Date Published: September 9, 2015
Publisher: Public Library of Science
Author(s): Denise K. Gessner, Birthe Gröne, Aline Couturier, Susann Rosenbaum, Sonja Hillen, Sabrina Becker, Georg Erhardt, Gerald Reiner, Robert Ringseis, Klaus Eder, Renzhi Han.
Lactating sows have been shown to develop typical signs of an inflammatory condition in the liver during the transition from pregnancy to lactation. Hepatic inflammation is considered critical due to the induction of an acute phase response and the activation of stress signaling pathways like the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), both of which impair animal´s health and performance. Whether ER stress-induced UPR is also activated in the liver of lactating sows and whether dietary fish oil as a source of anti-inflammatory effects n-3 PUFA is able to attenuate hepatic inflammation and ER stress-induced UPR in the liver of sows is currently unknown. Based on this, two experiments with lactating sows were performed. The first experiment revealed that ER stress-induced UPR occurs also in the liver of sows during lactation. This was evident from the up-regulation of a set of genes regulated by the UPR and numerically increased phosphorylation of the ER stress-transducer PERK and PERK-mediated phosphorylation of eIF2α and IκB. The second experiment showed that fish oil inhibits ER stress-induced UPR in the liver of lactating sows. This was demonstrated by decreased mRNA levels of a number of UPR-regulated genes and reduced phosphorylation of PERK and PERK-mediated phosphorylation of eIF2α and IκB in the liver of the fish oil group. The mRNA levels of various nuclear factor-κB-regulated genes encoding inflammatory mediators and acute phase proteins in the liver of lactating sows were also reduced in the fish oil group. In line with this, the plasma levels of acute phase proteins were reduced in the fish oil group, although differences to the control group were not significant. In conclusion, ER stress-induced UPR is present in the liver of lactating sows and fish oil is able to inhibit inflammatory signaling pathways and ER stress-induced UPR in the liver.
Lactation is a physiological state, which is characterized by a marked increase in energy and nutrient requirement for production of milk. In most mammals, this elevated energy and nutrient demand is met by an increase in food intake and a mobilisation of body´s energy stores, i.e., white adipose and muscle tissue [1–3]. To conserve energy and metabolic substrates for milk synthesis in the lactating mammary gland, most species develop a diversity of metabolic adaptations in the liver, such as a reduced oxidation of fatty acids through down-regulation of transcriptional regulators of genes involved in fatty acid utilization and export of triacylglycerols from the liver to the lactating mammary gland [4–9]. Besides metabolic adaptations, pathophysiologic conditions are commonly developing in the liver during the transition from pregnancy to lactation. For instance, in dairy cows a pro-inflammatory condition in the liver is arising in early lactation, which has been suggested to be associated with the development of fatty liver syndrome and ketosis [10,11]. While metabolic adaptations and pathophysiologic conditions developing during early lactation have been well studied in dairy cows, little is known about specific, corresponding adaptation processes in sows (Sus scrofa). Recently, we have observed that lactation induces also a pro-inflammatory condition in the liver of sows, as evidenced from activation of the key regulator of inflammation nuclear factor-kappa B (NF-κB) and up-regulation of genes encoding positive acute phase proteins (APPs), like haptoglobin (HP) and C-reactive protein (CRP) [12,13]. In line with this, earlier observations showed that the plasma levels of APPs, like HP and CRP, are elevated in sows one week after farrowing compared to late pregnancy . The hepatic production of APPs, which is mediated by pro-inflammatory cytokines and occurs in response to different stimuli including infections, tissue damage and stress , is regarded as detrimental in farm animals as it not only increases energy and amino acid requirement in the liver for the synthesis of positive APPs but also commonly impairs liver function . Moreover, the pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α generated during an inflammatory process are able to induce stress of the endoplasmic reticulum (ER), a state in which unfolded or misfolded proteins accumulate in the ER lumen [17,18]. ER stress leads to the activation of an adaptive response known as the unfolded protein response (UPR), which aims to restore ER homeostasis and functions by triggering three kinds of protective cellular responses: (i) up-regulation of ER chaperones to assist in the refolding of proteins; (ii) attenuation of protein translation, and (iii) degradation of misfolded proteins by the proteasome by a process called ER-associated degradation (ERAD) [19,20]. Moreover, an induction of the UPR leads to an enhancement of inflammation by activation of NF-κB, a stimulation of lipid biosynthesis and an induction of fibroblast growth factor (FGF) 21, which is a hormonal regulator of lipolysis and ketogenesis [21–23]. Besides these adverse effects, the UPR leads to an improvement of the antioxidant and cytoprotective capacity by activation of nuclear factor E2-related factor 2 (Nrf2) . In the case that ER stress-induced damage is too strong and homeostasis cannot be restored, the UPR can lead to cell death by the induction of apoptosis [25,26]. Recently, it has been observed that ER stress occurs in the liver of dairy cows during early lactation and it has been suggested that the concomitant UPR might be involved in the development of fatty liver syndrome and ketosis . Whether the inflammatory process in the liver observed in lactating sows also leads to ER stress and induction of UPR in the liver during lactation, however, is currently unknown and remains to be demonstrated.
