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During an escape from predators, many animals need to evaluate and choose a refuge within seconds. We investigated refuge choice in the amphibious blackspotted rockskipper, Entomacrodus striatus, in Moorea, French Polynesia. Rockskippers are small combtooth blennies that inhabit rocky beaches and jetties at the aquatic/terrestrial interface. They are conspicuous for their eponymous jumping to/from refugia among rocks when threatened. We have observed refugia with both multiple conspecifics and solitary fish in the field, and here tested whether fish choose refugia that are occupied by conspecifics in the laboratory. E. striatus chose unoccupied refugia on the opposite side of the experimental tank in 11/14 trials, a significantly greater number of times than they chose occupied refugia. In 3/14 trials, fish chose occupied refugia, indicating that refuge occupation does not prohibit their use by conspecifics. We hypothesize that chemical stress signals from the occupying fish deter most fish from choosing the same refuge., (© 2023 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

The hypothesis advanced is that endospanin, a highly conserved vesicle traffic protein in vertebrates, regulates leptin sensitivity in bone signaling. The effects of leptin on bones are well-studied but without consensus on whether the increases in leptin signaling stimulate bone gain or loss. The bone response may depend on leptin sensitivity, and endospanin is an established modulator of leptin sensitivity. An argument is advanced to develop zebrafish models for specific leptin signaling pathways. Zebrafish have well-developed molecular tools (e.g., CRISPR) and the advantage of non-destructive sampling of bones in the form of scales. Using these tools, experiments are described to substantiate the role of endospanin in zebrafish bone dynamics., Competing Interests: 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., (Copyright © 2022 Londraville, Tuttle, Liu and Andronowski.)

Response of orange-finned anemonefish Amphiprion chrysopterus and three-spot damselfish Dascyllus trimaculatus to red laser-pointer light was studied in Mo'orea, French Polynesia. Four magnificent anemones Heteractis magnifica and their resident fish were observed for typical behaviours (biting, chasing, hiding, posing, lunging and retreating) with and without exposure to laser-pointer light. Lunging behaviour increased significantly for both fish species upon exposure to laser-pointer light; none of the other behaviours changed significantly. We advance the hypothesis that orange-finned anemonefish and three-spot damselfish interpret laser pointer stimulation as a territorial threat., (© 2019 The Fisheries Society of the British Isles.)

We analyzed microarray expression data to highlight biological pathways that respond to embryonic zebrafish Leptin-a ( lepa ) signaling. Microarray expression measures for 26,046 genes were evaluated from lepa morpholino oligonucleotide "knockdown", recombinant Leptin-a "rescue", and uninjected control zebrafish at 72-hours post fertilization. In addition to KEGG pathway enrichment for phosphatidylinositol signaling and neuroactive ligand-receptor interactions, Gene Ontology (GO) data from lepa rescue zebrafish include JAK/STAT cascade, sensory perception, nervous system processes, and synaptic signaling. In the zebrafish lepa rescue treatment, we found changes in the expression of homologous genes that align with mammalian leptin signaling cascades including AMPK ( prkaa2 ), ACC ( acacb ), Ca 2+ /calmodulin-dependent kinase ( camkk2 ), PI3K ( pik3r1 ), Ser/Thr protein kinase B ( akt3 ), neuropeptides ( agrp2 , cart1 ), mitogen-activated protein kinase (MAPK), and insulin receptor substrate ( LOC794738 , LOC100537326 ). Notch signaling pathway and ribosome biogenesis genes respond to knockdown of Leptin-a. Differentially expressed transcription factors in lepa knockdown zebrafish regulate neurogenesis, neural differentiation, and cell fate commitment. This study presents a role for zebrafish Leptin-a in influencing expression of genes that mediate phosphatidylinositol and central endocrine signaling., Competing Interests: The authors declare there are no competing interests.

Over a decade passed between Friedman's discovery of the mammalian leptin gene (1) and its cloning in fish (2) and amphibians (3). Since 2005, the concept of gene synteny conservation (vs. gene sequence homology) was instrumental in identifying leptin genes in dozens of species, and we now have leptin genes from all major classes of vertebrates. This database of LEP (leptin), LEPR (leptin receptor), and LEPROT (endospanin) genes has allowed protein structure modeling, stoichiometry predictions, and even functional predictions of leptin function for most vertebrate classes. Here, we apply functional genomics to model hundreds of LEP, LEPR, and LEPROT proteins from both vertebrates and invertebrates. We identify conserved structural motifs in each of the three leptin signaling proteins and demonstrate Drosophila Dome protein's conservation with vertebrate leptin receptors. We model endospanin structure for the first time and identify endospanin paralogs in invertebrate genomes. Finally, we argue that leptin is not an adipostat in fishes and discuss emerging knockout models in fishes.

