High Methionine Research Articles

The β-hairpin from the Thermus thermophilus HB27 laccase works as a pH-dependent switch to regulate laccase activity

The multi-copper oxidase from the hyper-thermophilic bacteria Thermus thermophilus (Tth-MCO), has been previously characterized and described as an example of a laccase with low catalytic properties, especially when it is compared with the activity of fungal laccases, but it is active at high temperatures. Structurally, Tth-MCO has a unique feature: a β-hairpin near the T1Cu site, which is not present in any other laccases deposited at the PDB. This β-hairpin has an expected crystallographic behavior in solvent-exposed areas of a crystallized protein: lack of electron density, high B-values and several crystalline contacts with neighboring crystallographic copies; however, its dynamical behavior in solution and its biological implications have not been described. Here, we describe four new Tth-MCO crystallographic structures, and the β-hairpin behavior has been analyzed by molecular dynamics simulations, considering the effect of pH and temperature. The β-hairpin new crystallographic conformations described here, together with their dynamics, were used to understand the pH-restrained laccase activity of Tth-MCO against substrates as syringaldazine. Remarkably, there are insertions in laccases from Thermus and Meiothermus genus, sharing the same position and a methionine-rich composition of the Tth-MCO β-hairpin. This unique high methionine content of the Tth-MCO β-hairpin is responsible to coordinate, Ag+1 and Hg+1 in oxidative conditions, but Cu+1 and Cu+2 are not coordinated in crystallographic experiments, regardless of the redox conditions; however, Ag+1 addition does not affect Tth-MCO laccase activity against syringaldazine. Here, we propose that the pH-dependent β-hairpin dynamical behavior could explain, at least in part, the inefficient laccase activity displayed by Tth-MCO in acidic pH values.

High Methionine Diet-Induced Alzheimer's Disease like Symptoms Are Accompanied by 5-Methylcytosine Elevated Levels in the Brain.

Background Excessive or insufficient intake of methionine (Met) causes neuronal dysfunction, neurodegeneration, cerebrovascular dysfunction, vascular leakage, and short-term memory loss, which result in the occurrence of Alzheimer's disease- (AD-) like symptoms. Objective To determine the relationship between high methionine diets (HMD) induced AD-like symptoms and 5-methylcytosine (5-mC) level. Methods C57BL/6J mice were randomly divided into two groups: the control group (Maintain diets) and the model group (2% HMD). Mice were fed with 2% HMD for 9 weeks. Animals were weighed and food intake was recorded weekly. Open field test, nesting ability test, Y maze test, new object recognition test, and Morris water maze test were used to detect the motor, learning, and memory ability. Hematoxylin-eosin (HE) staining was used to observe the damage of cells in hippocampus and cortex. Immunofluorescence (IF) staining was used to detect the expression and distribution of amyloid-β 1-40 (Aβ1-40), amyloid-β 1-42 (Aβ1-42), and 5-methylcytosine (5-mC) in hippocampus and cortex. Western blotting (WB) was used to determine the expression of Aβ and DNA methyltransferases- (DNMTs-) related proteins in the cortex. Enzyme-linked immunosorbent assay (ELISA) was performed to detect homocysteine (Hcy) level (ELISA). Results Feeding of HMD decreased the body weight and food intake of mice. Behavioral testing revealed that HMD caused learning, memory, and motor ability impairment in the mice. HE staining results showed that HMD feeding caused damage of hippocampal and cortical neurons, along with disordered cell arrangement, and loss of neurons. Furthermore, HMD increased the contents of Aβ1-40, Aβ1-42, and 5-mC in the hippocampus and cortex. WB results showed that HMD increased the expression of Aβ production-related proteins, such as amyloid precursor protein (APP) and beta-secretase 1 (BACE1), and decreased the expression of Aβ metabolism-related protein in the cortex, including insulin-degrading enzyme (IDE) and neprilysin (NEP). Additionally, the decreased expression of DNA methyltransferase1 (DNMT1) was observed in HMD-treated mice, but there was no significant change of DNMT3a level. ELISA results showed that HMD increased the levels of Hcy in serum. Conclusion Our result suggested that the HMD can cause neurotoxicity, leading to AD-like symptoms in mice, which may be related to 5-mC elevated.

