Mark P. Richards

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.

Characterization of metallothionein isoforms. Comparison of capillary zone electrophoresis with reversed-phase high-performance liquid chromatography

The purpose of this study was to compare and contrast the separation of metallothionein (MT) isoforms by reversed-phase high-performance liquid chromatography (RP-HPLC) with capillary zone electrophoresis (CZE). RP-HPLC was performed on a Vydac C8 column eluted with a linear acetonitrile gradient. CZE was performed in a 57 cm x 75 microns I.D. fused-silica tube at an operating voltage of 30 kV. Phosphate buffer (10 mM) at pH 2.5, 7.0 and 11.0 was used for both separations. CZE at pH 2.5 resolved three distinct peaks of rabbit liver MT which were incompletely resolved at pH 7.0 or 11.0. RP-HPLC at pH 2.5 gave two peaks and the resolution was not as good as with CZE at the same pH. At pH 7.0 or 11.0, RP-HPLC of rabbit liver MT gave a single predominant peak of unresolved MT-1 and MT-2. Purified rabbit liver MT-1 and MT-2 were used to verify the identity of these peaks. In contrast, MT from horse kidney demonstrated three predominant peaks which were best resolved by CZE at pH 11.0, whereas RP-HPLC resolved only two peaks at pH 11.0 and 7.0. At pH 2.5, RP-HPLC of horse kidney MT gave three peaks, though two of the peaks were incompletely separated. We conclude that pH has a considerable impact on the resolution of MT isoforms by CZE and RP-HPLC and that it is possible to exploit changes in pH to optimize the separation of isoforms for a particular species of MT. When samples of human and sheep liver MT-1, both of which exhibit microheterogeneity, were subjected to CZE, a single predominant peak was observed at each pH value. RP-HPLC of human liver MT-1 at pH 2.5 yielded two peaks that were incompletely resolved. Purified chick liver MT and rat liver MT-1 and MT-2 gave a single predominant peak at all pH values on CZE. In contrast, pig liver MT-1 and MT-2 each exhibited multiple peaks when subjected to CZE, the number of which depended on the pH used to separate the MT. In conclusion, CZE, with its orthogonal selectivity, and RP-HPLC make an excellent combination for the separation and characterization of MT isoforms. Because CZE is rapid (run times typically < 10 min) and requires little sample (< 100 nl), MT samples can readily be analyzed by CZE in conjunction with RP-HPLC or other techniques in order to maximize the information obtained about the individual isoforms.

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.

Molecular cloning and expression of the turkey leptin receptor gene.

A cDNA encoding the long form of the turkey (Meleagris gallopavo) leptin receptor (LEPR) was cloned and sequenced. Turkey LEPR showed greater than 90% sequence identity at both the nucleotide and amino acid level with chicken LEPR. The LEPR gene (long form) encodes a protein of 1147 amino acids that has features similar to other LEPRs including: a signal peptide, a single transmembrane domain, and specific conserved motifs defining putative leptin-binding and signal transduction regions of the protein. In addition, a LEPR gene-related protein (LEPR-GRP) mRNA transcript was also identified and a portion of the corresponding cDNA containing the complete coding region was sequenced. The turkey LEPR-GRP gene encodes a 14-kDa (131 amino acids) protein that is distinct from LEPR. LEPR gene expression was quantified relative to beta-actin in total RNA samples isolated from various tissues of 3-week-old turkey poults. Expression of LEPR was highest in brain, spleen and lung tissue with lower levels of expression in kidney, pancreas, duodenum, liver, fat and breast muscle. In developing turkey embryos, expression of LEPR was highest in brain tissue throughout incubation (days 14-28). Expression of LEPR in embryonic liver tissue peaked at day 16 and then declined toward hatching (day 28). Yolk sac expression of LEPR declined from day 14 to day 20 and then increased toward hatching. Our findings clearly demonstrate the expression of LEPR and LEPR-GRP in different tissues during embryonic and post-hatch development. In conclusion, this is the first report to identify and characterize LEPR and LEPR-GRP gene homologues in the domestic turkey.

Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection.

