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1 Departments of Biochemistry and
2 Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
(Requests for offprints should be addressed to C H K Cheng; Email: chkcheng{at}cuhk.edu.hk)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies on the isolation of ghrelin from fish species have indicated that the function of ghrelin in stimulating GH release from the pituitary appears to be conserved from fish to mammals (Kojima et al. 1999, Kaiya et al. 2003b, 2003c). Moreover, it has been demonstrated that ghrelin is capable of stimulating food intake in goldfish (Unniappan et al. 2002). These findings indicate that a ghrelin/GHSR system is also operative in fish. However, the cognate GHSR has not been cloned and characterized from those fish species in which their ghrelins have been isolated or studied. To date, fish GHSR has only been reported in two species, the pufferfish Spheroides nephelus (Palyha et al. 2000) and the black seabream Acanthopagrus schlegeli (Chan & Cheng 2004), and is functionally characterized in black seabream only (Chan & Cheng 2004, Chan et al. 2004b). The putative ghrelin gene present in pufferfish, however, has not yet been isolated. We have recently identified and characterized two full-length GHSR cDNAs from the pituitary of the black seabream and demonstrated that these two receptor transcripts are derived from alternative splicing of a single GHSR gene (Chan & Cheng 2004). Herein we report the cloning and characterization of the ghrelin gene from the same fish species. Moreover, we have further demonstrated homologous upregulation of ghrelin expression in the seabream stomach. This work represents the first study on a teleost in which both the GHSR and ghrelin genes have been cloned.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial attempts to clone the seabream ghrelin cDNA failed because of the difficulty in designing effective primer pairs for reverse transcriptase (RT)-PCR owing to the small size of the peptide. A different approach was subsequently adopted and this involved the design of two overlapping forward degenerate primers from a single conserved region of ghrelin. A cDNA fragment towards the 3'-end of seabream ghrelin was obtained using these primers together with an oligo-dT primer in a 3'-rapid amplification of cDNA ends (RACE) approach. Subsequently, gene-specific reverse primers were designed from this partial sequence, allowing us to obtain the rest of the ghrelin cDNA using a library PCR approach. The experimental details are described below. Total RNA was first prepared from seabream stomach using TriPure Isolation Reagent (Roche Applied Science). First-strand cDNA was then made from 4 µg total RNA in the reverse transcription reaction. The reaction was carried out at 42 °C for 50 min using Superscript II RT (Invitrogen). Based on previously published fish ghrelin sequences (Unniappan et al. 2002, Kaiya et al. 2003c, Parhar et al. 2003), two degenerate primers, FGD and GTD (see Table 1
for their primer sequences), were designed and employed together with an oligo-dT primer in the first-round and nested PCR, respectively.
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), were designed from the partial cDNA sequence and employed to amplify from a seabream stomach cDNA library constructed in the pCMV·SPORT6 vector (Invitrogen) in order to obtain the 5'-end sequence of the ghrelin cDNA. SGR1 and SGR2 were used together with the primer M13-R in the first round and nested PCR, respectively. The PCR conditions were the same as described above except that the annealing temperature was at 56 °C. The nested PCR yielded a distinct product with a size of about 400 bp, which was subsequently cloned and sequenced. Finally, a pair of gene-specific primers, SGFL-F and SGFL-R (see Table 1), were designed and used to amplify the full-length ghrelin sequence from the seabream stomach cDNA library. This final confirmation step was essential to demonstrate that the composite sequence obtained from the two separate fragments truly represents the actual full-length ghrelin sequence. Also, the nucleotide sequence of each PCR product obtained above was confirmed by the sequencing of at least five independent clones in each case. Determination of the structural organization of the seabream ghrelin gene
Genomic DNA was extracted from seabream liver as previously described (Chan & Cheng 2004). Briefly, frozen seabream liver was homogenized in lysis buffer (10 mM Tris/HCl, pH 8.0, 50 mM EDTA and 0.5% SDS), followed by proteinase K digestion. The genomic DNA was then purified by phenol/chloroform extraction and ethanol precipitation. The gene-specific primers SGFL-F and SGFL-R were used to amplify the entire ghrelin gene in the genomic PCR. The PCR conditions were as follows: 5 min at 94 °C for initial denaturation, followed by 36 cycles of 1 min at 94 °C (denaturation), 1 min at 55 °C (annealing), and 2 min at 72 °C (extension), and then 7 min at 72 °C for the final extension. The PCR product was cloned and sequenced from both strands. The complete genomic sequence was obtained and confirmed by sequencing of at least five independent clones.