Preliminary analysis of immunoregulatory mechanism of hyperhomocysteinemia-induced brain injury in Wistar-Kyoto rats.

Hyperhomocysteinemia (HHcy) can be used as an independent risk factor for predicting cardiovascular disease, stroke and vitamin B12 deficiency. Patients with HHcy have elevated plasma homocysteine (Hcy) concentrations. Enhancing cerebrovascular permeability of substances such as Hcy and brain damage will synergistically increase the symptoms of hypertension, but the specific immune regulation mechanism is still not clear. The purpose of the present study was to preliminarily explore the immunomodulatory mechanism of brain damage caused by HHcy in Wistar-Kyoto (WKY) rats. A total of 60 WKYs were randomly divided into three groups: WKY control group (WKY-C group), WKY methionine group (WKY-M group) and WKY treatment group (WKY-T group; vitamin B6, B12 and folic acid were used as treatment), with 20 rats in each group. Physical examination of body weight, systolic blood pressure (SBP) and plasma Hcy content was performed routinely. The concentration of cytokines, including IL-6, IL-10, IL-17A and TGF-β, associated with T helper cell 17 (Th17) and regulatory T (Treg) cells and key regulator genes, including retinoic acid-related orphan receptor γ t (RORγt) and forkhead box P3 (FoxP3), were detected by ELISA, reverse transcription-quantitative PCR and western blotting. Th17/Treg lymphocytes were determined by flow cytometry. MRI scan was preliminarily used to detect the changes characteristic of the ischemic stroke. The results revealed that high methionine diets might have a significant effect on the body weight and SBP. The inflammatory response effect of Treg cells was significantly inhibited in the WKY-M group, and that of Th17 cells was upregulated when compared to the WKY-T group. Compared with the WKY-T group, the expression levels of IL-17A and RORγt in the WKY-M group were significantly upregulated, while the mRNA levels of FoxP3 in the WKY-M group were significantly downregulated. The diet intervention (including vitamins B6 and B12 and folic acid) could reduce the level of Hcy in the blood, but also reduce the inflammatory response and rectify the Treg/Th17 immune imbalance to ameliorate the brain tissue damage. In conclusion, the present study indicated that HHcy can promote inflammation by triggering Treg/Th17 immune imbalance to ameliorate the brain tissue damage.

A high methionine and low folate diet alters glucose homeostasis and gut microbiome

Hyperhomocysteinemia (HHcy) is considered as a risk factor for several complications, including cardiovascular and neurological disorders. A high methionine low folate (HMLF) diet chronically causes HHcy by accumulating homocysteine in the systemic circulation. Elevated Hcy level is also associated with the incidence of diabetes mellitus. However, very few studies focus on the impact of HMLF diet on glucose homeostasis, and that on gut microbiome profile. HHcy was induced by feeding C57BL/6 mice a HMLF diet for 8 weeks. The HMLF diet feeding resulted in a progressive body weight loss, and development of slight glucose intolerance and insulin resistance in HHcy mice. Notably, the HMLF diet alters the gut microbiome profile and increases the relative abundance of porphyromonadaceae family of bacteria in HHcy mice. These findings provide new insights into the roles of dysregulated glucose homeostasis and gut flora in the pathogenesis of HHcy-related complications.

Amino acid availability of processed Brassica carinata meals in hybrid striped bass Morone chrysops ♀ x M. saxatilis ♂

An earlier study showed that hybrid striped bass (Morone chrysops ♀ x M. saxatilis ♂) diets containing 20 % animal meals and up to 30 % aerobically fermented (AF) carinata meal (Brassica carinata, CM) followed by a single wash with water (AFCM) or 30 % CM double-washed with water without AF (WCM), resulted in similar growth. However feed utilization and Fulton’s condition factor of 30 % WCM were better. The current study investigated effects of AFCM and WCM on amino acid digestibilities using naive fish. Effects of AFCM and WCM on trypsin activity, and amino acid availability were determined using hybrid striped bass fed experimental diets for 127 and 148 days, respectively (after a growth trial). High (30 %) AFCM reduced all essential amino acids (EAA) and conditionally EAA (CEAA) except histidine, methionine and threonine. However, all EAA and CEAAs except lysine and cysteine were digested better in 30 %WCM than 30 % AFCM. High (30 %) CMs resulted in generally high serum free methionine and tryptophan, but less leucine for the first 9 h. However, 30 % WCM resulted in generally more serum free arginine and valine than 30 % AFCM. High CMs increased muscle free histidine. Generally, WCM resulted in more muscle lysine and valine than AFCM. Muscle methionine was increased by CMs. Both serum and muscle free total EAAs in hybrid striped bass fed 30 % WCM were elevated continuously over a longer period above the baseline levels. Using EAA:lysine, only muscle histidine were adequate for muscle synthesis over the 36-h period. Overall, amino acids were more available in WCM than AFCM.

