Avian Expression Patterns and Genomic Mapping Implicate Leptin in Digestion and TNF in Immunity, Suggesting That Their Interacting Adipokine Role Has Been Acquired Only in Mammals

In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokines mediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immune defense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledge about the function of avian leptin and TNF is limited and they are absent or lowly expressed in adipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped TNF and its syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC) region. This mapping position suggests that avian TNF has a role in regulating immune response. To test its possible interaction with leptin within the immune system and beyond, we compared the transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis of GenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in the brain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected and LPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistry staining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals, in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be unique to mammals.

Comparative omics and feeding manipulations in chicken indicate a shift of the endocrine role of visceral fat towards reproduction.

BACKGROUND: The mammalian adipose tissue plays a central role in energy-balance control, whereas the avian visceral fat hardly expresses leptin, the key adipokine in mammals. Therefore, to assess the endocrine role of adipose tissue in birds, we compared the transcriptome and proteome between two metabolically different types of chickens, broilers and layers, bred towards efficient meat and egg production, respectively. RESULTS: Broilers and layer hens, grown up to sexual maturation under free-feeding conditions, differed 4.0-fold in weight and 1.6-fold in ovarian-follicle counts, yet the relative accumulation of visceral fat was comparable. RNA-seq and mass-spectrometry (MS) analyses of visceral fat revealed differentially expressed genes between broilers and layers, 1106 at the mRNA level (FDR ≤ 0.05), and 203 at the protein level (P ≤ 0.05). In broilers, Ingenuity Pathway Analysis revealed activation of the PTEN-pathway, and in layers increased response to external signals. The expression pattern of genes encoding fat-secreted proteins in broilers and layers was characterized in the RNA-seq and MS data, as well as by qPCR on visceral fat under free feeding and 24 h-feed deprivation. This characterization was expanded using available RNA-seq data of tissues from red junglefowl, and of visceral fat from broilers of different types. These comparisons revealed expression of new adipokines and secreted proteins (LCAT, LECT2, SERPINE2, SFTP1, ZP1, ZP3, APOV1, VTG1 and VTG2) at the mRNA and/or protein levels, with dynamic gene expression patterns in the selected chicken lines (except for ZP1; FDR/P ≤ 0.05) and feed deprivation (NAMPT, SFTPA1 and ZP3) (P ≤ 0.05). In contrast, some of the most prominent adipokines in mammals, leptin, TNF, IFNG, and IL6 were expressed at a low level (FPKM/RPKM< 1) and did not show differential mRNA expression neither between broiler and layer lines nor between fed vs. feed-deprived chickens. CONCLUSIONS: Our study revealed that RNA and protein expression in visceral fat changes with selective breeding, suggesting endocrine roles of visceral fat in the selected phenotypes. In comparison to gene expression in visceral fat of mammals, our findings points to a more direct cross talk of the chicken visceral fat with the reproductive system and lower involvement in the regulation of appetite, inflammation and insulin resistance.

Mapping of leptin and its syntenic genes to chicken chromosome 1p.

BACKGROUND: Misidentification of the chicken leptin gene has hampered research of leptin signaling in this species for almost two decades. Recently, the genuine leptin gene with a GC-rich (~70%) repetitive-sequence content was identified in the chicken genome but without indicating its genomic position. This suggests that such GC-rich sequences are difficult to sequence and therefore substantial regions are missing from the current chicken genome assembly. RESULTS: A radiation hybrid panel of chicken-hamster Wg3hCl2 cells was used to map the genome location of the chicken leptin gene. Contrary to our expectations, based on comparative genome mapping and sequence characteristics, the chicken leptin was not located on a microchromosome, which are known to contain GC-rich and repetitive regions, but at the distal tip of the largest chromosome (1p). Following conserved synteny with other vertebrates, we also mapped five additional genes to this genomic region (ARF5, SND1, LRRC4, RBM28, and FLNC), bridging the genomic gap in the current Galgal5 build for this chromosome region. All of the short scaffolds containing these genes were found to consist of GC-rich (54 to 65%) sequences comparing to the average GC-content of 40% on chromosome 1. In this syntenic group, the RNA-binding protein 28 (RBM28) was in closest proximity to leptin. We deduced the full-length of the RBM28 cDNA sequence and profiled its expression patterns detecting a negative correlation (R = - 0.7) between the expression of leptin and of RBM28 across tissues that expressed at least one of the genes above the average level. This observation suggested a local regulatory interaction between these genes. In adipose tissues, we observed a significant increase in RBM28 mRNA expression in breeds with lean phenotypes. CONCLUSION: Mapping chicken leptin together with a cluster of five syntenic genes provided the final proof for its identification as the true chicken ortholog. The high GC-content observed for the chicken leptin syntenic group suggests that other similar clusters of genes in GC-rich genomic regions are missing from the current genome assembly (Galgal5), which should be resolved in future assemblies of the chicken genome.

