Homocysteine Transsulfuration Research Articles

Cystathionine β-Synthase Suppresses NLRP3 Inflammasome Activation via Redox Regulation in Microglia.

Aims: Cystathionine β-synthase (CBS) is essential for homocysteine (Hcy) transsulfuration, yielding cysteine as a common precursor of hydrogen sulfide (H2S), glutathione (GSH), and other sulfur molecules, which produce neuroprotective effects in neurological conditions. We previously reported a disruption of microglial CBS/H2S signaling in a Parkinson's disease (PD) mouse model. Yet, it remains unclear whether CBS affects nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome activity and other pathologies in PD. Results: Microglial CBS expression decreased after lipopolysaccharide (LPS) stimulation. Elevated GSSG (the oxidized GSH) content and decreased H2S generation were found in the brains of microglial cbs conditional-knockout (cbscKO) mice, whereas serum and brain Hcy levels remained unaltered. Moreover, microglial cbscKO mice were susceptible to NLRP3 inflammasome activation and dopaminergic neuron losses caused by LPS injection into the substantia nigra, whereas cbs overexpression or activation produced opposite effects. In vitro studies showed that cbs overexpression or activation suppressed microglial NLRP3 inflammasome activation and interleukin (IL)-1β secretion by reducing mitochondrial reactive oxygen species (mitoROS) level. Conversely, ablation of cbs enhanced NLRP3 expression and mitoROS generation and augmented microglial NLRP3 inflammasome activity in response to adenosine triphosphate challenge, which was blocked by the mitoROS scavenger. Innovation and Conclusion: The study demonstrated an elevated GSSG level and reduced H2S generation, which correlated with a susceptible status of microglia in the brain of cbscKO mice. Our findings reveal a critical role of CBS in restraining the microglial NLRP3 inflammasome by controlling redox homeostasis and highlight that activation or upregulation of CBS may become a potential strategy for PD treatment.

Overlapping sperm damages from vitamin B or D deficiency in mice: Insights into the role of clinical supplementations.

In this study, the effect of 14 weeks of standard diet (controls) or folate and vitamin B12-free diet (VBD group) or vitamin D-free diet (VDD group) were assessed on mice testicular function, and sperm function. Vitamin D deprivation caused increased body weight with no effect from VBD confirming the calcium-independent role of vitamin D on body weight homeostasis. The two deprivations caused convergent damages including decreased testosterone, worsened Johnson scores, tubular differentiation index and spermatogenesis index, and serious worsening of sperm parameters and of sperm functional tests (DNA methylation, protamination, DNA damage and lipid peroxidation). From a metabolic point of view, the damage from both models converged on the one carbon cycle (methylations) and the transsulfuration pathway (GSH and antioxidant defences) and increased circulating homocysteine, although with different mechanisms: VBD appeared to hamper methylations due to lower ability to regenerate homocysteine to methionine whereas VDD appeared to interfere with homocysteine transsulfuration to cysteine and, thereafter, GSH. VDD also caused a huge paradox increase of vitamin B12, which was likely in a non-functional form and warrants further investigation. These findings strongly endorse the potential benefit of combined folate/B12 and vitamin D supplementation in infertile patients.

Классическая гомоцистинурия: этиология, клинические проявления и методы лечения

Classic homocystinuria is a rare autosomal recessive disorder of methionine metabolism resulting from a deificiency of cystathionine beta-synthase that is involved in transsulfuration of homocysteine. The disorder is caused by mutations of CBS gene. Lack of enzyme is associated with accumulation of homocysteine that induce various toxicities, e.g. endothelial dysfunction and increased risk of thrombosis. Clinical manifestations of classic homocystinuria include ectopia lentis and/or severe myopia, skeletal abnormalities (marfanoid habitus, generalized osteoporosis and bone deformity), mental retardation, seizures, psychiatric and behavioral disorderts, venous and arterial thrombosis. Some patients with milder form of the disease can present only with thromboembolic events in adulthood. Diagnosis of classic homocystinuria should by established by biochemical (elevated plasma homocysteine and methionine and low cistathione) and genetic (homozygous and compound heterozygous mutations of CBS gene) tests. Low methionine diet, vitamin B6, folic acid and betaine anhydrous, a methylating agent, can be used to reduce homocysteine levels, to prevent progression of the homocystinuria and thromboembolic events.

