Methionine Restriction Prevents Lipopolysaccharide-Induced Acute Lung Injury via Modulating CSE/H2S Pathway

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) result in high mortality, whereas effective treatments are limited. Methionine restriction (MR) has been reported to offer various benefits against multiple pathological processes of organ injuries. However, it remains unknown whether MR has any potential therapeutic value for ALI/ARDS. The current study was set to investigate the therapeutic potential of MR on lipopolysaccharide (LPS)-induced ALI and its underlying mechanisms. We found that MR attenuated LPS-induced pulmonary edema, hemorrhage, atelectasis, and alveolar epithelial cell injuries in mice. MR upregulated cystathionine-gamma-lyase (CSE) expression and enhanced the production of hydrogen sulfide (H2S). MR also inhibited the activation of Toll-like receptors 4 (TLR4)/NF-κB/NOD-like receptor protein 3 (NLRP3), then reduced IL-1β, IL-6, and TNF-α release and immune cell infiltration. Moreover, the protective effects of MR on LPS-induced ALI were abrogated by inhibiting CSE, whereas exogenous H2S treatment alone mimicked the protective effects of MR in Cse−/− mice after LPS administration. In conclusion, our findings showed that MR attenuated LPS-induced lung injury through CSE and H2S modulation. This work suggests that developing MR towards clinical use for ALI/ARDS patients may be a valuable strategy.

Methionine Restriction Improves Gut Barrier Function by Reshaping Diurnal Rhythms of Inflammation-Related Microbes in Aged Mice

Age-related gut barrier dysfunction and dysbiosis of the gut microbiome play crucial roles in human aging. Dietary methionine restriction (MR) has been reported to extend lifespan and reduce the inflammatory response; however, its protective effects on age-related gut barrier dysfunction remain unclear. Accordingly, we focus on the effects of MR on inflammation and gut function. We found a 3-month methionine-restriction reduced inflammatory factors in the serum of aged mice. Moreover, MR reduced gut permeability in aged mice and increased the levels of the tight junction proteins mRNAs, including those of occludin, claudin-1, and zona occludens-1. MR significantly reduced bacterial endotoxin lipopolysaccharide concentration in aged mice serum. By using 16s rRNA sequencing to analyze microbiome diurnal rhythmicity during 24 h, we found MR moderately recovered the cyclical fluctuations of the gut microbiome which was disrupted in aged mice, leading to time-specific enhancement of the abundance of short-chain fatty acid-producing and lifespan-promoting microbes. Moreover, MR dampened the oscillation of inflammation-related TM7-3 and Staphylococcaceae. In conclusion, the effects of MR on the gut barrier were likely related to alleviation of the oscillations of inflammation-related microbes. MR can enable nutritional intervention against age-related gut barrier dysfunction.

A Study on How Methionine Restriction Decreases the Body’s Hepatic and Lipid Deposition in Rice Field Eel (Monopterus albus)

Methionine restriction reduces animal lipid deposition. However, the molecular mechanism underlying how the body reacts to the condition and regulates lipid metabolism remains unknown. In this study, a feeding trial was performed on rice field eel Monopterus albus with six isonitrogenous and isoenergetic feeds that included different levels of methionine (0, 2, 4, 6, 8, and 10 g/kg). Compared with M0 (0 g/kg), the crude lipid and crude protein of M. albus increased markedly in M8 (8 g/kg) (p < 0.05), serum (total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and non-esterified free fatty acids), and hepatic contents (hepatic lipase, apolipoprotein-A, fatty acid synthetase, total cholesterol, triglyceride, and lipoprteinlipase). However, in the serum, very-low-density lipoprotein and hepatic contents (hormone-sensitive triglyceride lipase, Acetyl CoA carboxylase, carnitine palmitoyltransterase, and mirosomal triglygeride transfer protein) decreased markedly in M8 (p < 0.05). The contents of hepatic C18:2n-6, C22:6n-3, and n-3PUFA in the M8 group were significantly higher than those in M0 (p < 0.05), and the contents of lipid droplets in M8 were higher than those in M0. Compared with M0, the hepatic gcn2, eif2α, hsl, mttp, ldlrap, pparα, cpt1, and cpt2 were remarkably downregulated in M8, while srebf2, lpl, moat2, dgat2, hdlbp, srebf1, fas, fads2, me1, pfae, and icdh were markedly upregulated in M8. Moreover, hepatic SREBP1 and FAS protein expression were upregulated significantly in M8 (p < 0.01). In short, methionine restriction decreased the lipid deposition of M. albus, especially for hepatic lipid deposition, and mainly downregulated hepatic fatty acid metabolism. Besides, gcn2 could be activated under methionine restriction.

