Remethylation Pathway Research Articles

32 Overview over methionine metabolism: Implications in swine nutrition and health

Abstract Methionine (Met) can be referred to as a functional amino acid (AA) because besides serving as a building block for protein bio-syntheses, it also performs many other biochemical and physiological functions in swine body, simply due to its unique chemical structure and cellular metabolism. As a sulfur-containing AA (SAA), a Met molecule, as its name indicates, contains a methyl group covalently bonded to a sulfur atom via a thioether bond. In terms of its functions, first of all, Met is the initiator for virtually all protein bio-syntheses. Secondly, Met can be easily oxidized to a sulfoxide via a covalent modification, and the resulting Met sulfoxide can be readily reduced back to Met. In the case of protein-bound Met, both Met and cysteine (Cys) residues are most susceptible to oxidation and this nature can protect many proteins from oxidative damages. The biochemical metabolism of Met involves transmethylation, remethylation, transsulfuration, and transamination pathways. The initial step of transmethylation is generation of S-adenosylmethionine (SAM) which is an essential methyl donor in a myriad of metabolic events that include the methylation of DNA, RNA, proteins, phospholipids, and some small molecules. These various methylations dictate the replicational, transcriptional and translational fidelities, mismatch repair, chromatin modelling, epigenetic modification, imprinting, as well as detoxification of xenobiotics, which are all topics of great interest and importance in swine health and production performance. SAM is also a key metabolite to produce homocysteine which can be remethylated back to Met via the remethylation pathway (known as methyl cycle). The transsulfuration pathway firstly converts homocysteine to cystathionine via a condensation with serine, and then cystathionine is cleaved to Cys plus α-ketobutyrate and ammonia. Transsulfuration pathway is irreversible, and that is why Met is an indispensable AA whose deficiency cannot be remedied by Cys, whereas Cys is dispensable when more Met is provided. Because of this metabolic interconversion between Met and Cys in the body, these two are generally grouped together as SAA (Met + Cys) in swine nutrition practice. That said, when tissue Met concentration is too great, Met can be catabolized via its transamination pathway which is minor and SAM-independent. Met toxicity is mainly caused by methanethiol and H2S, two major toxic metabolites in the transamination pathway. Synthesized from Met, a myriad of proteins and peptides are involved in the functioning of immune system for maintaining animal health. For example, SAM is also a precursor for synthesis of polyamines which can enhance immune cell proliferation during an immune challenge. Cys and its derivatives (e.g., glutathione and taurine), in addition to their antioxidative capacity, can ameliorate inflammation during an immune response. In short, Met is a unique SAA that has numerous indispensable biological functions for supporting swine health and production performance.

272 The Effect of Increasing Standardized Ileal Digestible Methionine Intake on Whole-Body Nitrogen Retention and Plasma Amino Acids, Homocysteine, and Glutathione of Gilts in Late Gestation

