Metabolism (sensu lato) can be defined as “the complex of physical and chemical processes involved in the maintenance of life” (32). Metabolic rate is a measure for the amount of energy used per unit of time by an organism, generally assessed as rate of oxygen consumed per hour [e.g., O₂ consumption in millimoles per hour (33, 34)]. In fish and other bradymetabolic animals (see Data Sheet S1 in Supplementary Material), and these are mostly ectotherms, the standard metabolic rate (SMR) and maximum metabolic rate (MMR) are generally used as ratios to define the “scope for activity” (35). SMR refers to the minimal rate of energy expenditure in an organism at rest. Basically, this equals the BMR in the tachymetabolic birds and mammals. However, SMR can be influenced by ambient temperature (36). MMR indicates the highest possible rate of energy expenditure, e.g., during sustained and aerobic maximal activity. In between these states, the routine metabolism is defined as energy expenditure during spontaneous activity in common life processes (35). Note that only the aerobic metabolism is taken into account when metabolic rate is measured via oxygen consumption.

The majority of animal life forms are ectothermic (see Data Sheet S1 in Supplementary Material for discussion on terminology). The environment determines the body temperature of ectotherms for the major part and, with that, the pace of biochemical reactions and rates of physiological processes. If we go back to the Magadi tilapia, we have seen, to the best of our knowledge, the upper limit of metabolic performance of an aquatic ectotherm. Note that the maximum performance of this fish comes to 35% of that of a similar sized mammal (31). After the water–land transition of vertebrates (~390–360 Mya) and the evolution of endothermy, new niches of the terrestrial environment became available. True endothermy (by this we do not mean regional endothermy, as we will discuss below) evolved independently in two different clades: (i) the diapsid clade that gave rise to extant birds and (ii) the synapsid clade that gave rise to the contemporary mammals (37). Animals that invest in endothermy (birds, mammals) can stay active independent of meteorological conditions, but do so at phenomenal cost [i.e., it requires lots of fuel (38)].

One could argue that ectotherms function at the mercy of ambient temperature; however, they are not passive and have the potential to regulate their body temperature. By shifting thermal preference and actively migrating toward warmer or colder environments, ectotherms are capable of sophisticated and adaptive behavioral thermoregulation (39, 40). To cope with challenges like infections or stressors (41–43), some fish migrate to warmer water, i.e., behavioral fever, a process that should be regarded as an intrinsic feature of ectothermic physiology.

Indeed, ectothermy comes with many benefits: it is energetically more economical, since the energy demand per unit mass, compared to an endothermic animal of the same size, is four to five times lower (44). Therefore, ectotherms require less time for foraging, and by doing so reduce their energy demand even more as a result of less locomotor activity. Moreover, as ectotherms need not invest in maintenance of a relatively high and constant body temperature, they can allocate more energy to growth and reproduction (44).

Here, we seem to reach a paradox, as endothermy also has advantages over ectothermy. Besides relative independence from ambient temperatures, energy metabolism is significantly enhanced in a warm body, which allows for high and fast muscular activity, fast metabolism, and growth during circadian and seasonal fluctuations. Notably, locomotion requires much more energy in terrestrial than in aqueous environments [the energetic cost for transport is about 10 times higher in terrestrial, running or walking, vertebrates than in equal-sized swimmers (45)], which might have been an important determinant in the evolution of endothermy. In addition, endothermy provides a stable thermal environment, securing optimal enzyme activities (46) and facilitating the ultimate parental care seen in mammals (47, 48).

What drove the evolution of endothermy? Three major hypotheses addressed this question. Crompton et al. (49) proposed that endothermy evolved in a two-step process: first, a more or less constant body temperature was acquired; next, the body temperature and metabolic rate increased. McNab (50) argued that early reptiles with a large body mass were de facto inertial homeotherms. Following an evolution of fur and decreasing body size, with a modest increase in mass-specific metabolic rate, inertial homeothermy became true endothermy. The most widely accepted theory, however, was put forward by Bennett and Ruben (38), who argued that higher body temperatures and endothermy have evolved secondarily to the selection on enhanced maximal aerobic capacity. This is known as the aerobic scope hypothesis. The evolution of endothermy is maybe the single most debated topic in comparative biology. The proposed hypotheses are not mutually exclusive. For this review, we focus on the aerobic scope hypothesis, as it conveniently joins aspects of metabolic demand and energy balance.

