C. M. Ashwell

Hormonal regulation of leptin expression in broiler chickens

Leptin, the polypeptide hormone encoded by the obese gene, is secreted by adipose tissue and has been shown to induce satiety and increase energy expenditure in mammals. In this study, we confirmed the presence of a leptin homolog in liver and adipose tissues of broiler chickens. Leptin expression was also detected in chicken embryonic liver and yolk sac. The effects of hormone treatment on leptin expression in chickens were also investigated. Leptin expression in the liver is increased by insulin and dexamethasone and decreased by glucagon and estrogen. There was no induction of leptin expression in adipose tissue by any treatment, whereas only estrogen decreased adipose expression. The differential effect on liver and adipose tissue suggests that adipocytes in chickens may be expressing leptin at a maximal rate or that its mechanism of expression regulation differs from liver. The localization of leptin expression and tissue-specific effects of hormone treatments on leptin expression observed in chickens may indicate a relationship between leptin and avian lipid metabolism.Leptin, the polypeptide hormone encoded by the obese gene, is secreted by adipose tissue and has been shown to induce satiety and increase energy expenditure in mammals. In this study, we confirmed the presence of a leptin homolog in liver and adipose tissues of broiler chickens. Leptin expression was also detected in chicken embryonic liver and yolk sac. The effects of hormone treatment on leptin expression in chickens were also investigated. Leptin expression in the liver is increased by insulin and dexamethasone and decreased by glucagon and estrogen. There was no induction of leptin expression in adipose tissue by any treatment, whereas only estrogen decreased adipose expression. The differential effect on liver and adipose tissue suggests that adipocytes in chickens may be expressing leptin at a maximal rate or that its mechanism of expression regulation differs from liver. The localization of leptin expression and tissue-specific effects of hormone treatments on leptin expression observed in chickens may indicate a relationship between leptin and avian lipid metabolism.

Leptin-induced decrease in food intake in chickens

The effect of intracerebroventricular (i.c.v.) injection of leptin was investigated using broiler and Single Comb White Leghorn (SCWL)-type chickens. These represent relatively fast- and slow-growing birds, respectively. The i.c.v. injection of leptin decreased food intake in both broilers and Leghorns in a dose-dependent manner. The most efficacious dose appeared to be 10 microg in both types of chickens. Water intake was generally not affected by leptin, indicating that this effect was not due to general malaise. It appears that leptin can act within the central nervous sytstem of birds to decrease food intake.

Genomic Regions Associated with Dermal Hyperpigmentation, Polydactyly and Other Morphological Traits in the Silkie Chicken

The Silkie chicken has been a model of melanoctye precursor and neural crest cell migration and proliferation in the developing embryo due to its extensive hyperpigmentation of dermal and connective tissues. Although previous studies have focused on the distribution and structure of the Silkies pigment or the general mechanisms by which this phenotype presents itself, the causal genetic variants have not been identified. Classical breeding experiments have determined this trait to be controlled by 2 interacting genes, the sex-linked inhibitor of dermal melanin (Id) and autosomal fibromelanosis (Fm) genes. Genome-wide single nucleotide polymorphism (SNP)-trait association analysis was used to detect genomic regions showing significant association with these pigmentation genes in 2 chicken mapping populations designed to segregate independently for Id and Fm. The SNP showing the highest association with Id was located at 72.3 Mb on chromosome Z and 10.3-13.1 Mb on chromosome 20 showed the highest association with Fm. Prior to this study, the linkage group to which Fm belonged was unknown. Although the primary focus of this study was to identify loci contributing to dermal pigmentation in the Silkie chicken, loci associated with various other morphological traits segregating in these populations were also detected. A single SNP in a highly conserved cis-regulatory region of Sonic Hedgehog was significantly associated with polydactyly (Po). Genomic regions in association with silkie feathering or hookless (h), feathered legs (Pti), vulture hock (V), rose comb (R), and duplex comb (D) were also identified.