Abstract Gestating gilts (n = 70; 166 ± 13 kg initial body weight at d 31 of gestation) were used to determine the dietary standardized ileal digestible (SID) Methionine (Met) content required to optimize whole-body nitrogen (N) retention versus the appearance of biomolecules derived via Met metabolism. On d 102 of gestation, gilts were randomly assigned to one of seven dietary treatments that provided between 50 and 150% of estimated SID Met requirements for late gestation gilts (0.10 to 0.30% SID Met). All other indispensable amino acids were supplied at least 20% above estimated requirements (NRC, 2012). A N balance (total urine collection and fecal grab sampling) was conducted for each gilt between days 109 and 112 of gestation, blood samples were collected on day 109 after a 24-h fasting period for analyses of plasma amino acids, homocysteine (Hcys), and reduced glutathione (GSH). Contrast statements were used to determine linear and quadratic effects of dietary SID Met levels. Linear and quadratic broken-line and polynomial quadratic models were used to determine the optimum level of SID Met for whole-body N retention and plasma concentrations of Hcys and GSH. Whole-body N retention increased with increasing dietary Met (linear and quadratic; P < 0.001; Table 1). Plasma concentrations of Hcys increased with increasing dietary Met (linear; P < 0.05). Glutathione tended to decrease and then increase at dietary SID Met greater than 0.20% (quadratic; P = 0.061; Table 2). Plasma glutamine increased with increasing dietary SID Met (linear; P < 0.01). Histidine increased then decreased at dietary SID Met greater than 0.20% (quadratic; P < 0.05), other amino acids were not influenced by dietary treatment. Linear and quadratic broken-line models described a lower breaking point for whole-body N retention, plasma Hcys, and plasma GSH compared with the quadratic polynomial model (0.154% and 0.155% vs. 0.226%, 0.185% and 0.188% vs. 0.328%, and 0.114% and 0.124% vs. 0.224% SID Met, respectively). The Bayesian information criterion (BIC) indicated that the quadratic polynomial model best fit the data versus the linear and quadratic broken-line models for plasma GSH (BIC 317.9 vs. 320.3 and 322.6, respectively; Graphic 1). However, the fits were similar among models for whole-body N retention (BIC 385.2 vs. 384.6 and 386.9, respectively; Graphic 2) and Hcys (BIC 465.5 vs. 465.6 and 467.9, respectively; Graphic 3). Based on the best fit (quadratic polynomial) model 0.226% SID Met is required to optimize whole-body N retention for late gestation gilts which is greater than that recommended by the NRC (2012; 0.20% SID Met). It appears that gilts have further capacity for Met transmethylation since the plasma Hcys breakpoint occurred at a greater dietary SID Met level than for whole-body N retention, though the impact on transsulfuration and remethylation pathways cannot be discounted. Therefore, greater feeding levels of SID Met might be required to maximize both protein and non-protein utilization of Met.

How Different Dietary Methionine Sources Could Modulate the Hepatic Metabolism in Rainbow Trout?

In aquafeeds in which plant proteins are used to replace fishmeal, exogenous methionine (Met) sources are demanded to balance the amino acid composition of diets and meet the metabolic fish requirements. Nonetheless, since different synthetic Met sources are commercially available, it is important to determine their bioavailability and efficacy. To address this issue, we conducted a two-month feeding trial with rainbow trout (Oncorhynchus mykiss), which were fed diets supplemented with five different forms of Met: Met-Met, L-Met, HMTBa, DL-Met, and Co DL-Met. No differences in growth performance were found in trout fed with different Met forms, but changes in the whole-body composition were found. In particular, Met-Met and L-Met promoted a significant body lipid reduction, whereas the protein retention was significantly increased in fish fed with HMTBa and Co DL-Met. The latter affected the hepatic Met metabolism promoting the trans-sulfuration pathway through the upregulation of CBS gene expression. Similarly, the L-Met enhanced the remethylation pathway through an increase in BHMT gene expression to maintain the cellular demand for Met. Altogether, our findings suggest an optimal dietary intake of all tested Met sources with similar promoting effects on fish growth and hepatic Met metabolism. Nevertheless, the mechanisms underlying these effects warrant further investigation.

The betaine-dependent remethylation pathway is a homocysteine metabolism pathway associated with the carnivorous feeding habits of spiders.

Homocysteine (Hcy) is a sulfur-containing amino acid derived from the essential amino acid methionine (Met). Circulating levels of Hcy in animals can be increased by feeding on Met-enriched diets, which is generally considered harmful. Spiders are one of the largest groups of obligate carnivores and feed on animals high in protein and Met. We analyzed the Hcy metabolism pathways in 18 species of 3 taxa (Mammalia, Insecta, and Arachnida) and found that the betaine-dependent remethylation pathway (BRP) was present in all carnivorous arachnid species and mammals but absent in insects and red spider mites. We then studied the Hcy metabolism pathway in Pardosa pseudoannulata. In P. pseudoannulata, Hcy is metabolized through the transsulfuration pathway, BRP, and S-methylmethionine-dependent remethylation pathway. Because of a prior duplication event of the betaine homocysteine S-methyltransferase (BHMT) gene in the BRP, BHMTa and BHMTb are present in tandem in the genome of P. pseudoannulata. The high expression levels of BHMTa and its high abundance in detoxification tissues indicate that it plays an important role in the BRP; the ability of BHMTa and BHMTb to remethylate Hcy using betaine as substrate was similar. Compared with other Hcy metabolic enzyme genes, BHMT responded quickly to the application of Hcy or betaine. In sum, the BRP is important in Hcy metabolism in P. pseudoannulata and in other spider species.