Bennett and Ruben (38) showed that the ratio between resting and maximal metabolic rates in vertebrate ectotherms and endotherms is roughly the same [on average 10 (51)], although the resting metabolic rate in endotherms is around 10 times higher than in ectotherms of similar body mass, i.e., the factorial aerobic scope is comparable for ectotherms and endotherms, but the absolute aerobic scope (the difference between resting and maximal metabolic rates) is much greater in endotherms. As a result, endotherms have, in general, more energy available for processes other than resting metabolism. The aerobic scope hypothesis holds aerobic metabolism at its center, which is important in sustained muscular activity. Still, a reptile can outrun a mammal, achieving great muscular power through anaerobic metabolism (52). This burst activity is time limited, as lactic acid builds up; some lizards and amphibians are highly tolerant to these metabolic waste products (53, 54).

Clarke and Pörtner (37) modified the original aerobic scope hypothesis: they regard a higher body temperature as the mechanism by which a greater aerobic scope was achieved, rather than as a consequence. They point out that an endotherm is not merely a warm ectotherm, since several processes are linked to the evolution of endothermy, such as the modification of mitochondrial membranes and heat retention (insulation by feathers, hair, and fat) mechanisms. In their view, the evolution of endothermy is considered a gradual process in which the driving force was selection for increased aerobic scope (37).

The magnitude of aerobic scope is greatly influenced by temperature. An optimal aerobic scope, associated with a species’ thermal niche specialization, is of vital importance for their performance and fitness [reviewed by Pörtner et al. (55)]. The first indication for thermal intolerance in ectotherms is a decreased aerobic scope due to a mismatch in oxygen supply and demand in tissues (56). Even before critical temperatures (here defined by the switch from aerobic to anaerobic metabolism) are reached, failure of circulatory and ventilatory systems occurs, which affects all higher functions (e.g., locomotor activity, behavior, growth, and reproduction) (57). Hence, even slight decreases in aerobic scope can result in lowered performance and enhanced mortality. The mechanisms that are crucial for aerobic scope and related to thermal intolerance are best studied in cold-adapted eurytherm and stenotherm fishes. These special adaptations allow them to withstand seasonal and permanent cold, respectively (58).

Eurythermal fishes are those that tolerate a wider thermal range than stenothermal fishes, and this relates to their ability to increase mitochondrial density or capacity in the cold to prevent hypoxia in tissues (58, 59). This is energetically costly, since they need to upregulate their SMR for mitochondrial maintenance. Stenotherm fishes have a permanently low SMR, associated with specialized cold-adapted mitochondria. These are found in extremely high densities in aerobic tissues, but do not increase overall aerobic capacity (60). Sidell (61) explained that the high density of enlarged mitochondria, seen in the icefishes lacking Hb and Mb, forms an interwoven membrane network, acting as a “lipid highway” for oxygen delivery. Selection for energy savings may have narrowed the thermal window for stenotherm fishes to survive with minimal aerobic capacity and energy expenditure, but allows them to preserve metabolic energy for processes like growth and reproduction, which are temporarily suspended in cold-acclimatized eurytherm fishes (55, 58).

Where is the heat coming from in an endotherm? During generation of ATP in the mitochondria, highly energetic electrons pass through the electron transfer chain and protons are pumped from the mitochondrial matrix into the intramembranous space. Following build-up of that gradient, protons flow back over the mitochondrial inner membrane and drive an H⁺-ATP synthase. As membranes are somewhat leaky for protons, some protons passively diffuse over the membrane and, without being used for ATP synthesis, emit energy as heat. Controlled uncoupling via the mitochondrial uncoupling proteins (UCPs), of the proton flux from ATP generation was thought to be the main source of heat in endotherms (62). However, more recent research shows that UCPs are not the exclusive actors in proton conductance, as the observed proton leak contributes proportionally and equally to the SMR in ectotherms and endotherms (63).

In mammals, five UCPs are present. In mammals UCP1 is responsible for non-shivering thermogenesis, whereas UCP2 and UCP3 are thought to be involved in maintaining the resting metabolism. In fish, we find orthologs of UCP1, UCP2, and UCP3, illustrating that the presence of UCPs is not restricted to endothermic animals (64, 65). In common carp (Cyprinus carpio), hepatic ucp1 transcript abundance was significantly downregulated in response to cold (opposing the function of UCP1 in mammals). Note that the fish liver is a primary storage site of energy. In red muscles, ucp3 is predominantly expressed and increases up to fivefold in response to fasting (65). As fasting leads to high lipid oxidation and reactive oxygen species, UCP3 is suggested to protect mitochondria and cells from damage via mild uncoupling activity or fatty acid anion export (66–68). The omnipresent UCP2 may play a similar scavenging role. Certainly, UCPs are evolutionary much older than originally thought; they were already present in fish ~420 Mya (65).

