- Brief communication
- Open Access
The stress signalling pathway nuclear factor E2-related factor 2 is activated in the liver of sows during lactation
- Susann Rosenbaum†1,
- Robert Ringseis†1,
- Sonja Hillen2,
- Sabrina Becker2,
- Georg Erhardt3,
- Gerald Reiner2 and
- Klaus Eder1Email author
https://doi.org/10.1186/1751-0147-54-59
© Rosenbaum et al.; licensee BioMed Central Ltd. 2012
- Received: 11 September 2012
- Accepted: 2 October 2012
- Published: 5 October 2012
Abstract
Background
It has recently been shown that the lactation-induced inflammatory state in the liver of dairy cows is accompanied by activation of the nuclear factor E2-related factor 2 (Nrf2) pathway, which regulates the expression of antioxidant and cytoprotective genes and thereby protects tissues from inflammatory mediators and reactive oxygen species (ROS). The present study aimed to study whether the Nrf2 pathway is activated also in the liver of lactating sows.
Findings
Transcript levels of known Nrf2 target genes, UGT1A1 (encoding glucuronosyltransferase 1 family, polypeptide A1), HO-1 (encoding heme oxygenase 1), NQO1 (encoding NAD(P)H dehydrogenase, quinone 1), GPX1 (encoding glutathione peroxidase), PRDX6 (encoding peroxiredoxin 6), TXNRD1 (encoding thioredoxin reductase 1), and SOD (encoding superoxide dismutase), in the liver are significantly elevated (between 1.7 and 3.1 fold) in lactating sows compared to non-lactating sows. The inflammatory state in the liver was evidenced by the finding that transcript levels of genes encoding acute phase proteins, namely haptoglobin (HP), fibrinogen γ (FGG), complement factor B (CFB), C-reactive protein (CRP) and lipopolysaccharide-binding protein (LBP), were significantly higher (2 to 8.7 fold) in lactating compared to non-lactating sows.
Conclusions
The results of the present study indicate that the Nrf2 pathway in the liver of sows is activated during lactation. The activation of Nrf2 pathway during lactation in sows might be interpreted as a physiologic means to counteract the inflammatory process and to protect the liver against damage induced by inflammatory signals and ROS.
Keywords
- Sow
- Liver
- Lactation
- Inflammation
- Nrf2 pathway
- Acute phase proteins
Findings
Typical characteristics for the lactation phase are dramatic increases in the energy and nutrient requirement of the organism, which are usually met by an elevation of food intake and a mobilisation of body’s energy stores [1, 2]. In dairy cows, lactation was also shown to induce immune and inflammatory responses in the liver [3, 4]. In contrast to extensive research in rodents and cows on the mechanisms underlying the lactation-induced metabolic and immunologic adaptations [3–6], only limited information is available in sows in this regard [7, 8]. In an attempt to improve the knowledge about metabolic and immunologic adaptations during lactation in sows we have recently analyzed the changes in the hepatic transcriptome of sows during lactation on a genome-wide level [9]. Besides the expected induction of energy-generating pathways in the liver during lactation, we found that lactation causes an induction of an inflammatory state as evidenced from the marked induction of several acute phase proteins in the liver of lactating sows [9]. Acute phase proteins are secreted from hepatocytes in response to inflammatory stimuli and are therefore established markers of inflammation in both human and veterinary clinical pathologies, which indicates that lactation induces an inflammatory state in the liver of sows, like in dairy cows [3, 4]. Interestingly, we have recently reported that the inflammatory state in the liver of dairy cows during lactation is accompanied by activation of the nuclear factor E2-related factor 2 (Nrf2) stress signalling pathway (unpublished observations). Nrf2 is a redox-sensitive transcription factor which upon activation by inflammatory stimuli but also reactive oxygen species (ROS), xenobiotics or electrophiles [10] induces the transcription of a large set of genes encoding various antioxidative proteins such as glutathione peroxidase 1 (encoded by GPX1), superoxide dismutase (encoded by SOD) and cytoprotective proteins [heme oxygenase 1 (encoded by HO-1), NAD(P)H dehydrogenase, quinone 1 (encoded by NQO1), peroxiredoxin 6 (encoded by PRDX6), thioredoxin reductase 1 (encoded by TXNRD1), glucuronosyltransferase 1 family, polypeptide A1 (encoded by UGT1A1)] and thereby protects tissues from inflammatory damage and neutralizes ROS produced under pro-inflammatory conditions [11]. In light of the observation that the lactation-induced inflammatory state in the liver of dairy cows is accompanied by activation of the Nrf2 pathway and in order to further improve knowledge about the mechanisms underlying the lactation-induced adaptations during lactation in sows, the present study aimed to investigate whether the Nrf2 pathway is activated in the liver of lactating sows.
