Cloning and characterization of the 5′-flanking region of the pig AgRP gene

Agouti-related peptide (AgRP), a brain neuropeptide generated by AgRP/neuropeptide Y (NPY) neurons, plays a vital role in the hypothalamic regulation of energy homeostasis. RT–PCR and real-time PCR were carried out in various tissues to detect the AgRP expression pattern in pigs. Our RT–PCR results showed that the pig AgRP gene was ubiquitously expressed in all examined tissues including heart, liver, spleen, lung, kidney, stomach, bladder, m. longissimus, belly fat, brain, large intestine, lymph, back fat, skin, and hypothalamus. Real-time quantitative PCR experiments revealed that it is in the hypothalamus with the highest expression of AgRP both in adult Lantang and Landrace pigs compared to the back fat and m.longissimus muscle and the cDNA level of AgRP in the hypothalamus of adult Chinese indigenous Lantang pig (fat-type) is significantly higher than that of Landrace pig (lean-type). To understand the regulation of the pig AgRP gene, the 5′-flanking region was isolated from a pig bacterial artificial chromosome library and used in a luciferase reporter assay. A positive cis-acting element for efficient AgRP expression was identified at nucleotides −501 to −479, by 5′-serial deletion of the promoter. Electrophoretic mobility-shift assays (EMSA) with competing oligonucleotides revealed that the critical region contained a cis-acting element for Neurogenic Differentiation (NeuroD), which is a member of the NeuroD family of basic-helix-loop-helix transcription factors. This element has not been reported in human or mouse AgRP genes. Our results indicated that NeuroD might be an essential regulatory factor for transcription of pig AgRP, providing an important clue about energy homeostasis regulation in the porcine and human brain.