Hypobaria-Induced Oxidative Stress Facilitates Homocysteine Transsulfuration and Promotes Glutathione Oxidation in Rats with Mild Traumatic Brain Injury.

Background:United States service members injured in combat theatre are often aeromedically evacuated within a few days to regional military hospitals. Animal and epidemiological research indicates that early exposure to flight hypobaria may worsen brain and other injuries. The mechanisms by which secondary exposure to hypobaria worsen trauma outcomes are not well elucidated. This study tested the hypothesis that hypobaria-induced oxidative stress and associated changes in homocysteine levels play a role in traumatic brain injury (TBI) pathological progression caused by hypobaria.Methods:Male Sprague Dawley rats were exposed to a 6 h hypobaria 24 h after mild TBI by the controlled cortical impact. Plasma and brain tissues were assessed for homocysteine levels, oxidative stress markers or glutathione metabolism, and behavioral deficits post-injury in the absence and presence of hypobaria exposure.Results:We found that hypobaria after TBI increased oxidative stress markers, altered homocysteine metabolism, and promoted glutathione oxidation. Increased glutathione metabolism was driven by differential upregulation of glutathione metabolizing genes. These changes correlated with increased anxiety-like behavior.Conclusion:These data provide evidence that hypobaria exposure after TBI increases oxidative stress and alters homocysteine elimination likely through enhanced glutathione metabolism. This pathway may represent a compensatory mechanism to attenuate free radical formation. Thus, hypobaria-induced enhancement of glutathione metabolism represents a potential therapeutic target for TBI management.

Faulty homocysteine recycling in diabetic retinopathy

BackgroundAlthough hyperglycemia is the main instigator in the development of diabetic retinopathy, elevated circulating levels of a non-protein amino acid, homocysteine, are also associated with an increased risk of retinopathy. Homocysteine is recycled back to methionine by methylenetetrahydrofolate reductase (MTHFR) and/or transsulfurated by cystathionine β-synthase (CBS) to form cysteine. CBS and other transsulfuration enzyme cystathionine-γ-lyase (CSE), through desulfuration, generates H2S. Methionine cycle also regulates DNA methylation, an epigenetic modification associated with the gene suppression. The aim of this study was to investigate homocysteine and its metabolism in diabetic retinopathy.MethodsHomocysteine and H2S levels were analyzed in the retina, and CBS, CSE and MTHFR in the retinal microvasculature from human donors with established diabetic retinopathy. Mitochondrial damage was evaluated in retinal microvessels by quantifying enzymes responsible for maintaining mitochondrial dynamics (fission-fusion-mitophagy). DNA methylation status of CBS and MTHFR promoters was examined using methylated DNA immunoprecipitation technique. The direct effect of homocysteine on mitochondrial damage was confirmed in human retinal endothelial cells (HRECs) incubated with 100 μM L-homocysteine.ResultsCompared to age-matched nondiabetic control human donors, retina from donors with established diabetic retinopathy had ~ 3-fold higher homocysteine levels and ~ 50% lower H2S levels. The enzymes important for both transsulfuration and remethylation of homocysteine including CBS, CSE and MTHFR, were 40–60% lower in the retinal microvasculature from diabetic retinopathy donors. While the mitochondrial fission protein, dynamin related protein 1, and mitophagy markers optineurin and microtubule-associated protein 1A/1B-light chain 3 (LC3), were upregulated, the fusion protein mitofusin 2 was downregulated. In the same retinal microvessel preparations from donors with diabetic retinopathy, DNA at the promoters of CBS and MTHFR were hypermethylated. Incubation of HRECs with homocysteine increased reactive oxygen species and decreased transcripts of mtDNA-encoded CYTB.ConclusionsCompromised transsulfuration and remethylation processes play an important role in the poor removal of retinal homocysteine in diabetic patients. Thus, regulation of their homocysteine levels should ameliorate retinal mitochondrial damage, and by regulating DNA methylation status of the enzymes responsible for homocysteine transsulfuration and remethylation, should prevent excess accumulation of homocysteine.

