Energy-dependent hypothalamic regulation of feed utilization in poultry

In recent years, genetic control of the mechanisms regulating energy homeostasis in birds has received considerable attention. Among the molecules thought to be important in this regulation is the glucose-dependent insulinotropic polypeptide (GIP). In response to feed intake, GIP stimulates pancreatic secretion of insulin and leads to an increased uptake of blood glucose by the liver. Also, GIP has been implicated in the gut-brain axis regulation of feeding behavior. Previous reports suggested that GIP was expressed exclusively in the enteroendocrine K cells of the intestine. However, recent studies have shown GIP messenger RNA (mRNA) and protein expression in the brain of mammals. Here we provide new evidence for the expression of a genetically-diverse GIP gene in the neuronal appetite center of an adult avian. A 1,000-clone cDNA library was constructed from enriched mRNA isolated from the guinea fowl (GF) hypothalamus and screened via PCR and restriction digestion. To further characterize the transcriptome of the GF hypothalamus, ultra-high throughput RNA sequencing was performed yielding approximately 9.5 million sequence reads representing 70% coverage of the GF hypothalamus transcriptome. From the clone library, a 349 nucleotide base GIP gene fragment, showing 89% similarity with its Gallus ortholog, was identified and sequenced. Nucleotide-to-amino acid (AA) translation of the +1 frame-shifted open reading frame yielded a 76 AA peptide sequence showing 79% similarity with the carboxy terminus of the Gallus form. Multiple sequence alignment of the GIP of GF against human, rat, mouse, swine, bovine and chicken GIP ortholog sequences (UniProt database) revealed two regions of high conservation (\u3e90%). The AA sequence motifs of GIP of GF were identified and further analyzed for functional site prediction using the Eukaryotic Linear Motif resource. The most pronounced conserved region detected was a 24 AA motif containing an N-glycosylation site, a glycosaminoglycan attachment site, and a proprotein convertase cleavage site. Across species, regions of low (\u3c60%) conservation exhibited higher variability in the avian-mammalian contrast, suggesting an evolutionary divergence in the GIP lineage. Consumption of high-caloric poultry diets led to up-regulation of GIP mRNA expression in the GF and broiler hypothalamus and small intestine. The potential for hypothalamic GIP secretion in birds is quite remarkable as it may provide further insight on hypothalamic regulation of appetite and nutrient utilization in poultry. Key words: hypothalamus, glucose-dependent insulinotropic polypeptide, nutrient utilization, feed efficiency, guinea fowl, poultr