Methionine synthase deficiency: Variable clinical presentation and benefit of early diagnosis and treatment.

Methionine synthase deficiency (cblG complementation group) is a rare inborn error of metabolism affecting the homocysteine re-methylation pathway. It leads to a biochemical phenotype of hyperhomocysteinemia and hypomethioninemia. The clinical presentation of cblG is variable, ranging from seizures, encephalopathy, macrocytic anemia, hypotonia, and feeding difficulties in the neonatal period to onset of psychiatric symptoms or acute neurologic changes in adolescence or adulthood. Given the variable and nonspecific symptoms seen in cblG, the diagnosis of affected patients is often delayed. Medical management of cblG includes the use of hydroxocobalamin, betaine, folinic acid, and in some cases methionine supplementation. Treatment has been shown to lead to improvement in the biochemical profile of affected patients, with lowering of total homocysteine levels and increasing methionine levels. However, the published literature contains differing conclusions on whether treatment is effective in changing the natural history of the disease. Herein, we present five patients with cblG who have shown substantial clinical benefit from treatment with objective improvement in their neurologic outcomes. We demonstrate more favorable outcomes in our patients who were treated early in life, especially those who were treated before neurologic symptoms manifested. Given improved outcomes from treatment of presymptomatic patients, cblG warrants inclusion in newborn screening.

Serum Homocysteine Level as A Marker of Erectile Dysfunction

Background: The Fourth International Consultation on Sexual Medicine defined erectile dysfunction (ED) as a consistent or recurrent inability to attain and/or maintain penile erection sufficient for sexual satisfaction homocysteine (Hcy) is a toxic, nonproteogenic sulfur-containing amino acid synthesized from dietary methionine in the liver and is metabolized either via trans-sulfuration or remethylation pathway. Objective: To investigate serum Hcy levels in ED patients as compared to those in healthy controls, and to explore its correlation with severity of ED. Patients and methods: This study consisted of 30 ED-affected men and 50 age-matched controls who were free from ED. All participants were recruited from the Andrology Outpatient Clinic of Mansoura University Hospital, between October 2019 and September 2020. Results: Serum Hcy was significantly higher in ED cases compared to controls (P 15 μmol/L). On the other hand, 6 out of 50 controls (12 %) had elevated level of Hcy. Elevation of Hcy levels was associated with an increased risk of ED, and that a cut-off value of 8.28 µmol/L was able to detect ED patients with a corresponding sensitivity and specificity of 90.0% and 78% respectively. Conclusion: Elevated Hcy is associated with ED and may represent an important risk factor and play a pathophysiologic role in ED. Hence, Hcy-lowering agents, such as folic acid and vitamin B12, seem reasonable choices for the prevention and management of this condition.

Increase of Homocysteinemia/Hydrogen Sulfide (Hcy/H2S) Ratio Raises Cardiovascular Injuries

Increased Homocysteine Levels (HHcy) is an independent risk factor for atherosclerosis. On the other hand, hydrogen sulfide (H2S) exerts a protection against cardiovascular injuries. On the contrary, accumulating evidences showed that downregulation of defective catabolism of HHcy, with reduced H2S synthesis, is involved in the pathogenesis of a variety of cardiovascular diseases. In that occurrence, the detrimental actions on cardiovascular structures performed by HHcy are added to the negative consequences of reduced H2S (in part unlike each HHcy) on cardiovascular system. Therefore, when the reduced re-methylation pathway of Hcy towards Met (resulting in HHcy) is contemporarily added to the decreased trans-sulfuration pathway (inducing a reduction of H2S synthesis) cardiovascular impairment significantly increases.