Expression of an uncoupling protein gene homolog in chickens

An avian uncoupling protein (UCP) gene homolog was recently sequenced from skeletal muscle and was proposed to have a role in thermogenesis in chickens, ducks and hummingbirds. Since mammalian UCP 2 and UCP 3 also appear to have functions associated with energy and substrate partitioning and body weight regulation, the purpose of this study was to further characterize chicken UCP under conditions of nutritional stress and/or leptin administration. Male 3-week-old chickens were starved for 24 or 48 h and then half of each group was refed for an additional 24 h. In a follow-up experiment, chickens were fed or starved for 48 h with or without leptin administration. Feed deprivation increased UCP mRNA expression in skeletal muscle by up to 260% (P<0.001), and in a time-dependent manner in pectoralis muscle. Refeeding for 24 h normalized muscle UCP mRNA levels. Leptin administration had no effect on muscle UCP. Chicken muscle UCP mRNA levels were highly correlated with plasma triglyceride and non-esterified fatty acid (NEFA) concentrations, and with circulating levels of insulin, insulin-like growth factor (IGF)-I and IGF-II. These results suggest that, as in mammals, avian UCP is up-regulated during feed deprivation and is highly correlated with increased fatty acid oxidation and flux into skeletal muscle.

Expressed Sequence Tag Analysis of Eimeria-Stimulated Intestinal Intraepithelial Lymphocytes in Chickens

Intraepithelial lymphocytes (IELs) play a critical role in protective immune response to intestinal pathogens such as Eimeria, the etiologic agent of avian coccidiosis. A list of genes expressed by intestinal IELs of Eimeria-infected chickens was compiled using the expressed sequence tag (EST) strategy. The 14,409 ESTs consisted of 1851 clusters and 7595 singletons, which revealed 9446 unique genes in the data set. Comparison of the sequence data with chicken DNA sequences in GenBank identified 125 novel clones. This EST library will provide a valuable resource for profiling global gene expression in normal and pathogen-infected chickens and identifying additional unique immune-related genes.

Effects of growth hormone and pair-feeding on leptin mRNA expression in liver and adipose tissue

Previous research has reported that elevations in circulating growth hormone (GH) levels in meat-type chickens depresses feed intake (FI) more than 30%. It is known that the product of the obese gene, leptin, functions to regulate FI and energy expenditure. To investigate the effect of GH on leptin gene expression, broiler chickens were infused with recombinant chicken GH. To separate any secondary effects of a GH-induced reduction in FI on leptin expression, groups of birds were pair-fed to an average level of voluntary intake similar to GH-treated birds, but received no GH treatment. GH treatment induced a dose-dependent increase in liver leptin gene expression, as measured by reverse transcriptase-polymerase chain reaction, whereas leptin expression in adipose tissue was unchanged. Conversely, in chickens pair-fed (feed-restricted) there was a decrease in leptin gene expression in both tissues. These results provide evidence of a direct effect of GH on leptin gene expression, which is independent of any effects on intake attributable to GH-treatment, and suggest differential regulation of leptin expression between adipose tissue and liver. The results of these experiments provide the first evidence of a relationship between GH and leptin in domestic birds.

Analysis of leptin gene expression in chickens using reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection.

Leptin is a peptide hormone product of the obese (ob) gene that functions in the regulation of appetite, energy expenditure and reproduction in animals and humans. We have developed a technique using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the analysis of chicken leptin (261 base pairs, bp) and beta-actin (612 bp) double-stranded DNA products from reverse transcription polymerase chain reaction (RT-PCR) assays. Amplicons were separated using a DB-1 coated capillary (27 cm x 100 microns I.D.) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethylcellulose (HPMC) in 1X TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 microgram/ml EnhanCE fluorescent intercalating dye. RT-PCR samples (1-2 microliters) were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic injection. Separations were completed in less than 6 min and the total time required per sample, including capillary conditioning, was 8 min. We have applied RT-PCR-CE-LIF to determine the effects of insulin and estrogen treatment on leptin gene expression relative to that of beta-actin in chicken liver and adipose tissue. In addition, we have constructed a chicken leptin mRNA competitor (234 bp amplicon) and evaluated it for use as an internal standard in the development of a quantitative-competitive RT-PCR assay. Our findings represent the first reported application of capillary electrophoresis to the analysis of leptin gene expression by RT-PCR.