Isolation of the 5'-flanking region of the seabream ghrelin gene by genome walking
The 5'-flanking region of the ghrelin gene was isolated using the Universal GenomeWalker Kit (BD Biosciences Clontech, Palo Alto, CA, USA). Five GenomeWalker libraries (independently generated by cutting with the following restriction enzymes: DraI, EcoRV, PvuII, ScaI, and StuI) were constructed according to the manufacturer’s instructions. First-round PCR was carried out using the adaptor primer 1 (AP1) and the gene-specific primer SGW-P (used in the first genome-walking experiment) or SGW-4 (used in the second genome-walking experiment; see Table 1). The PCR conditions were as follows: 3 min at 94 °C for initial denaturation, followed by 38 cycles of 30 s at 94 °C (denaturation), 30 s at 62 °C (annealing), and 3 min at 72 °C (extension) for 38 cycles, and then 7 min at 72 °C for final extension. Nested PCR was performed using the adaptor primer 2 (AP2) and the gene-specific primer SGW-N (used in the first genome-walking experiment) or SGW-5 (used in the second genome-walking experiment; see Table 1) under the same conditions except that the number of cycles was reduced to 35. The nucleotide sequences of the PCR products obtained above were confirmed by sequencing of at least five independent clones in each case.
Construction of reporter gene plasmids
The 5'-flanking region of the seabream ghrelin gene was amplified by PCR using SBG-PRO-F1 and SBG-PRO-R1 as primers and 700 ng seabream genomic DNA as the template. Subsequent to its sequence determination, the amplicon of the PCR was then cloned into the promoterless pGL3-Basic vector (Promega) to generate the construct p( – 2230/+1). Another reporter construct (p(+1/ – 2230)) containing the same 5'-flanking region as p( – 2230/+1) but in the reverse direction was made in parallel. Deletion constructs with successive removal of the 5'-region were generated by PCR using the primers SBG-PRO-F2, SBG-PRO-F3, SBG-PRO-F4 and SBG-PRO-R1 (Table 1) to generate the constructs p( – 1614/ +1), p( – 1422/+1), and p( – 1289/+1). Reporter constructs p( – 894/+1) and p( – 450/+1) were made by digesting the 5'-flanking region amplified from SBG-PRO-F1 and SBG-PRO-R1 with SacI and NheI respectively. All plasmid DNA preparations were isolated using the QIAprep Spin Miniprep Kit (Qiagen).
Cell culture, transfection and promoter assay
Cell culture materials were purchased from Invitrogen unless otherwise stated. Cell lines were purchased from ATCC (Manassas, VA, USA; NIH-3T3, HepG2, HEK-293, Caco-2) and RIKEN Bioresource Center (Ibaraki, Japan; ECC10, GAKS). NIH-3T3, HepG2, and HEK-293 cells were maintained at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Caco-2 cells were kept in DMEM at 37 °C containing non-essential amino acids and 10% FBS. GAKS cells were maintained at 37 °C in DMEM containing 5% FBS. ECC10 cells were maintained in RPMI containing 10% FBS at 37 °C. All media were supplemented with antibiotics (10 U/ml penicillin and 100 µg/ml streptomycin).
In the transfection experiments, Lipofectamine reagent (Invitrogen) was used as recommended by the manufacturer. Twenty hours before transfection, 1.5 x 105 cells/well were seeded onto 24-well tissue-culture plates. Five hundred ng seabream ghrelin promoter-reporter constructs and 50 ng pRL-CMV (for normalization of transfection efficiency) containing Renilla luciferase reporter gene were co-transfected into the cells in 250 µl serum-free medium. After 5 h of transfection, the reaction mixture was replaced with 20% FBS and the cells were incubated for 24 h. The firefly luciferase activities driven by various seabream ghrelin promoter regions were assayed using the Dual-Luciferase Assay System (Promega).
