Effect of oral administration of a single bolus of six different protein sources on digestive physiology of red seabream Pagrus major juveniles.

To reveal direct effects of various protein sources on digestive physiology of red seabream, Pagrus major (38.5 ± 0.4 g), six different protein sources of fishmeal (FM), soybean meal (SBM), corn gluten meal (CGM), soy protein concentrate (SPC), poultry by-product meal (PBM), and poultry-feather meal (PFM) were orally administered to fish (2 mg protein/g body weight) and sampled at 1.5 h and 3 h after administration. Gallbladder weight of fish administered FM, PBM, and PFM decreased after administration (p < 0.0001), while no difference was observed in the other ingredients compared to a non-protein sham control group, indicating that animal protein sources could more strongly stimulate bile secretion than plant protein sources in red seabream. Trypsin and chymotrypsin activity in the intestinal content markedly increased by the FM, SBM, and PFM administration (p < 0.0001). Lipase and amylase activity was also increased by FM and SBM but also by CGM for lipase and by PBM and PFM for amylase (p < 0.0001). These indicate that stimulation effect of the secretion of digestive enzymes is largely different among the protein sources. This might be due to the absorptive capacity of the protein source since intestinal absorption parameter genes (anpep, cpa, ggt1, and atp1a2) also increased by the FM, SBM, PBM or PFM (p < 0.05). In addition to the secretion levels of bile and digestive enzymes, gene expression levels of bile related genes (cyp7a1, cyp8b1, and shp) and digestion-regulating genes (casr and cck) were increased by the FM, SBM, PFM, and/or PBM administration, suggesting that animal proteins and SBM could be potent digestive stimulants compared to CGM and SPC. This study first revealed that single protein sources directly influence digestive enzyme secretion and bile secretion in fish. Information about the direct effect of each single source on digestive physiology could help to design feed formulation with less fishmeal.

Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters.

The high-affinity/low-capacity system Slc15a2 (PepT2) is responsible for the reuptake of di/tripeptides from the renal proximal tubule, but it also operates in many other tissues and organs. Information regarding PepT2 in teleost fish is limited and, to date, functional data are available from the zebrafish (Danio rerio) only. Here, we report the identification of two slc15a2 genes in the Atlantic salmon (Salmo salar) genome, namely slc15a2a and slc15a2b. The two encoded PepT2 proteins share 87% identity and resemble both structurally and functionally the canonical vertebrate PepT2 system. The mRNA tissue distribution analyses reveal a widespread distribution of slc15a2a transcripts, being more abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and the distal part of the gastrointestinal tract. The function of the two transporters was investigated by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp recordings of transport and presteady-state currents. Both PepT2a and PepT2b in the presence of Gly-Gln elicit pH-dependent and Na+ independent inward currents. The biophysical and kinetic analysis of the recorded currents defined the transport properties, confirming that the two Atlantic salmon PepT2 proteins behave as high-affinity/low-capacity transporters. The recent structures and the previous kinetic schemes of rat and human PepT2 qualitatively account for the characteristics of the two Atlantic salmon proteins. This study is the first to report on the functional expression of two PepT2-type transporters that operate in the same vertebrate organism as a result of (a) gene duplication process(es). KEY POINTS: Two slc15a2-type genes, slc15a2a and slc15a2b coding for PepT2-type peptide transporters were found in the Atlantic salmon. slc15a2a transcripts, widely distributed in the fish tissues, are abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and distal gastrointestinal tract. Amino acids involved in vertebrate Slc15 transport function are conserved in PepT2a and PepT2b proteins. Detailed kinetic analysis indicates that both PepT2a and PepT2b operate as high-affinity transporters. The kinetic schemes and structures proposed for the mammalian models of PepT2 are suitable to explain the function of the two Atlantic salmon transporters.

Regional Expression of npy mRNA Paralogs in the Brain of Atlantic Salmon (Salmo salar, L.) and Response to Fasting.

