[Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice].

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1ß, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1ß in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1ß, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1ß in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

Measuring bioavailability, utilization, and excretion of rumen-protected lysine in lactating cows using an isotope technique.

Supplementing a diet with rumen-protected amino acids (AAs) is a common feeding strategy for efficient production. For a cost-effective use of rumen-protected AA, the accurate bioavailability of rumen-protected amino acids should be known and their metabolism after absorption needs to be well understood. The current study determined the bioavailability, absorption, utilization, and excretion of rumen-protected Lys (RP-Lys). Four ruminally cannulated cows in a 4 × 4 Latin square design (12 d for diet adaptation; 5 or 6 d for total collections) received the following treatments: L0, a basal diet; L25, the basal diet and L-Lys infused into the abomasum to provide 25.9 g/d L-Lys; L50, the basal diet and L-Lys infused into the abomasum to provide 51.8 g/d L-Lys; and RPL, the basal diet supplemented with 105 g/d (as-is) of RP-Lys to provide 26.7 g of digestible Lys. During the last 5 or 6 d in each period, 15N-Lys (0.38 g/d) was infused into the abomasum for all cows to label the pool of AA, and the total collection of milk, urine, and feces were conducted. 15N enrichment of samples on d 4 and 5 were used to calculate the bioavailability and Lys metabolism. We used a model containing a fast AA turnover (≤ 5 d) and slow AA turnover pool (> 5 d) to calculate fluxes of Lys. The Lys flux to the fast AA turnover pool (absorbed Lys + Lys from the slow AA turnover pool to fast AA turnover pool) was calculated using 15N enrichment of milk Lys. The flux of Lys from the fast AA turnover pool to milk and urine was calculated using 15N transfer into milk and urine. Then, absorbed Lys was estimated by the sum of Lys flux to milk and urine assuming no net utilization of Lys by body tissues. Duodenal Lys flow was estimated by 15N enrichment of fecal Lys. The bioavailability of RP-Lys was calculated from duodenal Lys flows and Lys absorption for RPL. Increasing Lys supply from L25 to L50 increased Lys utilization for milk by 9 g/d but also increased urinary excretion by 10 g/d. For RPL, absorbed Lys was estimated to be 136 g/d where 28 g of absorbed Lys originated from RP-Lys. In conclusion, 68% of bioavailability was obtained for RP-Lys. The Lys provided from RP-Lys was not only utilized for milk protein (48%) but also excreted in urine (20%) after oxidation.

Methionine biosynthetic genes and methionine sulfoxide reductase A are required for Rhizoctonia solani AG1-IA to cause sheath blight disease in rice.

Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate-active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative-stress-enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double-stranded RNA-mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease-causing ability of Rs_MET13-silenced R. solani and facilitates its growth on 10 mM H2O2-containing minimal-media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA-silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray-induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice.

Architecture and regulation of filamentous human cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.

Dietary Methionine Restriction Improves Gut Health and Alters the Plasma Metabolomic Profile in Rats by Modulating the Composition of the Gut Microbiota.

Dietary methionine restriction (MetR) offers an integrated set of beneficial health effects, including delaying aging, extending health span, preventing fat accumulation, and reducing oxidative stress. This study aimed to investigate whether MetR exerts entero-protective effects by modulating intestinal flora, and the effect of MetR on plasma metabolites in rats. Rats were fed diets containing 0.86% methionine (CON group) and 0.17% methionine (MetR group) for 6 weeks. Several indicators of inflammation, gut microbiota, plasma metabolites, and intestinal barrier function were measured. 16S rRNA gene sequencing was used to analyze the cecal microbiota. The MetR diet reduced the plasma and colonic inflammatory factor levels. The MetR diet significantly improved intestinal barrier function by increasing the mRNA expression of tight junction proteins, such as zonula occludens (ZO)-1, claudin-3, and claudin-5. In addition, MetR significantly increased the levels of short-chain fatty acids (SCFAs) by increasing the abundance of SCFAs-producing Erysipclotxichaceae and Clostridium_sensu_stricto_1 and decreasing the abundance of pro-inflammatory bacteria Proteobacteria and Escherichia-Shigella. Furthermore, MetR reduced the plasma levels of taurochenodeoxycholate-7-sulfate, taurocholic acid, and tauro-ursodeoxycholic acid. Correlation analysis identified that colonic acetate, total colonic SCFAs, 8-acetylegelolide, collettiside I, 6-methyladenine, and cholic acid glucuronide showed a significant positive correlation with Clostridium_sensu_stricto_1 abundance but a significant negative correlation with Escherichia-Shigella and Enterococcus abundance. MetR improved gut health and altered the plasma metabolic profile by regulating the gut microbiota in rats.

