Mammalian Leptin Research Articles

Leptin- and cytokine-like unpaired signaling in Drosophila

Animals have evolved a multitude of signaling pathways that enable them to orchestrate diverse physiological processes to tightly regulate systemic homeostasis. This signaling is mediated by various families of peptide hormones and cytokines that are conserved across the animal kingdom. In this review, we primarily focus on the unpaired (Upd) family of proteins in Drosophila which are evolutionarily related to mammalian leptin and the cytokine interleukin 6. We summarize expression patterns of Upd in Drosophila and discuss the parallels in structure, signaling pathway, and functions between Upd and their mammalian counterparts. In particular, we focus on the roles of Upd in governing metabolic homeostasis, growth and development, and immune responses. We aim to stimulate future studies on leptin-like signaling in other phyla which can help bridge the evolutionary gap between insect Upd and vertebrate leptin and cytokines like interleukin 6.

Leptin signalling in teleost fish with emphasis in food intake regulation

Leptin, the product of the obese (ob or Lep) gene, was first cloned in teleost fish in 2005, more than a decade after its identification in mammals. This was because bony fish and mammalian leptins share a very low amino acid sequence identity, which suggests different functionality of the leptin system in fish compared to that of mammals. Indeed, major differences are evident between the mammalian and fish leptin system. Thus, for instance, mammalian leptin is synthesized and released by the adipose tissue in response to the amount of fat depots, while several tissues (mainly the liver) are the main sources of leptin in fish, whose determining factors of production are still unclear. In mammals, the main physiological role for leptin is its involvement in the maintenance of energy balance by decreasing food intake and increasing energy expenditure, although a wide variety of actions have been attributed to this hormone (e.g., regulation of lipid and carbohydrate metabolism, reproduction and immune functions). In fish, available literature also points towards a multifunctional nature for leptin, although knowledge on its functions is limited. In this review, we offer an overview of teleostean leptin structure and mechanism of action, and discuss the available knowledge on the role of this hormone in food intake regulation in teleost fish, aiming to provide a comparative overview between the functioning of the teleostean and mammalian leptin systems.

Leptin-a mediates transcription of genes that participate in central endocrine and phosphatidylinositol signaling pathways in 72-hour embryonic zebrafish (Danio rerio).

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), Ca2+/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.

Plasma mammalian leptin analogue predicts reproductive phenology, but not reproductive output in a capital-income breeding seaduck.

To invest in energetically demanding life history stages, individuals require a substantial amount of resources. Physiological traits, particularly those related to energetics, can be useful for examining variation in life history decisions and trade‐offs because they result from individual responses to environmental variation. Leptin is a protein hormone found in mammals that is proportional to the amount of endogenous fat stores within an individual. Recently, researchers have confirmed that a mammalian leptin analogue (MLA), based on the mammalian sequence of leptin, is present with associated receptors and proteins in avian species, with an inhibitory effect on foraging and body mass gain at high circulating levels. While MLA has been both quantified and manipulated in avian species, little is currently known regarding whether plasma MLA in wild‐living species and individuals is associated with key reproductive decisions. We quantified plasma MLA in wild, Arctic‐nesting female common eiders (Somateria mollissima) at arrival on the breeding grounds and followed them to determine subsequent breeding propensity, and reproductive phenology, investment, and success. Common eiders are capital‐income breeding birds that require the accumulation of substantial fat stores to initiate laying and successfully complete incubation. We found that females with lower plasma MLA initiated breeding earlier and in a shorter period of time. However, we found no links between plasma MLA levels and breeding propensity, clutch size, or reproductive success. Although little is still known about plasma MLA, based on these results and its role in influencing foraging behaviors and condition gain, plasma MLA appears to be closely linked to reproductive timing and is therefore likely to underlie trade‐offs surrounding life history decisions.