Analysis of ghrelin gene expression in seabream tissues by RT-PCR
First-strand cDNA was prepared from various tissues as described above. The gene-specific primers SGFL-F and SGFL-R were used in the RT-PCR to amplify the full-length ghrelin transcript. The PCR conditions were as follows: 5 min at 94 °C for initial denaturation, followed by 36 cycles of 1 min at 94 °C (denaturation), 1 min at 55 °C (annealing), and 1 min at 72 °C (extension), and then 7 min at 72 °C for the final extension. Mock RT controls were performed to exclude the possibility of genomic DNA contamination in the RNA preparations. Moreover, PCR using a pair of ß-actin primers, Actin-F and Actin-R (see Table 1), was also performed in parallel to indicate the quality of the cDNA templates synthesized. The PCR conditions were as follows: 5 min at 94 °C for initial denaturation, followed by 26 cycles of 30 s at 94 °C (denaturation), 30 s at 58 °C (annealing), and 30 s at 72 °C (extension), and then 7 min at 72 °C for the final extension.
Regulation of ghrelin expression in seabream stomach in vivo
Black seabream (body length, 24.3 ± 0.5 cm; body weight, 311.5 ± 21.7 g) of mixed sex purchased from a local marine fish farm were kept in fully aerated seawater at ambient temperature and fed ad libitum once daily with commercial diet. Fishes were injected with either synthetic ipamorelin (NeoMPS, Strasbourg, France) or recombinant seabream GH (GroPep, Thebarton, Australia) intraperitoneally once daily for 4 days. Physiological saline was injected in parallel as the control group. The fishes were then killed by decapitation and their stomachs removed and frozen in liquid nitrogen. The tissue was kept at –70 °C until further analysis.
RT-PCR was carried out to assess the expression of ghrelin in seabream stomach as described above. The number of cycles used in each case was determined first by a validation test in which the PCR was performed as described but terminated at different cycle numbers. A kinetic profile of the amount of PCR product generated at different cycle numbers was constructed and the cycle number to be adopted for the gene-expression assessment was chosen within the linear portion of the amplification curve. This validation step is essential to ensure that the amount of the PCR product reflects the amount of template in the original sample. The PCR products were then resolved on an 1.5% agarose gel and stained with ethidium bromide. The intensity of the amplicon was determined by densitometric scanning using Multi-Analyst software (Bio-Rad).
Statistical analyses
Where applicable data were analysed by one-way analysis of variance followed by Tukey’s test using PRISM software (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant when P< 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using degenerate primers (FGD and GTD) designed from the most conserved region of ghrelins (i.e. the core region of the mature peptide, G(S/T)SFLSP) together with an oligo-dT primer, a partial seabream ghrelin cDNA of about 800 bp was obtained in a 3'-RACE approach. Subsequently, two gene-specific reverse primers (SGR1 and SGR2) were designed and employed to isolate the 5'-end sequence from a stomach cDNA library by a library PCR approach. Finally, the full-length ghrelin cDNA (Fig. 1) was amplified from the same library using two specific primers (SGFL-F and SGFL-R) designed at both ends of the full-length sequence. The complete cDNA was found to encode 107 amino acids, with 56 bp in the 5'-untranslated region (UTR) and 564 bp in the 3'-UTR. Amino acid sequence comparison of the sea-bream ghrelin with other vertebrate ghrelins (Fig. 2) indicates that it shares the highest homology with the tilapia ghrelin (Fig. 3).
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Structural organization of the seabream ghrelin gene
The structural organization of the seabream ghrelin gene was determined by genomic PCR using the two gene-specific primers SGFL-F and SGFL-R. Sequence analysis of the amplified product (Fig. 4A) revealed that the ghrelin gene in black seabream consists of four exons and three introns (Fig. 4B). The DNA sequences of the exon/intron junctions are shown in Table 2. The exon/intron junctions were found to conform to the GT/AG rule (Chabot 1996). Exon 1 (170 bp) encodes the 5'-UTR and 38 amino acids (including the first 12 amino acids of the mature ghrelin peptide). Exon 2 (78 bp) is the smallest exon that encodes 26 amino acids (including the remaining eight amino acids of the mature ghrelin peptide). Exon 3 (112 bp) encodes 37 amino acids (including the putative obestatin peptide) and exon 4 (581 bp) encodes the last six amino acids and the 3'-UTR.
