Dietary Methionine Restriction Research Articles

EXTH-20. A MOUSE MODEL REVEALS VULNERABILITY OF IDH1-MUTATED GLIOMA TO FERROPTOSIS

Abstract The molecular classification of gliomas is currently based on the presence of IDH1 mutation, which is associated with better prognosis and longer survival. The hallmark of the IDH1 mutation is the production of D-2-Hydroxyglutarate with subsequent effects that are not fully understood. Ferroptosis, an iron-dependent mechanism of non-apoptotic cell death, is mostly triggered through blocking system Xc- or suppressing the antioxidant enzyme glutathione peroxidase 4 (GPX-4). In this study, we generated a mouse model of glioma harboring the IDH1(R132H) mutation in heterozygous condition, in combination with p53-deletion induced by the expression of PDGFA. We show that IDH1(R132H)-expressing mice survive longer compared to their wild-type counterparts, while histological analysis reveals characteristics of low-grade diffuse gliomas. We generated cell lines from primary tumors and analyzed their metabolic profile and response to mitochondrial stress and Ferroptosis. We treated them in vitro with RSL3, a GPX-4 inhibitor, alone or in combination with Cysteine and Methionine restriction. We show that IDH1(R132H)-expressing cells are far more sensitive to Ferroptosis in vitro. Finally, we show that convection-enhanced delivery of RSL3 in combination with dietary Cysteine and Methionine restriction in vivo, significantly prolongs survival of the IDH1(R132H)-expressing mice. Our findings suggest that Ferroptosis provides promising therapeutic potential worth exploring further and our mouse model could be used to test the efficacy of different treatments for IDH1(R132H) gliomas.

Safety and Feasibility of the Addition of a Radiosensitizing Methionine-Restricted Diet to Radiation Therapy

Methionine is an amino acid necessary for numerous processes critical for cell growth and survival. While normal cells can tolerate methionine deficiency, most cancer cells are methionine auxotrophs, requiring dietary intake since they cannot synthesize it. In vitro, methionine deficiency causes cancer cells to undergo cell cycle arrest and cell death, and in vivo a methionine restricted diet (MRD) enhances radiosensitization without significant adverse effects. Combining a MRD and radiation therapy (RT) to treat human malignancies has never been evaluated. The hypothesis of this Phase I study was that a MRD would be safe, and feasible to administer concurrently with curative-intent RT. Eligible patients included adults with any non-skin cancer malignancy undergoing standard radiation therapy without concurrent cytotoxic chemotherapy. The MRD consisted of low-protein cereals, grains, and breads; fruits; vegetables; margarines and oils; and simple carbohydrates. A clinical dietician developed a personalized meal plan with each subject to reduce methionine consumption to 5-10 mg/kg body weight/day, while maintaining adequate protein and caloric intake. An unlimited supply of a commercially available methionine-free protein supplement was provided to minimize hunger and weight loss. The MRD extended from 2 weeks before initiation of RT, through 2 weeks beyond completion of RT. The primary endpoint for safety was the rate of grade 3 or higher acute and late toxicities per CTCAE, over 12 months follow-up. Feasibility was assessed with a biweekly quantitative plasma amino acid panel during the MRD. The target accrual was 15 subjects. Over two years, 53 patients were offered enrollment, 9 subjects enrolled, 5 completed the MRD and RT, and 4 withdrew during the MRD. The table summarizes subjects' characteristics and outcomes. There was no grade 3 or higher adverse events attributable to the MRD. Methionine plasma levels varied over the course of treatment, and while no subject achieved the target of 13 μM, two nadired at 14 μM. The trial was closed early due to slow accrual and subjects' difficulty maintaining the diet. This study suggests a MRD is safe with thoracic or abdominopelvic RT, with toxicities comparable to those expected with RT alone. However, the diet was challenging, and unacceptable to most patients with cancer.

Increased Response to Immune Checkpoint Inhibitors with Dietary Methionine Restriction in a Colorectal Cancer Model.

Dietary methionine restriction (MR), defined as a reduction of methionine intake by around 80%, has been shown to reproducibly decrease tumor growth and synergize with cancer therapies. In this study, we combined DMR with immune checkpoint inhibitors (ICIs) in a model of colon adenocarcinoma. In vitro, we observed that MR increased the expression of MHC-I and PD-L1 in both mouse and human colorectal cancer cells. We also saw an increase in the gene expression of STING, a known inducer of type I interferon signaling. Inhibition of the cGAS-STING pathway, pharmacologically or with siRNA, blunted the increase in MHC-I and PD-L1 surface and gene expression following MR. This indicated that the cGAS-STING pathway, and interferon in general, played a role in the immune response to MR. We then combined dietary MR with ICIs targeting CTLA-4 and PD-1 in an MC38 colorectal cancer tumor model developed in immunocompetent C57BL/6 mice. The combination treatment was five times more effective at reducing the tumor size than ICIs alone in male mice. We noted sex differences in the response to dietary MR, with males showing a greater response than females. Finally, we observed an increase in membrane staining for the PD-L1 protein in MC38 tumors from animals who were fed an MR diet. MHC-I was highly expressed in all tumors and showed no expression difference when comparing tumors from control and MR-treated mice. These results indicated that MR increased PD-L1 expression both in vitro and in vivo and improved the response to ICIs in mice.

