Virginia Malloy

Methionine restriction increases blood glutathione and longevity in F344 rats.

Little is known about the biochemical mechanisms responsible for the biological aging process. Our previous results and those of others suggest that one possible mechanism is based on the loss of glutathione (GSH), a multifunctional tripeptide present in high concentrations in nearly all living cells. The recent finding that life‐long dietary restriction of the GSH precursor methionine (Met) resulted in increased longevity in rats led us to hypothesize that adaptive changes in Met and GSH metabolism had occurred, leading to enhanced GSH status. To test this, blood and tissue GSH levels were measured at different ages throughout the life span in F344 rats on control or Met‐restricted diets. Met restriction resulted in a 42% increase in mean and 44% increase in maximum life span, and in 43% lower body weight compared to controls (P < 0.001). Increases in blood GSH levels of 81% and 164% were observed in mature and old Met‐restricted animals, respectively (P < 0.001). Liver was apparently the source for this increase as hepatic GSH levels decreased to 40% of controls. Except for a 25% decrease in kidney, GSH was unchanged in other tissues. All changes in GSH occurred as early as 2 months after the start of the diet. Altogether, these results suggest that dramatic adaptations in sulfur amino acid metabolism occur as a result of chronic Met restriction, leading to increases in blood GSH levels and conservation of tissue GSH during aging.—Richie, J. P., Jr., Leutzinger, Y., Parthasarathy, S., Malloy, V., Orentreich, N., Zimmerman, J. A. Methionine restriction increases blood glutathione and longevity in F344 rats. FASEB J. 8, 1302‐1307 (1994)

Nutritional control of aging

For more than 60 years the only dietary manipulation known to retard aging was caloric restriction, in which a variety of species respond to a reduction in energy intake by demonstrating extended median and maximum life span. More recently, two alternative dietary manipulations have been reported to also extend survival in rodents. Reducing the tryptophan content of the diet extends maximum life span, while lowering the content of sulfhydryl-containing amino acids in the diet by removing cysteine and restricting the concentration of methionine has been shown to extend all parameters of survival, and to maintain blood levels of the important anti-oxidant glutathione. To control for the possible reduction in energy intake in methionine-restricted rats, animals were offered the control diet in the quantity consumed by rats fed the low methionine diet. Such pair-fed animals experienced life span extension, indicating that methionine restriction-related life span extension is not a consequence of reduced energy intake. By feeding the methionine restricted diet to a variety of rat strains we determined that lowered methionine in the diet prolonged life in strains that have differing pathological profiles in aging, indicating that this intervention acts by altering the rate of aging, not by correcting some single defect in a single strain.

Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction

Reduced 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.

Dietary methionine restriction enhances metabolic flexibility and increases uncoupled respiration in both fed and fasted states

Dietary methionine restriction (MR) is a mimetic of chronic dietary restriction (DR) in the sense that MR increases rodent longevity, but without food restriction. We report here that MR also persistently increases total energy expenditure (EE) and limits fat deposition despite increasing weight-specific food consumption. In Fischer 344 (F344) rats consuming control or MR diets for 3, 9, and 20 mo, mean EE was 1.5-fold higher in MR vs. control rats, primarily due to higher EE during the night at all ages. The day-to-night transition produced a twofold higher heat increment of feeding (3.0 degrees C vs. 1.5 degrees C) in MR vs. controls and an exaggerated increase in respiratory quotient (RQ) to values greater than 1, indicative of the interconversion of glucose to lipid by de novo lipogenesis. The simultaneous inhibition of glucose utilization and shift to fat oxidation during the day was also more complete in MR (RQ approximately 0.75) vs. controls (RQ approximately 0.85). Dietary MR produced a rapid and persistent increase in uncoupling protein 1 expression in brown (BAT) and white adipose tissue (WAT) in conjunction with decreased leptin and increased adiponectin levels in serum, suggesting that remodeling of the metabolic and endocrine function of adipose tissue may have an important role in the overall increase in EE. We conclude that the hyperphagic response to dietary MR is matched to a coordinated increase in uncoupled respiration, suggesting the engagement of a nutrient-sensing mechanism, which compensates for limited methionine through integrated effects on energy homeostasis.

Transcriptional response to aging and caloric restriction in heart and adipose tissue

Sustained caloric restriction (CR) extends lifespan in animal models but the mechanism and primary tissue target(s) have not been identified. Gene expression changes with aging and CR were examined in both heart and white adipose tissue (WAT) of Fischer 344 (F344) male rats using Affymetrix® RAE 230 arrays and validated by quantitative reverse transcriptase–polymerase chain reaction (qRT‐PCR) on 18 genes. As expected, age had a substantial effect on transcription on both tissues, although only 21% of cardiac age‐associated genes were also altered in WAT. Gene set enrichment analysis revealed coordinated small magnitude changes in ribosomal, proteasomal, and mitochondrial genes with similarities in aging between heart and WAT. CR had very different effects on these two tissues at the transcriptional level. In heart, very few age‐associated expression changes were affected by CR, while in WAT, CR suppressed a substantial subset of the age‐associated changes. Genes unaltered by aging but altered by CR were identified in WAT but not heart. Most interestingly, we identified a gene expression signature associated with mammalian target of rapamycin (mTOR) activity that was down‐regulated with age but preserved by CR in both WAT and heart. In addition, lipid metabolism genes, particularly those associated with peroxisome proliferator‐activated receptor γ (PPARγ)‐mediated adipogenesis were reduced with age but preserved with CR in WAT. These results highlight tissue‐specific differences in the gene expression response to CR and support a role for CR‐mediated preservation of mTOR activity and adipogenesis in aging WAT.