Age-Related Changes in Lipidome of Rat Frontal Cortex and Cerebellum Are Partially Reversed by Methionine Restriction Applied in Old Age

Lipids are closely associated with brain structure and function. However, the potential changes in the lipidome induced by aging remain to be elucidated. In this study, we used chromatographic techniques and a mass spectrometry-based approach to evaluate age-associated changes in the lipidome of the frontal cortex and cerebellum obtained from adult male Wistar rats (8 months), aged male Wistar rats (26 months), and aged male Wistar rats submitted to a methionine restriction diet (MetR)—as an anti-aging intervention—for 8 weeks. The outcomes revealed that only small changes (about 10%) were observed in the lipidome profile in the cerebellum and frontal cortex during aging, and these changes differed, in some cases, between regions. Furthermore, a MetR diet partially reversed the effects of the aging process. Remarkably, the most affected lipid classes were ether-triacylglycerols, diacylglycerols, phosphatidylethanolamine N-methylated, plasmalogens, ceramides, and cholesterol esters. When the fatty acid profile was analyzed, we observed that the frontal cortex is highly preserved during aging and maintained under MetR, whereas in the cerebellum minor changes (increased monounsaturated and decreased polyunsaturated contents) were observed and not reversed by MetR. We conclude that the rat cerebellum and frontal cortex have efficient mechanisms to preserve the lipid profile of their cell membranes throughout their adult lifespan in order to maintain brain structure and function. A part of the small changes that take place during aging can be reversed with a MetR diet applied in old age.

Methionine Restriction As a Novel Therapeutic Strategy for MLL (KMT2A)-Rearranged Acute Lymphoblastic Leukemia