Abstract 433: Hydrogen Sulfide Protects the Heart Against Homocysteine-Induced Remodeling by Regulating Autophagy and Pyroptosis

Hydrogen sulfide (H 2 S), a byproduct of homocysteine (Hcy) transsulfuration, protects against adverse cardiac remodeling in diabetic cardiomyopathy, hyperhomocysteinemia (HHcy), and other cardiomyopathies. However, the underlying molecular mechanisms are unclear. Pyroptosis is a caspase 1-mediated cell death mechanism that promotes remodeling. It is inhibited by autophagy, a lysosomal degradation process that maintains cellular homeostasis. We hypothesized that H 2 S supplementation inhibits Hcy-induced pyroptosis by increasing autophagy in the heart. We treated CBS +/- mice (a model of HHcy) and WT mice with SG1002 (slow-releasing H 2 S donor) or control diet for 14 weeks and performed deep sequencing of their hearts (LV tissue). Ingenuity Pathway Analysis revealed SG1002 downregulated cardiac hypertrophy genes, plausibly by altering upstream regulators involved in pyroptosis and autophagy (IL-1β and mTOR). Western blot results showed that SG1002 suppresses mTOR in CBS +/- hearts (WT: 2.1±0.83, CBS: 10±2.4, CBS+SG: 3.8±2.3, WT+SG: 0.96±0.39) to induce autophagy. SG1002 decreases IL-1β (WT: 0.61±0.08, CBS: 0.88±0.07, CBS+SG: 0.32±0.01, WT+SG: 0.38±0.03) and inhibits caspase-1 activity to suppress Hcy-induced pyroptosis. NF-kB p65, which activates pyroptosis, is also downregulated by SG1002. We conclude that H 2 S could alleviate cardiac remodeling by inducing autophagy and inhibiting NF-kB-mediated pyroptosis. This is the first report that H 2 S controls cross-talk of cardiac pyroptosis and autophagy.

Hydrogen sulfide lowers hyperhomocysteinemia dependent on cystathionine γ lyase S-sulfhydration in ApoE-knockout atherosclerotic mice.

Background and PurposeHydrogen sulfide donors can block the cardiovascular injury of hyperhomocysteinemia. H2S also lowers serum homocysteine in rats with mild hyperhomocysteinemia, but the pharmacological mechanism is unknown. The present study investigated the mechanism(s) involved.Experimental ApproachApoE‐knockout mice were fed a Paigen diet and L‐methionine in drinking water for 16 weeks to create a mouse model of atherosclerosis with hyperhomocysteinemia. H2S donors (NaHS and GYY4137) were administered by intraperitoneal injection. We also assayed the H2S produced (by methylene blue assay and mito‐HS [H2S fluorescence probe]), cystathionine γ lyase (CSE) mRNA and protein expression, and CSE sulfhydration and nitrosylation and its activity.Key ResultsH2S donor treatment significantly lowered atherosclerotic plaque area, macrophage infiltration, and serum homocysteine level in the mouse model of atherosclerosis with co‐existing hyperhomocysteinemia. mRNA and protein levels of CSE, a key enzyme catalyzing homocysteine trans‐sulfuration, were down‐regulated with hyperhomocysteinemia, and CSE catalytic activity was inhibited. All these effects were reversed with H2S donor treatment. Hyperhomocysteinemia induced CSE nitrosylation, whereas H2S sulfhydrated CSE at the same cysteine residues. Nitrosylated CSE decreased and sulfhydrated CSE increased its catalytic and binding activities towards L‐homocysteine. Mutation of C252, C255, C307, and C310 residues in CSE abolished CSE nitrosylation or sulfhydration and prevented its binding to L‐homocysteine.Conclusions and ImplicationsSulfhydration or nitrosylation of CSE represents a yin/yang regulation of catalysis or binding to L‐homocysteine. H2S donor treatment enhanced CSE sulfhydration, thus lowering serum L‐homocysteine, which contributed in part to the anti‐atherosclerosis effects in ApoE‐knockout mice with hyperhomocysteinemia.