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As a first step towards understanding the transcriptional regulation of the seabream ghrelin gene, the 5'-flanking region of the gene was also isolated in the present study. In the first genome-walking experiment, the longest PCR product (~800 bp) was obtained from the PvuII GenomeWalker library. In order to isolate a longer 5'-flanking region for transcriptional analysis, a second genome walking was performed using the two gene-specific primers SGW-4 (for first-round PCR) and SGW-5 (for nested PCR). Distinct PCR products of sizes of about 1500 bp were obtained from both the StuI and EcoRV GenomeWalker libraries. The PCR products from the StuI library were subsequently cloned and sequenced. From the two consecutive and overlapping genome walks, a 2230-bp 5'-flanking region of the seabream ghrelin gene was finally isolated. Sequence analysis using TFSEARCH software (http://molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html) revealed a number of putative transcription factor-binding sites on the 5'-flanking region of the seabream ghrelin gene (Fig. 5).
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The promoter activity of the 5'-flanking region of the seabream ghrelin gene was then tested by transfecting the plasmid p( – 2230/+1) into various cell lines and measuring the putative promoter-driven luciferase activity. A 6-fold increase of luciferase activity over and above the promoter-less control (pGL3-Basic) was recorded in the colon epithelial Caco-2 cells (Fig. 6A). This result suggested that the cloned 5'-flanking region represents the functional promoter of the seabream ghrelin gene. Moreover, this promoter activity was orientation-dependent as no detectable luciferase activity could be measured in Caco-2 cells transfected with the construct p(+1/ – 2230), which has an identical nucleotide sequence to p( – 2230/+1) but in a reverse orientation. Furthermore, the seabream ghrelin promoter is cell-type-specific as no promoter activity could be detected in the other cell lines originated from embryo fibroblast (NIH-3T3), liver (HepG2), kidney (HEK-293) and stomach (ECC10), nor from fish scale fibroblast (GAKS; Fig. 6B).
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Analysis of ghrelin gene expression in seabream tissues by RT-PCR
The expression of the ghrelin gene in various seabream tissues was analyzed by RT-PCR using a pair of gene-specific primers (SGFL-F and SGFL-R) that amplified the full-length transcript (941 bp). Using this pair of primers, which span three introns of the seabream ghrelin gene, we could only obtain a single distinct band of about 950 bp on the gel, indicating that there was no genomic DNA present in the first-strand cDNA samples since any such contamination would give rise to a much larger amplicon of 1771 bp. This result was in line with the mock RT-PCR controls where no bands were observed. Ghrelin mRNA expression was only detected in the stomach but not in all the other tissues examined including the brain, gill, intestine, kidney, liver and spleen (Fig. 7). A control PCR using two ß-actin primers (Actin-F and Actin-R) was also performed. The ß-actin transcripts (with the expected size of 212 bp) were detected in all the tissues examined (Fig. 7), indicating that the absence of a ghrelin transcript in most tissues could not be attributed to the quality of the cDNA templates.
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Hormonal regulation of ghrelin expression in seabream stomach was also studied in vivo. As shown in Fig. 8, ghrelin expression could be enhanced in fish injected with recombinant seabream GH or with the synthetic GH secretagogue ipamorelin in a dose-dependent manner. A significant increase (P< 0.05) in ghrelin expression was observed when 1 µg/g seabream GH or 100 ng/g ipamorelin were administered to the fish.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial attempts to design degenerate primers from known ghrelin sequences had encountered difficulties because there is only a single conserved region within the short sequence where primers could be reasonably designed. Thus the conventional method of RT-PCR using a pair of degenerate primers failed. Alternatively we have adopted a novel method in the present study. This entails the design and deployment of two overlapping forward degenerate primers from the single conserved region to obtain a cDNA fragment of the target by a nested 3'-RACE approach. This has successfully generated a cDNA fragment from which gene-specific reverse primers could be designed to enable the successful cloning of the 5'-end sequence using a library PCR approach in a nested manner. This method, which we have successfully employed in obtaining the full-length seabream ghrelin cDNA in the present study, could be equally applicable to other target genes where the design of degenerate primers is severely restricted by the limited conserved regions among the known sequences.
