Revisiting recommendations for methionine and cysteine in the light of their effects on oxidative stress and cell function

Sulfur amino acids participate in the control of oxidative status since they are involved in the synthesis of intracellular antioxidants, particularly glutathione⁽Reference Soeters, van de Poll and van Gemert^4, Reference Métayer, Seiliez and Collin^8, Reference Wu, Fang and Yang⁹⁾, and in the methionine sulfoxide reductase antioxidant system⁽Reference Métayer, Seiliez and Collin^8, Reference Moskovitz, Rahman and Strassman^10, Reference Levine, Mosoni and Berlett¹¹⁾. Using cysteine derivatives (i.e. N-acetylcysteine) or cysteine-rich dietary proteins has been shown to increase blood and/or intracellular glutathione, which may affect the thiol redox status⁽Reference Stipanuk, Dominy and Lee^6, Reference Mariotti, Simbelie and Makarios-Lahham^12–Reference Dröge¹⁴⁾. Interestingly, dietary cysteine may also improve glucose control and alleviate sucrose-induced insulin resistance⁽Reference Blouet, Mariotti and Azzout-Marniche^15–Reference Mariotti, Hermier and Sarrat¹⁷⁾. Moreover, several studies have reported a beneficial effect of cysteine supplementation at nutritional doses in the prevention of post-exercise oxidative stress in human subjects⁽Reference Tsakiris, Parthimos and Parthimos^18–Reference Lands, Grey and Smountas²¹⁾. There is also evidence that the cysteine/cystine redox couple acts as an extracellular thiol redox buffer (i.e. it represents the major plasma redox pool) and a modulator of cell function. In vitro studies have indicated that the extracellular cysteine/cystine redox potential affects proliferation and the p44/p42 mitogen-activated protein kinase pathway via a transforming growth factor α-dependent mechanism in enterocytes⁽Reference Jonas, Ziegler and Gu^22, Reference Nkabyo, Go and Ziegler²³⁾, the production of reactive oxygen species and expression of cell–cell adhesion molecules in endothelial cells⁽Reference Go and Jones²⁴⁾, and modulates reactive oxygen species-induced apoptosis in retinal pigment epithelial cells⁽Reference Jiang, Moriarty-Craige and Orr²⁵⁾ and Burkitt's lymphoma cells⁽Reference Banjac, Perisic and Sato²⁶⁾. In these studies, the effects of the cysteine/cystine redox potential as a thiol/disulfide redox couple were independent of intracellular glutathione status, suggesting a specific role of the extracellular redox environment in cell function⁽Reference Go and Jones²⁷⁾.

The cysteine/cystine redox potential in plasma has recently been shown to vary during the day, with a pattern influenced by meals which may reflect acute changes in plasma thiol redox state with dietary intake of sulfur amino acids⁽Reference Blanco, Ziegler and Carlson²⁸⁾. Interestingly, it has been found in rats that the cysteine/cystine redox potential was affected by both decrease and increase in the sulfur amino acid (l-methionine+l-cystine) content of the diet, in contrast to glutathione redox potential⁽Reference Nkabyo, Gu and Jones²⁹⁾. Taken together, these studies provide new insights into the roles of cysteine and dietary sulfur amino acids in controlling cell function through modulation of the extracellular redox environment. These findings also highlight the complexity of the underlying mechanisms and do not support any systematic benefit from supplementing sulfur amino acids. In agreement with this assumption, decreasing methionine ingestion could be recommended in some situations. For example, due to the role of the methionine sulfoxide reductase antioxidant system, lowering dietary methionine levels appears to decrease the sensitivity of proteins to oxidative damage, oxidative stress and reactive oxygen species production⁽Reference Sanz, Caro and Ayala^30, Reference Caro, Gómez and López-Torres³¹⁾. Possible dietary implications for humans were discussed in a recent review⁽Reference López-Torres and Barja³²⁾. Since the intake of proteins, and therefore methionine, is higher than required in humans in developed countries, the authors suggest that decreasing excessive consumption could be an efficient way to reduce tissue oxidative stress and improve healthy life span.