Age‐dependence of Methylation, Transsulfuration and Glutathione Synthesis Pathways in Human Frontal Cortex

Neuronal synthesis of the antioxidant glutathione (GSH) depends upon availability of cysteine, provided either by cellular uptake or by transsulfuration of homocysteine generated by the methionine cycle of methylation (Fig. 1). To evaluate age‐dependent changes in the relative contribution of these two cysteine sources, we measured redox and methylation metabolite levels in postmortem human frontal cortex from subjects across the lifespan. Levels of the transsulfuration intermediate cystathionine were significantly decreased in 60–80 yr‐old subjects, indicating that the balance between these two cysteine sources shifts to transsulfuration in older age. This shift was accompanied by decreased methionine and S‐adenosylmethionine, and an increase in homocysteine, indicating lower activity of the folate and B12‐dependent enzyme methionine synthase (MS), consistent with earlier finding of decrease MS expression with age. Cystathionine levels in 10 yr‐old autistic subjects were decreased, similar to those of 60 yr‐old subjects. In vitro studies with human neuronal cells showed that the pro‐inflammatory cytokine TNF‐alpha increased transsulfuration and inhibited MS activity in association with decreased cysteine uptake. Transsulfuration pathway enzymes cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL) both produce hydrogen sulfide, which inhibits oxidative respiration and increases glycolysis. This action decreases production of reactive oxygen species (ROS), limiting the demand for antioxidant. Together these observations illustrate how an age‐dependent increase in transsulfuration links antioxidant synthesis to methylation status in human brain. TNF‐alpha, whose levels increase with age, may be a regulator of transsulfuration, with important implications for epigenetic regulation during early brain development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correlation of Homocysteine Levels With Folate Acid, Cyanocobalamine, and Pyridoxine Serum Levels In Acute Infark Miocard Patients

Cardiovascular disease is the cause of the highest mortality rate in the world. Acute myocardial infarction is one of the 5 major manifestations of coronary heart disease and the highest mortality rate in Indonesia. Hyperhomocysteinemia is one of the risk factors for the occurrence of acute myocardial infarction. The metabolism of homocysteine is influenced by folate acid, cyanocobalamin, and pyridoxine. Enzimsistationin-β-synthetase and cystationin-γ-liase that catalyze the transsulfuration of homocysteine are highly depended on pyridoxine. This study aims to determine the association of homocysteine with folate acid, cyanocobalamin, and pyridoxine serum levels in acute myocardial infraction patients. Cross-sectional design study with 25 samples of acute myocardial infraction patients are selected randomly. The research was conducted from July to December 2016 in Biomedical laboratory Andalas University. Homocysteine, folate acid, cyanocobalamin, and pyridoxine levels were examined by the ELISA method. Data is analyzed by the Pearson correlation test. The mean result of examination of homocysteine level was 23.17±9.2 nmol/mL, pyridoxine level 128.44 ± 34.02 pmol/L, cyanocobalamine level 188.79 ± 10.23 pmol/L and folate acid level 2.72 ± pmol / L. Statistic analysis test found that there is a significant relationship of levels of homocysteine with pyridoxine (r = -0.530), but there is a very weak relationship with cyanocobalamine and folate acid (r = - 0.027 and - 0.003). There is a significant relationship of homocysteine levels with pyridoxine serum on acute myocardial infarction patients. And there is a very weak relationship of homocysteine levels with cyanocobalamine and folate acid.

313 - Age-dependent changes in human cortex antioxidant and methylation metabolic pathways and their implications for neurodegenerative disorders

Oxidative stress and impaired methylation, including increased levels of homocysteine (HCY), are well-recognized for their contribution to the pathogenesis of Alzheimer's disease (AD). Metabolic pathways for production of the antioxidant glutathione (GSH) and the methyl donor S-adenosylmethionine (SAM) are interrelated and connected by transsulfuration of homocysteine to cysteine (Fig. 1). These pathways are uniquely regulated with age in human brain and their dysfunction can result in neurological disorders manifested across the lifespan. Levels of antioxidant and methylation metabolites and individual vitamin B12 species were measured in postmortem human frontal cortex across the lifespan, along with mRNA levels of the folate and vitamin B12-dependent enzyme methionine synthase (MS), which converts homocysteine to methionine. In control subjects, expression of the MS gene (mtr) showed a remarkable 400-fold progressive decrease with age, in conjunction with alternative splicing that increases its sensitivity to oxidative stress inhibition. Levels of the MS cofactor methylcobalamin (methylB12) were >10-fold lower in 60-80 yo subjects vs. 0-20 yo subjects, accompanied by higher levels of HCY and lower levels of SAM, indicative of an age-dependent decrease in methylation. By contrast, levels of cystathionine decreased with age, indicative of increased transsufuration. Autism and schizophrenia subjects exhibited premature changes in MS expression, methylB12 and transsulfuration. Normal aging of human frontal cortex is associated with progressive changes in methylation and antioxidant metabolism, and deviations from this normal progression may be associated with AD. Providing metabolic support for brain methylation appears to be a promising approach to reducing the risk and progression of AD.

