Dietary sulfur amino acid restriction upregulates DICER to confer beneficial effects.

OBJECTIVE: Dietary restriction (DR) improves health and prolongs lifespan in part by upregulating type III endoribonuclease DICER in adipose tissue. In this study, we aimed to specifically test which missing dietary component was responsible for DICER upregulation. METHODS: We performed a nutrient screen in mouse preadipocytes and validated the results in vivo using different kinds of dietary interventions in wild type or genetically modified mice and worms, also testing the requirement of DICER on the effects of the diets. RESULTS: We found that sulfur amino acid restriction (i.e., methionine or cysteine) is sufficient to increase Dicer mRNA expression in preadipocytes. Consistently, while DR increases DICER expression in adipose tissue of mice, this effect is blunted by supplementation of the diet with methionine, cysteine, or casein, but not with a lipid or carbohydrate source. Accordingly, dietary methionine or protein restriction mirrors the effects of DR. These changes are associated with alterations in serum adiponectin. We also found that DICER controls and is controlled by adiponectin. In mice, DICER plays a role in methionine restriction-induced upregulation of Ucp1 in adipose tissue. In C. elegans, DR and a model of methionine restriction also promote DICER expression in the intestine (an analog of the adipose tissue) and prolong lifespan in a DICER-dependent manner. CONCLUSIONS: We propose an evolutionary conserved mechanism in which dietary sulfur amino acid restriction upregulates DICER levels in adipose tissue leading to beneficial health effects.

Prospective study of serum cysteine and cysteinylglycine and cancer of the head and neck, esophagus, and stomach in a cohort of male smokers.

BACKGROUND: The nonessential amino acid cysteine is known to be involved in many antioxidant and anticarcinogenic pathways. Cysteinylglycine is a pro-oxidant metabolite of glutathione and a precursor of cysteine. OBJECTIVE: To examine the relation between serum cysteine and cysteinylglycine and risk of gastric adenocarcinomas, esophageal squamous cell carcinomas, and head and neck squamous cell carcinomas, we conducted a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention study of male Finnish smokers aged 50-69 y at baseline. DESIGN: In total, 170 gastric adenocarcinomas, 68 esophageal squamous cell carcinomas, and 270 head and neck squamous cell carcinomas (identified from the Finnish Cancer Registry) were matched one-to-one with cancer-free control subjects on age and the date of serum collection. We calculated ORs and 95% CIs with the use of a multivariate-adjusted conditional logistic regression. RESULTS: Cysteine had a U-shaped association with gastric adenocarcinomas; a model that included a linear and a squared term had a significant global P-test (P = 0.036). Serum cysteinylglycine was inversely associated with adenocarcinomas of the gastric cardia (OR for above the median compared with below the median: 0.07; 95% CI: 0.01, 0.70; n = 38 cases) but not for other sites. Both cysteine and cysteinylglycine were not associated with esophageal squamous cell carcinoma or head and neck squamous cell carcinoma. CONCLUSIONS: We observed associations between serum cysteine and cysteinylglycine with upper gastrointestinal cancer risk. Future studies are needed to replicate these findings. This trial was registered at clininicaltrials.gov as NCT00342992.

Protective role of bacillithiol in superoxide stress and Fe-S metabolism in Bacillus subtilis.

Glutathione (GSH) serves as the prime thiol in most organisms as its depletion increases antibiotic and metal toxicity, impairs oxidative stress responses, and affects Fe and Fe-S cluster metabolism. Many gram-positive bacteria lack GSH, but instead produce other structurally unrelated yet functionally equivalent thiols. Among those, bacillithiol (BSH) has been recently identified in several low G+C gram-positive bacteria. In this work, we have explored the link between BSH and Fe-S metabolism in Bacillus subtilis. We have identified that B. subtilis lacking BSH is more sensitive to oxidative stress (paraquat), and metal toxicity (Cu(I) and Cd(II)), but not H2 O2 . Furthermore, a slow growth phenotype of BSH null strain in minimal medium was observed, which could be recovered upon the addition of selected amino acids (Leu/Ile and Glu/Gln), supplementation of iron, or chemical complementation with BSH disulfide (BSSB) to the growth medium. Interestingly, Fe-S cluster containing isopropylmalate isomerase (LeuCD) and glutamate synthase (GOGAT) showed decreased activities in BSH null strain. Deficiency of BSH also resulted in decreased levels of intracellular Fe accompanied by increased levels of manganese and altered expression levels of Fe-S cluster biosynthetic SUF components. Together, this study is the first to establish a link between BSH and Fe-S metabolism in B. subtilis.