Neuropeptide Y (NPY) is known as a potent orexigenic signal in vertebrates, but its role in Atlantic salmon has not yet been fully established. In this study, we identified three npy paralogs, named npya1, npya2, and npyb, in the Atlantic salmon genome. In silico analysis revealed that these genes are well conserved across the vertebrate's lineage and the mature peptide sequences shared at least 77% of identity with the human homolog. We analyzed mRNA expression of npy paralogs in eight brain regions of Atlantic salmon post-smolt, and the effect of 4 days of fasting on the npy expression level. Results show that npya1 was the most abundant paralog, and was predominantly expressed in the telencephalon, followed by the midbrain and olfactory bulb. npya2 mRNA was highly abundant in hypothalamus and midbrain, while npyb was found to be highest expressed in the telencephalon, with low mRNA expression levels detected in all the other brain regions. 4 days of fasting resulted in a significant (p < 0.05) decrease of npya1 mRNA expression in the olfactory bulb, increased npya2 mRNA expression in the midbrain and decreased npyb mRNA expression in the pituitary. In the hypothalamus, the vertebrate appetite center, expression of the npy paralogs was not significantly affected by feeding status. However, we observed a trend of increased npya2 mRNA expression (p = 0.099) following 4 days of fasting. Altogether, our findings provide a solid basis for further research on appetite and energy metabolism in Atlantic salmon.

Leptin receptor-deficient (knockout) zebrafish: Effects on nutrient acquisition.

In mammals, knockout of LEPR results in a hyperphagic, morbid obese, and diabetic phenotype, which supports that leptin plays an important role in the control of appetite and energy metabolism, and that its receptor, LEPR, mediates these effects. To date, little is known about the role(s) of lepr in teleost physiology. We investigated a zebrafish (Danio rerio) homozygous lepr knockout (lepr-/-) line generated by CRISPR/Cas9 in comparison to its wt counterpart with respect to nutrient acquisition, energy allocation, and metabolism. The metabolic characterization included oxygen consumption rate and morphometric parameters (yolk sac area, standard length, wet weight, and condition factor) as proxies for use and allocation of energy in developing (embryos, larvae, and juveniles) zebrafish and showed no particular differences between the two lines, in agreement with previous studies. One exception was found in oxygen consumption at 72 hpf, when zebrafish switch from embryonic to early larval stages and food-seeking behavior could be observed. In this case, the metabolic rate was significantly lower in lepr-/- than in wt. Both phenotypes showed similar responses, with respect to metabolic rate, to acute alterations (22 and 34 °C) in water temperature (measured in terms of Q10 and activation energy) compared to the standard (28 °C) rearing conditions. To assess lepr involvement in signaling the processing and handling of incoming nutrients when an exogenous meal is digested and absorbed, we conducted an in vivo analysis in lepr-/- and wt early (8 days post-fertilization) zebrafish larvae. The larvae were administered a bolus of protein hydrolysate (0%, 1%, 5%, and 15% lactalbumin) directly into the digestive tract lumen, and changes in the mRNA expression profile before and after (1 and 3 h) administration were quantified. The analysis showed transcriptional differences in the expressions of genes involved in the control of appetite and energy metabolism (cart, npy, agrp, and mc4r), sensing (casr, t1r1, t1r3, t1r2-1, t1r2-2, pept1a, and pept1b), and digestion (cck, pyy, try, ct, and amy), with more pronounced effects observed in the orexigenic than in the anorexigenic pathways, suggesting a role of lepr in their regulations. Differences in the mRNA levels of these genes in lepr-/-vs. wt larvae were also observed. Altogether, our analyses suggest an influence of lepr on physiological processes involved in nutrient acquisition, mainly control of food intake and digestion, during early development, whereas metabolism, energy allocation, and growth seem to be only slightly influenced.

The Melanocortin System in Atlantic Salmon (Salmo salar L.) and Its Role in Appetite Control.