Although most fishes are strictly ectothermic, there are (of course) also fish that attain a regional endothermy in important organs and tissues, such as muscles, eyes, visceral organs, and the brain (69). We follow the nomenclature proposed by Clarke and Pörtner (37) (see Data Sheet S1 in Supplementary Material), and name this process heterothermy. An exceptional group of teleosts capable of heterothermy are the Thunnini (tuna).

Tuna are obligate ram ventilators and they are not sufficiently buoyant, which means they have to swim continuously to maintain a constant water flow over their gills (to fuel their relatively high metabolic activity) and to prevent sinking (70). Tuna have a specialized red muscle (RM) that is constantly metabolically active to power this so-called cruise swimming. Since the byproduct of all metabolic processes is heat, tunas have developed a way to retain the heat that is generated by RM and use it to elevate their body temperature regionally (69).

In tuna, the circulation to and from RM is structurally arranged in so-called retia mirabilia (literally: wonderful nets) (71). In these parallel to one another organized vascular bundles with very thin vessel walls, arterial and venous vessels lay side by side with the blood flowing in opposite directions, so that they function as countercurrent heat exchangers. Hence, a stable thermal gradient is established, which warms the cool arterial blood coming from the gills. The heat conserved is used to warm other body parts (72). When tuna dive during predation from pelagic zones with high temperature (e.g., 20°C) to more benthic zones where temperatures may drop to around 4°C, the biochemistry of brains, eyes, and swimming muscles is guaranteed of a rather constant temperature.

Considering that the SMR of tuna is 2–10 times higher than in most other fish species of comparable size and activity (69), this demonstrates that metabolism, and more specifically metabolic rate, is at the basis of thermal regulation in both ecto- and endotherms. The production of heat and having heat-retaining mechanisms are equally important. In general, maintaining the temperature of (part of) the body above the ambient temperature, is energetically costlier than keeping the body at lower temperatures, due to higher standard or basal metabolic rates. However, it also results in enhanced ATP production, which in turn allows for higher locomotor activity, growth rate and, completing the circle, metabolic rate. To understand causality, one needs to take a closer look at the regulation of energy intake and expenditure, and a comparative endocrine view may facilitate this, as we will do in the next section. Yet, it is clear why temperature is regarded as the abiotic master factor (73).

Metabolism, thermoregulation, and aerobic performance are intertwined and have one nexus in common: they are all dependent on food intake, food being the source of chemical energy for these related processes. In general, and despite mismatches in energy intake and expenditure on the short term, on the long term, they are carefully balanced and regulated by several endocrine systems, which together guarantee energy homeostasis (74).

A plethora of research on mammals is available in this field, not in the least to gain a better understanding of disturbances in energy homeostasis, which are pivotal for the current obesity pandemic (75, 76). In a healthy animal, the control system that matches energy intake to energy expenditure is remarkably accurate. A striking example is put forward by Seeley and Woods (75), who calculated that it takes only a mismatch of 46 kJ per day (0.55% of the daily energy intake!) for an adult human male to gain a pound in a year. Such tight regulation of energy balance can only be achieved by an accurate integration of signal molecules from stored and currently available fuel, in accordance with internal set points reflecting energy availability.

Where these signals come from and how they contribute to the regulation of food intake is of ongoing research interest. It has long been recognized that specific hypothalamic nuclei are crucial in monitoring energy balance (77), and these seem to be mainly tuned by signals regarding the storage of fat (i.e., lipostatic regulation). Another theory about hypothalamic tuning of energy balance through signaling of carbohydrate storage [i.e., glucostatic regulation (78)] has largely been abandoned in the past decades, as it has become clear that neurons are well protected from fluctuating glucose levels and usually feeding takes place when plasma glucose levels are within normal physiological values (75).

The link between body-fat stores (adiposity) and food intake was formalized by Kennedy (79), when he postulated that signals distributed in proportion to the total amount of body fat influence the control of food intake by the brain. This lipostatic regulation is based on a negative feedback system involving communication on the total amount of fat by adiposity signals and the central nervous system (CNS) (80).