Genetics and Epigenetics of One-Carbon Metabolism Pathway in Autism Spectrum Disorder: A Sex-Specific Brain Epigenome?

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition affecting behavior and communication, presenting with extremely different clinical phenotypes and features. ASD etiology is composite and multifaceted with several causes and risk factors responsible for different individual disease pathophysiological processes and clinical phenotypes. From a genetic and epigenetic side, several candidate genes have been reported as potentially linked to ASD, which can be detected in about 10–25% of patients. Folate gene polymorphisms have been previously associated with other psychiatric and neurodegenerative diseases, mainly focused on gene variants in the DHFR gene (5q14.1; rs70991108, 19bp ins/del), MTHFR gene (1p36.22; rs1801133, C677T and rs1801131, A1298C), and CBS gene (21q22.3; rs876657421, 844ins68). Of note, their roles have been scarcely investigated from a sex/gender viewpoint, though ASD is characterized by a strong sex gap in onset-risk and progression. The aim of the present review is to point out the molecular mechanisms related to intracellular folate recycling affecting in turn remethylation and transsulfuration pathways having potential effects on ASD. Brain epigenome during fetal life necessarily reflects the sex-dependent different imprint of the genome-environment interactions which effects are difficult to decrypt. We here will focus on the DHFR, MTHFR and CBS gene-triad by dissecting their roles in a sex-oriented view, primarily to bring new perspectives in ASD epigenetics.

Clinical, Biochemical, Radiological, and Genetic Profile of Patients with Homocysteine Remethylation Pathway Defect and Spastic Paraplegia.

Objectives:The objective of this study is to describe the clinical, biochemical, radiological, and genetic profile of patients presenting with progressive spastic paraparesis due to homocysteine remethylation pathway defect.Methods:This was a retrospective study conducted by reviewing the medical records of patients with serum homocysteine levels >50 μmol/L between January 2015 and January 2019 at our hospital. We included patients presenting with progressive spastic paraparesis, having serum homocysteine >50 μmol/L with low or normal blood methionine suggesting disorders of homocysteine remethylation. Demographic details, clinical manifestations, biochemical abnormalities, neuroimaging findings, and genetic profile were analyzed.Results:A total of seven patients (M: F = 5:2) fulfilled the study eligibility criteria. The mean age at onset of the disease was 13.4 ± 2.4 years (range: 9–17 years). Spastic paraparesis was the presenting manifestation in 4/7 (57.1%) patients. Other manifestations included cognitive decline, poor scholastic performance, behavioral disturbances, seizures, and spastic bladder. Severe hyperhomocysteinemia (>100 μmol/L) was noted in 6/7 (85.7%) patients with median levels of serum homocysteine being 185.7 μmol/L (range: 85.78–338.5 μmol/L). Neuroimaging showed parieto-occipital predominant leukoencephalopathy in 5/7 (71.4%) and diffuse cerebral atrophy in 1/7 (14.2%). Genetic analysis in three patients revealed pathogenic missense variants c.459C >G (p.Ile153Met), c.973C >T (p.Arg325Cys), and c.1031G >T (p.Arg344Met) in MTHFR gene. All the patients received vitamin B12 (injection and oral), folic acid, and pyridoxine and two patients received betaine. At the last follow-up of a median duration of 12 months, there was a good clinical and biochemical response with reduction in the median value of serum homocysteine by 77.5 μmol/L.Conclusion:Evaluation of serum homocysteine and blood methionine in adolescents presenting with progressive spastic paraparesis gives clue to a treatable homocysteine remethylation disorders.

