Transcriptional and pathway analysis in the hypothalamus of newly hatched chicks during fasting and delayed feeding

Abstract

BackgroundThe hypothalamus plays a central role in regulating appetite and metabolism. However, the gene networks within the hypothalamus that regulate feed intake and metabolism, and the effects of fasting on those pathways are not completely understood in any species. The present experiment evaluated global hypothalamic gene expression in newly hatched chicks using microarray analysis to elucidate genes and pathways regulated by feeding, fasting, and delayed feeding. Ten groups of chicks were sampled over four days post-hatch, including fed, fasted, and 48 h fasted followed by access to feed for 4 h, 24 h, and 48 h. Hypothalamic samples were collected for microarray analysis (n = 4). Expression patterns of selected genes were confirmed by quantitative real-time PCR. Pathway analysis of the microarray results predicted a network of genes involved in neuropeptide or neurotransmitter signaling. To confirm the functionality of this predicted gene network, hypothalamic neurons from fed and fasted chicks were isolated and cultured in the presence of neuropeptide Y, somatostatin, α-melanocyte stimulating hormone, norepinephrine, and L-phospho-serine. Results confirmed functional relationships among members of the predicted gene network. Moreover, the effects observed were dependant upon the nutritional state of the animals (fed vs. fasted).ResultsDifferences in gene expression (≥ 1.6 fold) were detected in 1,272 genes between treatments, and of those, 119 genes were significantly (P < 0.05) different. Pathway Miner analysis revealed that six genes (SSTR5, NPY5R, POMC, ADRB2, GRM8, and RLN3) were associated within a gene network. In vitro experiments with primary hypothalamic neurons confirmed that receptor agonists involved in this network regulated expression of other genes in the predicted network, and this regulation within the network was influenced by the nutritional status and age of the chick.ConclusionsMicroarray analysis of the hypothalamus during different nutritional states revealed that many genes are differentially regulated. We found that functional interactions exist among six differentially regulated genes associated within a putative gene network from this experiment. Considering that POMC, an important gene in controlling metabolism, was central to this network, this gene network may play an important role in regulation of feeding and metabolism in birds.

Glucocorticoids Increase NPY Gene Expression via Hypothalamic AMPK Signaling in Broiler Chicks

“…Next, 2-mm bilateral cuts were made on either side of the midline, and the whole hypothalamus was removed according to the method described in Yuan et al (23). The cut was 4 -5 mm deep parallel to the base of the brain (24). After being snap frozen in liquid nitrogen, the hypothalamus tissue samples were stored at Ϫ80°C until RNA and protein extraction.…”

Regulation of the avian central melanocortin system and the role of leptin

“…This suggests a role for these pathways in the control of appetite within the hypothalamus. Although elements of the network such as the POMC and NPY5R genes are involved in the response to food deprivation in mammals, the network as a whole was uniquely observed in the Higgins et al (2010) study. There is also suggestive evidence for a role of hypothalamic energy sensing pathways in regulating central melanocortin signalling.…”

“…Changes in mRNA appear to reflect parallel changes in peptide because food deprivation increased the number of AGRP-immunoreactive cells in the arcuate nucleus of the ring dove (Streptopelia risoria) which were also elevated during the parental phase of the breeding cycle when the birds are in negative energy balance . For POMC, Higgins et al (2010) 2003; Song et al, 2012). Thus the changes in AGRP and POMC expression during fasting are in the direction predicted from the behavioural effects of their encoded peptides as part of a coordinated counter-regulatory response of hypothalamic appetite control peptides to restore lost energy stores (Phillips-Singh et al, 2003).…”

“…Most investigations have focused on short-term food deprivation, which increases AGRP mRNA (Phillips-Singh et al, 2003;Higgins et al, 2010;Song et al, 2012). Changes in mRNA appear to reflect parallel changes in peptide because food deprivation increased the number of AGRP-immunoreactive cells in the arcuate nucleus of the ring dove (Streptopelia risoria) which were also elevated during the parental phase of the breeding cycle when the birds are in negative energy balance .…”

“…Thus the changes in AGRP and POMC expression during fasting are in the direction predicted from the behavioural effects of their encoded peptides as part of a coordinated counter-regulatory response of hypothalamic appetite control peptides to restore lost energy stores (Phillips-Singh et al, 2003). Higgins et al (2010) used microarray analysis to explore patterns of gene expression in the hypothalamus of domestic chicks in relation to food deprivation. Pathway analysis software predicted that POMC lies within a network of six interrelated differentially-expressed genes and experimental evidence for the existence of the network was provided from in vitro experiments.…”