Characteristics of primers and primer performance data used for qPCR
Gene symbol | Forward primer (from 5’ to 3’) | Product size (bp) | NCBI GenBank | Slope | R2# | Efficiency* | M |
---|---|---|---|---|---|---|---|
Reverse primer (from 5’ to 3’) | |||||||
Reference genes | |||||||
RSP9 | GTCGCAAGACTTATGTGACC | 325 | XM_003356050 | -0.28 | 0.999 | 1.91 | 0.053 |
AGCTTAAAGACCTGGGTCT | |||||||
ATP5G1 | CAGTCACCTTGAGCCGGGCA | 94 | NM_001025218 | -0.30 | 0.998 | 1.99 | 0.054 |
TAGCGCCCCGGTGGTTTGC | |||||||
GSR | AGCGCGATGCCTACGTGAGC | 175 | AY368271 | -0.29 | 0.997 | 1.94 | 0.055 |
GGTACGCCGCCTGTGGCAAT | |||||||
ACTB | GACATCCGCAAGGACCTCTA | 205 | XM_003124280 | -0.32 | 0.992 | 2.1 | 0.064 |
ACATCTGCTGGAAGGTGGAC | |||||||
SHAS2 | GAAAAGGCTAACCTACCCTG | 218 | NM_214053 | -0.21 | 0.996 | 1.65 | 0.076 |
TGTTGGACAAGACCAGTTGG | |||||||
Target genes | |||||||
HP | ACAGATGACAGCTGCCCAAA | 188 | NM_214000 | -0.30 | 0.997 | 1.99 | |
CCGCACACTGCTTCACATTC | |||||||
FGG | GACATCTGTCTCCTACTGGA | 375 | NM_001244524 | -0.29 | 0.999 | 1.95 | |
CATGACACTTGTTCATCCAC | |||||||
CFB | CTCAACGCAAAGACCGCAAA | 106 | NM_001101824 | -0.29 | 0.998 | 1.96 | |
AAATGGGCCTGATGGTCTGG | |||||||
CRP | CCTTTGTCTTCCCCAAAGAG | 563 | NM_213844 | -0.28 | 0.999 | 1.91 | |
CACCTCGCCACTCATTTCAT | |||||||
LBP | ACCGCTCCCCAGTTGGCTTC | 406 | NM_001128435 | -0.29 | 0.999 | 1.96 | |
AGCGCGGCGGACACATTAGT | |||||||
NQO1 | CCAGCAGCCCGGCCAATCTG | 160 | NM_001159613 | -0.28 | 0.997 | 1.89 | |
AGGTCCGACACGGCGACCTC | |||||||
TXNRD1 | CTTTACCTTATTGCCCGGGT | 162 | NM_214154 | -0.30 | 0.999 | 1.98 | |
GTTCACCGATTTTGTTGGCC | |||||||
UGT1A1 | GATCCTTTCCTGCAACGCAT | 313 | XM_003483776 | -0.28 | 0.996 | 1.91 | |
GGAAGGTCATGTGATCTGAG | |||||||
HO-1 | AGCTGTTTCTGAGCCTCCAA | 130 | NM_001004027 | -0.30 | 0.998 | 1.98 | |
CAAGACGGAAACACGAGACA | |||||||
PRDX6 | GGCCGCATCCGTTTCCACGA | 280 | NM_214408 | -0.29 | 0.998 | 1.95 | |
ACTGGATGGCAAGGTCCCGACT | |||||||
SOD | TCCATGTCCATCAGTTTGGA | 250 | NM_001190422 | -0.27 | 0.998 | 1.88 | |
CTGCCCAAGTCATCTGGTTT | |||||||
GPX1 | GGCACAACGGTGCGGGACTA AGGCGAAGAGCGGGTGAGCA | 235 | NM_214201 | -0.29 | 0.998 | 1.96 |
Relative transcript levels of Nrf2 target genes in the liver of lactating and non-lactating sows on day 20 of lactation
Non-lactating (n = 10) | Lactating (n = 10) | P value | |
---|---|---|---|
UGT1A1 | 1 ± 0.52 | 1.79 ± 0.59* | 0.013 |
HO-1 | 1 ± 0.48 | 1.71 ± 0.42* | 0.007 |
NQO1 | 1 ± 0.49 | 2.91 ± 1.54* | 0.007 |
GPX1 | 1 ± 0.56 | 3.11 ± 1.19* | 0.001 |
PRDX6 | 1 ± 0.56 | 2.12 ± 0.78* | 0.008 |
TXNRD1 | 1 ± 0.28 | 2.99 ± 2.45* | 0.029 |
SOD | 1 ± 0.65 | 2.28 ± 1.01* | 0.007 |
MT1A | 1 ± 0.37 | 1.64 ± 1.26 | 0.190 |
Relative transcript levels of genes encoding acute phase proteins in the liver of lactating and non-lactating sows on day 20 of lactation
Non-lactating (n = 10) | Lactating (n = 10) | P value | |
---|---|---|---|
HP | 1 ± 0.89 | 2.58 ± 1.08* | 0.005 |
FGG | 1 ± 0.55 | 2.90 ± 0.66* | 0.001 |
CFB | 1 ± 0.52 | 2.01 ± 0.36* | 0.001 |
CRP | 1 ± 0.68 | 8.68 ± 4.20* | 0.001 |
LBP | 1 ± 0.71 | 5.82 ± 1.94* | 0.001 |
