Physical activity of mice on dietary sulfur amino acid restriction is influenced by age of diet initiation and biological sex.

Sulfur amino acid restriction (SAAR)-the reduction of methionine and cysteine concentrations either in the diet or by genetic manipulation-promotes health span and extends lifespan, but its effects on physical activity remain unclear. We investigated whether age of diet initiation and biological sex could influence physical activity in mice fed either a control diet (CF, 0.86% methionine w/w) or SAAR (0.12% methionine w/w). Quadriceps femoris muscle mass is smaller in SAAR than in CF mice. Young mice fed a chronic SAAR diet at 8 weeks of age exhibited improved wire hang and running wheel activities compared to young CF mice, while aged mice showed comparable results. The effects of chronic SAAR on physical activity was mildly influenced by sex as observed in middle-aged male SAAR mice who showed minor improvements than CF males while middle-aged females displayed no discernible effects. Muscle mass is minimally affected by changes in markers of protein synthesis, autophagy and atrophy. Improvements to physical activity in young SAAR mice could be partially attributed to increased skeletal muscle mitochondrial activity. Furthermore, SAAR in C2C12 myotubes increased citrate synthase protein expression and enhanced succinyl dehydrogenase enzyme activity compared to CF myotubes. Overall, our data reveal that SAAR can improve mouse physical activity without compromising muscle proteostasis. This is partially due to enhanced mitochondrial activity, but the effects are influenced by age of diet initiation and sex.

Cystine rather than cysteine is the preferred substrate for ß-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 ß-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 ß-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 ß-elimination of the thiol moiety of cysteine, as catalyzed by cystathionine ß-synthase or cystathionine γ-lyase. Another possibility for this H2S production is the cystathionine γ-lyase-catalyzed ß-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 ß-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 ß-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 ß-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.

Cysteine restriction-specific effects of sulfur amino acid restriction on lipid metabolism.

Decreasing the dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti-adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine- and cysteine-titrated diets, we demonstrate that the anti-adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. In mice, the magnitude of SAAR-induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age-at-onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. Controlled feeding of low-SAA, high-polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.

Bone Marrow Adiposity: Basic and Clinical Implications.

The presence of adipocytes in mammalian bone marrow (BM) has been recognized histologically for decades, yet, until recently, these cells have received little attention from the research community. Advancements in mouse transgenics and imaging methods, particularly in the last 10 years, have permitted more detailed examinations of marrow adipocytes than ever before and yielded data that show these cells are critical regulators of the BM microenvironment and whole-body metabolism. Indeed, marrow adipocytes are anatomically and functionally separate from brown, beige, and classic white adipocytes. Thus, areas of BM space populated by adipocytes can be considered distinct fat depots and are collectively referred to as marrow adipose tissue (MAT) in this review. In the proceeding text, we focus on the developmental origin and physiologic functions of MAT. We also discuss the signals that cause the accumulation and loss of marrow adipocytes and the ability of these cells to regulate other cell lineages in the BM. Last, we consider roles for MAT in human physiology and disease.

Dietary methionine restriction modulates renal response and attenuates kidney injury in mice.

Methionine restriction (MR) extends the lifespan across several species, such as rodents, fruit flies, roundworms, and yeast. MR studies have been conducted on various rodent organs, such as liver, adipose tissue, heart, bones, and skeletal muscle, to elucidate its benefits to the healthspan; however, studies of the direct effect of MR on kidneys are lacking. To investigate the renal effects of MR, we used young and aged unilateral nephrectomized and 5/6 nephrectomized (5/6Nx) mice. Our studies indicated that MR mice experienced polydipsia and polyuria compared with control-fed counterparts. Urine albumin, creatinine, albumin-to-creatinine ratio, sulfur amino acids, and electrolytes were reduced in MR mice. Kidneys of MR mice up-regulated genes that are involved in ion transport, such as Aqp2, Scnn1a, and Slc6a19, which indicated a response to maintain osmotic balance. In addition, we identified renoprotective biomarkers that are affected by MR, such as clusterin and cystatin C. Of importance, MR attenuated kidney injury in 5/6Nx mice by down-regulating inflammation and fibrosis mechanisms. Thus, our studies in mice show the important role of kidneys during MR in maintaining osmotic homeostasis. Moreover, our studies also show that the MR diet delays the progression of kidney disease.-Cooke, D., Ouattara, A., Ables, G. P. Dietary methionine restriction modulates renal response and attenuates kidney injury in mice.

Pleiotropic responses to methionine restriction.

