Comparative effects of dietary methionine and cysteine supplementation on redox status and intestinal integrity in immunologically challenged-weaned pigs

Sulfur-containing amino acids such as methionine and cysteine play critical roles in immune system and redox status. A body of evidence shows that metabolic aspects of supplemented Met and Cys may differ in the body. Therefore, the study aimed to investigate the effects of dietary Met and Cys supplementation in immunologically challenged weaned pigs. Forty weaned piglets (6.5 ± 0.3 kg) were randomly allocated to five treatment groups. The treatment included: (1) sham-challenged control (SCC), (2) challenged control (CC), (3) MET (CC + 0.1% DL-Met), (4) CYS (CC + 0.1% L-Cys), and (5) MET + CYS (CC + 0.1% DL-Met + 0.1% L-Cys). On day 7, all pigs were intramuscularly injected with either Escherichia coli O55:B5 lipopolysaccharides (LPS) or phosphate-buffered saline. Blood, liver, and jejunum samples were analyzed for immune response and redox status. The CC group had lower (P < 0.05) villus surface area and higher (P < 0.05) flux of 4-kDa fluorescein isothiocyanate dextran (FD4) than the SCC group. A lower (P < 0.05) glutathione (GSH) concentration was observed in the jejunum of pigs in the CC group than those in the SCC group. Dietary Cys supplementation increased (P < 0.05) villus surface area, GSH levels, and reduced (P < 0.05) the flux of FD4 in the jejunum of LPS-challenged pigs. Dietary Met supplementation enhanced (P < 0.05) hepatic GSH content. Pigs challenged with LPS in the MET group had lower serum IL-8 concentration than those in the CC group. There was a Met × Cys interaction (P < 0.05) in serum IL-4 and IL-8 concentrations, and Trolox equivalent antioxidant capacity. Dietary L-Cys supplementation restored intestinal integrity and GSH levels that were damaged by lipopolysaccharides administration. Dietary DL-Met supplementation improved hepatic GSH and reduced systemic inflammatory response, but antagonistic interaction with dietary L-Cys supplementation was observed in the inflammatory response and redox status.


Introduction
Methionine is a nutritionally indispensable amino acid in animals (Elango 2020). Apart from being a building block of proteins, Met possesses biological significance with the presence of methyl groups and sulfur atoms (Stipanuk 2004). Methionine undergoes the transmethylation pathway, becoming S-adenosylmethionine (SAM) that serves as a methyl donor for numerous metabolic processes including the synthesis of DNA and RNA and histone methylations (Stipanuk 2004). The SAM is also a substrate for polyamines, which are associated with the innate immune response and proliferation of intestinal epithelial cells (Ding et al. 2015;Martínez-López et al. 2008). Methionine serves as a precursor of Cys by transferring its sulfur to serine via the transsulfuration pathway (Stipanuk 2004). Cysteine is a rate-limiting substrate for the syntheses of glutathione (GSH) and taurine, which play pivotal roles in cellular redox status and osmoregulation . The catabolism of Cys can release H 2 S, a gasotransmitter that modulates the immune system (Magierowski et al. 2015).
The liver is the major organ for sulfur amino acid (SAA) metabolism in the body, with high activities of enzymes involved in transmethylation and transsulfuration pathways (Stipanuk 2004). The small intestine also expresses the enzymes to a lesser extent but plays a significant role in whole body SAA metabolism (Riedijk et al. 2007). Isotopic tracer research indicated that 20% and 25% of dietary Met and Cys were metabolized by the intestine, respectively (Riedijk et al. 2007;Bauchart-Thevret et al. 2011). This reflects the high demands for methyl donors, GSH, and Cys-rich protein (e.g., mucins and defensins) by the intestinal epithelial cells in response to exogenous toxins and pathogens (Bauchart-Thevret et al. 2009a;Rakhshandeh et al. 2020). During the acute phase of inflammation, SAA metabolism increases mainly because of the increased demand for acute-phase proteins and GSH (Rakhshandeh et al. 2019(Rakhshandeh et al. , 2020. Dietary SAA for protein deposition (growth) is redirected toward mounting an immune response, suggesting greater dietary SAA requirement for optimal growth (Litvak et al. 2013;Rakhshandeh et al. 2014). In this regard, dietary SAA supplementation could be a nutritional strategy to maintain the redox status and immune system (Shen et al. 2014;Su et al. 2018;Song et al. 2016), while minimizing body protein mobilization.
Dietary SAA content can be simply elevated by supplementing synthetic Met or Cys. However, a body of evidence shows that metabolic aspects of supplemented Met and Cys may differ in the body. The Cys for intestinal metabolism is mainly sourced from lumen absorption, showing no extraction from the arterial blood (Rémond et al. 2011). By contrast, Riedijk et al. (2007) reported that the intestine did not utilize first-pass Met, but did rely on arterial Met supply. Furthermore, an isotope tracer study (Bauchart-Thevret et al. 2009b) found that transmethylation and transsulfuration rates are suppressed under SAAdeficient conditions in pigs, suggesting that methionine is preserved for hepatic protein syntheses. In this context, it is postulated that the effects of supplemented Met and Cys on redox status and inflammation may differ in a tissue-and immune status-dependent manner. However, no studies have investigated comparative effects with respect to dietary Met and Cys supplementation. Thus, the objective of our study was to investigate the effects of dietary Met and Cys supplementation on systemic and intestinal redox status and inflammation in weaned pigs challenged with LPS.

