PPAR signaling pathway is a key regulator of metabolism of the intestine , which together with the liver are considered as important sites for lipid metabolism . In the present study, the lipid metabolism-related genes such as FABP1, FABP2, FABP3, FABP5, FABP6, LPL and APOA1 that mapped to PPAR signaling pathway were downregulated in LP group relative to PP group. FABP multigene can code for diversified kinds of FABPs such as liver-type FABP (encoded by FABP1), intestinal-type FABP (encoded by FABP2), heart-type FABP (encoded by FABP3), epidermal-type FABP (encoded by FABP5), and ileal-type FABP (encoded by FABP6) . These proteins display high-affinity binding for fatty acids and other hydrophobic ligands, facilitating the transport of lipids to the specific compartments of cells for storage or oxidation . Although FABPs share a highly conserved structure, each of them has its own sequence and exhibits distinct affinity for ligand preferences . Specifically, ileal-type FABP that located in the distal small intestine is regarded as the cytosolic receptor for bile acids, although it has a low binding affinity for fatty acids . Therefore, the reduced expression of FABP6 with the resultant downregulations of GO clusters of transport and transporter activity might suggest a compromised reabsorption of luminal bile acids into enterocytes , resulting in a disordered regulation of lipid metabolism of the laying hens in LP group. On the other hand, the decreased expression of FABP1, FABP2 and FABP3 with the relevant downregulation of GO cluster of lipid binding were deduced to induce a malabsorption of fatty acids in LP group, since the entry of them from the lumen across the apical side of enterocytes was highly dependent on the binding by FABPs . Analogously, it was indicated that the age-related decline in intestinal lipid uptake of rat is associated with a reduced abundance of FABPs .
The malabsorption of fatty acids in LP group could subsequently act on the nuclear receptors of PPARs, which were characterized by a DNA-binding domain and ligand-binding domains, allowing for interaction with their ligands encompassing a variety of lipid components such as fatty acids . When these ligands are delivered to the nucleus under the facilitation by FABPs, the PPARs are activated and heterodimerize with retinoid receptor, thus regulating the expression of downstream target genes by binding to PPAR response elements in their promoters . In this study, although no difference in the expression of PPARs was observed between groups, there might be reduced bindings of PPARs to the promoters of their downstream genes such as APOA1, LPL, FABP1, FABP3 and SCP2 in LP group [Additional file 4], leading to the corresponding reductions of these genes expression. APOA1, an essential structural and functional component of chylomicron, can be synthesized in the intestine . Chylomicron can transport the absorbed triglycerides to certain parenchymal tissues such as skeletal muscle where they can release free fatty acids for oxidation under the catalysis of LPL , an enzyme that is nonspecifically synthesized in the intestine and spread along the vascular mesh . Accordingly, the downregulations of APOA1 and LPL in LP group probably caused an inefficient utilization of dietary lipids that serve as a momentous energy source for animals, presumptively favoring the compromised performance of laying hens. Besides participating in the assembly of chylomicron, APOA1 together with APOA4 are the major functional components of very-low density lipoprotein and high density lipoprotein, being closely connected with various metabolic processes especially the cholesterol metabolism . Indeed, the current study showed that the downregulated expression of APOA1 and APOA4 induced reductions of cholesterol metabolism-related GO clusters such as regulation of intestinal cholesterol absorption, cholesterol transporter activity, very-low density lipoprotein particle, positive regulation of cholesterol esterification and reverse cholesterol transport, indicating perturbations of cholesterol absorption, transport and excretion of laying hens in LP group. Phosphatidylcholine-sterol O-acyltransferase catalyzes cholesterol esterification by promoting the binding of fatty acyl group from phospholipid in high density lipoprotein to the cell-derived cholesterol , a process necessary for the reverse cholesterol transport. Phospholipid efflux can be conjugated with the reverse cholesterol transport from peripheral tissues to the liver, where cholesterol can be transformed into bile acids and in turn excrete to the feces . Thus, the lower GO clusters of phosphatidylcholine-sterol O-acyltransferase activator activity and phospholipid efflux in LP group may exacerbate the impaired efflux of cholesterol, triggering cholesterol accumulation inside the body of laying hens in LP group.
FABP1 and FABP3 not only participate in modulation of absorption and storage of lipids, but also involved in fatty acid oxidation by promoting transport of them to mitochondria [34, 35]. SCP2 exhibits high affinity for many hydrophobic ligands such as fatty acids and acyl-CoA, mediating the transport of acyl-CoA to mitochondria for oxidation . Thereby, the downregulated expression of FABP1, FABP3 and SCP2 might cause an impairment of fatty acid oxidation in LP group, resulting in a lower production of substrates like NADH and FADH2 , from which the electrons could be less released and shuttled through respiratory chain. This might thus deteriorate the deficiency of oxidative phosphorylation of laying hens in LP group.
