The cocaine- and amphetamine-regulated transcript (CART), initially discovered as an unknown peptide isolated from ovine hypothalamus by Spiess et al. (1981), was found to be released in response to the psychomotor stimulants cocaine and amphetamine in rat brain by Douglass et al. (1995) and, thus, its name. The mammalian CART is known to be an anorectic peptide in mediating feeding in accordance with energy balance (Kristensen et al., 1998; Yang et al., 2005; Rogge et al., 2008) but is also involved in stress response, reward and drug addiction/abuse (Koylu et al., 2006).

The CART function and its involvement in appetite control is, however, not so clear for other vertebrates, including teleost fishes. In fish, one cart gene has been previously identified in several species, such as Atlantic cod (Gadus morhua) (Le et al., 2016), winter flounder (Pseudopleuronectes americanus) (MacDonald and Volkoff, 2009a), winter skate (Raja ocellata) (MacDonald and Volkoff, 2009b), Mexican tetra/blind cave fish (Astyanax mexicanus) (Penney and Volkoff, 2014), rainbow trout (Oncorhynchus mykiss) (Comesaña et al., 2018), channel catfish (Ictalurus punctatus) (Kobayashi et al., 2008), silver dollar (Metynnis argenteus), bucktooth tetra (Exodon paradoxus), black widow tetra (Gymnocorymbus ternetzi) (Butt et al., 2019), and Atlantic salmon (Salmo salar) (Murashita et al., 2009). In other teleost species, multiple cart transcripts have been identified in the brain including two genes in goldfish (Carassius auracius) (Volkoff and Peter, 2001), three in Ya-fish (Schizothorax prenanti) (Yuan et al., 2015), four in zebrafish (Danio rerio) (Nishio et al., 2012; Akash et al., 2014; Ahi et al., 2019), five in yellowtail (Seriola quinqueradiata) (Fukada et al., 2021), six in medaka (Oryzias latipes) (Murashita and Kurokawa, 2011), and seven in Senegalese sole (Solea senegalensis) (Bonacic et al., 2015).

The existence of multiple cart genes raises questions about their physiological function, and recent studies have suggested that maybe not all the paralogs are involved in the control of feed intake. The cart response to feeding has varied among studies, which can possibly be explained by differences in the method of analysis used, like in situ vs. qPCR, or varied methodological approaches like use of whole brain vs. specific brain regions and different development stages, and sampling time related with feeding or fasting. However, it might well be that Cart response to feeding is also species-specific. The differential expression patterns of cart genes in brain regions have been described in some teleost species such as goldfish (Volkoff and Peter, 2001), zebrafish (Nishio et al., 2012; Akash et al., 2014), yellowtail (Fukada et al., 2021), and Senegalese sole (Bonacic et al., 2015). The anorectic effect of cart has been reported for several teleost species, notably in zebrafish where two forms of hypothalamic cart mRNA expressions were downregulated after 5 days of fasting (Akash et al., 2014), and all four identified cart genes expression decreased in the brain after 3 days of fasting (Nishio et al., 2012). Similar cart anorectic effects have been observed in Ya-fish, where the mRNA expression of the three identified cart genes decreased in the hypothalamus after fasting and increased with refeeding (Yuan et al., 2015), while in yellowtail, cart1b and 2a mRNA expression decreased in the telencephalon and hypothalamus following 8 days of fasting (Fukada et al., 2021). Furthermore, whole brain analysis of cart mRNA expression showed that 6 days of fasting downregulated it’s expression in Atlantic salmon (Murashita et al., 2009), while 1 week of fasting decreased cart mRNA in silver dollar but did not affect the expression in bucktooth or black widow tetra (Butt et al., 2019). In medaka, cart2b (previously named cart ch3) expression decreased with 17 days of starvation (Murashita and Kurokawa, 2011).

Atlantic salmon is a key species in Norwegian aquaculture industry and a substantial portion of the production cost, more than 50%, goes to fish feed (Asche and Oglend, 2016). Furthermore, the extensive expansion of the fish industry combined with frequent overfeeding has contributed to potentially negative effects on the ecosystem (Strain and Hargrave, 2005). As such, the efficient utilization of fish feed is vital to ensure sustainable production with regards to the environment, fish welfare, and production costs. Thus, a clear understanding of the biological mechanisms that underly hunger and satiety, and thereby control feed intake, can be of great benefit to the salmon industry. This includes the neuro-endocrine players that modulate feeding and the identification of reliable biomarkers that can be used to assess appetite and potentially growth in fish trials. Moreover, a 2–4 days fasting is implemented prior to aquaculture practices like handling for vaccination, transportation, and harvest (Waagbø et al., 2017). In this regard, learning the impact of fasting will help to optimize the feed utilization, fish growth performance, and welfare. In the current study, we have identified 10 cart paralogs in the Atlantic salmon genome databases and analyzed their brain distribution as well as their response to 4 days of fasting. The results provide a basis for a more comprehensive understanding the role of Cart in the appetite control in Atlantic salmon.