Animals, experimental design, and diets
Forty female piglets (TN 70 × TN Tempo; Topigs Norsvin) with an initial body weight of 6.5 ± 0.3 kg were obtained from Glenlea Research Station at the University of Manitoba. Pigs were individually housed in pens and randomly allocated based on their body weight to five treatment groups. The treatments were: (1) sham-challenged control (SCC), (2) challenged control (CC), (3) CC + 0.1% DL -Met (MET), (4) CC + 0.1% L -Cys (CYS), and (5) CC + 0.1% DL -Met + 0.1% L -Cys (MET + CYS). One batch of basal diet was formulated to meet the NRC (National Research Council. Nutrient Requirements of Swine. 11th Revised Edition. Washington, DC: National Academies Press; 2012) requirement for standardized ileal digestible Met but contained 0.1% less than the requirement for SAA (Table 1). The levels of all other nutrients in the basal diet were equal to or over the NRC (National Research Council. Nutrient Requirements of Swine. 11th Revised Edition. Washington, DC: National Academies Press; 2012) requirement for 8 kg of body weight with 14.3 MJ/kg of metabolizable energy. Either 0.1% of DL -Met (MetAMINO, Evonik Nutrition & Care GmbH) or L -Cys (Sigma-Aldrich) was supplemented to the basal diet to make the MET and CYS diets, respectively, which supplied 100% of the NRC requirement for standardized ileal digestible SAA. The MET + CYS diet was prepared by supplementing the basal diet with 0.1% of DL -Met and 0.1% of L -Cys simultaneously.

Experimental procedure, sampling, and measurements
Piglets were allowed a 7-day adaptation period to their respective diets; thereafter, a single dose of Escherichia coli O55:B5 LPS (300 μg/mL; Sigma-Aldrich) was administered intramuscularly at 0.1 mL/kg body weight to all pigs in the CC, Met, Cys, and Met + Cys groups, whereas pigs in the SCC group were intramuscularly injected with an equal volume of sterilized phosphate buffered saline (PBS). All pigs and feeders were weighed on days 7 and 9 to calculate body weight gain and feed intake. Blood samples (5 mL) from the jugular veins of all pigs were collected into two vacutainer tubes to obtain whole blood (BD Vacutainer® spraycoated K2DETA Tubes) and serum (BD Vacutainer® Plus Plastic Serum Tubes) on days 6 (24 h pre-inoculation), 7 (5 h post-inoculation), and 8 (24 h post-inoculation). Rectal temperature was determined before each time of blood collection. On day 9, all pigs were euthanized by captive bolt following stresnil-xylazine (2:4 mg/kg) sedation to allow for tissue collection. A sample of jejunum was taken 2 m away from the ileocecal junction. The jejunum sample was rinsed with cold PBS. The sample for gene expression and protein analysis was immediately snap-frozen in liquid nitrogen, and then stored in a − 80 °C freezer. Another subsample of jejunum was immediately immersed in Krebs Ringer buffer and then immediately transported to the laboratory for ex vivo Ussing chamber analyses. Jejunum samples for histomorphology were immediately stored in 10% buffered formalin to fix the villi, crypts, and goblet cells. Samples for hepatic gene expression and protein analysis were collected from the middle of the right lobe of the liver. After sampling, the content of the gastrointestinal tract was removed and weighed to calculate the empty body weight. Jejunum and liver samples stored at − 80 °C were immersed in liquid nitrogen in a mortar and ground with a pestle for subsequent analyses for mRNA abundance and protein assay.

