ-rt ghrelin or rat ghrelin with similar kinetics and to the same extent (Fig. 6BGo).



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  		FIG. 6. Biological activity of rt ghrelin in rat models. A, Dose-response relationships of changes in [Ca2+]i concentrations after treatment of CHO-GHSR62 cells with octanoylated rt ghrelin, octanoylated des-VRQ-rt ghrelin, and rat ghrelin. The maximum value of fluorescence changes was used for data calculation. Values represent means ± SEM (n = 3). B, Time-course of changes in plasma GH levels after iv injection of octanoylatd des-VRQ-rt ghrelin or rat ghrelin into male Sprague Dawley rats. Due to variation in the initial baseline levels, values (means ± SEM; n = 5) are expressed in terms of the ratio of each time point to the initial level (des-VRQ-rt ghrelin, 141.7 ± 8.8 ng/ml; rat ghrelin, 118.8 ± 19.1 ng/ml; saline, 201.9 ± 62.4 ng/ml).

 
Intraperitoneal injection of rt ghrelin into rainbow trout
Plasma GH levels increased 30 min after ip injection of des-VRQ-rt ghrelin (Fig. 7AGo). The increased levels of GH were maximal 1 h after injection and gradually decreased to basal levels 6 h after injection (Fig. 7BGo). Conversely, no changes in plasma PRL (Fig. 8Go, A and B) and SL (Fig. 8Go, C and D) were observed at each dose during the 6-h experimental period. Octanoylated rt ghrelin demonstrated similar effects (data not shown).



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  		FIG. 7. Changes in plasma levels of GH after ip injection of rt ghrelin. Changes seen 0.5–3 h after injection (A) and 1–6 h after injection (B) are presented. The data result from synthetic octanoylated des-VRQ-rt ghrelin. Similar changes were seen in the administration of rt ghrelin (data not shown). Significant difference is expressed by *, P < 0.05; **, P < 0.01, compared with the values of saline injection at each time point.

 


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  		FIG. 8. Changes in plasma levels of PRL and SL after ip injection of rt ghrelin. Changes seen 0.5–3 h after injection (A and C) and 1–6 h after injection (B and D) are presented. The data result from synthetic octanoylated des-VRQ-rt ghrelin. Similar changes were seen in the administration of rt ghrelin (data not shown). No significant difference was seen compared with the values of saline injection at each time point.

 
In vitro study using trout pituitary
We examined whether ghrelin isolated in rainbow trout stimulates the release of adenohypophyseal hormones using organ-cultured rainbow trout pituitary. In this experiment, we examined only des-VRQ-rt ghrelin because similar effects were seen between rt ghrelin and des-VRQ-rt ghrelin in in vivo study (Figs. 7Go and 8Go). A significant increase in the release of GH was observed during a 24-h incubation at a dose of 1 nM (Fig. 9Go). GH release was maximal after treatment with 10 nM; GH release was inversely related to ghrelin dose at 100 nM. In these samples, no significant changes in the release of PRL and SL were observed as demonstrated in in vivo study (Fig. 9Go).



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  		FIG. 9. Effects of rt ghrelin on the release of GH, PRL, and SL from the rainbow trout pituitary in vitro. Synthetic octanoylated des-VRQ-rt ghrelin was used for this assay. Values are expressed as mean ± SEM (n = 5). Significant difference is expressed by **, P < 0.01; ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that ghrelin is present in rainbow trout and that it can alter GH levels in vivo and in vitro. This is in agreement with previous in vivo or in vitro studies that demonstrated that ip injected peptidyl GHS, KP-102, stimulates the release of GH in the tilapia (16), or rat, eel, and tilapia ghrelins stimulate the release of GH from the tilapia pituitary (18, 19, 22). Furthermore, we show for the first time in fish that peripheral administration of ghrelin stimulates the release of GH. The effects of ghrelin were specific for the release of GH but not that of PRL and SL both in vivo and in vitro. This differs from previous observations in the tilapia pituitary (22). It is possible that GHS-R is not present in PRL and SL cells in rainbow trout. It needs to be tested further for other aspects, for example PRL or SL synthesis.