Molecular cloning, characterization and evolutionary analysis of leptin gene in Chinese giant salamander, Andrias davidianus

AbstractLeptin is an important hormone possessing diverse physiological roles in mammals and teleosts. However, it has been characterized only in a few amphibian species, and its evolutions are still under debate. Here, the full length of the leptin (Adlep) cDNA of Chinese giant salamander (Andrias davidianus), an early diverging amphibian species, is characterized and according to the results of the primary sequence analysis, tertiary structure reconstruction and phylogenetic analysis is confirmed to be an ortholog of mammalian leptin. An intron was identified between the coding exons of A. davidianus leptin, which indicated that the leptin is present in the salamander genome and contains a conserved gene structure in vertebrates. Adlep is widely distributed but expression levels vary among different tissues, with highest expression levels in the muscle. Additionally, the leptin receptor and other genes were mapped to three known leptin signaling pathways, suggesting that the leptin signaling pathways are present in A. davidianus. Phylogenetic topology of leptins are consistent with the generally accepted evolutionary relationships of vertebrates, and multiple leptin members found in teleosts seem to be obtained through a Cluopeocephala-specific gene duplication event. Our results will lay a foundation for further investigations into the physiological roles of leptin in A. davidianus.

Central injection of a synthetic chicken partial leptin peptide does not affect food intake in chicks.

Leptin is an adipose tissue-derived hormone in mammals that plays an important role in whole body energy balance via its inhibitory effects on food intake mediated through the hypothalamus. Chicken leptin has a low sequence homology to mammalian leptin and its role in appetite regulation is not reported; hence the objective of this study was to determine effects of central injection of chicken leptin on food and water intake and associated behaviors in chicks. Chicks were intracerebroventricularly injected with 0 (vehicle), 0.3, 1.0, or 3.0 nmol of a synthetic chicken leptin partial peptide and food and water intake were monitored. There were no effects observed and a second experiment was conducted to evaluate food and water intake at higher doses; after injection of 0, 2.5, 5.0, or 10.0 nmol leptin. Again, there were no effects on food or water intake. In the third experiment, behaviors were analyzed during the first 30 min post-injection of vehicle or 10 nmol leptin. At 5 min post-injection, vehicle-injected chicks spent more time sitting than leptin-injected chicks. A wide dose range was evaluated however, the absence of an effect on food intake or behavior suggests that the chicken leptin peptide that was tested does not mediate effects on appetite in the brain and that chicken leptin likely has a different physiological role in birds than in mammals.

On the Molecular Evolution of Leptin, Leptin Receptor, and Endospanin.

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.

Insights into kisspeptin- and leptin-signalling on GnRH mRNA expression in hypothalamic organ cultures of immature pikeperch Sander lucioperca

Two types of gonadotropin-releasing hormones (GnRH) were identified as gnrh1 and gnrh2 in pikeperch Sander lucioperca. The administration of rodent leptin on hypothalamic organ cultures of immature pikeperch resulted in significantly elevated levels of gnrh2, but not in gnrh1 mRNA, whereas kisspeptin-10 administration did not affect gnrh1 or gnrh2 expression. These results represent preliminary insights into leptin-GnRH-signaling on a hypothalamic level in fish, potentially coupling fat metabolism and the activation of the reproductive axis during puberty. Mammalian leptin and kisspeptin-10, however, failed to initiate a consistent response in pikeperch and their use cannot be recommended.

Short Communication Molecular conservation of the mammalian leptin protein.

In this study, we comparatively assessed multiple sequences of the leptin protein from different animal species to establish new insights into conservation degree of biological sequences and evolutionary biology among mammals using computational biology tools. First, amino acid sequences of the leptin protein from Homo sapiens (human, P41159), Sus scrofa (wild pig, Q29406), Felis catus (domestic cat, Q29406), Rattus norvegicus (rat, P50596), and Mus musculus (mouse, P41160) were randomly searched in the high-quality annotated and non-redundant protein sequence database UniProtKB/Swiss-Prot. A dendogram showing the evolutionary relationships among specimens was constructed from the sequences of interest using the Mega 6.0 software with the neighbor-joining method. The resulting tree presenting the evolutionary relationships among specimens inferred from amino acid sequences of the leptin protein in mammals demonstrated 2 main branches: 1 cluster including the rat and mouse species (0.02) and a second cluster containing both wild pig and domestic cat species grouped in a sub-branch (0.04 and 0.06, respectively), linking them to the human sequence (0.08). These findings were reinforced by comparing estimates of evolutionary divergence among leptin sequences analyzed. Based on comparative analyses of multiple sequence alignments in the present study, there was a stronger conservation degree of the leptin protein in evolutionarily close species and several conservative changes along the sequences of interest, revealing information regarding the evolutionary biology among mammals.

