Met Restriction Research Articles

Effects of methionine restriction and methionine hydroxy analogs on intestinal inflammation and physical barrier function in mice

Methionine (Met) restriction in the diet can reduce intestinal permeability by modulating the expression of intestinal tight proteins leading to enhance the integrity of the intestinal epithelium. Furthermore, our previous study observed that 2-hydroxy-4-(methylthio)butanoate (HMTBA) increased Tight junction protein 1 (ZO-1) mRNA expression in IPEC-J2 when compared with L-Met. This increment may be related to the decline in RNA N6-methyladenosine (m6A) modification level. Nonetheless, the impact of HMTBA utilization as a source of Met and the effects of combining it with Met restriction on the intestinal epithelial barrier in vivo remains uncertain. We constructed the methionine restrict model and Salmonella typhimurium-induced damage model, and then detected the intestinal damage by real-time qPCR, Immunofluorescence staining, and Elisa, and used LC-MS/MS to detect the m6A level and Met Metabolites. Results exhibited that dietary Met restraint, either through Met or HMTBA as the exclusive Met source, could drastically mitigate intestinal barrier dysfunction and the inflammation caused by S. typhimurium. The underlying mechanism involved in the impact of low HMTBA and Met concentration on intestinal barrier function alteration seems to be associated with changes in Met metabolism, along with m6A RNA or 5-methylcytosine (m5C) DNA level modification. The HMTBA addition to normal levels of Met, apart from enhancing the growth rate, fails to prevent S. typhimurium-induced damage to the intestinal barrier function.

Changes in growth performance, plasma metabolite concentrations, and myogenic gene expression in growing pigs fed a methionine-restricted diet.

Background: Methionine (Met) is usually the second or third limiting amino acid in swine diets and plays vital roles in promoting the growth, especially, the muscle growth of pigs. This research evaluated the effects of dietary Met restriction on the growth performance, plasma metabolite concentrations, and myogenic gene expression in growing pigs. Materials and methods: Eight genes in two families (myogenic regulatory factor family and myocyte enhancer factor 2 family) were selected for the analysis. Twenty individually penned barrows (crossbred, 23.6 ± 2.4 kg) were randomly allotted to two dietary treatments (n = 10). A diet based on corn and soybean meal (Diet 1, Met-restricted) was formulated to meet or exceed the energy and nutrient requirements, except for Met. Diet 2 (Met-adequate) was formulated by adding crystalline DL-Met to Diet 1 to meet the Met requirement. During the 4-week feeding trial, average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were measured. Immediately before and after the feeding trial, blood was sampled via jugular venipuncture for plasma nutrient metabolite analysis, while Longissimus dorsi muscle were sampled via aseptic biopsy for gene expression analysis. Data were analyzed with Student t-test. Results: Pigs fed the Met-restricted diet had lower ADG and G:F (P < 0.01). Plasma Met, cysteine, and taurine concentrations were lower (P < 0.05), while glycine and histidine concentrations were higher (P < 0.05), in pigs fed the Met-restricted diet. Furthermore, the pigs fed the Met-restricted diet tended to express less myogenic factor 6 (Myf6) and myocyte enhancer factor 2D (Mef2D) mRNA in longissimus dorsi muscle (P < 0.09). Conclusion: Given the fact that Myf6, assisted by Mef2D, is involved in myocyte differentiation, this study suggests that the reduced growth performance in the Met-restricted pigs may be associated with a reduced muscle cell differentiation.