The seabream ghrelin cDNA encodes a total of 107 amino acids, with the predicted mature peptide consisting of 20 amino acids (Fig. 1). Protein sequence alignment of different vertebrate ghrelins (Fig. 2) reveals that the seabream ghrelin shares the highest homology with the tilapia counterpart, as indicated by the phylogenetic tree shown in Fig. 3. This finding of a close evolutionary relationship between seabream and tilapia is in agreement with phylogenetic analysis of other fish hormones such as the type II form of gonadotropin-releasing hormone (Ikemoto & Park 2003). In fact, a similar evolutionary relationship among teleost species is observed in the present and other studies (Zhu et al. 1999, Nilsson et al. 2001, Ikemoto & Park 2003), indicating that seabream is more closely related to tilapia than to rainbow trout, and that goldfish is more distantly related to these fish species (Fig. 3). The structural organization of the seabream ghrelin gene (Fig. 4) is also similar to that of tilapia (Parhar et al. 2003). The sizes of the cloned seabream and tilapia ghrelin genes are 1771 and 1416 bp, respectively. Both genes are consisted of four exons and three introns. This pattern is in fact similar to that of goldfish, in which the genome size is only 980 bp (Unniappan et al. 2002). However, the genomic organization in rainbow trout (Kaiya et al. 2003a) is different from that in other known fish species. It is consisted of five exons and four introns, which is more similar to that of the mouse ghrelin gene (Tanaka et al. 2001).
It is interesting to note that gene duplication has been observed for some components of the somatotropic axis in certain teleosts. For example, it has been reported that tilapia has two GH genes (Ber & Daniel 1993). Cardoso et al.(2003) also suggested the presence of two GH-releasing hormone receptor genes in the Fugu rubripes genome. Furthermore, we have recently reported the identification of two GH receptors from the black seabream (Jiao et al. 2006). From the present study however, there is no evidence for the existence of multiple ghrelin genes in seabream. Whether ghrelin gene duplication occurred during teleost evolution awaits further investigation. In this context though, it is interesting to note that we have previously shown that there exists only a single gene for GHSR in the seabream genome (Chan & Cheng 2004).
Although the ghrelin genes have been previously identified from a number of fish species, little is known about their promoter structures and functions. The transcriptional regulation of the teleostean ghrelin genes therefore remains poorly understood. To date, the 5'-flanking regions of the ghrelin genes from human and rat only have been isolated and characterized (Kishimoto et al. 2003, Kanamoto et al. 2004, Wei et al. 2005). Hence in the present study we have isolated a 2230-bp 5'-flanking region of the seabream ghrelin gene (Fig. 5) as a first step towards understanding its transcriptional regulation. Our results revealed that, similar to the human situation (Kishimoto et al. 2003), there is neither a GC box nor a CAAT box present in the isolated 5'-flanking region. A putative transcription start site is located at position –56 (Fig. 5), corresponding to the most 5'-end of the ghrelin transcripts (Fig. 1) identified from the seabream stomach cDNA library. Although a TATATAA element was found in the human ghrelin gene, deletion and mutation studies have shown that it is not important for promoter activity (Kishimoto et al. 2003). However, the seabream ghrelin gene does not contain any TATA-like element upstream of the putative transcription start site (Fig. 5). These observations therefore suggest that both mammalian and fish ghrelin genes are TATA-less. Incidentally this is the same as in the GHSR genes (Kaji et al. 1998, Petersenn et al. 2001, Tanaka et al. 2003, Yeung et al. 2004). However, the 5'-flanking region of the seabream ghrelin gene is found to contain a number of putative transcription factor-binding sites (Fig. 5) different from the human counterpart (Kishimoto et al. 2003), indicating that they are probably regulated in different manners. The functional analysis of the seabream ghrelin promoter gave further support to this notion since its cell-type specificity is different from that of human. The human ghrelin promoter was active in ECC10 cells (Kishimoto et al. 2003) but the seabream ghrelin promoter was totally inactive in this cell type. Instead, the seabream promoter exhibited strong activity in human colon epithelial adenocacinoma Caco-2 cells (Fig. 6). Limited by the unavailability of intestine-derived cell line from teleost species, it is not known whether the seabream promoter is also active in the same tissue type in fish. Nevertheless, no promoter activity was recorded in the fish cell line GAKS derived from scale epthelial cells.
