Effect of endoplasmic reticulum stress on metabolic and stress signaling and kidney-specific functions in Madin-Darby bovine kidney cells.

Recent studies demonstrated induction of endoplasmic reticulum (ER) stress in tissues of cows after parturition, but knowledge about the effect of ER stress on important cellular processes, such as critical signaling and metabolic pathways, in cattle is scarce. Thus, the present study aimed to investigate the effect of ER stress induction on nuclear factor-κB (NF-κB), nuclear factor E2-related factor 2 (Nrf2), and sterol regulatory element-binding protein (SREBF1) pathway in Madin-Darby bovine kidney (MDBK) cells, a widely used in vitro model in ruminant research. To consider the kidney origin of MDBK cells, the effect on renal distal tubular cell-specific functions, such as transport processes and regulation of 1,25(OH)2D3 levels, was also studied. Treatment of MDBK cells with 2 different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), strongly induced ER stress as evident from induction of ER stress target genes, increased phosphorylation of PKR-like ER kinase, and enhanced splicing of X-box binding protein 1. The TM decreased the protein concentration of NF-κB p50 and the mRNA levels of the NF-κB target genes. Likewise, TG decreased the mRNA concentration of tumor necrosis factor and tended to decrease NF-κB p50 protein and mRNA levels of NF-κB target genes. The mRNA levels of most of the Nrf2 target genes investigated were reduced by TG and TM in MDBK cells. Both ER stress inducers reduced the mRNA levels of SREBF1 and its target genes in MDBK cells. Interestingly, TG decreased, but TM increased the mRNA level of the Ca2+ binding protein calbindin 1, whereas the mRNA level of the plasma membrane Ca2+-transporting ATPase 1 remained unchained. The mRNA level of the cytochrome P450 component 24A1 involved in 1α-hydroxylation of 25(OH)D3 was strongly elevated, whereas the mRNA level of the cytochrome P450 component 27A1 catalyzing the breakdown of 1,25(OH)2D3 was markedly reduced by both ER stress inducers. The concentration of 1,25(OH)2D3 in the supernatant of MDBK cells was increased by approximately 15% by both TG and TM. The present study indicates that under conditions of ER stress, critical signaling pathways, such as NF-κB, Nrf2, and SREBF1, are inhibited, whereas the formation of 1,25(OH)2D3 is stimulated in bovine MDBK cells. Future studies are necessary to clarify the physiological relevance of these findings.

Effects of body condition, monensin, and essential oils on ruminal lipopolysaccharide concentration, inflammatory markers, and endoplasmatic reticulum stress of transition dairy cows.

Evidence exists that dairy cows experience inflammatory-like phenomena in the transition period. Rumen health and alterations in metabolic processes and gene networks in the liver as the central metabolic organ might be key factors for cows' health and productivity in early lactation. This study made use of an animal model to generate experimental groups with different manifestations of postpartal fat mobilization and ketogenesis. In total, 60 German Holstein cows were allocated 6 wk antepartum to 3 high-body condition score (BCS) groups (BCS 3.95) and 1 low-BCS group (LC; BCS 2.77). High-BCS cows were fed an antepartal forage-to-concentrate ratio of 40:60 on dry matter basis, in contrast to 80:20 in the LC group, and received a monensin controlled-release capsule (HC/MO), a blend of essential oils (HC/EO), or formed a control group (HC). We evaluated serum haptoglobin, kynurenine, tryptophan, ruminal lipopolysaccharide concentration and mRNA abundance of nuclear factor kappa B (NF-κB), nuclear factor E2-related factor 2 (Nrf2), and endoplasmatic reticulum stress-induced unfolded protein response (UPR) target genes in liver biopsy samples from d -42 until +56 relative to calving. Nearly all parameters were highly dependent on time, with greatest variation near calving. The ruminal lipopolysaccharide concentration and evaluated target genes were not generally influenced by antepartal BCS and feeding management. The kynurenine-to-tryptophan ratio was higher in LC than in HC/MO treatment on d 7. Ruminal lipopolysaccharide concentration was higher in HC/MO than in the HC group, but not increased in HC/EO group. Abundance of UPR target gene X-box binding protein 1 was higher in HC/MO than in HC/EO group on d 7. Hepatic mRNA abundance of Nrf2 target gene glutathione peroxidase 3 was higher, whereas expression of NF-κB target gene haptoglobin tended to be higher in LC than in HC/EO cows. The HC/MO cows showed the most prominent increase in the abundance of glutathione peroxidase 3 and haptoglobin after calving in comparison to antepartal values. Results indicate the presence of inflammatory-like phenomena near calving. Simultaneously, alterations in UPR and Nrf2 target genes with antioxidative properties and haptoglobin occurred, being most prominent in LC and HC/MO group.