Leptin in teleostean fish, towards the origins of leptin physiology

Teleostean leptin was first cloned in 2005, more than a decade after the discovery of mammalian leptin. The reason for this delay lies in the very poor primary sequence conservation (∼13–25%) between mammalian and fish leptins. These low sequence conservations indicate a high degree of molecular evolvability and warrant a search for different and original functions of leptin in teleosts. Indeed, new and original insights are obtained because of the unique phylogenetic position of teleostean fish as the earliest vertebrates and because of their ectothermy, which means that teleosts are more flexible in changing their metabolism than mammals and leptin could play a role in this flexibility. Research during the last decade reveals that leptin is a truly pleiotropic hormone in fish and mammals alike, with functions among others in the regulation of food intake and body weight, development, but also in the regulation of the stress axis and acclimation processes to for instance low oxygen levels in the water. In this review, we provide an overview of the teleostean leptin work done in the last ten years, and demonstrate that the power of a comparative approach leads to new insights on the origins of leptin physiology.

Production, gene structure and characterization of two orthologs of leptin and a leptin receptor in tilapia

Full-length cDNA encoding two leptin sequences (tLepA and tLepB) and one leptin receptor sequence (tLepR) were identified in tilapia (Oreochromis niloticus). The full-length cDNA of tLepR was 3423bp, encoding a protein of 1140 amino acid (aa) which contained all functionally important domains conserved among vertebrate leptin receptors. The cDNAs of tLepA and tLepB were 486bp and 459bp in length, encoding proteins of 161 aa and 152 aa, respectively. Modeling the three-dimensional structures of tLepA and tLepB predicted strong conservation of tertiary structure with that of human leptin, comprised of four helixes. Using synteny, the tLeps were found near common genes, such as IMPDH1 and LLRC4. The cDNA for tLepA and tLepB was cloned and synthetic cDNA optimized for expression in Escherichia coli was prepared according to the cloned sequence. The tLepA- and tLepB-expressing plasmids were transformed into E. coli and expressed as recombinant proteins upon induction with nalidixic acid, found almost entirely in insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and purified to homogeneity by anion-exchange chromatography. In the case of tLepA, the fraction eluted contained a mixture of monomers and dimers. The purified tLepA and tLepB monomers and tLepA dimer showed a single band of ∼15kDa on an SDS–polyacrylamide gel in the presence of reducing agent, whereas the tLepA dimer showed one band of ∼30kDa in the absence of reducing agent, indicating its formation by S–S bonds. The three tLeps were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor (hLepR), but their activity was four orders of magnitude lower than that of mammalian leptin. Furthermore, the three tLeps were biologically active in promoting STAT-LUC activation in COS7 cells transfected with the identified tLepR but not in cells transfected with hLepR. tLepA was more active than tLepB. Low or no activity likely resulted from low identity (9–22%) to mammalian leptins. In an in vivo experiment in which tilapia were fed ad libitum or fasted, there was no significant difference in the expressions of tLepA, tLepB or tLepR in the brain between the two groups examined both by real-time PCR and RNA next generation sequencing. In conclusion, in the present report we show novel, previously unknown sequences of tilapia leptin receptor and two leptins and prepare two biologically active recombinant leptin proteins.

Discovery and characterization of the first genuine avian leptin gene in the rock dove (Columba livia).