The processes of lipid deposition and utilization, via the gene leptin (Lep), are poorly understood in taxa with varying degrees of adipose storage. This study examines how these systems may have adapted in marine aquatic environments inhabited by cetaceans. Bowhead (Balaena mysticetus) and beluga whales (Delphinapterus leucas) are ideal study animals-they possess large subcutaneous adipose stores (blubber) and undergo bi-annual migrations concurrent with variations in food availability. To answer long-standing questions regarding how (or if) energy and lipid utilization adapted to aquatic stressors, we quantified variations in gene transcripts critical to lipid metabolism related to season, age, and blubber depth. We predicted leptin tertiary structure conservation and assessed inter-specific variations in Lep transcript numbers between bowheads and other mammals. Our study is the first to identify seasonal and age-related variations in Lep and lipolysis in these cetaceans. While Lep transcripts and protein oscillate with season in adult bowheads reminiscent of hibernating mammals, transcript levels reach up to 10 times higher in bowheads than any other mammal. Data from immature bowheads are consistent with the hypothesis that short baleen inhibits efficient feeding. Lipolysis transcripts also indicate young Fall bowheads and those sampled during Spring months limit energy utilization. These novel data from rarely examined species expand the existing knowledge and offer unique insight into how the regulation of Lep and lipolysis has adapted to permit seasonal deposition and maintain vital blubber stores.

Cetacean evolution was shaped by an extraordinary land-to-sea transition in which the ancestors of whales became fully aquatic. As part of this transition, these mammals evolved unusually thick blubber which acts as a metabolic reservoir as well as an insulator and provides buoyancy and streamlining. This study describes blubber stratification and correlates it to seasonal variation, feeding patterns, and ontogeny in an arctic-adapted mysticete, the bowhead whale (Balaena mysticetus). Bowheads are unique among mammals for possessing the largest known blubber stores. We found that adipocyte numbers in bowheads, like other mammals, do not vary with season or feeding pattern but that adipocyte size and structural fiber densities do vary with blubber depth., (© 2015 Wiley Periodicals, Inc.)

As we approach the end of two decades of leptin research, the comparative biology of leptin is just beginning. We now have several leptin orthologs described from nearly every major clade among vertebrates, and are moving beyond gene descriptions to functional studies. Even at this early stage, it is clear that non-mammals display clear functional similarities and differences with their better-studied mammalian counterparts. This review assesses what we know about leptin function in mammals and non-mammals, and gives examples of how these data can inform leptin biology in humans., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The melanocortin receptors (MCRs) are a family of G protein-coupled receptors that are activated by melanocortin ligands derived from the proprotein, proopiomelanocortin (POMC). During the radiation of the gnathostomes, the five receptors have become functionally segregated (i.e. melanocortin 1 receptor (MC1R), pigmentation regulation; MC2R, glucocorticoid synthesis; MC3R and MC4R, energy homeostasis; and MC5R, exocrine gland physiology). A focus of this review is the role that ligand selectivity plays in the hypothalamus/pituitary/adrenal-interrenal (HPA-I) axis of teleosts and tetrapods as a result of the exclusive ligand selectivity of MC2R for the ligand ACTH. A second focal point of this review is the roles that the accessory proteins melanocortin 2 receptor accessory protein 1 (MRAP1) and MRAP2 are playing in, respectively, the HPA-I axis (MC2R) and the regulation of energy homeostasis by neurons in the hypothalamus (MC4R) of teleosts and tetrapods. In addition, observations are presented on trends in the ligand selectivity parameters of cartilaginous fish, teleost, and tetrapod MC1R, MC3R, MC4R, and MC5R paralogs, and the modeling of the HFRW motif of ACTH(1-24) when compared with α-MSH. The radiation of the MCRs during the evolution of the gnathostomes provides examples of how the physiology of endocrine and neuronal circuits can be shaped by ligand selectivity, the intersession of reverse agonists (agouti-related peptides (AGRPs)), and interactions with accessory proteins (MRAPs)., (© 2014 Society for Endocrinology.)

Leptin is a pleiotropic protein best known for regulation of appetite and fat storage in mammals. While many leptin orthologs have been identified among vertebrates, an authentic leptin in birds has remained elusive and controversial. Here we identify leptin sequence from the Peregrine falcon, Falco peregrinus (pfleptin), and identify sequences from two other birds (mallard and zebra finch), and 'missing' vertebrates (elephant shark, alligator, Indian python, Chinese soft-shelled turtle, and coelacanth). The pattern of genes surrounding leptin (snd1, rbm28) is syntenic between the falcon and mammalian genomes. Phylogenetic analysis of all known leptin protein sequences improves our understanding of leptin's evolution. Structural modeling of leptin orthologs highlights a highly conserved hydrophobic core in the four-helix cytokine packing domain. A docked model of leptin with the leptin receptor for Peregrine falcon reveals several conserved amino acids important for the interaction and possible coevolution of leptin with its receptor. We also show for the first time, an authentic avian leptin sequence that activates the JAK-STAT signaling pathway. These newly identified sequences, structures, and tools for avian leptin and its receptor will allow elucidation of the function of these proteins in feral and domestic birds.