Rescue of Methionine Dependence by Cobalamin in a Human Colorectal Cancer Cell Line.

Methionine dependence is a characteristic of most cancer cells where they are unable to proliferate when the essential amino acid methionine is replaced with its precursor homocysteine in the growing media. Normal cells, on the other hand, thrive under these conditions and are referred to as methionine-independent. The reaction that adds a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine is catalyzed by the enzyme methionine synthase with the cofactor cobalamin (vitamin B12). However, decades of research have shown that methionine dependence in cancer is not due to a defect in the activity of methionine synthase. Cobalamin metabolism has been tied to the dependent phenotype in rare cell lines. We have identified a human colorectal cancer cell line in which the cells regain the ability to proliferation in methionine-free, L-homocystine-supplemented media when cyanocobalamin is supplemented at a level of 1 µg/mL. In human SW48 cells, methionine replacement with L-homocystine does not induce any measurable increase in apoptosis or reactive oxygen species production in this cell line. Rather, proliferation is halted, then restored in the presence of cyanocobalamin. Our data show that supplementation with cyanocobalamin prevents the activation of the integrated stress response (ISR) in methionine-deprived media in this cell line. The ISR-associated cell cycle arrest, characteristic of methionine-dependence in cancer, is also prevented, leading to the continuation of proliferation in methionine-deprived SW48 cells with cobalamin. Our results highlight differences between cancer cell lines in the response to cobalamin supplementation in the context of methionine dependence.

Dietary supplementation of methionine, lysine, and tryptophan as possible modulators of growth, immune response, and disease resistance in striped catfish (Pangasius hypophthalmus).

The present study investigated the potential role of different essential amino acids (AA) in striped catfish (Pangasius hypophthalmus). Fish (initial weight = 17.91±0.27 g, n = 260) were fed with eight isonitrogenous (30%), and isolipidic diets (6%) formulated to include different combinations of tryptophan (Trp), methionine (Met), and lysine (Lys) (T0: Zero AA, T1: Trp, T2: Lys, T3: Met, T4: Trp+Met, T5: Lys+Trp, T6: Met+Lys, T7: Lys+Trp+Met) for eight weeks. The dose of amino acid supplementation, whether individually or in combination, was 5g of each amino acid per kg of diet. The trial comprised eight treatments, with each treatment consisted of three replicates (n = 10/replicate). At the end of the growth experiment, the highest total body weight, crude protein, digestive enzymatic activity, immune response, and amino acids level were observed in treatments supplemented with amino acids compared to T0. After the growth experiment, fish in all treatments were exposed to Staphylococcus aureus (5×105 CFU/ml). For bacterial challenge trial, the T0 treatment was designated as positive (+ve T0) and negative control (-ve T0). Following the S. aureus challenge, fish fed with amino acids showed a better response to reactive oxygen species and lipid peroxidation, as indicated by the increased levels of catalase and superoxide dismutase. Conversely, the concentration of malondialdehyde gradually decreased in all treatments compared to the +ve T0 treatment. It is concluded that supplementation of amino acids improved the growth, protein content, and immunocompetency against S. aureus in striped catfish. The most favorable outcomes in striped catfish were shown by fish supplemented with T7 diet. These essential amino acids hold potential as efficient supplements for use in the intensive aquaculture for striped catfish.

A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Relative biological efficacy of methionine hydroxy analogue-free acid compared to dl-methionine in the broiler chickens.