White blood cell profiles and serum cytokine
The whole blood samples were analyzed for total white blood cell (WBC) counts using an automatic blood analyzer (Advia 2120i). The proportions of WBC profiles were determined from the stained blood smears based on WBC morphology using light microscopy at 100× magnification. The concentrations of each WBC were calculated based on the proportion and total WBC concentrations.

RNA isolation, complementary DNA synthesis, and quantitative real-time PCR
A TRIzol Plus RNA purification kit (Invitrogen Canada Inc.) was used to isolate RNA from 80 mg of the ground jejunum. The quantity and quality of the isolated RNA were evaluated using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific). The integrity of the total RNA was confirmed by agarose gel electrophoresis. A total of 2 μg of total RNA was used to synthesize cDNA using a high-capacity cDNA synthesis kit (Applied Biosystems) following the supplier's protocol. Quantitative real-time PCR was performed in duplicate reactions, using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories), as described by Waititu et al. (2017). For qPCR amplification, negative controls were prepared by replacing the cDNA with nuclease-free water. A melt Table 1 Composition of experimental diets, as-fed basis (g/kg) 1 Basal diet was supplemented with either 0.1% DL -methionine or L -cysteine at the expense of cornstarch for Met and Cys diets, respectively. 0.1% of each DL -methionine and L -cysteine were supplemented to basal diet for Met + Cys diet 2 Supplied per kilogram of diet: vitamins A, 2200 IU; vitamin D 3, 220 IU; vitamin E, 16 IU; vitamin K, 0.5 mg; thiamine, 1.5 mg; riboflavin, 4 mg; niacin, 30 mg; pantothenic acid, 12 mg; vitamin B 12 , 0.02 mg; folic acid, 0.3 mg; copper, 6 mg as copper sulfate; iodine, 0.14 mg as calcium iodate; iron, 100 mg as ferrous sulfate; manganese, 4 mg as manganese oxide; selenium, 0.3 mg as sodium selenite; zinc, 100 mg as zinc oxide; biotin 0.2 mg 3 Standardized ileal digestible  curve analysis was performed with a temperature gradient of 0.1 °C/s from 70 to 95 °C to confirm that only specific products were amplified. Pairs of primers for each gene were designed and checked for target identity using the National Center for Biotechnology Information database (Supplemental Table 1). Each set of primers used for qPCR amplification confirmed that their efficiencies were between 90 and 110%.

Histomorphology measurement
After fixation in 10% buffered formalin, the specimens were embedded in paraffin and cut into 5 μm sections. Each section was dewaxed and immersed sequentially in xylene, 95% ethanol, and 100% ethanol for 5 min; this sequential immersion was repeated twice. After rinsing with water, the sections were stained with 0.5% periodic acid solution for 5 min, followed by Schiff reagent staining for 10 min. The sections were counterstained with hematoxylin for 10 s and then dehydrated in alcohol, cleared, and mounted on slides for viewing with an Axio Scope A1 microscope coupled with an Infinity 2 digital camera (Lumenera Corporation). Images from all the circular basolateral membranes of the specimens were captured for evaluation of villi and crypts. Villus heights, villus width, and crypt depth were measured for all the distinguishable villi and crypts in the captured images using ImageJ software (National Institutes of Health) and averaged for each pig. Villus absorptive surface area was calculated using the formula: Villus absorptive surface area = 2π × (average villus width/2) × villus height. The numbers of goblet cells on the corresponding villi were counted manually.

Redox status indicators
The Trolox equivalent antioxidant capacity (TEAC) of the liver sample was measured using a colorimetric Antioxidant Assay kit (Sigma-Aldrich) according to the supplier's manual with Trolox as a standard. Total GSH and oxidized GSH (GSSG) of jejunum and liver samples were measured using a Glutathione Colorimetric Detection kit (Thermo Fisher Scientific). The reduced GSH content was calculated by subtracting GSSG from the total GSH. The superoxide dismutase (SOD) activities of the jejunum and liver samples were measured using a SOD Determination kit (Thermo Fisher Scientific), and then SOD concentrations were calculated using a SOD standard curve. Aliquots of each isolated protein were taken for the analysis of their protein content using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific); then, the measured TEAC and concentrations of GSH and SOD were corrected for the unit of protein content.