Since adipose tissue is poorly innervated by the peripheral nervous system, research has been directed mainly to humoral signals. Adiposity signals (see below) should fulfill three criteria, viz.,: they should circulate in proportion to the total body fat, they should reach specific nuclei within the CNS [i.e., cross the blood–brain barrier (BBB)], and they should produce predictable changes in energy balance by altering food intake and energy expenditure dose-dependently (74).

In the 1980s, ample evidence identified insulin as a major adiposity signal (81, 82). Porte and Woods (81) showed that intracerebroventricular (icv) infusion of insulin in baboons induced drastic weight loss and decreased food intake dose-dependently. In additional studies, insulin receptors were found in key brain areas involved in control of food intake, including the hypothalamic arcuate nucleus (ARC, see below) (83, 84). Insulin is shuttled over the BBB via receptor-mediated transport (85). Insulin, produced by pancreatic β-cells, is best known for its involvement in carbohydrate metabolism; in response to high plasma glucose levels, insulin enhances the uptake of glucose by adipocytes, liver, and skeletal muscle cells, storing energy as glycogen or triglycerides (86). Insulin circulates in proportion to the amount of adipose tissue, although its levels fluctuate greatly when food is ingested and absorbed, in accordance with its hypoglycemic actions. However, baseline insulin levels and the magnitude of the fluctuations following food intake are directly proportional to body adiposity (87).

A second adiposity signal, leptin, a type-I α-helical cytokine, is encoded by the obese (ob) gene and was discovered in mice in 1994 (88) and has gained unparalleled momentum in research on energy homeostasis. It is a potent anorexigenic hormone produced in peripheral white fat and meets the aforementioned criteria for an adiposity signal. In addition, receptors for leptin are present in brain areas involved in the regulation of food intake, especially in the ARC (75).

Two subpopulations of neurons in the ARC integrate peripheral signals regarding energy homeostasis: the “anabolic” population expresses neuropeptide Y and agouti-related peptide, is orexigenic, and inhibited by leptin (89), whereas the “catabolic” population expresses pro-opiomelanocortin and cocaine and amphetamine regulated transcript, is anorexigenic, and stimulated by leptin (90). In line with this model, leptin administration in rodents was shown to decrease body mass dose dependently (91, 92). During fasting, plasma leptin levels fall dramatically and over prolonged periods of food deprivation, changes in the activities of the gonadal, adrenal, and thyroid axes are observed (93). Postprandially, leptin does not increase significantly, nor does it lead to termination of a meal by itself, indicating that leptin is largely involved in long-term regulation of feeding behavior and energy balance (94).

Leptin administration is an effective treatment for obesity in mice and humans with genetic leptin deficiency (95–97). However, in most cases of obesity, circulating leptin levels are already high and leptin therapy is not effective. Indeed, leptin resistance and the consequent lack of anorexic signaling in the ARC is commonly associated with obesity (98). Interestingly, leptin resistance in the ARC does not cause obesity, but it contributes to its persistence, as it develops secondarily after adiposity and body mass increase (99). More recent research revealed that vagal afferent neurons (VAN), apart from their role in meal termination via short-term gut–brain signaling, are involved in the long-term regulation of food intake (100–102). The authors showed that leptin resistance developed in VAN, before hypothalamic leptin signaling became disturbed (100). Moreover, when the leptin receptor is knocked out in VAN specifically, mice displayed increased food intake, body mass, and adiposity, indicating that the absence of leptin signaling in VAN is an important factor in the onset of hyperphagia and obesity (101).

Both adiposity signals (insulin and leptin) exert their function centrally through signaling via the ARC neurons, which results in reduced food intake and increased energy expenditure (103, 104). Arguably, leptin, for a while thought to be the panacea to beat obesity (105), received major attention in obesity research, whereas the role of insulin was less appreciated. This might be due to the opposing actions of insulin in peripheral tissues and the CNS; its hypoglycemic and anabolic function (energy storage) peripherally; and its catabolic effect (steering energy expenditure) in the hypothalamus (106). In the periphery, however, obesity eventually leads to inflammation of adipose tissue, and the following interplay between leptin signaling and the inflammatory cytokines secreted by macrophages seems to contribute to insulin resistance (107).