Cord blood glutathione depletion in preterm infants: correlation with maternal cysteine depletion.

BACKGROUND: Depletion of blood glutathione (GSH), a key antioxidant, is known to occur in preterm infants. OBJECTIVE: Our aim was to determine: 1) whether GSH depletion is present at the time of birth; and 2) whether it is associated with insufficient availability of cysteine (cys), the limiting GSH precursor, or a decreased capacity to synthesize GSH. METHODOLOGY: Sixteen mothers delivering very low birth weight infants (VLBW), and 16 mothers delivering healthy, full term neonates were enrolled. Immediately after birth, erythrocytes from umbilical vein, umbilical artery, and maternal blood were obtained to assess GSH [GSH] and cysteine [cys] concentrations, and the GSH synthesis rate was determined from the incorporation of labeled cysteine into GSH in isolated erythrocytes ex vivo, measured using gas chromatography mass spectrometry. PRINCIPAL FINDINGS: Compared with mothers delivering at full term, mothers delivering prematurely had markedly lower erythrocyte [GSH] and [cys] and these were significantly depressed in VLBW infants, compared with term neonates. A strong correlation was found between maternal and fetal GSH and cysteine levels. The capacity to synthesize GSH was as high in VLBW as in term infants. CONCLUSION: The current data demonstrate that: 1) GSH depletion is present at the time of birth in VLBW infants; 2) As VLBW neonates possess a fully active capacity to synthesize glutathione, the depletion may arise from inadequate cysteine availability, potentially due to maternal depletion. Further studies would be needed to determine whether maternal-fetal cysteine transfer is decreased in preterm infants, and, if so, whether cysteine supplementation of mothers at risk of delivering prematurely would strengthen antioxidant defense in preterm neonates.

Alpha-lipoic acid induces elevated S-adenosylhomocysteine and depletes S-adenosylmethionine.

Lipoic acid is a disulfhydryl-containing compound used in clinical medicine and in experimental models as an antioxidant. We developed a stable isotope dilution capillary gas chromatography/mass spectrometry assay for lipoic acid. We assayed a panel of the metabolites of transmethylation and transsulfuration 30 min after injecting 100 mg/kg lipoic acid in a rat model. Lipoic acid values rose 1000-fold in serum and 10-fold in liver. A methylated metabolite of lipoic acid was also detected but not quantitated. Lipoic acid injection caused a massive increase in serum S-adenosylhomocysteine and marked depletion of liver S-adenosylmethionine. Serum total cysteine was depleted but liver cysteine and glutathione were maintained. Serum total homocysteine doubled, with increases also in cystathionine, N,N-dimethylglycine, and alpha-aminobutyric acid. In contrast, after injection of 2-mercaptoethane sulfonic acid, serum total cysteine and homocysteine were markedly depleted and there were no effects on serum S-adenosylmethionine or S-adenosylhomocysteine. We conclude that large doses of lipoic acid displace sulfhydryls from binding sites, resulting in depletion of serum cysteine, but also pose a methylation burden with severe depletion of liver S-adenosylmethionine and massive release of S-adenosylhomocysteine. These changes may have previously unrecognized deleterious effects that should be investigated in both human disease and experimental models.

Low plasma taurine concentration in Newfoundland dogs is associated with low plasma methionine and cyst(e)ine concentrations and low taurine synthesis.

