Methionine Restriction Research Articles

Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer.

In recent years, various forms of caloric restriction (CR) and amino acid or protein restriction (AAR or PR) have shown not only success in preventing age-associated diseases, such as type II diabetes and cardiovascular diseases, but also potential for cancer therapy. These strategies not only reprogram metabolism to low-energy metabolism (LEM), which is disadvantageous for neoplastic cells, but also significantly inhibit proliferation. Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumour types, with over 600,000 new cases diagnosed annually worldwide. With a 5-year survival rate of approximately 55%, the poor prognosis has not improved despite extensive research and new adjuvant therapies. Therefore, for the first time, we analysed the potential of methionine restriction (MetR) in selected HNSCC cell lines. We investigated the influence of MetR on cell proliferation and vitality, the compensation for MetR by homocysteine, the gene regulation of different amino acid transporters, and the influence of cisplatin on cell proliferation in different HNSCC cell lines.

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.

Synergy of Combining Methionine Restriction and Chemotherapy: The Disruptive Next Generation of Cancer Treatment.

All cancer cell types are methionine-addicted, which is termed the Hoffman effect. Cancer cells, unlike normal cells, cannot survive without large amount of methionine. In general, when methionine is depleted, both normal cells and cancer cells synthesize methionine from homocysteine, but cancer cells consume large amounts of methionine and they cannot survive without exogenous methionine. For this reason, methionine restriction has been shown to be effective against many cancers in vitro and in vivo. Methionine restriction arrests cancer cells in the S/G2-phase of the cell cycle. Cytotoxic agents that act in the S/G2-phase are highly effective when used in combination with methionine restriction due to the cancer cells being trapped in S/G2-phase, unlike normal cells which arrest in G1/G0-phase. Combining methionine restriction and chemotherapeutic drugs for cancer treatment is termed the Hoffman protocol. The efficacy of many cytotoxic agents and molecular-targeted drugs in combination with methionine restriction has been demonstrated. The most effective method of methionine restriction is the administration of recombinant methioninase (rMETase), which degrades methionine. The efficacy of rMETase has been reported in mice and human patients by oral administration. The present review describes studies on anticancer drugs that showed synergistic efficacy in combination with methionine restriction, including rMETase administration. It is proposed that the next disruptive generation of cancer chemotherapy should employ current therapy in combination with methionine restriction for all cancer types.

Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids

Methionine restriction (MR) provides metabolic benefits in many organisms. However, mechanisms underlying the MR-induced effect remain incompletely understood. Here, we show in the budding yeast S. cerevisiae that MR relays a signal of S-adenosylmethionine (SAM) deprivation to adapt bioenergetic mitochondria to nitrogenic anabolism. In particular, decreases in cellular SAM constrain lipoate metabolism and protein lipoylation required for the operation of the tricarboxylic acid (TCA) cycle in the mitochondria, leading to incomplete glucose oxidation with an exit of acetyl-CoA and α-ketoglutarate from the TCA cycle to the syntheses of amino acids, such as arginine and leucine. This mitochondrial response achieves a trade-off between energy metabolism and nitrogenic anabolism, which serves as an effector mechanism promoting cell survival under MR.

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.

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 3093: Synthesis and bioevaluation of LAT-1 efflux agonists for the treatment of pancreatic cancers