The complementary, and sometimes redundant, roles of leptin and insulin in the central regulation of energy homeostasis are best illustrated in a condition known as diabetic hyperphagia. When pancreatic β-cells become non-functional, complete depletion of insulin leads to strong hyperglycemia and an inability to store energy peripherally, with considerable weight loss as a result. However, without the catabolic insulin action in the hypothalamus and the co-occurring decrease in plasma leptin levels, increased compensatory food intake is initiated, counteracting energy wasting and preventing more rapid weight loss (106). Administration of either leptin or insulin to the CNS in rats with streptozotocin-induced diabetes blocks this compensatory hyperphagia almost completely (108, 109).

In fact, one can state that leptin and insulin are equally important adiposity signals involved in a negative feedback loop, as has been shown in many studies before (Figure 1) (75, 103, 110, 111). We focus on leptin mainly in this review, because leptin received the lion’s share of attention in recent research; however, we recognize the significance of the undervalued role of insulin. As Seeley and Woods (75) point out, insulin provides the brain with information not only about fat storage (long-term energy) but also about glucose availability (short-term energy). From this perspective, although indirectly, glucostatic regulation of food intake is indeed taking place. Thus, insulin signaling to the brain could provide the link that integrates glucostatic and lipostatic peripheral signals, allowing for a precise monitoring and accurate regulation of energy balance.

The insulin signaling pathway and its role in energy metabolism is evolutionary conserved and serves fundamentally the same physiological functions from invertebrates to mammals; it is found in phylogenetically distant invertebrate species, such as Caenorhabditis elegans and Drosophila melanogaster, where it is involved in energy storage (106, 112–114). Interestingly, these animals express homologs of key players in the insulin signal transduction pathway, but do not seem to actively regulate carbohydrate fluxes. They do regulate fat stores: insulin appears to limit energy storage in C. elegans (mutants for insulin signal transduction had increased fat deposits), which indicates a catabolic function of this protein in early evolution (106).

In elasmobranchs (mostly carnivores), protein and lipid metabolism are the primary energy sources (115), and although infusion of mammalian insulin resulted in severe prolonged hypoglycemia in spiny dogfish (Squalus acanthias) (over 15 times lower than time-zero levels), these fish did not exhibit any symptoms of illness (116). Apparently, strict glucose regulation is not as vital in these animals as it is in later vertebrates. The structure of insulin and its receptor is also highly conserved among vertebrates (117). Fish insulins, as their mammalian orthologs, consist of an α- and β-chain, linked by disulfide bridges (118); when we align the zebrafish an human insulin genes, we find a sequence of 60%.

As in mammals, it is important to distinguish between peripheral and central functions of insulin. The role of insulin in the periphery as an anabolic hormone has gained more attention, as it was long, but erroneously thought that fish were glucose intolerant [reviewed in Ref. (117)]. He argues that metabolic rate is a factor commonly overlooked in the studies performed on glucose clearance in fish. Acknowledging the fact that the metabolic rate of fish is about 10 times lower than in mammals, it may not come as a surprise that glucose turnover is rather slow (119). For several fish, it was shown that amino acids are a more potent stimulator of insulin secretion than glucose (120, 121), and fish are in general much slower in clearing a glucose load, compared to mammals (117). Notwithstanding, hyperglycemia does induce hyperinsulinemia in vivo (122–124). Significant differences between species exist: in the herbivorous carp glucose is more potent in stimulating insulin secretion than it is in carnivorous species, such as trout (Oncorhynchus mykiss) and sea bream (Sparus aurata) (125).

Contrasting results have been found in studies that address the central role of insulin. Fish brains are likely insulin-sensitive organs, as they contain insulin receptors (126–128). In channel catfish (Ictalurus punctatus), 24 h of icv injection of insulin appeared to have no effect on food intake (128). In contrast, icv administration of insulin in rainbow trout elicited a reduction of food intake after 26 h (129). Moreover, it has been shown in rainbow trout that insulin levels drop dramatically after 6 weeks of fasting, a response also seen in mammals (130). These conflicting outcomes could be the result of the longer administration time in the trout study or, more likely, central insulin effects could be species dependent. Progressive insight suggests that fish, like mammals, have specialized neurons involved in glucose sensing, which modulate the hypothalamic neurocircuitry controlling food intake (131).

