Physiologic Responses to Dietary Sulfur Amino Acid Restriction in Mice Are Influenced by Atf4 Status and Biological Sex.

BACKGROUND: Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). OBJECTIVE: We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. METHODS: Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. RESULTS: We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. CONCLUSIONS: Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

Glutathione Deficiency and Alterations in the Sulfur Amino Acid Homeostasis during Early Postnatal Development as Potential Triggering Factors for Schizophrenia-Like Behavior in Adult Rats.

Impaired glutathione (GSH) synthesis and dopaminergic transmission are important factors in the pathophysiology of schizophrenia. Our research aimed to assess the effects of l-buthionine-(S,R)-sulfoximine (BSO), a GSH synthesis inhibitor, and GBR 12909, a dopamine reuptake inhibitor, administered alone or in combination, to Sprague-Dawley rats during early postnatal development (p5-p16), on the levels of GSH, sulfur amino acids, global DNA methylation, and schizophrenia-like behavior. GSH, methionine (Met), homocysteine (Hcy), and cysteine (Cys) contents were determined in the liver, kidney, and in the prefrontal cortex (PFC) and hippocampus (HIP) of 16-day-old rats. DNA methylation in the PFC and HIP and schizophrenia-like behavior were assessed in adulthood (p90-p93). BSO caused the tissue-dependent decreases in GSH content and alterations in Met, Hcy, and Cys levels in the peripheral tissues and in the PFC and HIP. The changes in these parameters were accompanied by alterations in the global DNA methylation in the studied brain structures. Parallel to changes in the global DNA methylation, deficits in the social behaviors and cognitive functions were observed in adulthood. Only BSO + GBR 12909-treated rats exhibited behavioral alterations resembling positive symptoms in schizophrenia patients. Our results suggest the usefulness of this neurodevelopmental model for research on the pathomechanism of schizophrenia.

Dietary Sulfur Amino Acid Restriction and the Integrated Stress Response: Mechanistic Insights.

Dietary sulfur amino acid restriction, also referred to as methionine restriction, increases food intake and energy expenditure and alters body composition in rodents, resulting in improved metabolic health and a longer lifespan. Among the known nutrient-responsive signaling pathways, the evolutionary conserved integrated stress response (ISR) is a lesser-understood candidate in mediating the hormetic effects of dietary sulfur amino acid restriction (SAAR). A key feature of the ISR is the concept that a family of protein kinases phosphorylates eukaryotic initiation factor 2 (eIF2), dampening general protein synthesis to conserve cellular resources. This slowed translation simultaneously allows for preferential translation of genes with special sequence features in the 5' leader. Among this class of mRNAs is activating transcription factor 4 (ATF4), an orchestrator of transcriptional control during nutrient stress. Several ATF4 gene targets help execute key processes affected by SAAR such as lipid metabolism, the transsulfuration pathway, and antioxidant defenses. Exploration of the canonical ISR demonstrates that eIF2 phosphorylation is not necessary for ATF4-driven changes in the transcriptome during SAAR. Additional research is needed to clarify the regulation of ATF4 and its gene targets during SAAR.

Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Splanchnic tissues respond differently when piglets are offered a diet 30 % deficient in total sulfur amino acid for 10 days.

PURPOSE: A deficient total sulfur amino acid (TSAA) supply has been reported to differently affect the amino acid composition of tissues, but limited information is available about its effects on the morphology and metabolic properties of splanchnic tissues. METHODS: The amino acid composition, protein metabolism, glutathione concentration of the liver, proximal and distal jejunum, ileum and kidneys, and intestinal architecture were compared in 42-day-old piglets pair-fed either a diet deficient (TSAA-; 28 % deficiency) or sufficient (TSAA+) in TSAA for 10 days. RESULTS: The supply of TSAA had no effect on tissue weights, but influenced the amino acid composition in a tissue-dependent manner. Compared with animals receiving diet TSAA+, the concentrations of Met and Ser were higher in liver protein of TSAA- animals while the Cys concentration in protein was lower in the liver but higher in the distal jejunum. The TSAA supply had no effect on protein synthesis and proteolytic activities of tissues. Villus width and surface, and crypt surface were lower in the proximal jejunum of TSAA- versus TSAA+ pigs. Crypt surface in the ileum of TSAA- pigs was higher. Pigs receiving diet TSAA- had lower GSH and GSSG concentrations in the liver and proximal jejunum, but the GSH/GSSG ratio was decreased only in the liver. CONCLUSIONS: A greater nutritional priority appears to be given to splanchnic tissues so that its growth and protein metabolism can be maintained when the TSAA supply is limiting. The amino acid composition, glutathione status, and intestinal mucosa architecture are affected in a tissue-dependent manner.