BACKGROUND: In the broiler's diets based on corn-soya bean meal, methionine (Met) and cystine (Cys), known as sulphur amino acids (SAAs), are the first limiting indispensable amino acids because of their limited presence, which are supplemented with different synthetic sources. Evaluation of the biological effectiveness of these sources can be important in their correct replacement, especially in the starter and growth diets. OBJECTIVES: The current study was done to assess the relative biological efficacy (RBE) of liquid Met hydroxy analogue-free acid (MHA-FA) in comparison with dl-Met (dl-Met) based on broiler performance traits at different levels of digestible SAA in the 1-11 (starter) and 11-25 (grower) days of age periods. METHODS: Two experiments were developed with treatments consisting of a basal diet without Met addition that met the nutrient and energy requirements of broilers with the exception of SAAs (Met + Cys) and five increasing Met doses for both sources (dl-Met and/or MHA-FA), resulting in digestible SAA concentrations from 0.62% to 1.02% of diet in the starter period (Trial 1) and 0.59% to 0.94% of diet in the grower period (Trial 2). The multi-linear regression model and slope ratio method were employed to calculate the RBE of MHA-FA compared with dl-Met for measured variables. RESULTS: In both experiments, the results obtained during the starter and grower periods with the different Met supplementations show significant growth responses to digestible SAAs levels. By increasing dietary dl-Met and/or MHA-FA levels, the growth performance traits and immune responses were improved (quadratic; p < 0.05). The RBE of MHA-FA compared to dl-Met on an equimolar basis was estimated 66%-89% (59%-79% on a weight-to-weight basis). CONCLUSIONS: It is concluded that the RBE of MHA-FA in comparison with dl-Met depends on broiler chicken age and what attribute is being evaluated.

Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02 g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

Beyond the Bowl: Understanding Amino Acid Requirements and Digestibility to Improve Protein Quality Metrics for Dog and Cat Foods.

The determination of amino acid (AA) requirements for mammals has traditionally been done through nitrogen (N) balance studies, but this technique underestimates AA requirements in adult animals. There has been a shift toward researchers using the indicator amino acid oxidation (IAAO) technique for the determination of AA requirements in humans, and recently in dogs. However, the determination of AA requirements specific to adult dogs and cats at maintenance is lacking and the current requirements outlined by the National Research Council are based on a dearth of data and are likely underreporting the requirements of indispensable AA (IAA) for the population. To ensure the physiological requirements of our cats and dogs are met, we need methods to accurately and precisely measure digestibility. In vivo methods, such as ileal cannulation, are most commonly used, however, due to ethical considerations, we are moving away from animal models and toward in vitro methods. Harmonized static digestion models have the potential to replace in vivo methods but work needs to be done to have these methods more accurately represent the gastrointestinal tract (GIT) of cats and dogs. The Digestible IAA Score (DIAAS) is one metric that can help define protein quality for individual ingredients or mixed diets that uses AA SID estimates and ideally those can be replaced with in vitro AA digestibility estimates. Finally, we need accurate and reliable laboratory AA analyses to measure the AA present in complete diets, especially those used to quantify methionine (Met) and cysteine (Cys), both often limiting AAs in cat and dog diets. Together, this will guide accurate feed formulation for our companion animals to satisfy requirements while avoiding over-supplying protein, which inevitably contributes to excess N excretion, affecting both the environment and feed sustainability.

A novel methionine nanoparticle in broiler chickens: Bioavailability and requirements.