Another difference between fish and human ghrelin genes is that there is only one transcription start site identified in seabream that is different from the situation in human where two transcription start sites were found (Kanamoto et al. 2004, Wei et al. 2005). Moreover, studies have suggested that E-box elements are involved in the promoter activation of the human ghrelin gene (Kanamoto et al. 2004) but no E-box elements were found in the seabream ghrelin promoter. Although ghrelin expression in both human and rat could be modulated by upstream stimulatory factor and glucagon stimulation, the regulation of ghrelin expression varied significantly between human and rat even though a 65% sequence identity was found between the core promoter regions of the human and rat ghrelin genes (Wei et al. 2005). These observations further suggest that the regulatory mechanisms of ghrelin expression are very different among species. Further studies on the transcriptional control of ghrelin expression in fish are highly warranted.
The expression of ghrelin and its receptor (GHSR) have been demonstrated in a number of human cells and tissues including immune cells, thyroid gland, pancreas, spleen, myocardium, adrenal gland, and ovary (Hattori et al. 2001, Gnanapavan et al. 2002, Volante et al. 2002, Gaytan et al. 2003). Their expression is also found in the mouse kidney (Mori et al. 2000) and embryo (Kawamura et al. 2003), and rat testis (Tena-Sempere et al. 2002, Barreiro et al. 2003) and adrenal (Barreiro et al. 2002, Andreis et al. 2003). These findings indicate that ghrelin is widely expressed and may play important physiological roles in different mammalian tissues. However, a recent study has shown that ghrelin is not critically required for the viability, fertility, growth, appetite, bone density, and fat deposition in a ghrelin-null mouse model (Sun et al. 2003). In spite of the diverse physiological functions that have been proposed for ghrelin in mammals, the ghrelin-null mice are visibly healthy and could reproduce normally (Sun et al. 2003). This study suggested that compensatory mechanisms probably accommodate some of the actions of ghrelin in the mutant mice. By analyzing ghrelin-deficient mice, another study (Wortley et al. 2004) has also indicated that endogenous ghrelin is not an essential regulator of food intake. However, it has been demonstrated that ghrelin plays a prominent role in determining the type of metabolic substrate employed for maintenance of energy balance (Wortley et al. 2004). More recently, another hormone called obestatin was found to be encoded by the ghrelin gene (Zhang et al. 2005). Obestatin appears to act as an anorexic hormone by decreasing food intake via activation of an orphan G-protein-coupled receptor GPR39. It has therefore been suggested that the reason why mutant mice with a deletion of the ghrelin gene do not exhibit impaired appetite or growth is that these animals lack both the orexigenic ghrelin and the anorexic obestatin (Zhang et al. 2005). Although no report has been published so far on the identification of teleostean obestatin, our sequence alignment shown in Fig. 2 indicates a putative region of low similarity to mammalian obestatin in the teleostean ghrelin prepropeptide, suggesting that an orthologue of mammalian obestatin might be present in fish. Further studies to substantiate this possibility are highly warranted. Previous studies have indicated that the function of ghrelin in stimulating GH release from the pituitary appears to be conserved from fish to mammals (Kaiya et al. 2003b, 2003c, Parhar et al. 2003). In particular, ghrelin has been shown to be capable of stimulating food intake in goldfish (Unniappan et al. 2002). The expression of ghrelin mRNA has also been demonstrated in the kidney and gill of some fish species (Kaiya et al. 2003b, 2003c), suggesting that it may have an autocrine or paracrine function in these organs. In the present study, ghrelin was found to be expressed significantly in the seabream stomach (Fig. 7). This result is similar to the findings in Nile tilapia (Parhar et al. 2003), rainbow trout (Kaiya et al. 2003a), eel (Kaiya et al. 2003b), Mozambique tilapia (Kaiya et al. 2003c) and catfish (Kaiya et al. 2005), but somewhat different from that in goldfish (Unniappan et al. 2002, Unniappan & Peter 2005). Similar to the situation in Nile tilapia (Parhar et al. 