38 Developmental competence of bovine cumulus–oocyte complexes collected from cows fed rumen-protected methionine and lysine

Supplementation of rumen-protected amino acids (RPAA) has proven to be an effective tool to supply limiting AA in dairy diets. Methionine and lysine are the two most limiting AA for lactating dairy cows. Recently, it has been shown that methionine supplementation seems to affect pre-implantation embryos collected from superovulated cows enhancing their developmental competence because there is strong evidence that endogenous lipid reserves serve as an energy substrate (Acosta et al. 2016 Theriogenology 85, 1669–1679). Moreover, higher concentrations of methionine were determined in the follicular fluid of the first dominant follicle postpartum in cows supplemented with rumen-protected methionine and rumen-protected choline from 21 days before calving to 30 days postpartum and it was assumed that higher methionine concentrations in the follicular fluid could affect oocyte quality (Acosta et al. 2017 Theriogenology 96, 1–9). There is no information available so far regarding the effect of a combined methionine and lysine supplementation (each rumen-protected) on oocyte quality. Therefore, the objective of this study was to evaluate the effect of a combined methionine and lysine supplementation during early to mid-lactation on the developmental competence of oocytes collected from lactating dairy cows (days 0 to 100 p.p.). Thirty pregnant multiparous German Holstein dairy cows were grouped 3 weeks before their expected calving date, receiving identical diets. After calving, they were randomly allocated to 2 groups fed a total mixed ration supplemented with (N=14 cows; RPAA) or without (N=16 cows; CON) LysiGEMTM (encapsulated lysine; Kemin Industries) and Metasmart DryTM (isopropyl ester of the hydroxylated analogue of methionine adsorbed onto a silicon dioxide carrier; Adisseo). Starting from 45 days p.p., animals from both groups were submitted to an ovum pickup (OPU) session once a week for at least 8 weeks. Collected cumulus–oocyte complexes (COC) were subjected to a standard invitro production (IVP) protocol (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502–10) including IVM, IVF, and invitro culture (IVC). Cleavage and developmental rates up to the morula/blastocyst stage were recorded on Days 3, 7, and 8. In total, 1211 follicles have been aspirated from RPAA animals compared with 1413 from CON animals, from which 742 and 885 COC were collected, respectively. The calculated recovery rate based on the number of aspirated follicles and collected COC was similar for both groups (61.3±29.4% vs. 62.6±33.5%). Cleavage and developmental rates based on 240 (RPAA group) and 299 (CON group) COC also showed similar results [RPAA: 84.1±5.9% (202/240), 18.3±4.4% (44/240), 18.8±4.7% (45/240); CON: 81.9±8.6% (245/299), 15.4±8.9% (46/299), 16.7±8.4% (50/299)]. In conclusion, supplementation of RPAA (methionine and lysine) had no beneficial effect on the developmental competence of COC obtained from these animals compared with those collected from cows fed the diet without RPAA supplementation.

ER stress—induced adipocytes secrete-aldo-keto reductase 1B7—containing exosomes that cause nonalcoholic steatohepatitis in mice

