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). The augment of TNF-α and IL-1 release has been observed immediately after LPS recognition by immune cells, and these cytokines increase the synthesis of prostaglandins, which act on the hypothalamus, elevating the thermoregulatory set point (Abbas et al. 2017). 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). LPS 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) 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). The GSH has a short lifespan, and its turnover and synthesis is accelerated after immune stimulation (Rakhshandeh et al. 2019). 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 intake of SAA 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. (Castellano et al. 2015) in which a Met-deficient diet lowered the level of GSH in the muscle in growing pigs. Cys 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. The GSS is a homodimeric enzyme that catalyzes the condensation of γ-glutamylcysteine and glycine, forming a molecule of GSH (Yin et al. 2016). Luo et al. (Luo et al. 1998) postulated that the depletion of GSH pool in skeletal muscle was associated with decreased activity of GSS in humans after surgical trauma. This suggests that the elevated GSH level in the jejunum may be attributed to the upregulation of the GSS-encoding gene in pigs fed the Cys-supplemented diet.
GSR is the key antioxidant enzyme that maintains the GSH:GSSG ratio. A higher ratio is desirable, suggesting a higher reducing potential (Bhattacharyya et al. 2014). Our study showed that Cys supplementation also upregulated GSR gene expression in the jejunum, which 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 GSH levels was not shown in the liver, but greater 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. However, there is disagreement with the effect of Cys supplementation on hepatic GSH. Lin and Yin (Lin and Yin 2008) reported that cysteine supplementation to water via N-acetyl cysteine, S-ethyl cysteine, S-propyl cysteine, or Cys restored the lowered hepatic GSH 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). Pro-inflammatory cytokines trigger the cascade of mitogen-activated 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 supplemented Cys may have sequestered the oxidants via GSH preservation in the intestine, 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. CBS catalyzes the conversion of homocysteine into cystathionine as the first irreversible step of the transsulfuration pathway (Stipanuk 2004). 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). However, contrary to the effect of dietary Cys supplementation, dietary Met supplementation with upregulated CBS-gene expression did not restore the GSH levels and intestinal integrity in the jejunum. 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 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 content 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 cystine 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 NH3 and H2S 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.