Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1

Ferroptosis is a type of programmed necrosis triggered by iron-dependent lipid peroxidation. We investigated the role of B-cell translocation gene 1 (BTG1) in cystine and methionine deficiency (CST/Met (–))-mediated cell death. CST/Met (–) depleted reduced and oxidized glutathione in hepatocyte-derived cells, increased prostaglandin-endoperoxide synthase 2 expression, and promoted reactive oxygen species accumulation and lipid peroxidation, as well as necrotic cell death. CST/Met (–)-mediated cell death and lipid peroxidation was specifically inhibited by pretreatment with ferroptosis inhibitors. In parallel with cell death, CST/Met (–) blocked global protein translation and increased the expression of genes associated with the integrated stress response. Moreover, CST/Met (–) significantly induced BTG1 expression. Using a BTG1 promoter-harboring reporter gene and siRNA, activating transcription factor 4 (ATF4) was identified as an essential transcription factor for CST/Met (–)-mediated BTG1 induction. Although knockout of BTG1 in human HAP1 cells did not affect the accumulation of reactive oxygen species induced by CST/Met (–), BTG1 knockout significantly decreased the induction of genes associated with the integrated stress response, and reduced lipid peroxidation and cell death in response to CST/Met (–). The results demonstrate that CST/Met (–) induces ferroptosis by activating ATF4-dependent BTG1 induction.

Methionine Deficiency Affects Liver and Kidney Health, Oxidative Stress, and Ileum Mucosal Immunity in Broilers

Methionine (Met) is the first limiting amino acid in broiler diets, but its unclear physiological effects hamper its effective use in the poultry production industry. This study assessed the effect of a Met-deficient (MD) diet on chicken liver and kidney health, exploring the associated mechanisms of antioxidant capacity and ileum mucosal immunity. Seventy-two broilers were administered either the control diet (0.46% Met in starter diet, 0.36% Met in grower diet) or the MD diet (0.22% Met in starter diet, 0.24% Met in grower diet). Liver and kidney samples were collected every 14 days for anatomical, histological, and ultrastructural analyses, accompanied by oxidative stress assessment. Meanwhile, T- and B-lymphocyte abundance and essential cytokine gene expression were measured in the ileum, the center of the gut–liver–kidney axis. Signs of kidney and liver injury were observed morphologically in the MD group at 42 days of age. Furthermore, aspartate aminotransferase, alanine aminotransferase, creatinine, and uric acid levels were decreased in the MD group compared with the control group, accompanied by decreased superoxide dismutase activity, increased malondialdehyde content, decreased numbers of T and B lymphocytes, and decreased cytokine expression in the ileum, such as IL-2, IL-6, LITAF, and IFN-γ. These results suggest that MD can induce kidney and liver injury, and the injury pathway might be related to oxidative stress and intestinal immunosuppression.

Dietary Methionine Deficiency Enhances Genetic Instability in Murine Immune Cells

Both cell and animal studies have shown that complete or partial deficiency of methionine inhibits tumor growth. Consequently, the potential implementation of this nutritional intervention has recently been of great interest for the treatment of cancer patients. Unfortunately, diet alteration can also affect healthy immune cells such as monocytes/macrophages and their precursor cells in bone marrow. As around half of cancer patients are treated with radiotherapy, the potential deleterious effect of dietary methionine deficiency on immune cells prior to and/or following irradiation needs to be evaluated. Therefore, we examined whether modulation of methionine content alters genetic stability in the murine RAW 264.7 monocyte/macrophage cell line in vitro by chromosomal analysis after 1-month culture in a methionine-deficient or supplemented medium. We also analyzed chromosomal aberrations in the bone marrow cells of CBA/J mice fed with methionine-deficient or supplemented diet for 2 months. While all RAW 264.7 cells revealed a complex translocation involving three chromosomes, three different clones based on the banding pattern of chromosome 9 were identified. Methionine deficiency altered the ratio of the three clones and increased chromosomal aberrations and DNA damage in RAW 264.7. Methionine deficiency also increased radiation-induced chromosomal aberration and DNA damage in RAW 264.7 cells. Furthermore, mice maintained on a methionine-deficient diet showed more chromosomal aberrations in bone marrow cells than those given methionine-adequate or supplemented diets. These findings suggest that caution is warranted for clinical implementation of methionine-deficient diet concurrent with conventional cancer therapy.