Metabolic adaptations to methionine restriction that benefit health and lifespan in rodents.

Restriction of dietary methionine by 80% slows the progression of aged-related diseases and prolongs lifespan in rodents. A salient feature of the methionine restriction phenotype is the significant reduction of adipose tissue mass, which is associated with improvement of insulin sensitivity. These beneficial effects of MR involve a host of metabolic adaptations leading to increased mitochondrial biogenesis and function, elevated energy expenditure, changes of lipid and carbohydrate homeostasis, and decreased oxidative damage and inflammation. This review summarizes observations from MR studies and provides insight about potential mediators of tissue-specific responses associated with MRs favorable metabolic effects that contribute to health and lifespan extension.

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 β-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.

Inhibition of Prostate Cancer in Rats by the Administration of Dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) reduces the growth of normal and neoplastic cells by inhibition of glucose-&phosphate dehydrogenase (G6PD) and NADH oxidase, thereby reducing the amount of NADPH, NADH, and pentose phosphates available for biosynthetic processes.’ Pharmacological administration of DHEA in laboratory animals prevents the formation of spontaneous breast cance? and inhibits the development of carcinogen-induced colon and lung carcinoma?^^ In vitro, this steroid also acts as a protectant against the cytotoxic and transforming effects of DMBA and aflatoxin B on cultured cells. Although these studies clearly illustrate the preventive effect of DHEA on the onset of tumors, its direct effects on tumor growth rate have not been adequately explored. We have examined this question using the Dunning R3327-H prostate adenocarcinoma, a slow-growing, transplantable, androgen-dependent tumor in the Copenhagen rat. Pretreatment of these rats with DHEA before S.C. tumor implant did not influence the incidence of “tumor take”; however, the growth rate of the tumor was markedly reduced (FIG. 1). In previous studies,24 the preventive effects of DHEA were accompanied by inhibitory effects on weight gain. Since inhibition of weight gain is generally known to delay the onset of cancer, it is difficult to assess whether the effect of DHEA was due to a direct action on the tumor or an indirect effect via inhibition of lipogenesis.’ In this study, neither food consumption nor weight gain was affected (FIG. 2), suggesting that the observed anti-tumor effect was more likely due to a direct effect on prostate cancer. When G6PPD activity was measured in DHEA-treated rats, tumoral and liver G6PD was reduced by 15% and 34%, respectively (data not shown). This miniscule effect on tumoral G6PD cannot possibly account for the 65% reduction of prostate cancer observed in this study. As a pro-hormone, DHEA can be converted to either androgens or estrogens depending upon the tissue. While it stimulates the growth of the normal rat

A Comparative Study of the Effects of Percutaneously Administered 5α‐Reductase and Androgen Receptor Inhibitors on Hamster Sebaceous Glands

There is a need for the development of potent and locally active antiandrogens because the presence of side effects makes the systemic administration of antiandrogens unacceptable for the treatment of acne, hirsutism, and male pattern baldness. The antagonistic activities of these compounds in the skin are expressed through the inhibition of the metabolic conversion of testosterone to dihydrotestosterone (DHT) or the prevention of DHT binding to specific receptor proteins. Although the relative potencies of systemically administered 5a-reductase and androgen receptor blockers have already been intensively studied, there is little data concerning their relative potencies when applied topically. In this study, the topical antiandrogenic activity of progesterone (P), a potent 5a-reductase inhibitor, and two receptor antagonists, namely cyproterone acetate (CA) and spironolactone (SL), were compared using the androgen-sensitive ear skin sebaceous glands of the Syrian hamsters.’.’

Sulfur amino acid restriction–induced changes in redox‐sensitive proteins are associated with slow protein synthesis rates

The mechanisms underlying life span extension by sulfur amino acid restriction (SAAR) are unclear. Cysteine and methionine are essential for the biosynthesis of proteins and glutathione (GSH), a major redox buffer in the endoplasmic reticulum (ER). We hypothesized that SAAR alters protein synthesis by modulating the redox milieu. Male F344‐rats were fed control (CD: 0.86% methionine without cysteine) and SAAR diets (0.17% methionine without cysteine) for 12 weeks. Growth rates, food intake, cysteine and GSH levels, proteins associated with redox status and translation, and fractional protein synthesis rates (FSRs) were determined in liver. Despite a 40% higher food intake, growth rates for SAAR rats were 27% of those fed CD. Hepatic free cysteine in SAAR rats was 55% compared with CD rats. SAAR altered tissue distribution of GSH, as hepatic and erythrocytic levels were 56% and 196% of those in CD rats. Lower GSH levels did not induce ER stress (i.e., unchanged expression of Xbp1s, Chop, and Grp78), but activated PERK and its substrates eIF2‐α and NRF2. SAAR‐induced changes in translation‐initiation machinery (higher p‐eIF2‐α and 4E‐BP1, and lower eIF4G‐1) resulted in slower protein synthesis rates (53% of CD). Proteins involved in the antioxidant response (NRF2, KEAP1, GCLM, and NQO1) and protein folding (PDI and ERO1‐α) were increased in SAAR. Lower FSR and efficient protein folding might be improving proteostasis in SAAR.