We used antisense morpholino oligonucleotide technology to knockdown leptin-(A) gene expression in developing zebrafish embryos and measured its effects on metabolic rate and cardiovascular function. Using two indicators of metabolic rate, oxygen consumption was significantly lower in leptin morphants early in development [<48 hours post-fertilization (hpf)], while acid production was significantly lower in morphants later in development (>48 hpf). Oxygen utilization rates in <48 hpf embryos and acid production in 72 hpf embryos could be rescued to that of wildtype embryos by recombinant leptin coinjected with antisense morpholino. Leptin is established to influence metabolic rate in mammals, and these data suggest leptin signaling also influences metabolic rate in fishes.

Leptin is the primary hormone in mammals that regulates adipose stores. Arctic adapted cetaceans maintain enormous adipose depots, suggesting possible modifications of leptin or receptor function. Determining expression of these genes is the first step to understanding the extreme physiology of these animals, and the uniqueness of these animals presents special challenges in estimating and comparing expression levels of mRNA transcripts. Here, we compare expression of two model genes, leptin and leptin-receptor gene-related product (OB-RGRP), using two quantitative real-time PCR (qPCR) methods: "relative" and "absolute". To assess the expression of leptin and OB-RGRP in cetacean tissues, we first examined how relative expression of those genes might differ when normalized to four common endogenous control genes. We performed relative expression qPCR assays measuring the amplification of these two model target genes relative to amplification of 18S ribosomal RNA (18S), ubiquitously expressed transcript (Uxt), ribosomal protein 9 (Rs9) and ribosomal protein 15 (Rs15) endogenous controls. Results demonstrated significant differences in the expression of both genes when different control genes were employed; emphasizing a limitation of relative qPCR assays, especially in studies where differences in physiology and/or a lack of knowledge regarding levels and patterns of expression of common control genes may possibly affect data interpretation. To validate the absolute quantitative qPCR methods, we evaluated the effects of plasmid structure, the purity of the plasmid standard preparation and the influence of type of qPCR "background" material on qPCR amplification efficiencies and copy number determination of both model genes, in multiple tissues from one male bowhead whale. Results indicate that linear plasmids are more reliable than circular plasmid standards, no significant differences in copy number estimation based upon background material used, and that the use of ethanol precipitated, linearized plasmid preparation produce the most reliable results.

Leptin is a circulating protein which regulates dietary intake through binding the leptin receptor. Numerous labs have used known structures and mutagenesis to study this binding process in common animal models (human, mouse and rat). Understanding this binding process in other vertebrate species will allow for a better understanding of leptin and leptin receptor function. The binding site between leptin and leptin receptor is highly conserved in mammals as confirmed through sequence alignments mapped onto structures of both leptin and leptin receptor. More variation in this interaction is found in lizard and frog sequences. Using our models, we show that the avian leptin sequences have far less variation in the binding site than does the leptin receptor. This analysis further suggests that avian leptins are artifactual. In fish, gene duplication events have led to the expression of multiple leptin proteins. These multiple leptin proteins have variation in the regions interacting with leptin receptor. In zebrafish and the Japanese rice fish, we propose that leptin A has a higher binding energy than does B. Differing binding energies are evidence of either divergent functions, different binding confirmations, or other protein partners of leptin B., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Using morpholino antisense oligonucleotide (MO) technology, we blocked leptin A or leptin receptor expression in embryonic zebrafish, and analyzed consequences of leptin A knock-down on fish development. Embryos injected with leptin A or leptin receptor MOs (leptin A or leptin receptor morphants) had smaller bodies and eyes, undeveloped inner ear, enlarged pericardial cavity, curved body and/or tail and larger yolk compared to control embryos of the same stages. The defects persisted in 6-9 days old larvae. We found that blocking leptin A function had little effect on the development of early brain (1 day old), but differentiation of both the morphant dorsal brain and retinal cells was severely disrupted in older (2 days old) embryos. Despite the enlarged pericardial cavity, differentiation of cardiac cells appeared to be similar to control embryos. Formation of the morphants' inner ear is also severely disrupted, which corroborates existing reports of leptin receptor expression in inner ear of both zebrafish and mammals. Co-injection of leptin A MO and recombinant leptin results in partial rescue of the wild-type phenotype. Our results suggest that leptin A plays distinct roles in zebrafish development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