347-OR: Methionine Restriction Differentially Protects against High-Fat Diet-Induced Neuroinflammation and Central Insulin Resistance in Male and Female Mice

High-fat diets (HFD) promote central inflammation and insulin resistance (IR). Methionine restriction (MR) improves peripheral insulin sensitivity, lipid metabolism, and cognitive function, at least in part by increasing fibroblast growth factor 21 (FGF21). However, the ability of MR to protect against neuroinflammation and central IR during adolescence has not been examined. Moreover, whether MR depends on FGF21 to protect against HFD-induced changes in central insulin signaling or neuroinflammation is unknown. We sought to determine whether MR protects against HFD-induced neuroinflammation and central IR during adolescence in males and females and whether MR’s effects are dependent on FGF21. Male and female wild-type (WT) and FGF21 knockout (KO) mice, aged 5 to 6 wk, were fed normal (18.9% fat-derived kcal) or high-fat (60% fat-derived kcal) versions of control (0.86% methionine) or MR (0.17% methionine) diets for 5 wk. To investigate insulin action, ITTs were performed after 4 wk on the diet, and saline or insulin (0.6U/kg body weight) was administered 15 min prior to sacrifice. MR significantly decreased weight gain of HFD-fed mice regardless of sex or genotype (p < 0.05). MR increased serum FGF21 of all males and HFD-fed females (p < 0.05), and strongly tended to increase FGF21 in female mice on the normal-fat diet. HFD feeding tended to promote inflammatory gene expression in the hippocampus of WT males, while MR trended to mitigate inflammation independent of FGF21. MR also significantly reduced fasting blood glucose and improved peripheral insulin response in an FGF21-dependent fashion (p < 0.05). Though, neither HFD nor MR had a notable impact on markers of IR in the hypothalamus or hippocampus. Our findings indicate that short-term MR improves metabolic parameters of adolescent mice fed normal or high-fat diets. In addition, MR acts in an FGF21-independent and sex-dependent manner to improve neuroinflammation. Disclosure H.L.Lail: None. R.Lawrence: None. D.Wanders: None.

Intrinsic catalytic properties of histone H3 lysine-9 methyltransferases preserve monomethylation levels under low S-adenosylmethionine

S-adenosylmethionine (SAM) is the methyl donor for site-specific methylation reactions on histone proteins, imparting key epigenetic information. During SAM-depleted conditions that can arise from dietary methionine restriction, lysine di- and tri-methylation are reduced while sites such as Histone-3 lysine-9 (H3K9) are actively maintained, allowing cells to restore higher-state methylation upon metabolic recovery. Here, we investigated if the intrinsic catalytic properties of H3K9 histone methyltransferases (HMTs) contribute to this epigenetic persistence. We employed systematic kinetic analyses and substrate binding assays using four recombinant H3K9 HMTs (i.e., EHMT1, EHMT2, SUV39H1, and SUV39H2). At both high and low (i.e., sub-saturating) SAM, all HMTs displayed the highest catalytic efficiency (kcat/KM) for monomethylation compared to di- and trimethylation on H3 peptide substrates. The favored monomethylation reaction was also reflected in kcat values, apart from SUV39H2 which displayed a similar kcat regardless of substrate methylation state. Using differentially methylated nucleosomes as substrates, kinetic analyses of EHMT1 and EHMT2 revealed similar catalytic preferences. Orthogonal binding assays revealed only small differences in substrate affinity across methylation states, suggesting that catalytic steps dictate the monomethylation preferences of EHMT1, EHMT2, and SUV39H1. To link invitro catalytic rates with nuclear methylation dynamics, we built a mathematical model incorporating measured kinetic parameters and a time course of mass spectrometry-based H3K9 methylation measurements following cellular SAM depletion. The model revealed that the intrinsic kinetic constants of the catalytic domains could recapitulate invivo observations. Together, these results suggest catalytic discrimination by H3K9 HMTs maintains nuclear H3K9me1, ensuring epigenetic persistence after metabolic stress.