200 Reduced growth performance of pigs fed methionine deficient diet may be associated with their reduced muscle cell differentiation

Abstract Methionine (Met) is the second or third limiting AA in typical swine diets and plays important roles in promoting the growth, especially, the muscle growth of pigs. This research studied the effects of dietary Met restriction on growth performance and myogenic gene expression in growing pigs. Eight genes in two families, including myogenic regulatory factor family comprising myogenic differentiation 1 (MyoD1), myogenic factor 5 (Myf5), myogenin (MyoG) and myogenic factor 6 (Myf6), and myocyte enhancer factor 2 (Mef2) family comprising Mef2A, Mef2B, Mef2C and Mef2D, were selected for analysis. Individually penned barrows (crossbred, 23.6±2.4 kg) were randomly assigned to two dietary treatments. A basal diet (Diet 1; Met-deficient) was formulated to contain 0.22% standardized ileal digestible (SID) Met and 0.52% SID Met+Cys but to meet the NRC (2012) recommendations for other nutrients. Crystalline DL-Met was added to the basal diet to generate Diet 2 adequate in Met (0.37% SID Met, 0.67% SID Met+Cys). During the four-week ad libitum feeding trial, ADG, ADFI and G:F were measured. Immediately before and after the trial, muscle samples were collected from the longissimus dorsi using a standard biopsy protocol. Total RNA was extracted from the muscle samples (TRIzol Reagent; Invitrogen) and subjected to cDNA reverse-transcription (QuantiTect Reverse Transcription Kit; Qiagen). Transcribed cDNA were used for qPCR analysis (Rotor-Gene Q System; Qiagen). Comparative ΔΔCT method was used for quantitation. Data were analyzed with Student’s T-test. Pigs fed Diet 1 (vs. Diet 2) had a lower ADG and G:F (P&amp;lt; 0.01). Before the feeding trial, all the tested genes had comparable mRNA levels between the two treatments (P &amp;gt;0.17). After the trial, Diet 1 pigs showed tendency for lower levels of Myf6 and Mef2D mRNA (P&amp;lt; 0.09). These results suggest that the reduced ADG and G:F is associated with the possibly-reduced muscle cell differentiation in pigs fed Met-deficient diet.

In Vitro Influence of Extracts from Snail Helix aspersa Müller on the Colon Cancer Cell Line Caco-2.

Colorectal cancer is the third most widely diagnosed cancer. Extracts from snails may modulate growth and development of colorectal cancer cells. The objective of this study was to determine the chemical composition of tissues derived from Helix aspersa Müller and red-ox properties of tissue extracts. Then, the influence of extracts and their fractions of different molecular weights on viability of Caco-2 cells was examined. Tissue lyophilisates contained antioxidants that could be important in the prevention of colorectal cancer. Moreover, we confirmed the presence of a wide array of compounds that might be used in treatment of this disease. The decrease of cell viability after the application of extracts from lyophilized mucus and foot tissues was affirmed. The effect of extract from mucus could be related to the content of some proteins and peptides, proper essential amino acids (EAA)/non-essential amino acids (NEAA) ratio, Met restriction and the presence of Cu, Ca, Zn, Se. The influence of the extract from foot tissues could be assigned additionally to the presence of eicosapentaenoic, α-linolenic, linoleic and γ-linolenic acids. The opposite effect was demonstrated by extract from lyophilized shells which increased cell viability. Further studies are needed to know whether dietary supplying of H. aspersa Müller tissues can be used as an approach in colorectal cancer management.

Enhancement of the Antitumor Effect of Methotrexate on Colorectal Cancer Cells via Lactate Calcium Salt Targeting Methionine Metabolism

ABSTRACTMethionine (Met) is involved in one-carbon de novo nucleotide synthesis and is an essential amino acid for cell survival. The impact of lactate calcium salt (CaLa) on the Met metabolism was investigated to evaluate the enhanced antitumor effect of methotrexate (MTX) on colorectal cancer (CRC) cells. Met dependency relating to homocysteine (Hcy) and betaine was investigated in human CRC cells (HCT-116 and HT-29) using a viability assay and liquid chromatography-mass spectrometry. Expression of betaine transporter-1 (BGT-1) following treatment with MTX alone or with CaLa was determined by Western blot. Enhanced antitumor effect due to malfunction of Met synthesis was confirmed. CRC cell viability decreased in Met-restricted medium, but was maintained after Hcy and betaine treatment while overcoming Met restriction. BGT-1 expression was downregulated following the treatment of dose-increased CaLa, whereas there was no effect on BGT-1 expression after MTX treatment. CaLa in combination with MTX induced reduced Met synthesis when CRC cell viability was reduced. The results indicated that CaLa-mediated BGT-1 downregulation inhibits Met synthesis by disrupting betaine homeostasis. CaLa raised the antitumor effect of MTX via secondary role in the inhibition of the de novo nucleotide synthesis. Combination therapy of MTX and CaLa could maximize the effectiveness of CRC treatment.

