Koji Murashita

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

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 (rt-leptin) 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.

Leptin and leptin receptor genes in Atlantic salmon: Cloning, phylogeny, tissue distribution and expression correlated to long-term feeding status.

The present study reports the complete coding sequences for two paralogues for leptin (sLepA1 and sLepA2) and leptin receptor (sLepR) in Atlantic salmon. The deduced 171-amino acid (aa) sequence of sLepA1 and 175 aa sequence for sLepA2 shows 71.6% identity to each other and clusters phylogenetically with teleost Lep type A, with 22.4% and 24.1% identity to human Lep. Both sLep proteins are predicted to consist of four helixes showing strong conservation of tertiary structure with other vertebrates. The highest mRNA levels for sLepA1 in fed fish (satiation ration=100%) were observed in the brain, white muscle, liver, and ovaries. In most tissues sLepA2 generally had a lower expression than sLepA1 except for the gastrointestinal tract (stomach and mid-gut) and kidney. Only one leptin receptor ortholog was identified and it shares 24.2% aa sequence similarity with human LepR, with stretches of highest sequence similarity corresponding to domains considered important for LepR signaling. The sLepR was abundantly expressed in the ovary, and was also high in the brain, pituitary, eye, gill, skin, visceral adipose tissue, belly flap, red muscle, kidney, and testis. Fish reared on a rationed feeding regime (60% of satiation) for 10 months grew less than control (100%) and tended to have a lower sLepA1 mRNA expression in the fat-depositing tissues visceral adipose tissue (p<0.05) and white muscle (n.s.). sLepA2 mRNA levels was very low in these tissues and feeding regime tended to affect its expression in an opposite manner. Expression in liver differed from that of the other tissues with a higher sLepA2 mRNA in the feed-rationed group (p<0.01). Plasma levels of sLep did not differ between fish fed restricted and full feeding regimes. No difference in brain sLepR mRNA levels was observed between fish fed reduced and full feeding regimes. This study in part supports that sLepA1 is involved in signaling the energy status in fat-depositing tissues in line with the mammalian model, whereas sLepA2 may possibly play important roles in the digestive tract and liver. At present, data on Lep in teleosts are too scarce to allow generalization about how the Lep system is influenced by tissue-specific energy status and, in turn, may regulate functions related to feed intake, growth, and adiposity in fish. In tetraploid species like Atlantic salmon, different Lep paralogues seems to serve different physiological roles.

Genomic characterization of multiple leptin genes and a leptin receptor gene in the Japanese medaka, Oryzias latipes.

We comprehensively surveyed leptin (LEP) and leptin receptor (LEPR) genes in medaka, Oryzias latipes and identified two LEP (mLEP-A and mLEP-B) genes and one LEPR (mLEPR) gene. The gene arrangement around both mLEPs in medaka chromosomes 6 and 23 were well conserved with human chromosome 7q31 including LEP. This means that both mLEP-A and mLEP-B are orthologs of human LEP and paralogs derived from whole-genome duplication early in the teleost lineage. The expression of mLEP-A mRNA was relatively high in the liver, and mLEP-B was expressed in the brain and eye. The 3-D modeling of both mLEP-A and mLEP-B protein showed conservation of the four-helix structure that is characteristic in vertebrate leptin. Human LEPR and leptin receptor overlapping the transcript (LEPROT) genes are continuously located on chromosome 1p31. In contrast, medaka LEPR and LEPROT are located on chromosomes 4 and 17, respectively, but both genomic regions showed genomic synteny with the human genome around the LEPR on chromosome 1p31. This result could mean that the medaka chromosome regions around the LEPR and LEPROT are paralogous genomic regions derived from whole-genome duplication, and that the overlapping gene of LEPR and LEPROT was subsequently lost in the medaka genome.

A homologous salmonid leptin radioimmunoassay indicates elevated plasma leptin levels during fasting of rainbow trout

The present study was conducted to establish a homologous radioimmunoassay (RIA) for quantifying plasma leptin (Lep) levels in salmonid species, and to study Lep levels in relation to nutritional status. A part of the Lep peptide, a 14 amino acid long sequence, identical between a Salmo and an Oncorhynchus species was synthesised. Polyclonal antibodies were raised in rabbit against this antigen and both were subsequently used in the development of a RIA protocol for assessing plasma Lep levels. The limit of detection of the assay was 0.3 nM, and intra- and interassay coefficient of variation (CV) were 8.4% and 13%, respectively. Apart from Atlantic salmon and rainbow trout, the assay exhibits measuring parallelism for a range of fish species, including arctic char, Atlantic cod and turbot, suggesting that the established RIA is useful for quantifying Lep levels in several fish species. The RIA indicates that Lep is found in salmonid plasma at levels of 0.5-5 nM, which is comparable with other peptide hormones, and well within the measuring range of the RIA. A study of fed and fasted rainbow trout showed elevated plasma Lep levels during fasting. In addition there was no correlation between Lep levels and condition factor. These data suggest that the relation between circulating Lep levels and energy status differs from that in mammals. While Lep is linked to energy balance, it may not act as an adiposity signal in salmonids, possibly pointing to functional divergence among ectothermic and endothermic vertebrates.