Evaluation of tracer labelled methionine load test in vitamin B-12 deficient adolescent women.

BackgroundMethionine loading test (MLT) has been used primarily to identify defects in transsulfuration of homocysteine in cystathionine beta synthase deficiency. It may not be as useful to evaluate remethylation pathway, in vitamin B-12 and folate deficiencies.ObjectiveWe used tracer isotope labelled MLT to interrogate transsulfuration and remethylation independently in vitamin B-12 deficiency.DesignWe studied vitamin B-12 deficient women with a tracer labelled MLT before and eleven months after treatment with vitamin B-12. The fractional contribution of [13C]homocysteine to breath CO2 was used as a measure of transsulfuration, and difference in the intracellular enrichment of [13C]methionine and that of [C2H3]methionine as a measure of remethylation of homocysteine. Combined pre- and post-treatment results were analyzed to investigate the association between plasma vitamin B-12 concentrations and measures of homocysteine metabolism.ResultsThe subjects were 17 years old, with a BMI of 19.4 kg/m2. Treatment with vitamin B-12, 2µg/day increased plasma B-12 from 93 (78.7, 106.2) [median (25th, 75th centiles)] to 161.5 (125.5, 226.2) pmol/L; 44% were below <150pmol/L after treatment. Fasting homocysteine concentration was significantly lower and that of cysteine higher in subjects with B-12 levels >150pmol/L. The tracer estimated transsulfuration of homocysteine was lower and remethylation higher with B-12 levels >150pmol/L when compared with those <150pmol/L.ConclusionsThe tracer labelled MLT in combination with fasting parameters is a robust way to estimate parameters of methionine metabolism and can be used in the field where prime-constant rate infusion studies cannot be done efficiently.

Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship With Insulin Resistance.

Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown. To investigate whole body methionine and homocysteine kinetics and the effects of insulin in essential hypertension. Eight hypertensive male subjects and six male normotensive controls were infused with l-[methyl-2H3,1-13C]methionine for 6 hours. In the last 3 hours a euglycemic, hyperinsulinemic clamp was performed. Steady-state methionine and homocysteine kinetics were determined in postabsorptive and hyperinsulinemic conditions. Postabsorptive hypertensive subjects had elevated homocysteine concentrations (+30%, P = 0.035) and slightly (by 15% to 20%) but insignificantly lower methionine rates of appearance (Ras) (P = 0.07 to P = 0.05) and utilization for protein synthesis (P = 0.06) than postabsorptive normotensive controls. Hyperinsulinemia suppressed methionine Ra and protein synthesis, whereas it increased homocysteine trans-sulfuration, clearance, and methionine transmethylation (the latter only in the normotensive subjects). However, in the hypertensive subjects trans-sulfuration was significantly lower (P < 0.05) and increased ~50% less [by +1.59 ± 0.34 vs +3.45 ± 0.52 µmol/kg lean body mass (LBM) per hour, P < 0.005] than in normotensive controls. Homocysteine clearance through trans-sulfuration was ~50% lower in hypertensive than in normotensive subjects (P < 0.005). In the hypertensive subjects, insulin-mediated glucose disposal was ~45% lower (460 ± 44 vs 792 ± 67 mg/kg LBM per hour, P < 0.0005) than in normotensive controls and was positively correlated with the increase of trans-sulfuration (P < 0.0015). In subjects with essential hypertension, hyperhomocysteinemia is associated with decreased homocysteine trans-sulfuration and probably represents a feature of insulin resistance.

Exendin-4 regulates redox homeostasis in rats fed with high-fat diet.

Non-alcoholic fatty liver disease (NAFLD) is associated with increased plasma homocysteine level, which is caused by down-regulation of hepatic cystathionine beta-synthase (CBS) activity. CBS catalyzes the first step in the transsulfuration of homocysteine to cysteine, which contributes ∼50% of the cysteine required for hepatic biosynthesis of glutathione (GSH), the most abundant antioxidant in cells. As the glucagon-like peptide-1 (GLP-1) receptor agonists (e.g. exendin-4) effectively reverse hepatic steatosis, the effect of exendin-4 on both homocysteine and redox status was investigated in the livers of rats fed with high-fat diet (HFD). It was found that HFD down-regulated CBS protein expression, which was probably due to induction of rno-miR-376c expression in the liver. The level of GSH was markedly reduced, whereas the level of malonydialdehyde, an indicator of lipid peroxidation, was significantly increased in the livers of rats fed with HFD. Exendin-4 treatment increased hepatic CBS protein and GSH levels, and reduced malonydialdehyde level in hyperlipidemic rats. Our findings suggest that GLP-1 receptor agonists have beneficial effects on redox homeostasis in NAFLD.

