Major appetite regulating factors

Central orexigenic factors

Agouti-related protein (or peptide, AgRP)
AgRP is a peptide released by hypothalamic NPY/AgRP neurons and is an endogenous antagonist of the melanocortin receptors MC3R and MC4R. AgRP plays a crucial role in the regulation of energy balance, as it increases food intake, by antagonizing the effects of the anorexigenic POMC product, α-melanocyte-stimulating hormone (α-MSH) (Sohn, 2015; Takeuchi, 2016).
In fish, AgRP has been identified in several species, including teleosts (e.g., goldfish Carassius auratus Cerdá-Reverter and Peter, 2003 and zebrafish Danio rerio Song et al., 2003, Atlantic salmon Salmo salar Murashita et al., 2009a, and seabass Dicentrarchus labrax Agulleiro et al., 2014, pufferfish Takifugu rubripes Klovins et al., 2004; Kurokawa et al., 2006), who have two genes products (AgRP1 and AgRP2; Cérda-Reverter et al., 2011) and Holocephali (Chimaeriforme, elephant fish Callorhinchus milii Västermark and Schioth, 2011).
AgRP appears to act as an orexigenic factor in Cypriniformes, as fasting increases hypothalamic AgRP expression in goldfish (Cerdá-Reverter and Peter, 2003), zebrafish (Song et al., 2003), and Ya fish Schizothorax prenanti (Wei et al., 2013). In addition, transgenic zebrafish overexpressing AgRP exhibit obesity, increased growth and adipocyte hypertrophy (Song and Cone, 2007). GH-transgenic common carp Cyprinus carpio, which display increased food intake, have higher hypothalamic AgRP1 mRNA expression levels than non-transgenic fish, further suggesting an orexigenic action (Zhong et al., 2013). However, this is contradicted by another study in carp showing that brain AgRP mRNA expression decreases after fasting and increases after re-feeding (Wan et al., 2012). In seabass (Perciforme), long-term fasting increases hypothalamic expression of AgRP1 but decreases that of AgRP2 (Agulleiro et al., 2014), suggesting an isoform-specific orexigenic action.
Within Salmoniformes, there is conflicting data with regards to the actions of AgRP. In Arctic charr Salvelinus alpinus, non-feeding fish have higher brain AgRP expression levels than feeding fish (Striberny et al., 2015) and transgenic coho salmon Oncorhynchus kisutch, which display increased feeding, have higher brain AgRP1 levels of mRNA than wild-type fish (Kim et al., 2015), suggesting an orexigenic role for AgRP. However, in Atlantic salmon, AgRP-1 brain mRNA levels decrease after fasting (Murashita et al., 2009a) and increase after feeding (Valen et al., 2011), rather pointing to an anorexigenic role.

Galanin
Galanin is a peptide expressed in both central nervous system and GIT, that regulates diverse physiological functions in mammals, including arousal/sleep, feeding, energy metabolism, and reproduction (Merchenthaler, 2010). Galanin and its receptors have been identified in a number of fish species (see review in Mensah et al., 2010). Central injections of galanin stimulate feeding in Cypriniformes (both goldfish de Pedro et al., 1995; Volkoff and Peter, 2001b, and tench, Tinca tinca Guijarro et al., 1999). In goldfish, brain galanin mRNA expression is not affected by fasting but increases post-prandially in unfed fish (Unniappan et al., 2004) and in zebrafish, fasting up-regulates brain mRNA expression of galanin receptors (Li et al., 2013). These data suggest that the galanin system is involved in the regulation of feeding in Cypriniformes, and perhaps other fish.

Melanin concentrating hormone (MCH)
Melanin concentrating hormone is a peptide originally isolated from the pituitary of chum salmon (Oncorhynchus keta) as a hormone involved in body color change (Kawauchi et al., 1983). MCH was later isolated in mammals and shown to stimulate feeding (Qu et al., 1996). In fish, the role of MCH as an appetite regulator is still unclear.