Sulfur amino acids, particularly cysteine, may also act on cell function as precursors of hydrogen sulfide (H₂S)⁽Reference Stipanuk³³⁾. First considered as a toxic gas, H₂S is now recognised for its physiological function as the third (with NO and CO) gaseous transmitter in the body. In the brain, H₂S may act as a neuromodulator and protect neurons from oxidative stress⁽Reference Abe and Kimura^34–Reference Kimura and Kimura³⁸⁾. H₂S has been shown to directly activate K_ATP channels in vascular smooth muscle cells and insulin-secreting cells⁽Reference Zhao, Zhang and Lu^39–Reference Yang, Yang and Jia⁴¹⁾, thus promoting vasorelaxation and inhibiting insulin secretion⁽Reference Zhao, Zhang and Lu^39–Reference Kaneko, Kimura and Kimura⁴⁵⁾. Although little is known regarding the production of endogenous H₂S in response to changes in cysteine concentrations, it is important to consider the possibility that sulfur amino acids might affect cell function through modulation of H₂S and not to ignore the possible consequences in terms of nutrition. Similarly, it is necessary to consider other products of sulfur amino acid metabolism such as sulfates. Sulfate is the predominant endproduct of sulfur amino acid catabolism, and a relationship has been reported between sulfate excretion and sulfur amino acid intake⁽Reference Hamadeh and Hoffer^46, Reference Hamadeh and Hoffer⁴⁷⁾. Sulfates contribute to glycosaminoglycan synthesis and detoxification processes through reactions depending on 3′-phosphoadenosine-5′-phosphosulfate, and these sulfation reactions account for 27 % of inorganic sulfate turnover in humans⁽Reference Hoffer, Hamadeh and Robitaille⁴⁸⁾. In rats, ingesting a sulfur-deficient diet decreases plasma sulfate and elimination of the analgesic acetaminophen, with concomitant increase in its toxication⁽Reference Gregus, Kim and Madhu⁴⁹⁾, highlighting the importance of adequate methionine and/or cysteine intake for detoxification processes. Nevertheless, sulfates have long been thought to increase urinary excretion of Ca⁽Reference Hu, Zhao and Parpia^50, Reference Whiting and Draper⁵¹⁾; a recent study suggests that sulfur amino acids counterbalance the positive association between protein intake and bone mineral density⁽Reference Thorpe, Mojtahedi and Chapman-Novakofski⁵²⁾. These positive and negative effects of sulfates should be taken into account when establishing recommendations for sulfur amino acid intake.

Potential function of sulfur amino acids in controlling gene expression

Amino acids are recognised to act as modulators of signal transduction pathways that control gene transcription and translation⁽Reference Jefferson and Kimball^53–Reference Jousse, Averous and Bruhat⁵⁵⁾. Some in vitro experiments performed in mammals⁽Reference Shigemitsu, Tsujishita and Miyake^56, Reference Stubbs, Wheelhouse and Lomax⁵⁷⁾ and avian species⁽Reference Tesseraud, Bigot and Taouis⁵⁸⁾ have suggested that methionine may have such a signal function by inducing the activation of an intracellular kinase involved in the control of mRNA translation, i.e. the p70 S6 kinase (p70S6K, also called S6K1). In addition, amino acid availability regulates the mammalian general control non-depressible 2/eukaryotic initiation factor 2α (eIF2α) pathway that is thought to be affected by tRNA deacylation in the case of amino acid depletion⁽Reference Kimball and Jefferson^59–Reference Tremblay, Lavigne and Jacques⁶¹⁾. By influencing the phosphorylation of eIF2α, this pathway affects (1) the first step in the initiation of mRNA translation through the formation of eIF2-GTP, and (2) amino acid-controlled gene expression through the induction of activating transcription factor 4 (ATF4). Indeed, there is evidence that ATF4 can bind to the amino acid response element in the promoters of genes such as CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and asparagine synthetase, thereby up-regulating their expression⁽Reference Kilberg, Pan and Chen^54, Reference Averous, Bruhat and Jousse⁶²⁾. In relation to the present review, CHOP and asparagine synthetase are over-expressed when cells are deprived of a particular amino acid, for instance leucine, lysine, phenylalanine and methionine⁽Reference Jousse, Bruhat and Ferrara^63, Reference Bruhat, Jousse and Wang⁶⁴⁾. Different mechanisms are probably involved in such regulation since CHOP expression is strongly induced in response to methionine deprivation but only slightly affected by cysteine, asparagine or histidine deprivation, whereas asparagine synthetase expression is induced equally whatever the amino acid depleted⁽Reference Kilberg, Pan and Chen^54, Reference Jousse, Bruhat and Ferrara⁶³⁾. Cysteine restriction may induce the expression of several genes via the mammalian general control non-depressible 2 protein kinase/ATF4-dependent integrated stress response pathway to cope with oxidative and chemical stresses⁽Reference Lee, Dominy and Sikalidis⁶⁵⁾. It has also been reported that homocysteine (a metabolite of methionine) increases the expression of vascular endothelial growth factor by a mechanism involving ATF4⁽Reference Roybal, Yang and Sun⁶⁶⁾. It is of note that CHOP can interfere with C/EBP α/β expression and function, thus regulating adipogenesis, which could provide a mechanism through which amino acids have an effect on the pathogenesis of insulin resistance, as proposed by Tremblay et al. ⁽Reference Tremblay, Lavigne and Jacques⁶¹⁾. Moreover, methionine and cysteine are potent inhibitors of insulin-stimulated glucose transport in muscle cells⁽Reference Tremblay and Marette⁶⁷⁾. These results have been reported in non-physiological conditions, and the importance of a p70 S6 kinase-dependent regulatory loop in the development and/or the maintenance of insulin resistance remains to be determined⁽Reference Hinault, Van Obberghen and Mothe-Satney⁶⁸⁾. More information is therefore needed on the potential adverse effects of sulfur amino acid supplementation before use for nutritional purposes.