For this study, two trials with sows were performed in accordance with established guidelines for the care and handling of laboratory animals and were approved by the local Animal Welfare Authorities (Regierungspräsidium Giessen; permission no: GI 19/3-No. 29/2010).
We have recently reported that the liver of lactating sows develops typical signs of an inflammatory condition, such as activation of NF-κB and up-regulation of genes encoding APPs, as a consequence of the metabolic and physiologic adaptations occurring during the transition from pregnancy to lactation . Hepatic inflammation and the associated acute phase response are critical because it impairs performance of farm animals and it results in systemically elevated levels of inflammatory mediators (APPs, cytokines) and ROS. Both, inflammatory mediators and ROS are well known stimulators of ER stress that is known to induce the adaptive UPR . One key finding of the present study is that ER stress-induced UPR occurs also in the liver of sows during lactation. This was evident from the up-regulation of a number of genes regulated by the UPR including the ER chaperones HSP90B1 and HSPA5, the protein disulfide isomerase PDIA4, the key regulator of ER-stress induced apoptosis DDIT3, the apoptotic protein CASP3, and ATF4, which is the main regulator of DDIT3 and ERAD components. All these genes are downstream target genes of the three ER stress transducers inositol requiring 1 (IRE1), PKR-like ER kinase (PERK) and activating factor 6 (ATF6), and, thus, these genes are considered reliable markers of ER stress . Moreover, we found that phosphorylation of the ER stress transducer PERK and phosphorylation of the ER stress targets eIF2α and IκB were increased, at least numerically, in the liver of lactating sows. Collectively, these results strongly suggest that, like in high-yielding dairy cows , ER stress and the induction of the UPR occur in the liver of lactating sows. Like in the liver, at least some of the UPR-regulated genes considered were found to be up-regulated in skeletal muscle of lactating sows, which indicates that the occurrence of ER stress-induced UPR during lactation is not restricted to the liver but is present also in non-hepatic tissues.
Our study shows for the first time that, like in dairy cows, ER stress-induced UPR is present in the liver and skeletal muscle of sows during lactation, and dietary fish oil is able to inhibit, at least in the liver, ER stress-induced UPR and inflammatory and stress signaling pathways, which are involved in the induction of ER stress. The occurrence of ER stress in the liver during lactation indicates that the metabolic and physiologic changes occurring during the transition from pregnancy to lactation represent cellular stress that might be detrimental to health and performance of sows. At least in dairy cows it has been suggested that the ER stress-induced UPR contributes to the pathophysiologic conditions commonly observed in the liver of periparturient cows , such as fatty liver and ketosis, which are considered critical with regard to milk and reproductive performance. Although little is known about the occurrence of liver-associated diseases and its relevance for milk and reproductive performance in lactating sows, it is assumed that feeding fish oil is a useful dietary strategy to improve health and performance of lactating sows.