Abstract The purpose of this report is to describe the discovery and development of the world’s first L-type amino acid transporter 1 (LAT-1) efflux agonist. LAT-1 is used for the import of amino acid substrates (leucine, phenylalanine, and methionine) and LAT-1 expression has been showed to be increased in pancreatic ductal adenocarcinoma (PDAC) cells. LAT-1 is a promising target for the treatment of rapidly growing cancers, such as PDAC, in part, due to its key role in methionine import. Methionine is required for various cellular processes, including the generation of S-adenosylmethionine (SAM) and the maintenance of intracellular polyamine pools, which are essential for PDAC growth. Indeed, the biosynthesis of the higher polyamines (spermidine and spermine) requires the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM), illustrating the molecular connection between methionine and polyamine pools. LAT-1 inhibition, such as with the known LAT-1 inhibitor JPH203, has shown efficacy in suppressing tumor growth in pancreatic cancer cell lines. While the early pre-clinical data is promising, competitive inhibitors of LAT-1 may have limited potency in the presence of high extracellular levels of LAT-1 substrates. In contrast, LAT-1 efflux agonists are insensitive to extracellular LAT-1 substrates. These novel compounds reverse the flow of substrates through LAT-1, effectively depleting cells of key intracellular amino acids, rather than blocking the import of extracellular substrates like methionine. The chiral piperazine (1), our lead efflux agonist, was discovered by a high throughput screen and was shown to deplete intracellular methionine, leucine and phenylalanine pools by LC-MS as well as decrease the synthesis of the higher polyamines (spermidine and spermine), a process which is reliant upon methionine as a precursor. A structure activity relationship (SAR) analysis of the four possible diastereomers of 1 was performed. The performance of these diastereomers and other derivatives were evaluated by LC-MS, HPLC, and Western blot. These molecules were shown to increase 3H-leucine efflux, decrease intracellular polyamine, SAM, and S-adenosylhomocysteine (SAH) levels, decrease phosphatidylethanolamine levels and induce the autophagic marker LC3-II. In summary, this novel method of methionine restriction allows one to alter intracellular methionine levels in a dose-dependent manner and provides a novel way to control pancreatic cancer cell growth. Citation Format: Holly Moots, Houssine Ikhlef, Vandana Sekhar, Otto Phanstiel. Synthesis and bioevaluation of LAT-1 efflux agonists for the treatment of pancreatic cancers [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 3093.

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.

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.

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.

Methionine Restriction Increases Insulin Sensitivity in Type-2 Diabetes via miRNA Activation

Methionine Restriction (MR) causes a higher level of circulating and hepatic fibroblastic growth factor 21 (FGF21). This leads to metabolic phenotypes, including increased energy expenditure, insulin sensitivity, and extended lifespan. Previous studies on obese mice have concluded that dietary MR in a high-fat regimen prevents hyperglycemia and improves glucose homeostasis, thus preventing type-2 diabetes, a multifactorial metabolic disease characterized by high blood glucose levels and cell insulin resistance. Recent experiments have shown that cells’ response to dietary MR includes changes in methylation of DNA promoters that activate or repress microRNAs (miRNAs), which are small endogenous nucleotide sequences and contain 18-22 base pairs that control gene expression for lipid metabolism. Considering that the disruption of miRNA levels affects insulin resistance, miRNA potentially plays a role in MR to increase insulin sensitivity for type-2 diabetes. In this paper, we investigate the mechanism of MR influencing the expression level of miRNA-15b to promote insulin sensitivity in obese organisms. Using our in-vitro model, we measured the expression of miRNA-15b in adipocytes cultured in MR and control conditions. Additionally, we compared insulin sensitivity and free fatty acid (FFA) metabolite levels between obese mice on control and MR diets. Taken together, we were able to verify the positive effects of MR in reducing hepatic fatty acid production, decreasing blood glucose levels, and increasing insulin sensitivity. However, miRNA-15b downregulates cells’ insulin signaling pathway and insulin sensitivity. Therefore, we proposed potential influences of MR on other miRNAs in reducing lipid cell differentiation and enhancing insulin sensitivity for future investigation.

Methionine Restriction Increases Exosome Production and Secretion in Breast Cancer Cells.