Ex vivo Ussing chamber analyses
A modified Ussing chamber (VCC-MC6; Physiologic Instruments Inc.) was used to study gut permeability across the jejunal epithelial tissues. The serosal and muscle layers of the jejunum were removed using microforceps, and the epithelial tissues were placed in the tissue holder with an aperture of 1 cm 2 . Each holder was mounted in a two-coupled chamber containing pairs of current (Ag wire) and voltage (Ag/AgCl pellet) electrodes housed in 3% agar bridges and filled with 3 M KCl. The samples for Ussing chamber analysis were processed quickly, within 15 min postmortem, to ensure the retention of the electrophysiological properties of the jejunal samples. Both mucosal and serosal chambers were bathed in 5 mL of Krebs-Ringer buffer containing 115 mM NaCl, 2.4 mM K 2 HPO 4 , 0.4 mM KH 2 PO 4 , 1.2 mM CaCl 2 , 1.2 mM MgCl 2 , 25 mM NaHCO 3 , 1 µm indomethacin, and 10 mM D-glucose. The bathing medium in the chambers was continuously aerated with a mixture of 95% O 2 and 5% CO 2 and maintained at 37 °C in a water bath. The electrode potential and the solution resistance were corrected before the tissues were mounted in the chamber. The clamps were connected to the software (Physiological Instruments) for automatic data collection and calculation. After a 10 min period to allow the establishment of electrophysiological equilibrium, the transepithelial electrical resistance (TEER) was recorded at 15 min intervals over a 1 h period. The flux of 4-kDa fluorescein isothiocyanate dextran (FD4) was measured by adding 0.1 mg/mL of FD4 (Sigma-Aldrich) to the mucosal chamber at the end of the equilibrium period, and 2 mL of the serosal buffer was sampled at 60 min and transferred to a light protection tube. The optical density of the sample was read at 450 nm with the emission wavelength set at 540 nm.

Calculations and statistical analyses
The geometric means of glyceraldehyde 3-phosphate dehydrogenase and hypoxanthine-guanine phosphoribosyltransferase expression were used to normalize the transcriptional levels for immune cytokines and enzymes associated with SAA metabolism. The relative expression was expressed as a ratio of the target gene to the SCC group gene, using the formula 2 −ΔΔCt according to Livak and Schmittgen (Livak and Schmittgen 2001), where ΔΔCt = (Ct target -Ct geometric mean ) treatment -(Ct target -Ct geometric mean ) SCC . All data were analyzed using the MIXED procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC, USA) with each animal used as an experimental unit. The model included Met, Cys, and their interaction. The LSMEANS statement with the Tukey-adjusted PDIFF option was used to calculate and separate the mean values for each treatment. In addition, the orthogonal contrast was used to test the effect of LPS challenge (SCC vs. CC). Results were considered significant at P < 0.05 and tendencies were observed at 0.05 ≤ P < 0.10.

Growth performance and rectal temperature
Dietary Met or Cys supplementation did not affect growth performance and rectal temperature during the pre-challenge and post-challenged periods (Table 2). However, during the post-challenge period, pigs in the CC group showed lower (P < 0.01) body weight gain (g/day), feed intake (g/day), and gain:feed than those in the SCC group. Pigs in the CC group had a higher (P < 0.05) change (%) in rectal temperature at 5 h post-challenge but not at 24 h post-challenge than pigs in the SCC group (Table 2).

Organ weight, jejunum morphology, and ex vivo Ussing chamber analyses
There were no differences in organ weights between pigs in the SCC and CC groups (Table 3). There were Met × Cys interaction (P < 0.05) effects on the weight of the gastrointestinal tract and portal-drained viscera. LPS-challenged control pigs had lower (P < 0.05) villus height, villus width, villus absorptive surface area, and crypt depth than sham-challenged control pigs. Dietary Cys supplementation increased (P < 0.05) villus width and villus absorptive surface area (Table 3). There were trends (P < 0.10) toward interactive effects on the number of goblet cells and villus height to crypt depth ratio. No difference was observed in TEER among the treatments. A tendency (P < 0.10) toward the higher FD4 flux in the CC group compared with the SCC group was observed (Fig. 1). Dietary Cys supplementation tended to decrease (P < 0.10) FD4 flux.

