Ipamorelin and GH in vivo upregulate stomach-specific ghrelin expression in black seabream
Background
Ghrelin is a stomach-derived peptide that drives growth hormone (GH) release through the GHSR (growth hormone secretagogue receptor). Understanding how ghrelin gene expression is controlled is central to dissecting the somatotropic axis in vertebrates, yet promoter architecture and tissue-specific regulation in teleost fish remained poorly defined. The authors had previously cloned two GHSR subtypes from black seabream (Acanthopagrus schlegeli), making this species an ideal single-species model for mapping the full ghrelin/GHSR signalling system. A gap existed in linking receptor characterisation to endogenous ligand gene structure and transcriptional control in non-mammalian vertebrates.
Study Design
Ghrelin cDNA was isolated from black seabream (Acanthopagrus schlegeli) stomach using a conserved coding-region PCR strategy. Genomic structure was resolved by genomic PCR. A 2230-bp 5'-flanking region was obtained by genome walking, cloned upstream of a luciferase reporter in pGL3-Basic, and transfected into multiple cell lines to assess promoter activity. Tissue expression was profiled across stomach, brain, gill, intestine, kidney, liver and spleen by RT-PCR. Regulatory response was tested by administering GH or ipamorelin to fish in vivo and measuring stomach ghrelin expression.
Results
The full-length seabream ghrelin cDNA encodes a 107-amino-acid prepropeptide, with a predicted mature peptide of 20 amino acids (GSSFLSPSQKPQNRGKSSRV). Embedded within the cDNA is a putative fish orthologue of the mammalian obestatin peptide. The ghrelin gene spans four exons and three introns, sharing structural organisation with tilapia and goldfish but differing from rainbow trout. The 2230-bp 5'-flanking region lacks both a GC box and a CAAT box, paralleling the human ghrelin gene promoter architecture, yet harbours a distinct set of putative transcription factor-binding sites, indicating divergent cis- and trans-acting regulatory elements.
Luciferase reporter activity was detected only in colon-derived Caco-2 cells, confirming tissue-specific promoter function;
RT-PCRshowed ghrelin expression exclusively in the stomach across all tissues examined.
In vivo administration of either GH or ipamorelin enhanced stomach ghrelin expression, mirroring the previously reported homologous upregulation of the seabream GHSR gene.
Key Findings
- Mature seabream ghrelin peptide is 20 amino acids: GSSFLSPSQKPQNRGKSSRV
- Prepropeptide precursor encodes 107 amino acids including a putative obestatin orthologue
- Gene structure: 4 exons, 3 introns — conserved with tilapia/goldfish, distinct from rainbow trout
- 2230-bp 5'-flanking region drives luciferase activity only in Caco-2 colon-derived cells
- In vivo GH and ipamorelin administration each enhance stomach-specific ghrelin expression
Why It Matters
Both ghrelin and its receptor are now fully characterised in a single teleost species, enabling controlled mechanistic studies of the ghrelin/GHSR axis without cross-species extrapolation. The finding that ipamorelin enhances endogenous ghrelin transcription in vivo suggests a positive-feedback loop between GH secretagogue administration and ghrelin gene expression — a dynamic relevant to anyone using ipamorelin to modulate the GH axis. Clinicians and researchers should note that ghrelin upregulation after ipamorelin dosing could amplify downstream GH pulses beyond the receptor-level effect alone. The identification of a putative obestatin sequence in fish also opens comparative avenues for studying appetite and energy-balance peptides across vertebrates.