Activation of stress signalling pathways in the liver of sows during lactation. Activation of Nrf2 and NF-κB during lactation is mediated by various stimuli including reactive oxygen species (ROS), bacterial lipopolysaccharide (LPS) and cytokines, which are known activators of both, Nrf2 and NF-κB. Activation of Nrf2 leads to up-regulation of classical Nrf2 target genes such as glutathione peroxidase 1 (encoded by GPX1), superoxide dismutase (encoded by SOD) and cytoprotective proteins [heme oxygenase 1 (encoded by HO-1), NAD(P)H dehydrogenase, quinone 1 (encoded by NQO1), peroxiredoxin 6 (encoded by PRDX6), thioredoxin reductase 1 (encoded by TXNRD1), glucuronosyltransferase 1 family, and polypeptide A1 (encoded by UGT1A1)]. Acivation of NF-κB results in the induction of acute phase proteins such as haptoglobin (encoded by HP), fibrinogen γ (encoded by FGG), complement factor B (encoded by CFB), C-reactive protein (encoded by CRP) and lipopolysaccharide-binding protein (encoded by LBP). Activation of stress signalling pathways during lactation in sows might be interpreted as a physiologic means to counteract the inflammatory process and to protect the liver against deleterious effects of inflammatory signals and ROS, which are released at elevated levels as a consequence of the metabolic and immunologic adaptations occurring during the transition from pregnancy to lactation.
Notes
Declarations
Acknowledgements
This study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft; Grant no. ED 70/9-1).
Authors’ Affiliations
References
- Trayhurn P, Douglas JB, McGuckin MM: Brown adipose tissue thermogenesis is 'suppressed' during lactation in mice. Nature. 1982, 298: 59-60. 10.1038/298059a0.View ArticlePubMedGoogle Scholar
- Williamson DH: Regulation of metabolism during lactation in the rat. Reprod Nutr Dev. 1986, 26: 597-603. 10.1051/rnd:19860409.View ArticlePubMedGoogle Scholar
- Loor JJ, Dann HM, Everts RE, Oliveira R, Green CA, Guretzky NA, Rodriguez-Zas SL, Lewin HA, Drackley JK: Temporal gene expression profiling of liver from periparturient dairy cows reveals complex adaptive mechanisms in hepatic function. Physiol Genomics. 2005, 23: 217-226. 10.1152/physiolgenomics.00132.2005.View ArticlePubMedGoogle Scholar
- Loor JJ, Dann HM, Guretzky NA, Everts RE, Oliveira R, Green CA, Litherland NB, Rodriguez-Zas SL, Lewin HA, Drackley JK: Plane of nutrition prepartum alters hepatic gene expression and function in dairy cows as assessed by longitudinal transcript and metabolic profiling. Physiol Genomics. 2006, 27: 29-41. 10.1152/physiolgenomics.00036.2006.View ArticlePubMedGoogle Scholar
- Gutgesell A, Ringseis R, Schmidt E, Brandsch C, Stangl GI, Eder K: Downregulation of peroxisome proliferator-activated receptor α and its coactivators in liver and skeletal muscle mediates the metabolic adaptations during lactation in mice. J Mol Endocrinol. 2009, 43: 241-250. 10.1677/JME-09-0064.View ArticlePubMedGoogle Scholar
- Bionaz M, Loor JJ: Gene networks driving bovine mammary protein synthesis during the lactation cycle. Bioinform Biol Insights. 2011, 5: 83-98.PubMed CentralPubMedGoogle Scholar