The melanocortin system is a key neuroendocrine network involved in the control of food intake and energy homeostasis in vertebrates. Within the hypothalamus, the system comprises two main distinct neuronal cell populations that express the neuropeptides proopiomelanocortin (POMC; anorexigenic) or agouti-related protein (AGRP; orexigenic). Both bind to the melanocortin-4 receptor (MC4R) in higher order neurons that control both food intake and energy expenditure. This system is relatively well-conserved among vertebrates. However, in Atlantic salmon (Salmo salar L.), the salmonid-specific fourth round whole-genome duplication led to the presence of several paralog genes which might result in divergent functions of the duplicated genes. In the current study, we report the first comprehensive comparative identification and characterization of Mc4r and extend the knowledge of Pomc and Agrp in appetite control in Atlantic salmon. In silico analysis revealed multiple paralogs for mc4r (a1, a2, b1, and b2) in the Atlantic salmon genome and confirmed the paralogs previously described for pomc (a1, a2, and b) and agrp (1 and 2). All Mc4r paralogs are relatively well-conserved with the human homolog, sharing at least 63% amino acid sequence identity. We analyzed the mRNA expression of mc4r, pomc, and agrp genes in eight brain regions of Atlantic salmon post-smolt under two feeding states: normally fed and fasted for 4 days. The mc4ra2 and b1 mRNAs were predominantly and equally abundant in the hypothalamus and telencephalon, the mc4rb2 in the hypothalamus, and a1 in the telencephalon. All pomc genes were highly expressed in the pituitary, followed by the hypothalamus and saccus vasculosus. The agrp genes showed a completely different expression pattern from each other, with prevalent expression of the agrp1 in the hypothalamus and agrp2 in the telencephalon. Fasting did not induce any significant changes in the mRNA level of mc4r, agrp, or pomc paralogs in the hypothalamus or in other highly expressed regions between fed and fasted states. The identification and wide distribution of multiple paralogs of mc4r, pomc, and agrp in Atlantic salmon brain provide new insights and give rise to new questions of the melanocortin system in the appetite regulation in Atlantic salmon.

Hypothalamic agrp and pomc mRNA Responses to Gastrointestinal Fullness and Fasting in Atlantic Salmon (Salmo salar, L.).

The orexigenic agouti-related protein (AgRP) and the anorexigenic pro-opiomelanocortin (POMC) are crucial players in the control of feed intake in vertebrates, yet their role in teleosts has not been fully established. Triplicate groups of Atlantic salmon (Salmo salar) post smolts were subjected to (1) fasting for 3 days (fast) and (2) normal feeding (fed), resulting in a significant (p < 0.05) upregulation of hypothalamic agrp1 transcripts levels in the fast group. Moreover, the mRNA abundance of agrp1 was significantly (p < 0.05) correlated with the stomach dry weight content. Corresponding inverse patterns were observed for pomca2, albeit not statistically significant. No significant differences were found for the other paralogues, agrp2 and pomca1 and b, between fed and fast groups. The significant correlation between stomach fullness and agrp1 mRNA expression suggests a possible link between the stomach filling/distension and satiety signals. Our study indicates that hypothalamic agrp1 acts as an orexigenic signal in Atlantic salmon.

Identification and characterization of the Atlantic salmon peptide transporter 1a.