Abstract 5330: Methioninase-loaded erythrocytes: a promising drug for L-methionine restriction therapy in cancer

Abstract Several studies reported a complete dependence of different tumor cells on L-methionine (Met), an essential amino acid, for maintaining their high proliferation rate while normal cells keep their ability to grow on Met-deprived medium (Cavuoto and Fenech, 2012). Met restriction appearing as an important strategy for cancer treatment, different therapeutic approaches using either Met restricted regimens or Met-catabolizing enzymes were investigated. Phase I and II clinical trials associating dietary Met restriction with chemotherapy were conducted with cancer patients (Thivat et al., 2009; Durando et al, 2010). A 40% reduction of plasmatic methionine was achieved with a 1-day Met-free diet per month. Interesting long-duration of disease stabilization was observed in some patients. However this diet is extremely difficult to maintain. In order to obtain longer Met depletion in plasma, enzyme therapy using Methionine gamma-lyase (MGL), a pyridoxal-dependent enzyme that specifically degrades Met into alpha-ketobutyrate, ammonia and thiols, was explored. MGL enzyme isolated from Pseudomonas putida was selected as preferred candidate for the development of therapy. However, preclinical studies conducted in primates showed a very short half-life of the active form of MGL enzyme (2h) due to a rapid in vivo dissociation of the pyridoxal (PLP) cofactor (Yang et al., 2004). To overcome this problem of PLP-dissociation, we encapsulated the active MGL into erythrocytes, natural pool of PLP in the body and evaluated PK-PD parameters of product over a 5-day period in a mouse model. Once entrapped into erythrocytes, the MGL enzyme maintained more than 30% of its specific activity after 5 days in circulation. A 60-70% Met depletion was achieved 15 min post-injection and then depletion was stabilized at 40% over the 5 day-period of the study. In comparison, administration of the free MGL at similar doses led to a rapid loss of the active form in several hours. Met was totally deprived 15 min post-injection but plasmatic Met level returned to baseline by 24h. Increasing two-fold the concentration of free methioninase did not improved the in vivo Met depletion. In conclusion, we report that methioninase-loaded-erythrocytes can lower Met concentration in mouse plasma for at least 5 days and may be an innovative strategy to kill tumors by met-starvation. Citation Format: Fabien Gay, Julie Bes, Vanessa Bourgeaux, Yann Godfrin. Methioninase-loaded erythrocytes: a promising drug for L-methionine restriction therapy in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2015-5330

Methionine restriction extends lifespan of Drosophila melanogaster under conditions of low amino-acid status

Reduced methionine (Met) intake can extend lifespan of rodents, but whether this regimen represents a general strategy for regulating aging has been controversial. Here we report that Met restriction extends lifespan in both fruit flies and yeast, and that this effect requires low amino acid status. Met restriction in Drosophila mimicks the effect of dietary restriction and is associated with decreased reproduction. However, under conditions of high amino acid status, Met restriction is ineffective and the trade-off between longevity and reproduction is not observed. Overexpression of InRDN or Tsc2 inhibits lifespan extension by Met restriction, suggesting the role of TOR signaling in the Met control of longevity. Overall, this study defines the specific roles of Met and amino acid imbalance in aging and suggests that Met restiction is a general strategy for lifespan extension.