In this study, we report the identification and characterization of 10 cart paralogs in Atlantic salmon, here named cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b, and cart4. This is the first study to analyze all the aforementioned paralogs physiological role in Atlantic salmon appetite control, while the previously reported Atlantic salmon Cart by Murashita et al. (2009) is here denoted as Cart2b (b1 and b2). Furthermore, the mRNA sequences of the qPCR amplicons confirmed that none of salmon cart genes are pseudogenes. The deduced amino acid sequences showed a highly divergent N-terminal AA residues whereas the C-terminal showed a high degree of conservation. Among Cart paralogs, the potential proteolytic processing sites of dibasic residues KR and KK (Kuhar and Yoho, 1999; Rogge et al., 2008) are conserved only in Cart2, possibly cleaving it into two different size products as previously suggested by Murashita et al. (2009). In previous studies, Kuhar and Yoho (1999) identified four immunoreactive CART peptides from human and five from rat hypothalamus varying from 4 to 10 kDa. The predicted cleavage sites for Atlantic salmon Cart (Figure 2) also suggest possible multiple Cart peptides. The 3 cysteine disulfide bonds between Cys⁸² and Cys¹⁰⁰, Cys⁸⁸ and Cys¹⁰⁸, Cys¹⁰², and Cys¹¹⁵ were predicted for human CART C-termini (UNIPROT⁷). These six C-terminal cysteines are conserved in all the Atlantic salmon Cart, except Cart4 where only 5 cysteine residues are present. The absence of C- terminal cysteine bond among the homologs has not been reported in teleosts before. The three disulfide bridges are key residual structures that are essential for the protein 3-dimensional structure and its function in biological activities such as food intake (Ludvigsen et al., 2001; Maixnerová et al., 2007). The Atlantic salmon Cart sequence alignment clearly show that Cart4 structure potentially differs from the other salmon’s Carts and human CART. This suggests that Atlantic salmon Cart4 may differ in functionality from the other Carts and may likely be ruled out from its role in appetite regulation. It would also be possible that Cart4 is non-functional or has adopted new function and this warrants further studies. In teleost cart mRNA expression in peripheral tissues like eye, gill, ovary, testis has also been reported suggesting Cart may also have other roles (Volkoff and Peter, 2001; Murashita et al., 2009). Interestingly, the multiple sequence alignment of Cart prepropeptides revealed also Cart4 in northern pike, rainbow trout, Atlantic herring and Cart2 in Atlantic cod lack of one or more Cysteine residue in the C-termini (Supplementary Figure 1).

The evolutionary history of cart depicted in the phylogenetic tree shows that the Ts 3R genome duplication resulted in the presence of 7 cart genes in species like Senegalese sole, Mexican tetra, Northern pike, etc., and the Ss 4R genome duplication resulted in 10 cart genes in the Atlantic salmon, including sub types cart1b1 and 1b2, cart2b1 and 2b2, cart3a1 and 3a2. The divergence of the cart gene in salmonids opens the possibility that the gene products might have adopted to new functions (neofunctionalization) or partitioning the ancestral function among the paralogs (sub functionalization) during genome evolution reviewed by Volff (2005). Furthermore, the fact that Atlantic salmon and other teleosts have multiple cart genes while only one is present in mammals’, open up interesting perspectives for comparative research on evolutionary adaptations of gene function and the control of appetite and energy homeostasis.

To date, there are only a few studies exploring the full cart gene repertoire in teleost species. These studies include the identification of seven cart paralogous genes in Senegalese sole (Bonacic et al., 2015), six genes in medaka (Murashita and Kurokawa, 2011), and five yellowtail (Fukada et al., 2021). Thus, a revision of the teleost genome databases and nomenclature of cart genes is essential in further studies exploring the functional role of Cart in these species.