Methionine addiction is the elevated requirement for exogenous methionine for growth and survival of cancer cells, termed the Hoffman effect. Methionine-addicted cancer cells synthesize normal or excess amounts of methionine but still need an external source of methionine. Methionine restriction (MR) by either a methionine-free medium or in vivo by a low-methionine diet or by methioninase, selectively arrests cancer cells in the late S/G2 cell cycle phase, but not normal cells. The present study focuses on the comparison of production and secretion of exosomes by cancer cells under MR and normal conditions. MDA-MB-231 cells (triple-negative breast cancer), containing exosomes labeled with CD63-GFP (CD63-GFP exosomes), were visualized by fluorescence microscopy. MDA-MB-231 cells expressing exosome-specific CD63-GFP were cultured in methionine-containing (MET+) or in methionine-free (MET-) DMEM conditions. Exosomes were isolated from conditioned medium of cultured MDA-MD-231 cells by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and Western blotting. When MDA-MB-231-CD63-GFP cells were cultured under MR conditions, they arrested their growth and CD63-GFP-expressing exosomes were strongly increased in the cells. MR resulted in approximately a 2-fold increase in exosome production and secretion per cell, even though cell growth was arrested. Methionine restriction thus resulted in elevated exosome production and secretion per surviving cell. Exosome production and secretion in the cancer cells increased under MR, suggesting a relation between MR and exosome production and secretion.

Methionine restriction and cancer treatment: a systems biology study of yeast to investigate the possible key players.

Dietary restriction, mainly carbon and/or methionine restriction are among the upcoming supporting interventions along with chemotherapy in various cancers. Although dietary restriction has been proven to be beneficial, the main cellular machineries affected by its administration lacks deeper information considerably, a notable pitfall in its use as a personalized nutritional approach. In this study, cellular effects of methionine restriction on a yeast model are explored via systems biology approaches. The methionine biosynthesis network, constructed by integrating interaction data with gene ontology terms, was analysed topologically, and proved to be informative about the intertwined relationship of methionine biosynthesis and cancer. Experimentally, effects of methionine restriction on the yeast model were explored in vivo, with transcriptome analyses. The integrative analysis of the transcriptional data together with the reconstructed network gave insight into cellular machineries such as TOR, MAPK, and sphingolipid-mediated signaling cascades as the mostly responsive cellular pathways in the methionine-restricted cases with Sch9p (functional orthologue of mammalian S6 kinase) being placed at the intersection of these signaling routes.

Rapid Reduction of CEA and Stable Metastasis in an NRAS-mutant Rectal-Cancer Patient Treated With FOLFIRI and Bevacizumab Combined With Oral Recombinant Methioninase and a Low-Methionine Diet Upon Metastatic Recurrence After FOLFIRI and Bevacizumab Treatment Alone.

The choice of chemotherapy agents for RAS-mutant colorectal cancer is limited, and prognosis is poor compared to RAS-wild-type colorectal cancer. The purpose of the present study was to evaluate the effectiveness of methionine restriction combined with chemotherapy in a patient with NRAS-mutant rectal cancer. A 59-year-old female was diagnosed with lung-metastatic recurrence of NRAS-mutant rectal cancer two and a half years after resection of the primary tumor. She started chemotherapy, which consisted of fluorouracil, irinotecan (FOLFIRI), and bevacizumab, in October 2020. Eight months later, stereotactic body radiation therapy (SBRT) was performed to treat the lung metastases. She stopped chemotherapy at this point and had blood tests and computed tomography (CT) scans regularly. Her CEA level increased to 139.91 ng/ml and her lung metastasis became larger by September 2022. Therefore, she was reintroduced to FOLFIRI and bevacizumab in October 2022, and also started a low-methionine diet and oral recombinant methioninase (o-rMETase) as a supplement. After starting the combination therapy with o-rMETase, a low-methionine diet, FOLFIRI, and bevacizumab, blood CEA levels very rapidly decreased and were almost within the normal limits five months later. CT findings showed the lung metastasis did not grow. Methionine restriction comprising o-rMETase and a low-methionine diet combined with first-line chemotherapy was effective in a patient with NRAS-mutant rectal cancer in which metastasis had re-occurred after first-line chemotherapy alone.