Abstract 3792: Specific amino acid restriction differentially induces autophagy in DU145 and PC3 human prostate cancer cell lines

Abstract We previously found that selective amino acid restriction differentially induces death of DU145 and PC3 human prostate cancer cells. Herein we examined the hypothesis that this differential cell death was associated with induction of autophagy. By detecting the immunofluorescence of microtubule-associated protein 1A/1B light chain 3 (LC3) by confocal microscopy, this study shows that selective amino acid restriction also differentially induces autophagy in DU145 and PC3 human prostate cancer cell lines. In DU145 cells, restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), and methionine (Met) induces autophagy. Addition of pyruvate significantly increases Tyr/Phe restriction-induced autophagy and significantly reduces Met and Gln restriction-induced autophagy. All of the amino acid restrictions induce cell death in DU145 cells. Pyruvate, a major metabolite of glucose metabolism, significantly reduces Tyr/Phe and Gln restriction-mediated cell death, but not Met restriction-medicated cell death. Tyr/Phe and Gln restriction induces autophagy in PC3 cells. Addition of pyruvate does not alter Tyr/Phe restriction-induced autophagy but significantly increases Met and Gln restriction-induced autophagy. Only Met restriction induces cell death in this cell line, and pyruvate addition reverses this effect. In summary, the present study indicates 1) cell line specific differences between DU145 and PC3 cells in the induction of autophagy by specific amino acid restriction, 2) lack of association between induction of autophagy and cell death, and 3) differential effects of pyruvate on autophagy and cell death. (This study was supported by grant R01CA101035 from the National Cancer Institute). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3792. doi:10.1158/1538-7445.AM2011-3792

Dietary glycine supplementation mimics lifespan extension by dietary methionine restriction in Fisher 344 rats

Dietary methionine (Met) restriction (MR) extends lifespan in rodents by 30–40% and inhibits growth. Since glycine is the vehicle for hepatic clearance of excess Met via glycine N-methyltransferase (GNMT), we hypothesized that dietary glycine supplementation (GS) might produce biochemical and endocrine changes similar to MR and also extend lifespan. Seven-week-old male Fisher 344 rats were fed diets containing 0.43% Met/2.3% glycine (control fed; CF) or 0.43% Met/4%, 8% or 12% glycine until natural death. In 8% or 12% GS rats, median lifespan increased from 88 weeks (w) to 113 w, and maximum lifespan increased from 91 w to 119 w v CF. Body growth reduction was less dramatic, and not even significant in the 8% GS group. Dose-dependent reductions in several serum markers were also observed. Long-term (50 w) 12% GS resulted in reductions in mean (±SD) fasting glucose (158 ± 13 v 179 ± 46 mg/dL), insulin (0.7 ± 0.4 v 0.8 ± 0.3 ng/mL), IGF-1 (1082 ± 128 v 1407 ± 142 ng/mL) and triglyceride (113 ± 31 v 221 ± 56 mg/dL) levels compared to CF. Adiponectin, which increases with MR, did not change in GS after 12 w on diet. We propose that more efficient Met clearance via GNMT with GS could be reducing chronic Met toxicity due to rogue methylations from chronic excess methylation capacity or oxidative stress from generation of toxic by-products such as formaldehyde. This project received no outside funding.

Abstract 39: Specific amino acid restriction affects glucose metabolism and induces oxidative damage of mitochondria in prostate cancer cells

Abstract The purpose of this study was to examine how specific amino acid restriction targets mitochondria to induce apoptosis in DU145 and PC3 prostate cancer cells. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), and methionine (Met) differentially modulates glucose metabolism, the activity of pyruvate dehydrogenase (PDH) and antioxidant status in the mitochondria of these two cell lines. In DU145 cells, Gln and Met restriction but not Tyr/Phe restriction, increase glucose consumption and decrease lactate production. All the examined restrictions increase mitochondrial PDH activity and reactive oxygen species (ROS). Gln and Met restriction increase mitochondrial activity of glutathione peroxidase (GPx). Tyr/Phe and Met restriction increase mitochondrial activity of manganese superoxide dismutase (SOD) in the first 2 days of the restriction, and the activity returns to basal level on day 4 of restriction. Moreover, all the examined restrictions decrease reduced glutathione (GSH) and cause mitochondrial DNA damage. In PC3 cells, all of the examined restrictions reduce glucose consumption and lactate production; however, they do not alter mitochondrial PDH activity. Gln restriction increases accumulation of ROS and elevates mitochondrial GPx activity. Tyr/Phe restriction increases mitochondrial activity of SOD during the entire restriction period. Although all the restrictions decrease GSH in PC3 cells, they do not cause mitochondrial DNA damage. The data indicate that specific amino acid dependency differentially regulates glucose metabolism, oxidation-reduction reactions of mitochondria, and oxidative damage in mitochondria of these two cell lines. This partially explains how selective amino acid restriction targets mitochondria to induce apoptosis. (The project described was supported by Award Number R01CA101035 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 39.