Whether insulin has similar functions in fish as in mammals in communicating adiposity status to the CNS is not evident. In studies on rainbow trout, insulin stimulates lipogenesis in the liver and, more recently, in white mesenteric adipose tissue (132, 133). Traditionally, researchers considered the fish liver to be the main lipogenic tissue (134). However, Polakof et al. (133) showed that in rainbow trout fed a carbohydrate-rich diet and provided with insulin, lipogenesis in adipose tissue was significantly enhanced. The researchers hypothesized that the increased lipogenic capacity helps to regulate glycemia when excess carbohydrates need to be processed. In addition, these results are indicative for the utilization of lipids, stored in white adipose tissue, as an energy source. In juvenile chinook salmon, circulating insulin levels increased with a high-fat diet, and body adiposity clearly reduced food intake compared to fish that were fed a low-fat diet (135). However, no direct association between insulin levels and adiposity was found, but insulin levels correlated with dietary fat content.

More than a decade after the breakthrough identification of the mammalian ob gene and its protein product leptin (1994), the first non-mammalian ortholog was characterized in a teleostean fish [common carp (136, 137)] and many other fish species followed, including zebrafish (Danio rerio) (138), Atlantic salmon (Salmo salar) (139), and tiger pufferfish (Takifugu rubripes) (140). More recently, the first non-vertebrate functional leptin homolog was identified in Drosophila (141). The Drosophila cytokine Unpaired 2 has structural and functional similarities with mammalian leptin, is secreted by the fat body and signals via a similar intracellular messenger signaling pathway to reduce growth and alter energy metabolism. The canonical paper by Zhang et al. (88) addressed the ob gene as “evolutionary conserved” after demonstrating its presence in evolutionary distant species like Drosophila and eel (Anguilla rostrata LeSueur). However, once fish leptin was cloned and sequenced in 2004 (Huising et al.; accession numbers AJ868357 and AJ868356), the primary sequence identity between fish leptins and mammalian leptins turned out to be less than 25% (136), at variance with the earlier proposed “evolutionary conservation.” However, conserved gene structure, phylogenetic analyses, the conservation of essential cysteine residues, and tertiary structure, as well as synteny, firmly demonstrated true orthology of teleostean leptins (136, 138, 140). This indicates that if one wants to study the original function(s) of leptin(s), fish should be addressed.

The organization of hypothalamic nuclei involved in energy metabolism and food intake is conserved throughout the vertebrate lineage (142, 143), yet reports on leptin’s physiological role(s) have not been consistent for different fish species, which may not be surprising considering the vast number of species and niche adaptations. Most studies, however, recognize the organs and tissues with a high-fat content (liver or muscle) as the main expression sites for leptin in fish, not adipose tissue like in mammals (136, 138–140, 144, 145). In contrast with mammals, the liver is one of the major energy depots (glycogen and fat) in fish, together with muscle tissue and mesenteric fat (146). Moreover, lipid catabolism is the main source of energy in many fish species (147). Next to being the primary energy storage site, hypodermic fat in endotherms serves a second function, viz. insulation (148). No such insulation is required in an ectotherm, which then may explain differential leptin functioning (see below).

In some fish, leptin inhibits food intake through an interaction with hypothalamic orexigenic and anorexigenic genes, as it does in mammals. For example, in goldfish, administration of heterologous (murine) leptin caused a decrease in food intake, with lower doses needed when applied centrally than when applied peripherally to exert the same effect (149). In rainbow trout, recombinant homologous leptin injected intraperitoneally (144) and recombinant human leptin, administered icv, reduces food intake (150).

In common carp, two leptin paralogs were characterized initially, designated leptin-I and leptin-II, which, given their similarity, are probably the result of the tetraploidization following the “recent” cyprinid WGD ~16 Mya (11, 136, 138). These were later renamed leptin-a paralogs. The common carp leptin-a paralog did not respond to 6 weeks of fasting, nor to feeding to satiation, which feeding regime caused the experimental fish to grow twice as fast as controls. Only transient postprandial increase and decrease were observed in hepatic leptin-a-II and leptin-a-I mRNA, respectively (136).