Safety and feasibility of the addition of a radiosensitizing methionine-restricted diet to radiation therapy.

e15087 Background: Methionine is an amino acid necessary for cell growth and survival. Normal cells can tolerate methionine deficiency, but most cancer cells are methionine auxotrophs, requiring dietary intake since they cannot synthesize it. In vitro, methionine deficiency causes cancer cells to undergo cell cycle arrest and cell death, and in vivo a methionine restricted diet (MRD) enhances radiosensitization without significant adverse effects. Combining a MRD and radiation therapy (RT) for human malignancies has never been evaluated. The hypothesis of this Phase I study was that a MRD would be safe, and feasible to administer concurrently with curative-intent RT. Methods: Eligible patients included adults with any non-skin cancer malignancy undergoing standard RT without concurrent cytotoxic chemotherapy. The MRD consisted of low-protein cereals, grains, and breads; fruits; vegetables; margarines and oils; and simple carbohydrates. A clinical dietician developed a personalized meal plan with each subject to reduce methionine consumption to 5-10 mg/kg body weight/day, while maintaining adequate protein and caloric intake. An unlimited supply of a commercially available methionine-free protein supplement was provided to minimize hunger and weight loss. The MRD extended from 2 weeks before initiation of RT, through 2 weeks beyond completion of RT. The primary endpoint for safety was the rate of grade 3 or higher acute and late toxicities per CTCAE, over 12 months follow-up. Feasibility was assessed with a biweekly quantitative plasma amino acid panel during the MRD. The target accrual was 15 subjects. Results: Over two years, 53 patients were offered enrollment, 9 subjects enrolled, 5 completed the MRD and RT, and 4 withdrew during the MRD. The table summarizes subjects’ characteristics and outcomes. There was no grade 3 or higher adverse events attributable to the MRD. Methionine plasma levels varied over the course of treatment, and while no subject achieved the target of 13 μM, two nadired at 14 μM. The trial was closed early due to slow accrual and subjects’ difficulty maintaining the diet. Clinical trial information: NCT03574194 .Conclusions: This study suggests a MRD is safe with thoracic or abdominopelvic RT, with toxicities comparable to those expected with RT alone. However, the diet was challenging, and unacceptable to most patients with cancer. [Table: see text]

Cystine rather than cysteine is the preferred substrate for β-elimination by cystathionine γ-lyase: implications for dietary methionine restriction

Dietary methionine restriction (MR) increases longevity by improving health. In experimental models, MR is accompanied by decreased cystathionine β-synthase activity and increased cystathionine γ-lyase activity. These enzymes are parts of the transsulfuration pathway which produces cysteine and 2-oxobutanoate. Thus, the decrease in cystathionine β-synthase activity is likely to account for the loss of tissue cysteine observed in MR animals. Despite this decrease in cysteine levels, these tissues exhibit increased H2S production which is thought to be generated by β-elimination of the thiol moiety of cysteine, as catalyzed by cystathionine β-synthase or cystathionine γ-lyase. Another possibility for this H2S production is the cystathionine γ-lyase-catalyzed β-elimination of cysteine persulfide from cystine, which upon reduction yields H2S and cysteine. Here, we demonstrate that MR increases cystathionine γ-lyase production and activities in the liver and kidneys, and that cystine is a superior substrate for cystathionine γ-lyase catalyzed β-elimination as compared to cysteine. Moreover, cystine and cystathionine exhibit comparable Kcat/Km values (6000 M−1 s−1) as substrates for cystathionine γ-lyase-catalyzed β-elimination. By contrast, cysteine inhibits cystathionine γ-lyase in a non-competitive manner (Ki ~ 0.5 mM), which limits its ability to function as a substrate for β-elimination by this enzyme. Cysteine inhibits the enzyme by reacting with its pyridoxal 5′-phosphate cofactor to form a thiazolidine and in so doing prevents further catalysis. These enzymological observations are consistent with the notion that during MR cystathionine γ-lyase is repurposed to catabolize cystine and thereby form cysteine persulfide, which upon reduction produces cysteine.

Transcriptome analysis of the gene expression of M. iliotibialis lateralis affected by dietary methionine restriction.