Correction to: Mapping of leptin and its syntenic genes to chicken chromosome 1p.

CORRECTION: After the publication of this work [1] an error was noticed in one of the author surnames. The author name Leif Anderson should be spelt as Leif Andersson.

Quack leptin.

BACKGROUND: A LEP transcript up-regulated in lungs of ducks (Anas platyrhynchos) infected by avian influenza A virus was recently described in the Nature Genetics manuscript that reported the duck genome. In vertebrates, LEP gene symbol is reserved for leptin, the key regulator of energy balance in mammals. RESULTS: Launching an extensive search for this gene in the genome data that was submitted to the public databases along with duck genome manuscript and extending this search to all avian genomes in the whole-genome shotgun-sequencing database, we were able to report the first identification of coding sequences capable of encoding the full leptin protein precursor in wild birds. Gene structure, synteny and sequence-similarity (up to 54% identity and 68% similarity) to reptilian leptin evident in falcons (Falco peregrinus and cherrug), tits (Pseudopodoces humilis), finches (Taeniopygia guttata) and doves (Columba livia) confirmed that the bird leptin was a true ortholog of its mammalian form. Nevertheless, in duck, like other domestic fowls the LEP gene was not identifiable. CONCLUSION: Lack of the LEP gene in poultry suggests that birds that have lost it are particularly suited to domestication. Identification of an intact avian gene for leptin in wild birds might explain in part the evolutionary conservation of its receptor in leptin-less fowls.

Primordial germ cells in the dorsal mesentery of the chicken embryo demonstrate left-right asymmetry and polarized distribution of the EMA1 epitope.

Despite the importance of the chicken as a model system, our understanding of the development of chicken primordial germ cells (PGCs) is far from complete. Here we characterized the morphology of PGCs at different developmental stages, their migration pattern in the dorsal mesentery of the chicken embryo, and the distribution of the EMA1 epitope on PGCs. The spatial distribution of PGCs during their migration was characterized by immunofluorescence on whole-mounted chicken embryos and on paraffin sections, using EMA1 and chicken vasa homolog antibodies. While in the germinal crescent PGCs were rounded and only 25% of them were labeled by EMA1, often seen as a concentrated cluster on the cell surface, following extravasation and migration in the dorsal mesentery PGCs acquired an elongated morphology, and 90% exhibited EMA1 epitope, which was concentrated at the tip of the pseudopodia, at the contact sites between neighboring PGCs. Examination of PGC migration in the dorsal mesentery of Hamburger and Hamilton stage 20-22 embryos demonstrated a left-right asymmetry, as migration of cells toward the genital ridges was usually restricted to the right, rather than the left, side of the mesentery. Moreover, an examination of another group of cells that migrate through the dorsal mesentery, the enteric neural crest cells, revealed a similar preference for the right side of the mesentery, suggesting that the migratory pathway of PGCs is dictated by the mesentery itself. Our findings provide new insights into the migration pathway of PGCs in the dorsal mesentery, and suggest a link between EMA1, PGC migration and cell-cell interactions. These findings may contribute to a better understanding of the mechanism underlying migration of PGCs in avians.

Induction of leptin resistance through direct interaction of C-reactive protein with leptin.