Skeletal muscles respond differently when piglets are offered a diet 30% deficient in total sulfur amino acid for 10 days.

PURPOSE: Although amino acids (AA) are required for growth, little is known about the effect of a deficient AA supply on the composition and the contractile and metabolic properties of skeletal muscles. METHODS: Protein metabolism, oxidative catabolism, glutathione system, and fiber-type composition of the longissimus (LM), rhomboideus (RM), and semitendinous (SM) muscles were compared between 42-day-old piglets pair-fed for 10 days either with a diet with a 28% deficient supply of total sulfur AA (TSAA-) or with a diet with a sufficient supply of total sulfur AA (TSAA+). RESULTS: The relative weight, protein mass, and protein synthesis of LM were 10-32% lower in TSAA- pigs compared with TSAA+ pigs, while RM and SM were not affected by the TSAA supply. The TSAA supply affected the AA composition of muscles. Concentrations of Met and branched-chain AA were, respectively, 7 and 3% lower in TSAA- pigs compared with TSAA+ pigs. The His concentration was 30% higher in LM and SM in TSAA- pigs compared with TSAA+ pigs and unaffected in RM. The activity of citrate synthase was 14% higher in all three muscles of TSAA- pigs. In these pigs, the ß-hydroxy-acyl-CoA dehydrogenase activity was 20% higher in RM compared with TSAA+ pigs while that of lactate dehydrogenase was 21% lower in LM. Total and reduced glutathione concentrations were more than 70% greater in RM than in LM or SM, and these concentrations were approximately 10% lower in TSAA- pigs than in TSAA+ pigs. CONCLUSIONS: Results of this study indicate that a TSAA deficiency affects muscle properties in a muscle-dependent manner increasing the oxidative capacity of RM and reducing growth and glycolytic metabolism of LM.

Changes in body composition in broilers by a sulfur amino acid deficiency during growth.

In the factorial approach, amino acid (AA) requirements are determined using the AA composition of retained protein, which is assumed to be constant. However, this hypothesis may not be valid because the AA composition of body protein can be affected by the diet. The objective of this study was to quantify the changes in chemical body composition of broilers receiving diets either deficient (TSAA-) or sufficient (TSAA+) in TSAA. Diet TSAA+ was formulated according to the Ross recommendation. Diet TSAA- provided 36% true digestible Met:Lys and 64% true digestible TSAA:Lys, which were, respectively, 34 and 22% lower compared with diet TSAA+. Performance and tissue weight gain between 7 and 42 d of age were not affected by the TSAA supply. In TSAA- chickens, protein gain was lower in the carcass (P < 0.01) and tended to be lower in the empty body (P = 0.06) and pectoralis major muscle (P = 0.10). Compared with TSAA+ chickens, lipid gain in TSAA- chickens was 78% greater in the pectoralis muscle (P < 0.001), 28% greater in abdominal fat (P < 0.05), and 10% greater in the carcass (P = 0.10). In the pectoralis muscle, there was a tendency for an increase in the redness value (a*; P = 0.10). The TSAA supply affected the AA composition of tissues and tissue gain, but the Met and Cys concentrations were changed only in the offal (P = 0.08). The deficient TSAA supply resulted in an increase in the Ser concentration in the empty body, carcass, and pectoralis muscle (P < 0.05). In contrast, it resulted in a decrease in the concentrations of Lys and Glu in the empty body, of Phe, Tyr, Gly, and Glu in the pectoralis muscle, and of Ala in the offal (P < 0.05). This indicates that although chickens cope with a TSAA deficiency predominantly by changing the protein and lipid concentration in the body, the AA composition is also affected. This calls into question the use of a constant ideal AA profile in poultry nutrition.