Our results revealed that all cart paralogs were expressed at different mRNA levels in the eight brain regions analyzed in this study. Among all the cart genes, cart2b was the most abundant paralog, with predominant expression in the OB followed by TEL, MB, HYP, and SV. A similar pattern has also been reported in Senegalese sole (Bonacic et al., 2015) and yellowtail (Fukada et al., 2021). In teleosts, Cart has been suggested to play a role in processing olfactory information (Akash et al., 2014; Porter et al., 2017) including antiviral immune response (Sepahi et al., 2019). However, the Atlantic salmon cart2b expression in the OB and a possible link to olfaction and/or immune response is not clear and needs to be further investigated. The cart2b was the only gene that was affected by 4 days of fasting, and a significantly higher expression was observed in the fasted group compared to the fed group in the Atlantic salmon specifically in OB, MB, and HYP. Hypothalamus is considered as a center of appetite control in vertebrates (Volkoff, 2016; Rønnestad et al., 2017; Soengas et al., 2018), and in fish this is supported by Ye et al. (2020) who reported that in grass carp the hypothalamus and telencephalon might be involved in feeding and reproduction. Hence, in Atlantic salmon, hypothalamic cart2b might play a vital role in appetite regulation, with fasting maybe inducing shutting down hunger. This is in line with observations in Siberian sturgeon (Acipenser baerii) (Zhang et al., 2018), where cart mRNA in whole brain increased after 3–17 days of food deprivation. Interestingly, our finding is contrary to several previous studies that demonstrate the anorectic function of Cart. In goldfish, cart form I (alias cart2b) mRNA level declined with 96 h food deprivation in telencephalon, hypothalamus, and olfactory bulb while cart form II (alias cart2a) mRNA level declined only in the olfactory bulb. In the same study, cart2b transcript levels increased 2 h following a meal in olfactory bulb and hypothalamus whereas no postprandial changes were observed in cart2a (Volkoff and Peter, 2001; Volkoff et al., 2005). However, in yellowtail cart2b expression in OB, TEL, HYP was upregulated in fish fasted for 8 days, though not significantly, while cart3a was significantly upregulated in OB, TEL, and PT of the fasted group (Fukada et al., 2021). The brain cart mRNA expression in response to fasting varied depending on species, and even within the same order as for example, in silver dollar, fasting induced a decrease in cart expression while for black widow tetra and bucktooth no effect was observed (Butt et al., 2019). Forebrain cart mRNA expression levels were significantly higher in cod fish held at 2∘C than in those held at either 11 or 15∘C (Kehoe and Volkoff, 2008) suggesting Cart mediates temperature- induced changes in appetite during the seasonal changes in the lifespan of this species. In addition, the stomach from fish acclimated to 2∘C had less food. Taking into consideration that these fish were handfed to satiety, the results suggested Cart acts as a satiety factor in this species (Kehoe and Volkoff, 2008). Previous studies investigating the role of cart in appetite regulation in Atlantic cod (Kehoe and Volkoff, 2007, 2008) and Atlantic salmon (Murashita et al., 2009) using a single cart paralog suggested an anorectic role for the neuropeptide. Indeed, in Atlantic salmon, 6 days of fasting resulted in decrease of brain cart2b (previously named cart) (Murashita et al., 2009). In medaka, 17 days of fasting also downregulated brain cart2b (previously named cart ch3) (Murashita and Kurokawa, 2011). However, both studies have used whole brain in the gene expression analysis. Accordingly, cart2b seemed to be involved in appetite regulation but the available data on its action are contradictory since orexigenic, anorexigenic or no action have been reported depending on whether the analyses were performed at brain region level (for e.g., hypothalamus) or on the whole brain and/or the fasting time used. Besides, two different studies in zebrafish reported that cart2a and cart3a (previously named cart2 and cart4, respectively, Akash et al., 2014) were downregulated in response to short-term fasting within hypothalamic region and cart2b (previously named cart3, Ahi et al., 2019) was downregulated in brain after 7 days of fasting. The involvement of Cart2 in appetite regulation in Atlantic salmon is further supported by the fact that this is the only Cart paralog that has the same potential proteolytic sites as in mammals. These studies show the diversity between species and importance of long-term vs. short-term testing of the Cart neuropeptide and emphasizes the need for more sampling time points to uncover the specific role of Cart paralogs in appetite regulation in Atlantic salmon.

The cart1a and 1b show quite similar mRNA expression profile and cart1, cart2a, and cart3a are highly expressed specially in the MB, suggesting that these cart paralogs might share the same functional role. Comparably, in Senegalese sole, Bonacic et al. (2015) observed that cart1, cart2a, and cart3a were highly expressed in mesencephalon which is the equivalent region to the MB in our study. The cart1a and 1b are primarily expressed in MB followed by PT in Atlantic salmon brain, which indicate that these genes might be involved in functions linked to these brain regions, such as vision and feeding for MB, and energy metabolism, organ development and reproduction for PT. Ye et al. (2020) showed that in grass carp the optic tectum seems to play an important role in the visual system and feeding. The potential role of cart in reproduction is reported in chicken, where pituitary CART peptide expression and secretion is regulated in the hypothalamic-pituitary-gonadal (HPG) axis through gonadotropin-releasing hormone (GnRH), and GnRH administration increased plasma CART (Mo et al., 2019). In this study, the cart2a, 2b, and 4 mRNA expression in hypothalamus suggests that these might be involved in appetite control in Atlantic salmon. We further speculate that they might relay the signals relevant to feeding at different time points. However, further studies are needed to unravel the actual molecular interaction and functionalization among cart paralogs.