Introduction: Methionine (Met) is an important amino acid related to the development of skeletal muscle. This study investigated the effects of dietary Met restriction on the gene expression of M. iliotibialis lateralis. Methods: A total of 84 day-old broiler chicks (Zhuanghe Dagu) with a similar initial body weight (207.62 ± 8.54 g) were used in this study. All birds were divided into two groups (CON; L-Met) based on the initial body weight. Each group consisted of six replicates with seven birds per replicate. The experimental period was 63 days (phase 1, days 1-21; phase 2, days 22-63). According to the nutritional requirements of Zhuanghe Dagu chickens, we provided a basal diet (0.39% Met levels during phase 1 and 0.35% Met levels during phase 2, as-fed basis) to the birds in the CON group, while we provided a Met-restricted diet (0.31% Met levels during phase 1 and 0.28% Met levels during phase 2, as-fed basis) to the birds in the L-Met group. The growth performance of broiler chicks and their M. iliotibialis lateralis development parameters were measured on days 21 and 63. Results and Discussion: In this study, dietary Met restriction did not affect the growth performance of broiler chicks but hindered the development of M. iliotibialis lateralis at both sampling timepoints. On the final day, three birds selected from each group (three from CON and three from L-Met) were used to obtain M. iliotibialis lateralis samples from leg muscle for further transcriptome analysis. Transcriptome analysis revealed that dietary Met restriction significantly upregulated 247 differentially expressed genes (DEGs) and downregulated 173 DEGs. Additionally, DEGs were mainly enriched in 10 pathways. Among DEGs, we observed that dietary Met restriction downregulated the expression of CSRP3, KY, FHL1, LMCD1, and MYOZ2 in M. iliotibialis lateralis. Therefore, we considered that dietary Met restriction had negative effects on the development of M. iliotibialis lateralis, and CSRP3, KY, FHL1, LMCD1, and MYOZ2 may serve as potential functional genes involved in this process.

Abstract 1095: Exploiting altered methionine metabolism to overcome treatment resistance in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive adult brain tumor and is uniformly fatal due to resistance to standard therapies such as radiation (RT) and chemotherapy. Our group and others have identified altered metabolism as a key mediator of GBM RT resistance. Methionine is an essential sulfur-containing amino acid that cells use to synthesize antioxidants, polyamines and S-adenosyl methionine (SAM), which drives intracellular methylation reactions. Methionine uptake is dramatically elevated in GBM compared to normal brain, but what GBMs use this methionine for, and whether it governs GBM treatment resistance, is unknown. Here, we find that RT acutely increases the levels of numerous methionine-related metabolites in multiple RT-resistant GBM models. To interrogate metabolic pathway activity, we used 13C5 methionine stable isotope tracing to show that GBMs respond to RT by activating the conversion of methionine to SAM, which is dependent on signaling through the DNA damage response. We developed in vivo methionine stable isotope tracing techniques to confirm these findings in orthotopic PDX models of GBM. Blocking the conversion of methionine to SAM, through pharmacologic inhibition of methionine adenosyltransferase 2A (MAT2A), slowed the repair of RT-induced DNA damage and increased cell death in GBM models following RT. These effects were especially pronounced in GBM models lacking the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP). Pharmacologic inhibition of MAT2A in flank and orthotopic in vivo GBM models depleted SAM levels and slowed tumor growth when combined with RT. Combining MAT2A inhibition with dietary methionine restriction and RT slowed GBM tumor growth even further. Together, our work demonstrates a new signaling link between DNA damage and methionine-driven SAM synthesis in GBM. Inhibiting SAM synthesis slows the repair of RT-induced DNA damage and augments RT efficacy. This therapeutic strategy may be especially effective in GBMs defective in methionine salvage and spare normal cortex in which methionine salvage is active. Citation Format: Navyateja Korimerla, Kari-Wilder Romans, Peter Kalev, Ayesha Kothari, Nathan Qi, Charles Evans, Maureen Kachman, Marc L Hyer, Katya Marjon, Taryn Sleger, Daniel R Wahl. Exploiting altered methionine metabolism to overcome treatment resistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1095.