Leptin, the key regulator of mammalian energy balance, has been at the center of a great controversy in avian biology for the last 15 years since initial reports of a putative leptin gene (LEP) in chickens. Here, we characterize a novel LEP in rock dove (Columba livia) with low similarity of the predicted protein sequence (30% identity, 47% similarity) to the human ortholog. Searching the Sequence-Read-Archive database revealed leptin transcripts, in the dove's liver, with 2 noncoding exons preceding 2 coding exons. This unusual 4-exon structure was validated by sequencing of a GC-rich product (76% GC, 721 bp) amplified from liver RNA by RT-PCR. Sequence alignment of the dove leptin with orthologous leptins indicated that it consists of a leader peptide (21 amino acids; aa) followed by the mature protein (160 aa), which has a putative structure typical of 4-helical-bundle cytokines except that it is 12 aa longer than human leptin. Extra residues (10 aa) were located within the loop between 2 5'-helices, interrupting the amino acid motif that is conserved in tetrapods and considered essential for activation of leptin receptor (LEPR) but not for receptor binding per se. Quantitative RT-PCR of 11 tissues showed highest (P < .05) expression of LEP in the dove's liver, whereas the dove LEPR peaked (P < .01) in the pituitary. Both genes were prominently expressed in the gonads and at lower levels in tissues involved in mammalian leptin signaling (adipose; hypothalamus). A bioassay based on activation of the chicken LEPR in vitro showed leptin activity in the dove's circulation, suggesting that dove LEP encodes an active protein, despite the interrupted loop motif. Providing tools to study energy-balance control at an evolutionary perspective, our original demonstration of leptin signaling in dove predicts a more ancient role of leptin in growth and reproduction in birds, rather than appetite control.

Inhibitory Mechanism of Signal Transduction through Chicken Leptin Receptor by Suppressor of Cytokine Signaling 3 (SOCS3)

Leptin is an adipocyte-derived hormone involved in the regulation of feeding behavior and energy homeostasis in vertebrates. We recently reported that leptin activates the JAK-STAT signaling pathway through the chicken leptin receptor (chLEPR). However, the molecular inhibitory mechanism by suppressor of cytokine signaling 3 (SOCS3), observed in mammalian leptin signaling, has not been elucidated in avian species. Therefore, the role of chicken SOCS3 (chSOCS3) in signal transduction through the chLEPR was analyzed in this study. Leptin increases SOCS3 mRNA expression in chicken hepatoma cells, LMH, and also activates the chSOCS3 gene promoter in the chLEPR-expressing cells. Overexpression of chSOCS3 inhibited leptin-induced signaling by blocking phosphorylation of JAK2 and subsequent activation of STAT3 similar to that observed in mammals. Signaling inhibited by chSOCS3 was not restored in the chLEPR mutated docking site of SOCS3. In addition, mutation of Phe25 in the kinase inhibitory region of chSOCS3 abolished SOCS3 activity via the wild chLEPR. The present study indicates that SOCS3 is a negative feedback regulator of leptin signaling in chickens as well as in mammals. However, the inhibitory mechanism in chickens may differ slightly from that observed in mammals.

Leptin protects host cells from Entamoeba histolytica cytotoxicity by a STAT3-dependent mechanism.

The adipocytokine leptin links nutritional status to immune function. Leptin signaling protects from amebiasis, but the molecular mechanism is not understood. We developed an in vitro model of ameba-host cell interaction to test the hypothesis that leptin prevents ameba-induced apoptosis in host epithelial cells. We demonstrated that activation of mammalian leptin signaling increased cellular resistance to amebic cytotoxicity, including caspase-3 activation. Exogenous expression of the leptin receptor conferred resistance in susceptible cells, and leptin stimulation enhanced protection. A series of leptin receptor signaling mutants showed that resistance to amebic cytotoxicity was dependent on activation of STAT3 but not the Src homology-2 domain-containing tyrosine phosphatase (SHP-2) or STAT5. A common polymorphism in the leptin receptor (Q223R) that increases susceptibility to amebiasis in humans and mice was found to increase susceptibility to amebic cytotoxicity in single cells. The Q223R polymorphism also decreased leptin-dependent STAT3 activation by 21% relative to that of the wild-type (WT) receptor (P = 0.035), consistent with a central role of STAT3 signaling in protection. A subset of genes uniquely regulated by STAT3 in response to leptin was identified. Most notable were the TRIB1 and suppressor of cytokine signaling 3 (SOCS3) genes, which have opposing roles in the regulation of apoptosis. Overall apoptotic genes were highly enriched in this gene set (P < 1E-05), supporting the hypothesis that leptin regulation of host apoptotic genes via STAT3 is responsible for protection. This is the first demonstration of a mammalian signaling pathway that restricts amebic pathogenesis and represents an important advance in our mechanistic understanding of how leptin links nutrition and susceptibility to infection.

