SummaryReduced dietary methionine intake (0.17% methionine, MR) and calorie restriction (CR) prolong lifespan in male Fischer 344 rats. Although the mechanisms are unclear, both regimens feature lower body weight and reductions in adiposity. Reduced fat deposition in CR is linked to preservation of insulin responsiveness in older animals. These studies examine the relationship between insulin responsiveness and visceral fat in MR and test whether, despite lower food intake observed in MR animals, decreased visceral fat accretion and preservation of insulin sensitivity is not secondary to CR. Accordingly, rats pair fed (pf) control diet (0.86% methinone, CF) to match the food intake of MR for 80 weeks exhibit insulin, glucose, and leptin levels similar to control-fed animals and comparable amounts of visceral fat. Conversely, MR rats show significantly reduced visceral fat compared to CF and PF with concomitant decreases in basal insulin, glucose, and leptin, and increased adiponectin and triiodothyronine. Daily energy expenditure in MR animals significantly exceeds that of both PF and CF. In a separate cohort, insulin responses of older MR animals as measured by oral glucose challenge are similar to young animals. Longitudinal assessments of MR and CF through 112 weeks of age reveal that MR prevents age-associated increases in serum lipids. By 16 weeks, MR animals show a 40% reduction in insulin-like growth factor-1 (IGF-1) that is sustained throughout life; CF IGF-1 levels decline much later, beginning at 112 weeks. Collectively, the results indicate that MR reduces visceral fat and preserves insulin activity in aging rats independent of energy restriction.

Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism, but little is known about its nutritional regulation. Dietary methionine restriction in rats decreases hepatic Scd1 mRNA and protein, increases energy expenditure, and decreases fat-pad mass/body-weight% (FM/BW%). In humans, plasma concentrations of the methionine product, cysteine, are associated with obesity. To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats. MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels. Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that anti-obesity effects of MR are caused by low cysteine and that dietary sulfur amino acid composition contributes to SCD1 regulation.

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Background/Aims: Methionine restriction (MR) is a dietary intervention that increases lifespan, reduces adiposity and improves insulin sensitivity. These effects are reversed by supplementation of the MR diet with cysteine (MRC). Genomic and metabolomic studies were conducted to identify potential mechanisms by which MR induces favorable metabolic effects, and that are reversed by cysteine supplementation. Methods: Gene expression was examined by microarray analysis and TaqMan quantitative PCR. Levels of selected proteins were measured by Western blot and metabolic intermediates were analyzed by mass spectrometry. Results: MR increased lipid metabolism in inguinal adipose tissue and quadriceps muscle while it decreased lipid synthesis in liver. In inguinal adipose tissue, MR not only caused the transcriptional upregulation of genes associated with fatty acid synthesis but also of Lpin1, Pc, Pck1 and Pdk1, genes that are associated with glyceroneogenesis. MR also upregulated lipolysis-associated genes in inguinal fat and led to increased oxidation in this tissue, as suggested by higher levels of methionine sulfoxide and 13-HODE + 9-HODE compared to control-fed (CF) rats. Moreover, MR caused a trend toward the downregulation of inflammation-associated genes in inguinal adipose tissue. MRC reversed most gene and metabolite changes induced by MR in inguinal adipose tissue, but drove the expression of Elovl6, Lpin1, Pc, and Pdk1 below CF levels. In liver, MR decreased levels of a number of long-chain fatty acids, glycerol and glycerol-3-phosphate corresponding with the gene expression data. Although MR increased the expression of genes associated with carbohydrate metabolism, levels of glycolytic intermediates were below CF levels. MR, however, stimulated gluconeogenesis and ketogenesis in liver tissue. As previously reported, sulfur amino acids derived from methionine were decreased in liver by MR, but homocysteine levels were elevated. Increased liver homocysteine levels by MR were associated with decreased cystathionine β-synthase (CBS) protein levels and lowered vitamin B6 and 5-methyltetrahydrofolate (5MeTHF) content. Finally, MR upregulated fibroblast growth factor 21 (FGF21) gene and protein levels in both liver and adipose tissues. MRC reversed some of MR’s effects in liver and upregulated the transcription of genes associated with inflammation and carcinogenesis such as Cxcl16, Cdh17, Mmp12, Mybl1, and Cav1 among others. In quadriceps muscle, MR upregulated lipid metabolism-associated genes and increased 3-hydroxybutyrate levels suggesting increased fatty acid oxidation as well as stimulation of gluconeogenesis and glycogenolysis in this tissue. Conclusion: Increased lipid metabolism in inguinal adipose tissue and quadriceps muscle, decreased triglyceride synthesis in liver and the downregulation of inflammation-associated genes are among the factors that could favor the lean phenotype and increased insulin sensitivity observed in MR rats.