- Ringseis R, Heller K, Kluge H, Eder K: mRNA expression of genes involved in fatty acid utilization in skeletal muscle and white adipose tissues of sows during lactation. Comp Biochem Physiol A Mol Integr Physiol. 2011, 158: 450-454. 10.1016/j.cbpa.2010.12.007.View ArticlePubMedGoogle Scholar
- Theil PK, Labouriau R, Sejrsen K, Thomsen B, Sørensen MT: Expression of genes involved in regulation of cell turnover during milk stasis and lactation rescue in sow mammary glands. J Anim Sci. 2005, 83: 2349-2356.PubMedGoogle Scholar
- Rosenbaum S, Ringseis R, Hillen S, Lange S, Erhardt G, Reiner G, Eder K: Genome-wide transcript profiling indicates induction of energy-generating pathways and an adaptive immune response in the liver of sows during lactation. Comp Biochem Physiol D Genomics Prot. 2012, doi: http://dx.doi.org/10.1016/j.cbd.2012.09.001Google Scholar
- Kaspar JW, Niture SK, Jaiswal AK: Nrf2:INrf2 (Keap1) signaling in oxidative stress. Free Radic Biol Med. 2009, 47: 1304-1309. 10.1016/j.freeradbiomed.2009.07.035.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim J, Cha YN, Surh YJ: A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders. Mutat Res. 2010, 690: 12-23. 10.1016/j.mrfmmm.2009.09.007.View ArticlePubMedGoogle Scholar
- Keller J, Ringseis R, Koc A, Lukas I, Kluge H, Eder K: Supplementation with L-carnitine downregulates genes of the ubiquitin proteasome system in the skeletal muscle and liver of piglets. Animal. 2012, 6: 70-78. 10.1017/S1751731111001327.View ArticlePubMedGoogle Scholar
- Sidwell CG, Salwin H, Benca M, Mitchell JH: The use of thiobarbituric acid as a measure of fat oxidation. J Am Oil Chem Soc. 1954, 31: 603-606. 10.1007/BF02545589.View ArticleGoogle Scholar
- Jung KA, Kwak MK: The Nrf2 system as a potential target for the development of indirect antioxidants. Molecules. 2010, 15: 7266-7291. 10.3390/molecules15107266.View ArticlePubMedGoogle Scholar
- Morris DG, Waters SM, McCarthy SD, Patton J, Earley B, Fitzpatrick R, Murphy JJ, Diskin MG, Kenny DA, Brass A, Wathes DC: Pleiotropic effects of negative energy balance in the postpartum dairy cow on splenic gene expression: repercussions for innate and adaptive immunity. Physiol Genomics. 2009, 39: 28-37. 10.1152/physiolgenomics.90394.2008.PubMed CentralView ArticlePubMedGoogle Scholar
- Wagner AE, Ernst I, Iori R, Desel C, Rimbach G: Sulforaphane but not ascorbigen, indole-3-carbinole and ascorbic acid activates the transcription factor Nrf2 and induces phase-2 and antioxidant enzymes in human keratinocytes in culture. Exp Dermatol. 2009, 19: 137-144.View ArticlePubMedGoogle Scholar
- Ernst IM, Wagner AE, Schuemann C, Storm N, Höppner W, Döring F, Stocker A, Rimbach G: Allyl-, butyl- and phenylethyl-isothiocyanate activate Nrf2 in cultured fibroblasts. Pharmacol Res. 2011, 63: 233-240. 10.1016/j.phrs.2010.11.005.View ArticlePubMedGoogle Scholar
- Elmore RG, Martin CE, Berg JN: Absorption of Escherichia coli endotoxin from the mammary glands and uteri of early post-partum sows and gilts. Theriogenology. 1978, 10: 439-445. 10.1016/0093-691X(78)90133-4.View ArticleGoogle Scholar
Copyright
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.