Background: MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is a rare but aggressive subset that represents 5% of childhood ALL cases, and accounts for about 70% of infant leukemias. While overall survival in these young children is around 50%, after relapse, MLLr ALL becomes an almost incurable disease, highlighting the urgent clinical need for new strategies for this patient group. The histone methyl transferase function of the MLL fusion protein complex requires the methionine metabolite s-adenosylmethionine (SAM) as methyl donor, suggesting a selective sensitivity of MLL-r ALL for perturbations in methionine availability. Recent studies in solid tumor models suggest clinical utility of methionine restricted diets or oral administration of methionine depleting enzyme Methionine Gamma Lyase (MGL) to be safe and effective. Therefore, we explored the effect of methionine restriction (MR) as a potential, new therapy for MLLr ALL. Methods: We compared the effects of MR on metabolic activity and viability between MLLr and non-MLLr pre-BCP ALL cell lines using enzymatic depletion, small molecule inhibitors targeting methionine metabolism, and restrictive culture conditions. To identify intrinsic metabolic differences between MLLr and non-MLLr cells and explore how MR impinges on their metabolic state, we performed global metabolomics on MLLr SEM cells and non-MLLr NALM6 cells cultured with complete depletion of methionine. Additionally, we used RNA sequencing to assess the global effects of MR on gene expression, and a CRISPR/Cas9-based reverse genetic screen to identify sensitizers towards MR. Results were validated in vitro using targeted knockouts and small-molecule inhibitors, as well as in vivo using a 95% methionine restricted diet. Immunocompromised mice were engrafted with MLLr SEM cells and 7 days after transplantation, mice were randomized to control or 95% MR diet. Leukemia progression was monitored by flowcytometric detection of human lymphocytes in the blood. Results: We observed that depletion of methionine reduces metabolic activity in almost all BCP-ALL (B-ALL) cell lines, however, only in MLLr B-ALL cell lines was rapid apoptosis induced (Figure 1A). Global metabolic profiling revealed significant basal metabolic differences, of note being SAM, whose levels were approximately 5-fold higher in MLLr SEM cells compared to non-MLLr NALM6 cells. Consistent with this, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells (Figure 1A). Metabolomic profiling also highlighted different salvage mechanisms at play in NALM6 cells, with the folate cycle and polyamine synthesis pathway being activated upon MR. Together, these results indicate that MLLr B-ALL cells are selectively sensitive to MR. In line with this, RNASeq data showed significant decreased expression of several known MLL fusion target genes such as PROM1, HOXA10, and MEIS1 in response to MR. To obtain further insight into the pathways involved in the response to MR and to identify potential therapeutic targets that further sensitize cells to MR, we performed a CRISPR/Cas9-based screen. This identified three members of the Bromodomain- and extra-terminal domain (BET) family as potential modifiers of the response to MR in SEM cells. Indeed, RNAseq analysis showed that Myc activity as a proxy of BRD4 function, was strongly suppressed by MR. Finally, preliminary results show the efficacy of dietary intervention alone on leukemia progression. We observe with 95% MR diet, significant delays on leukemic growth (Figure 1B). Moreover, the MR diet was well tolerated, as indicated by minimal weight loss after two months. Although further studies are needed, we anticipate that targeting epigenetic regulators or use of conventional therapies in combination with MR would further potentiate this effect. Conclusions: MLLr leukemic cells have an increased dependency on S-adenosylmethionine and therefore show increased vulnerability to methionine depletion. Limiting methionine availability, either by enzymatic methionine depletion or dietary restriction could provide a novel therapeutic option for this patient group, particularly when combined with other therapies. The availability of an FDA approved methionine-free formula facilitates rapid translation to clinical practice, particularly in infants. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Cysteine Restriction in Murine L929 Fibroblasts as an Alternative Strategy to Methionine Restriction in Cancer Therapy

Methionine restriction (MetR) is an efficient method of amino acid restriction (AR) in cells and organisms that induces low energy metabolism (LEM) similar to caloric restriction (CR). The implementation of MetR as a therapy for cancer or other diseases is not simple since the elimination of a single amino acid in the diet is difficult. However, the in vivo turnover rate of cysteine is usually higher than the rate of intake through food. For this reason, every cell can enzymatically synthesize cysteine from methionine, which enables the use of specific enzymatic inhibitors. In this work, we analysed the potential of cysteine restriction (CysR) in the murine cell line L929. This study determined metabolic fingerprints using mass spectrometry (LC/MS). The profiles were compared with profiles created in an earlier work under MetR. The study was supplemented by proliferation studies using D-amino acid analogues and inhibitors of intracellular cysteine synthesis. CysR showed a proliferation inhibition potential comparable to that of MetR. However, the metabolic footprints differed significantly and showed that CysR does not induce classic LEM at the metabolic level. Nevertheless, CysR offers great potential as an alternative for decisive interventions in general and tumour metabolism at the metabolic level.

Metabolomics Based on 1H-NMR Reveal the Regulatory Mechanisms of Dietary Methionine Restriction on Splenic Metabolic Dysfunction in Obese Mice