Brain glutathione as a target for aetiological factors in neurolathyrism and konzo.

Both neurolathyrism and konzo are associated with the nutritional dependence of human populations on a single plant food. These diseases express themselves as chronic disorders of upper motor neurones, leading to signs and symptoms that characterise amyotrophic lateral sclerosis (motor neurone disease). The plant food associated with neurolathyrism is grass pea, which contains the neurotoxic ß-N-oxalyl-α,ß-diaminopropionic acid (ß-ODAP). The plant food associated with konzo is cassava, which may contain significant concentrations of cyanogenic glycosides and their degradation products. A monotonous diet of grass pea is likely to generate nutritional deficiencies; it is proposed that one of these, plasma methionine deficiency, may predispose neurones to the neurotoxic effects of ß-ODAP. Subjects suffering from konzo also have low concentrations of plasma methionine as a result of a dietary deficiency of this amino acid. However, the plasma cystine concentration is also compromised because cyanide released from cyanogenic glycosides in cassava probably reacts with plasma cystine non-enzymatically. The product of this reaction is 2-imino-4-thiazolidine carboxylic acid. Since both plasma methionine and cystine are used for glutathione synthesis it seems likely that one common feature that leads to motor neurone death in neurolathyrism and konzo is the depletion of glutathione in the central nervous system.

Grass pea and neurolathyrism: farmers' perception on its consumption and protective measure in North Shewa, Ethiopia.

Neurolathyrism in Ethiopia is caused by food dependency on grass pea (Lathyrus sativus L.). In the study area, a large proportion of the farmers are growing grass pea since it can withstand harsh environments. Socio-economic factors (poverty; lack of money to buy other food legumes) and environmental problems (such as water logging and frost hazards) influence consumption of grass pea. Most of the respondents have the idea that some chemical contained in grass pea causes a health problem. Different processing and preparation methods are used to prepare grass pea into different food forms. The major processing methods include washing and soaking, as the farmers apply these methods mainly because they assume that the chemical that causes lathyrism, scientifically known as ß-ODAP (ß-N-oxalyl-L-α,ß-diaminopropionic acid) is reduced through washing and soaking. The farmers adopt different strategies to avoid the problem of lathyrism such as avoiding consumption of grass pea in the form that they suspect to cause the problem, blending/mixing with other crops, applying different processing/detoxification methods. Since grass pea is consumed with a fear of lathyrism, future research should concentrate either on developing grass pea varieties with safe level of ß-ODAP content or improving the traditional/indigenous processing methods.

The nutritive value of grasspea (Lathyrus sativus) and allied species, their toxicity to animals and the role of malnutrition in neurolathyrism.

The safe use of grasspea (Lathyrus sativus) and allied species (L. cicera, L. clymenum and L. ochrus) requires a better understanding of the factors that are involved in the development of neurolathyrism. A suitable animal model is needed. The nutritional quality, seed chemical composition, the role of malnutrition, synergistic action of antinutritional factors, the toxicity of both seed and forage to animals, metabolism and tissue distribution of the toxic amino acid beta-N-oxalyl-alpha,beta-L-diaminopropionic acid (ODAP) in mammals are reviewed. Malnutrition is not necessary for the development of neurolathyrism, however, the supply of sulfur amino acids by Lathyrus spp. is limited by the combined action of several antinutritional factors and the low inherent levels in the seeds. Metabolism or excretion of ODAP and clearance from the central nervous system appear to function well under normal circumstances, while species differences exist. Interruptions to these processes and excessive concurrent demands for reduced sulfur amino acids are likely to be conducive to the onset of neurotoxicity.

