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

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.