Richard L. Londraville

Leptin in Teleost Fishes: An Argument for Comparative Study

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.

Murine leptin injections increase intracellular fatty acid-binding protein in green sunfish (Lepomis cyanellus).

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 leptins action may be slower in ectotherms due to their lower metabolic rate.

Expression of leptin receptor gene in developing and adult zebrafish.

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.

Comparative endocrinology of leptin: assessing function in a phylogenetic context.

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.

Leptin and leptin receptor: Analysis of a structure to function relationship in interaction and evolution from humans to fish

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.

Discovery of the Elusive Leptin in Birds: Identification of Several ‘Missing Links’ in the Evolution of Leptin and Its Receptor

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.

Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish

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.

Up-regulation of cadherin-2 and cadherin-4 in regenerating visual structures of adult zebrafish.

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.

Molecular evolution of GPCRs: Melanocortin/melanocortin receptors.

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).

Cold acclimation increases fatty acid-binding protein concentration in aerobic muscle of striped bass, Morone saxatilis

Fatty acid-binding protein was isolated from aerobic skeletal muscle of the striped bass Morone saxatilis. FABP from striped bass (MS-FABP) has a molecular mass of 14,800 Da (as estimated by SDS-PAGE) and binds long-chain fatty acids with 1:1 molar stoichiometry and with micromolar affinity. Binding dissociation constants, as estimated by liposome assay, were 0.63 ± 0.44, 0.82 ± 0.12, and 1.60 ± 0.44 μM for palmitic (16:0), oleic (18:1), and palmitoleic (16:1) acids, respectively. No significant difference was detected in MS-FABPs binding affinity among these fatty acids or those estimated by competitive displacement assay (22:6, 20:5, and 18:0). Acclimation of striped bass from 25°C to 5°C caused an increase in FABP concentration in aerobic skeletal muscle (5°C = 1.44 ± 0.12 mg/g wet weight; warm = 1.04 ± 0.11 mg/g wet weight). The magnitude of this increase matches the predicted decrease in diffusion coefficient of FABP at 5°C. Previous studies, however, report that fatty acid oxidation is not only maintained but increased in cold-acclimated fish. We speculate that FABP may increase flux of fatty acids through β-oxidation (in cold-acclimated fish) by an increase in the percent of FABP molecules carrying fatty acid.