Nonalcoholic steatohepatitis (NASH) is an increasingly prevalent liver disease linked to obesity and associated complications. Endoplasmic reticulum (ER) stress provokes dysfunction in lipid metabolism, which often leads to a progression of obesity-induced hepatic steatosis to NASH. However, the underlying mechanisms in which ER stress in adipose tissue induces hepatic pathology remain elusive. Here, we used male C57BL/6J mice to develop an animal model of NASH induced by a high fat (HFD) diet and methionine- and choline-deficient (MCD) diets. Using a gene-silencing approach with a recombinant lentiviral vector and extensive LC-MS/MS–based proteomics and lipidomics, we demonstrate that the ER stress–induced adipocyte–secreted exosome (ATEx) orchestrates lipid dynamics in the liver. We also noted that ATEx causes hepatic steatosis, inflammation, and fibrosis that lead to NASH through initial accumulation of glycerol and triglycerides in hepatocytes. We also determined that aldo-keto-reductase 1B7 (Akr1b7), a key mediator in liver lipid metabolism, is involved in ATEx–mediated NASH induction. Of note, Akr1b7 deficiency in ER stress–induced ATEx strongly protected the murine liver against HFD and MCD–induced NASH. Our results indicated that ER stress–induced, adipocyte-secreted ATEx triggers NASH by delivering exosomal AKR1B7 to, and elevating glycerol level, in hepatocytes. These findings suggest potential therapeutic strategie that target ATEx to prevent or manage obesity-induced NASH.

Met125 is essential for maintaining the structural integrity of calmodulin\u2019s C-terminal domain

We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations. CaM is a Ca2+-sensitive regulatory protein that interacts with numerous targets. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species under conditions of cell stress and age-related muscle degeneration. CaM oxidation alters regulation of a host of CaM’s protein targets, emphasizing the importance of understanding the mechanism of CaM oxidation in muscle degeneration and overall physiology. It has been shown that the M125Q CaM mutant can mimic the functional effects of methionine oxidation on CaM’s regulation of the calcium release channel, ryanodine receptor (RyR). We report here that the M125Q mutation causes a localized unfolding of the C-terminal lobe of CaM, preventing the formation of a hydrophobic cluster of residues near the EF-hand Ca2+ binding sites. NMR analysis of CaM oxidation by peroxide offers further insights into the susceptibility of CaM’s Met residues to oxidation and the resulting structural effects. These results further resolve oxidation-driven structural perturbation of CaM, with implications for RyR regulation and the decay of muscle function in aging.

Methionine Supplementation Affects Metabolism and Reduces Tumor Aggressiveness in Liver Cancer Cells.

Liver cancer is one of the most common cancer worldwide with a high mortality. Methionine is an essential amino acid required for normal development and cell growth, is mainly metabolized in the liver, and its role as an anti-cancer supplement is still controversial. Here, we evaluate the effects of methionine supplementation in liver cancer cells. An integrative proteomic and metabolomic analysis indicates a rewiring of the central carbon metabolism, with an upregulation of the tricarboxylic acid (TCA) cycle and mitochondrial adenosine triphosphate (ATP) production in the presence of high methionine and AMP-activated protein kinase (AMPK) inhibition. Methionine supplementation also reduces growth rate in liver cancer cells and induces the activation of both the AMPK and mTOR pathways. Interestingly, in high methionine concentration, inhibition of AMPK strongly impairs cell growth, cell migration, and colony formation, indicating the main role of AMPK in the control of liver cancer phenotypes. Therefore, regulation of methionine in the diet combined with AMPK inhibition could reduce liver cancer progression.

Histone methylation status of H3K4me3 and H3K9me3 under methionine restriction is unstable in methionine-addicted cancer cells, but stable in normal cells

Methionine addiction is a fundamental and general hallmark of cancer. Methionine addiction prevents cancer cells, but not normal cells from proliferation under methionine restriction (MR). Previous studies reported that MR altered the histone methylation levels in methionine-addicted cancer cells. However, no study has yet compared the status of histone methylation status, under MR, between cancer cells and normal cells. In the present study, we compared the histone methylation status between cancer cells and normal fibroblasts of H3K4me3 and H3K9me3, using recombinant methioninase (rMETase) to effect MR. Human lung and colon cancer cell lines and human normal foreskin fibroblasts were cultured in control medium or medium with rMETase. The viability of foreskin fibroblasts was approximately 10 times more resistant to rMETase than the cancer cells in vitro. Proliferation only of the cancer cells ceased under MR. The histone methylation status of H3K4me3 and H3K9me3 under MR was evaluated by immunoblotting. The levels of the H3K4me3 and H3K9me3 were strongly decreased by MR in the cancer cells. In contrast, the levels of H3K4me3 and H3K9me3 were not altered by MR in normal fibroblasts. The present results suggest that histone methylation status of H3K4me3 and H3K9me3 under MR was unstable in cancer cells but stable in normal cells and the instability of histone methylation status under MR may determine the high methionine dependency of cancer cells to survive and proliferate.