Design and application of a polyclonal peptide antiserum for the universal detection of leptin protein

An epitope-specific polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from the coding sequence reported for the porcine leptin gene (GenBank Accession No. U59894). This peptide contains a core sequence comprised of eight amino acids (GLDFIPGL) that is totally conserved in all leptin proteins studied to date. Purified recombinant human, mouse, rat, pig, and chicken leptin proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto PVDF membranes. Western blots were developed employing the leptin-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The peptide antiserum was found to be highly specific for leptin which exhibited an estimated molecular weight of about 16 kDa for all species analyzed. The sensitivity of the Western blot assay was not sufficient to permit the direct detection of leptin in chicken serum or plasma. However, with this assay we were able to detect native leptin protein in an enriched fraction prepared from chicken plasma using a combination of gel filtration and ion exchange column chromatography. Slot blots indicated a potential application of the immunostaining technique for quantitative analysis of leptin protein. Finally, the peptide antiserum was successfully employed to localize leptin protein by immunohistochemical staining of thin sections prepared from adipose (chicken and pig) and liver (chicken) tissue samples. This study is the first to report a polyclonal peptide antiserum that apparently recognizes intact leptin protein, both native and recombinant, regardless of the species of origin.

Quantitative analysis of leptin mRNA using competitive reverse transcription polymerase chain reaction and capillary electrophoresis with laser‐induced fluorescence detection

Leptin, the protein hormone product of the obese (ob) gene, functions in the regulation of appetite, energy expenditure, and reproduction in animals and humans. Since changes in the level of circulating leptin can have marked physiological consequences, it is important to be able to accurately quantify leptin gene expression. Toward this goal, we have constructed a chicken leptin RNA competitor and successfully employed it as an internal standard in the development of a quantitative‐competitive reverse transcription polymerase chain reaction (QC‐RT‐PCR) assay for leptin mRNA. Capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF) was utilized for the separation and analysis of chicken leptin target (261 bp) and competitor (234 bp) dsDNA products from QC‐RT‐PCR assay samples. Leptin amplicons were separated using a DB‐1 coated capillary (27 cm × 100 μm ID) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethyl cellulose (HPMC) in 1 × TBE (89 mM Tris‐base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 μg/mL EnhanCE™ fluorescent intercalating dye. Samples were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic injection. QC‐RT‐PCR/CE‐LIF was used to quantify leptin mRNA in liver and adipose tissue from 8‐week‐old male and female broiler chickens. This study is the first report of quantitative analysis of leptin gene expression using QC‐RT‐PCR/CE‐LIF.

Prehatch intestinal maturation of turkey embryos demonstrated through gene expression patterns

Some of the challenges faced by neonatal turkeys include weakness, reduced feed intake, impaired growth, susceptibility to disease, and mortality. These symptoms may be due to depleted energy reserves after hatch and an immature digestive system unable to replenish energy reserves from consumed feed. To better understand enteric development in turkeys just before hatch, a new method was used to identify the patterns of intestinal gene expression by utilizing a focused microarray. The duodenums of 24 turkey embryos were sampled on embryonic day (E)20, E24, E26, and hatch (E28). The RNA populations of 96 chosen genes were measured at each time point, from which 81 significantly changed (P < 0.01). These genes were clustered by gene expression pattern similarity into 4 groups. The expression pattern of hormone receptors revealed that intestinal tissues may be less responsive to growth hormone, insulin, glucagon, and triiodothyronine during the last 48 h before hatch, when developmental emphasis switches from cell proliferation to functional maturation. Based on gene expression patterns, we concluded that at hatch, poults should have the capacity to 1) digest disaccharides but not oligopeptides, due to increased expression of sucrase-isomaltase but decreased expression of aminopeptidases and 2) absorb monosaccharides and small peptides due to high expression of sodium-glucose cotransporter-4 and peptide transporter-1.