Many other examples demonstrate that sulfur amino acids are able to regulate the expression of numerous genes, thereby controlling nutrient metabolism and cell functions. Methionine supply has been shown (1) to affect growth hormone-induced insulin-like growth factor I gene expression in ovine hepatocytes⁽Reference Stubbs, Wheelhouse and Lomax⁵⁷⁾ and (2) to regulate the expression of an important gene controlling ubiquitin-proteasome-dependent proteolysis (i.e. E3 ubiquitin ligase atrogin-1) in avian fibroblasts⁽Reference Tesseraud, Métayer-Coustard and Boussaid⁶⁹⁾. Cysteine and cysteine derivatives (for example, N-acetylcysteine, or N-acetylcysteine amide) can modulate the activity of NF-κB⁽Reference Mihm, Ennen and Pessara^70–Reference Lee, Kim and Park⁷³⁾, which induces the expression of many genes that are involved in cell survival and proliferation, and in the regulation of immune and inflammatory responses (for reviews, see Barnes & Karin⁽Reference Barnes and Karin⁷⁴⁾, Chen et al. ⁽Reference Chen, Castranova and Shi⁷⁵⁾, Grimble⁽Reference Grimble⁷⁶⁾ and Wek et al. ⁽Reference Wek, Jiang and Anthony⁶⁰⁾). The activation pathways of this transcription factor involve the thiol redox status that responds to antioxidants including cysteine (see the section ‘Revisiting recommendations for methionine and cysteine in the light of their effects on oxidative stress and cell function’ above). Despite the clear theoretical importance of sulfur amino acids in immune function, studies directly linking methionine and cysteine intake and immune function are still needed⁽Reference Grimble⁷⁶⁾. Sulfur amino acids are also recognised to control genes involved in lipid metabolism, including cholesterol 7α-hydroxylase and apoA-I⁽Reference Oda⁷⁷⁾, which could be in favour of sulfur amino acid supplementation in cases of abnormal lipid metabolism. Nevertheless, high methionine intake can promote the development of atheromatous disorder⁽Reference Troen, Lutgens and Smith⁷⁸⁾, indicating that any such supplementation has to be undertaken with care to prevent atherosclerosis. The underlying mechanisms are still unclear. Methionine is the precursor of homocysteine (Fig. 1) and elevated levels of plasma homocysteine (hyperhomocysteinaemia) have been associated with occlusive vascular disease⁽Reference Gerhard and Duell⁷⁹⁾. However, Troen et al. ⁽Reference Troen, Lutgens and Smith⁷⁸⁾ have shown in mice that the atherogenic effect of methionine exists even in the absence of hyperhomocysteinaemia. High plasma homocysteine levels appear not to be independently atherogenic as recently reported⁽Reference Zhou, Werstuck and Lhoták^80, Reference Liu, Wang and Guo⁸¹⁾. Vitamin B supplementation (its effect on methionine metabolism is discussed in the section ‘Role of sulfur amino acids in the methylation processes’ below) does not prevent CVD in humans despite lowering homocysteine⁽Reference Jamison, Hartigan and Kaufman^82, Reference Albert, Cook and Gaziano⁸³⁾, similarly challenging the role of homocysteine as an independent cardiovascular risk factor.

Role of sulfur amino acids in the methylation processes

In addition to the above roles, sulfur amino acids have another major function since methionine is a source of the methyl groups needed for all biological methylation reactions, including methylation of DNA and histones, a process that influences chromatin structure and gene expression⁽Reference Egger, Liang and Aparicio^84–Reference Waterland⁸⁹⁾. DNA methylation generally occurs at cytosines within cytosine-guanine dinucleotides (CpG). The CpG sites are dispersed throughout the genome, with specific CpG-rich regions called CpG islands. The methylation status of cytosine residues within CpG dinucleotides and in the context of CpG islands determines which genes are active or not, and plays an important role in maintaining gene silencing that is needed for tissue- and development-specific gene expression, epigenetic mechanisms involved in the establishment and maintenance of gene expression patterns, and genomic imprinting.