Betaine insufficiency and reduced betaine dependent remethylation are associated with hyperhomocysteinemia, oxidative stress and mitochondrial damage in early Polycystic Kidney Disease

Polycystic kidney disease (PKD) is the most common monogenic kidney disorder and an important cause of end stage renal disease. Vascular abnormalities are the most important non‐cystic complication and contribute to renal disease progression. Homocysteine (Hcy)‐mediated endothelial dysfunction (ED) and oxidative stress are evident early on, but the underlying mechanisms remain unknown. We hypothesized that abnormalities in Hcy metabolism are responsible for the increased levels, leading to Hcy‐induced ED from early stages of PKD.MethodsWe investigated the Hcy metabolic pathway (transulfuration/remethylation fig. 1) and downstream effects ‐main sources of renal tubular (RT) and endothelial cell (EC) reactive oxygen species (ROS), NADPH oxidase 4, (NOX4) and mitochondria‐ in 4‐week‐old PCK and Sprague‐Dawley (SD) control rats (n=12 each). Kidneys were harvested, frozen in liquid nitrogen, or preserved in formalin for metabolomics and ex‐vivo studies. Twenty‐four‐hour urine and terminal blood samples were collected for metabolite analysis and chemistries. Plasma folic acid, vitamin B12, and Hcy were determined by ELISA. Hcy pathway metabolites (betaine, dimethylglycine) were determined by 1HNMR. Endothelial NO (eNOS) and oxidative stress were assessed by double immunofluorescence staining for CD31/eNOS and CD31/NOX4, respectively. RT and EC mitochondrial structure was assessed by electron microscopy.ResultsSerum creatinine and BUN remained unchanged, yet kidney weight/body weight ratio were increased in PCK. eNOS immunoreactivity was lower in PCK vs SD. Plasma Hcy and urine excretion of methyl donor betaine were elevated in PCK, and Hcy positively correlated with urine betaine. Plasma betaine was similar between the groups, but its tissue concentration was lower in PCK, which may lead to betaine insufficiency, impaired betaine dependent remethylation, and hyperhomocysteinemia. Contrarily, tissue glutathione concentration was higher in PCK vs SD, and plasma folate and vitamin B12 were similar, arguing against a defect in the transsulfuration or folate‐dependent remethylation pathways. RT NOX4 expression and immunoreactivity were increased in PCK compared to SD, in association with renal oxidative stress. PCK had increased number of CD31/NOX4 positive cells in peritubular capillaries, indicating increased EC ROS production. Collecting duct and EC mitochondria were swollen and showed cristae remodeling.ConclusionIn PKD, early elevation in Hcy is likely related to betaine insufficiency and impaired betaine‐dependent remethylation may be responsible for the early elevation of Hcy. NOX4 and mitochondria abnormalities may be important contributors to oxidative stress and disease severity. Our data suggests these may not only be early vascular disease markers, but also play an important pathogenic role in vascular complications associated with PKD, and contribute to renal disease progression.Support or Funding InformationR25‐DK101405, NIDDK P30 DK090728, NIDDK P30 DK 106912This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Betaine-rich sugar beet molasses protects from homocysteine-induced reduction of survival in Caenorhabditis elegans.

Homocysteine (Hcy) in humans represents a blood-borne biomarker which predicts the risk of age-related diseases and mortality. Using the nematode Caenorhabditis elegans, we tested whether feeding betaine-rich sugar beet molasses affects the survival under heat stress in the presence of Hcy, in spite of a gene loss in betaine-homocysteine methyltransferase. Knockdown of the genes relevant for remethylation or transsulfuration of Hcy was achieved by RNA interference (RNAi). Survival assay was conducted under heat stress at 37°C and Hcy levels were determined by enzyme-linked immunosorbent assay. Addition of 500mg/l betaine-rich sugar beet molasses (SBM) prevented the survival reduction that was caused by exposure to Hcy at 37°C. Although SBM was no longer capable of reducing Hcy levels under RNAi versus homologues for 5, 10-methylenetetrahydrofolate reductase or cystathionine-β-synthase, it still enabled the survival extension by SBM under exposure to Hcy. In contrast, RNAi for the small heat shock protein hsp-16.2 or the foxo transcription factor daf-16 both prevented the extension of survival by betaine-rich molasses in the presence of Hcy. Our studies demonstrate that betaine-rich SBM is able to prevent survival reduction caused by Hcy in C. elegans in dependence on hsp-16.2 and daf-16 but independent of the remethylation pathway.