In Cypriniformes, early immunoreativity (ir) studies in goldfish showed the presence of MCH in neuron populations related to the regulation of feeding and of sleep and arousal (Huesa et al., 2005). In goldfish, central injections of MCH decrease feeding but have no effect on locomotor activity (Shimakura et al., 2006), anti-MCH serum treatments increase feeding (Matsuda et al., 2007a), and the number of certain hypothalamic neuronal cell bodies containing MCH-ir decreases in fasted fish (Matsuda et al., 2007a), altogether suggesting an anorexigenic role for MCH in this species. However, in Ya fish, MCH hypothalamic mRNA expression is higher in fasted compared to fed fish, suggesting an orexigenic role (Wang et al., 2016). Data on Gadiformes and Pleuronectiformes also seem to suggest an appetite-stimulating role for MCH: MCH brain mRNA levels increase during fasting in both Atlantic cod Gadus morhua (Tuziak and Volkoff, 2013a) and winter flounder Pseudopleuronectes americanus (Tuziak and Volkoff, 2012), and in cod fed diets with relatively high amounts of plant (camelina) material (Tuziak et al., 2014). In starry (Platichthys stellatus; Kang and Kim, 2013b), olive (Paralichthys olivaceus; Kang and Kim, 2013a) and Barfin (Verasper moseri; Takahashi et al., 2004) flounders, fish placed in light backgrounds have enhanced appetite and growth, which is concomitant with increased expression levels of MCH mRNA and/or numbers of MCH neurons in the brain. However, in medaka Oryzias latipes, transgenic fish overexpressing MCH have normal growth and feeding behavior (Qu et al., 1996) and in the scalloped hammerhead shark Sphyrna lewini, hypothalamic MCH mRNA levels are not affected by fasting (Mizusawa et al., 2012), suggesting little or no role of MCH in feeding regulation of Beloniformes and sharks.
Neuronal relationship between MCH- and NPY-containing neurons have been shown in goldfish (Matsuda et al., 2009) and MCH treatment increases orexin mRNA expression and decreases NPY mRNA expression in cultured goldfish forebrain slices (Matsuda et al., 2009), suggesting an interaction of MCH with appetite regulators in goldfish. Similarly, in red-bellied piranha Pygocentrus nattereri, orexin and MCH co-localize in pituitary and brain (Suzuki et al., 2007), and in Barfin flounder, close contacts are seen between orexin- and MCH-ir cell bodies and fibers in the hypothalamus, suggesting an interaction between the two systems and a possible role for MCH in the modulation of locomotion and feeding (Amiya et al., 2008).

Neuropeptide Y (NPY)
Neuropeptide Y (NPY) belongs to the NPY family of peptides, which also includes, peptide YY and pancreatic polypeptide (PP) (Holzer et al., 2012). Originally isolated from mammalian brain extracts (Tatemoto et al., 1982), NPY is one of the most abundant neuropeptides within the brain and has a major regulatory role in energy homeostasis and food intake (Loh et al., 2015).
