Feeding behavior and body energy homeostasis are intimately connected with the brain (Kuenzel et al., 1999; Richards, 2003; Richards and Proszkowiec-Weglarz, 2007; Bungo et al., 2011), in particular with the infundibular nucleus of the hypothalamus (Bungo et al., 2011). Here, the hypothalamic melanocortin system contains two different populations of neurons which can modulate feed intake through the secretion of various neuropeptides. Briefly, a reduction of feed consumption is mediated by the α-melanocyte stimulating hormone, released by the proopiomelanocortin neurons (POMC), and the cocaine- and amphetamine-regulated transcript (CART). On the other hand, neuropeptide Y (NPY) and agouti-related protein can stimulate appetite and increase feed intake by repressing the melanocortin anorexigenic effect (Bungo et al., 2011). Differences in the expression levels of these neuropeptides and some feeding-related genes have been reported in the hypothalamus of chickens (Sintubin et al., 2014) and quails (Blankenship et al., 2016) divergently selected for RFI and FE, respectively. However, other factors such as leptin (Dridi et al., 2005) and several gut hormones (Honda et al., 2017) can affect central feed intake regulation and hence energy homeostasis in chickens.
1.6.3.1. Focus on Neuropeptide Y (NPY)
This research topic was developed during my 5-month research period at the Center of Excellence for Poultry Science – University of Arkansas. This study was carried out to characterize the expression of neuropeptide Y in chicken muscle and assess its effects on mitochondria dynamics.
NPY is an orexigenic 36-amino acid peptide mainly expressed in the hypothalamus of chickens (Kuenzel et al., 1987). While the distribution of NPY and its receptors in mammals is well
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documented and defined, there is a paucity of scientific information regarding these aspects in avian (non-mammalian vertebrates) species. Indeed, NPY expression was only recently reported in other chicken tissues beside the brain, such as liver and skeletal muscle (Gao et al., 2017). In addition, although the role of hypothalamic NPY on the regulation of feeding behavior is well known (Saneyasu et al., 2011; Newmyer et al., 2013), its effect on physiological and metabolic features in peripheral tissues is still not well defined. Therefore, due to the important functions of NPY on overall energy homeostasis and hence on feed efficiency in chickens, a study was conducted to investigate whether NPY and its receptors are expressed in the skeletal muscle, the main site for thermogenesis in poultry species, and then to investigate its potential role in regulating the expression of mitochondrial biogenesis-, function-, and dynamics-related genes.
For the characterization, 6 birds showing the same age, gender and genetic line, fed the same diet and raised in the same environmental conditions, were humanely euthanized to collect breast and leg muscle, and hypothalamus. Quail leg muscle and quail myoblast (QM7) cells were also used for the characterization. Total mRNA was extracted (Trizol method) according to the manufacturer’s recommendations, and its integrity and quality were evaluated using 1% agarose gel electrophoresis. RNA concentration and purity were assessed for each sample by Take 3 Micro-Volume Plate using Synergy HT multi-mode micro plate reader (BioTek,Winooski, VT). Then, 1 μg of RNA was reverse transcribed using qScript cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD) and the expression of NPY and its receptors was assessed with conventional PCR.
To understand the physiological role of NPY in chicken muscle, QM7 cells were treated either with 0 (CON), 1 (NPY1) or 100 (NPY100) nM of recombinant NPY. Total mRNA was extracted from treated QM7 cells as described above, and RT-qPCR analysis performed to determine the relative expression of NPY and its receptors as well as that of mitochondrial biogenesis-, function-, and dynamics-related genes. Briefly, real-time quantitative PCR (Applied Biosystems 7500 Real-Time PCR system) was performed using 5 μL of 10X diluted cDNA, 0.5 μM
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of each forward and reverse primer, and SYBR Green Master Mix (ThermoFisher Scientific, Rockford, IL) for a total 25 μL reaction. The qPCR cycling conditions were the same reported by Lassiter et al. (2014) and 18S gene was used as housekeeping gene. The 2−ΔΔCt method (Schmittgen and Livak, 2008) was used to determine the relative expression of target genes considering the untreated group as calibrator.
From this study emerged that NPY is expressed in both chicken breast and leg muscles, quail leg muscle, and in QM7 cells, as shown in Figure 1.6.1. The mRNA expression of NPY and its receptors in QM7 cells was reduced by the NPY1 treatment while was increased with the NPY100 (Figure 1.6.2). In addition, the treatment with recombinant NPY altered the mRNA expression of mitochondrial biogenesis-, function-, and dynamics-related genes in QM7 cells (Figure 1.6.3).
Figure 1.6.1 Characterization of NPY and its receptors in chicken and quail tissues (hypothalamus and muscles) through conventional PCR.
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Figure 1.6.2 mRNA expression of NPY and its receptors in QM7 cells treated with recombinant NPY.
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Figure 1.6.3 mRNA expression of mitochondrial biogenesis-, function-, and dynamics-related genes in QM7 cells treated with recombinant NPY.
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In addition, an in vivo study was performed. 10-d-old chicks were intraperitoneally injected with different dosages of NPY (0, 60, 120, 240 nM NPY). The results (data not showed) indicated that NPY treatments stimulated feed but not water consumption. Similarly, the treatments determined significant changes in NPY expression in blood as well as in the expression of NPY, NPY receptors, and mitochondria genes, in breast and leg muscle. Furthermore, we observed significant changes in the expression of NPY and related receptors according to the feeding state of the birds (fasted vs. fed). Ongoing analysis on mitochondria bioenergetics processes are defining the role of NPY on metabolic aspects of muscle mitochondria. However, due to the deadline anticipation for submitting the PhD thesis (occurred for the first time in this academic year), the results of the bioenergetics analysis, currently ongoing, have not been included in this dissertation.
Taken together, these results suggest that:
NPY is expressed in chicken breast and leg muscle,
NPY regulates its own system,
NPY may play a pivotal role in mitochondria function and dynamics.
Therefore, due to its important functions on feed intake and mitochondria activity in muscle tissue, NPY could be considered a crucial factor involved in energy homeostasis processes and thereby in FE in broiler chickens.