Symposium 2015: Diet, Sulfur Amino Acids, and Healthspan
September 20–22, 2015, Tarrytown, NY
- Gene P Ables, Orentreich Foundation for the Advancement of Science
Methionine restriction beyond lifespan extension
Dietary methionine restriction (MR) extends lifespan in rodents and several other species. In yeasts, roundworms, drosophila, and human cells, MR extends lifespan via various intracellular regulatory mechanisms. In rodents, MR induces adiposity resistance, improves hepatic glucose metabolism, preserves cardiac function, and reduces body size, which could affect the onset of age-related diseases.
Recent studies have shown that MR affected hormones and enzymes such as Fgf21, Scd1, Ucp1, adiponectin, leptin, CBS, and IGF1 at the gene and protein levels, potentially altering physiology. The beneficial effects of MR could be explained by increased antioxidant substrates that reduce mitochondrial oxidative stress, as has been shown in the liver, heart, kidney, and brain of rats and pigs. Studies have demonstrated that MR can reduce reactive oxygen species that damage cells and promote cancer progression. Cancer metabolism is hindered by reduced levels of methionine, which are in high demand for protein synthesis. Recent studies demonstrate that either MR or targeting specific genes in the methionine cycle can induce cell apoptosis while decreasing proliferation in several cancer models. The complete mechanism of how MR acts on the cell cycle during cancer has not been completely elucidated, but activation of cell cycle inhibitor p21 may be one mode of action. Epigenetic mechanisms, such as methylation and non-coding RNAs are also possible downstream effectors of MR, and ongoing studies at OFAS should help to elucidate some of these mechanisms. Cumulative evidence for altered methylation of DNA and histones in the context of natural aging also suggests a role for epigenetics in MR-induced benefits.
Despite evidence that changes in dietary methionine can affect epigenetics, it remains to be investigated whether this is a mechanism in MR. The focus of this review is to consolidate research on MR and its involvement in metabolism, cancer, and epigenetics.
- Sean Adams, University of Arkansas for Medical Sciences
Intersections between fat metabolism and amino acids
Arkansas Children’s Nutrition Center and Department of Pediatrics,
University of Arkansas for Medical Sciences, Little Rock, AR
Fine-tuning the contributions of fat, carbohydrate, and amino acid catabolism toward generation of ATP to power cells is an important aspect of physiological homeostasis. Mitochondrial fuel management involves a balance between types and rates of fuel delivery, ATP demand, and enzyme regulation by metabolites sensitive to redox status (i.e., NADH/NAD+ ratio), reactive oxygen species (ROS) generation, or tricarboxylic acid (TCA) cycle capacity relative to fuel delivery (i.e., acetyl-CoA). Efficiency of mitochondrial oxidation of fuels may be defined as their relative flux toward full combustion vs. partial catabolism, and less efficient mitochondrial oxidation of any fuel leads to accumulation of specific upstream metabolites and derivatives; this contributes to a metabolite “signature” reflected in tissue or blood.
An interesting illustration of these concepts is the interplay between mitochondrial fatty acid oxidation (FAO) and amino acid catabolism. The mitochondrial branched-chain ketoacid dehydrogenase complex (BCKDC), for instance, is a primary regulator of BCAA and cysteine oxidative catabolism, and the enzyme is inhibited under conditions of increased FAO. This may explain how essential amino acids are partially “spared” from oxidation during fasting. Higher FAO also drives increased ROS generation, and this may, in theory, alter glutathione generation and cysteine-cystine dynamics. As another example, efficient, complete FAO may be attenuated when anaplerosis (i.e., from metabolism of select amino acids delivering net carbon to the TCA cycle) is limited, as hypothesized in the “anaplerotic stress” model of insulin resistance and type 2 diabetes mellitus. This may present an even greater metabolic challenge since FAO can also drive cataplerosis (loss of net carbon from the TCA cycle). In summary, it is clear that there are several intersections between metabolism of amino acids and fatty acids, and a greater understanding of this cross-talk should provide clues regarding key regulators of metabolic (patho)physiology.