Abstract 689: Cystathionine-Beta-Synthase Deficiency Induces Cardiac Hypertrophy and Contributes to Diminished Fatty Acid Oxidation in Mice with Diet-Induced Obesity

Obesity-related cardiac lipid accumulation is associated with lipotoxicity and dysfunction. Cysteine is required for the synthesis of the antioxidant glutathione, which can be supplied by the transsulfuration of homocysteine by cystathionine-beta-synthase (Cbs). Cbs+/- mice with diet-induced obesity had greater glucose intolerance and lipotoxicity in the heart. Our objective was to determine the functional effects and mechanisms of cardiac lipotoxicity in Cbs+/- mice with diet-induced obesity. Cbs+/- and Cbs+/+ mice were fed a control diet or a high-fat diet (HFD) from weaning for 20 weeks. As expected, Cbs+/+ and Cbs+/- mice fed the HFD had greater final body weights, visceral (retroperitoneal and epididymal) and subcutaneous (inguinal) adiposity compared to mice fed the control diet. Cbs+/- mice had greater heart weights accompanied by higher concentrations of long chain polyunsaturated fatty acids arachidonic acid, 20:4n6 (AA) and docosahexaenoic acid, 22:6n3 (DHA) in the heart compared to Cbs+/+ mice. Mice fed the HFD had higher AA, but lower DHA concentrations in the heart compared to mice fed the control diet, with the greatest effect in Cbs+/- mice. Isolated working hearts revealed a reduced heart rate and cardiac output in Cbs+/- mice fed the control diet compared to Cbs+/+ mice. Independent of diet, Cbs+/- mice also had reduced aortic flow compared to Cbs+/+ mice, with a higher coronary flow only in Cbs+/- mice fed the HFD. Working hearts also revealed that Cbs+/- mice had lower palmitate oxidation rates compared to Cbs+/+ mice, with higher palmitate oxidation and glycolysis rates in mice fed the HFD compared to mice fed the control diet. Cbs+/- mice had a lower ratio of phosphorylated-AMP activated protein kinase alpha (AMPKα)/AMPKα expression (regulator of cellular energy) in heart compared to Cbs+/+ mice, and this occurred to a greater extent in those fed the HFD. Furthermore, we observed a higher ratio of collagen type 1(COL1α1)/ collagen type 3 (COL3α1) expression (implicated in myocardial stiffness) in heart, only in Cbs+/- mice fed the HFD. Collectively, these findings suggest that cardiac lipotoxicity in Cbs+/- mice with obesity is associated with cardiac hypertrophy, impaired cardiac fatty acid metabolism, and cardiac dysfunction.

Dysfunctional Cardiac Fatty Acid Metabolism in Cystathionine‐beta‐Synthase +/‐ Mice with Obesity

Obesity‐related cardiac lipid accumulation is associated with toxicity and dysfunction. Cysteine is required for the synthesis of the antioxidant glutathione, supplied by the transsulfuration of homocysteine by cystathionine‐beta‐synthase (Cbs). Cbs+/‐ mice with diet‐induced obesity have reduced cardiac glutathione, and greater glucose intolerance and cardiac lipotoxicity. Our objective was to determine the functional effects and mechanisms of cardiac lipotoxicity in Cbs+/‐ mice with diet‐induced obesity. Cbs+/‐ and Cbs+/+ mice were fed a control‐diet (CD) or a high‐fat diet (HFD) from weaning for 20 weeks. Cbs+/‐ mice fed CD had less visceral adiposity compared to Cbs+/+ mice. Ex vivo working heart assessments revealed greater palmitate oxidation in mice fed the HFD than CD. Independent of diet, Cbs+/‐ mice had reduced palmitate oxidation compared to Cbs+/+ mice. Cbs+/‐ mice had larger hearts and more arachidonic acid (AA) and docosahexaenoic acid (DHA) concentrations in the heart than Cbs+/+ mice. Mice fed the HFD had higher AA, but lower DHA concentrations in the heart than mice fed the CD; effects greatest in Cbs+/‐ mice. A lower ratio of phosphorylated‐AMPKα/AMPKα expression was observed in Cbs+/‐ than Cbs+/+ mice, and this occurred to a greater extent in those fed the HFD. Cbs+/‐ mice with diet‐induced obesity have altered cardiac fatty acid metabolism and larger hearts, further supporting a role for Cbs in obesity‐related cardiac lipotoxicity.Supported by NSERC and CIHR

