Enrichment of Autophagy and Proteosome Pathways in Breast Muscle of Feed Efficient Pedigree Male Broilers

Background: Feed efficiency (FE) is an important genetic trait in poultry and livestock. Autophagy (self-eating) and proteosomes are cellular processes that remove damaged cell components (e.g., proteins, organelles). As evidence of extensive protein oxidation was observed in Pedigree Male (PedM) broilers exhibiting a low FE (LFE) phenotype compared to a high FE (HFE) phenotype, the main goal of this study was to assess gene and protein expression of the autophagy and proteosome pathways in breast muscle obtained in PedM broilers exhibiting HFE and LFE phenotypes.Methods: Feed efficiency was calculated as weight gain divided by feed intake gain in individual PedM broilers that were measured between 6 and 7 weeks of age. Targeted gene expression was conducted on breast muscle using quantitative real-time polymerase chain reaction (qPCR) to determine mRNA expression of genes associated with the autophagy pathway; AMP-activated protein kinase alpha 1 (AMPKα1), mammalian target of rapamycin (mTOR), Beclin 1, and autophagy genes (Atg) 3, Atg7, and Atg16L1. Binomial distribution analysis was conducted on transcriptomic and data obtained by RNAseq and shotgun proteomics, respectively on the same set of tissues for genes associated with autophagy, vacuole formation, and proteosome expression.Results: Greater efficiency was attained in the HFE PedM broilers by greater weight gain on the same amount of feed consumed resulting in FEs of 0.65 ± 0.01 and 0.46 ± 0.01 in the HFE and LFE phenotypes, respectively. Targeted mRNA expression analysis revealed significant (P < 0.05) elevations in AMPKa1, mTOR, Atg16L1, and Atg7 and a marginal (P = 0.07) elevation in Beclin1. Binomial distribution analysis transcriptomic and proteomic data revealed significant skews favoring autophagy-, vacuole-, and proteosome-related genes in the HFE phenotype. These results indicate that the autophagy and proteosome expression is enhanced in the HFE compared to the LFE pedigree male broiler phenotype suggesting that protein and organelle quality control may be enhanced in high feed efficiency

Orexin regulates mitochondrial dynamics in avian muscle

The growing obesity epidemic has sparked numerous studies on the identification of molecular signatures that regulate energy homeostasis using different experimental animal models. Orexin, which acts via two G-protein coupled receptors, orexin receptor 1 and 2, has been originally identified as feeding-related hypothalamic neuropeptide that regulate energy balance in mammals. Recently, using chicken, non-mammalian species that are characteristically hyperglycemic and prone to obesity, we made a breakthrough by identifying the orexin system in avian muscle and unraveling its effect on mitochondrial dynamics and function. Therefore, understanding orexin signaling and function may help to identify novel therapeutic opportunities for treating metabolic disorders related to mitochondrial dysfunction

Tissue distribution, gender- and genotype-dependent expression of autophagy-related genes in avian species.

As a result of the genetic selection of broiler (meat-type breeders) chickens for enhanced growth rate and lower feed conversion ratio, it has become necessary to restrict feed intake. When broilers are fed ad libitum, they would become obese and suffer from several health-related problems. A vital adaptation to starvation is autophagy, a self-eating mechanism for recycling cellular constituents. The autophagy pathway has witnessed dramatic growth in the last few years and extensively studied in yeast and mammals however, there is a paucity of information in avian (non-mammalian) species. Here we characterized several genes involved in autophagosome initiation and elongation in Red Jungle fowl (Gallus gallus) and Japanese quail (coturnix coturnix Japonica). Both complexes are ubiquitously expressed in chicken and quail tissues (liver, leg and breast muscle, brain, gizzard, intestine, heart, lung, kidney, adipose tissue, ovary and testis). Alignment analysis showed high similarity (50.7 to 91.5%) between chicken autophagy-related genes and their mammalian orthologs. Phylogenetic analysis demonstrated that the evolutionary relationship between autophagy genes is consistent with the consensus view of vertebrate evolution. Interestingly, the expression of autophagy-related genes is tissue- and gender-dependent. Furthermore, using two experimental male quail lines divergently selected over 40 generations for low (resistant, R) or high (sensitive, S) stress response, we found that the expression of most studied genes are higher in R compared to S line. Together our results indicate that the autophagy pathway is a key molecular signature exhibited gender specific differences and likely plays an important role in response to stress in avian species

Effect of heat stress on the hypothalamic expression profile of water homeostasis‐associated genes in low‐ and high‐water efficient chicken lines