- Holly Brown-Borg, University of North Dakota
Impact of dietary methionine on aging: Dependence on growth hormone status
Department of Basic Sciences, University of North Dakota School of Medicine & Health Sciences, Grand Forks, ND
Growth hormone plays a major role in aging and longevity in mammals. A lack of GH signaling (either GH deficiency or GH resistance) results in extended healthspans and lifespans in mice. Stress resistance is also greater when somatotropic signaling is low. In contrast, high plasma GH levels have been shown to accelerate aging-related dysfunction, decrease stress resistance, and shorten lifespan. Altering dietary methionine has also been shown to affect survival in rodents. Our studies show that methionine restriction extends lifespan in mice with normal or high levels of GH but not in mice that are GH deficient or GH resistant. These data indicate that intact GH signaling is necessary to discriminate variations of this amino acid emanating from the diet. Metabolomic data supports this hypothesis, showing that the lack of GH signaling may buffer the changes in various metabolic pathways normally impacted by changing dietary methionine levels. Thus, reduced somatotropic signaling may reprogram metabolism by shifting resources away from growth and proliferation and more towards stress resistance and cytoprotection.
- Rochelle Buffenstein, University of Texas Health Science Center
Maintenance of glutathione redox status in the naked mole-rat heart under conditions of high oxidative stress
Department of Physiology and the Sam and Ann Barshop Institute for Aging and Longevity Studies,
University of Texas Health Science Center at San Antonio, San Antonio, TX
Unlike all other mammals studied to date, not only can the naked mole-rat (NMRs) achieve an extraordinary lifespan but it is also able to maintain cardiovascular function for at least 75% of this extraordinary longevity. Oxidative stress is largely implicated in both the age-associated decline in cardiovascular function already evident in middle age and in cardiovascular disease. Therefore we sought to test the hypothesis that resistance to oxidative stress allows the NMR to well maintain cardiovascular function.
We treated NMRs and mice with a large bolus of doxorubicin (DOX; 20 mg/kg), a potent cardiac oxidative stressor. Echocardiography showed that 7 days after DOX treatment mice had a significant 25% decline in cardiac contractility, whereas NMRs maintained heart function. We found that DOX caused an increase in reduced glutathione (GSH) levels in NMR hearts, but no change in those of mice. Oxidized glutathione levels (GSSG) were conversely maintained in NMR hearts, but increased in hearts of mice. As a result, the GSH:GSSG ratio declined in mice but was maintained in NMRs with DOX, indicating a preservation of redox status in NMR hearts. Furthermore, DOX caused no change in glutathione S-transferase [GST] activity in mouse hearts, but induced a significant increase in GST activity in the hearts of NMRs.
Taken together, these data indicate that the NMR heart has a robust glutathione antioxidant capacity to stave off the damaging effects of oxidative stress allowing for the maintenance of cardiac function, and this likely plays a large role in the species’ trait of healthy cardiovascular aging.
- Maria Figueiredo-Pereira, City University of New York
Prostaglandin J2: A potential target for halting inflammation-induced neurodegeneration
We are addressing how prostaglandins, which are bioactive products of inflammation, contribute to neurodegenerative disorders including Alzheimer’s (AD) and Parkinson’s (PD) diseases. Prostaglandins are produced from arachidonic acid via cyclooxygenases, which are enzymes that play a major role in inflammation. Epidemiological studies show that chronic treatment with low levels of cyclooxygenase inhibitors (i.e., NSAIDs) lowers the risk of developing AD and PD. So far, NSAIDs are the only approved clinical drugs that prevent or delay the onset of these disorders. Unfortunately, inhibiting cyclooxygenases with NASAIDs blocks the synthesis of downstream neuroprotective as well as neurotoxic prostaglandins, thus producing adverse side effects. New therapeutic strategies that neutralize the effects of specific neurotoxic prostaglandins downstream from cyclooxygenases could have a great impact on treating these devastating neurodegenerative disorders with fewer negative side effects.
We chose to focus our studies on prostaglandin J2 (PGJ2) because it is by far the most neurotoxic when compared to prostaglandins A1, D2, and E2. Unlike other prostaglandins, PGJ2 and its metabolites have a cyclopentenone ring with reactive α,β-unsaturated carbonyl groups. These carbonyl groups form covalent Michael adducts with free thiols present in specific cysteine residues within proteins. Electrophiles, such as PGJ2, that bind to key protein cysteine(s) are regarded as playing an important role in determining whether neurons will live or die.
I will discuss our in vitro and in vivo studies showing that PGJ2 induces pathological processes relevant to neurodegenerative disorders. Furthermore, we found that increasing intracellular cAMP with the lipophilic peptide PACAP27 counteracts some of the detrimental effects induced by PGJ2. In conclusion, there is a need for new approaches that target neurotoxic prostaglandins such as PGJ2, downstream of cyclooxygenases. These approaches hold great promise for treating neurodegenerative disorders by preventing inflammation-induced neurodegeneration without affecting the beneficial effects of neuroprotective prostaglandins.