The Level of Methionine Residues in Storage Proteins Is the Main Limiting Factor of Protein-Bound-Methionine Accumulation in Arabidopsis Seeds.

The low level of methionine, an essential sulfur-containing amino acid, limits the nutritional quality of seeds. Two main factors can control the level of protein-bound methionine: the level of free methionine that limits protein accumulation and the methionine residues inside the storage proteins. To reveal the main limiting factor, we generated transgenic Arabidopsis thaliana seed-specific plants expressing the methionine-rich sunflower seed storage (SSA) protein (A1/A2). The contents of protein-bound methionine in the water-soluble protein fraction that includes the SSA in A1/A2 were 5.3- and 10.5-fold, respectively, compared to control, an empty vector (EV). This suggests that free methionine can support this accumulation. To elucidate if the level of free methionine could be increased further in the protein-bound methionine, these lines were crossed with previously characterized plants having higher levels of free methionine in seeds (called SSE). The progenies of the crosses (A1S, A2S) exhibited the highest level of protein-bound methionine, but this level did not differ significantly from A2, suggesting that all the methionine residues of A2 were filled with methionine. It also suggests that the content of methionine residues in the storage proteins is the main limiting factor. The results also proposed that the storage proteins can change their content in response to high levels of free methionine or SSA. This was assumed since the water-soluble protein fraction was highest in A1S/A2S as well as in SSE compared to EV and A1/A2. By using these seeds, we also aimed at gaining more knowledge about the link between high free methionine and the levels of metabolites that usually accumulate during abiotic stresses. This putative connection was derived from a previous analysis of SSE. The results of metabolic profiling showed that the levels of 29 and 20 out of the 56 metabolites were significantly higher in SSE and A1, respectively, that had higher level of free methionine, compared A1S/A2S, which had lower free methionine levels. This suggests a strong link between high free methionine and the accumulation of stress-associated metabolites.

Hypermethioninemia Leads to Fatal Bleeding and Increased Mortality in a Transgenic I278T Mouse Model of Homocystinuria.

Severely elevated plasma homocysteine and methionine lead to thromboembolic events and strokes in homocystinuric (HCU) patients. Mouse models of HCU failed to exhibit prothrombotic phenotype, presumably due to lack of hypermethioninemia. We evaluated the impact of hypermethioninemia together with hyperhomocysteinemia on murine HCU phenotype and compared the efficacy of the current and novel therapies for HCU. High methionine intake decreased survival of I278T mice, which died from intestinal bleeding with hepatic and pancreatic failure. I278T mice on normal or increased methionine intake developed endothelial dysfunction, but paradoxically demonstrated delayed occlusion in an induced arterial thrombosis model. RNA-seq analysis suggested that expression of coagulation factor XI (FXI) is downregulated in livers of I278T mice. Indeed, plasma concentrations of FXI were decreased in I278T mice on normal diet and further reduced by increased methionine intake. Dietary methionine restriction normalized the observed phenotype. Similarly, treatment with OT-58, a novel enzyme therapy for HCU, corrected the phenotype in I278T mice regardless of their dietary methionine intake. Hypermethioninemia does not contribute to prothrombotic phenotype in murine HCU. Downregulation of FXI may contribute to the lack of prothrombotic tendency in I278T mice. Methionine restriction or treatment with OT-58 corrects vascular disease in the I278T mouse model of HCU.

Hydrogen Sulfide Alleviates Anxiety, Motor, and Cognitive Dysfunctions in Rats with Maternal Hyperhomocysteinemia via Mitigation of Oxidative Stress.

Hydrogen sulfide (H2S) is endogenously produced from sulfur containing amino acids, including homocysteine and exerts neuroprotective effects. An increase of homocysteine during pregnancy impairs fetal growth and development of the offspring due to severe oxidative stress. We analyzed the effects of the H2S donor—sodium hydrosulfide (NaHS) administered to female rats with hyperhomocysteinemia (hHcy) on behavioral impairments and levels of oxidative stress of their offspring. Rats born from females fed with control or high methionine diet, with or without H2S donor injections were investigated. Rats with maternal hHcy exhibit increased levels of total locomotor activity and anxiety, decreased muscle endurance and motor coordination, abnormalities of fine motor control, as well as reduced spatial memory and learning. Oxidative stress in brain tissues measured by activity of glutathione peroxidases and the level of malondialdehyde was higher in rats with maternal hHcy. Concentrations of H2S and the activity and expression of the H2S generating enzyme—cystathionine-beta synthase—were lower compared to the control group. Administration of the H2S donor to females with hHcy during pregnancy prevented behavioral alterations and oxidative stress of their offspring. The acquisition of behavioral together with biochemical studies will add to our knowledge about homocysteine neurotoxicity and proposes H2S as a potential agent for therapy of hHcy associated disorders.