Vitamin B-12 and liver activity and expression of methionine synthase are decreased in fetuses with neural tube defects

The risk of neural tube defects (NTDs) is influenced by nutritional factors and genetic determinants of one-carbon metabolism. A key pathway of this metabolism is the vitamin B-12- and folate-dependent remethylation of homocysteine, which depends on methionine synthase (MS, encoded by MTR), methionine synthase reductase, and methylenetetrahydrofolate reductase. Methionine, the product of this pathway, is the direct precursor of S-adenosylmethionine (SAM), the universal methyl donor needed for epigenetic mechanisms. This study aimed to evaluate whether the availability of vitamin B-12 and folate and the expression or activity of the target enzymes of the remethylation pathway are involved in NTD risk. We studied folate and vitamin B-12 concentrations and activity, expression, and gene variants of the 3 enzymes in liver from 14 NTD and 16 non-NTD fetuses. We replicated the main findings in cord blood from pregnancies of 41 NTD fetuses compared with 21 fetuses with polymalformations (metabolic and genetic findings) and 375 control pregnancies (genetic findings). The tissue concentration of vitamin B-12 (P=0.003), but not folate, and the activity (P=0.001), transcriptional level (P=0.016), and protein expression (P=0.003) of MS were decreased and the truncated inactive isoforms of MS were increased in NTD livers. SAM was significantly correlated with MS activity and vitamin B-12. A gene variant in exon 1 of GIF (Gastric Intrinsic Factor gene) was associated with a dramatic decrease of liver vitamin B-12 in 2 cases. We confirmed the decreased vitamin B-12 in cord blood from NTD pregnancies. A gene variant of GIF exon 3 was associated with NTD risk. The decreased vitamin B-12 in liver and cord blood and decreased expression and activity of MS in liver point out the impaired remethylation pathway as hallmarks associated with NTD risk. We suggest evaluating vitamin B-12 in the nutritional recommendations for prevention of NTD risk beside folate fortification or supplementation.

Vitamin B12 , folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation.

Vitamin B12 (cobalamin, Cbl) is a nutrient essential to human health. Due to its complex structure and dual cofactor forms, Cbl undergoes a complicated series of absorptive and processing steps before serving as cofactor for the enzymes methylmalonyl-CoA mutase and methionine synthase. Methylmalonyl-CoA mutase is required for the catabolism of certain (branched-chain) amino acids into an anaplerotic substrate in the mitochondrion, and dysfunction of the enzyme itself or in production of its cofactor adenosyl-Cbl result in an inability to successfully undergo protein catabolism with concomitant mitochondrial energy disruption. Methionine synthase catalyzes the methyl-Cbl dependent (re)methylation of homocysteine to methionine within the methionine cycle; a reaction required to produce this essential amino acid and generate S-adenosylmethionine, the most important cellular methyl-donor. Disruption of methionine synthase has wide-ranging implications for all methylation-dependent reactions, including epigenetic modification, but also for the intracellular folate pathway, since methionine synthase uses 5-methyltetrahydrofolate as a one-carbon donor. Folate-bound one-carbon units are also required for deoxythymidine monophosphate and de novo purine synthesis; therefore, the flow of single carbon units to each of these pathways must be regulated based on cellular needs. This review provides an overview on Cbl metabolism with a brief description of absorption and intracellular metabolic pathways. It also provides a description of folate-mediated one-carbon metabolism and its intersection with Cbl at the methionine cycle. Finally, a summary of recent advances in understanding of how both pathways are regulated is presented.