Although taurine is not dietarily essential for dogs, taurine deficiency and dilated cardiomyopathy (DCM) are sporadically reported in large-breed dogs. Taurine status and husbandry were examined in 216 privately owned Newfoundlands, a giant dog breed with high incidence of idiopathic DCM (1.3-2.5%). Plasma taurine concentration was positively correlated (P < 0.01) with plasma cyst(e)ine (r = 0.37) and methionine (r = 0.35) concentrations and was similar across age, sex, neutering status, body weight, and body-condition scores. Plasma taurine concentration was low (< or =40 micromol/L) in 8% of dogs. Dogs with low plasma taurine were older, less active, had more medical problems and treatments, and had lower plasma albumin, cyst(e)ine, tryptophan, and alpha-amino-n-butyric acid concentrations than the other dogs (P < 0.05). Of 9 taurine-deficient, clinically evaluated dogs, 3 had DCM that was reversed by taurine supplementation and 1 had retinal degeneration. When given a diet apparently adequate in sulfur amino acids (5.4 g/kg) for 3 wk, 6 Newfoundlands (52.5 +/- 2.3 kg, 3.5-7 y), compared with 6 Beagles (13.2 +/- 2.3 kg, 5.5 y), had lower (P < 0.01) concentrations of plasma taurine (49 +/- 16 vs. 97 +/- 25 micromol/L) and cyst(e)ine and blood glutathione, lower (P < 0.01) de novo taurine synthesis (59 +/- 15 vs. 124 +/- 27 mg x kg(-0.75) x d(-1)), and greater (P < 0.05) fecal bile acid excretion (1.7 +/- 0.2 vs. 1.4 +/- 0.2 micromol/g). Newfoundlands would appear to have a higher dietary sulfur amino acid requirement than Beagles, a model breed used in nutrient requirement determinations.

Adequate range for sulfur-containing amino acids and biomarkers for their excess: lessons from enteral and parenteral nutrition.

The adequacy range of dietary requirements of specific amino acids in disease states is difficult to determine. In health, several techniques are available allowing rather precise quantification of requirements based on growth of the organism, rises in plasma concentration, or increases in the oxidation of marker amino acids during incremental administration of the amino acid under study. Requirements may not be similar in disease with regard to protein synthesis or with regard to specific functions such as scavenging of reactive oxygen species by compounds including glutathione. Requirements for this purpose can be assessed only when such a function can be measured and related to clinical outcome. There is apparent consensus concerning normal sulfur amino acid (SAA) requirements. WHO recommendations amount to 13 mg/kg per 24 h in healthy adults. This amount is roughly doubled in artificial nutrition regimens. In disease or after trauma, requirements may be altered for methionine, cysteine, and taurine. Although in specific cases of congenital enzyme deficiency, prematurity, or diminished liver function, hypermethionemia or hyperhomocysteinemia may occur, SAA supplementation can be considered safe in amounts exceeding 2-3 times the minimal recommended daily intake. Apart from some very specific indications (e.g., acetaminophen poisoning), the usefulness of SAA supplementation is not yet established. There is a growing body of data pointing out the potential importance of oxidative stress and resulting changes in redox state in numerous diseases including sepsis, chronic inflammation, cancer, AIDS/HIV, and aging. These observations warrant continued attention for the potential role of SAA supplementation. In particular, N-acetylcysteine remains promising for these conditions.

Oxidative stress and ageing: is ageing a cysteine deficiency syndrome?

Reactive oxygen species (ROS) are constantly produced in biological tissues and play a role in various signalling pathways. Abnormally high ROS concentrations cause oxidative stress associated with tissue damage and dysregulation of physiological signals. There is growing evidence that oxidative stress increases with age. It has also been shown that the life span of worms, flies and mice can be significantly increased by mutations which impede the insulin receptor signalling cascade. Molecular studies revealed that the insulin-independent basal activity of the insulin receptor is increased by ROS and downregulated by certain antioxidants. Complementary clinical studies confirmed that supplementation of the glutathione precursor cysteine decreases insulin responsiveness in the fasted state. In several clinical trials, cysteine supplementation improved skeletal muscle functions, decreased the body fat/lean body mass ratio, decreased plasma levels of the inflammatory cytokine tumour necrosis factor alpha (TNF-alpha), improved immune functions, and increased plasma albumin levels. As all these parameters degenerate with age, these findings suggest: (i) that loss of youth, health and quality of life may be partly explained by a deficit in cysteine and (ii) that the dietary consumption of cysteine is generally suboptimal and everybody is likely to have a cysteine deficiency sooner or later.

