Identification of differentially expressed proteins at four growing stages in chicken liver

ABSTRACT : Because of high growth rate and large deposition of fat in the abdomen, the chicken has been used as a model organism for understanding lipid metabolism, fattening and growing. In this study, differentially expression of proteins in chicken liver, one of the important organs for lipid metabolism, has been investigated at four different growing stages. After separation of proteins using two-dimensional electrophoresis (2-DE), more than 700 protein spots were detected. Among them, 13 growing stage specific proteins in chicken liver were selected and further investigated by matrix-assisted laser adsorptions ionization-time of flight mass spectrometry (MALDI-TOF MS). Of these, 12 proteins were matched to existing proteins based on a database search. The identified fat-related proteins in this study were fatty acid synthase (FASN) and malic enzyme (ME1). These proteins were more highly expressed at week 32 than at other weeks. In order to confirm the differential expression, one of the proteins, FASN, was confirmed by western blotting. The identified proteins will give valuable information on biochemical roles in chicken liver, especially for lipid metabolism. (Key Words : Chicken Liver, Different Growing Stages, MALDI-TOF MS, 2-DE)

INTRODUCTION

Chicken (Gallus gallus) has been regarded as a valuable model organism, especially for studies of vertebrate development, since the success of embryo manipulations in vitro (Stern, 2004 and 2005) and facilitated from the success in chicken genome sequencing (Hillier et al., 2004). Besides, 70 million tons of poultry meat and 47 million tons of eggs were consumed per year in the world. Among the poultry meat, 85% was the chicken meat and 96% was the chicken eggs (Arthur and Albers, 2003). Therefore, the chicken meat and eggs are the valuable protein sources for human.

Along with the recent development of molecular genetic techniques, proteomics is one of the powerful technologies, mainly because this technique allows us to investigate the final products of the metabolism, proteins. Previous results indicated that there were very less correlations between the mRNA expression levels and the amount of target proteins (Futcher et al., 1999; Gygi et al., 1999). In order to understand the expressed proteins from genes, protein-protein interactions and modified proteins in a given environment, proteomics becomes the essential part of many researches (Pandey and Mann, 2000).

Until recently, a number of proteomics research has been carried out in livestock species, including cattle, pig and chicken (Kelly et al., 2006; Jung et al., 2007; Kim et al., 2007). Proteomics can also be widely applied in order to find proteins that are controlling endocrine system mechanism, embryo development and organism developmental changes (Naaby-Hansen et al., 2001). People can anticipate the phenotypic changes by investigation of the diverse changes in metabolites (Fiehn, 2002; Weckwerth, 2003; Dettmer and Hammock, 2004). In case of chicken, Doherty et al. (2004) investigated chicken skeletal muscle proteomes at the specified time points after hatching and identified a number of biologically important proteins. Also, the chicken muscles between layer and broiler breeds were compared by proteomics approach in order to identify economically important growth related proteins (Jung et al., 2007). However, only the comparison of chicken muscles was not enough for the understanding the complex biological process in the muscle. There are also big problems for abdominal fat in adult chicken and the breeding strategy has been aimed to decrease the abdominal fat contents and increase the feed efficiency. In the chicken organs, liver is the main lipogenic tissue, where adipose tissue is also primarily responsible for lipogenesis in mammals (Kanai et al., 1997). After few days of hatching, the majority of the accumulated lipids in the liver of birds is esterified cholesterol, which is stored within lipolysosomes in the hepatocytes (Kanai, 1989; Kanai et al., 1994). Previously, differentially expressed genes in chicken liver from two divergent chicken lines were investigated (Ding et al., 2008). In the present study, we investigated the proteomes in chicken liver at different growing stages in order to identify biologically important fat-related proteins.

MATERIALS AND METHODS

Experimental animals

Liver samples from two female birds of White Leghorn breed were obtained at 0, 10, 21 and 32 weeks of age. These samples were collected from National Institute of Animal Science (NIAS), Seong-hwan, Korea and were immediately stored at liquid nitrogen until use.

Sample preparation

Two hundred mg of frozen liver samples were mixed with 200 il of solution containing 0.3% sodium dodecyl sulfate (SDS) (GE Healthcare, Sweden), 50 mM Tris-HCl pH 8.0, 1 mM phenylmethylsulfonyl fluoride (PMSF) (Roche, Germany), 50 x Protease inhibitor (Roche, Germany), 200 mM dithiothreitol (DTT) (GE Healthcare, Sweden). These mixtures were homogenized by sonication (Hielscher, Germany) and then centrifuged at 15,000 g for 10 min at 4[degrees]C. Obtained supernatants were incubated with solution containing 40 U DNase I (Roche, Germany), 14 U RNase A (Roche, Germany), 50 mM Tris-HCl pH 8.0, 0.1 mM Mg[Cl.sub.2] in ice for 50 min. After centrifugation at 4[degrees]C with 15,000 g for 15 min, the supernatants were used for further analysis. Protein concentration of each sample was determined according to Bradford assay (Bradford, 1976).