Methionine restriction (MR) has been reported to have many beneficial health effects, including stress resistance enhancement and lifespan extension. However, the effects of MR on the splenic metabolic dysfunction induced by obesity in mice remain unknown. This study aimed to investigate the scientific problem and clarify its possible mechanisms. C57BL/6J mice in the control group were fed a control diet (0.86% methionine, 4.2% fat) for 34 weeks, and others were fed a high-fat diet (0.86% methionine, 24% fat) for 10 weeks to establish diet-induced obese (DIO) mouse models. Then, the obtained DIO mice were randomly divided into two groups: the DIO group (DIO diet), the DIO + MR group (0.17% methionine, 24% fat) for 24 weeks. Our results indicated that MR decreased spleen weight, and spleen and plasma lipid profiles, promoted lipid catabolism and fatty acid oxidation, glycolysis and tricarboxylic acid cycle metabolism, and improved mitochondrial function and ATP generation in the spleen. Moreover, MR normalized the splenic redox state and inflammation-related metabolite levels, and increased plasma levels of immunoglobulins. Furthermore, MR increased percent lean mass and splenic crude protein levels, activated the autophagy pathway and elevated nucleotide synthesis to maintain protein synthesis in the spleen. These findings indicate that MR can ameliorate metabolic dysfunction by reducing lipid accumulation, oxidative stress, and inflammation in the spleen, and the mechanism may be the activation of autophagy pathway.

To Identify and Verify the Role and Crucial Genes of Methionine Restriction in Inhibiting the Progression of Colon Cancer Cells

Purpose: Study the potential mechanism of methionine restriction (MR) inhibiting colon cancer (CC).Materials and Methods: The differential expression genes (DEGs) of the CC cell line HCT116 after MR restriction treatment in the GSE72131 and GSE103602 data sets were identified, and functional enrichment analysis was performed. Furthermore, the DEGs of CC in the TCGA database were identified, and the MR-related DEGs were combined to establish transcriptional regulation (TR), competing endogenous RNA (ceRNA), and protein-protein interaction (PPI) networks, and identify key genes from it. Then, in vitro experiments were performed to analyze the effects of MR on the proliferation, invasion, migration, and apoptosis of HCT116 cells, and to verify the effects of MR on the expression of key genes identified.Results: In HCT116 cells, 1181 genes regulated by MR were identified, and their functional enrichment suggests that they are closely related to tumor progression. Combined with the DEGs of CC, a total of 330 dysregulated genes of CC were affected by MR. 6 hub genes (E2F1, MIR17HG, FANCI, HJURP, KPNA2, KIF15) were obtained from the TR, ceRNA, and PPI network constructed based on this, and the results were consistent with the analysis by rt-PCR. In vitro studies have shown that MR can inhibit the proliferation and metastasis of CC cells and promote apoptosis.Conclusion: MR may inhibit the progress of CC by down-regulating the expression of E2F1, MIR17HG, FANCI, HJURP, KPNA2, and KIF15.

A genetic model of methionine restriction extends Drosophila health- and lifespan

Loss of metabolic homeostasis is a hallmark of aging and is characterized by dramatic metabolic reprogramming. To analyze how the fate of labeled methionine is altered during aging, we applied 13C5-Methionine labeling to Drosophila and demonstrated significant changes in the activity of different branches of the methionine metabolism as flies age. We further tested whether targeted degradation of methionine metabolism components would “reset” methionine metabolism flux and extend the fly lifespan. Specifically, we created transgenic flies with inducible expression of Methioninase, a bacterial enzyme capable of degrading methionine and revealed methionine requirements for normal maintenance of lifespan. We also demonstrated that microbiota-derived methionine is an alternative and important source in addition to food-derived methionine. In this genetic model of methionine restriction (MetR), we also demonstrate that either whole-body or tissue-specific Methioninase expression can dramatically extend Drosophila health- and lifespan and exerts physiological effects associated with MetR. Interestingly, while previous dietary MetR extended lifespan in flies only in low amino acid conditions, MetR from Methioninase expression extends lifespan independently of amino acid levels in the food. Finally, because impairment of the methionine metabolism has been previously associated with the development of Alzheimer’s disease, we compared methionine metabolism reprogramming between aging flies and a Drosophila model relevant to Alzheimer’s disease, and found that overexpression of human Tau caused methionine metabolism flux reprogramming similar to the changes found in aged flies. Altogether, our study highlights Methioninase as a potential agent for health- and lifespan extension.