Quercetin Increases Hepatic Homocysteine Remethylation and Transsulfuration in Rats Fed a Methionine-Enriched Diet.

This study was aimed at investigating the effects of quercetin on mRNA expression and activity of critical enzymes in homocysteine metabolism in rats fed a methionine-enriched diet. Rats were fed for 6 weeks the following diets, that is, control, 0.5% quercetin, 1.0% methionine, and 1.0% methionine plus 0.5% quercetin diets. Serum homocysteine was significantly increased after methionine treatment and decreased after the addition of quercetin. The mRNA expression of methionine synthase was significantly increased after methionine or methionine plus quercetin supplementation, while its enzymatic activity was significantly increased after methionine plus quercetin supplementation. The mRNA expression and enzymatic activity of cystathionine β-synthase and cystathionine γ-lyase were upregulated after quercetin, methionine, or quercetin plus methionine treatment and a more significant increase was observed for hepatic cystathionine β-synthase in the methionine plus quercetin treated rats, suggesting an interaction between methionine and quercetin. Meanwhile, hepatic ratio of S-adenosylmethionine to S-adenosylhomocysteine was significantly decreased in response to methionine supplementation and normalized after the addition of quercetin. It is concluded that quercetin reduces serum homocysteine by increasing remethylation and transsulfuration of homocysteine in rats exposed to a methionine-enriched diet.

Abstract 278: Enhanced Cystathionine ß-Synthase Nitration in Diet-Induced Hyperhomocysteinemic Rats Accelerates the Progression of Hyperhomocysteinemia

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases,which may be caused by genetic deficiencies in enzymes responsible for remethylation or transsulfuration of homocysteine, nutritional deficiencies in vitamins, or excessive intakes of methionine. Since transsulfuration pathway is the only irreversible pathway in homocysteine metabolism, its key enzyme cystathionine β-synthase is vital in terminal removement of homocysteine. In addition, Studies have observed that enhanced nitrative stress in hyperhomocysteinemic rats could induce cardiovascular injuries, and peroxynitrite stimulation in vitro resulting in decreased bioactivity of cystathionine β-synthase. Therefore, to determine the contribution of nitrative stress to progression of hyperhomocysteinemia, and how it is related to cystathionine β-synthase, we used three groups of rats (Con with normal diet, HHcy with 2.5% methionine diet, HHcy+FeTMPyP with 2.5% methionine diet +FeTMPyP) to observe the effects of nitrative stress on cystathionine β-synthase. Results showed that in diet-induced hyperhomocysteinemic rats, tHcy levels and nitrative stress increased, surprisingly, pretreatment with peroxynitrite scavenger FeTMPyP ameliorated the level of tHcy as well as nitrative stress. Further experiments showed cystathionine β-synthase bioactivity in HHcy rats was less potent than other groups yet described, and the level of cystathionine β-synthase nitration was the most significant,while the entire trends reversed after FeTMPyP pretreatment. In conclusion, these results highlight for the first time that in diet-induced hyperhomocysteinemic rats, cystathionine β-synthase bioactivity reduction is not just a traditional etiologic factor, but rather an outcome of hyperhomocysteinemia, and resulting from the high level of nitrative stress caused by hyperhomocysteinemia, the cystathionine β-synthase nitration plays a mutual role in hyperhomocysteinemia development via causing the metabolic disorder of homocysteine. These findings may shed a novel light on the homocystine-lowering target of hyperhomocysteinemia.