Maternal One-Carbon Metabolism and Infant DNA Methylation between Contrasting Seasonal Environments: A Case Study from The Gambia

BackgroundThe periconceptional period is a time in which environmentally induced changes to the epigenome could have significant consequences for offspring health. Metastable epialleles (MEs) are genomic loci demonstrating interindividual variation in DNA methylation with intraindividual crosstissue correlation, suggesting that methylation states are established in the very early embryo before gastrulation. In our previous Gambian studies, we have shown that ME methylation states in the offspring are predicted by maternal concentrations of certain nutritional biomarkers around the time of conception. ObjectiveWe aimed to assess whether the profile of maternal biomarker predictors of offspring methylation differs between rainy and dry seasons in a population of rural Gambians, using a larger set of 50 recently identified MEs. MethodsWe measured 1-carbon biomarkers in maternal plasma back-extrapolated to conception, and cytosine-phosphate-guanine (CpG) methylation at 50 ME loci in their infants’ blood at a mean age of 3.3 mo (n = 120 mother-child pairs). We tested for interactions between seasonality and effects of biomarker concentrations on mean ME methylation z score. We used backward stepwise linear regression to select the profile of nutritional predictors of methylation in each season and repeated this analysis with biomarker principal components (PCs) to capture biomarker covariation. ResultsWe found preliminary evidence of seasonal differences in biomarker-methylation associations for folate, choline, and homocysteine (interaction P values ≤0.03). Furthermore, in stratified analyses, biomarker predictors of methylation changed between seasons. In the dry season, vitamin B-2 and methionine were positive predictors. In the rainy season, however, choline and vitamin B-6 were positive predictors, and folate and vitamin B-12 were negative predictors. PC1 captured covariation in the folate metabolism cycle and predicted methylation in dry season conceptions. PC2 represented the betaine remethylation pathway and predicted rainy season methylation. ConclusionsUnderlying nutritional status may modify the association between nutritional biomarkers and methylation, and should be considered in future studies.

Betaine reduces \u03b2-amyloid-induced paralysis through activation of cystathionine-\u03b2-synthase in an Alzheimer model of Caenorhabditis elegans

BackgroundThe neurodegenerative disorder Alzheimer’s disease is caused by the accumulation of toxic aggregates of β-amyloid in the human brain. On the one hand, hyperhomocysteinemia has been shown to be a risk factor for cognitive decline in Alzheimer’s disease. On the other hand, betaine has been demonstrated to attenuate Alzheimer-like pathological changes induced by homocysteine. It is reasonable to conclude that this is due to triggering the remethylation pathway mediated by betaine-homocysteine-methyltransferase. In the present study, we used the transgenic Caenorhabditis elegans strain CL2006, to test whether betaine is able to reduce β-amyloid-induced paralysis in C. elegans. This model expresses human β-amyloid 1–42 under control of a muscle-specific promoter that leads to progressive, age-dependent paralysis in the nematodes.ResultsBetaine at a concentration of 100 μM was able to reduce homocysteine levels in the presence and absence of 1 mM homocysteine. Simultaneously, betaine both reduced normal paralysis rates in the absence of homocysteine and increased paralysis rates triggered by addition of homocysteine. Knockdown of cystathionine-β-synthase using RNA interference both increased homocysteine levels and paralysis. Additionally, it prevented the reducing effects of betaine on homocysteine levels and paralysis.ConclusionOur studies show that betaine is able to reduce homocysteine levels and β-amyloid-induced toxicity in a C. elegans model for Alzheimer’s disease. This effect is independent of the remethylation pathway but requires the transsulfuration pathway mediated by cystathionine-β-synthase.

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4–8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [13C1]methionine and [2H3-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 μg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70–80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.

Circular RNAs profiling in the cystathionine-β-synthase mutant mouse reveals novel gene targets for hyperhomocysteinemia induced ocular disorders