Although reports for NPY-like ir in fish brain and other tissues appear in the 1980's (e.g., Osborne et al., 1985; Danger et al., 1990), the first fish NPY cDNAs were reported in goldfish and the electric ray Torpedo marmorata (elasmobranch, Torpediniformes; Blomqvist et al., 1992). One of the first studies showing the role of NPY in regulating in fish was that of Silverstein et al., showing by in situ hybridization (ISH) that, in chinook salmon (Oncorhynchus tshawytscha) and coho salmon, NPY-like mRNA signal areas were greater in fasted than fed fish (Silverstein et al., 1998). The first in vivo injection studies were performed in goldfish (Lopez-Patino et al., 1999; de Pedro et al., 2000; Narnaware et al., 2000) and channel catfish Ictalurus punctatus (Silverstein and Plysetskaya, 2000). Since then, NPY has been one of the most studied appetite-regulating hormones in fish. It has been cloned and/or shown to regulate feeding in several groups, including Characiformes (Pereira et al., 2015), Cypriniformes [(e.g., goldfish, zebrafish (Yokobori et al., 2012), blunt snout bream Megalobrama amblycephala (Xu et al., 2016), grass carp Ctenopharyngodon idellus (Jin et al., 2015), Jian carp (Cyprinus carpio) (Tang et al., 2014), Ya fish (Wei et al., 2014)], Gadiformes (Atlantic cod Kortner et al., 2011; Tuziak et al., 2014); Gonorynchiformes (milkfish Chanos chanos, Lin et al., 2016); Perciformes (yellowtail Seriola quinqueradiata Hosomi et al., 2014, Astatotilapia burtoni Grone et al., 2012, cunner Tautogolabrus adspersus Babichuk and Volkoff, 2013, orange-spotted grouper Epinephelus coioides Tang et al., 2013, sea bass Leal et al., 2013, mandarin fish, Siniperca chuatsi Sun et al., 2014, cobia Rachycentron canadum Van Nguyen et al., 2013, gourami Trichogaster pectoralis Boonanuntanasarn et al., 2012); Pleuronectiformes (olive flounder Wang et al., 2015, winter flounder MacDonald and Volkoff, 2009a, Brazilian flounder Paralichthys orbignyanus Campos et al., 2012), Salmoniformes (e.g., rainbow trout Oncorhynchus mykiss Aldegunde and Mancebo, 2006, Atlantic salmon Valen et al., 2011; Kim et al., 2015), Siluriformes (channel catfish, Peterson et al., 2012; Schroeter et al., 2015); Tetraodontiformes (tiger puffer Takifugu rubripes Kamijo et al., 2011) as well as elasmobranchs [(e.g., winter skate Leucoraja ocellata, Rajiforme (MacDonald and Volkoff, 2009b) and spotted catshark (Scyliorhinus canicula, Carcharhiniforme) Mulley et al., 2014)] and holocephalans (elephant fish Chimaeriformes; Larsson et al., 2009). The majority of these studies indicate that NPY has a widespread distribution and is present in both brain and intestinal tract, that it acts as an orexigenic factor and that its expression is affected by feeding and fasting.

Orexin
Orexins (also called hypocretins) are neuropeptides originally isolated in rats (Sakurai, 2014), that have since been identified in several fish species. The first direct evidence of an orexigenic action of orexins was shown via intracerebroventricular (ICV) injections in goldfish (Volkoff et al., 1999). As in mammals (Tsujino and Sakurai, 2009; Sakurai, 2014), orexins increase not only appetite and feeding behavior but also locomotor activity and reward-seeking/foraging behavior in fish (Panula, 2010).
In both goldfish (Volkoff et al., 1999; Nakamachi et al., 2006; Facciolo et al., 2011) and zebrafish (Danio rerio) (Yokobori et al., 2011) (Cypriniformes), and cavefish (Astyanax mexicanus) (Characiforme) (Penney and Volkoff, 2014), orexin injections increase searching/feeding behaviors. In orange-spotted grouper (Perciforme), intraperitoneal (IP) orexin injections increase hypothalamic mRNA expression levels of NPY, a major appetite stimulator (Yan et al., 2011), further suggesting an orexigenic role. However, in ornate wrasse (Thalassoma pavo) (Perciforme), orexin IP injections induce increases in locomotion but decreases in feeding (Facciolo et al., 2009), suggesting that the major role of orexin might be induction of hyperactivity rather than increasing food ingestion. Indeed, in goldfish, hypothalamic orexin mRNA expression levels peak when fish are active prior to a scheduled meal (Hoskins and Volkoff, 2012) and in zebrafish, increased locomotor activity is associated with increased activity of hypothalamic orexin neurons (Naumann et al., 2010) and larvae overexpressing orexin are hyperactive (Woods et al., 2014). Similarly, orexin expression decreases post-feeding in Characiformes [cavefish (Wall and Volkoff, 2013), dourado (Salminus brasiliensis) (Volkoff et al., 2016) and pacu (Piaractus mesopotamicus) (Volkoff et al., 2017)] and is higher at mealtime in orange-spotted grouper (Yan et al., 2011) and tilapia (Chen et al., 2011) (Perciformes), as well as Atlantic cod (Gadiforme) (Xu and Volkoff, 2007). In cod, orexin levels are also higher during daylight hours, when animals are active (Hoskins and Volkoff, 2012).