How HIV-1 causes AIDS: implications for prevention and treatment.

HIV-1 encodes for one of the human glutathione peroxidases. As a consequence, as it is replicated, its genetic needs cause it to deprive HIV-1 seropositive individuals not only of glutathione peroxidase, but also of the four basic components of this selenoenzyme, namely selenium, cysteine, glutamine, and tryptophan. Eventually this depletion process causes severe deficiencies of all these substances. These, in turn, are responsible for the major symptoms of AIDS which include immune system collapse, greater susceptibility to cancer and myocardial infarction, muscle wasting, depression, diarrhea, psychosis and dementia. As the immune system fails, associated pathogenic cofactors become responsible for a variety of their own unique symptoms. Any treatment for HIV/AIDS must, therefore, include normalization of body levels of glutathione, glutathione peroxidase, selenium, cysteine, glutamine, and tryptophan. Although various clinical trials have improved the health of AIDS patients by correcting one or more of these nutritional deficiencies, they have not, until the present, been addressed together. Physicians involved in a selenium and amino-acid field trial in Botswana, however, are reporting that this nutritional protocol reverses AIDS in 99% of patients receiving it, usually within three weeks.

Effects of cysteine on the pharmacokinetics and pharmacodynamics of intravenous and oral azosemide in rats with protein-calorie malnutrition.

The effects of cysteine on the pharmacokinetics and pharmacodynamics of azosemide were investigated after intravenous (10 mg/kg) and oral (20 mg/kg) administration to male Sprague-Dawley rats fed on 23% protein diet (control rats), and 5% protein diet with (rats with PCMC) or without (rats with PCM) oral cysteine (250 mg/kg, twice daily for the fourth week) for 4 weeks. After intravenous administration to rats with PCMC, some pharmacokinetic parameters restored fully or more than the level of control rats; the time-averaged nonrenal clearance (2.70 versus 2.32 ml/min/kg) and apparent volume of distribution at steady state (160 versus 189 ml/kg) were comparable to those in control rats, however, the terminal half-life (34.7 versus 57.2 min) and mean residence time (73.3 versus 99.3 min) were significantly shorter, area under the plasma concentration-time curve from time zero to time infinity (AUC, 1930 versus 2680 microg min/ml) was significantly smaller, and time-averaged renal (2.24 versus 1.21 ml/min/kg) and total body (CL, 4.98 versus 3.65 ml/min/kg) clearances were significantly faster than those in control rats. This could be mainly due to significantly faster renal clearance and at least partly due to increased cytochrome P450 1A2 activity by cysteine supplementation. After intravenous administration to rats with PCMC, the total amount of 8-hr urinary excretion of unchanged azosemide was significantly greater (457 versus 305 microg/g body weight), however, the 8-hr urine output (15.3 versus 31.1 ml/g kidney) was not significantly different between control rats and rats with PCMC. This could be due to the fact that urine output seemed to reach an upper plateau from 10 mg/kg dose of azosemide in rats.

Glutathione and immune function.

The immune system works best if the lymphoid cells have a delicately balanced intermediate level of glutathione. Even moderate changes in the intracellular glutathione level have profound effects on lymphocyte functions. Certain functions, such as the DNA synthetic response, are exquisitely sensitive to reactive oxygen intermediates and, therefore, are favoured by high levels of the antioxidant glutathione. Certain signal pathways, in contrast, are enhanced by oxidative conditions and favoured by low intracellular glutathione levels. The available evidence suggests that the lymphocytes from healthy human subjects have, on average, an optimal glutathione level. There is no indication that immunological functions such as resistance to infection or the response to vaccination may be enhanced in healthy human subjects by administration of glutathione or its precursor amino acid cysteine. However, immunological functions in diseases that are associated with a cysteine and glutathione deficiency may be significantly enhanced and potentially restored by cysteine supplementation. This factor has been studied most extensively in the case of human immunodeficiency virus (HIV)-infected patients who were found to experience, on average, a massive loss of S equivalent to a net loss of approximately 4 g cysteine/d. Two randomized placebo-controlled trials have shown that treatment of HIV-infected patients with N-acetyl-cysteine caused in both cases a significant increase in all immunological functions under test, including an almost complete restoration of natural killer cell activity. It remains to be tested whether cysteine supplementation may be useful also in other diseases and conditions that are associated with a low mean plasma cystine level and impaired immunological functions.