Acyl modification
The major form of acyl modification in all the known mammalian and nonmammalian ghrelins is n-octanoic acid or n-decanoic acid (1, 18, 19, 20, 21, 28). In the present study, saturated octanoylated and decanoylated ghrelins were isolated, but the amounts were relatively low. Most of the isolated ghrelin in rainbow trout was modified by unsaturated n-decanoic acid. Additional forms of ghrelin were also isolated with other types of acyl modifications including unsaturated octanoylated forms. In purified chicken ghrelin, several different types of acyl modifications were also observed (Kaiya, H., unpublished observations). In the bullfrog, eel, and chicken, octanoylated and decanoylated ghrelins were isolated in similar amounts (18, 20, 21), but decanoylated ghrelin was the major form in the tilapia (19). Several types of posttranscriptional modification have been reported in human ghrelin (28). The mechanisms governing the acylation of ghrelin are still unknown, but a condition of feeding may influence the type and amount of acyl modification in the ghrelin polypeptide.

Peptide structure
Peptide sequencing led to the identification of two ghrelin isoforms that differ in the deletion of three amino acids (Q13V14R15). This was confirmed from the predicted peptide sequence resulting from translation of two isolated cDNAs; we have designated the shortened form as preproghrelin-2. Analysis of the full-length ghrelin gene suggests that mRNAs of two different lengths are generated by alternative splicing of intron 2. Similar alternative splicing of the ghrelin gene has been reported in the production of rat des-Gln14-ghrelin (29), but not in other fish species (18, 19). In the analysis of partial sequence of the goldfish ghrelin gene (17) and the eel ghrelin gene (Kaiya, H., unpublished observation), there was no tandem AG-GT sequence in intron 2 as seen in the rainbow trout gene. In a recent deposited ghrelin gene of the tilapia, Oreochromis niloticus, such a sequence is also not found (DDBJ/EMBL/GenBank accession no. AB104860; Parhar, I. S., unpublished observation). This is the first case to show the presence of ghrelin splice variants in fish.

Amide and nonamide structure
Rainbow trout ghrelin has an amide structure at its C terminus; a similar modification is seen in other fish including eel, goldfish, and tilapia, but not in mammalian, chicken, or bullfrog ghrelin (1, 17, 18, 19, 20, 21, 28). It is most likely that the C-terminal amide structure is a conserved characteristic in fish ghrelin. Amidated eel ghrelin did not potentiate the release of GH in rats in vivo, and this effect was the same as nonamidated rat ghrelin (18). Rat and tilapia ghrelins seem to have no difference in their ability to stimulate GH release in the tilapia (19, 22). To date, the importance of the amide structure in ghrelin remains unclear.

Nonamidated rt ghrelin and des-VRQ-rt ghrelin, designated as rt ghrelin-Gly and des-VRQ-rt ghrelin-Gly, were isolated in this study. Although a glycine residue within the amidation signal originally contributes to the formation of the amide structure, this residue was found at the C terminus of nonamidated ghrelins. This type of ghrelin molecule was not isolated in the previous purification of eel or tilapia ghrelin. There appears to be a species-specific amidation of ghrelin in the stomach of fish. We did not examine their biological activities in rainbow trout. These findings need to be further studied for their biological significance, including possible different activities compared with amidated rt ghrelin.

Gene structure
We determined the partial structure of the rt ghrelin gene. The overall gene length is 3720 bp and is similar to that of the mouse ghrelin gene (3748 bp) (30), but it is longer than that of the goldfish ghrelin gene (980 bp) (17). The rt ghrelin gene is organized with five exons and four introns. This arrangement is the same as the rat and mouse genes, but not the goldfish gene (17) and the recent reported tilapia ghrelin gene (Parhar, I. S., unpublished observation), which each have four exons and three introns. The composition is also similar to those of the rat and mouse genes, in which a noncoding 25-bp short exon 1 was found (30).

mRNA expression
Ghrelin is primarily expressed in the stomach in all species previously examined. Although goldfish lack a stomach, the intestine is the major site of ghrelin production (17). These findings suggest that ghrelin is primarily synthesized in gastrointestinal tracts in vertebrates. In the present study, high levels of ghrelin mRNA expression were observed in the stomach, consistent with findings in other species. Expression of ghrelin mRNA has also been detected throughout the intestinal tract of different species of fish; the local role of ghrelin in the intestine is not known. Ghrelin mRNA expression was also observed in the brain and hypothalamus. Central ghrelin, produced in the hypothalamic Arc in the rat, seems to participate in the regulation of GH secretion from the pituitary (8). In addition to GH regulation, centrally administered ghrelin modifies feeding behavior in rats (5, 31). In fish, intracerebroventricular injection of octanoylated goldfish ghrelin into the goldfish stimulated food intake (17). In addition to stomach-derived ghrelin, hypothalamic ghrelin may also regulate pituitary function and feeding in rainbow trout.