Sulfur amino acids deficiency caused by grass pea diet plays an important role in the toxicity of L-ß-ODAP by increasing the oxidative stress: studies on a motor neuron cell line.

Neurolathyrism is a motor neuron disease caused by the overconsumption of grass pea (Lathyrus sativus L.) containing L-ß-ODAP. The precise mechanism to cause motor neuron degeneration has yet to be elucidated, but should agree with the epidemiological backgrounds. Considering the amino acid content of the legume, and the epidemiological link with prolonged unbalanced nutrition, the shortage of sulfur amino acids methionine and cysteine could affect the toxicity of L-ß-ODAP. We analyzed the effect of these amino acids in the media on the toxicity using primary motor neuron culture and a motor neuron cell line NSC-34. Deprivation of both methionine and cysteine exacerbated the toxicity of L-ß-ODAP by 66% compared to the complete medium. The glutathione content of these cells was greatly decreased in sulfur amino acid-deprived medium. L-ß-ODAP further lowered the content in the deprived media to be 32-44% of the controls compared to normal media being 62-74%. The increased motor neuron toxicity in this medium was neutralized by the addition of reduced glutathione ethyl ester or N-acetylcysteine suggesting the importance of the mitochondrial oxidative stress induced by L-ß-ODAP under sulfur amino acid-deficient conditions.

Hepatic oxidative stress in fructose-induced fatty liver is not caused by sulfur amino acid insufficiency.

Fructose-sweetened liquid consumption is associated with fatty liver and oxidative stress. In rodent models of fructose-mediated fatty liver, protein consumption is decreased. Additionally, decreased sulfur amino acid intake is known to cause oxidative stress. Studies were designed to test whether oxidative stress in fructose-sweetened liquid-induced fatty liver is caused by decreased ad libitum solid food intake with associated inadequate sulfur amino acid intake. C57BL6 mice were grouped as: control (ad libitum water), fructose (ad libitum 30% fructose-sweetened liquid), glucose (ad libitum 30% glucose-sweetened water) and pair-fed (ad libitum water and sulfur amino acid intake same as the fructose group). Hepatic and plasma thiol-disulfide antioxidant status were analyzed after five weeks. Fructose- and glucose-fed mice developed fatty liver. The mitochondrial antioxidant protein, thioredoxin-2, displayed decreased abundance in the liver of fructose and glucose-fed mice compared to controls. Glutathione/glutathione disulfide redox potential (E(h)GSSG) and abundance of the cytoplasmic antioxidant protein, peroxiredoxin-2, were similar among groups. We conclude that both fructose and glucose-sweetened liquid consumption results in fatty liver and upregulated thioredoxin-2 expression, consistent with mitochondrial oxidative stress; however, inadequate sulfur amino acid intake was not the cause of this oxidative stress.

On the biomarkers and mechanisms of konzo, a distinct upper motor neuron disease associated with food (cassava) cyanogenic exposure.

Konzo is a self-limiting central motor-system disease associated with food dependency on cassava and low dietary intake of sulfur amino acids (SAA). Under conditions of SAA-deficiency, ingested cassava cyanogens yield metabolites that include thiocyanate and cyanate, a protein-carbamoylating agent. We studied the physical and biochemical modifications of rat serum and spinal cord proteins arising from intoxication of young adult rats with 50-200mg/kg linamarin, or 200mg/kg sodium cyanate (NaOCN), or vehicle (saline) and fed either a normal amino acid- or SAA-deficient diet for up to 2 weeks. Animals under SAA-deficient diet and treatment with linamarin or NaOCN developed hind limb tremors or motor weakness, respectively. LC/MS-MS analysis revealed differential albumin carbamoylation in animals treated with NaOCN, vs. linamarin/SAA-deficient diet, or vehicle. 2D-DIGE and MALDI-TOF/MS-MS analysis of the spinal cord proteome showed differential expression of proteins involved in oxidative mechanisms (e.g. peroxiredoxin 6), endocytic vesicular trafficking (e.g. dynamin 1), protein folding (e.g. protein disulfide isomerase), and maintenance of the cytoskeleton integrity (e.g. α-spectrin). Studies are needed to elucidate the role of the aformentioned modifications in the pathogenesis of cassava-associated motor-system disease.