Activation of transsulfuration pathway by salvianolic acid a treatment: a homocysteine-lowering approach with beneficial effects on redox homeostasis in high-fat diet-induced hyperlipidemic rats

BackgroundElevated homocysteine is a cardiovascular risk factor in hyperlipidemia. Transsulfuration pathway provides an endogenous pathway for homocysteine conversion to antioxidant glutathione (GSH). Salvianolic acid A (Sal A) contains two molecules of caffeic acid and one molecule of danshensu that is capable of enhancing homocysteine transsulfuration, which led to the hypothesis that Sal A has activatory effect on transsulfuration pathway and this effect may have beneficial effects on both homocysteine and redox status in hyperlipidemia.Methods and resultsTo test this hypothesis, we developed a rat model of hyperlipidemia induced by high-fat diet for 16 weeks, during which rats were treated with 1 mg/kg salvianolic acid A (Sal A) for the final 4 weeks. Activities of key enzymes and metabolite profiling in the transsulfuration pathway revealed that hyperlipidemia led to elevated plasma homocysteine levels after 16-week dietary treatment, which was associated with reduced activities of homocysteine transsulfuration enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The impaired transsulfuration pathway prevented homocysteine transsulfuration to cysteine, resulting in cysteine deficiency and subsequent reduction in GSH pool size. The redox status was altered in the setting of hyperlipidemia as indicated by GSH/GSSG ratio. Sal A treatment increased hepatic CBS and CSE activities, which was associated with reduced accumulation in circulating homocysteine levels and attenuated decline in hepatic cysteine content in hyperlipidemic rats. Sal A also led to an increase in GSH pool size, which subsequently caused a restored GSH/GSSG ratio. The activatory effect of Sal A on CBS was also observed in normal rats and in in vitro experiment.ConclusionOur results suggest that activation of transsulfuration pathway by Sal A is a promising homocysteine-lowering approach that has beneficial effects on redox homeostasis in hyperlipidemic settings.

Dysfunctional Cardiac Lipid Metabolism in Cystathionine-Beta-Synthase Deficient Mice With Diet-Induced Weight Gain

Obesity is characterized by ectopic cardiac lipid deposition. Excess cardiac lipid accumulation is associated with lipotoxicity and eventually cardiac dysfunction. The molecular mechanisms linking obesity and the development of cardiac lipotoxicity are unknown. Cysteine is a sulfur-containing amino acid required for synthesis of the antioxidant glutathione. Cysteine is supplied by the diet or the transsulfuration of homocysteine by cystathionine-beta-synthase (Cbs). We previously reported mice heterozygous for targeted disruption of Cbs (Cbs +/-) with diet-induced weight gain and glucose intolerance have enhanced cardiac lipotoxicity. We hypothesize that Cbs +/- mice diet-induced weight gain have altered myocardial fatty acid uptake, activation, and oxidation. To address our hypothesis, Cbs +/+ and Cbs +/- mice were fed a high-fat diet (HFD) from weaning for 20 weeks to induce weight gain and glucose intolerance. Cbs +/- mice fed HFD had higher fasting blood glucose concentrations (P<0.01) and greater glucose intolerance (P<0.01) than Cbs +/+ mice fed the HFD. Cbs +/+ and Cbs +/- mice fed the HFD had higher fasting insulin concentrations (P<0.01) compared to mice fed chow diet. Cbs +/- mice had lower total adiponectin concentrations (P<0.06) in plasma compared to Cbs +/+ mice. An interaction was observed between diet and genotype for high molecular weight (HMW) adiponectin concentrations in plasma, with lower HMW adiponectin concentrations (P<0.01) in Cbs +/- mice than Cbs +/+ mice fed chow diet. When assessing the heart, Cbs +/- mice had higher heart weights (P<0.01) compared to Cbs +/+ mice. Interventricular septum thickness in Cbs +/+ and Cbs +/- fed HFD was greater during both end systole (P<0.01) and diastole (P<0.01). Left ventricle performance was also higher for measurements such as ejection fraction (P<0.01) and fractional shortening (P<0.01) than in chow fed mice. This was accompanied by increased expression of markers of fatty acid uptake (CD36, P<0.01); activation (long-chain acyl CoA synthetase 1, P<0.01); and oxidation (carnitine palmitoyltransferase 1 and acyl CoA oxidase, P<0.01) in heart. These findings demonstrate a novel role for Cbs in the pathology of cardiac lipotoxicity.

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H2S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H2S in kidney fibrosis remains to be defined. Here, we investigated the role of H2S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H2S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H2S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H2S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H2S in UO-induced kidney fibrosis, suggesting that H2S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.