All organisms face tradeoffs with regard to how limited energy resources should be invested. When is it most favorable to grow, to reproduce, how much lipid should be allocated to storage in preparation for a period of limited resources (e.g., winter), instead of being used for growth or maturation? These are a few of the high consequence fitness "decisions" that represent the balance between energy acquisition and allocation. Indeed, for animals to make favorable decisions about when to grow, eat, or reproduce, they must integrate signals among the systems responsible for energy acquisition, storage, and demand. We make the argument that leptin signaling is a likely candidate for an integrating system. Great progress has been made understanding the leptin system in mammals, however our understanding in fishes has been hampered by difficulty in cloning fish orthologs of mammalian proteins and (we assert), underutilization of the comparative approach.

Interactions of leptin and leptin receptors play crucial roles during animal development and regulation of appetite and energy balance. In this study we analyzed expression pattern of a zebrafish leptin receptor gene in both developing and adult zebrafish using in situ hybridization and Q-PCR methods. Zebrafish leptin receptor message (lepr) was detected in all embryonic and larval stages examined, and in adult zebrafish. In embryonic zebrafish, lepr was mainly expressed in the notochord. As development proceeded, lepr expression in the notochord decreased, while its expression in several other tissues, including the trunk muscles and gut, became evident. In both larval and adult brains, large lepr expressing cells were detected in similar regions of the hindbrain. In adult zebrafish, lepr expression was also observed in several other brain regions including the hypothalamic lateral tuberal nucleus, the fish homolog of the arcuate nucleus. Q-PCR experiments confirmed lepr expression in the adult fish brain, and also showed lepr expression in several adult tissues including liver, muscle and gonads. Our results showed that lepr expression was both spatially and temporally regulated., ((c) 2009 Elsevier Inc. All rights reserved.)

Physiological colour change was investigated in the blackspotted rockskipper Entomacrodus striatus in Moorea, French Polynesia. Fish colour cycled with significant autocorrelation over the 30 min observation period and was not affected by observation temperature (27 and 31 degrees C). Cycling depended most on dark and yellow pigments (as assayed by separation of colours via software), and therefore, it was hypothesized that short-term cycling was driven by melanophores and xanthophores.

Cadherin cell adhesion molecules play crucial roles in vertebrate development. Most studies have focused on examining the functions of classical type I cadherins (e.g., cadherin-2) in the development of vertebrates. Little information is available concerning the function of classical type II cadherins (e.g., cadherin-7) in vertebrate development. We have previously shown that cadherin-7 mRNA exhibits a dynamic expression pattern in the central nervous system and notochord in embryonic zebrafish. To gain insight into the role of cadherin-7 in the formation of these structures, we analyzed their formation in zebrafish embryos injected with cadherin-7-specific antisense morpholino oligonucleotides (MO). Notochord development was severely disrupted in MO-injected embryos, whereas gross defects in the development of the central nervous system were not detected in MO-injected embryos. Our results thus demonstrate that cadherin-7 plays an important role in the normal development of the zebrafish notochord.

Cadherin cell adhesion molecules exhibit unique expression patterns during development of the vertebrate central nervous system. In this study, we obtained a full-length cDNA of a novel zebrafish cadherin using reverse transcriptase-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of this molecule is most similar to the published amino acid sequences of chicken and mammalian cadherin7 (Cdh7), a member of the type II cadherin subfamily. cadherin7 message (cdh7) expression in embryonic zebrafish was studied using in situ hybridization and RT-PCR methods. cdh7 expression begins at about 12h postfertilization (hpf) in a small patch in the anterior neural keel, and along the midline of the posterior neural keel. By 24 hpf, cdh7 expression in the brain shows a distinct segmental pattern that reflects the neuromeric organization of the brain, while its expression domain in the spinal cord is continuous, but confined to the middle region of the spinal cord. As development proceeds, cdh7 expression is detected in more regions of the brain, including the major visual structures in the fore- and midbrains, while its expression domain in the hindbrain becomes more restricted, and its expression in the spinal cord becomes undetectable. cdh7 expression becomes reduced in 3-day old embryos. Our results show that cdh7 expression in the zebrafish developing central nervous system is both spatially and temporally regulated.

