br Expression of adiponectin mRNA in chickens

Expression of adiponectin mRNA in chickens We first sought to determine the nucleotide sequence of the chicken adiponectin cDNA. The open reading frame of the chicken adiponectin cDNA consists of 735 nucleotides that was 65–68% homologous to various mammalian adiponectin cDNAs (Maddineni et al., 2005, Yuan et al., 2006). In contrast, chicken adiponectin mRNA sequence shares a high degree of identity with other DHT such as Meleagris gallopavo (93%; Accession# XR_118340.1), Anas platyrhynchos (86%; Accession# GU236315.1), Anser anser (89%; EU370686.1), and Taeniopygia guttata (82%; XM_002188735.1). Using RT-PCR and northern analysis, we detected chicken adiponectin mRNA transcript in adipose tissue, liver, anterior pituitary gland, diencephalon, skeletal muscle, liver, kidney, ovary, and spleen, but not in blood (Maddineni et al., 2005). In support of our data, adiponectin gene and/or protein expression has been detected in skeletal muscle (Ding et al., 2004, Punyadeera et al., 2005), pituitary gland (Rodriguez-Pacheco et al., 2007), and testis (Caminos et al., 2008) in mammals. The concentration of adiponectin mRNA was found to be the highest in adipose tissue followed by liver, anterior pituitary, diencephalon, kidney and skeletal muscle. We also found that adiponectin mRNA quantity was significantly decreased following a 48h food deprivation in adipose tissue, liver, and anterior pituitary gland but not in diencephalon (Maddineni et al., 2005).
Biochemical properties of adiponectin Structurally, adiponectin consists of four distinct domains that include a signal peptide at the N-terminus followed by a short variable region, a collagenous domain, and a C-terminal globular domain (Shapiro and Scherer, 1998). During biosynthesis, the 30kDa adiponectin monomers are assembled into oligomers and multimers and secreted as three distinct isoforms denoted as low (LMW), medium (MMW), and heavy molecular weight (HMW) isoforms in mammals (Tsao et al., 2002, Tsao et al., 2003, Waki et al., 2003). The LMW adiponectin isoform represents a homotrimer of three adiponectin monomers, while the MMW isoform is a hexamer composed of two homotrimers and the HMW adiponectin isoform is likely to contain 18 or more adiponectin monomers (Tsao et al., 2003). We began to characterize the biochemical properties of chicken adiponectin using anti-chicken adiponectin antibodies developed in our laboratory, as our attempts to utilize anti-mammalian adiponectin antibody to detect chicken adiponectin failed, possibly due to poor homology of chicken and mammalian adiponectin. Our studies revealed that under non-reducing and non-heat-denaturing conditions, adiponectin exists predominantly as a polymer with a molecular mass of approximately 720kDa in chicken plasma, adipose tissue, and skeletal muscle (Fig. 1A). Traces of an oligomeric adiponectin isoform of approximately 242kDa was detected in leghorn chickens or 4-week-old broiler chickens (Fig. 1B). Gel filtration of chicken plasma or adipose tissue protein extract followed by Western analysis of the fractions under native conditions confirmed that chicken adiponectin is predominantly a HMW isoform of mass equal to or greater than 669kDa. (Hendricks et al., 2009). Chicken plasma and adipose tissue extracts were then subjected to electrophoresis under reducing conditions with or without reducing agent and/or heating. Omission of heat and reducing agent resulted in a major immunoreactive band that is >191kDa, which is representative of the HMW isoform, as well as a less intense 64kDa oligomeric adiponectin isoform in both plasma and adipose tissue extracts (Fig. 1C). Treatment with a reducing agent, however, led to a complete disappearance of the HMW isoform, which is reduced primarily to a 64kDa oligomer in plasma, and to a 64kDa oligomer as well as a 30kDa monomer in the adipose tissue extract (Fig. 1C). In contrast to chicken, human and mouse adiponectin exists as three different molecular mass species (67, 136, and >300kDa) termed LMW, MMW, and HMW isoforms, respectively (Waki et al., 2003). The presence of HMW adiponectin isoforms in chicken plasma and tissues, without significant amounts of LMW and MMW oligomeric forms is enigmatic. The mechanism(s) that aid in the assembly of this single, major HMW multimeric isoform in the chicken is currently unknown. However, the amino acid sequence as well as post-translational modifications of chicken adiponectin combined with physiological factor(s) unique to chickens may favor such unique multimerization.