Effects of polyphenol-rich plant products from grape or hop as feed supplements on the expression of inflammatory, antioxidative, cytoprotective and endoplasmic reticulum stress-related genes and the antioxidative status in the liver of piglets.

Recent studies have shown that supplementation of plant products rich in polyphenols exerts anti-inflammatory effects in the small intestine and improves feed conversion in piglets. This study aimed to investigate whether dietary polyphenols have also anti-inflammatory and cytoprotective effects in the liver of piglets. For this end, relative mRNA concentrations of eight genes involved in proinflammatory pathways, eight genes involved in the antioxidative and cytoprotective system, six genes of phase I and phase II metabolism and 15 genes of the unfolded protein response (triggered by stress of the endoplasmic reticulum) in the liver of pigs fed diets supplemented with either 1% of grape seed and grape marc meal extract (GME) or 1% spent hops (SH) as sources of polyphenols were determined. Relative mRNA concentrations of almost all these genes, with few exceptions, in the liver of pigs supplemented with GME or SH did not differ from those in the liver of control piglets. Gene expression data were validated by consideration of concentrations of some selected proteins of these pathways which also did not differ between piglets supplemented with GME or SH and control piglets. Moreover, concentrations of thiobarbituric acid-reactive substances and tocopherols as well as the total antioxidant capacity in liver and plasma did not differ between pigs supplemented with either GME or SH and control piglets. Overall, this study shows that supplementation of GME or SH as sources of polyphenols does not influence hepatic pathways linked to inflammation, the antioxidant and cytoprotective system, stress of the endoplasmic reticulum and the xenobiotic system in healthy piglets.

Potential of plant polyphenols to combat oxidative stress and inflammatory processes in farm animals.

Polyphenols are secondary plant metabolites which have been shown to exert antioxidative and antiinflamma tory effects in cell culture, rodent and human studies. Based on the fact that conditions of oxidative stress and inflammation are highly relevant in farm animals, polyphenols are considered as promising feed additives in the nutrition of farm animals. However, in contrast to many studies existing with model animals and humans, potential antioxidative and antiinflammatory effects of polyphenols have been less investigated in farm animals so far. This review aims to give an overview about potential antioxidative and antiinflammatory effects in farm animals. The first part of the review highlights the occurrence and the consequences of oxidative stress and inflammation on animal health and performance. The second part of the review deals with bioavailability and metabolism of polyphenols in farm animals. The third and main part of the review presents an overview of the findings from studies which investigated the effects of polyphenols of various plant sources in pigs, poultry and cattle, with particular consideration of effects on the antioxidant system and inflammation.

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells.

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the mutated SRE at -38 of the NIS 5'-flanking region showed that in vitro-translated nSREBP-1c and nSREBP-2 bind only the wild-type but not the mutated SRE at -38 of NIS. Collectively, the present results from cell culture experiments with human mammary epithelial MCF-7 cells and from genetic studies show for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells. Future studies are necessary to clarify if the regulation of NIS expression and iodide uptake by SREBP also applies to the lactating bovine mammary epithelium.

Efficacy of DL-methionine hydroxy analogue-free acid in comparison to DL-methionine in growing male white Pekin ducks.

