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

Abstract

Background: The 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, alpha-melanocyte stimulating hormone, norepinephrine, and L-phospho-serine. Results confirmed functional relationships among members of the predicted gene network. Moreover, the effects observed were dependent upon the nutritional state of the animals (fed vs. fasted).

Results: Differences in gene expression (> or = 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.

Conclusions: Microarray 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.

Figure 1

Relative levels of mRNA for selected genes upregulated by fasting as determined by microarray and qRT-PCR. Data were normalized to the housekeeping gene phosphoglycerate kinase 1 (PGK1), and are expressed relative to mRNA levels at hatch, which are set to the value of 1. Gene expression higher than 1 indicates upregulation compared to hatch, while gene expression levels less than 1 indicate downregulation compared to hatch. Chicks were either fully fed (FF), not fed (NF) or delayed fed (DF). Treatment groups are designated as follows: hatch (H), fed for 24 h (24FF), fasted for 24 h (24NF), fed for 48 h (48FF), fasted for 48 h (48NF), fasted for 48 h then fed for 4 h (4DF), fed for 72 h (72FF), fasted for 48 h then fed for 24 h (24DF), fed for 96 h (96FF), fasted for 48 h then fed for 48 h (48DF). Genes evaluated were: neuropeptide Y receptor 5 (NPY5R), aromatase (CYP19A), deiodinase II (DIO2), FK506 binding protein 51 (FKBP51), somatostatin receptor 5 (SSTR5), and coagulation factor C (COCH). Black bars are microarray results, and white bars are qRT-PCR results. Values within a single method with differing letters are significantly different (P < 0.05).

Figure 2

Relative levels of mRNA for selected genes downregulated by fasting as determined by microarray and qRT-PCR. Data were normalized to the housekeeping gene phosphoglycerate kinase 1 (PGK1), and are expressed relative to mRNA levels at hatch, which are set to the value of 1. Gene expression higher than 1 indicates upregulation compared to hatch, while gene expression levels less than 1 indicate downregulation compared to hatch. Treatment groups are designated as follows: hatch (H), fed for 24 h (24FF), fasted for 24 h (24NF), fed for 48 h (48FF), fasted for 48 h (48NF), fasted for 48 h then fed for 4 h (4DF), fed for 72 h (72FF), fasted for 48 h then fed for 24 h (24DF), fed for 96 h (96FF), fasted for 48 h then fed for 48 h (48DF). Genes evaluated were: fatty acid binding protein 7 (FABP7), hemoglobin alpha (HBA), pro-opiomelanocortin (POMC), oxysterol 7-alpha-hydroxylase (CYP39A1), protein kinase C iota (PRKCI), and sal-like 3 (SALL3). Black bars are microarray results, and white bars are qRT-PCR results. Values within a single method with differing letters are significantly different (P < 0.05).

Figure 3

Relative levels of mRNA for selected genes associated with feeding and fasting in the hypothalamus as determined by microarray and qRT-PCR. Data were normalized to the housekeeping gene phosphoglycerate kinase 1 (PGK1), and are expressed relative to mRNA levels at hatch, which are set to the value of 1. Gene expression higher than 1 indicates upregulation compared to hatch, while gene expression levels less than 1 indicate downregulation compared to hatch. Treatment groups are designated as follows: hatch (H), fed for 24 h (24FF), fasted for 24 h (24NF), fed for 48 h (48FF), fasted for 48 h (48NF), fasted for 48 h then fed for 4 h (4DF), fed for 72 h (72FF), fasted for 48 h then fed for 24 h (24DF), fed for 96 h (96FF), fasted for 48 h then fed for 48 h (48DF). Genes evaluated were neuropeptide Y (NPY), agouti-related protein (AGRP), leptin receptor (LEPR), corticotropin releasing hormone (CRH), growth hormone releasing hormone (GHRH), thyrotropin releasing hormone (TRH), and melanocortin receptor 4 (MC4R). Black bars are microarray results, and white bars are qRT-PCR results. Values within a single method with differing letters are significantly different (P < 0.05).

Figure 4

Diagram of associated genes derived from the online Pathway Miner tool for clustering genes associated within a pathway. Associated genes are neuropeptide Y receptor 5 (NPY5R), metabotropic glutamate receptor 8 (GRM8), somatostatin receptor 5 (SSTR5), pro-opiomelanocortin (POMC), relaxin 3 (RLN3), and beta-2-adrenergic receptor (ADRB2). Gene expression data generated by the microarray revealed that POMC exhibited reduced expression in fasted chicks, and the other associated genes were expressed at increased levels in fasted chicks.

Figure 5

Relative levels of mRNA determined by qRT-PCR for selected genes associated with feeding and fasting in the hypothalamus. Data were normalized to the housekeeping gene phosphoglycerate kinase 1 (PGK1), and are expressed relative to mRNA levels in the 24 h fed group (24FF). Treatment groups are designated as follows: fed for 24 h (24FF), fasted for 24 h (24NF), fed for 48 h (48FF), and fasted for 48 h (48NF). Values with differing letters are significantly different (P < 0.05). Genes evaluated were: neuropeptide Y (NPY), deiodinase II (DIO2), FK506 binding protein 51 (FKBP51), thyrotropin releasing hormone (TRH), aromatase (CYP19A1), leptin receptor (LEPR), pro-opiomelanocortin (POMC), fatty acid binding protein 7 (FABP7), and beta-2-adrenergic receptor (ADRB2).

Figure 6

Gene expression in dispersed hypothalamic neurons isolated from chicks following feeding or fasting for 24 or 48 h and treated with receptor agonists in culture. Following feeding or fasting of chicks for 24 or 48 h, the hypothalamus was collected and neurons were dispersed and cultured in 12-well culture dishes in neurobasal medium for 24 h. Receptor agonists [α- melanocyte stimulating hormone (aMSH), neuropeptide Y (NPY), somatostatin (SRIF), norepinephrine (NE), or O-phospho-L-serine (LSOP, a group III metabotropic receptor agonist] were added to the medium following 24 h of incubation, and neurons were incubated for an additional 24 h. Cells were collected, and RNA was extracted for analysis by qRT-PCR. Gene expression was evaluated for pro-opiomelanocortin (POMC), melanocortin 4 receptor (MC4R), neuropeptide Y (NPY), beta-2-adrenergic receptor (ADRB2), and metabotropic glutamate receptor 8 (GRM8). The first column compares the relative gene expression levels between fed and fasted chicks within each time point (24 h - white bars, 48 h - black bars). Gene expression levels in treated neurons from chicks fed (black bars) or fasted (white bars) for 24 h are represented in the 2nd column, and levels from chicks fed (black bars) or fasted (white bars) for 48 h are in the 3rd column. Asterisks indicate significant differences (P < 0.05) within time points for the control graphs (column 1) or within time and nutritional status (fed or fasted) (columns 2 and 3).

Figure 7

Summary of changes in gene expression of primary hypothalamic neurons obtained from chicks following feeding or fasting for 24 or 48 h and treated in cell culture. Gray boxes represent receptors and white boxes represent the receptor agonists utilized for treatment in culture. Lines indicate a significant effect on a specific gene following addition of the agonist in culture as compared with the non-treated control within the same group (fed or fasted) and time point (24 or 48 h). Lines ending in arrows indicate an increase in gene expression level, and lines ending with a bar indicate a reduction in gene expression level due to treatment.