Peptide transporter 1 (PepT1) mediates the uptake of dietary di-/tripeptides in vertebrates. However, in teleost fish gut, more than one PepT1-type transporter might operate, because of teleost-specific whole gen(om)e duplication event(s) that occurred during evolution. Here, we describe a novel teleost di-/tripeptide transporter, i.e., the Atlantic salmon (Salmo salar) peptide transporter 1a [PepT1a; or solute carrier family 15 member 1a (Slc15a1a)], which is a paralog (77% similarity and 64% identity at the amino acid level) of the well-described Atlantic salmon peptide transporter 1b [PepT1b, alias PepT1; or solute carrier family 15 member 1b (Slc15a1b)]. Comparative analysis and evolutionary relationships of gene/protein sequences were conducted after ad hoc database mining. Tissue mRNA expression analysis was performed by quantitative real-time PCR, whereas transport function analysis was accomplished by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp measurements. Atlantic salmon pept1a is highly expressed in the proximal intestine (pyloric ceca ≈ anterior midgut > midgut >> posterior midgut), in the same gut regions as pept1b but notably ~5-fold less abundant. Like PepT1b, Atlantic salmon PepT1a is a low-affinity/high-capacity system. Functional analysis showed electrogenic, Na+-independent/pH-dependent transport and apparent substrate affinity (K0.5) values for Gly-Gln of 1.593 mmol/L at pH 7.6 and 0.076 mmol/L at pH 6.5. In summary, we show that a piscine PepT1a-type transporter is functional. Defining the role of Atlantic salmon PepT1a in the gut will help to understand the evolutionary and functional relationships among peptide transporters. Its functional characterization will contribute to elucidate the relevance of peptide transporters in Atlantic salmon nutritional physiology.

Appetite-Controlling Endocrine Systems in Teleosts.

Mammalian studies have shaped our understanding of the endocrine control of appetite and body weight in vertebrates and provided the basic vertebrate model that involves central (brain) and peripheral signaling pathways as well as environmental cues. The hypothalamus has a crucial function in the control of food intake, but other parts of the brain are also involved. The description of a range of key neuropeptides and hormones as well as more details of their specific roles in appetite control continues to be in progress. Endocrine signals are based on hormones that can be divided into two groups: those that induce (orexigenic), and those that inhibit (anorexigenic) appetite and food consumption. Peripheral signals originate in the gastrointestinal tract, liver, adipose tissue, and other tissues and reach the hypothalamus through both endocrine and neuroendocrine actions. While many mammalian-like endocrine appetite-controlling networks and mechanisms have been described for some key model teleosts, mainly zebrafish and goldfish, very little knowledge exists on these systems in fishes as a group. Fishes represent over 30,000 species, and there is a large variability in their ecological niches and habitats as well as life history adaptations, transitions between life stages and feeding behaviors. In the context of food intake and appetite control, common adaptations to extended periods of starvation or periods of abundant food availability are of particular interest. This review summarizes the recent findings on endocrine appetite-controlling systems in fish, highlights their impact on growth and survival, and discusses the perspectives in this research field to shed light on the intriguing adaptations that exist in fish and their underlying mechanisms.

Characterization of differentially expressed genes in liver in response to the rearing temperature of rainbow trout Oncorhynchus mykiss and their heritable differences.

To characterize thermal-responsive genes in fish, firstly, juvenile rainbow trout were reared in four different temperature conditions (average temperatures were 10, 14, 18, and 22 °C, respectively) and differentially expressed genes were identified. Gene expression in the liver was analyzed by the differential display method, followed by validation using real-time PCR. Subsequently, to examine whether the identified genes show heritable differences, the gene expression levels were compared among juveniles of three genetically distinct lines of rainbow trout (a strain and two closed colonies) by rearing at two different temperature conditions (average 14 and 22 °C). By rearing at 22 °C, growth retardation was observed compared with fish reared at 14 and 18 °C, and six genes were identified as differentially expressed genes in response to the rearing temperature in the gene expression analyses. With the increase in rearing temperature, gene expressions of a complement C1q and two ribosomal proteins were significantly up-regulated. On the other hand, three metabolic genes (betaine homocysteine methyltransferase, triosephosphate isomerase, and glucose-6-phosphatase) were down-regulated, indicating a metabolic depression due to high temperature. In the subsequent analyses, in response to the rearing temperature (14 and 22 °C), there was a trend that the complement C1q and glucose-6-phosphatase genes showed different expression patterns among the three rainbow trout lines, suggesting heritable differences in these genes. Our study provides information on thermal-responsive genes in fish, and we anticipate it will facilitate further investigation in the thermal biology of fish.

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae.

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6-11, 8-11 and 6-9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40-50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception-increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10-12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.

Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation.

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.