The mechanisms underlying leptin resistance are still being defined. We report here the presence in human blood of several serum leptin-interacting proteins (SLIPs), isolated by leptin-affinity chromatography and identified by mass spectrometry and immunochemical analysis. We confirmed that one of the major SLIPs is C-reactive protein (CRP). In vitro, human CRP directly inhibits the binding of leptin to its receptors and blocks its ability to signal in cultured cells. In vivo, infusion of human CRP into ob/ob mice blocked the effects of leptin upon satiety and weight reduction. In mice that express a transgene encoding human CRP, the actions of human leptin were completely blunted. We also found that physiological concentrations of leptin can stimulate expression of CRP in human primary hepatocytes. Recently, human CRP has been correlated with increased adiposity and plasma leptin. Thus, our results suggest a potential mechanism contributing to leptin resistance, by which circulating CRP binds to leptin and attenuates its physiological functions.

Attenuation of food intake in chicks by an inverse agonist of cannabinoid receptor 1 administered by either injection or ingestion in hydrocolloid carriers.

The cannabinoid receptor (CB(1)) was studied primarily in mammals where it was found to comprise a link between reward processes and addictive behavior such as food consumption. The purpose of this study was twofold: first to characterize the effect of the chicken CB(1) receptor inverse agonist AM251 on food intake, and second, to establish a stress-free approach for application of AM251 to birds using hydrocolloid carriers, which can be mixed with food. A single administration of AM251 by intravenous injection (at 0.85 or 5 mg kg(-1)BW) or by ingestion of hydrocolloid carriers entrapping AM251 at a concentration of 5 mg kg(-1)BW led to a transient attenuation of food intake. The consequent reduced cumulative food intake and BW were observed in the treated chicks for at least 7h post-administration, with no gender differences. Circulating levels of AM251, assessed by LC-MS following 48 h of continuous feeding with hydrocolloid carriers containing 50mg AM 251 kg(-1) BW day(-1), were physiologically significant at 186 ± 73 pmol ml(-1). It is concluded that unlike some other factors, which act differently in birds compared to mammals such as ghrelin, CB(1) inverse agonists attenuate food intake in chicks similar to its effect in mammals. In addition, the new approach for administration of AM251 to birds in hydrocolloid carriers could provide a simple and stress-free tool for prolonged studies of this control mechanism in birds.

Time-Restricted Feeding in Commercial Layer Chickens Improves Egg Quality in Old Age and Points to Lack of Adipostat Activity in Chickens.

In mammals, time-restricted feeding (TRF) with no caloric restriction provides health benefits and extends longevity, usually with a minor (∼3%) or no reduction in total food consumption. In the current study, a TRF regimen of 6 h free access to food (08:00-14:00 h) was applied to Leghorn chickens from 25 to 86 weeks of age; control birds ate freely during the light hours (06:00-20:00 h). Unexpectedly, the TRF-treated birds consumed, on average, 11.7% less food than the controls. This was manifested by an average reduction of 9.6% in body weight, 2.6-fold in visceral fat accumulation, and 6.5% in egg weight. Hen-housed egg production was reduced by 3.6% in the TRF group compared with the control, along the first 40 weeks of the follow-up (P < 0.05), and changed into a tendency of 0.7% higher egg production thereafter. Several parameters of egg quality showed significant improvement (P < 0.05) in the TRF group compared with the controls. A comparison of diurnal patterns of feed consumption revealed a higher rate of hourly consumption in the TRF group and increased consumption before dark in the control group. In conclusion, the reduced feed intake in response to the TRF treatment and loss in visceral fat accumulation supports the lack of a strong adipostat activity in chickens and different appetite regulation mechanisms compared with mammals. Therefore, future TRF studies in chickens should be adjusted by extending the ad libitum time window. The lower feed intake by the TRF-treated chickens compared with the ad libitum-fed controls seems to reduce the efficiency of egg production. Nevertheless, the improved egg quality and persistence of egg lay at the older age suggest that similarly to mammals, the TRF treatment delayed at least some of the negative impacts associated with advanced age.