Etiology of Konzo, epidemic spastic paraparesis associated with cyanogenic glycosides in cassava: role of thiamine deficiency?

Konzo is a syndrome of symmetrical, non-progressive, non-remitting spastic paraparesis occurring in epidemic and endemic forms in several countries in Africa, invariably associated with monotonous consumption of inadequately processed bitter cassava roots (Manihot esculenta) with very minimal protein supplementation. Despite numerous epidemiological, clinical and biochemical studies by authors in several countries aimed at elucidating the etiological mechanisms of Konzo, the etiology remains unknown. High cyanide consumption with low dietary sulfur intake due to almost exclusive consumption of insufficiently processed bitter cassava roots was proposed as the cause of Konzo, but there has been no evidence of a causal association between cyanide consumption and Konzo. In this paper a new etiological mechanism of thiamine deficiency is presented, based on detailed review of the epidemiological, clinical and biochemical features of Konzo. It is postulated that in Konzo patients, a severe exacerbation of thiamine deficiency results from the inactivation of thiamine that occurs when, in the absence of dietary sulfur-containing amino acids; the sulfur in thiamine is utilized for the detoxification of cyanide consumed in improperly processed bitter cassava. Thiamine is known to be rendered inactive when the sulfur in its thiazole moiety is combined with hydrogen cyanide. This hypothesis may stimulate studies examining the role of thiamine in the etiology of Konzo, and may lead to the formulation of strategies for the prevention and treatment of this debilitating disease.

Acetaminophen elimination half-life in humans is unaffected by short-term consumption of sulfur amino acid-free diet.

Sulfation and glutathione (GSH) conjugation are important pathways for elimination of acetaminophen (APAP). Previous studies in rodents show that limitation of dietary sulfur amino acids (SAAs) reduces biosynthesis of 3'-phosphoadenosine-5'-phosphosulfate, the precursor for sulfation, and GSH, the precursor for the mercapturatic acid pathway. The amount of SAA needed for the metabolism of two doses of APAP is equivalent to 62% of the recommended dietary allowance (RDA) for SAA in humans. A decrease in the activity of these metabolic pathways could lead to decreased elimination of the reactive metabolite of APAP and possibly affect risk of APAP use. To determine whether intake of a SAA-deficient diet alters APAP metabolism, a pilot clinical study with a double-blind, cross-over design was performed. Subjects received the RDA for SAA for 3 days for equilibration. After admission to the clinical research unit, subjects were given a chemically defined diet with 100 or 0% of the RDA for SAA for 2 days. On day 3, two doses of APAP (15 mg/kg) or placebo were given successively within a 6-h interval. Plasma samples were collected at baseline and hourly for 12 h, and two 6-h urine aliquots were collected during this time course. The data show that SAA limitation 1) did not change the pattern of APAP metabolites in plasma or urine and 2) did not alter APAP pharmacokinetics. Thus, the results show that 2 days of diet completely devoid of SAA have no effect on APAP metabolism or disposition in healthy humans.

Oxidation of plasma cysteine/cystine and GSH/GSSG redox potentials by acetaminophen and sulfur amino acid insufficiency in humans.