This bioassay evaluated the bioavailability (RBV) of a novel nanoparticle of methionine (nano-Met) relative to DL-methionine (DL-Met), and estimated methionine requirements for both sources in starting broilers. Five supplemental levels (0.05, 0.10, 0.15, 0.20, and 0.25% of diet) of DL-Met or nano-Met were added to a basal diet containing 0.35% standardized ileal digestible (SID) methionine to create 11 experimental diets, including a basal diet and 10 experimental diets containing 0.40, 0.45, 0.50, 0.55, and 0.60% SID-Met, respectively. A total of 825 one-day-old male Ross 308 birds were randomly assigned to 11 treatments with 5 pen replicates and 15 birds each. Body weight gain (BWG), breast meat yield (BMY), and thigh meat yield (TMY) increased (P < 0.001) while feed conversion ratio (FCR) and malondialdehyde (MDA) concentration in meat samples decreased (P < 0.001) with increasing dietary methionine. Based on the slope-ratio method, the RBV of nano-Met relative to DL-Met for BWG, FCR, and TMY were 102 (48-155%; R2 = 0.71), 134 (68-201%; R2 = 0.77), and 110% (27-193%; R2 = 0.55), respectively. Considering the statistical accuracy of the spline models, the estimated values of DL-Met for maximum BWG and nano-Met for maximum TMY were 0.578% and 0.561%, respectively, which were statistically higher than those recommended for commercial settings. The highest effect size of supplemental methionine was on MDA (ƞ2p = 0.924), followed by FCR (ƞ2p = 0.578), BMY (ƞ2p = 0.575), BWG (ƞ2p = 0.430), and TMY (ƞ2p = 0.332), suggesting the potent antioxidant properties of methionine. Our findings suggest that reducing the particle size of DL-Met to nanoparticles could be a promising strategy to enhance the efficiency of methionine supplementation in broilers, an idea that requires further investigation in future research.

Methionyl-Methionine Dipeptide Enhances Mammogenesis and Lactogenesis by Suppressing the Expression of a Novel Long Noncoding RNA MGPNCR to Inhibit eIF4B Dephosphorylation.

Previous research has demonstrated that in pregnant mice deficient in l-methionine (Met), the mixture of the dipeptide l-methionyl-l-methionine (Met-Met) with Met was more effective than Met alone in promoting mammogenesis and lactogenesis. This study aimed to investigate the role of a novel long noncoding RNA (lncRNA), named mammary gland proliferation-associated lncRNA (MGPNCR), in these processes. Transcriptomic analysis of mammary tissues from Met-deficient mice, supplemented either with a Met-Met/Met mixture or with Met alone, revealed significantly higher MGPNCR expression in the Met group compared to the mixture group, a finding recapitulated in a mammary epithelial cell model. Our findings suggested that MGPNCR hindered mammogenesis and milk protein synthesis by binding to eukaryotic initiation factor 4B (eIF4B). This interaction promoted the dephosphorylation of eIF4B at serine-422 by enhancing its association with protein phosphatase 2A (PP2A). Our study sheds light on the regulatory mechanisms of lncRNA-mediated dipeptide effects on mammary cell proliferation and milk protein synthesis. These insights underscore the potential benefits of utilizing dipeptides to improve milk protein in animals and potentially in humans.

Role of folate receptor α in the partial rejuvenation of dentate gyrus cells: Improvement of cognitive function in 21-month-old aged mice.

Neuronal aging may be, in part, related to a change in DNA methylation. Thus, methyl donors, like folate and methionine, may play a role in cognitive changes associated to neuronal aging. To test the role of these metabolites, we performed stereotaxic microinjection of these molecules into the dentate gyrus (DG) of aged mice (an average age of 21 month). Folate, but not S-Adenosyl-Methionine (SAM), enhances cognition in aged mice. In the presence of folate, we observed partial rejuvenation of DG cells, characterized by the expression of juvenile genes or reorganization of extracellular matrix. Here, we have also tried to identify the mechanism independent of DNA methylation, that involve folate effects on cognition. Our analyses indicated that folate binds to folate receptor α (FRα) and, upon folate binding, FRα is transported to cell nucleus, where it is acting as transcription factor for expressing genes like SOX2 or GluN2B. In this work, we report that a FRα binding peptide also replicates the folate effect on cognition, in aged mice. Our data suggest that such effect is not sex-dependent. Thus, we propose the use of this peptide to improve cognition since it lacks of folate-mediated side effects. The use of synthetic FRα binding peptides emerge as a future strategy for the study of brain rejuvenation.