The present study was performed to assess the bioefficacy of DL-methionine hydroxy analogue-free acid (MHA) in comparison to DL-methionine (DLM) as sources of methionine for growing male white Pekin ducks in the first 3 wk of life. For this aim, 580 1-day-old male ducks were allocated into 12 treatment groups and received a basal diet that contained 0.29% of methionine, 0.34% of cysteine and 0.63% of total sulphur containing amino acids or the same diet supplemented with either DLM or MHA in amounts to supply 0.05, 0.10, 0.15, 0.20, and 0.25% of methionine equivalents. Ducks fed the control diet without methionine supplement had the lowest final body weights, daily body weight gains and feed intake among all groups. Supplementation of methionine improved final body weights and daily body weight gains in a dose dependent-manner. There was, however, no significant effect of the source of methionine on all of the performance responses. Evaluation of the data of daily body weight gains with an exponential model of regression revealed a nearly identical efficacy (slope of the curves) of both compounds for growth (DLM = 100%, MHA = 101%). According to the exponential model of regression, 95% of the maximum values of daily body weight gain were reached at methionine supplementary levels of 0.080% and 0.079% for DLM and MHA, respectively. Overall, the present study indicates that MHA and DLM have a similar efficacy as sources of methionine for growing ducks. It is moreover shown that dietary methionine concentrations of 0.37% are required to reach 95% of the maximum of daily body weight gains in ducks during the first 3 wk of life.

Effect of dietary fish oil on the expression of genes involved in lipid metabolism in liver and skeletal muscle of lactating sows.

This study investigated the hypothesis that dietary supplementation of fish oil as a source of n-3 polyunsaturated fatty acids (PUFA) influences the expression of target genes of sterol regulatory element-binding proteins (SREBP)-1 and (SREBP)-2 involved in triacylglycerol (TAG) synthesis and fatty acid and cholesterol metabolism in the liver, and moreover activates the expression of target genes of peroxisome proliferation-activated receptor (PPAR)-α involved in TAG and fatty acid catabolism in liver and skeletal muscle. Twenty lactating sows were fed a control diet or a fish oil diet with either 50 g of a mixture of palm oil and soya bean oil (4:1, w/w) or fish oil per kg. The diet of the fish oil group contained 19.1 g of n-3 PUFA (mainly 20:5 n-3 and 22:6 n-3) per 100 g of total fatty acids, while the diet of the control group contained 2.4 g of n-3 PUFA (mainly 18:3 n-3) per 100 g of total fatty acids. The fish oil group had reduced relative mRNA concentrations of various target genes of SREBP-1 involved in fatty acid and TAG synthesis in comparison with the control group (p < 0.05). Relative mRNA concentrations of target genes of PPARα involved in fatty acid catabolism in both liver and muscle, and mRNA concentrations of target genes of SREBP-2 involved in cholesterol synthesis and uptake were not influenced by fish oil supplementation. Concentrations of cholesterol and TAG in plasma, fat content of milk and weight gains of litters during the suckling period were not different between the two groups of sows. In conclusion, this study suggests that fish oil has only minor effects on hepatic lipid metabolism, which are non-critical with respect to milk production in sows.

The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation.

During the periparturient phase, cows are typically in an inflammation-like condition, and it has been suggested that inflammation associated with the development of stress of the endoplasmic reticulum (ER) in the liver contributes to the development of fatty liver syndrome and ketosis. In the present study, we investigated the hypothesis that feeding grape seed and grape marc meal extract (GSGME) as a plant extract rich in flavonoids attenuates inflammation and ER stress in the liver of dairy cows. Two groups of cows received either a total mixed ration as a control diet or the same total mixed ration supplemented with 1% of GSGME over the period from wk 3 prepartum to wk 9 postpartum. Dry matter intake during wk 3 to 9 postpartum was not different between the 2 groups. However, the cows fed the diet supplemented with GSGME had an increased milk yield and an increased daily milk protein yield. Cows supplemented with GSGME moreover had a significantly reduced mRNA abundancy of fibroblast growth factor (FGF) 21, a stress hormone induced by various stress conditions, in the liver in wk 1 and 3 postpartum. In contrast, mRNA abundances of a total of 3 genes involved in inflammation and 14 genes involved in ER stress response, as well as concentrations of triacylglycerols and cholesterol, in liver samples of wk 1 and 3 postpartum did not differ between the 2 groups. Overall, this study shows that supplementation of GSGME did not influence inflammation or ER stress in the liver but increased milk yield, an effect that could be due to effects on ruminal metabolism.