Glutathione peroxidase regulation of reactive oxygen species level is crucial for in vitro plant differentiation.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is overexpressed in plants under abiotic and biotic stress conditions that mediate oxidative stress. To study its biological role and its ability to confer stress resistance in plants, we tried to obtain transgenic plants overexpressing citrus (Citrus sinensis) PHGPx (cit-PHGPx). All attempts to obtain regenerated plants expressing this enzyme constitutively failed. However, when the enzyme's catalytic activity was abolished by active site-directed mutagenesis, transgenic plants constitutively expressing inactive cit-PHGPx were successfully regenerated. Constitutive expression of enzymatically active cit-PHGPx could only be obtained when transformation was based on non-regenerative processes. These results indicate that overexpression of the antioxidant enzyme PHGPx interferes with shoot organogenesis and suggests the involvement of reactive oxygen species (ROS) in this process. Using transgenic tobacco (Nicotiana tabacum) leaves obtained from plants transformed with a beta-estradiol-inducible promoter, time-dependent induction of cit-PHGPx expression was employed. A pronounced inhibitory effect of cit-PHGPx on shoot formation was found to be limited to the early stage of the regeneration process. Monitoring the ROS level during regeneration revealed that upon cit-PHGPx induction, the lowest level of ROS correlated with the maximal level of shoot inhibition. Our results clearly demonstrate the essential role of ROS in the early stages of in vitro shoot organogenesis and the possible involvement of PHGPx in maintaining ROS homeostasis at this point.

Avian Leptin: Bird's-Eye View of the Evolution of Vertebrate Energy-Balance Control.

Discovery of the satiety hormone leptin in 1994 and its characterization in mammals provided a key tool to deciphering the complex mechanism governing adipose tissue regulation of appetite and energy expenditure. Surprisingly, despite the perfectly logical notion of an energy-storing tissue announcing the amount of fat stores using leptin signaling, alternate mechanisms were chosen in bird evolution. This conclusion emerged based on the recent discovery and characterization of genuine avian leptin - after it had been assumed missing by some, and erroneously identified by others. Critical evaluation of the past and present indications of the role of leptin in Aves provides a new perspective on the evolution of energy-balance control in vertebrates; proposing a regulation strategy alternative to the adipostat mechanism.

Leptin inhibits cell growth of human vascular smooth muscle cells.

Elevated leptin levels are thought to contribute to the individual cardiovascular risk, however, the role of leptin in the pathogenesis of atherosclerosis remains unclear. The aim of our study was to elucidate the effects of leptin on growth of human vascular smooth muscle cells (VSMC) and leptin receptor expression. By establishing a new quantitative real-time PCR for leptin receptor (ObR) isoforms we showed that the short isoforms of ObR were expressed in a 10- to 27-fold excess compared to the long isoform in cultured human VSMCs. Incubation of VSMCs with 100 ng/ml leptin downregulated the short isoforms significantly, whereas the long isoform was not influenced. Increasing leptin concentrations of 50 and 100 ng/ml significantly reduced the cell number of VSMCs compared to untreated controls. Our findings suggest a role for leptin in vascular smooth muscle cell growth, associated to a downregulation of leptin receptor isoforms.

Correspondence on Lovell et al.: identification of chicken genes previously assumed to be evolutionarily lost.

Through RNA-Seq analyses, we identified 137 genes that are missing in chicken, including the long-sought-after nephrin and tumor necrosis factor genes. These genes tended to cluster in GC-rich regions that have poor coverage in genome sequence databases. Hence, the occurrence of syntenic groups of vertebrate genes that have not been observed in Aves does not prove the evolutionary loss of such genes.Please see related Research article: http://dx.doi.org/10.1186/s13059-014-0565-1 and Please see response from Lovell et al: https://www.dx.doi.org/10.1186/s13059-017-1234-y.

The melanocortin circuit in obese and lean strains of chicks.