Ghrelin, cholecystokinin, and peptide YY in Atlantic salmon (Salmo salar) : Molecular cloning and tissue expression

Gastrointestinal (GI) peptide hormones, ghrelin (GHRL), cholecystokinin (CCK), and peptide YY (PYY) genes were identified in Atlantic salmon, Salmo salar. Full-length cDNAs encoding two isoforms of GHRL (GHRL-1 and GHRL-2), two isoforms of CCK (CCK-L and CCK-N) and peptide YY (PYY) cDNA were obtained. The GHRL-1 and GHRL-2 genes encoded proteins of 111- and 108-amino acids, respectively. Both types of GHRL were mainly expressed in the stomach, but also weakly expressed in the pyloric caeca, mid-gut, adipose tissue, and testis. The CCK-L and CCK-N genes encoded preproproteins of 132- and 140-amino acids, respectively. Both types of CCK were strongly expressed in the brain and comparatively weakly expressed in other tissues, including the digestive tract. In the digestive tract, CCK-L was mainly expressed in the pyloric caeca and hind-gut, while CCK-N was only expressed in the pyloric caeca. The PYY gene encoded for 97-amino acid residues and was mainly expressed in the brain and anterior part of the intestine, including the pyloric caeca. In an experiment, we demonstrated that 6 days starvation led to, increased GHRL-1 mRNA levels in the GI tract (stomach), while there no significant changes in expression levels for the other hormones in the GI tract. This suggests an orexigenic role for GHRL-1 in Atlantic salmon. These data contribute to elucidate the functional relationships among teleost gastrointestinal peptide hormones.

Leptin and ghrelin in anadromous Arctic charr: Cloning and change in expressions during a seasonal feeding cycle

Anadromous (sea-migrating) Arctic charr (Salvelinus alpinus) display pronounced seasonal variations in food intake and growth and is an interesting model for studying mechanisms of appetite regulation. In this study cDNAs encoding for ghrelin (GHRL) and leptin (LEP) in Arctic charr were cloned, after which stomach GHRL and liver LEP mRNA expressions were examined by qPCR during a seasonal feeding cycle of semi-wild anadromous Arctic charr. The fish were captured as they returned from summer feeding in seawater and transferred to an indoor tank where they were fed in excess until October the year after. Growth rate was low in late winter, increased in late spring and reached a peak during summer, and then declined during autumn, when the fish became sexually mature. The changes in growth rate were associated with corresponding changes in the proportion of fish that had been eating at each sampling date, and whole body lipid status. Stomach GHRL mRNA expression was high in late winter, decreased to a nadir in mid-summer and increased again to a high level in early autumn. Liver LEP mRNA remained low during winter, spring and early summer, after which there was a gradual, 7-fold increase until October. The seasonal changes in ghrelin and leptin support a role of these hormones in the long-term regulation of energy homeostasis in the anadromous Arctic charr. It cannot be excluded, however, that the increase in liver leptin expression during autumn is related to sexual maturation.

Characterization, tissue distribution, and regulation of agouti-related protein (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) in Atlantic salmon (Salmo salar).

Key peptide hormones involved in the control of appetite in vertebrates were identified, their genes characterized and their regulation studied in Atlantic salmon: two agouti-related proteins (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY). The AgRP-1 and AgRP-2 genes encode prepro-proteins of 142- and 117-amino acids, respectively. The deduced AgRP-2 protein has 10 cysteine residues in the C-terminal polycysteine domain, while the AgRP-1 lacks the 6th and 7th cysteine residues observed in other species. AgRP-1 was principally expressed in the pituitary and skin, while AgRP-2 was highly expressed in the mid-gut, red muscle and gonads. The CART gene, encoding 118-amino acids, was strongly expressed in the brain and eye. In addition to salmon CART, we identified three to six variants of the CART gene in lower vertebrates by mining available databases. The salmon NPY gene, encoding 100-amino acids, was mainly expressed in the brain and eye. AgRP-1 and CART mRNA levels in the brain decreased after 6 days of fasting while AgRP-2 and NPY showed no significant change, suggesting that AgRP-1 and CART are involved in feeding regulation in Atlantic salmon. The identification of multiple variants of these appetite-regulating genes emphasizes the importance to further investigate the complex regulation of these genes.