Cystathionine-β-synthase (CBS) gene encodes L-serine hydrolyase which catalyzes β-reaction to condense serine with homocysteine (Hcy) by pyridoxal-5′-phosphate helps to form cystathionine which in turn is converted to cysteine. CBS resides at the intersection of transmethylation, transsulfuration, and remethylation pathways, thus lack of CBS fundamentally blocks Hcy degradation; an essential step in glutathione synthesis. Redox homeostasis, free-radical detoxification and one-carbon metabolism (Methionine-Hcy-Folate cycle) require CBS and its deficiency leads to hyperhomocysteinemia (HHcy) causing retinovascular thromboembolism and eye-lens dislocation along with vascular cognitive impairment and dementia. HHcy results in retinovascular, coronary, cerebral and peripheral vessels' dysfunction and how it causes metabolic dysregulation predisposing patients to serious eye conditions remains unknown. HHcy orchestrates inflammation and redox imbalance via epigenetic remodeling leading to neurovascular pathologies. Although circular RNAs (circRNAs) are dominant players regulating their parental genes' expression dynamics, their importance in ocular biology has not been appreciated. Progress in gene-centered analytics via improved microarray and bioinformatics are enabling dissection of genomic pathways however there is an acute under-representation of circular RNAs in ocular disorders. This study undertook circRNAs' analysis in the eyes of CBS deficient mice identifying a pool of 12532 circRNAs, 74 exhibited differential expression profile, ∼27% were down-regulated while most were up-regulated (∼73%). Findings also revealed several microRNAs that are specific to each circRNA suggesting their roles in HHcy induced ocular disorders. Further analysis of circRNAs helped identify novel parental genes that seem to influence certain eye disease phenotypes.

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.

Lean Body Mass Harbors Sensing Mechanisms that Allow Safeguarding of Methionine Homeostasis.

Protein-depleted states generate allosteric inhibition of liver cystathionine β-synthase (CBS), which governs the first enzymatic step of the transsulfuration cascade, resulting in upstream accretion of homocysteine (Hcy) in body fluids. A similar Hcy increase may arise from normal hepatocytes undergoing experimentally-induced impairment of betaine-homocysteine methyltransferase (BHTM) activity or from components of lean body mass (LBM) submitted to any inflammatory disorder. LBM comprises a composite agglomeration of extrarenal tissues characterized by naturally occurring BHTM inactivity. As a result of cellular injury, LBM releases high concentrations of Hcy into the extracellular space, contrasting with the disruption of normal remethylation pathways. Hyperhomocysteinemia acts as a biomarker, reflecting the severity of insult and operating as an alarm signal. Elevated Hcy levels constitute a precursor pool recognized by a CBS coding region that reacts to meet increased methionine requirements in LBM tissues, using its enhanced production in hepatocytes. Preservation of methionine homeostasis benefits from its high metabolic priority and survival value.

The Association of Thyroid Function With Maternal and Neonatal Homocysteine Concentrations.

High homocysteine concentrations are associated with maternal pregnancy complications and low birth weight, jaundice, and cerebrovascular accidents in neonates. Thyroid hormone may interfere with homocysteine metabolism via stimulation of vitamin B12- and folate-dependent processes and via effects on enzymes of the remethylation pathway. Investigating the associations of maternal and neonatal thyroid function with homocysteine during pregnancy and after delivery, respectively. Within Generation R study, a population-based prospective cohort, we studied the associations of maternal and neonatal thyroid stimulating hormone (TSH) and free thyroxine (FT4) with homocysteine, folate, and vitamin B12 concentrations using multiple linear regression analyses. TSH, FT4, homocysteine, folate, and vitamin B12 concentrations were determined in early pregnancy (<18 weeks; N = 1094 women without folic acid supplementation) and in cord blood of 4475 neonates. In neonates, there was a positive association of FT4 with homocysteine and an inverse association of TSH with homocysteine. The associations attenuated after adjustment for folate and vitamin B12 concentration (β change: for FT4, 0.00559 ± 0.001, P < 0.0001, to 0.00310 ± 0.001, P = 0.015; and for TSH, -0.00165 ± 0.001, P = 0.005, to -0.00086 ± 0.001, P = 0.11). In mothers, there was a positive association of FT4 with homocysteine (P = 0.026) but no association of FT4 with folate or vitamin B12 (P ≥ 0.08). Higher thyroid function is associated with higher homocysteine concentrations in pregnant women and in neonates. These data provide new insights into the effects of thyroid hormone on folate- and vitamin B12-dependent processes during early growth and development.