Bioavailable level and source of cysteine determine protein quality of a commercial enteral product: adequacy of tryptophan but deficiency of cysteine for rats fed an enteral product prepared fresh or stored beyond shelf life.

Young rats were used in bioassays designed to assess the protein quality and tryptophan as well as cystine adequacy of the enteral product TwoCal-HN that was either freshly prepared or had been stored (nonrefrigerated) in a warehouse for 10 mo (i.e., beyond shelf life). Based upon supplementation studies, cystine was observed to be the first-limiting amino acid in both fresh and expired TwoCal-HN, and tryptophan was not second limiting. Protein efficiency ratio (PER) of expired, but not fresh, TwoCal-HN was lower than that of the casein control diet, but with cystine supplementation, PER of the TwoCal-HN products was equal to or greater than the PER of the casein control. With a diet containing 10 g protein/100 g that also contained energy-furnishing ingredients simulating TwoCal-HN, maximal growth enhancement occurred with a supplement of 1 g cystine/kg diet. Both glutathione and N-acetyl-L-cysteine were observed to be equivalent to an isomolar level of L-cystine in stimulating growth. Using a chemically defined amino acid diet that was singly deficient in tryptophan, bioavailability of tryptophan was determined for casein, fresh TwoCal-HN, expired TwoCal-HN and D-tryptophan. Slope-ratio bioefficacy values relative to L-tryptophan (weight gain regressed on supplemental tryptophan intake) indicated that none of the experimental sources of tryptophan had bioavailabilities different from 100%. The results indicated that tryptophan did not deteriorate, as measured analytically or biologically, as a result of storing TwoCal-HN beyond shelf life.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione deficiency and peripheral metabolism of thyroid hormones during dietary cysteine deprivation in rats.

1. For a period of 24 d, young rats received a diet containing 120 g casein/kg or the same basic diet supplemented with 1.93 g cysteine/kg. 2. The thyroxine (T4) turnover was decreased in rats receiving the cysteine-deficient diet compared with that of rats on the supplemented diet. Moreover, the extrathyroidal T4 pool and T4 disposal rate decreased. 3. Cysteine deprivation also decreased the peripheral metabolism of 3,5,3'-triiodothyronine (T3). The T3 distribution space, extrathyroidal pool of T3 and T3 disposal rate were diminished. 4. In vitro, deiodination of T4 in liver homogenate assayed with endogenous glutathione (GSH) demonstrated decreased T3 production rates in the case of cysteine deficiency. This difference was minimized by the addition of GSH in amounts sufficient to saturate the reaction kinetics. In the light of this finding, GSH is probably involved in the promotion of certain thyroidal problems induced by a cysteine-deficient diet.

Maturation of a hypermodified nucleoside in transfer RNA.

E. coli C6 rel- met- cys- was cultured in a fully supplemented medium and in media lacking cysteine or methionine. tRNA isolated from the three cultures containted, respectively, a normal complement of modified nucleosides; a deficiency in thiolated nucleosides and a deficiency in methylated nucleosides. Both sulfur-deficient tRNA and methyl-deficient tRNA contained large amounts of N-6- (delta-2-isopentenyl) adenosine and small amounts of the 2-methylthio derivative. Methyl-deficient tRNA contained, in addition a large amount of a cytokinin active, differently modified nucleoside that is believed to be a sulfur derivative of N6-(delta-2-isopentenyl) adenosine. The structure of this compound is unknown. When methly-deficient tRNA and the precusor the tRNA-Tyr su3-+ A25 were enzymatically methylated in vitro, methyl groups were incorporated into derivatives of isopentenyladenosine. These results indicate that the biosynthesis of the 2-methylthio derivative of isopentenyladenosine may occur in a sequential manner, i.e., thiolation of isopentenyladenosine followed by methylation.