Being converted to S-adenosylmethionine, the major biological methylating agent, methionine provides the methyl groups that are needed for cellular methylation reactions. However, in order to adapt dietary methionine intake effectively, it is important to keep in mind the specific features of the methyl metabolism that is more generally involved in methylation regulation. S-adenosylmethionine is converted to S-adenosylhomocysteine following methyl donation, and to homocysteine (reactions of transmethylation; Fig. 1). Homocysteine can be remethylated into methionine through two separate pathways: (1) the folate-dependent remethylation pathway that involves 5,10-methyl tetrahydrofolate; (2) the folate-independent remethylation pathway in which the methyl group is provided by betaine. Disorders of methyl balance lead to several diseases (liver disease, CVD, closure of the neural tube, synthesis of creatine, cancer), illustrating the key role played by methylation reactions⁽Reference Brosnan, da Silva and Brosnan^90, Reference Williams and Schalinske⁹¹⁾. Whether DNA methylation is increased by high levels of methionine is still unclear, since, for example, excess methionine may impair DNA methylation by inhibiting remethylation of homocysteine⁽Reference Dunlevy, Burren and Chitty⁹²⁾. These findings need further investigation to quantify their physiological relevance, particularly using more physiological amounts of methionine. In addition, based on the methionine cycle, nutritional factors affecting the supply of S-adenosylmethionine and/or removal of homocysteine (for example, dietary choline, betaine, folic acid and vitamin B₁₂) potentially affect DNA methylation and must therefore be taken into account when assessing the supply of methionine. Mathematical models have been developed to consider the complexity of methionine metabolic pathways, and they will be useful to complement and help guide laboratory studies⁽Reference Reed, Nijhout and Sparks^93, Reference Reed, Nijhout and Neuhouser⁹⁴⁾.

Recent in vivo studies have demonstrated opposite diet-related changes in DNA methylation according to the tissue studied: long-term administration of a folate/methyl-deficient diet in rats results in progressive hypomethylation of DNA in the liver⁽Reference Pogribny, Ross and Wise⁹⁵⁾, but an increase in DNA methylation in the brain⁽Reference Pogribny, Karpf and James⁹⁶⁾. Although the underlying mechanisms and significance of these findings are not understood, insufficient supply of the nutrients involved in methyl metabolism clearly affects methylation reactions and concomitantly alters patterns of gene expression⁽Reference Pogribny, Karpf and James⁹⁶⁾. This function of methyl group donors represents another mechanism though which sulfur amino acids can regulate gene expression (see section ‘Potential function of sulfur amino acids in controlling gene expression’ above) with potentially both negative and positive consequences, for example, either unsuitable dysregulation of gene expression or conversely restoration of appropriate locus-specific DNA methylation using therapeutic pro-methylation diets⁽Reference Waterland⁸⁹⁾. Epigenetic regulation thus plays an important role in the potential development (or in contrast prevention) of cancer, CVD, type 2 diabetes and obesity. For example, the availability of methyl group donors regulates the expression of genes involved in the development of the digestive tract through a mechanism dependent on DNA methylation, at least for the homeobox gene CDX1, which could explain the protective role attributed to folates in colon cancer⁽Reference Lu, Freund and Muller⁹⁷⁾.

An increasing number of reviews have highlighted the importance of perinatal nutrition (i.e. nutrition in early life), since aberrant methyl metabolism in utero is linked with certain disorders (for reviews, see Ulrey et al. ⁽Reference Ulrey, Liu and Andrews⁸⁵⁾, Waterland⁽Reference Waterland⁸⁹⁾, Rees et al. ⁽Reference Rees, Wilson and Maloney⁹⁸⁾, Waterland & Michels⁽Reference Waterland and Michels⁸⁷⁾ and Nafee et al. ⁽Reference Nafee, Farrell and Carroll⁸⁸⁾). For example, a murine metastable epiallele (axin fused, Axin^Fu) exhibits epigenetic plasticity to the maternal diet, since supplementation with methyl group donors before and during pregnancy increases offspring DNA methylation at Axin^Fu(Reference Waterland, Dolinoy and Lin⁹⁹⁾. As discussed by some authors⁽Reference Waterland^89, Reference Rees, Wilson and Maloney^98, Reference Rees¹⁰⁰⁾, manipulating the sulfur amino acid content of the early diet may induce chronic changes in cell functions that have implications for long-term health. More recent findings have provided evidence that changes in the supply of methionine and specific B vitamins such as B₁₂ during the periconception period can lead to widespread epigenetic changes in DNA methylation in offspring, and modify adult health-related phenotypes⁽Reference Sinclair, Allegrucci and Singh¹⁰¹⁾. For instance, adult offspring exhibit changes in body composition (fatness), immune function and insulin response. Interestingly, these effects were obtained with modest early dietary intervention (changes within physiological ranges), indicating the importance of adequate dietary methyl group supply for metabolic programming and the need for further studies to define the level of intake. Such studies will certainly provide a rationale for nutritional advice in the future.