Mitochondria are the main site for oxidizing nutrients such as fatty acids to generate ATP via oxidative phosphorylation. This is accomplished by the respiratory chain in the inner mitochondrial membrane , comprising five complexes including complex I (NADH-CoQ dehydrogenase), complex II (succinate-CoQ dehydrogenase), complex III (reduced CoQ-cytochrome c reductase), complex IV (cytochrome C oxidase) and complex V (ATP synthase) (Additional file 5). These enzyme complexes are indispensable for the proton-coupled electron transfer during oxidative phosphorylation . The gastrointestinal tract is known as an intense metabolic activity tissue with a high demand for free energy due to its roles in multiple physiological actions, accounting for as much as 15-25% of the whole energy requirement of birds . Consequently, mitochondrial dysfunction could restrict nutrient absorption and metabolism, therefore favoring the declined performance of laying hens. Indeed, it was verified that feed efficiency of chickens was positively correlated with the activities of respiratory chain complexes of the intestine [40, 41]. In this study, the expression of complex I subunits (NDUFA1, NDUFA8, NDUFB2, NDUFB9 and NDUFS6), complex III subunit (UQCR9), and complex V subunits (ATP5H, ATP5I, ATP5J, ATP5L and ATP6V1G1), together with the GO clusters in association with electron transport chain coupling such as ATP synthesis coupled proton transport, mitochondrial proton-transporting ATP synthase complex, and hydrogen ion transmembrane transporter activity were all downregulated in LP group, implying a structural disorder of respiratory chain with a subsequent hypofunction of oxidative phosphorylation in LP group. Similarly, it was reported that aging induced reduced expression of the subunits of respiratory chain complexes (III, IV and V) in the brain of mice , as well as the subunits of all the respiratory chain complexes in rat heart . We also observed that the intestine from LP group had a reduced ATP level and a lower activity of Na+/K+-ATPase, a major ion pump in basolateral membrane of enterocytes and drives the co-absorption of sodium with selected nutrients , confirming a disturbance of intestinal mitochondria to supply energy for laying hens in LP group. This could inevitably obstruct various metabolic processes with energy expenditure such as active transport of nutrients, presumably conducing to the impaired performance of laying hens in the late phase of production.
GSTs are encoded by GST multigene family and largely divided into groups of GST A (α), M (µ), P (π), O (ω), T (θ), D (δ), S (σ), K (κ) and Z (ζ) on the bases of biochemical and structural properties [45, 46]. GSTs are broadly spread in various cell compartments inside the body, among which GST A, M, P, K and Z can reside in the mitochondria . As a crucial group of multifunctional enzymes within the body, GSTs assist with the maintenance of cellular glutathione level and play a vital role in modulating glutathione metabolism [46, Additional file 6], because they are the antioxidant enzymes with glutaredoxin-like and glutathione reductase-like activities and also associated with increased protein glutathionylation, an important modification in response to cellular redox status. These could protect respiratory chain complexes against oxidative stress [47, 48]. Specifically, GSTA3 is found to exist in the mitochondria and capable to clear various peroxidation products , while GSTM2 protects against mitochondrial dysfunction by acting on V-type proton ATPase . GSTO1 can also be directly involved in glutathionylation of mitochondrial ATP synthase that defends against oxidative stress [50, 51]. The present study revealed that the gene expression of GSTO1, GSTM2 and GSTA3 and the activity of GST were all decreased in LP group. Similarly, the expression of GSTs in the visceral organs (liver and lung) of rats was reported to be decreased due to aging . Besides, the intestine from LP group had a reduced activity of SOD, a key line of antioxidant enzyme defense systems against reactive oxygen species . A decreased T-AOC coupled with an increased MDA content were also detected in LP group as compared to PP group. These findings demonstrated that the layer intestine from LP group may undergo an aggravation of oxidative stress. In support of this view, we also observed downregulations of several GO clusters related to oxidation resistance such as hydrogen peroxide catabolic process, removal of superoxide radicals, glutathione transferase activity, and antioxidant activity in LP group. Since mitochondria in the intestinal tissue is highly sensitive to oxidative stress that can lead to an inactivation of respiratory chain enzymes , the depressed oxidation resistance of the intestine presumably induced an inefficiency of energy production of laying hens in LP group . This was in accordance with the finding that oxidative stress-induced disorder of energy production via the dysfunctional mitochondria plays a fundamental role in age-related processes .
In addition to involving in antioxidative activities, GSTs also represent a major cellular defense system in response to environmental hazards, as they can detoxify both endogenous and exogenous compounds such as pharmaceuticals and environmental pollutants by catalyzing the conjugation of glutathione with these compounds containing electrophilic centers, thus forming more soluble, non-toxic peptide derivatives to be excreted from the body . The intestine is the primary site exposed to dietary xenobiotics that are chemical compounds foreign to the animal organism without nutritional value and considered as potential toxins , promoting the generation of cellular free radicals . However, there were enzyme systems such as GSTs capable of biotransformation of xenobiotics in the intestine, which consequently influenced the overall bioavailability of these chemicals . In this study, the reduced expression of GSTO1, GSTM2 and GSTA3 in LP group mediated a decreasing trend of pathway of metabolism of xenobiotics by cytochrome P450, being disadvantageous for detoxifying certain hazardous xenobiotics such as benzopyrene, naphthalene and aflatoxin [Additional file 7], potentially resulting in an oxidative stress in the intestine with a resultant compromise of intestinal functionality of laying hens in LP group .