Evidence for Positive Selection on the Leptin Gene in Cetacea and Pinnipedia

The leptin gene has received intensive attention and scientific investigation for its importance in energy homeostasis and reproductive regulation in mammals. Furthermore, study of the leptin gene is of crucial importance for public health, particularly for its role in obesity, as well as for other numerous physiological roles that it plays in mammals. In the present work, we report the identification of novel leptin genes in 4 species of Cetacea, and a comparison with 55 publicly available leptin sequences from mammalian genome assemblies and previous studies. Our study provides evidence for positive selection in the suborder Odontoceti (toothed whales) of the Cetacea and the family Phocidae (earless seals) of the Pinnipedia. We also detected positive selection in several leptin gene residues in these two lineages. To test whether leptin and its receptor evolved in a coordinated manner, we analyzed 24 leptin receptor gene (LPR) sequences from available mammalian genome assemblies and other published data. Unlike the case of leptin, our analyses did not find evidence of positive selection for LPR across the Cetacea and Pinnipedia lineages. In line with this, positively selected sites identified in the leptin genes of these two lineages were located outside of leptin receptor binding sites, which at least partially explains why co-evolution of leptin and its receptor was not observed in the present study. Our study provides interesting insights into current understanding of the evolution of mammalian leptin genes in response to selective pressures from life in an aquatic environment, and leads to a hypothesis that new tissue specificity or novel physiologic functions of leptin genes may have arisen in both odontocetes and phocids. Additional data from other species encompassing varying life histories and functional tests of the adaptive role of the amino acid changes identified in this study will help determine the factors that promote the adaptive evolution of the leptin genes in marine mammals.

Expression of leptin-like peptide (LLP) mRNA in channel catfish (Ictalurus punctatus) is induced by exposure to Edwardsiella ictaluri but is independent of energy status

Leptin is a key pleiotropic cytokine involved in regulation of energy homeostasis and immunity in mammals. In channel catfish, the presence of a partial messenger RNA sequence that encodes a leptin-like peptide (LLP) has been identified and investigated. The objectives of the present studies were to clone and characterize full-length catfish LLP gene, examine tissue expression of LLP mRNA, and determine effects of prolonged fasting and exposure to Edwardsiella ictaluri (E. ictaluri), the bacteria that causes enteric septicemia in catfish, on LLP mRNA expression. Full-length catfish LLP gene was sequenced by genome walking and by 5′- and 3′-RACE. Catfish LLP gene contained three exons with the coding region located in exons 2 and 3. The amino acid sequence of the channel catfish LLP shared very low sequence similarities with leptin of other fish species or the mammalian leptin (24–49%). Using real-time polymerase chain reaction, LLP mRNA expression was detected in various tissues including brain, stomach, spleen, heart, liver, and trunk kidney and was especially high in the liver and trunk kidney. Expression of LLP mRNA in liver and brain was similar between fish that were fasted for 30days and those that received feed daily for 30days (P>0.10). Expression of LLP mRNA was increased in liver, spleen, and trunk kidney within 48h post-exposure to E. ictaluri compared to unexposed fish (P<0.05). Based on the results of the current studies, amino acid sequence of catfish LLP is highly dissimilar to mammalian and fish leptin. Unlike in most mammals, catfish LLP expression is independent of energy status. However, the expression of catfish LLP is increased after exposure to pathogenic bacteria, which is similar to mammals. Further investigations are required to clearly define the biological function and regulation of catfish LLP.

Leptin‐like immunoreactivity in the muscle of juvenile sea bass (Dicentrarchus labrax)

The mammalian hormone, leptin, is mainly synthesized in adipose tissue along with other tissues. Leptin plays a role in numerous processes such as in the control of food intake, homeostasis, immune function and reproduction. In this study, we detected and localized leptin immunoreactivity to the muscle of early juvenile sea bass (Dicentrarchus labrax) by Western blot analysis and immunohistochemistry. A leptin immunopositive band with a molecular weight of approximately 16 kDa, corresponding to mammalian leptin, was identified in trunk skeletal muscle homogenate. Furthermore, leptin immunopositive cells were detected in the endomysium of skeletal muscular fibers. These cells showed immunostained cytoplasmic granules and roundish and oval nuclei. The most intense immunostaining was observed in the endomysial space among the superficial red muscular fibers of the trunk. These findings suggest that in early juvenile sea bass, leptin is mostly produced by skeletal muscles. Therefore, during the developmental stage lacking adipose tissue, skeletal muscles can be considered an important source of leptin. As already suggested in mammals, we can hypothesize the potential roles of leptin not only in energy expenditure for muscle contraction but also during muscle differentiation and growth.