Concentrations of retinol and tocopherols in the milk of cows supplemented with conjugated linoleic acid.

This study was performed to investigate the hypothesis that supplementation of conjugated linoleic acid (CLA) changes the concentrations of retinol and tocopherols in the milk of cows. To investigate this hypothesis, Holstein cows received daily from 3 weeks ante-partum to 14 weeks post-partum either 172 g of a CLA-free rumen-protected control fat (control group, n = 20) or the same amount of a rumen-protected CLA fat, supplying 4.3 g of cis-9, trans-11 CLA and 3.8 g of trans-10, cis-12 CLA per d (CLA group, n = 20). Milk samples (collected at weeks 1, 3, 5, 8 and 11 of lactation) were analysed for retinol, α- and γ-tocopherol concentrations. Milk of cows supplemented with CLA had higher concentrations of retinol (+34%), α-tocopherol (+44%) and γ-tocopherol (+21%) than milk of control cows (p < 0.05). The daily output of these vitamins via milk was also greater in cows of the CLA group than in cows of the control group (+36, 50 and 24% for retinol, α-tocopherol and γ-tocopherol, respectively, p < 0.05). In agreement with higher concentrations of tocopherols, concentrations of thiobarbituric acid-reactive substances, determined in milk of week 5, were lower in cows of the CLA group than in control cows, indicative of a lower susceptibility of milk lipids to peroxidation. Plasma concentrations of retinol and α-tocopherol, determined at 1 and 5 weeks post-partum, were not different between the two groups of cows. In conclusion, this study shows that supplementing dairy cows with a moderate amount of CLA causes an increase of the concentrations of vitamins A and E in the milk and results in an increased output of those vitamins via milk. These effects might be beneficial with respect to the nutritional value of dairy products and the susceptibility of milk fat to oxidative deterioration.

Molecular insights into the mechanisms of liver-associated diseases in early-lactating dairy cows: hypothetical role of endoplasmic reticulum stress.

The transition period represents the most critical period in the productive life of high-yielding dairy cows due to both metabolic and inflammatory stimuli, which challenge the liver and predispose dairy cows to develop liver-associated diseases such as fatty liver and ketosis. Despite the fact that all high-yielding dairy cows are affected by marked metabolic stress due to a severe negative energy balance (NEB) during early lactation, not all cows develop liver-associated diseases. Although the reason for this is largely unknown, this indicates that the capacity of the liver to cope with metabolic and inflammatory challenges varies between individual high-yielding dairy cows. Convincing evidence exists that endoplasmic reticulum (ER) stress plays a key role in the development of fatty liver, and it has been recently shown that ER stress occurs in the liver of high-yielding dairy cows. This indicates that ER stress may be involved in the development of liver-associated diseases in dairy cows. The present review shows that the liver of dairy cows during early lactation is exposed to several metabolic and inflammatory challenges, such as non-esterified fatty acids, tumour necrosis factor α, interleukin-1ß, reactive oxygen species and lipopolysaccharides, which are known inducers of ER stress. Thus, ER stress may represent a molecular basis for fatty liver development and account for the frequent occurrence of fatty liver and ketosis in high-yielding dairy cows. Interindividual differences between dairy cows in the activation of hepatic stress response pathways, such as nuclear factor E2-related factor 2, which is activated during ER stress and reduces the sensitivity of tissues to oxidative and inflammatory damage, might provide an explanation at the molecular level for differences in the capacity to cope with pathological inflammatory challenges during early lactation and the susceptibility to develop liver-associated diseases between early-lactating dairy cows with similar NEB and milk yield.

Expression of target genes of nuclear factor E2-related factor 2 in the liver of dairy cows in the transition period and at different stages of lactation.