Characterizing leptin's structure and function in mammals has been the subject of thousands of studies since 1994. Recently, the study of leptin has expanded to include its distribution in non-mammalian taxa, and the role that leptin plays in the reproductive axis. We demonstrated in a previous study that Sceloporus undulatus, fence lizards (ectotherms), express a leptin-like protein. In the current study we quantified seasonal variation in this putative leptin among free-ranging fence lizards from two populations characterized by early and late reproductive maturation (after one or two years, respectively). Immunoblots were performed on whole blood samples to detect leptin and estimate its titer. Leptin titers were higher in the reproductive population of S. undulatus (early maturing: 2.5+/-0.2 microg/mL; late-maturing 2.2+/-0.3 microg/mL; mean+/-2 S.E.), but both populations showed the same seasonal pattern. Leptin titers were lowest in fall when fat stores are expected to be highest (spring: 2.6+/-0.3 microg/mL; summer: 2.6+/-0.3; microg/mL; fall: 1.8+/-0.3 microg/mL), consistent with findings of seasonal variation in free-ranging mammals. Our data support previous work asserting that lizards express leptin and that it has a similar physiological function in endotherms and ectotherms. Our long-term goal is to use leptin to manipulate age at maturity and to test fundamental questions in the evolution of life-history strategies.

Zebrafish is a good model organism for the study of vertebrate development due to numerous experimental advantages. Here we show that zebrafish are also suitable for the study of cellular and molecular mechanisms underlying tissue regeneration in the vertebrate central nervous system. In this report we describe simple protocols to introduce injuries to the adult zebrafish visual structures and analyze cadherin-2 expression in the regenerating visual system using immunocytochemistry and immunoblotting.

We tested the hypothesis that the physiological strategy for acclimating to low body temperature is similar among closely related fish. Largemouth bass (Micropterus salmoides), green sunfish (Lepomis cyanellus), bluegill sunfish (Lepomis macrochirus), black crappie (Pomonix nigromaculatus), and white crappie (Pomonix annularis), all members of the family Centrarchidae, were acclimated to 5 degrees and 25 degrees C. Morphometric variables (total mass, total length, organ masses) and enzyme activities (hexokinase; lactate dehydrogenase; and cytochrome oxidase in heart, liver, and muscle) were measured in 5 degrees C- and 25 degrees C-acclimated fish at 5 degrees and 25 degrees C assay temperatures. Each species displayed a distinct physiological response to cold acclimation that differed among tissues. These data suggest that the response to cold acclimation is highly variable within families. Our findings are consistent with other studies suggesting that acclimation responses are labile and may evolve independently even among closely related species.

Green sunfish (Lepomis cyanellus) were injected daily with either murine leptin, phosphate-buffered saline (PBS), or simply handled without injection for 14 days. At the end of the experiment, fish were assayed for intracellular indicators of fatty acid metabolism. Intracellular fatty acid-binding protein (FABP) expression in heart ventricle was twofold higher in the leptin treated group (87.2+/-5.6 Leptin; 47.2+/-6.8 PBS; 28.9+/-3.9 Handled; percent relative expression, Prob.>F<0.001). Two other indicators of intracellular fat metabolism, carnitine palmitoyl transferase activity (CPT) in liver and 3-hydroxyacyl-CoA dehydrogenase (HOAD) in heart were not significantly different among groups, although the trend is for higher values in the leptin treatment (CPT: 0.23+/-0.04 Leptin, 0.11+/-0.04 PBS, 0.10+/-0.03 Handled; U/gm wet weight; Prob.>F=0.08; HOAD: 1.34+/-0.28 Leptin, 0.76+/-0.12 PBS, 0.86+/-0.25 Handled; U/gm wet weight; Prob.>F=0.18). Percent change in total weight, body fat (as a percent of dry weight), cardiosomatic index, and hepatosomatic index were not significantly different among treatments. These results suggest that fish respond to murine leptin injections by increasing fat metabolism, however many of the hallmarks of leptin treatment in mammals (loss of total weight and body fat) were not observed. This lack of response may be due to incompatibility of mouse leptin with fish receptors or an inadequate dose of leptin. We also suggest that leptin's action may be slower in ectotherms due to their lower metabolic rate.

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. In this study, we examined expression of zebrafish cadherin-2 (Cdh2, N-cadherin) and cadherin-4 (Cdh4, R-cadherin) in the visual system of adult zebrafish after eye or optic nerve lesions using immunocytochemistry and immunoblotting. Both Cdh2 and Cdh4 immunoreactivities were specifically up-regulated in regenerating retina and/or the optic pathway. Furthermore, temporal expression patterns of these two cadherins were distinct during the regeneration of the injured tissues. Cadherins have been shown to regulate axonal outgrowth in the developing nervous system, but this is the first report, to our knowledge, of increased cadherin expression associated with axonal regeneration in the vertebrate central nervous system. Our results suggest that both Cdh2 and Cdh4 may be important for regeneration of injured retinal ganglion cell axons.