Quantitative PCR analysis clearly demonstrated that ghrelin mRNA, although the level was very low, was expressed in all tissues examined. Recent reports demonstrate the expression of ghrelin mRNA in the spleen of goldfish (17) or in the kidney and gills of the eel and tilapia (18, 19). Expression of ghrelin mRNA was demonstrated in the kidney, glomerulus, and renal cells of rodents (32) and in the kidney of humans (33). Autocrine or paracrine action of ghrelin is assumed, but a role(s) in these tissues and cells remains unclear. In fish, the kidney and gills are the major osmoregulatory organs, and the functions are governed by several endocrine or autocrine factors (34, 35). Ghrelin may play a role(s) in the functional regulation of these organs. A recent paper reported that ghrelin potently inhibited water drinking in eels acclimated in seawater (36), suggesting ghrelin may be related to osmoregulation in fish.

Bioactivity
Shepherd et al. (16) reported that, in the tilapia, ip injection of KP-102, a peptidyl-GHS, stimulated the release of GH after 6 h of injection. In contrast, plasma GH levels increased 30 min after injection of ghrelin with the same ip injection in the present study, and the increased levels returned to basal levels 6 h after injection. The release of GH in the rat occurs rapidly (within 5 min) after iv injection of ghrelin. The alteration of GH levels within 30 min after injection did not examine, but we have presented similar evidence here in the rainbow trout. Date et al. (11) have reported the involvement of the vagal nerve in conveying a peripheral ghrelin signal to the brain in the rat. A similar neural pathway may exist in rainbow trout.

The present study demonstrated that rt ghrelin stimulates the release of GH in rainbow trout when administered to explanted rainbow trout pituitaries. Similarly, a recent paper reported that tilapia ghrelin stimulated the release of GH from their pituitaries (19). These data indicate that ghrelin plays a role as a GHS in fish. In cultured whole pituitary of rainbow trout, a significant increase in the release of GH was observed at 1 nM after 24-h incubation. In other studies, a relatively short period of time for incubation was sufficient to induce the release of GH by ghrelin from the whole pituitary (18, 19, 22), but ghrelin could still stimulate the release of GH after 24-h incubation as shown by Riley et al. (22). Our results are consistent with these findings and suggest a direct effect of ghrelin on rainbow trout pituitary.

In addition to GH regulation, PRL secretion was potently stimulated by rat, eel, and tilapia ghrelins in the cultured tilapia pituitary (18, 19, 22). Similar effects on PRL secretion have been observed in human in vivo and bullfrog in vitro (9, 20, 37). In contrast, no effects on the release of PRL and another pituitary hormone, SL, were observed in the present in vivo and in vitro studies. It is possible that GHS-R is not present in PRL and SL cells in rainbow trout. For clarifying this issue, further aspects need to be examined, for example, PRL or SL synthesis or expression of ghrelin receptor in these cells.

In summary, ghrelin mRNA is found in high levels in the stomach of rainbow trout, and ghrelin can be isolated from the stomach. Four isoforms of ghrelin are produced through the amidation of alternatively spliced ghrelin gene products. Furthermore, multiple types of acylation were observed, but all ghrelins were biologically active, as demonstrated by the assay using GHSR62 cells expressed rat GHS-R. The rt ghrelin gene is comprised of five exons and four introns, an arrangement identical to the rat and mouse ghrelin genes. Rainbow trout ghrelin stimulated the release of GH, but not PRL and SL both in vivo and in vitro. There are species-specific differences in the effects of ghrelin on the release of pituitary hormones in fish. Physiological functions of ghrelin in fish other than secretory regulation of pituitary hormone will be the next subject.


   Acknowledgments
 
We greatly appreciate Yasuo Kitajima, Masaru Matsumoto, and Yoshiharu Minamitake, the Daiichi Suntory Pharma Co., Ltd., Institute for Medicinal Research and Development for synthesizing ghrelin peptides.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.Kai., K.K., and H.Kaw.), a Grant-in-Aid for the Promotion of Fundamental Studies in Health Science from the Organization for Pharmaceutical Safety and Research of Japan, and a grant from Takeda Science Foundation (IBN 01-33714).

Abbreviations: Arc, Arcuate nuclei; BCM, basic culture medium; [Ca2+]i, intracellular calcium concentration; CHO, Chinese hamster ovary; CM, carboxymethyl; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; GSP, gene-specific primer; PRL, prolactin; rt ghrelin, rainbow trout ghrelin; RACE, rapid amplification of the cDNA ends; RP, reverse-phase; RT, reverse transcriptase; SL, somatolactin.

Received August 19, 2003.

Accepted for publication September 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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