Abstract With climate change, selection for water efficiency and heat resilience are vitally important. We undertook this study to determine the effect of chronic cyclic heat stress (HS) on the hypothalamic expression profile of water homeostasis‐associated markers in high (HWE)‐ and low (LWE)‐water efficient chicken lines. HS significantly elevated core body temperatures of both lines. However, the amplitude was higher by 0.5–1°C in HWE compared to their LWE counterparts. HWE line drank significantly less water than LWE during both thermoneutral (TN) and HS conditions, and HS increased water intake in both lines with pronounced magnitude in LWE birds. HWE had better feed conversion ratio (FCR), water conversion ratio (WCR), and water to feed intake ratio. At the molecular level, the overall hypothalamic expression of aquaporins (AQP8 and AQP12), arginine vasopressin (AVP) and its related receptor AVP2R, angiotensinogen (AGT), angiotensin II receptor type 1 (AT1), and calbindin 2 (CALB2) were significantly lower; however, CALB1 mRNA and AQP2 protein levels were higher in HWE compared to LWE line. Compared to TN conditions, HS exposure significantly increased mRNA abundances of AQPs (8, 12), AVPR1a, natriuretic peptide A (NPPA), angiotensin I‐converting enzyme (ACE), CALB1 and 2, and transient receptor potential cation channel subfamily V member 1 and 4 (TRPV1 and TRPV4) as well as the protein levels of AQP2, however it decreased that of AQP4 gene expression. A significant line by environment interaction was observed in several hypothalamic genes. Heat stress significantly upregulated AQP2 and SCT at mRNA levels and AQP1 and AQP3 at both mRNA and protein levels, but it downregulated that of AQP4 protein only in LWE birds. In HWE broilers, however, HS upregulated the hypothalamic expression of renin (REN) and AVPR1b genes and AQP5 proteins, but it downregulated that of AQP3 protein. The hypothalamic expression of AQP (5, 7, 10, and 11) genes was increased by HS in both chicken lines. In summary, this is the first report showing improvement of growth performances in HWE birds. The hypothalamic expression of several genes was affected in a line‐ and/or environment‐dependent manner, revealing potential molecular signatures for water efficiency and/or heat tolerance in chickens

Relative expression of autophagosome initiation-related genes in various tissues of R ans S male Japonica quail lines.

<p>Total RNA from each tissue was DNAse-treated, reverse transcribed, and subjected to real-time quantitative PCR. Sample were run in duplicate, and the average threshold cycle (Ct) values were determined for the target and houskeeping genes. Relative quantity of autophagy genes was determined by the 2^−ΔΔCt method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112449#pone.0112449-Davis1" target="_blank">[58]</a>. Data are presented as mean ± SEM (n = 6 for each line and each tissue). * Line-matched differences among tissues (*<i>P</i><0.05). Different letters indicate tissue-matched differences among Lines (a–c, difference between tissues within R line and α-ε indicate differences between tissues within S line).</p

Characterization of autophagosome initiation- (a) and elongation-related genes (b) in various tissues of male and female Red Jungle Fowl (<i>Gallus gallus</i>) by RT-qPCR as described in materials and methods.

<p>Signals were visualized by agarose gel electrophoresis.</p

Comparison of relative expression of autophagosome elongation-related genes in various tissues of R and S male Japonica quail lines.

<p>Total RNA from each tissue was DNAse-treated, reverse transcribed, and subjected to real-time quantitative PCR. Sample were run in duplicate, and the average threshold cycle (Ct) values were determined for the target and houskeeping genes. Relative quantity of autophagy genes was determined by the 2^−ΔΔCt method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112449#pone.0112449-Davis1" target="_blank">[58]</a>. Data are presented as mean ± SEM (n = 6 for each line and each tissue). * Genotype-matched differences among tissues (*<i>P</i><0.05 and ***<i>P</i><0.001). Different letters indicate tissue-matched differences among genotype (a, b, difference between tissues within R line and α-β indicate differences between tissues within S line). 28.</p

Phylogenetic relationships among chicken autophagy-related genes and their mammalian orthologs were inferred using the neighbor-joining method in MUSCLE alignment and MEGA6.

<p>Scale bar indicates the substitution rate per residue. Genbank accession numbers are included in the phenogram and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112449#pone-0112449-t002" target="_blank">Table 2</a>.</p

Comparison of relative expression of autophagosome initiation-related genes in various tissues of male and female Red Jungle Fowl.

<p>Total RNA from each tissue was DNAse-treated, reverse transcribed, and subjected to real-time quantitative PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112449#s2" target="_blank">material and methods</a>. Samples were run in duplicate, and the average threshold cycle (Ct) values were determined for the target and houskeeping genes. Relative quantity of autophagy genes was determined by the 2^−ΔΔCt method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112449#pone.0112449-Davis1" target="_blank">[58]</a>. Data are presented as mean ± SEM (n = 6 for each gender and each tissue). * Sex-matched differences among tissues (*<i>P</i><0.05 and **<i>P</i><0.01). Different letters indicate tissue-matched differences among gender (a–e, difference between tissues within female and α-δ indicate differences between male tissues).</p