There has been a dramatic expansion of DNA sequence information compiled over the past several years for a variety of eukaryotic and prokaryotic genomes. Accompanying this increase in knowledge of genomic structure and organization has been a growing interest in studying the function of individual genes including regulation of their expression. A number of methods such as Northern blotting, ribonuclease protection assay, and hybridization arrays have been developed to analyze gene expression at the transcriptional (mRNA) level. Although quantitative estimates of mRNA transcripts can be obtained from each of these methods, oftentimes they lack sufficient sensitivity or the methodology is too costly or too labor-intensive to be applied to the analysis of a large number of samples. The most sensitive method for analyzing gene expression at the mRNA level involves the combination of reverse transcription and polymerase chain reaction (RT-PCR). However, in order to provide accurate quantitative estimates of gene expression, a rapid and efficient method is required for separation and detection of the double-stranded DNA (dsDNA) products of RT-PCR. Recent advances in capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) have made this method suitable for the automated analysis of large numbers of RT-PCR samples. An overview of the application of CE/LIF to quantitative analysis of gene expression by RT-PCR is presented along with selected protocols and examples. Both relative-quantitative (RQ) and quantitative-competitive (QC) approaches to RT-PCR are discussed in conjunction with the use of CE/LIF for rapid and accurate quantitative analysis of PCR products.

Dietary protein regulates in vitro lipogenesis and lipogenic gene expression in broilers.

The purpose of this experiment was to determine the possible relationship between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 21 or 30% protein ad libitum. In addition, another group of birds was fed on a regimen consisting of a daily change in the dietary protein level (12 or 30%). This latter group was further subdivided such that one-half of the birds received each level of protein on alternating days. Birds were sampled from 28 to 30 days of age. Measurements taken included in vitro lipogenesis, malic enzyme activity the expression of the genes for malic enzyme, fatty acid synthase and acetyl coenzyme carboxylase. In vitro lipogenesis and malic enzyme activity were inversely related to dietary protein levels (12-30%) and to acute changes from 12 to 30%. In contrast, expression of malic enzyme, fatty acid synthase and acetyl CoA carboxylase genes were constant over a dietary protein range of 12-21%, but decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out, however, that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.

Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to isolate, characterize and quantitate liver and kidney metallothionein (MT) isoforms from a variety of animal species. The isoMTs were eluted from a radially compressed C18 column with a neutral sodium phosphate buffer and detected by UV absorbance (214 nm). Rabbit liver and horse kidney MTs were each found to be comprised of seven distinct isoforms. Pig liver and kidney MTs each exhibited three predominant isoforms, two of which were found to be subspecies of the MT-2 isoform. Rat liver MT was characterized by a less complex isoform pattern with only two major isoforms corresponding to the MT-1 and MT-2 isoMTs. Similarly, avian liver MT exhibited a distinct isoform pattern characterized by a low degree of complexity with the MT-2 isoform much more abundant than the MT-1 species. It was possible to apply the RP-HPLC separation to the resolution of individual MT isoforms from complex mixtures such as heat-treated cytosol. A standard curve was constructed using purified turkey hen liver MT-2 which demonstrated excellent linear correlation between integrated peak area and the quantity of MT injected onto the column. Recovery of MT from RP-HPLC was estimated to exceed 90%. Liver tissue from chicks injected on consecutive days with a dose of zinc was assayed for both MT-1 and MT-2 isoforms using the RP-HPLC analysis of cytosol samples. The MT-2 isoform was found to be preferentially expressed in response to zinc induction.

Separation of metallothionein isoforms by micellar electrokinetic capillary chromatography

Current techniques for the separation and quantification of metallothionein isoforms have limited value for routine analysis. Isoforms having a similar charge have been separated successfully using reversed-phase HPLC but this technique suffers from a slow sample turnover time. The use of the surfactant sodium dodecyl sulphate for the separation of metallothionein isoforms by micellar electrokinetic capillary chromatography (MECC) is described. The charge-different isoforms MT-1 and MT-2 from rats, rabbits and sheep were separated within 9-12 min. In addition, a varying degree of heterogeneity was observed in purified samples of human MT-1, rat MT-2, rabbit MT-1, rabbit MT-2 and sheep MT-1. The behaviour of chicken MT was different from that of any other species. The separation of sheep liver extracts indicated the potential of MECC as the basis for a quantitative assay for both charge-different and charge-similar metallothionein isoforms.

The avian proglucagon system.

Understanding how the proglucagon system functions in maintaining glycemic control and energy balance in birds, as well as defining its specific roles in regulating metabolism, gastrointestinal tract function and food intake requires detailed knowledge of the components that comprise this system. These include proglucagon, a precursor protein from which glucagon and two glucagon-like peptide hormones (GLP-1 and GLP-2) are derived, and the membrane bound G-protein-coupled receptors that specifically bind glucagon, GLP-1 and GLP-2 to mediate their individual physiological actions. Another key feature of the proglucagon system that is important for regulating its activity in different tissues involves post-translational processing of the proglucagon precursor protein and the individual peptide hormones derived from it. Currently, there is limited information about the proglucagon system in birds with the majority of that coming from studies involving chickens. By summarizing what is currently known about the proglucagon system in birds, this review aims to provide useful background information for future investigations that will explore the nature and actions of this important hormonal system in different avian species.

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.