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.

Tissues from bony fish were screened with anti-mouse leptin antibodies to detect the presence of the fat-regulating hormone in fishes. Low molecular-weight (16 kDa) immunoreactive bands were detected in blood, brain, heart and liver of green sunfish (Lepomis cyanellus), bluegill sunfish (Lepomis macrochirus), largemouth bass (Micropterus salmoides), white crappie (Pomonix annularis), channel catfish (Ictalurus punctatus), and rainbow trout (Oncorhynchus mykiss). To further verify that we had identified leptin, the response of fish "leptin" was measured in fed and fasted green sunfish. Fed sunfish had approximately threefold higher concentration of leptin in blood than did fasted sunfish (fed vs. fasted; 0.599 +/- 0.03 microg/microl vs. 0.196 +/- 0.04 microg/microl; P > F = 0.0001), which is consistent with mammalian models of leptin function. Brain leptin concentration is also positively correlated with percent body fat in white crappie and bluegill. Based upon electrophoretic mobility, immunoreactivity, response to fasting, and correlation with adiposity, we believe we have the first evidence for leptin expression in an ectotherm.

Leptin is a hormone that regulates energy expenditure and body mass in mammals, and it has attracted considerable attention because of its potential in treating human obesity. Comprehensive data from both pathological and non-pathological systems strongly support a role for leptin in regulating energy metabolism, in thermoregulation and in regulating the onset of puberty. We report here that daily injections of recombinant murine leptin in fence lizards (Sceloporus undulatus) produce phenotypic effects similar to those observed when leptin injections are given to mice. Lizards injected with leptin had body temperatures 0.6 degrees C higher, ate 30 % less food and showed a 14 % reduction in activity rates, and females showed a 2. 5-fold increase in resting metabolic rates, compared with lizards injected with vehicle only (phosphate-buffered saline). We also detected native lizard leptin using an immunoassay. Our results indicate that leptin is expressed in ectotherms and may be conserved both functionally and structurally. In the wake of unprecedented research activity on the role of leptin as a cause of, and potential treatment for, human obesity, we believe that other applications of leptin research have been ignored. For example, the response of lizards to leptin injection in our study has important implications for two broad areas of research in evolutionary biology: the evolution of age at first reproduction and of endothermy. We argue that research in these areas, previously limited to comparative approaches, may now benefit from experimental manipulations using leptin.

A variety of circulating fuels can support the work of the teleost gill. Previous work indicates, however, that unlike other aerobic tissues from teleosts, the gill may have a limited capacity to oxidize fatty fuels. We determined capacities for catabolism of carbohydrate, fatty acids, and amino acids in four species of temperate marine or euryhaline teleosts representing distinct lineages. In addition, we assessed the capacity for fatty acid oxidation in the gill from an Antarctic species. Activities of rate-limiting or regulatory enzymes from pathways of energy metabolism were measured at physiological temperatures (15 degrees or 1 degrees C). In the temperate species, ATP yields from glucose are 3- to 30-fold greater (varying with species) than ATP yields from a monounsaturated fatty acid, while ATP generation from glutamate is 2-50 times greater than similar capacities for the lipid fuel. Like the temperate species, capacity for beta-oxidation of fatty acids is limited in the Antarctic species. A positive linear correlation between activities of citrate synthase (central pathway of oxidative metabolism) and hexokinase (glycolysis) adds further support to the hypothesis that glucose is a preferred metabolic fuel in gill. Our results also demonstrate that fatty acid-binding protein is present in the gill of teleost fishes. It is likely that this protein plays a more important role facilitating anabolic pathways in lipid metabolism rather than fatty acid oxidation in the gill of teleost fishes., (Copyright 1999 Wiley-Liss, Inc.)

We have cloned a group of cDNAs that encodes the skeletal ryanodine receptor isoform (RyR1) of fish from a blue marlin extraocular muscle library. The cDNAs encode a protein of 5,081 amino acids with a calculated molecular mass of 576,302 Da. The deduced amino acid sequence shows strong sequence identity to previously characterized RyR1 isoforms. An RNA probe derived from a clone of the full-length marlin RyR1 isoform hybridizes to RNA preparations from extraocular muscle and slow-twitch skeletal muscle but not to RNA preparations from fast-twitch skeletal or cardiac muscle. We have also isolated a partial RyR clone from marlin and toadfish fast-twitch muscles that shares 80% sequence identity with the corresponding region of the full-length RyR1 isoform, and a RNA probe derived from this clone hybridizes to RNA preparations from fast-twitch muscle but not to slow-twitch muscle preparations. Western blot analysis of slow-twitch muscles in fish indicates the presence of only a single high-molecular-mass RyR protein corresponding to RyR1. [3H]ryanodine binding assays revealed the fish slow-twitch muscle RyR1 had a greater sensitivity for Ca2+ than the fast-twitch muscle RyR1. The results indicate that, in fish muscle, fiber type-specific RyR1 isoforms are expressed and the two proteins are physiologically distinct.