Effect of dietary cassava peel meal supplemented with methionine and multienzyme on hemo-biochemical indices, digestibility, and antioxidants in rabbits

BackgroundThe effects of cassava peel meal (CPM) supplemented with methionine (MET) and multienzyme (ENZ) was studied using 240 5-week-old rabbits in a completely randomized design arranged in 2 × 2 × 2 factorial (2 CPM levels, 0 and 350 g/kg; 2 methionine levels, 5.6 and 8.3 g/kg; and 2 enzyme levels, 0 and 0.5 g/kg).ResultsThe CPM inclusion reduced (P < 0.05) leucine, lysine, valine, and threonine digestibility, while the digestibility of leucine, lysine, and valine increased (P < 0.05) by high methionine level. Multienzyme increased leucine, lysine, and valine digestibility. CPM reduced (P < 0.01) red blood cell (RBC) count and the total serum protein. Higher methionine supplementation increased the serum total protein by 9.2%. The CPM inclusion increased bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Methionine and enzyme supplementation increased albumin (ALB) but reduced creatinine (CRE). Superoxide dismutase, glutathione peroxidase, and catalase activity level increased (P < 0.01) with methionine and enzyme supplementation.ConclusionCassava peel meal could be included in growing rabbits’ diets at 350 g/kg level with methionine at 32.53% higher than requirement and 0.5-g/kg multienzyme supplementation.

2136-PUB: GLP-1 Agonist Exendin-4 Protects Endothelial Cells against Hyperhomocysteinemia by Alleviating ER Stress and Enhancing Protein Folding Machinery

Hyperhomocysteinemia (HHcy) is a risk factor for higher mortality in type 2 diabetic patients. HHcy induces endothelial dysfunction, which precedes overt hyperglycemia in type 2 diabetic patients. In addition, endothelial dysfunction predisposes diabetic patients to cardiovascular disorders, such as atherosclerotic cardiovascular diseases and retinopathy. HHcy induces oxidative stress and endoplasmic reticulum (ER) stress to impair endothelial cells. GLP-1 agonist exendin-4 was shown to attenuate ER stress in endothelial cells although detailed mechanism remains elusive. The current study intends to reveal the beneficial effects of exendin-4 on HHcy-impaired endothelial cells. HHcy was induced by feeding C57BL/6 mice a high methionine low folate diet for 12 weeks. To assess endothelial function, mouse aortae and mesenteric arteries were mounted on wire myograph to determine acetylcholine-induced endothelium-dependent relaxations (EDRs). OGTT and ITT were conducted to study in vivo glucose metabolism. Dihydroethidium staining and lucigenin-enhanced chemiluminescence were used to measure ROS production. The ER stress markers expression was measured by biochemical assays. The role of ER chaperone was studied by adenoviral overexpression and siRNA-mediated knockdown. A 4-week subcutaneous injection of exendin-4 significantly improved glucose tolerance, insulin sensitivity and EDRs in arteries of diet-induced HHcy mice. Exendin-4 inhibited ROS production and ER stress markers expression in mouse aortae and human endothelial cells. Such inhibitions were ablated by compound C, an AMPK inhibitor. By AMPK activation, exendin-4 up-regulated endothelial expression of ER oxidoreductase (ERO1α), an ER chaperone. In short, through AMPK-dependent upregulation of ERO1α in endothelial cells, GLP-1 agonist exendin-4 lowers HHcy-induced ER stress and oxidative stress to restore endothelial function. Disclosure C. Cheng: None. J. Luo: None. C. Lau: None. X. Tian: None. Y. Huang: None. Funding Health and Medical Research Fund (05162906); Early Career Scheme (24122318); Hong Kong Research Grants Council (14112919); Collaborative Research Fund (C4024-16W)