Leptin reduces Atlantic salmon growth through the central pro-opiomelanocortin pathway

Leptin (Lep) is a key factor for the energy homeostasis in mammals, but the available data of its role in teleosts are not conclusive. There are large sequence differences among mammalian and teleost Lep, both at the gene and protein level. Therefore, in order to characterize Lep function in fish, the use of species-specific Lep is crucial. In this study, the cDNA sequence of salmon leptin a1 (lepa1) was used to establish a production protocol for recombinant salmon LepA1 (rsLepA1) in Escherichia coli, that enabled a final yield of 1.7 mg pure protein L⁻¹ culture. The effects of 20-day administration of rsLepA1 on growth and brain neuroendocrine peptide gene expression [npy, cart, agrp (-1 and -2), pomc (-a1, -a2, -a2s, and -b)] were studied in juvenile, immature Atlantic salmon (96.5±2.1g) fed a commercial diet to satiation. Intraperitoneal osmotic pumps were used to deliver rsLepA1 at four different concentrations (calculated pumping rates were 0, 0.1, 1.0 and 10 ng g⁻¹ h⁻¹). In the highest dosage group (10 ng g⁻¹ h⁻¹), the growth rate was significantly reduced, and pomc-a1 gene expression was higher than in controls. The results support the lipostatic hypothesis and suggest that sLepA1 reduces growth in Atlantic salmon by affecting food intake through the central pro-opiomelanocortin pathway.

Postprandial effects on appetite-related neuropeptide expression in the brain of Atlantic salmon, Salmo salar

Following feeding of a single meal to Atlantic salmon, the temporal changes in the brain mRNA expression of neuropeptide y (npy), cocaine-amphetamine regulated transcript (cart), peptide yy (pyy), two isoforms of agouti-related protein (agrp), two isoforms of cholecystokinin (cck), and four isoforms of proopiomelanocortin (pomc) were assessed by q-PCR. In the course of 24h post-feeding (hpf), several of the brain neuropeptides displayed changes in mRNA expression compared to an unfed control group, indicating that food intake and processing affect the regulation of expression of these genes in Atlantic salmon. Expression of cart, cck-l, pomc-a1 and pomc-b all increased within 3h of feeding, while most of the feed was still in the stomach, suggesting that these neuropeptides play central anorexigenic roles similar to those described in higher vertebrates, including determining meal intervals. On the other hand, the npy and agrp isoforms which have been described as playing orexigenic roles in mammals, showed an opposite response in salmon and both were elevated in the first 3h after feeding. The different isoforms of cck, agrp and pomc had different mRNA expression patterns, which indicate specific roles related to feeding regulation. The minimal effect of feeding and digestion on pyy expression in the brain indicates that PYY plays a minor role in the central control of short-term food intake in Atlantic salmon.

Genomic characterization and tissue distribution of leptin receptor and leptin receptor overlapping transcript genes in the pufferfish, Takifugu rubripes

Full-length cDNAs encoding the leptin receptor (tfLEPR), leptin receptor overlapping transcript (tfLEPROT) and leptin receptor overlapping transcript-like 1 (tfLEPROTL1) were cloned and sequenced from the pufferfish, Takifugurubripes. The tfLEPR gene encoded an 1116-amino acid protein that includes almost all functionally important domains conserved among vertebrate LEPR such as three fibronectin type III domains, the immunoglobulin (Ig) C2-like domain and a pair of repeated tryptophan/serine motifs. The tfLEPR mRNA was abundantly expressed in the pituitary and ovary and moderately expressed in brain, eye, heart, kidney, liver and testis. Both tfLEPROT and tfLEPROTL1 genes encoded a 130-amino acid protein. Human LEPR gene shares the first and second exons with the LEPROT gene, and they are continuously located on chromosome 1p31. In contrast, TakifuguLEPR and LEPROT were located at different regions of the chromosome. However, both Takifugu regions showed genomic synteny with the human genome around LEPR gene on chromosome 1p31. This result could mean that the Takifugu chromosomes around LEPR and LEPROT genes are paralogous genomic regions derived from genome duplication early in the teleost lineage and the overlapping LEPR and LEPROT genes were subsequently lost.