This report provides the first evidence for the existence of two distinct types of fatty acid-binding protein (FABP) in cardiac tissue of vertebrates. Four species of Antarctic teleost fish (Chaenocephalus aceratus, Cryodraco antarcticus, Gobionotothen gibberifrons and Notothenia coriiceps) exhibited two FABP mRNAs of 1. 0 kb and 0.8 kb, which we have termed Hh-FABP and Had-FABP (isolated from Heart tissue, with similarity to mammalian heart-type FABP or mammalian adipose-type FABP respectively). These FABP types appear to be products of distinct genes. Both FABP transcripts were abundant in cardiac and aerobic pectoral muscle. However, relative abundance of the two types varied distinctly among other tissues such as kidney, brain, spleen and white muscle. Neither FABP type was expressed in liver or intestine. The coding regions of Hh-FABP and Had-FABP cDNAs from the same species are only approximately 60% identical with one another. However, homologues of each FABP species, which exhibit >98% identity to their respective types, were isolated from three other Antarctic teleosts. Phylogenetic analysis of aligned amino-acid sequences places Hh-FABP with other vertebrate heart-type FABPs, and Had with adipose/cutaneous FABPs. Expression of two distinct FABPs in cardiac tissue of Antarctic teleosts may be related to their ability to both utilize fatty acid as the primary metabolic fuel and to store lipid intracellularly.

The important intracellular oxygen-binding protein, myoglobin (Mb), is thought to be absent from oxidative muscle tissues of the family of hemoglobinless Antarctic icefishes, Channichthyidae. Within this family of fishes, which is endemic to the Southern Ocean surrounding Antarctica, there exist 15 known species and 11 genera. To date, we have examined eight species of icefish (representing seven genera) using immunoblot analyses. Results indicate that Mb is present in heart ventricles from five of these species of icefish. Mb is absent from heart auricle and oxidative skeletal muscle of all species. We have identified a 0.9-kb mRNA in Mb-expressing species that hybridizes with a Mb cDNA probe from the closely related red-blooded Antarctic nototheniid fish, Notothenia coriiceps. In confirmation that the 0.9-kb mRNA encodes Mb, we report the full-length Mb cDNA sequence of the ocellated icefish, Chionodraco rastrospinosus. Of the eight icefish species examined, three lack Mb polypeptide in heart ventricle, although one of these expresses the Mb mRNA. All species of icefish retain the Mb gene in their genomic DNA. Based on phylogeny of the icefishes, loss of Mb expression has occurred independently at least three times and by at least two distinct molecular mechanisms during speciation of the family.

Binding affinity and binding-pocket polarity is determined for intracellular fatty acid-binding protein (FABP) from aerobic muscle of Chaenocephalus aceratus, the Antarctic icefish, and from rat heart. FABPs bind fatty acids via weak-bond forces (both ionic and hydrophobic), and these bond forces are temperature sensitive, yet FABPs are present in animals whose body temperatures range over nearly 40 degrees C. To investigate FABP's sensitivity to body temperature, fatty acid binding affinity (Kd) was determined for both rat heart-FABP and icefish heart-FABP at two physiological temperatures (0 degrees C or 37 degrees C). Saturated and unsaturated fatty acids (16:0 and 16:1), delivered in model membranes (liposomes) whose composition is typical of either Antarctic fish (16:0/22:6 phosphatidylcholine) or mammals (bovine-heart phosphatidylcholine) were examined. Incubation at 0 degree C or 37 degrees C dose not significantly affect Kd for rat heart FABP, regardless of liposome composition or fatty acid ligand (Kd = 0.686 +/- 0.127 - 1.129 +/- 0.356 microM at 0 degree C, 0.775 +/- 0.307 - 1.605 +/- 0.427 microM at 37 degrees C). Incubation temperature significantly affects icefish FABP's affinity for 16:1 (0.626 +/- 0.093 microM at 37 degrees C vs. 1.896 +/- 0.343 microM at 0 degree C for fatty acid presented in Antarctic fish liposomes; 0.331 +/- 0.101 microM at 37 degrees C vs. 0.949 +/- 0.121 microM at 0 degree C for bovine heart liposomes) but not 16:0. Kd is not significantly different between FABPs under any set of conditions (with one exception: Kd is significantly lower in rat FABP vs. icefish FABP for 16:0 at 0 degree C for fatty acids delivered in bovine heart liposomes). Although Kd values are largely equivalent between the two FABPs, relative contributions from ionic vs. hydrophobic weak-bond forces are different between the two animals. Rat heart FABP has a binding pocket that is significantly more nonpolar than that of icefish FABP (as measured by quantum yield of the bound fluorescent fatty-acid analogue (PA-DPH); Q = 0.067 +/- 0.008 vs. 0.034 +/- 0.005 at 0 degree C, 0.030 +/- 0.003 vs. 0.019 +/- 0.002 at 37 degrees C). This suggests that rat-heart FABP realizes a micromolar Kd with a greater reliance upon hydrophobic interactions than does icefish FABP.