Reduction of food intake is an early response of fish when exposed to stress in general (18) and hypoxia in particular (151, 152); in mammals, leptin levels are known to increase in hypoxic conditions (153). Indeed, the mammalian ob gene contains hypoxia response element (HRE) sites which can be bound by HIF-1α, a transcription factor that regulates the expression of hypoxia-sensitive genes (153–155). Analyses of the promotor region of zebrafish leptin and the leptin receptor (lepr) revealed putative HRE in both genes (156). Therefore, a follow-up study with common carp was done to elucidate the relation between leptin, hypoxia, and the hypothalamic regulation of food intake. It was shown that hepatic leptin-a-I, leptin-a-II, and lepr expression in common carp is indeed stimulated in hypoxic conditions, which is congruent with a reduction in food intake (156). This fits with a well-known strategy among fishes to deal with hypoxia (152, 157, 158), since appetite suppression leads to precious energy and oxygen savings by reducing the cost of specific dynamic action (i.e., the metabolic energy cost of digestion) (159). These results were subsequently confirmed by a study in zebrafish, in which chronic hypoxia and HIF-1α induced a rise in hepatic leptin mRNA levels (160).

The Bernier laboratory (2012) compared hypothalamic gene expression between hypoxic and fasted (pair-fed to the hypoxic groups) carp, which led to the suggestion that during hypoxia, leptin counteracts the suppression of pomc and upregulation of agrp, characteristic for fasted carp (136), since agrp levels were not affected in hypoxia and the suppression of pomc was attenuated (156). These data provide grounds for the involvement of leptin in re-establishing energy balance during chronic hypoxia and indicate a broader physiological role for leptin beyond the signaling of nutrient status.

After the initial cloning of leptin in carp, multiple leptin paralogs were found in zebrafish (138) and in the Japanese ricefish [Oryzias latipes, aka medaka (145)] and named leptin-a and leptin-b. Indeed, also in common carp a leptin-b paralog was found (167) and the first characterized carp leptins were renamed leptin-a-I and leptin-a-II. The leptin-a and leptin-b paralogs most likely find their origin in the third, fish-specific, genome duplication, as they are found in evolutionary distant species, such as zebrafish (Cypriniformes) and medaka (Beloniformes), that shared their last common ancestor ~296 Mya (168).

However, reflecting the diversity among teleostean fishes, leptin phylogeny appears to be even more complex (Figure 2). Also in the genome of salmonids, four leptin paralogs (leptin-aI/II and leptin-bI/II) are present. These paralogs originated as a result of the salmonid WGD 88–103 Mya (13, 139, 169). In more modern fish, e.g., the Tiger puffer (Tetraodon family), only a single leptin gene is found, which suggests that this lineage experienced genome reduction, after the split from the Beloniformes ~186 Mya (170).

As some of the leptin paralogs share very low primary amino acid sequence identity even within a species [e.g., zebrafish leptin-a and leptin-b amino acid identity is only 24% (138)], it is likely that these leptin genes acquired different functionality (neofunctionalization or sub-functionalization) (165). Testimony to this reasoning is that leptin-a and leptin-b are expressed differentially (at least spatially); leptin-a is mainly expressed in the liver (138, 139), while the ovary is the main expression site for leptin-b (138). In addition, calculations on binding energy suggest that in both medaka and zebrafish, leptin-a has a higher binding energy for the leptin receptor than leptin-b (172).

Surprisingly, in all currently available teleostean genomes only one lepr gene is found (165). In salmonids [Atlantic salmon; Rønnestad et al. (139), and rainbow trout; Gong et al. (173)], several splice variants have been identified, but, as in the mammalian situation, only one of these variants contains the full length sequence required for intracellular signal transduction. In Atlantic salmon, four truncated and one full length leptin receptor variants were identified, the latter of which was ubiquitously expressed, including in gills, gonads, and brain (139), whereas in rainbow trout at least three splice variants gave rise to functional circulating leptin binding proteins (LepBP1, LepBP2, and LepBP3) (173). Together, these findings suggest a complex mode of leptin functioning in teleostean fish, with both inter- and intraspecies differences in the interaction of leptin (paralogs) with the leptin receptor, and in the modulation of endocrine and/or paracrine signaling pathways.

In the past 5 years, research on leptin functioning in teleostean fish has been carried out not only in the field of feeding and appetite regulation (156, 174–177) but has extended to other endocrine regulatory systems, including sexual maturation (169, 178), energy expenditure and metabolic rate (179, 180), osmotic adaptation (181, 182), and glucose homeostasis (183), all of which we will discuss below. In addition, the involvement of leptin in reproduction (184), the growth hormone (GH)–insulin-like growth factor axis [GH–IGF axis (185)], hypothalamic neurotensin networks (186), and the immune system (187) has been studied.