Methionine restriction (MR) extends lifespan across different species. The main responses of rodent models to MR are well-documented in adipose tissue (AT) and liver, which have reduced mass and improved insulin sensitivity, respectively. Recently, molecular mechanisms that improve healthspan have been identified in both organs during MR. In fat, MR induced a futile lipid cycle concomitant with beige AT accumulation, producing elevated energy expenditure. In liver, MR upregulated fibroblast growth factor 21 and improved glucose metabolism in aged mice and in response to a high-fat diet. Furthermore, MR also reduces mitochondrial oxidative stress in various organs such as liver, heart, kidneys, and brain. Other effects of MR have also been reported in such areas as cardiac function in response to hyperhomocysteinemia (HHcy), identification of molecular mechanisms in bone development, and enhanced epithelial tight junction. In addition, rodent models of cancer responded positively to MR, as has been reported in colon, prostate, and breast cancer studies. The beneficial effects of MR have also been documented in a number of invertebrate model organisms, including yeast, nematodes, and fruit flies. MR not only promotes extended longevity in these organisms, but in the case of yeast has also been shown to improve stress tolerance. In addition, expression analyses of yeast and Drosophila undergoing MR have identified multiple candidate mediators of the beneficial effects of MR in these models. In this review, we emphasize other in vivo effects of MR such as in cardiovascular function, bone development, epithelial tight junction, and cancer. We also discuss the effects of MR in invertebrates.

Short term methionine restriction increases hepatic global DNA methylation in adult but not young male C57BL/6J mice.

Despite well-documented evidence for lifespan extension by methionine restriction (MR), underlying mechanisms remain unknown. As methionine can alter S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), the substrate and product of DNA methyltransferase-1 (DNMT1), we hypothesized that MR diet alters DNA methylation. Young (8-week-old) and adult (1-year-old) male C57BL/6J mice were fed diets with different levels of methionine (0.12%-MR, 0.84%-CD) for 12weeks. Functional indicators of DNA methylation, including global methylation (GM), gene-specific methylation (GSM) and LINE-1 methylation; and biochemical factors affecting DNA methylation, SAH, SAM, and DNMT1 were assessed in different tissues. MR altered DNA methylation depending on the age of intervention. While MR had no effect on hepatic GM in young animals, it increased GM by 27% over CD in adults (p<0.01). In comparison with young animals, hepatic GM levels were 17% lower in CD adults (p<0.05), but not different in MR adults. The MR-induced increase in hepatic GM was associated with a 38% decrease in SAH levels in adults (p<0.001), with SAH and GM levels being negatively correlated (r2=0.33, p<0.001). No changes were observed in DNMT protein levels in liver. In adipose tissue, MR caused a 6% decline in GM in adults (p<0.05), a corresponding 2-fold increase in SAH (p<0.05), and a 2-fold decrease in DNMT1 (p<0.01). MR caused both increases and decreases in GSM of liver and adipose. No changes were observed in LINE-1. Together, these findings provide evidence for protective effects of MR diet on hepatic DNA hypomethylation in adults, apparently mediated by SAH. These findings also indicate that altered DNA methylation might be playing a role in benefits conferred by MR diet.

Methionine restriction beyond life-span extension.

Dietary methionine restriction (MR) extends life span across species via various intracellular regulatory mechanisms. In rodents, MR induces resistance against adiposity, improves hepatic glucose metabolism, preserves cardiac function, and reduces body size, all of which can affect the onset of age-related diseases. Recent studies have shown that MR-affected biomarkers, such as fibroblast growth factor 21, adiponectin, leptin, cystathionine ß synthase, and insulin-like growth factor 1, can potentially alter physiology. The beneficial effects of MR could be explained in part by its ability to reduce mitochondrial oxidative stress. Studies have revealed that MR can reduce reactive oxygen species that damage cells and promote cancer progression. It has been demonstrated that either MR or the targeting of specific genes in the methionine cycle could induce cell apoptosis while decreasing proliferation in several cancer models. The complete mechanism underlying the actions of MR on the cell cycle during cancer has not been fully elucidated. Epigenetic mechanisms, such as methylation and noncoding RNAs, are also possible downstream effectors of MR; future studies should help to elucidate some of these mechanisms. Despite evidence that changes in dietary methionine can affect epigenetics, it remains unknown whether epigenetics is a mechanism in MR. This review summarizes research on MR and its involvement in metabolism, cancer, and epigenetics.

Effects of methionine restriction and endurance exercise on bones of ovariectomized rats: a study of histomorphometry, densitometry, and biomechanical properties.

To investigate the effects of dietary methionine restriction (MetR) and endurance exercise on bone quality under a condition of estrogen deficiency, female Sprague-Dawley rats (36-wk-old) were assigned to a sham surgery group or one of five ovariectomized groups subjected to interventions of no treatment (Ovx), endurance exercise (Exe), methionine restriction (MetR), methionine restriction plus endurance exercise (MetR + Exe), and estrogen treatment (Est). Rats in the exercise groups were subjected to a treadmill running regimen. MetR and control diets contained 0.172 and 0.86% methionine, respectively. After the 12-wk intervention, all animals were killed, and serum and bone tissues were collected for analyses. Compared with estrogen treatment, MetR diet and endurance exercise showed better or equivalent efficiency in reducing body weight gain caused by ovariectomy (P < 0.05). Whereas only the Est group showed evidence for reduced bone turnover compared with the Ovx group, MetR diet and/or endurance exercise demonstrated efficiencies in downregulating serum insulin, leptin, triglyceride, and thiobarbituric acid reactive substances (P < 0.05). Both the Exe and MetR groups showed higher femoral cortical and total volumetric bone mineral density (vBMD), but only the Exe and Est groups preserved cancellous bone volume and/or vBMD of distal femora (P < 0.05) compared with the Ovx group. After being normalized to body mass, femora of the MetR and MetR + Exe groups had relatively higher bending strength and dimension values followed by the Sham, Exe, and Est groups (P < 0.05). In conclusion, both MetR diet and endurance exercise improved cortical bone properties, but only endurance exercise preserved cancellous bone under estrogen deficiency.