Systemic intracellular analysis for balancing complex biosynthesis in a transcriptionally deregulated Escherichia coli l-Methionine producer.

l-Methionine (l-Met) has gained remarkable interest due to its multifaceted and versatile applications in the fields of nutrition, pharmaceuticals and clinical practice. In this study, the fluxes of the challenging l-Met biosynthesis in the producer strain Escherichia coli (E. coli) DM2853 were fine-tuned to enable improved l-Met production. The potential bottlenecks identified in sulfur assimilation and l-Met synthesis downstream of O-succinyl-l-homoserine (OSHS) were addressed by overexpressing glutaredoxin 1 (grxA), thiosulfate sulfurtransferase (pspE) and O-succinylhomoserine lyase (metB). Although deemed as a straightforward target for improving glucose-to-Met conversion, the yields remained at approximately 12%-13% (g/g). Instead, intracellular l-Met pools increased by up to four-fold with accelerated kinetics. Overexpression of the Met exporter ygaZH may serve as a proper valve for releasing the rising internal Met pressure. Interestingly, the export kinetics revealed maximum saturated export rates already at low growth rates. This scenario is particularly advantageous for large-scale fermentation when product formation is ideally uncoupled from biomass formation to achieve maximum performance within the technical limits of large-scale bioreactors.

The restoration of hippocampal nerve de-myelination by methylcobalamin relates with the enzymatic regulation of homocysteine level in a rat model of moderate grade hepatic encephalopathy.

This article describes how methylcobalamin (MeCbl) restores nerve myelination in a moderate- grade hepatic encephalopathy (MoHE) model of ammonia neurotoxicity. The comparative profiles of myelin basic protein (MBP), homocysteine (Hcy) and methionine synthase (MS: a MeCbl- dependent enzyme) activity versus nerve myelination status were studied in the hippocampus of the control, the MoHE (developed by administering 100 mg/kg bw thioacetamide i.p. for 10 days) and the MoHE rats treated with MeCbl (500 µg/kg BW i.p.) for 7 days. Compared to those of control rats, the hippocampal CA1 and CA3 regions of the MoHE rats showed significantly lower myelinated areas and MBP immunostaining. This coincided with the deranged myelin layering in TEM images, decreased MBP protein and its transcript levels in hippocampus of MoHE rats. However, all these parameters recovered to normal levels after MeCbl treatment. MeCbl is a cofactor of MS that catalyzes the conversion of Hcy to methionine as a feeder step of methylation reactions. We observed significantly increased serum and hippocampal Hcy levels in MoHE rats, however, these levels were restored to control values with a concordant activation of MS due to MeCbl treatment. A significant recovery in neurobehavioral impairments in the MoHE rats due to MeCbl treatment was also observed. These findings suggest that MoHE pathogenesis is associated with deranged nerve myelination in the hippocampus and that MeCbl treatment is able to restore it mainly by activating MS, a MeCbl-dependent Hcy-metabolizing enzyme.

Methionine Supplementation Alleviates the Germ Cell Apoptosis Increased by Maternal Caffeine Intake in a C. elegans Model.

Caffeine (1,3,7-trimethylxanthine) is a widely consumed bioactive substance worldwide. Our recent study showed that a reduction in both reproduction and yolk protein production (vitellogenesis) caused by caffeine intake were improved by vitamin B12 supplementation, which is an essential co-factor in methionine metabolism. In the current study, we investigated the role of methionine in the reproduction of caffeine-ingested animals (CIAs). We assessed the effect of methionine metabolism on CIAs and found that caffeine intake decreased both methionine levels and essential enzymes related to the methionine cycle. Furthermore, we found that the caffeine-induced impairment of methionine metabolism decreased vitellogenesis and increased germ cell apoptosis in an LIN-35/RB-dependent manner. Interestingly, the increased germ cell apoptosis was restored to normal levels by methionine supplementation in CIAs. These results indicate that methionine supplementation plays a beneficial role in germ cell health and offspring development by regulating vitellogenesis.

High methionine diet mediated oxidative stress and proteasome impairment causes toxicity in liver.

