Introduction

Chronic subclinical levels of disease occur frequently in intensive animal production systems and have been shown to compromise the efficiency of nutrient utilization for production(Colditz, 2002). Animals reared in these environments display reduced appetiteand growth when compared to healthy animals (Le Floc'h et al., 2004) as a result of immune system stimulation (ISS) and the subsequent release of pro-inflammatory cytokines. Dietary nutrients, in particular amino acids, that may be used for growth and production are thus redirected towards supporting the immune response (Le Floc'h et al., 2004). The activation of certain metabolic pathways important in sustaining the immune system leads to specific changes in amino acid requirements. Previous research has shown that during ISS, the liver increases production of a wide range of immune peptides and proteins, including glutathione (GSH) and the acute phase protein albumin. Both of these compounds are particularly rich in cysteine content, a conditionally essential amino acid. Opportunities to influence the response of growing animals to ISS relate to the speed with which recovery from ISS is initiated. According to Reeds and Jahoor (2001), nutritional intervention may prove most useful to accelerate recovery. Thus the increased production of immune peptides may result in an increased need for dietary sulfur amino acid (SAA) intake. The objective of this paper is to review existing scientific literature on the effects of ISS on muscle protein catabolism and whole-body amino acid metabolism, with a focus on SAAs. Ideally, this review will assist in the design of future experiments that will aid in the development of SAA supplementation, thereby minimizing the negative effects of ISS on animal production efficiency.

ISS and Pro-inflammatory Cytokines

Cytokines are a large family of molecules produced by various cells of the body, but mainly immune cells in response to infection (Reeds and Jahoor, 2001). Disease states, both clinical and subclinical, result in the release of pro-inflammatory cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor-a(TNF-a),which are known to be involved in the regulation of protein metabolism. IL-1 and TNF-a inhibit muscle protein synthesis while inducing muscle protein loss. Further, IL-1 is responsible for the induction of fever and anorexia, as well as acute phase protein (APP) synthesis in synergy with IL-6 (Le Floc'h et al., 2004). APPs are a family of unrelated proteins produced by the liver whose plasma concentration increases (positive APPs) or decreases (negative APPs) in response to pro-inflammatory cytokines,and act to affect one or more stages of inflammation. Cytokines can also signal pro-inflammatory genes encoding cytokines, cytokine receptors, cell adhesion molecules, APPs, and growth factors through nuclear transcription factor κB andactivator protein 1 (Dinarello, 2000). The end result is an altered biochemistry that will ensure the support of the immune system with nutrients from within the body (Figure 1) (Grimble, 2006).

Figure1. The impact of cytokines on protein metabolism. Le Floc'h et al., 2004.

The Effects of ISS on Muscle Protein Catabolism

Despite the skeletal muscle protein catabolism observed during ISS, plasma amino acid concentrations are decreased (Melchior et al., 2004). This can be explained by an increase in amino acid utilization during infection to provide energy and nutrients for immune cell proliferation, to serve as substrates for molecules involved in inflammation, as well as the use of certain amino acids in specific metabolic pathways related to host defenses (Melchior et al., 2004). Melchior et al. (2004) injected pigs with Complete Freund's Adjuvant (CFA), a model that mimics conditions observed in conventional farms. CFA induces chronic protein and amino acid metabolism disruptions without inducing severe clinical disorders or mortality. The authors noted that the plasma amino acid concentrations in these animals were decreased more in the fed state compared to the fasted state, suggesting that amino acids supplied through the diet were more rapidly metabolized. However, due to cytokine-induced anorexia, mobilization of skeletal muscle protein occurs in order to supply the amino acids to the visceral organs for energy and host defenses (Obled, 2002;Breuille et al., 2006). In fact, skeletal muscle exhibits inducible expression of receptors for pro-inflammatory cytokines that results in reduced amino acid uptake, inhibition of protein synthesis, and increased protein breakdown of the skeletal muscle (Colditz, 2004). Nevertheless, the pattern of amino acids required for the modified metabolic pathways may differ from those released by skeletal muscle proteolysis (Le Floc'h et al., 2004), as the amino acid composition of muscle is fixed such that any amino acid can become limiting (Reeds and Jahoor, 2001). Typically, limiting amino acids are those whose dietary supply most closely matches the animal's metabolic needs. Under normal physiological circumstances, this is generally a nutritionally essential aminoacid. However, during a diseased state, this may not necessarily apply. Amino acids such as glutamine and cysteine, which are not traditionally essential, become at least potentially limiting (Reeds and Jahoor, 2001). Thus, skeletal muscle proteolysis leads to an excess of non-limiting amino acids, while amino acids required for the immune system become limiting (Le Floc'h et al., 2004). It is therefore hypothesized that nutritional supplementation of specific amino acids would increase the availability of substrates for the synthesis of proteins and peptides needed during ISS (Grimble and Grimble, 1998; Reeds and Jahoor, 2001; Obled,2002).