Fasting increases orexin brain mRNA expression in Cypriniformes (goldfish Abbott and Volkoff, 2011 and zebrafish Yokobori et al., 2011), Characiformes (cavefish Wall and Volkoff, 2013, dourado Volkoff et al., 2016, pacu Volkoff et al., 2017, and red-bellied piranha Volkoff, 2014a), and Pleuronectiformes (winter flounder Buckley et al., 2010 and Barfin flounder Amiya et al., 2012). In the mouth-brooding Astatotilapia burtoni (Perciforme), brain orexin mRNA levels increase in non-feeding females carrying eggs (Grone et al., 2012). In Atlantic cod (Gadiforme), orexin brain expression levels are higher in fish fed low rations than in fish fed high rations (Xu and Volkoff, 2007) or in fish fed the 30% camelina (plant) meal diet compared to fish fed a control (fish) diet (Tuziak et al., 2014), suggesting an effect of food quality and quantity on orexin expression. However, torpid cunner (Peciforme, labridae) undergoing a long-term fasting have low brain and gut orexin expression levels (Babichuk and Volkoff, 2013; Hayes and Volkoff, 2014), but this decrease might be due to a toprpor-induced general metabolic shutdown.
Anatomical studies provide further evidence for a role of orexin in nutrient digestion/abrorption and growth. In several fish species, e.g., pirapitinga (Piaractus brachypomus) (Characiforme) (Volkoff, 2015a), cunner (Perciforme) (Hayes and Volkoff, 2014) and rainbow trout (Salmoniforme) (Varricchio et al., 2015), orexin mRNA/protein expression is high in the gastrointestinal tract, suggesting a role of the orexin system in regulating feeding and digestive processes. Among Perciformes, in Japanese sea perch (Lateolabrax japonicus), orexin-like ir is present in pituitary GH-containing cells, suggesting a control of growth by the orexin system (Suzuki et al., 2007) and in Cichlasoma dimerus, orexin-ir fibers are present in both hypothalamus and in pituitary, suggesting a neuroendocrine control of pituitary secretions (Pérez Sirkin et al., 2013).
In addition to teleosts, orexin has been examined in the primitive bony fish birchir Polypterus senegalus and rope fish Erpetoichthys calabaricus (Chondrosteans, Polypteriformes) for which the brain orexin ir patterns are similar to that of other fish examined (López et al., 2014) and in the Chondrichthyan winter skate (Rajiforme), in which fasting increases hypothalamic orexin expression (MacDonald and Volkoff, 2010).
Overall, it appears that in all fish species studied to date, orexin is related to both food intake and appetitive/searching behavior and perhaps to growth.

Anorexigenic factors

CART
CART is a peptide which transcript expression is regulated by administration of cocaine or amphetamine in rodents (Vicentic and Jones, 2007; Subhedar et al., 2014) and amphetamine in goldfish (Volkoff, 2013). CART acts as an anorexigenic factor in mammals (Larsen and Hunter, 2006), and was first identified and shown to be anorexigenic in goldfish (Volkoff and Peter, 2000, 2001a).