Abstract 2839: Methionine restriction radiosensitizes KRAS mutant rectal cancer

Abstract Purpose/Objective(s): Radiation therapy (RT) is the standard of care in patients with locally advanced rectal cancer, but not all patients respond equally to RT. KRAS-mutated (KRASmut) cancer cells are highly radioresistance and require higher levels of methionine, an essential amino acid, compared to KRAS wildtype (KRASwt) and normal cells, and diet restriction of methionine has been shown to potentiate the effects of radiation in vivo. We hypothesized that methionine restriction (MR) could radiosensitize KRASmut rectal cancer cells. Materials/Methods: HCT116 KRASG13D/+ and SW48 KRASwt colorectal carcinoma isogenic cell lines and their isogenic derivative cell lines HCT 116 KRAS+/- and SW48 KRASG13D were obtained from Horizon Discovery Ltd (Cambridge, UK). Radiation clonogenic assays, immunofluorescence, and western blot analysis of key DNA damage response proteins were performed in the KRASmut vs. KRASwt colorectal cells treated with ionizing radiation (IR, 2-6 Gy) at increasing time points following pre-treatment with methionine control (MC) (200 µM) or MR (40-fold reduction in methionine, 5 µM) media for 48 hours. One hundred thousand luciferase-expressing KRASwt or KRASmut SW48 cells were implanted orthotopically in the rectum of athymic nude mice. After confirmation of tumor formation at one-week post-injection, the mouse diet was changed to either an MC (0.84% methionine w/w) or MR (0.12% methionine w/w) diet for a total of two weeks. Rectal tumors were treated on an Xstrahl small animal radiation research platform (SARRP) to a dose of 25 Gy in 5 daily fractions (1 cm x 1 cm collimator, isocenter at anal verge) during the second week of diet modification. Tumor growth was measured via bioluminescence on IVIS Lumina every two weeks for four weeks post-radiation. Results: MR significantly increased radiosensitization compared to MC in both HCT116 and SW48 cells in vitro. KRASmut cells showed a 20-30% increase in radiation in the dose enhancement ratio compared to the KRASwt cells. The protein expression and nuclear foci per cell of the DNA damage marker γH2AX were significantly higher in the KRASmut cells compared to KRASwt cells following 5 Gy of RT at 6 hours. Protein expression of homologous recombination pathway proteins RAD51 and the CtBP interacting protein (CtIP) were higher at baseline in the KRASmut cells than KRASwt. MR substantially reduced the expression of these proteins following RT. Mice harboring SW48 KRASmut rectal tumors treated with 5 Gy x 5 RT and MR showed a significant increase in the complete response rate compared to MC (80% vs. 10%, p=0.02). In the mice harboring SW48 KRASwt rectal tumors, there was no significant difference in response rate between MR vs. MC (40% vs. 30%, p=NS). Conclusion: Our study indicates a novel strategy to radiosensitize resistant KRAS-mutated rectal cancer through MR. Studies testing the mechanism of KRAS-dependent methionine-mediated DNA repair is ongoing. Citation Format: Adam R. Wolfe, Sarita Garg, Stetson Van Matre, Robin Eluvathingal, Oscar Zuniga, Henrique Rodrigues, Nükhet Aykin-Burns, Isabelle Racine Miousse. Methionine restriction radiosensitizes KRAS mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2839.

Dietary Methionine Restriction Improves Gastrocnemius Muscle Glucose Metabolism through Improved Insulin Secretion and H19/IRS-1/Akt Pathway in Middle-Aged Mice

Methionine restriction (MR) improves glucose metabolism. In skeletal muscle, H19 is a key regulator of insulin sensitivity and glucose metabolism. Therefore, this study aims to reveal the underlying mechanism of H19 upon MR on glucose metabolism in skeletal muscle. Middle-aged mice were fed MR diet for 25 weeks. Mouse islets β cell line β-TC6 cells and mouse myoblast cell line C2C12 cells were used to establish the apoptosis or insulin resistance model. Our findings showed that MR increased B-cell lymphoma-2 (Bcl-2) expression, deceased Bcl-2 associated X protein (Bax), cleaved cysteinyl aspartate-specific proteinase-3 (Caspase-3) expression in pancreas, and promoted insulin secretion of β-TC6 cells. Meanwhile, MR increased H19 expression, insulin Receptor Substrate-1/insulin Receptor Substrate-2 (IRS-1/IRS-2) value, protein Kinase B (Akt) phosphorylation, glycogen synthase kinase-3β (GSK3β) phosphorylation, and hexokinase 2 (HK2) expression in gastrocnemius muscle and promoted glucose uptake in C2C12 cells. But these results were reversed after H19 knockdown in C2C12 cells. In conclusion, MR alleviates pancreatic apoptosis and promotes insulin secretion. And MR enhances gastrocnemius muscle insulin-dependent glucose uptake and utilization via the H19/IRS-1/Akt pathway, thereby ameliorating blood glucose disorders and insulin resistance in high-fat-diet (HFD) middle-aged mice.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism

Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.