White blood cells profile and mRNA gene expression in jejunum and liver
There were no differences in WBC concentrations between the SCC and CC groups at 24 h pre-challenge (Table 4). However, pigs in the CC group had lower (P < 0.05) concentrations of total WBC, neutrophils, lymphocytes, and monocyte, neutrophils but higher (P < 0.05) band cell concentration than those in the SCC group at 5 h post challenge. Dietary Met supplementation increased (P < 0.05) lymphocyte content but decreased (P < 0.05) monocyte content at 24 h post challenge.
The CC group tended to have a greater (P < 0.10) mRNA abundance of TNF-α and lower (P < 0.10) mRNA  was a trend towards a higher (P < 0.10) glutathione-disulfide reductase (GSR) mRNA abundance in the jejunum of pigs fed Cys-supplemented diets. An interactive effect (P < 0.05) of Met × Cys was observed for IL-1β gene expression in the jejunum. Pigs fed Met-supplemented diets showed higher (P < 0.05) mRNA abundance of CBS in the liver. Furthermore, dietary Met supplementation tended to increase (P < 0.10) the abundance of mRNA encoding methionine adenosyltransferase 1A (MAT1A) in the liver. Pigs in the Met group had greater (P < 0.05) mRNA abundance of CBS in the liver. Met × Cys interaction effect (P < 0.05) was observed in the mRNA abundance of IL-6 and TNF-α in the liver.

Serum cytokine concentrations and oxidative stress indicators in jejunum and liver
Pigs in the CC group had higher (P < 0.05) serum TNF-α and IL-8 concentrations than those in the SCC group (Table 6). The CC group tended to have higher (P < 0.10) serum IL-1 receptor antagonist concentration than the SCC group. Pigs in the Met group had lower (P < 0.05) serum IL-8 concentration than those in the CC group. However, there were interactions (P < 0.05) of Met × Cys on serum IL-4 and IL-8 concentrations. Similarly, trends (P < 0.10) were found for the interaction on serum IL-1α, IL-2, and IL-10.
The CC group showed lower (P < 0.05) the levels of total GSH as well as the reduced form of GSH in the jejunum than the SCC group (Fig. 2). Dietary Cys supplementation elevated (P < 0.05) levels of total GSH and reduced form of GSH in the jejunum. A tendency (P < 0.10) for the increased GSH:GSSG in the jejunum was observed in pigs fed Cys supplemented diets. A tendency (P < 0.10) was observed for a lower total GSH level in the liver of pigs in CC than those in SCC (Fig. 3). Dietary Met supplementation increased (P < 0.05) levels of total GSH and reduced form of GSH in the liver. There were trends (P < 0.10) for a Met × Cys interactive effects on TEAC and SOD content in the liver. Table 4 White blood cell (WBC) concentrations (× 10 6 / mL) in nursery pigs fed diets supplemented with either DL -methionine or L -cysteine in response to LPS administration SCC sham-challenged control, CC challenged control, Met methionine-supplemented diet, Cys cysteinesupplemented diet supplemented diet, Met + Cys methionine and cysteine-supplemented diet. Values are least-squares means, n = 8 per treatment 1 No effects of Met, Cys, and the interaction were observed (P > 0.10) 2 Met, effect of methionine supplementation; Cys, effect of cysteine supplementation; M × C, effect of interaction between methionine and cysteine supplementation a SCC vs. CC (P < 0.05)

