Characterization of the Two CART Genes (CART1 and CART2) in Chickens (Gallus gallus)

Abstract

Cocaine- and amphetamine-regulated transcript (CART) peptide is implicated in the control of avian energy balance, however, the structure and expression of CART gene(s) remains largely unknown in birds. Here, we cloned and characterized two CART genes (named cCART1 and cCART2) in chickens. The cloned cCART1 is predicted to generate two bioactive peptides, cCART1(42-89) and cCART1(49-89), which share high amino acid sequence identity (94-98%) with their mammalian counterparts, while the novel cCART2 may produce a bioactive peptide cCART2(51-91) with 59% identity to cCART1. Interestingly, quantitative RT-PCR revealed that cCART1 is predominantly expressed in the anterior pituitary and less abundantly in the hypothalamus. In accordance with this finding, cCART1 peptide was easily detected in the anterior pituitary by Western blot, and its secretion from chick pituitaries incubated in vitro was enhanced by ionomycin and forskolin treatment, indicating that cCART1 is a novel peptide hormone produced by the anterior pituitary. Moreover, cCART1 mRNA expression in both the pituitary and hypothalamus is down-regulated by 48-h fasting, suggesting its expression is affected by energy status. Unlike cCART1, cCART2 is only weakly expressed in most tissues examined by RT-PCR, implying a less significant role of cCART2 in chickens. As in chickens, 2 or more CART genes, likely generated by gene and genome duplication event(s), were also identified in other non-mammalian vertebrate species including coelacanth. Collectively, the identification and characterization of CART genes in birds helps to uncover the roles of CART peptide(s) in vertebrates and provides clues to the evolutionary history of vertebrate CART genes.

Immunohistochemical localization of cocaine‐ and amphetamine‐regulated transcript peptide (CARTp) in the brain of the pigeon (Columba livia) and zebra finch (Taeniopygia guttata)

“…Western blot analysis showed an intense band around 16 kDa and a smaller band around 14 kDa for the pigeon. While the expected size for functional fragments of CARTp is around 5 kDa (Cai et al, ), bands at higher molecular weight are consistent with what has been found in other vertebrates (see Discussion; Lázár et al, ; Roubos et al, ). The antibody used was raised against the rat CART peptide, which shows a high degree of identity with the chicken and budgerigar ( Melopsittacus undulates ) putative signal peptides, differing in only three amino acids (98% matching; Cai et al, ).…”

“…B–E). This is consistent with the high degree of identity (96–98%) between the amino acid sequence of the CART peptide of birds and mammals (Cai et al, ).…”

“…In birds the evidence for a role of CARTp in regulating feeding behavior is scarce. As mentioned before, intracerebroventricular injection of CARTp alters feeding behavior in chickens (Tachibana et al, ), and recently Cai et al () showed that fasting downregulates CART mRNA in the hypothalamus of chickens. Our results provide further support to a role of CARTp in mediating the regulation of feeding behavior in birds.…”

“…These two bands most likely correspond to the CART pre‐pro‐peptide and CART pre‐peptides. We only used pigeon brain for this analysis, but Cai et al () have shown that the amino acid sequence of chickens and budgerigars CARTp is 100% identical, suggesting that the CARTp sequence is highly conserved among birds.…”

“…In birds, the CART gene is present, and the peptide encoded by it is similar to that in other vertebrates (Murashita et al, ; Cai et al, ). Furthermore, injection of human CARTp into the central nervous system of chicks ( Gallus domesticus ) inhibits food intake (Tachibana et al, ), suggesting that CARTp is expressed and active in the brain of birds.…”