The first international mini-symposium on methionine restriction and lifespan.

It has been 20 years since the Orentreich Foundation for the Advancement of Science, under the leadership Dr. Norman Orentreich, first reported that low methionine (Met) ingestion by rats extends lifespan (Orentreich et al., 1993). Since then, several studies have replicated the effects of dietary methionine restricted (MR) in delaying age-related diseases (Richie et al., 1994; Miller et al., 2005; Ables et al., 2012; Sanchez-Roman and Barja, 2013). We report the abstracts from the First International Mini-Symposium on Methionine Restriction and Lifespan held in Tarrytown, NY, September 2013. The goals were (1) to gather researchers with an interest in MR and lifespan, (2) to exchange knowledge, (3) to generate ideas for future investigations, and (4) to strengthen relationships within this community. The presentations highlighted the importance of research on cysteine, growth hormone (GH), and ATF4 in the paradigm of aging. In addition, the effects of dietary restriction or MR in the kidneys, liver, bones, and the adipose tissue were discussed. The symposium also emphasized the value of other species, e.g., the naked mole rat, Brandt's bat, and Drosophila, in aging research. Overall, the symposium consolidated scientists with similar research interests and provided opportunities to conduct future collaborative studies (Figure 3).

Methionine restriction prevents the progression of hepatic steatosis in leptin-deficient obese mice.

OBJECTIVE: This study investigated the effects of dietary methionine restriction (MR) on the progression of established hepatic steatosis in the leptin-deficient ob/ob mouse. MATERIAL/METHODS: Ten-week-old ob/ob mice were fed diets containing 0.86% (control-fed; CF) or 0.12% methionine (MR) for 14 weeks. At 14 weeks, liver and fat were excised and blood was collected for analysis. In another study, blood was collected to determine in vivo triglyceride (TG) and very-low-density lipoprotein (VLDL) secretion rates. Liver histology was conducted to determine the severity of steatosis. Hepatic TG, free fatty acid levels, and fatty acid oxidation (FAO) were also measured. Gene expression was analyzed by quantitative PCR. RESULTS: MR reversed the severity of steatosis in the ob/ob mouse. This was accompanied by reduced body weight despite similar weight-specific food intake. Compared with the CF group, hepatic TG levels were significantly reduced in response to MR, but adipose tissue weight was not decreased. MR reduced insulin and HOMA ratios but increased total and high-molecular-weight adiponectin levels. Scd1 gene expression was significantly downregulated, while Acadvl, Hadha, and Hadhb were upregulated in MR, corresponding with increased ß-hydroxybutyrate levels and a trend toward increased FAO. The VLDL secretion rate was also significantly increased in the MR mice, as were the mRNA levels of ApoB and Mttp. The expression of inflammatory markers, such as Tnf-α and Ccr2, was also downregulated by MR. CONCLUSIONS: Our data indicate that MR reverses steatosis in the ob/ob mouse liver by promoting FAO, increasing the export of lipids, and reducing obesity-related inflammatory responses.

Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis.

Hepatic steatosis is often associated with insulin resistance and obesity and can lead to steatohepatitis and cirrhosis. In this study, we have demonstrated that hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), two enzymes critical for lipolysis in adipose tissues, also contribute to lipolysis in the liver and can mobilize hepatic triglycerides in vivo and in vitro. Adenoviral overexpression of HSL and/or ATGL reduced liver triglycerides by 40-60% in both ob/ob mice and mice with high fat diet-induced obesity. However, these enzymes did not affect fasting plasma triglyceride and free fatty acid levels or triglyceride and apolipoprotein B secretion rates. Plasma 3-beta-hydroxybutyrate levels were increased 3-5 days after infection in both HSL- and ATGL-overexpressing male mice, suggesting an increase in beta-oxidation. Expression of genes involved in fatty acid transport and synthesis, lipid storage, and mitochondrial bioenergetics was unchanged. Mechanistic studies in oleate-supplemented McA-RH7777 cells with adenoviral overexpression of HSL or ATGL showed that reduced cellular triglycerides could be attributed to increases in beta-oxidation as well as direct release of free fatty acids into the medium. In summary, hepatic overexpression of HSL or ATGL can promote fatty acid oxidation, stimulate direct release of free fatty acid, and ameliorate hepatic steatosis. This study suggests a direct functional role for both HSL and ATGL in hepatic lipid homeostasis and identifies these enzymes as potential therapeutic targets for ameliorating hepatic steatosis associated with insulin resistance and obesity.