Discussion
After weaning, animals are vulnerable to enteric disease due to their immature digestive and immune systems and commonly undergo inflammation and oxidative stress (Moeser et al. 2017;Pluske et al. 2018). Lipopolysaccharide or endotoxin is the structural component of bacteria and activates the innate immune system (Wassenaar and Zimmermann 2018). Purified LPS has been widely used in different nutritional studies to elucidate the roles of nutrients and test nutritional strategies against inflammation and oxidative stress (Waititu et al. 2016;Hou et al. 2012). Thus, in the present study, LPS was administered to pigs to mimic the postweaning immune status and to investigate the effects of dietary Met or Cys supplementation under the status. The pathogenesis of LPS has been well documented. LPS serves as a ligand for toll-like receptor 4/myeloid differentiation factor 2 on immune cells, which initiates the cascade of innate immune systems (Wang et al. 2016). This corresponds with our results on febrile response, upregulation of the gene encoding TNF-α in the jejunum, and serum TNF-α concentration in LPS-administered pigs. Immune stimulation dramatically redistributes WBC profile (Abbas et al. 2017). Lipopolysaccharides administration induces IL-8 and IL-1 receptor antagonist to recruit neutrophils to the infection site and to regulate the inflammatory response by IL-1, respectively (Hoffmann et al. 2002;Arend et al. 1998;Yoshimura et al. 1997). It has been well documented that TNF-α and IL-6 elevation that results from LPS activates the differentiation of myeloid progenitor cells into neutrophils (Soler-Rodriguez et al. 2000;Ai and Udalova 2020). This is consistent with our results showing increased band neutrophils in CC group pigs compared to the SCC group pigs immediately after LPS administration. Furthermore, inflammation increases vascular permeability and facilitates the infiltration of leukocytes (e.g., neutrophils and monocytes) Table 5 Relative mRNA abundance (2 −∆∆Ct ) in jejunum and livers of nursery pigs fed diets supplemented with either DL -methionine or L -cysteine in response to LPS administration SCC sham-challenged control, CC challenged control, Met methionine-supplemented diet, Cys cysteinesupplemented diet supplemented diet, Met + Cys methionine and cysteine-supplemented diet. Values are least-squares means, n = 8 per treatment IL interleukin, TNF-α tumor necrosis factor-alpha, MAT methionine adenosyltransferase, CBS cystathionine beta synthase, CES cystathionine gamma-lyase, GPx1 glutathione peroxidase 1, GSR glutathione-disulfide reductase, GSS glutathione synthetase 3 Met, effect of methionine supplementation; Cys, effect of cysteine supplementation; M × C, effect of interaction between methionine and cysteine supplementation a SCC vs. CC (P < 0.10) into tissues (Abbas et al. 2017). This may explain the lower concentration of circulating WBC in CC compared with SCC. A previous study also found leukopenia in pigs following LPS administration (Huntley et al. 2018;Rakhshandeh et al. 2012). Changes in leukocyte profile and cytokine levels together with lower body weight gain indicated that LPS administration in our study successfully stimulated the immune system. Under inflammatory conditions, multiple enzymes in immune cells, including lipoxygenase, myeloperoxidase, nitric oxide synthase, and cyclooxygenase, and the respiratory burst of phagocytes are stimulated, which results in greater release of reactive oxygen species (Bhattacharyya et al. 2014) than under normal physiologic conditions. In this regard, LPS administration commonly increases the production of oxidants above the level that systemic antioxidants can sequester (Abdel-Salam et al. 2014;Hou et al. 2012). This aligns with our findings demonstrating lower TEAC in the liver of the CC group than in SCC.
The liver is the major site where LPS is detoxified by Kupffer cells; it has a high rate of oxidant production (Yao et al. 2016). Glutathione is an endogenous nonenzymatic antioxidant and plays a key role in maintaining cellular redox status (Bhattacharyya et al. 2014). Unlike other studies in which GSH levels in tissue are maintained or elevated following LPS injection (Rakhshandeh et al. 2019;Malmezat et al. 2000), the present study showed a lower GSH concentration in the liver and intestine in the CC group. This may be associated with the lower feed intake and SAA deficiency after LPS administration. There may have been a lack of SAA for a higher rate of GSH turnover. This is partially supported by a study by Castellano et al. (2015) in which a Met-deficient diet lowered the level of GSH in the muscle in growing pigs.
Cysteine is the rate-limiting substrate for GSH synthesis, and the plasma Cys flux increases mainly for the higher GSH turnover when the immune system is stimulated in pigs (Rakhshandeh et al. 2020). This reflects that dietary Cys supplementation may help replenish the depleted GSH under oxidative stress conditions. Previous studies found that Cys supplementation via L -Cys or N-acetylcysteine enhanced the GSH levels in the intestines of immune-stimulated pigs (Song et al. 2016;Hou et al. 2012). This is consistent with our finding of the elevated GSH level in the jejunum of pigs fed a Cys-supplemented diet. Luo et al. (1998) postulated that the depletion of GSH pool in skeletal muscle was associated with decreased activity of GSS, an enzyme that catalyzes the GSH synthesis from the condensation of γ-glutamylcysteine and glycine , in humans after surgical trauma. This suggests that the elevated GSH level in the jejunum in the present study may be attributed to the upregulation of the GSS-encoding gene in pigs fed the Cys-supplemented diet.