Two CART isoforms have been identified in goldfish (Volkoff and Peter, 2001a) and common carp (Wan et al., 2012), and 4 in zebrafish (Akash et al., 2014) whereas, to date, only one form has been isolated for grass carp (Zhou et al., 2013; Liu et al., 2014), Characiformes [pirapitinga (serrasalmidae) (Volkoff, 2015a), pacu (serrasasalmidae) (Volkoff et al., 2017) and dourado (characidae) (Volkoff et al., 2016), red bellied piranha (serrasalmidae) (Volkoff, 2014a)], Salmoniformes [Atlantic salmon (Murashita et al., 2009a), rainbow trout (Figueiredo-Silva et al., 2012), Arctic charr (Striberny et al., 2015) and lake trout (Salvelinus namaycush) (Volkoff et al., 2007)], Siluriformes (channel catfish Kobayashi et al., 2008), Gadiformes (Atlantic cod Kehoe and Volkoff, 2007), Perciformes (cunner Babichuk and Volkoff, 2013), winter flounder (MacDonald and Volkoff, 2009a) and Atlantic halibut (Hippoglossus hippoglossus) (Gomes et al., 2015) (Pleuronectiformes), venomous toadfish Thalassophryne nattereri (Batrachoidiforme) (Magalhaes et al., 2006), rainbow smelt (Osmerus mordax) (Osmeriforme), pufferfishes (Takifugu rubripes and Tetraodon nigroviridis, Tetraodontiforme) and stickleback Gasterosteus aculeatus (Gasterosteiforme) (cited in Murashita et al., 2009a). However, six forms of CART have been identified in the medaka (Beloniforme) (Murashita and Kurokawa, 2011) and seven forms in Senegalese sole Solea senegalensis (Pleuronectiforme), the highest number of CART genes reported to date in a vertebrate species (Bonacic et al., 2015). The only elasmobranch CART identified to date is that of winter skate (Rajiforme) (MacDonald and Volkoff, 2009b).
CART injections induce a decrease in food intake and an increase in locomotion in goldfish (Volkoff and Peter, 2000) and enhance responsiveness to sensory stimuli in zebrafish larvae (Woods et al., 2014), suggesting that CART is involved in feeding/searching behaviors in cyprinids.
Fasting/food restriction decreases CART brain expression in Cypriniformes (goldfish Volkoff and Peter, 2001a, zebrafish Nishio et al., 2012; Guillot et al., 2016 and common carp, Wan et al., 2012), most Characiformes (red-bellied piranha Volkoff, 2014a, and pacu Volkoff et al., 2017), most Salmoniformes (Atlantic salmon, Murashita et al., 2009a; Kousoulaki et al., 2013, rainbow trout Figueiredo-Silva et al., 2012), Atlantic cod (Kehoe and Volkoff, 2007), cunner (Perciforme) (Babichuk and Volkoff, 2013), medaka (CART3) (Murashita and Kurokawa, 2011), and Siluriformes (channel catfish Kobayashi et al., 2008, African sharptooth catfish Clarias gariepinus Subhedar et al., 2011), suggesting an anorexigenic role for CART in teleost fish. Postprandial increases in CART brain expression have been shown in Senegalese sole (CART1a, CART 2a and CART4) (Bonacic et al., 2015), pacu (Volkoff et al., 2017), dourado (Volkoff et al., 2016), channel catfish (Peterson et al., 2012) but not in cod (Kehoe and Volkoff, 2007).
However, in Arctic charr, CART hypothalamic expression is similar throughout the seasonal feeding cycles (Striberny et al., 2015) and fasting does not affect CART expression in either dourado (Volkoff et al., 2016), winter flounder (MacDonald and Volkoff, 2009a) or Atlantic halibut larvae (Gomes et al., 2015), and in lake trout, fish exposed to the pesticide tebufenozide and control fish have similar food intakes, despite higher CART mRNA brain expression levels in exposed fish (Volkoff et al., 2007). In winter skate, 2 weeks of fasting have no effects on brain CART expression (MacDonald and Volkoff, 2009b), suggesting that CART might not have a major feeding-regulating role in elasmobranchs.
CART expression does not appear to be affected by diet, as in both cod fed a camelina (plant) diet (Tuziak et al., 2014) or rotifers or zooplankton (Katan et al., 2016) and pacu fed soybean concentrate (Volkoff et al., 2017), similar CART brain expression are seen between experimental and control fish.
Overall, there is a large interspecific variation in the number of forms and responses to fasting in the CART system in fish, although most studies tend to show that CART is mostly a central factor that might act as an appetite inhibitor.