Variations in plasma sulfur amino acid (SAA) pools are associated with disease risks, but little information is available about the factors affecting plasma SAA pools. Drug metabolism by glutathione (GSH) and sulfate conjugation can, in principle, represent a quantitatively important burden on SAA supply. The present study was designed to determine whether therapeutic doses of acetaminophen (APAP) alter SAA metabolism in healthy human adults. A double-blind, crossover design incorporating four treatment periods with diets providing 100% of the recommended dietary allowance (RDA) for SAA without or with APAP (15 mg/kg) and 0% RDA for SAA without or with APAP, in randomized order. After a 3-day equilibration period, chemically defined diets with 100 or 0% RDA for SAA were given for 2 complete days. On day 3, APAP or placebo was given in two successive doses (6-h interval), and timed plasma samples were collected. With SAA intake at 100% RDA, APAP administration oxidized the plasma cysteine/cystine redox potential (E(h)CySS) but not the plasma GSH/GSSG redox potential (E(h)GSSG). The extent of oxidation caused by APAP was similar to that seen with 0% SAA and no APAP. However, APAP administration with 0% SAA did not cause further oxidation beyond APAP or 0% SAA alone. In contrast, an oxidation of the plasma E(h)GSSG was apparent for SAA insufficiency only with APAP. The results suggest a need to evaluate possible effects of APAP in association with SAA insufficiency as a contributing factor in disease risk.

Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.

We recently showed that the developing gut is a significant site of methionine transmethylation to homocysteine and transsulfuration to cysteine. We hypothesized that sulfur amino acid (SAA) deficiency would preferentially reduce mucosal growth and antioxidant function in neonatal pigs. Neonatal pigs were enterally fed a control or an SAA-free diet for 7 days, and then whole body methionine and cysteine kinetics were measured using an intravenous infusion of [1-(13)C;methyl-(2)H(3)]methionine and [(15)N]cysteine. Body weight gain and plasma methionine, cysteine, homocysteine, and taurine and total erythrocyte glutathione concentrations were markedly decreased (-46% to -85%) in SAA-free compared with control pigs. Whole body methionine and cysteine fluxes were reduced, yet methionine utilization for protein synthesis and methionine remethylation were relatively preserved at the expense of methionine transsulfuration, in response to SAA deficiency. Intestinal tissue concentrations of methionine and cysteine were markedly reduced and hepatic levels were maintained in SAA-free compared with control pigs. SAA deficiency increased the activity of methionine metabolic enzymes, i.e., methionine adenosyltransferase, methionine synthase, and cystathionine beta-synthase, and S-adenosylmethionine concentration in the jejunum, whereas methionine synthase activity increased and S-adenosylmethionine level decreased in the liver. Small intestine weight and protein and DNA mass were lower, whereas liver weight and DNA mass were unchanged, in SAA-free compared with control pigs. Dietary SAA deficiency induced small intestinal villus atrophy, lower goblet cell numbers, and Ki-67-positive proliferative crypt cells in association with lower tissue glutathione, especially in the jejunum. We conclude that SAA deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.

Thiol/disulfide redox status is oxidized in plasma and small intestinal and colonic mucosa of rats with inadequate sulfur amino acid intake.

Low molecular weight thiol/disulfide redox pools are dependent upon extracellular cysteine (Cys) availability. We determined whether dietary sulfur amino acid (SAA) deficiency induces oxidative stress in vivo, as determined by redox state of major thiol/disulfide couples in plasma [Cys/cystine (CySS)] and intestinal mucosa [glutathione (GSH)/glutathione disulfide (GSSG)]. Rats were fed isocaloric, isonitrogenous semipurified diets: either SAA-adequate (control), SAA-deficient, or SAA-supplemented, pair-fed to intake of the SAA-deficient group. Reference rats consumed standard rat food ad libitum. After 7 d, plasma and gut mucosal samples were analyzed for Cys, CySS, GSH and GSSG, and the redox potentials of Cys/CySS and GSH/GSSG were determined. Mean daily food intake in the pair-fed rats was similar (approximately one-half of reference-rat intake). Body weight decreased in all pair-fed groups, but rats fed the SAA-deficient diet lost significantly more body weight. Dietary SAA deficiency decreased GSH concentrations in both plasma and gut mucosa, increased plasma GSSG, and oxidized plasma and gut mucosal GSH/GSSG redox and plasma Cys/CySS redox. SAA supplementation resulted in a more reducing plasma Cys/CySS redox potential. Reference rats exhibited similar tissue and plasma GSH/GSSG redox as rats that ate semipurified SAA-adequate rat food, which provided similar net SAA intake. Our in vivo data show that inadequate dietary SAA intake oxidizes the thiol/disulfide redox status in rat-gut mucosa and plasma. Such oxidation of redox pools is associated with oxidative stress and the onset or progression of several pathological conditions. Thus, dietary SAA deficiency could contribute to the progression of disease by causing an oxidation of these components.