Dietary methionine restriction attenuates cognitive dysfunction associated with sleep deprivation by promoting production of hydrogen sulfide, upregulating expression of BDNF and suppressing neuro-inflammation in rats

IntroductionIn this study, we aimed to study the potential role of H2S and the application of dietary methionine restriction (DMR) in the treatment of cognitive dysfunction in sleep deprived rat models.Material and methodsThe rat groups were established as a Control group, a sleep deprivation (SD) group, a SD + NaSH group and a SD + DMR group. Behavioral data was studied via Morris water maze test. TUNEL assay, real-time PCR, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were performed to study the differences of cognitive impairment related genes and proteins in different rat groups.ResultsPath length and escape latency were higher in the sleep deprived rats compared with the control rats, which were subsequently recovered by the treatment of NaSH or DMR. Also, DMR most significantly recovered the cognitive impairment and neuron status of sleep deprived rats compared with the administration of NaSH. The elevated level of hippocampal Iba1 and H2S production was recovered by NaSH and DMR, and the expressions of hippocampal phenotypic-related genes including NOS, CD68, CD32 and CD206 mRNA also showed similar trend as hippocampal Iba1. Meanwhile, the increased relative expression of IL-6 and IL-4 were recovered by DMR in sleep deprived rats, while the reduced level of hippocampal brain-derived neurotrophic factor (BDNF) and cystathionine γ-lyase (CSE) were elevated by the treatment of NaSH and MDR, with MDR exhibiting the most significant effect.ConclusionsThe application of DMR could attenuate cognitive dysfunction in sleep deprivation rats by upregulating the production of H2S and BDNF expression and alleviating neuro-inflammation responses.

Cystathionine Beta-Synthase Deficiency: Three Consecutive Cases Detected in 40 Days by Newborn Screening in Emilia Romagna (Italy) and a Comprehensive Review of the Literature.

Cysthiatonine beta-synthase (CBS) deficiency (CBSD) is an autosomal recessive rare disorder caused by variations on CBS that leads to impaired conversion of homocysteine (Hcy) to cystathionine. Marked hyperhomocysteinemia is the hallmark of the disease. The administration of pyridoxine, the natural cofactor of CBS, may reduce total plasma Hcy. Patient phenotype is classified on pyridoxine responsivity in two groups: pyridoxine-responsive and non-responsive patients. Ectopia lentis, bone deformities, developmental delay, and thromboembolism are the classic signs and symptoms of the disease. Early diagnosis and treatment impact patients' natural history. Therapy aims to lower promptly and maintain Hcy concentrations below 100 μmol/L. Depending on the patient's phenotype, the treatment goals could be obtained by the administration of pyridoxine and/or betaine associated with a methionine-restricted diet. CBSD could be diagnosed in the early days of life by expanded newborn screening (ENS), however, the risk of false negative results is not negligible. In Emilia-Romagna (Italy), during the first 10 years of screening experience, only three cases of CBSD identified have been diagnosed, all in the last two years (incidence 1:118,000 live births). We present the cases and a comprehensive review of the literature to emphasize the role of ENS for early diagnosis of CBSD and its potential pitfalls, reiterating the need for a more effective method to screen for CBSD.

Dietary methionine restriction alleviates oxidative stress and inflammatory responses in lipopolysaccharide-challenged broilers at early age.

In this study, we investigated the effect of dietary methionine restriction (MR) on the antioxidant function and inflammatory responses in lipopolysaccharide (LPS)-challenged broilers reared at high stocking density. A total of 504 one-day-old male Arbor Acre broiler chickens were randomly divided into four treatments: 1) CON group, broilers fed a basal diet; 2) LPS group, LPS-challenged broilers fed a basal diet; 3) MR1 group, LPS-challenged broilers fed a methionine-restricted diet (0.3% methionine); and 4) MR2 group, LPS-challenged broilers fed a methionine-restricted diet (0.4% methionine). LPS-challenged broilers were intraperitoneally injected with 1mg/kg body weight (BW) of LPS at 17, 19, and 21days of age, whereas the CON group was injected with sterile saline. The results showed that: LPS significantly increased the liver histopathological score (p < 0.05); LPS significantly decreased the serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity at 3h after injection (p < 0.05); the LPS group had a higher content of Interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF)-α, but a lower content of IL-10 than the CON group in serum (p < 0.05). Compared with the LPS group, the MR1 diet increased catalase (CAT), SOD, and T-AOC, and the MR2 diet increased SOD and T-AOC at 3h after injection in serum (p < 0.05). Only MR2 group displayed a significantly decreased liver histopathological score (p < 0.05) at 3h, while MR1 and MR2 groups did so at 8h. Both MR diets significantly decreased serum LPS, CORT, IL-1β, IL-6, and TNF-α contents, but increased IL-10 content (p < 0.05). Moreover, the MR1 group displayed significantly increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), CAT, and GSH-Px at 3 h; the MR2 group had a higher expression of Kelch-like ECH-associated protein 1 (Keap1), SOD, and GSH-Px at 8h (p < 0.05). In summary, MR can improve antioxidant capacity, immunological stress, and liver health in LPS-challenged broilers. The MR1 and MR2 groups experienced similar effects on relieving stress; however, MR1 alleviated oxidative stress more rapidly. It is suggested that precise regulation of methionine levels in poultry with stress may improve the immunity of broilers, reduce feed production costs, and increase production efficiency in the poultry industry.