Effect of Dietary Methionine Deficiency Followed by a Re-Feeding Phase on the Hepatic Antioxidant Activities of Lambs

Our objective was to investigate the effect of methionine restriction and resuming supply on liver antioxidant response in lambs. The concentrations of methionine and its metabolites and the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2), a redox sensitive factor, were detected after methionine restriction treatment for 50 days and methionine supply recovery for 29 days. The expression of glutathione (GSH) S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were characterized at the level of transcription and translation. Methionine restriction can directly change the content of methionine and its metabolites in plasma and liver, and affect the redox state of lambs by activating the Nrf2 signaling pathway. Liver tissue can adapt to oxidative environment by upregulating the expression of antioxidant enzymes such as GSH-Px and SOD. Moreover, it was found that there was a lag effect in the recovery of metabolism after methionine supplementation.

Low Protein/low Methionine/high Carbohydrate Diets Induce Hyperphagia, Increase Energy Expenditure and FGF21, but Modestly Affect Adiposity in Female BalbC Mice (OR09-01-19)

Objectives Low-protein diets are reported to induce hyperphagia to fulfil protein needs but at the expense of energy balance with a risk to gain in adiposity. However, different studies did not show body fat gain because an increased energy expenditure partly compensated the increase in energy intake and prevents the gain in adiposity. The present study evaluated in mice the consequence of protein restricted diets combined with protein quality (milk protein versus soy protein with slight methionine deficiency) on energy balance and adiposity and the role of FGF21 in the response to the protein restricted diets. Methods The study investigated in female BalbC mice the behavioural, metabolic and phenotypic responses to 8 weeks feeding a very low (3% energy - P3), moderately low (6% - P6) or adequate (20% - P20) dietary protein diet and evaluated if methionine scarcity, using soy protein (S) vs casein (C), affected these responses. Food intake, body weight, adiposity (assessed by DEXA), were measured throughout the study and body composition determined at the end of the study. Plasma, liver, muscle, adipose tissue and hypothalamus samples were collected for nutrient, hormones and gene expression measurements. Results Decreasing dietary casein from 20% to 3% increased energy intake, slightly increased adiposity, and this was exacerbated with methionine-deficient soy protein (figure 1). Lean body mass was reduced in 3% casein fed mice but preserved in all 6% fed mice. The effect on fat mass was limited because energy expenditure was also increased (figure 2). In plasma, low protein diets decreased IGF-1 and increased FGF21 that was related to protein level, protein to carbohydrate ratio and methionine content in the diet (figure 3). Insulin response to an oral glucose test was reduced in soy and low-protein fed mice. Low-protein diets did not affect Ucp1 but increased Fgf21 in brown adipose tissue and Fgf21, Fas, and Cd36 in the liver. In the hypothalamus, Npy was increased and Pomc was decreased only in 3% casein fed mice. Conclusions Reducing dietary protein and protein quality increases both energy intake and energy expenditure resulting only in slight increase in adiposity. In this process FGF21 is probably a signal that responds to a combination of protein restriction and carbohydrate content of the diet. Funding Sources Institut National de la Recherche Agronomique, AgroParisTech. Supporting Tables, Images and/or Graphs

Effects of dietary methionine deficiency followed by replenishment on the growth performance and carcass characteristics of lambs

Twelve pairs of male twin lambs were used to assess the effects of dietary methionine (Met) deficiency followed by replenishment on lamb growth performance and carcass characteristics. All lambs were weaned at 7 days of age and divided into the Control (CON) group and Met deficiency (MD) group. From 8 to 56 days of age, the lambs in the CON group were fed a milk replacer and starter feed containing 0.91% and 0.60% Met, respectively, whereas the lambs in the MD group were fed with a milk replacer and starter feed containing 0.21% and 0.20% Met, respectively. All lambs were fed a starter feed containing 0.60% Met from 57 to 84 days of age. Six twin pairs were slaughtered at 56 and 84 days of age, and their organ weights and carcass traits were measured. During 8 to 56 days of age, the Met-deficient diet decreased (P < 0.05) Met intake, average daily gain, feed conversion ratio, shrunk bodyweight, empty bodyweight, hot carcass weight, and the apparent digestibility of crude protein, ether extract and neutral detergent fibre; however, no differences were detected in dressing percentage or in the percentage of visceral organ weight to shrunk bodyweight between the groups (P > 0.05). During the follow-up Met replenishment stage, no differences in growth performance, nutrient digestibility, carcass characteristics, and percentages of visceral organs to shrunk bodyweight were found between the groups (P > 0.05). In conclusion, dietary Met deficiency in early life retarded the growth and development of lambs. Growth rate was not retarded during the 28 days of subsequent Met replenishment, but the difference in bodyweight between the groups remained.