The Effects of ISS on SAA Requirements

Despite a major decrease in skeletal muscle protein synthesis, a significant increase in whole body protein synthesis occurs due to anabolic responses observed in numerous organs and tissues, particularly the liver (Obled, 2002). In fact, liver protein synthesis represents 33% of whole body protein synthesis during sepsis compared to 17% in healthy rats (Breuille et al., 2006). In a recent review of the modificationsof protein and amino acid metabolism during inflammation and ISS (Le Floc'h etal., 2004), the authors noted that the requirement of sulfur amino acids,particularly cysteine, is important in septic patients. In addition, a substantial increase in cysteine content in the liver was observed in septic rats (+79% compared to control rats) (Breuille et al., 2006). This is in part due to the fact that cysteine is important for the synthesis of acute phase proteins. During ISS, total liver protein synthesis increases with preferential secretion of acute phase proteins. These proteins appear earlier than specific antibodies, and play numerous roles in modulating the immune system through their anti-oxidant, anti-inflammatory, and anti-bacterial activities (Le Floc'het al., 2004; Melchior et al, 2004). One report (Reeds et al., 1994) examined the amount of muscle proteolysis required to supply amino acids for a "typical" acute phase response in adult humans. It was concluded that in order to increase APPs by 850 mg/kg body weight, 1980 mg/kg body weight of muscle protein would need to be mobilized, due to the mismatch between the amino acid composition of APPs and muscle proteins. Further, it was observed that cysteine was among the amino acids released in quantities closest to the theoretical requirements for APP synthesis, and thus would be expected to be limiting for supporting the immune response unless adequately supplemented in the diet (Grimble and Grimble,1998). While albumin is considered to be a negative APP in most livestock species, the fall in albumin concentration during ISS is apparently the consequence of an increased fractional rate of degradation rather than a lower rate of synthesis (Breitkreutz et al., 2000; Reeds and Jahoor, 2001). Since albumin contains 6% cysteine, compared with 1 to 2% for most proteins (Breuilleet al., 2006) and its synthesis persists in the face of limited amino acid supplies, it has the potential to create a substantial deficit of SAAs.

More importantly, cysteine is essential for the synthesis of GSH (Le Floc'h et al., 2004). As a response to stress and infection, an organism will activate the production of free radical products in an attempt to neutralize viruses and bacteria (Reeds and Jahoor, 2001). However, this often results in oxidative stress. Glutathione, a tri-peptide consisting of glutamate, glycine and cysteine, is central to the peroxidative protection system (Figure 2). It serves several vital functions including detoxifying electrophiles, scavenging free radicals, and modulating critical cellular processes related to immune function (Malmezat, 2000; Lu,2008). By bearing a free and readily oxidizable sulfhydryl group, it is able to maintain the redox potential of the cell (Roth, 2007). As GSH conjugation to free radicals irreversibly consumes intracellular GSH, severe oxidative stress can deplete cellular GSH content (Lu, 2008). Recent evidence suggests that whole body GSH increases 4-fold in septic rats in as early as 2 days post-infection with E. coli (Breuille et al., 2006).

Figure2. The structure of glutathione or g-glutamylcysteinyl-glycine.Lu, 2008.

It is important to note that the rate of GSH synthesis is largely determined by two factors: glutamate cysteine ligase,the rate-limiting enzyme in the synthesis of GSH, and cysteine availability (Lu, 2008). The latter is regulated through membrane transport of cysteine, cysteine and methionine intake, as well as the trans-sulfuration pathway (Lu,2008). This pathway allows the enzymatic conversion of methionine to homocysteine and further into cysteine (Figure 3) and is particularly active in the liver (Lu, 2008). Several studies have noted that ISS and inflammation lead to a reduction in cysteine catabolism and an increase in cysteine trans-sulfuration from methionine (Malmezat 1998, 2000). The author concludes that this pathway is the likely mechanism to help preserve the availability of cysteine for GSH synthesis, underlining its importance to host defenses. Despite the organism's ability to synthesize cysteine during ISS, this is probably not sufficient to respond to the increased demands (Malmezat, 2000). Thus, it is clear that as ISS depletes GSH, a substantial increase in GSH production is needed to maintain cellular function, thereby increasing the need for both cysteine and methionine intake.

Figure3. Hepatic methionine (Met) metabolism and GSH synthesis. Lu, 2008.

Conclusions and Implications

Many studies have theorized that SAA supplementation would benefit individuals subject to ISS (Breitkreutz et al., 2000; Malmezat, 2000; Le Floc'het al., 2004; Grimble, 2006). One study examining the effects of diets supplemented with different amino acids noted that diets containing cysteine significantly reduced body weight loss, muscle wasting, and urinary nitrogen excretion in septic rats (Breuille et al., 2006). The authors stated, "The data implicated cysteine as the most limiting amino acid to combat infection." Reeds and Jahoor (2001) explained that amino acid supplementation should target the later stages of the immune response in an effort to support the normal response rather than preventing it. With the current knowledge of the effects of ISS on SAA requirements, it is plain to see that the supplementation of these nutrients during ISS will help accelerate recovery while minimizing the negative impact of a normal immune response. In doing so, production efficiencies can be maintained while upholding animal welfare. In an animal production setting, this supplementation can decrease nutrient losses into the environment by more closely matching the animal's nutrient requirements, thus reducing excess catabolism and subsequent excretion of amino acids. In addition, meat product quality can be improved, as the rate of recovery from an immune response can be accelerated and the use of antibiotics reduced.

Yet it is important to note that our understanding of the impact ofISS on SAA requirements is not complete. Immune cell function appears to be sensitive to a range of intracellular sulfhydryl compounds (Grimble, 2006) and while many studies have elucidated the effects of these compounds on the immune response, the precise mechanism is unknown. Further, the optimal diet must be determined for each species, since over-supplementation can lead to adverse and even toxic effects. It has been shown that excess levels of cysteine intake can lead to feed refusal, which would negate the effect of supplementation (Breuille et al., 2006). Moreover, heightened inflammatory stress has been interlinked with homocysteine accumulation (Grimble, 2006). Some researchers state that caution should be taken when supplementing methionine, as conditions that raise the concentration of homocysteine may modulate immune function. However, this is not supported by a strong scientific basis. Thus, definitive studies need to be performed to determine the optimal ratio of SAAs in the diet to minimize the adverse effects of ISS, and the precise mechanism for how this supplementation affects the acceleration of the immune response.

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