Dietary Methionine Restriction Alleviates Hyperglycemia in Pigs with Intrauterine Growth Restriction by Enhancing Hepatic Protein Kinase B Signaling and Glycogen Synthesis

BackgroundIndividuals with intrauterine growth restriction (IUGR) are prone to developing type 2 diabetes mellitus (T2DM). Dietary methionine restriction (MR) improves insulin sensitivity and glucose homeostasis in individuals with normal birth weight (NBW). ObjectiveThis study investigated the effects of MR on plasma glucose concentration and hepatic and muscle glucose metabolism in pigs with IUGR. MethodsThirty female NBW and 60 same-sex spontaneous IUGR piglets (Landrace × Yorkshire) were selected. After weaning (day 21), the piglets were fed diets with adequate methionine (NBW-CON and IUGR-CON) or 30% less methionine (IUGR-MR) (n = 6). At day 180, 1 pig with a body weight near the mean of each replication was selected for biochemical analysis. ResultsThe IUGR-CON group showed 41.6%, 68.6%, and 67.1% higher plasma glucose concentration, hepatic phosphoenolpyruvate carboxykinase activity, and glucose-6-phosphatase activity, respectively, than the NBW-CON group (P < 0.05). Muscle glycogen content and glycogen synthase activity were 36.9% and 38.8% lower, respectively, in the IUGR-CON than the NBW-CON group (P < 0.05), respectively, and there was decreased hepatic and muscle protein kinase B phosphorylation in the IUGR-CON group (P < 0.05). Compared with the IUGR-CON pigs, the IUGR-MR pigs had 28.7% lower plasma glucose concentrations (P < 0.05), which were similar to those of the NBW-CON pigs (P ≥ 0.05). The hepatic glycogen content and glycogen synthase activity of the IUGR-MR pigs were 62.9% and 50.8% higher than those of the IUGR-CON pigs (P < 0.05) and 53.5% and 84.3% higher than the NBW-CON pigs (P < 0.05), respectively. The IUGR-MR pigs’ hepatic and muscle protein kinase B phosphorylation was higher than that of the IUGR-CON pigs (P < 0.05) and similar to that of the NBW-CON pigs (P ≥ 0.05). ConclusionsMR attenuates hyperglycemia in IUGR pigs by enhancing hepatic protein kinase B signaling and glycogen synthesis, implying a potential nutritional strategy to prevent type 2 diabetes mellitus in IUGR offspring.

Dietary Methionine and Total Sulfur Amino Acid Restriction in Healthy Adults.

Dietary restriction of methionine (Met) and cysteine (Cys) delays the aging process and aging-related diseases, improves glucose and fat metabolism and reduces oxidative stress in numerous laboratory animal models. Little is known regarding the effects of sulfur amino acid restriction in humans. Thus, our objectives were to determine the impact of feeding diets restricted in Met alone (MetR) or in both Met and Cys (total sulfur amino acids, SAAR) to healthy adults on relevant biomarkers of cardiometabolic disease risk. A controlled feeding study. We included 20 healthy adults (11 females/9 males) assigned to MetR or SAAR diet groups consisting of three 4-wk feeding periods: Control period; low level restriction period (70% MetR or 50% SAAR); and high level restriction period (90% MetR or 65% SAAR) separated by 3-4-wk washout periods. No adverse effects were associated with either diet and level of restriction and compliance was high in all subjects. SAAR was associated with significant reductions in body weight and plasma levels of total cholesterol, LDL, uric acid, leptin, and insulin, BUN, and IGF-1, and increases in body temperature and plasma FGF-21 after 4 weeks (P<0.05). Fewer changes occurred with MetR including significant reductions in BUN, uric acid and 8-isoprostane and an increase in FGF-21 after 4 weeks (P<0.05). In the 65% SAAR group, plasma Met and Cys levels were significantly reduced by 15% and 13% respectively (P<0.05). These results suggest that many of the short-term beneficial effects of SAAR observed in animal models are translatable to humans and support further clinical development of this intervention.

Dietary Methionine Restriction Promotes Fat Browning and Attenuates Hepatic Lipid Accumulation in High-Choline-Fed Mice Associated with the Improvement of Thyroid Function.