Intracellular fatty acid-binding proteins (FABPs) are small (15 kDa), highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. Fifteen isoforms have been identified and characterized to date. Members of the FABP protein family share many common characteristics: they have a common "clamshell" tertiary structure despite significant variation in primary structure; they bind their ligands with 1:1 molar stoichiometry and micromolar affinity (with the exception of liver type FABP, which binds two fatty acids per FABP); they are ubiquitously expressed, yet are found at greatest concentration in tissues with high capacities to oxidize or store lipids; and their level of expression changes with conditions that affect overall lipid metabolism. Because of the ligands bound by FABP and FABP's tissue distribution, it is thought that FABPs play a role in the intracellular metabolism of lipids; however, a precise function(s) for FABP has not been defined. This review highlights contributions from researchers studying FABP in nonmammalian systems. FABPs isolated from birds, fish, amphibians, insects, and flatworms have similar structures to mammals in FABPs, yet they are expressed in systems with unique lipid metabolisms. Because mammals are closely related and have relatively invariant fatty acid metabolisms, non-mammals provide alternative systems that may reveal the elusive function(s) of this protein family.

Intracellular fatty acid-binding protein is purified and characterized from aerobic skeletal muscle of the Antarctic icefish Chaenocephalus aceratus. Molecular mass of C. aceratus FABP (CA-FABP) is 14,936 Da as estimated by electrospray mass spectrometry. CA-FABP is expressed at an intracellular concentration of 0.984 +/- 0.115 mg CA-FABP g-1 wet weight aerobic muscle and binds 0.859 +/- 0.013 moles oleic acid per mole of protein at a physiological temperature of 0 degrees C. Dissociation constants (KdS for various fatty acid ligands range from 1.38 to 2.71 microM; KdS are not significantly different among palmitic acid (16:0), palmitoleic acid (16:1), and oleic acid (18:1). Competition assays reveal that CA-FABP does not have preferential affinity for the very-long-chain, polyunsaturated fatty acids that are common in Antarctic fish (e.g., docosahexaenoic acid; 22:6). Partial amino acid sequence from CA-FABP aligns with mammalian heart-type FABPs with as high as 74% identity. These data are strikingly similar to mammalian values, yet they are derived from an organism that is distant from mammals in terms of phylogeny, body temperature, and physiology. This suggests that the FABP family is conserved not only in primary sequence, but also in its physiological properties.

Mitochondrial volume density (Vv [mit]) distributions were measured with a test pattern of concentric rings centered upon randomly chosen capillaries in oxidative skeletal muscle cells of two Antarctic fishes, Trematomus newnesi and Notothenia gibberifrons. Vv(mit) in both species was highest in the ring closest to the capillary, minimal further from the capillary (at a distance that was characteristic for each species), and rose in the annuli furthest from the capillary. Plots of Vv(mit) against total area between each ring and the central capillary fit the form of a second-order polynomial (r2 greater than 0.9). If PO2 or blood-borne metabolite concentration predicates the pattern of Vv(mit) distribution, minimal Vv(mit) is at the same position as the minimum in concentration or gaseous partial pressure of capillary-supplied commodities. This minimum is the boundary between cylinders of tissue being supplied by adjacent capillaries, and thus delineates the maximal diffusion-distance for capillary-supplied commodities. Maximal diffusion-distance (microns) for T. newnesi = 26.23 +/- 1.64; N. gibberifrons = 21.45 +/- 0.51. For O2, maximal diffusion-distance conventionally is referred to as Krogh's radius, R. With an easily obtained estimate of numerical capillary density, these R values can be used to calculate a capillary tortuosity constant (c[k,0]) and capillary length density (Jv[c,f]). c(k,0) values were also determined using an established method, and R and Jv(c,f) values calculated from these values did not significantly differ from values determined from mitochondrial distributions. Mitochondrial distribution analysis may more accurately reflect changes in capillary blood flow and heterogeneity of diffusion and solubility constants within muscle than currently existing techniques. Similar distributions of Vv(mit) reported for several species of vertebrates suggest wide applicability of the method.