Citrus reticulata Blanco peel extract ameliorates hepatic steatosis, oxidative stress and inflammation in HF and MCD diet-induced NASH C57BL/6 J mice

Excessive lipid deposition, oxidative stress and inflammation in liver tissues are regarded as crucial inducers of nonalcoholic steatohepatitis (NASH), which is the most frequent chronic liver disease and closely related to obesity and insulin resistance. In this work, the preventive and therapeutic effects of Citrus reticulata Blanco (Jizigan) peel extract (JZE) on NASH induced by high fat (HF) diet and methionine choline-deficient (MCD) diet in C57BL/6 mice were investigated. We found that daily supplementation of JZE with an HF diet effectively ameliorated glucose tolerance and insulin resistance. In addition, the key indexes of lipid profiles, oxidative stress, hepatic steatosis and inflammatory factors were also ameliorated in both NASH mouse models. Furthermore, JZE treatment activated nuclear factor erythroid-2-related factor 2 (Nrf2) in the livers of diet- induced NASH mice. Our study suggests that JZE might alleviate NASH via the activation of Nrf2 signaling and that citrus Jizigan could be used as a dietary therapy for NASH and related metabolic syndrome.

Elevated CO2 improves glucosinolate metabolism and stimulates anticancer and anti-inflammatory properties of broccoli sprouts

Sprouting process enhances plant bioactive compounds. Broccoli (Brassica oleracea L) sprouts are well known for their high levels of glucosinolates (GLs), amino acids, and antioxidants, which offer outstanding biological activities with positive impacts on plant metabolism. Elevated CO2 (eCO2, 620 ppm) was applied for 9 days to further improve nutritive and health-promoting values of three cultivars of broccoli sprouts i.e., Southern star, Prominence and Monotop. eCO2 improved sprouts growth and induced GLs accumulation e.g., glucoraphanin, possibly through amino acids production e.g., high methionine and tryptophan. There were increases in myrosinase activity, which stimulated GLs hydrolysis to yield health-promoting sulforaphane. Interestingly, low levels of ineffective sulforaphane nitrile were detected and positively correlated with reduced epithiospecifier protein after eCO2 treatment. High glucoraphanin and sulforaphane levels in eCO2 treated sprouts improved the anticarcinogenic and anti-inflammatory properties of their extracts. In conclusion, eCO2 treatment enriches broccoli sprouts with health-promoting metabolites and bioactivities.

Association of Homocysteine, Methionine, and MTHFR 677C>T Polymorphism With Rate of Cardiovascular Multimorbidity Development in Older Adults in Sweden

Strong evidence links high total serum homocysteine (tHcy) and low methionine (Met) levels with higher risk of ischemic disease, but other cardiovascular (CV) diseases may also be associated with their pleiotropic effects. To investigate the association of serum concentrations of tHcy and Met with the rate of CV multimorbidity development in older adults and to explore the role of methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism in this association. The Swedish National Study on Aging and Care in Kungsholmen is a cohort study of randomly selected individuals aged 60 years or older. The present study included data on 1969 individuals with complete information and without CV diseases at baseline, collected from the baseline examination (2001-2004) to the fourth follow-up (2013-2016). Data analysis was conducted from January to May 2019. Concentrations of tHcy and Met were measured from nonfasting venous blood samples. The Met:tHcy ratio was considered a possible indicator of methylation activity. MTHFR status was dichotomized as any T carriers vs noncarriers. The number of CV diseases at each wave was ascertained based on medical interviews and records, laboratory test results, and drug data. Linear mixed models were used to study the association of baseline tHcy and Met levels and the rate of CV multimorbidity development, adjusting for sociodemographic characteristics, CV risk factors, chronic disease burden, and drug use. Of 1969 participants, most were women (1261 [64.0%]), with a mean (SD) age of 70.9 (9.8) years; 1703 participants (86.6%) had at least a high school level of education. Baseline measurements of serum tHcy, Met, and the Met:tHcy ratio were associated with the rate of CV disease accumulation (tHcy: β = 0.023 per year; 95% CI, 0.015 to 0.030; P < .001; Met: β = -0.007 per year; 95% CI, -0.013 to -0.001; P = .02; Met:tHcy ratio: β = -0.017 per year; 95% CI, -0.023 to -0.011; P < .001). The association between low Met concentrations and the rate of CV multimorbidity development was restricted to the group with CT/TT alleles of MTHFR (β = 0.023 per year; 95% CI, 0.006 to 0.041; P = .009). Results remained largely significant when individual CV diseases were removed from the total count 1 at a time (eg, ischemic heart disease, tHcy: β = 0.023 per year; 95% CI, 0.013 to 0.027; P < .001; Met: β = -0.006 per year; 95% CI, -0.011 to -0.0003; P = .04; Met:tHcy ratio: β = -0.015 per year; 95% CI, -0.020 to -0.009; P < .001). In this study, high tHcy and low Met levels were associated with faster CV multimorbidity development in older age. The interactive association of Met concentrations and MTHFR polymorphism, together with the association found for the Met:tHcy ratio, point toward the relevance of impaired methylation in the pathogenesis of CV aging.