Methionine restriction (MR) limits age-related adiposity in Fischer 344 (F344) rats. To assess the mechanism of adiposity resistance, the effect of MR on adipose tissue (AT) 11b-hydroxysteroid dehydrogenase-1 (11b-HSD1) was examined. MR induced 11b-HSD1 activity in all ATs, correlating with increased tissue corticosterone. However, an inverse relationship between 11b-HSD1 activity and adipocyte size was observed. Because dietary restriction controls lipogenic and lipolytic rates, MR's effects on lipogenic and lipolytic enzymes were evaluated. MR increased adipose triglyceride lipase and acetyl-coenzyme A carboxylase (ACC) protein levels but induced ACC phosphorylation at serine residues that render the enzyme inactive, suggesting alterations of basal lipolysis and lipogenesis. In contrast, no changes in basal or phosphorylated hormone-sensitive lipase levels were observed. ACCphosphorylated sites were specific for AMP-activated protein kinase (AMPK); therefore, AMPK activation was evaluated. Significant differences in AMPKa protein, phosphorylation, and activity levels were observed only in retroperitoneal fat from MR rats. No differences in protein kinase A phosphorylation and intracellular cAMP levels were detected. In vitro studies revealed increased lipid degradation and a trend toward increased lipid synthesis, suggesting the presence of a futile cycle. In conclusion, MR disrupts the lipogenic/lipolytic balance, contributing importantly to adiposity resistance in F344 rats.-Perrone, C.

BackgroundDietary methionine restriction (MR) improves healthspan in part by reducing adiposity and by increasing insulin sensitivity in rodent models. The purpose of this study was to determine whether MR inhibits tumor progression in breast cancer xenograft model and breast cancer cell lines.MethodsAthymic nude mice were injected with MCF10AT1 cells in Matrigel® and fed a diet containing either 0.86 % methionine (control fed, CF), or 0.12 % methionine (MR) for 12 weeks. Plasma amino acid concentrations were measured by UPLC, and proliferation and apoptosis were examined using RT-PCR, immunohistochemistry, and Cell Titer 96® Aqueous One Solution Cell Proliferation assay.ResultsMice on the MR diet had reduced body weight and decreased adiposity. They also had smaller tumors when compared to the mice bearing tumors on the CF diet. Plasma concentrations of the sulfur amino acids (methionine, cysteine, and taurine) were reduced, whereas ornithine, serine, and glutamate acid were increased in mice on the MR diet. MR mice exhibited decreased proliferation and increased apoptosis in cells that comprise the mammary glands and tumors of mice. Elevated expression of P21 occurred in both MCF10AT1-derived tumor tissue and endogenously in mammary gland tissue of MR mice. Breast cancer cell lines MCF10A and MDA-MB-231 grown in methionine-restricted cysteine-depleted media for 24 h also up-regulated P21 and P27 gene expression, and MDA-MB-231 cells had decreased proliferation.ConclusionMR hinders cancer progression by increasing cell cycle inhibitors that halt cell cycle progression. The application of MR in a clinical setting may provide a delay in the progression of cancer, which would provide more time for conventional cancer therapies to be effective.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2367-1) contains supplementary material, which is available to authorized users.

Methionine restriction (MR) is one of only a few dietary manipulations known to robustly extend healthspan in mammals. For example, rodents fed a methionine-restricted diet are up to 45% longer-lived than control-fed animals. Tantalizingly, ongoing studies suggest that humans could enjoy similar benefits from this intervention. While the benefits of MR are likely due, at least in part, to improved cellular stress tolerance, it remains to be determined exactly how MR extends organismal healthspan. In previous work, we made use of the yeast chronological lifespan (CLS) assay to model the extension of cellular lifespan conferred by MR and explore the genetic requirements for this extension. In these studies, we demonstrated that both dietary MR (D-MR) and genetic MR (G-MR) (i.e., impairment of the cell’s methionine biosynthetic machinery) significantly extend the CLS of yeast. This extension was found to require the mitochondria-to-nucleus retrograde (RTG) stress signaling pathway, and was associated with a multitude of gene expression changes, a significant proportion of which was also dependent on RTG signaling. Here, we show work aimed at understanding how a subset of the observed expression changes are causally related to MR-dependent CLS extension. Specifically, we find that multiple autophagy-related genes are upregulated by MR, likely resulting in an increased autophagic capacity. Consistent with activated autophagy being important for the benefits of MR, we also find that loss of any of several core autophagy factors abrogates the extended CLS observed for methionine-restricted cells. In addition, epistasis analyses provide further evidence that autophagy activation underlies the benefits of MR to yeast. Strikingly, of the many types of selective autophagy known, our data clearly demonstrate that MR-mediated CLS extension requires only the autophagic recycling of mitochondria (i.e., mitophagy). Indeed, we find that functional mitochondria are required for the full benefit of MR to CLS. Finally, we observe substantial alterations in carbon metabolism for cells undergoing MR, and provide evidence that such changes are directly responsible for the extended lifespan of methionine-restricted yeast. In total, our data indicate that MR produces changes in carbon metabolism that, together with the oxidative metabolism of mitochondria, result in extended cellular lifespan.

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