This study aims to explore the influences of a methionine-restricted diet (MRD) on fat browning and hepatic lipid accumulation in mice fed with a high-choline diet (HCD) and their possible mechanisms. ICR mice were randomly divided into three groups and fed with a normal diet (0.86% methionine + 0.20% choline, ND), HCD (0.86% methionine + 1.20% choline), or MRD (0.17% methionine + 1.20% choline) for 90 consecutive days. We found that MRD reduced body weight and fat mass; increased heat production and ambulatory locomotor activity; reduced hepatic and plasma lipid levels, hepatic fatty infiltration area, and adipocyte volume in white and brown adipose tissue; promoted fat browning, especially upregulated gene and protein expression levels of uncoupling protein 1 (UCP1); and promoted fat catabolism and inhibited fat anabolism in the liver and adipose tissue. Moreover, MRD increased antioxidant defenses and reduced inflammatory cytokine levels in the thyroid, blood, and liver. Furthermore, MRD improved thyroid morphological structure, promoted the synthesis and secretion of thyroid hormones, and enhanced the actions of thyroid hormones on its receptor organs (liver and adipose tissue). These findings suggested that MRD promoted fat browning and attenuated hepatic lipid accumulation in HCD mice associated with the improvement of thyroid function.

Long-term prognosis of 35 patients with methionine adenosyltransferase deficiency based on newborn screening in China.

Methionine adenosyltransferase deficiency (MATD) is a rare metabolic disorder caused by mono- or biallelic MAT1A mutations that are not yet well understood. Of the 4,065,644 neonates screened between November 2010 and December 2021, 35 individuals have been diagnosed with an estimated incidence of 1: 116,161 by a cutoff value of methionine 82.7μmol/L and follow-up over 11years. MATD patients with autosomal recessive (AR) type had higher clinical and genetic heterogeneity than those with autosomal dominant (AD) type. Fifteen unrelated AD patients harbored one well-known dominant variant, c.791G>A or c.776C>T, and were clinically unaffected with a mean plasma methionine (Met) value <300μmol/L. Twenty AR cases have unique genotypes and presented a wide range of clinical abnormalities from asymptomatic to white matter lesions. Of them, 10 AR patients displayed severe manifestations, such as verbal difficulty, motor delay, development delay, and white matter lesions, with mean Met >500μmol/L and thereby were treated with a methionine-restricted diet alone or in combination with betaine, folate, or vitamin B6, and were healthy finally. Neurological abnormalities were evidenced in two patients (P16 and P27) with Met values >800μmol/L by MRI scan. Neurological abnormalities were reversed here by liver transplantation or by the determination of S-adenosylmethionine supplementation. Additionally, 38 variants of MAT1A were distributed within patients and carriers, of which 24 were novel and mostly predicted to be damaged. Our findings with an extensive clinical and genetic dataset provided new insights into its diagnosis and treatment and will be helpful for its optimal management in the future.

Dietary Methionine Restriction Alleviates Choline-Induced Tri-Methylamine-N-Oxide (TMAO) Elevation by Manipulating Gut Microbiota in Mice

Dietary methionine restriction (MR) has been shown to decrease plasma trimethylamine-N-oxide (TMAO) levels in high-fat diet mice; however, the specific mechanism used is unknown. We speculated that the underlying mechanism is related with the gut microbiota, and this study aimed to confirm the hypothesis. In this study, we initially carried out an in vitro fermentation experiment and found that MR could reduce the ability of gut microbiota found in the contents of healthy mice and the feces of healthy humans to produce trimethylamine (TMA). Subsequently, mice were fed a normal diet (CON, 0.20% choline + 0.86% methionine), high-choline diet (H-CHO, 1.20% choline + 0.86% methionine), or high-choline + methionine-restricted diet (H-CHO+MR, 1.20% choline + 0.17% methionine) for 3 months. Our results revealed that MR decreased plasma TMA and TMAO levels in H-CHO-diet-fed mice without changing hepatic FMO3 gene expression and enzyme activity, significantly decreased TMA levels and expression of choline TMA-lyase (CutC) and its activator CutD, and decreased CutC activity in the intestine. Moreover, MR significantly decreased the abundance of TMA-producing bacteria, including Escherichia-Shigella (Proteobacteria phylum) and Anaerococcus (Firmicutes phylum), and significantly increased the abundance of short-chain fatty acid (SCFA)-producing bacteria and SCFA levels. Furthermore, both MR and sodium butyrate supplementation significantly inhibited bacterial growth, down-regulated CutC gene expression levels in TMA-producing bacteria, including Escherichia fergusonii ATCC 35469 and Anaerococcus hydrogenalis DSM 7454 and decreased TMA production from bacterial growth under in vitro anaerobic fermentation conditions. In conclusion, dietary MR alleviates choline-induced TMAO elevation by manipulating gut microbiota in mice and may be a promising approach to reducing circulating TMAO levels and TMAO-induced atherosclerosis.