Leptin mRNA and Protein Immunoreactivity in Adipose Tissue and Liver of Rainbow Trout (Oncorhynchus mykiss) and Immunohistochemical Localization in Liver

Leptin is an important modulator of energy balance and metabolism in mammals, but for evolutionary older vertebrates like fish, the first reports on leptin expression were only recently characterized and the functional role scarcely. In this study, we demonstrated leptin immunoreactivity in liver tissue of rainbow trout (Oncorhynchus mykiss) by immunohistochemistry using three different polyclonal antibodies against mammalian leptin. Immunoreactivity was observed in hepatocytes and also in parts of the biliary system. Using Western blot, we detected an immunoreactive band of about 16 kDa in serum and visceral adipose tissue (AT) of rainbow trout. The presence of leptin in fish AT has been doubted in other studies. Besides the immunoreactivity, leptin mRNA was detected in trout AT albeit not in all animals sampled. Our observations add further evidence to the concept of AT being a source of leptin in trouts. Moreover, the cellular localization of leptin immunoreactivity in liver opens up new vistas for understanding the functional role of leptin in teleosts.

Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution

We describe duplicate leptin genes in zebrafish (Danio rerio) that share merely 24% amino acid identity with each other and only 18% with human leptin. We were also able to retrieve a second leptin gene in medaka (Oryzias latipes). The presence of duplicate leptin genes in these two distantly related teleosts suggests that duplicate leptin genes are a common feature of teleostean fishes. Despite low primary sequence conservation, we are confident in assigning orthology between mammalian and zebrafish leptins for several reasons. First, both zebrafish leptins share their characteristic gene structure and display key features of conserved synteny with mammalian leptin genes. Secondly, the cysteine residues that make up leptin's single disulphide bridge are equally spaced in mammalian and zebrafish leptins and are unique among all members of the class-I helical cytokine family. Thirdly, the zebrafish leptins cluster with other fish leptins and mammalian leptins in phylogenetic analysis, supported by high bootstrap values. Within the leptin cluster, leptin-b forms a separate clade with the leptin-b orthologue from medaka. Finally, our prediction of the tertiary structures shows that both leptins conform to the typical four alpha-helix bundle structure of the class-I alpha-helical cytokines. The zebrafish leptins are differentially expressed; the liver shows high leptin-a expression (in concordance with what we observed for carp leptins), while leptin-b is expressed at much lower levels, which are downregulated further upon fasting. The finding of duplicate leptin genes in teleosts adds to our understanding of the evolution of leptin physiology in the early vertebrate lineage.

Chicken leptin receptor is functional in activating JAK–STATpathway in vitro

Leptin is a cytokine-like hormone that regulates food intake and energy homeostasis via its interaction with the leptin receptor (LEPR) located in the target tissues. Leptin-dependent signal transduction pathways have been well characterised in mammals but less is known about them in other vertebrates. In birds, although the existence of the LEPR has been confirmed, the identity of the natural ligand for the LEPR is controversial and the signalling cascade is not fully understood either. Here, we describe the in vitro expression of chicken LEPR (chLEPR), which can mediate the leptin signal. Murine leptin specifically bound with the chLEPR, which initiated the activation of luciferase in chLEPR-expressing cells. Leptin stimulation led to phosphorylation of signal transducers and activators of transcription 3 (STAT3) via chLEPR, and Janus kinase(-2) (JAK(-2)) inhibitor partially blocked leptin-induced luciferase activation in CHO-K1 cells stably expressing chLEPR (CHO-chLEPR). RNA interference for chLEPR reduced the induction rate of luciferase activity by leptin in CHO-chLEPR cells. Furthermore, we found that leptin phosphorylated STAT3 and increased luciferase activity in LMH cells, a chicken hepatoma cell line, transiently expressing chLEPR. These results strongly suggest that the chLEPR is functional in activating the JAK-STAT pathway, which may indicate that the LEPR expressed in chicken tissues is capable of binding endogenous ligand as well as exogenous mammalian leptin, leading to physiological actions.