Homologue gene of bile acid transporters ntcp, asbt, and ost-alpha in rainbow trout Oncorhynchus mykiss: tissue expression, effect of fasting, and response to bile acid administration.

Bile acid transporters belonging to the SLC10A protein family, Na+ taurocholate cotransporting polypeptide (NTCP or SLC10A1), apical sodium-dependent bile salt transporter (ASBT or SLC10A2), and organic solute transporter alpha (Ost-alpha) have been known to play critical roles in the enterohepatic circulation of bile acids in mammals. In this study, ntcp, asbt, and ost-alpha-1/-2 cDNA were cloned, their tissue distributions were characterized, and the effects of fasting and bile acid administration on their expression were examined in rainbow trout Oncorhynchus mykiss. The structural characteristics of Ntcp, Asbt, and Ost-alpha were well conserved in trout, and three-dimensional structure analysis showed that Ntcp and Asbt were similar to each other. Tissue distribution analysis revealed that trout asbt was primarily expressed in the hindgut, while ntcp expression occurred in the brain, and ost-alpha-1/-2 was mainly expressed in the liver or ovary. Although asbt and ost-alpha-1 mRNA levels in the gut increased in response to fasting for 4 days, ost-alpha-1 expression in the liver decreased. Similarly, bile acid administration increased asbt and ost-alpha-1 expression levels in the gut, while those of ntcp and ost-alpha-2 in the liver decreased. These results suggested that the genes asbt, ntcp, and ost-alpha are involved in bile acid transport in rainbow trout.

Postprandial response and tissue distribution of the bile acid synthesis-related genes, cyp7a1, cyp8b1 and shp, in rainbow trout Oncorhynchus mykiss.

In mammals, cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) are rate-limiting enzymes in bile acid synthesis. In addition, a small heterodimer partner (SHP) is also known to inhibit bile acid synthesis via the suppression of CYP7A1 and CYP8B1 expression. However, little information is currently available regarding primary structure of the genes involved in bile acid synthesis in fish. We therefore cloned cyp7a1, cyp8b1 and shp genes from rainbow trout and obtained cDNAs encoding two isoforms each of Cyp7a1 (-1 and -2), Cyp8b1 (-1 and -2) and Shp (-1 and -2). Both cyp7a1-1 and -2 encoded proteins of 512 amino acids. Trout cyp7a1-1 was expressed not only primarily in the kidney, pyloric caecum and mid-gut, but also weakly in the liver, eye, gill and ovary. cyp7a1-2 was highly expressed in the liver, pyloric caecum and mid-gut. cyp8b1-1 and -2, which encoded proteins of 512 and 509 amino acids, respectively, were principally expressed in the liver. Both shp-1 and -2, which encoded proteins of 288 and 290 amino acids, respectively, were strongly expressed in the liver, but shp-2 was also highly expressed in the gallbladder and digestive tract. The temporal changes in the expression of cyp7a1-1/-2, cyp8b1-1/-2 and shp-1/-2 in the liver were assessed after consumption of a single meal. Expression of cyp7a1-1/-2 and cyp8b1-1/-2 increased within 3h post feeding (hpf) when the stomach was still approximately 84% full and the gallbladder was almost completely empty. Although the expression of shp-1 did not change after feeding, the expression pattern of shp-2 was inversely related to the expression patterns of cyp7a1-1/-2 and cyp8b1-1/-2. Specifically, shp-2 expression decreased until 3 hpf before returning to initial levels at 24 hpf. These findings suggest that Cyp7a1s/8b1s and Shp-2 function antagonistically in bile acid synthesis in rainbow trout.

Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae.

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.

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.

Multiple cocaine- and amphetamine-regulated transcript (CART) genes in medaka, Oryzias latipes: cloning, tissue distribution and effect of starvation.