Agonists of membranal melanocortin 3 and 4 receptors (MC3/4Rs) are known to take part in the complex control mechanism of energy balance. In this study, we compared the physiological response to an exogenous MC3/4R agonist and the hypothalamic expression of proopic melanocortin (POMC) gene, encoding few MC3/4R ligands, between broiler and layer chicken strains. These strains, representing the two most prominent commercial strains of chickens grown for meat (broilers) and egg production (layers), differ in their food intake, fat accumulation, and reproductive performance and, therefore, form a good model of obese and lean phenotypes, respectively. A single i.v. injection of the synthetic peptide melanotan-II (MT-II; 1 mg/kg body weight) into the wing vein of feed-restricted birds led to attenuation of food intake upon exposure to feeding ad libitum in both broiler and layer chickens. A study of the POMC mRNA encoding the two prominent natural MC3/4R agonists, alpha-MSH and ACTH, also revealed a general similarity between the strains. Under feeding conditions ad libitum, POMC mRNA levels were highly similar in chicks of both strains and this level was significantly reduced upon feed restriction. However, POMC mRNA down-regulation upon feed restriction was more pronounced in layers than in broilers. These results suggest: (i) a role for MC3/4R agonists in the control of appetite; (ii) that the physiological differences between broilers and layers are not related to unresponsiveness of broiler chickens to the satiety signal of MC3/4R ligands. Therefore, these findings suggest that artificial activation of this circuit in broiler chicks could help to accommodate with their agricultural shortcomings of overeating, fattening, and impaired reproduction.

Identification of the Long-Sought Leptin in Chicken and Duck: Expression Pattern of the Highly GC-Rich Avian leptin Fits an Autocrine/Paracrine Rather Than Endocrine Function.

More than 20 years after characterization of the key regulator of mammalian energy balance, leptin, we identified the leptin (LEP) genes of chicken (Gallus gallus) and duck (Anas platyrhynchos). The extreme guanine-cytosine content (∼70%), the location in a genomic region with low-complexity repetitive and palindromic sequence elements, the relatively low sequence conservation, and low level of expression have hampered the identification of these genes until now. In vitro-expressed chicken and duck leptins specifically activated signaling through the chicken leptin receptor in cell culture. In situ hybridization demonstrated expression of LEP mRNA in granular and Purkinje cells of the cerebellum, anterior pituitary, and in embryonic limb buds, somites, and branchial arches, suggesting roles in adult brain control of energy balance and during embryonic development. The expression patterns of LEP and the leptin receptor (LEPR) were explored in chicken, duck, and quail (Coturnix japonica) using RNA-sequencing experiments available in the Short Read Archive and by quantitative RT-PCR. In adipose tissue, LEP and LEPR were scarcely transcribed, and the expression level was not correlated to adiposity. Our identification of the leptin genes in chicken and duck genomes resolves a long lasting controversy regarding the existence of leptin genes in these species. This identification was confirmed by sequence and structural similarity, conserved exon-intron boundaries, detection in numerous genomic, and transcriptomic datasets and characterization by PCR, quantitative RT-PCR, in situ hybridization, and bioassays. Our results point to an autocrine/paracrine mode of action for bird leptin instead of being a circulating hormone as in mammals.

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.

Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

Lack of leptin activity in blood samples of Adélie penguin and bar-tailed godwit.

Unsuccessful attempts to identify the leptin gene in birds are well documented, despite the characterization of its receptor (LEPR). Since leptin and LEPR have poor sequence conservation among vertebrates, we speculated that a functional assay should represent the best way to detect leptin in birds. Using a leptin bioassay that is based on activation of the chicken LEPR in cultured cells, blood samples from wild birds with extreme seasonal variation in voluntary food intake and fat deposition (Adélie penguins and bar-tailed godwits) were tested for leptin activity. In these experiments, blood samples collected during the pre-incubation and the chick-rearing periods of Adélie penguins, and during the migratory flight and refueling stages of bar-tailed godwits, were found to contain no detectable leptin activity, while the sensitivity of the assay to activation by human blood samples from donor subjects representing a variety of body mass indices and fat contents was clearly demonstrated. These results suggest that in birds, an alternative control mechanism to that of mammals operates in the communication between the body fat tissues and the central control on energy homeostasis.

Monitoring leptin activity using the chicken leptin receptor.

We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.