The feeding regime (continuously or meal-based) and the period in between last feed and sampling are also reflected in the expression of neuropeptides regulating appetite in the brain. Most probably, there is a higher variation in voluntary feed intake when the fish are fed continuously. The macronutrient composition of the feed and the period of fasting are also important factors as it can influence the species digestive physiology and gut evacuation time, and consequently affect the appetite control. Fukada et al. (2021) reported that in yellowtail, cart2a showed a tendency to decrease in hypothalamus with fasting and increased with refeeding when the sampling was performed 3 h after feeding. Similarly, in our study, 3 days of fasting resulted a tendency to downregulation of cart2a in HYP in fasted group compared to the fed group (sampled 2 h after last feed).

The cart3b expression revealed a completely different profile compared to other paralogs with BS as its primary site of expression of this paralog in the brain. This is in line with particularly high expression levels detected in the spinal cord in Senegalese sole (Bonacic et al., 2015). Though there was no significant difference in cart3b mRNA expression between the fed and fasted group, the decreasing trend with fasting in BS shows a potential anorexigenic role. We speculate that satiety information generated during a meal, largely conveyed via afferent fibers of vagus nerves first to the hindbrain from the upper gastrointestinal tract in mammals (reviewed in Schwartz et al., 2000), seems to be conserved in fish species. The BS region which contains Mauthner neurons (MN), specific multifunctional neurons in fish, receives afferent information from vestibular, auditory, and visual analyzers and can integrate the afferent activity. In Chinese sleeper (Perccottus glehni), the MN responded to severe environmental conditions like deficit of nutrients/energy substrates, oxygen and temperature changes by modifying the structure of the cell and through different pathways for the maintenance of the energy balance (Santalova et al., 2018). Whether this link between cart3b and MN is also seen in Atlantic salmon brain under starvation is unknown and would be an interesting topic for further studies.

Cart has also been suggested to be involved in other physiological processes like stress response, anxiety, depression, reproduction (Koylu et al., 2006), and immune response (Burgos et al., 2019) in mammals.

The interlink between CART peptide and other stress related peptides in the hypothalamic-pituitary-adrenal (HPA) axis have been somewhat studied in mammals and the findings show that intracerebroventricular CART injection increases hypothalamic corticotropin-releasing factor (crf) mRNA levels and CRF release. In rats, centrally administered CART increased adrenocorticotropic hormone and corticosterone in circulation and exerted dose-dependent increase in anxiety-related behaviors (reviewed in Koylu et al., 2006). Similarly, in fish under stressed conditions the mechanisms involving food intake are deregulated. Consequently, the regular signals controlling appetite in the brain are altered, resulting in changes in the expression of the appetite-related neuropeptides and reduction in food intake (reviewed in Conde-Sieira et al., 2018). In zebrafish, exposure to acute handling stress induced increased expression of cart and pro-opiomelanocortin (pomc) as central keys in the appetite control (Cortés et al., 2018), but the response of appetite regulating neuropeptides to stress can differ in salmonids (reviewed in Conde-Sieira et al., 2018). In our study, both experimental groups of fish were maintained throughout the experiment under same rearing conditions, even during sampling both groups went through the same procedures. The effect of handling stress in the appetite regulating neuropeptides genes expression in Atlantic salmon brain still remains to be uncovered.

This study identifies 10 cart paralogs in Atlantic salmon brain with distinct distribution profile. Among the paralogs cart2b responded to 4 days of fasting by increase in mRNA expression in hypothalamus, olfactory bulb and midbrain while cart3b showed a trend of decline in brain stem. The difference in key residual structure of Cart4 suggesting potentially different function other than appetite regulation. The Cart paralogs 1a, 1b, 2a, and 3a might likely have potential different roles in the regional integration of appetite (relaying) signals in the hypothalamus, telencephalon, and brain stem, and/or other functions in the olfactory bulb and midbrain. To uncover their specific role in appetite regulation in Atlantic salmon more sampling time points will be required. Moreover, the complex molecular interactions with other peptide hormones need to be untangled to understand the potential role of Cart in appetite regulation in Atlantic salmon.

The datasets presented in this study can be found in Supplementary Table 3.

The animal study was reviewed and approved by the Local Representative of Animal Welfare at Department of Biological Sciences, University of Bergen, Norway, in accordance with Norwegian Animal Research Authority regulations.

TK, AG, KM, IT, FL, and IR conceived and designed the study. AG, KM, and IR conducted the experiment. AG, TK, KM, and FL contributed to the sampling. TK and AG did the primers design and phylogenetic analysis. TK and IT did preparatory lab work and qPCR. TK performed statistical analysis and drafted the manuscript. All authors contributed to the interpretation of the data, writing of the manuscript, read and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.