Tissue glutathione and cysteine levels in methionine-restricted rats

Objective Previously, we demonstrated that lifelong methionine (Met) restriction (MR) increases lifespan, decreases the incidence of aging-related diseases, increases blood glutathione (GSH) levels, and prevents loss of GSH during aging in rats. Our present objective was to elucidate the effects of MR on GSH metabolism and transport by determining the time course and nature of GSH and cysteine changes in blood and other tissues in young and mature rats. Methods Male F-344 rats were placed on control (0.86% Met) or MR (0.17% Met) defined amino acid diets at age 7 wk and killed at different times thereafter. MR was also initiated in adult (12-mo-old) rats. Results Throughout the first 2 mo of MR, blood GSH levels increased 84% and liver GSH decreased 66% in relation to controls. After this period, liver GSH levels remained constant through at least 6 mo. GSH levels also decreased in the pancreas (80%) and kidney (22%) but remained unchanged in other tissues examined after 11 wk of MR. The increase in blood GSH was evident as soon as 1 wk after initiating MR and reached a plateau by 6 wk. A similar increase in erythrocyte GSH levels was observed when MR was administered to mature adult rats. Fasting decreased liver GSH in controls but had no further effect in MR animals. By 1 mo, cysteine levels had decreased in all tissues except brain. Conclusion These results suggest that adaptive changes occur in the metabolism of Met, cysteine, and/or GSH as a result of MR in young and adult rats. These early metabolic changes lead to conservation of GSH levels in most extrahepatic tissues and increased GSH in erythrocytes by depleting liver GSH to a critical level.

Specific Amino Acid Dependency Regulates Invasiveness and Viability of Androgen-Independent Prostate Cancer Cells

Androgen-independent prostate cancer is resistant to therapy and is often metastatic. Here we studied the effect of deprivation of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met), in vitro on human DU145 and PC3 androgen-independent prostate cancer cells, and on nontumorigenic human infant foreskin fibroblasts and human prostate epithelial cells. Deprivation of the amino acids similarly inhibited growth of DU145 and PC3 cells, arresting the cell cycle at G0/G1. Met and Tyr/Phe deprivation induces apoptosis in DU145, but only Met deprivation induces apoptosis in PC3 cells. The growth of normal cells is inhibited, but no apoptosis is induced by amino acid deprivation. Tyr/Phe deprivation inhibits expression and phosphorylation of focal adhesion kinase (FAK) and extracellular-regulated kinase (ERK) in DU145 but not PC3 or normal cells. Met deprivation inhibits phosphorylation but not protein expression of FAK and ERK in PC3. Therefore, apoptosis of DU145 and PC3 cells by amino acid restriction is FAK and ERK dependent. Tyr/Phe and Met deprivation inhibits invasion of DU145 and PC3, but Gln deprivation only inhibits invasion of DU145 cells. This indicates that the inhibition of invasion is not dependent on induction of apoptosis. The inhibition of invasion by Tyr/Phe restriction in DU145 and Met restriction in PC3 is consistent with the inhibition on FAK/ERK signaling. The inhibition of Tyr/Phe restriction in PC3 and Gln restriction in DU145 is not associated with inhibition of FAK/ERK. This indicates that FAK/ERK-dependent and independent pathways are modulated by specific amino acid restriction. This study shows the potential for specific amino acid restriction to treat prostate cancer.