Pro-opiomelanocortin (POMC) family of peptides
Proopiomelanocortin (POMC) is a common precursor that is processed post-translationally to generate melanocortin peptides [α-, β-, and γ-melanocyte-stimulating hormone (α-, β-, γ-MSH)], adrenocorticotropic hormone (ACTH) and other hormones that include β-endorphin (β-END) and β-lipotropic hormone (β-LPH) (Adan et al., 2006; Takahashi, 2016). POMC is mainly produced in the vertebrate pituitary, but is also found in brain, in particular the arcuate nucleus (ARC) of the hypothalamus. Receptors for melanocortin peptides include five subtypes (MC1R- MC5R) (Takahashi, 2016). In mammals, POMC and α-MSH have been shown to be involved in the regulation of appetite and energy homeostasis: POMC neurons suppress appetite by releasing α-MSH, which is an agonist at the anorectic melanocortin-4 receptor (MC4R) (Adan et al., 2006; Cone, 2006; Sohn, 2015).
Teleost fish lack γ-MSH and the POMC gene encodes an extra MSH (δ-MSH) in elasmobranchs (Cérda-Reverter et al., 2011). Fish POMC was first identified in Salmoniformes (Kawauchi, 1983; Kitahara et al., 1988) and Cypriniformes (Arends et al., 1998), followed by the identification of several forms in other fish species. As in other vertebrates, fish POMC is mainly expressed in the pituitary gland, but also within the lateral tuberal nucleus, which is equivalent to the mammalian ARC (Cérda-Reverter et al., 2011). POMC, α-MSH and the MC4R have been shown to regulate feeding in a few fish species.
In goldfish, fasting does not seem to affect hypothalamic POMC mRNA expression levels (Cerdá-Reverter et al., 2003), but ICV administration of [Nle4, d-Phe7]- α-MSH, a melanocortin agonist, inhibits food intake (Cerdá-Reverter et al., 2003), suggesting the melanocortin system participates in central regulation of food intake in Cypriniformes (Cerdá-Reverter et al., 2003). In addition, ICV injections of a MSH (MC4R) receptor agonist (melanotan II) suppress hypothalamic NPY expression (Kojima et al., 2010), and hypothalamic α-MSH-containing neurons are in close contact to NPY-containing nerve fibers, suggesting that the anorexigenic actions of the melanocortin system are mediated in part by an inhibition of the NPY system. In zebrafish larvae, although early ISH studies could not detect fasting-induced changes in hypothalamic POMC transcript levels (Song et al., 2003), more recent qPCR studies indicate that POMCa expression decreases in starved fish (Shanshan et al., 2016). In addition, GH-transgenic zebrafish, who have increased feeding, display down-regulation of POMC (Dalmolin et al., 2015), consistent with an anorexigenic role for POMC-derived peptides in Cypriniformes.
Similarly, in salmonids, POMC/α-MSH appears to have an anorexigenic role. In coho salmon, IP injections of α-MSH decrease food intake (White et al., 2016), in rainbow trout, fasting induces a decrease in hypothalamic expression of POMC-A1 (but not POMC-A2 or POMC-B) (Leder and Silverstein, 2006), and in Atlantic salmon, expression of both POMC-A1 and POMC-B increase after feeding (Valen et al., 2011). Interestingly, α-MSH treatment does not affect feeding of GH-transgenic coho salmon (White et al., 2016), despite similar hypothalamic POMC and MC4R mRNA expression levels compared to non-transgenic fish (Kim et al., 2015), suggesting that the actions of α-MSH might be inhibited by high expression levels of GH and/or AgRP.
In both olive (Kang and Kim, 2015) and Barfin flounder (Takahashi et al., 2005) (Pleuronectiformes), pituitary POMC-C (isoforms 1, 2, and 3) mRNAs are not affected by fasting, suggesting pituitary POMC might not directly related to appetite regulation. However, in fasted halibut larvae, whole brain POMC-C mRNA expression is higher in unfed fish 30 min after re-feeding compared to continuously fed fish (Gomes et al., 2015), suggesting a short-term regulation of appetite. Given the small number of studies available, and the variation in experimental protocols (adults vs. larvae, pituitary vs. brain, long-term vs. short-term feeding), conclusions are difficult to drawn regarding the role of POMC in flatfish.