Our study showed that Cys supplementation upregulated GSR gene expression in the jejunum. Given that GSR catalyzes the reduction of GSSG to GSH, the upregulation may have led to higher GSH:GSSG. This is consistent with previous studies that found dietary Cys supplementation increased GSR activity in rats and pigs (Lee et al. 2013;Song et al. 2016). The benefit of Cys supplementation on Table 6 Serum cytokine concentrations (ng/mL) in pigs fed diets supplemented with supplemented with either DLmethionine or L -cysteine at 24 h after LPS administration SCC sham-challenged control, CC challenged control, Met methionine-supplemented diet, Cys cysteinesupplemented diet supplemented diet, Met + Cys methionine and cysteinine-supplemented diet. Values are least-squares means, n = 8 per treatment 2 Met, effect of methionine supplementation; Cys, effect of cysteine supplementation; M × C, effect of interaction between methionine and cysteine supplementation a, b SCC vs. CC (P < 0.05 and P < 0.10, respectively) GSH levels was not shown in the liver in the present study, but greater hepatic TEAC was observed. The enhanced GSH levels in the jejunum by dietary Cys supplementation were likely to sequester the intestinal oxidants and save the hepatic antioxidant pool, leading to greater TEAC in the liver. This is supported by previous studies by Lin and Yin (2008) in which cysteine supplementation to water via N-acetyl cysteine, S-ethyl cysteine, S-propyl cysteine, or Cys restored the hepatic GSH depletion due to a high-fat diet in mice. Similarly, sucrose-induced hepatic GSH depletion was restored by consuming a Cys-rich protein diet in rats (Blouet et al. 2007). By contrast, the hepatic GSH content was not affected by 1% or 2% of dietary Cys supplementation in rats (Lee et al. 2013). Thus, it seems that the effect of Cys supplementation on hepatic GSH content appears to vary depending on the source of Cys and physiologic state. It has been revealed that inflammation and redox signaling systemically govern the fate and permeability of intestinal epithelial cells, which are relevant to intestinal integrity (Vereecke et al. 2011;Circu and Aw 2012). Proinflammatory cytokines trigger the cascade of mitogenactivated protein kinase signaling and lead to apoptosis of enterocytes on the villus, mainly at the tip (Parker et al. 2019). Furthermore, the expression of myosin light-chain kinase that causes cytoskeletal contraction is activated under inflammatory conditions, thereby increasing intestinal permeability (Al-Sadi et al. 2009). A lower reducing potential in GSH-GSSG couple is associated with the apoptosis of intestinal epithelial cells and disruption of tight junction protein (Rao 2008;Circu and Aw 2012). In this regard, impaired intestinal integrity has been consistently shown after LPS administration (Hou et al. 2012;Song et al. 2016). This is consistent with our results in which pigs in the CC group showed shorter and narrower villi and increased FD4 flux than those in the SCC group. Higher FD4 flux indicates greater intestinal paracellular permeability, suggesting the disruption of tight junction proteins (Wijtten et al. 2011). In the present study, Cys supplementation seemed to restore the villus absorptive surface area of the jejunum and paracellular permeability. The GSH levels, elevated due to Cys supplementation may have sequestered the oxidants more efficiently, thereby preventing their attacks on intestinal integrity. This is in line with previous studies in which dietary Cys supplementation through L -Cys or N-acetylcysteine maintained the villus height (Hou et al. 2012) and FD4 flux (Song et al. 2016) against LPS administration.
Cysteine is nutritionally regarded as a dispensable amino acid because it can be synthesized de novo from Met via the transsulfuration pathway (Stipanuk 2004). Transsulfuration of Met occurs mainly in the liver, but in the small intestine to a lesser extent (Riedijk et al. 2007). Thus, it was generally hypothesized that dietary Met can fulfill the SAA requirement for protein deposition and GSH or taurine pool in the liver as well as intestine. In the present study, Met supplementation upregulated CBS-encoding genes in the jejunum and liver. This may be associated with the increase in transmethylation of Met surplus and the accumulation of SAM (Chen et al. 2014). Indeed, the expression of CBS is positively regulated by the content of SAM (Sbodio et al. 2019). Despite the upregulated expression of transmethylation-associated genes, dietary Met supplementation did not restore GSH levels and intestinal integrity in the jejunum, in contrast to dietary Cys supplementation. This suggests that the conversion of supplemental Met into Cys via the transsulfuration pathway was not as efficient as supplemental Cys in the intestine. Our study showed that LPS administration suppressed the expression of the CSE gene in the