Repression by oxidative stress of iNOS and cytokine gene induction in macrophages results from AP-1 and NF-kappaB inhibition mediated by B cell translocation gene-1 activation.

Previously, we reported that oxidative stress caused by sulfur amino acid deficiency (SD) induces B cell translocation gene-1 (Btg-1), which belongs to the Apro family, in hepatocytes. In view of the impairment of immune function by protein restriction that causes SD, this study investigated whether SD or other oxidative stress inhibits iNOS and cytokine expression and induces Btg-1 in macrophages and explored the causal relationship of Btg-1 induction and repression of the genes. When macrophages were incubated in sulfur amino acid-deprived medium, lipopolysaccharide induction of iNOS, TNFalpha, IL-1beta, and IL-6 was significantly decreased compared to control. Because AP-1 and NF-kappaB are the common transcription factors that regulate the genes encoding iNOS and cytokines, we examined AP-1 and NF-kappaB DNA binding activities and transactivation of the iNOS gene containing the DNA binding elements. Induction of the reporter gene pGL-miNOS-1588 comprising the -1.6 kb iNOS promoter in lipopolysaccharide-activated macrophages was inhibited 30-70% by SD or treatment with pro-oxidants, including tert-butylhydroxyquinone, buthionine sulfoximine, and 3-morpholinosydnonimine. Oxidative stress increased Btg-1 mRNA. SD-induced oxidative stress activated Btg-1 in macrophages, as evidenced by nuclear translocation of endogenous or green fluorescent protein-tagged Btg-1, which localized in the cytoplasm in the resting state. Expression of Btg-1 inhibited lipopolysaccharide-inducible AP-1 and NF-kappaB activities, repressing transactivation of the target gene pGL-miNOS-1588. These results provide evidence that oxidative stress induced by SD or pro-oxidants inhibits the expression of iNOS and cytokines in macrophages with Btg-1 activation and that the gene repression by oxidative stress may result from Btg-1-mediated inhibition of AP-1 and NF-kappaB activities.

Sulfur amino acid restriction induces the pi class of glutathione S-transferase expression in primary rat hepatocytes.

The regulation of genes by amino acids is attracting increasing attention. In the present study, we investigated the restriction of expression of the pi class of glutathione S-transferase (GST Yp) by sulfur amino acids. Hepatocytes isolated from male Sprague-Dawley rats were cultured with L-15-based medium containing low (LSAA; 0.1 mmol/L L-methionine and 0.1 mmol/L L-cysteine) or high (HSAA; 0.5 mmol/L L-methionine and 0.2 mmol/L L-cysteine) amounts of sulfur amino acids for up to 6 d. Cellular protein contents did not differ between LSAA- and HSAA-treated cells over the entire period. In contrast, glutathione concentrations were suppressed by the LSAA medium and on d 6 were only 20% of those of HSAA-treated cells (P < 0.05). As shown by immunoblot analysis, GST Yp protein levels were greater in LSAA-treated cells than in HSAA-treated cells (P < 0.05). The induction of GST Yp by L-methionine and L-cysteine restriction was not affected by insulin and dexamethasone, but the latter suppressed GST Yp expression (P < 0.05). LSAA increased GST Yp mRNA levels and GST activity toward ethacrynic acid (P < 0.05). GST Yp induction occurred only in cells with a limited supply of L-methionine; restriction of L-isoleucine, L-leucine, L-lysine, and L-phenylalanine had no significant effect. In contrast with the induction of GST Yp, the expression of the GST isoforms Ya and Yb was not changed by amino acid restriction. In conclusion, hepatic GST Yp gene expression is upregulated by a limited availability of sulfur amino acids.