In the liver of dairy cows, the production of cytokines is enhanced during the periparturient phase, which in turn leads to inflammation and an impairment of hepatic function. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that controls the transcription of genes encoding various antioxidative and cytoprotective proteins. In the present study, we investigated the hypothesis that Nrf2 is activated in the liver of dairy cows during the periparturient phase to protect the liver against the deleterious effects of cytokines and reactive oxygen species. Therefore, we determined relative mRNA abundances of TNF (encoding tumor necrosis factor-α), various acute phase proteins and several Nrf2 target genes in liver biopsy samples of 20 dairy cows at each time point from 3 wk antepartum to 1, 5, and 14 wk postpartum. We observed an increase in mRNA abundances of TNF and acute-phase proteins [serum amyloid A 3 (SAA3), haptoglobin (HP), and C-reactive protein (CRP)] from 3 wk antepartum to 1 wk postpartum, indicative of a proinflammatory condition. Messenger RNA abundances of various Nrf2 target genes with antioxidative or cytoprotective functions [glutathione peroxidase 3 (GPX3); microsomal glutathione S-transferase 3 (MGST3); superoxide dismutase (SOD1); catalase (CAT); metallothioneins 1A, 1E, and 2A (MT1A, MT1E, and MT2A, respectively); NAD(P)H dehydrogenase, quinone 1 (NQO1); heme oxygenase 2 (HMOX2); and UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1)] were also greatly increased from 3 wk antepartum to 1 wk postpartum. From 1 wk postpartum to later lactation, mRNA abundances of all the Nrf2-target genes considered declined but remained at levels that were higher than those in 3 wk antepartum. No correlations were found, however, between plasma concentrations of nonesterified fatty acids or ß-hydroxybutyrate and mRNA abundances of Nrf2 target genes, indicating that a negative energy balance might not have been the main factor responsible for upregulation of those genes in the liver during early lactation. In conclusion, this study provides additional evidence that the periparturient phase in dairy cows is associated with a proinflammatory condition in the liver. Moreover, it is shown for the first time that the transition from pregnancy to lactation leads to a strong upregulation of Nrf2 target genes with antioxidative or cytoprotective properties, which might be another physiologic means to prevent the liver against damage by the inflammation process and an increased generation of reactive oxygen species.

Inhibition of the pro-inflammatory NF-κB pathway by a grape seed and grape marc meal extract in intestinal epithelial cells.

In pigs and other monogastric animal, the weaning phase is commonly accompanied by an increased susceptibility to gut disorders such as diarrhoea owing to the induction of an inflammatory process in the intestine during weaning. Given the unfavourable effects of intestinal inflammation on feed consumption, digestive capacity of the intestine and growth of animals, controlling intestinal inflammation is a reasonable approach for the maintenance of performance characteristics of livestock animals. Therefore, this study aimed to study the anti-inflammatory potential of a commercial polyphenol-rich grape seed (GS) and grape marc (GM) meal-based feed additive in a well-established in vitro intestinal epithelium model (polarized Caco-2 cells). The anti-inflammatory potential was evaluated by studying the effect of an ethanolic extract obtained from the GS and GM meal-based feed additive (GSGME) on the pro-inflammatory transcription factor NF-κB, which is considered to play a key role in the induction of weaning-associated intestinal inflammation. The highest non-cytotoxic concentrations of the ethanolic GSGME dose dependently reduced TNFα-induced NF-κB transactivation and decreased TNFα-induced mRNA levels of the NF-κB target genes IL-1ß, IL-8, MCP-1 and CXCL1 in Caco-2 intestinal cells (p < 0.05). No effect of the ethanolic GSGME was observed on the cytoprotective Nrf2 pathway in Caco-2 cells as evidenced by an unaltered Nrf2 transactivation and unchanged mRNA levels of Nrf2 target genes, such as GPX-2, NQO1, CYP1A1 and UGT1A1. In conclusion, this study shows that an ethanolic GSGME exerts anti-inflammatory effects in intestinal cells under in vitro conditions. Thus, polyphenol-rich GSGM meal-based feed additives may be useful for the inhibition or prevention of inflammatory processes in the intestine of livestock animals, in particular during states with inappropriate NF-κB activation in the intestinal tissue, such as the weaning phase. Future studies are warranted to prove the in vivo anti-inflammatory potential of GSGM meal-based feed additives.