Methionine (Met) rich diet inducing oxidative stress is reported to alter many organs. Proteasome as a regulator of oxidative stress can be targeted. This study was performed to investigate if excessive methionine supplementation causes hepatotoxicity related to proteasome dysfunction under endogenous oxidative stress in rats. Male Wistar albino rats (n = 16) were divided into controls and treated groups. The treated rats (n = 08) received orally L-methionine (1 g/kg/day) for 21 days. Total homocysteine (tHcy), total oxidant status (TOS), total antioxidant status (TAS), hepatic enzymes levels: aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), with total bilirubin (TBil), albumin (Alb), and C-reactive protein (CRP) were determined in plasma by biochemical assays. Liver supernatants were used for malondialdehyde (MDA), protein carbonyls (PC), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), 20S proteasome activities and their subunits expression, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) evaluation by appropriate methods and light microscopy for liver histological examination. Methionine treatment increased homocysteine, TOS, oxidative stress index (OSI), MDA and PC but decreased TAS, GSH, CAT, SOD, GPx with the 20S proteasome activities and their ß subunits expression. Liver proteins: AST, ALT, LDH, ALP, TBil and CRP were increased but Alb was decreased. Liver histology was also altered. An increase in liver TNF-α and IL-6 levels were observed. These findings indicated that methionine supplementation associated oxidative stress and proteasome dysfunction, caused hepatotoxicity and inflammation in rat. Further investigations should be to better understand the relation between methionine, oxidative stress, proteasome, and liver injuries.

Tenascin-X is increased with decreased expression of miR-378a-5p and miR-486-5p in mice fed a methionine-choline-deficient diet that induces hepatic fibrosis.

We previously reported that tenascin-X (Tnxb) aggravates hepatic fibrosis in mice fed a high-fat and high-cholesterol diet with high levels of phosphorus and calcium (HFCD). In this study, we investigated Tnxb expression in livers with fibrosis caused by administration of a methionine-chorine-deficient (MCD) diet in mice. Whole transcriptome analysis showed that Tnxb was one of the genes with increased expression in livers of MCD diet-fed mice compared with that in livers of normal diet (ND)-fed mice. In microarray and subsequent microRNA (miRNA) network analyses, miR-378a-5p and miR-486-5p were identified in livers of MCD diet-fed mice as downregulated miRNAs, which have their predicted target sites in the 3' untranslated region of Tnxb mRNA and might suppress the translation of Tnxb mRNA. RT-qPCR analyses of livers of MCD diet-fed mice compared with livers of ND-fed mice verified the upregulation of Tnxb and fibrosis-triggering genes and conversely the downregulation of miR-378a-5p and miR-486-5p. Overexpression of miR-378a-5p and miR-486-5p resulted in decreased level not only of the FLAG-tagged fibrinogen-like domain of Tnxb protein (FLAG-mTNX-FG) but also of endogenous Tnxb protein in murine cultured cells. These results indicate that expression of Tnxb is regulated by miR-378a-5p and miR-486-5p in hepatic fibrosis following MCD diet feeding.

The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress.

Amino acid restriction induces cellular stress and cells often respond via the induction of autophagy. Autophagy or 'self-eating' enables the recycling of proteins and provides the essential amino acids needed for cell survival. Of the naturally occurring amino acids, methionine restriction has pleiotropic effects on cells because methionine also contributes to the intracellular methyl pools required for epigenetic controls as well as polyamine biosynthesis. In this report, we describe the chemical synthesis of four diastereomers of a methionine depletion agent and demonstrate how controlled methionine efflux from cells significantly reduces intracellular methionine, S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH), and polyamine levels. We also demonstrate that human pancreatic cancer cells respond via a lipid signaling pathway to induce autophagy. The methionine depletion agent causes the large amino acid transporter 1 (LAT1) to preferentially work in reverse and export the cell's methionine (and leucine) stores. The four diastereomers of the lead methionine/leucine depletion agent were synthesized and evaluated for their ability to (a) efflux 3H-leucine from cells, (b) dock to LAT1 in silico, (c) modulate intracellular SAM, SAH, and phosphatidylethanolamine (PE) pools, and (d) induce the formation of the autophagy-associated LC3-II marker. The ability to modulate the intracellular concentration of methionine regardless of exogenous methionine supply provides new molecular tools to better understand cancer response pathways. This information can then be used to design improved therapeutics that target downstream methionine-dependent processes like polyamines.