The neuropeptide cocaine- and amphetamine-regulated transcript (CART) is important in the regulation of food intake in mammals and fish. The tissue distributions of six CART cDNAs (cart ch3, ch4, ch6, ch9, ch11, and ch22) from medaka, Oryzias latipes, were cloned and the effect of starvation on their expression was examined. As in other species, medaka cart ch3, ch4, ch6, ch9, and ch22 consisted of three exons, while medaka cart ch11 contained four. The six cysteine residues at the C-terminal end of the CART motif and three-dimensional structure were well conserved in all medaka CART peptides. Tissue distribution analysis revealed that cart ch3, ch4, ch6, ch11, and ch22 were primarily expressed in the brain, but that the highest rates of cart ch9 expression occurred in the skin, suggesting different functions among the homologous genes. Although CART ch3 mRNA levels decreased in response to 17 days starvation, these levels were restored by re-feeding. However, the finding that the five other CART mRNAs did not respond to starvation suggests that only CART ch3 has an anorexigenic function in medaka.

Ontogeny of energy homeostatic pathways via neuroendocrine signaling in Atlantic salmon.

Leptin and ghrelin are known to regulate energy homeostasis via hypothalamic neuropeptide signaling in mammals. Recent studies have discovered that these hormones exist in teleosts, however, very little is known concerning their role during teleost ontogeny. Here, we have examined the steady state levels of leptins, ghrelins, their target neuropetides and several growth factors during Atlantic salmon development. Initial experiments revealed differential expression of leptin genes and ghrelin isoforms during embryogenesis. In larvae, equal upregulation of ghrl1 and ghrl2 was observed just prior to exogenous feeding while a surge of lepa1 occurred one week after first-feeding. Subsequent dissection of the embryos and larvae showed that lepa1, cart, pomca1, and agrp are supplied as maternal transcripts. The earliest zygotic expression was observed for lepa1 and cart at 320 day degrees. By 400 day degrees, this expression was localized to the head and coincided with upregulation of ghrl2 and npy. Over the hatching period growth factor signaling predominated. The ghrelin surge prior to first-feeding was exclusively localized in the internal organs and coincided with upregulation of npy and agrp in the head and agrp in the trunk. One week after exogenous feeding was established major peaks were detected in the head for lepa1 and pomca1 with increasing levels of cart, while lepa1 was also significantly expressed in the trunk. By integrating theses data into an ontogenetic model, we suggest that the mediation of Atlantic salmon energy homeostatic pathways via endocrine and neuropeptide signaling retains putative features of the mammalian system.

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.

Molecular cloning and functional expression of atlantic salmon peptide transporter 1 in Xenopus oocytes reveals efficient intestinal uptake of lysine-containing and other bioactive di- and tripeptides in teleost fish.

Atlantic salmon (Salmo salar L.) is one of the most economically important cultured fish and also a key model species in fish nutrition. During digestion, dietary proteins are enzymatically cleaved and a fraction of degradation products in the form of di- and tripeptides translocates from the intestinal lumen into the enterocyte via the Peptide Transporter 1 (PepT1). With this in mind, a full-length cDNA encoding the Atlantic salmon PepT1 (asPepT1) was cloned and functionally characterized. When overexpressed in Xenopus laevis oocytes, asPepT1 operated as a low-affinity/high-capacity transport system, and its maximal transport activity slightly increased as external proton concentration decreased (varying extracellular pH from 6.5 to 8.5). A total of 19 tested di- and tripeptides, some with acknowledged bioactive properties, some containing lysine, which is conditionally growth limiting in fish, were identified as well transported substrates, with affinities ranging between approximately 0.5 and approximately 1.5 mmol/L. Analysis of body tissue distribution showed the highest levels of asPepT1 mRNA in the digestive tract. In particular, asPepT1 mRNA was present in all segments after the stomach, with higher levels in the pyloric caeca and midgut region and lower levels in the hindgut. Depriving salmon of food for 6 d resulted in a approximately 70% reduction of intestinal PepT1 mRNA levels. asPepT1 will allow systematic in vitro analysis of transport of selected di- and tripeptides that may be generated in Atlantic salmon intestine during gastrointestinal transit. Also, asPepT1 will be useful as a marker to estimate protein absorption function along the intestine under various physiological and pathological conditions.

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.