jejunum. Considering CSE is an enzyme that catalyzes cystathionine to Cys, this suppression may be an attributing factor for the lack of effects of Met supplementation on the GSH content in the jejunum. However, the Met supplementation enhanced the hepatic GSH levels. Given that transsulfuration is the sole pathway of Met catabolism (Stipanuk 2004), supplemental Met that bypassed the intestine appeared to SCC sham-challenged control, CC challenged control, Met methio-nine-supplemented diet, Cys cysteine-supplemented diet, Met + Cys methionine and cysteine-supplemented diet, GSH glutathione, GSSG oxidized GSH. * , † Indicate the difference between SCC and CC (P < 0.05 and P < 0.10, respectively). Values are mean ± SEM, n = 8 per treatment 1 3 undergo transsulfuration in the liver, where the transsulfuration enzymes are more active than in the intestine (Riedijk et al. 2007). This postulation is further supported by an isotope tracer study, in which no first-pass metabolism of dietary Met was observed in the gut, although 20% of dietary Met was extracted from the artery (Riedijk et al. 2007). The authors suggested that dietary Met was prioritized for its metabolism in the liver. In this context, LPS administration in the present study may have increased the synthesis of proteins including GSH and acute-phase protein in the liver, reducing the Met efflux into the bloodstream from where the intestine mainly sources dietary Met.
The MAT1A catalyzes the conversion of Met to SAM (Martínez-López et al. 2008). Dietary Met supplementation with greater MAT1A gene expression may have increased the SAM synthesis of the liver. A growing body of evidence indicates that SAM modulates the hepatic inflammatory response. In vitro studies found that exogenous SAM supplementation increased IL-6 and IL-10 production in Kupffer cells of LPS-treated rats by activating adenosine receptors as a process of liver regeneration (Song et al. 2005(Song et al. , 2004. This possibly explains the increase in the expression of genes encoding IL-6 and IL-10 in the livers of pigs that consumed the Met-supplemented diet. Considering that the LPS is mainly sequestered by Kupffer cells, the activated immune response in the liver by Met supplementation appeared to reduce the circulating inflammatory cytokines (e.g., IL-2, IL-4, and IL-8), suggesting ameliorated systemic inflammation.
However, antagonistic effects of Met + Cys supplementations were observed in inflammatory responses, redox status, and intestinal morphology. The independent benefits of either Met or Cys supplementation were not observed when both Met and Cys were supplemented together. The reason for this is unclear, but one possible explanation is that excessive Cys and its metabolites may have caused adverse effects in the intestine and liver. Cys can be easily oxidized to disulfide cysteine in the extracellular matrix, which is reduced back by endogenous antioxidants (Bhattacharyya et al. 2014). Thus, this suggests that excessive Cys paradoxically causes depletion of antioxidants, causing oxidative stress (Dilger and Baker 2008). These toxic effects were usually observed at extreme levels, but also observed with a small quantity of Cys in a prooxidant-prevailing condition. Lin and Yin (Lin and Yin 2008) reported that Cys supplementation (1 g/L of water) increased the concentration of a lipid peroxidation marker, malondialdehyde, and decreased catalase activity in the liver of mice fed a high-fat diet, although it increased hepatic GSH synthesis. The Cys metabolism pathway is determined by its cellular concentration, which regulates the activities of associated enzymes. Under a high Cys condition, cysteine dioxygenase is downregulated by increasing the Cys catabolism pathway, whereas γ-glutamyl cysteine synthetase is suppressed, which increases the catabolism of Cys and reduces GSH synthesis (Kwon and Stipanuk 2001). In this regard, dietary Met + Cys supplementation may have increased the rate of Cys catabolism, generating NH 3 and H 2 S that were relevant to systemic inflammation (Regina et al. 1993). However, further study is warranted to elucidate the antagonistic relations between Met and Cys supplementation.
In conclusion, the current study demonstrated the tissue-dependent benefits of dietary Met or Cys supplementation. Dietary Cys supplementation replenished the GSH depletion caused by LPS administration and enhanced the GSH/GSSH couple upregulating GSR gene expression in the jejunum. This benefit in the jejunum appeared to save the hepatic antioxidants, elevating the hepatic antioxidant capacity without alteration in the hepatic GSH level. Cys supplementation restored jejunal integrity that was impaired by LPS administration. By contrast, the dietary Met supplementation did not alter the jejunal GSH level, but it did enhance hepatic GSH levels and inflammatory responses against LPS injection, resulting in a reduction in serum pro-inflammatory cytokines. However, antagonistic relationships between Met and Cys supplementation were found with regard to inflammatory responses and redox status. Taken together, either Met or Cys should be carefully chosen with the consideration of target tissues when supplementing diets with SAA against inflammation and oxidative stress in an immune-stimulated condition in pigs.