The effect of different dietary ratios of lysine and arginine in diets with high or low methionine levels on oxidative and epigenetic DNA damage, the gene expression of tight junction proteins and selected metabolic parameters in Clostridium perfringens-challenged turkeys

Two experiments were performed to investigate the effect of different ratios of arginine (Arg) to lysine (Lys) in diets with low (30% Lys; Experiment 1) and high (45% Lys; Experiment 2) methionine (Met) levels on selected metabolic parameters, oxidative and epigenetic DNA damage, and the mechanisms underlying intestinal barrier integrity in turkeys challenged with Clostridium perfringens. In each experiment, 108 one-day-old Hybrid Converter female turkeys were placed in 6 pens (18 birds per pen) and reared for 42 days. At 34, 36 and 37 days of age, half of the birds were subjected to C. perfringens challenge. A 3 × 2 factorial design with three levels of Arg relative to Lys (90, 100 and 110%; Arg90, Arg100 and Arg110, respectively) and C. perfringens infection (−, +) was employed. Challenging birds with C. perfringens increased lipid oxidation and the oxidation and methylation of DNA of intestinal mucosa, and down-regulated the activities of DNA-repairing enzymes. Neither the dietary treatment nor the challenge affected the markers of liver function or metabolism. Arg110 diets with the high Met level induced DNA oxidation and methylation whereas these processes were downregulated in birds fed Arg90 diets. The results indicate that Arg90 diets with high Met levels have a beneficial influence on the indicators of intestinal barrier integrity in turkeys with necrotic enteritis (NE). Despite the analyzed amino acid ratios interacted with the systems responsible for the maintenance of gut integrity in the host organism, this dietary intervention probably enabled birds to cope with NE.

Supplemental methionine and stocking density affect antioxidant status, fatty acid profiles, and growth performance of broiler chickens.

Broilers stocked in high densities may be prone to oxidative and inflammatory insults, resulting in impaired health status, growth performance, and meat quality. This study was to determine if 30% extra supplemental dl-methionine alleviated or prevented those adverse effects of a higher stocking density in broiler chickens. A total of 560 male Cornish Cross cockerels (day old) were divided into four groups: two stocking densities (9 and 12 birds/m2) and two supplementations of methionine (grower: 2.90 or 3.77 g/kg and finisher: 2.60 or 3.38 g/kg). Growth performance was recorded weekly. Blood and tissues were sampled at the end of each period. High stocking density decreased (P < 0.05) body weight and growth performance of growers and (or) finishers. Those differences were partially attenuated by the extra methionine supplementation. The high methionine elevated (P < 0.05) glutathione (GSH) concentration in the thigh at both ages (> 24%). The high stocking density elevated (>28%, P < 0.05) glutathione concentration in the plasma, breast, and thigh of growers, but decreased (P < 0.05) it in the liver of growers and thigh of finishers. Interaction effects (P < 0.05) between dietary methionine and stocking density were found on activities of the antioxidant enzyme glutathione S-transferase in the liver of growers and breast, thigh, and adipose tissue of finishers. The interaction effect was also found on activities of glutathione peroxidase and superoxide dismutase in the thigh of growers. The extra methionine decreased (P < 0.05) hepatic gene expression of heat shock protein 90 (18%) and thigh and breast malondialdehyde concentrations of the finishers (35%). In conclusion, the 30% extra dl-methionine supplementation was able to partially mitigate adverse effects caused by the higher stocking density and to improve the redox status of the broilers.