2003), we did not detect ghrelin expression in all the other tissues examined. This might be due to a low expression level beyond the detection limit of RT-PCR. Actually, in eel and Mozambique tilapia, the authors could only detect ghrelin expression in the brain and gill of Mozambique tilapia and in other tissues of the eel using an extended number of PCR cycles. When a more sensitive method of real-time PCR was used, ghrelin expression could be detected in various tissues at very low levels in catfish (Kaiya et al. 2005). Further investigations on the expression of ghrelin using a more sensitive and quantitative method would be warranted to study the tissue-specific and developmental pattern of expression in seabream. In light of our previous finding that the seabream GHSR is mainly expressed in the central nervous system but not in the peripheral tissues (Chan & Cheng 2004), it is envisaged that ghrelin secreted from the seabream stomach might have to traverse the blood–brain barrier to exert its physiological actions on the brain.
It has been demonstrated that ghrelin expression in the mammalian stomach is regulated by nutritional and energy status (Kim et al. 2003, Hanada et al. 2004, Sanchez et al. 2004). Moreover, it was found that the ghrelin expression in the rodent stomach was upregulated by estradiol, leptin and insulin (Toshinai et al. 2001, Matsubara et al. 2004), and downregulated in hyperthyroidism (Caminos et al. 2002). In fish, ghrelin expression was reported to be regulated by feeding conditions in which starvation increased ghrelin mRNA content in goldfish (Unniappan et al. 2004). This regulation of expression is sex-dependent in that an elevated ghrelin mRNA level could only be detected in the female cichlid fish (Parhar et al. 2003). However, information regarding its regulation of expression by other hormones is not available in any fish species. In the present study, we have demonstrated that ghrelin expression in the seabream stomach could be upregulated by both GH and the synthetic GH secretagogue ipamorelin (Fig. 8). This is indeed the first report on the homologous regulatory nature of this peptide in teleosts. While the physiological significance of this homologous upregulation in fish is not understood at present, conflicting results have been reported in mammals. Qi et al.(2003) have reported that GH exerts a negative-feedback effect on stomach ghrelin production in rats. On the other hand, however, Nass et al.(2004) have provided evidence that chronic changes in peripheral GH levels do not affect ghrelin stomach mRNA expression in mice.
Taking into consideration that ipamorelin is a potent GH secretagogue of GH secretion in seabream (Chan et al. 2004a) and that injection of GH in vivo could also upregulate ghrelin expression in the seabream stomach, it is possible that this homologous upregulation is GH-mediated. However, the other possibility that ipamorelin exerts a direct action in stimulating ghrelin expression in stomach could not be excluded since both ghrelin and GHSR could be detected in the seabream stomach (Chan & Cheng 2004). Further studies are needed to dissect the exact role of ipamorelin and GH in controlling the expression of ghrelin in the fish stomach. Interestingly, we have previously reported that the seabream GHSR promoter could be enhanced by another GH secretagogue (Yeung et al. 2004), indicating that this homologous upregulation mechanism is applicable to both the ligand and the receptor of the ghrelin/GHSR system in seabream.
In summary, we have isolated the cDNA and gene for ghrelin in a fish species in which its cognate receptor has been identified. Moreover, we also isolated and characterized the first ghrelin gene promoter in teleost. Similar to its receptor, the expression of seabream ghrelin could be enhanced by a GH secretagogue analogue. The identification of both the GHSR and ghrelin genes from seabream would facilitate our subsequent studies on the physiological functions and regulation of the fish ghrelin/GHSR system.
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Acknowledgements |
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References |
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Received in final form 17 December 2005
Accepted 2 February 2006
Made available online as an Accepted Preprint 2 February 2006
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