- Vadim Gladyshev, Harvard Medical School
Understanding control of lifespan through comparative genomics and methionine status
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Understanding the mechanisms that control lifespan is among the most challenging biological problems. Many complex human diseases are associated with aging, which is both the most significant risk factor and the process that drives the development of these diseases. The aging process can be regulated during evolution. For instance, mammals are characterized by >100-fold difference in lifespan, which can both increase and decrease during evolution. We employ this diversity in mammalian lifespan and the associated life-history traits to shed light on mechanisms that regulate species lifespan. For this, we utilize methods of comparative genomics to examine genomes of short- and long-lived species and carry out analysis of lifespan across a panel of mammals.
We sequenced the genomes of several mammals of exceptional lifespan, including mole rats and microbats, and identified genes that may contribute to their longevity. We also carried out analyses of gene expression and metabolites across a large panel of mammals. These studies point to both lineage-specific and common adaptations to longevity involving various pathways. One pathway that emerges as relevant to the control of lifespan is methionine availability. Indeed, reduced methionine intake can extend lifespan in rodents by mimicking dietary restriction, but whether this regimen represents a general strategy for regulating aging has been controversial. We found that methionine restriction can extend lifespan of both fruit flies and yeast, but this effect is dependent on the status of other amino acids. Under certain conditions, methionine restriction mimicked the effect of dietary restriction and was associated with decreased reproduction, whereas under other conditions, it was ineffective, and the regulation of lifespan was uncoupled from reproduction. These studies provide insights into the roles of methionine in aging and suggest a strategy for lifespan extention by methionine restriction. It is our hope that a better understanding of molecular mechanisms of mammalian lifespan control will lead to a better understanding of human diseases of aging.
- Tsang-hai Huang, National Cheng Kung University
Dietary restrictions, bone density, and bone quality
1Laboratory of Exercise, Nutrition, and Bone Biology, Institute of Physical Education, Health and Leisure Studies, National Cheng Kung University, Tainan, Taiwan;
2Orentreich Foundation for the Advancement of Science, Inc, Cold Spring, NY
Caloric restriction (CR), protein restriction (PR), or specific amino-acid (e.g., methionine restriction, MetR) as different dietary restrictions have been well-proven regarding their comprehensive benefits in metabolism and health. Based on bone densitometric measurements, dietary restrictions caused weight loss accompanied by reduced areal bone mineral density (aBMD), bone mass, and/or bone size, which are considered harmful to bone health. However, in the wake of improvements in bone densitometric instruments (e.g., high resolution X-ray tomography), dietary restrictions were verified to cause reductions in bone mass/size rather than volumetric bone mineral density (vBMD). Furthermore, according to the concept of bone quality, bone health consists of diverse indices rather than simply being represented by densitometric measurements. Indeed, there is evidence showing that moderate dietary restrictions do not impair intrinsic bone material properties in spite of the reduced whole bone strength due to absolute smaller bone size.
In the present review, we integrated reports from traditional densitometric measurements, metabolic status assays (e.g., energy metabolism, oxidative stresses, and inflammatory responses), and biomaterial analyses, and provided a revised concept regarding the effects of CR, PR, and MetR on the skeleton.
- Joseph Kemnitz, University of Wisconsin
Diet, calorie restriction, and aging in rhesus monkeys
2Wisconsin National Primate Research Center,
3Department of Medicine (Geriatrics) and 4Department of Nutritional Sciences, University of Wisconsin-Madison,
5Geriatric Research, Education, and Clinical Center, Wm. S. Middleton Memorial Veterans Hospital, Madison, WI
We have been assessing the effects of moderate calorie restriction (CR) on health and lifespan in rhesus macaques since 1989. Beginning when the monkeys were young adults (~10 years of age; median lifespan is ~26 years), all subjects (30 females and 46 males) were fed a purified diet containing 15% protein (lactalbumin), 10% fat (corn oil), and 65% carbohydrate (sucrose, starch, and dextrin), supplemented daily with fresh fruit and vegetables. Control monkeys (C, initially n=38, 3 currently surviving) were given enough food to allow ad libitum access for ~8 hr/day. Restricted monkeys (R, initially n=38, 10 currently surviving) were fed 20-30% less than their individualized baseline intake, subsequently adjusted for changes in intake by C except to safeguard health. Food intake was measured daily, and regular assessments of body mass and composition, metabolic rate and physical activity, and glucose tolerance and insulin sensitivity were made.
MR imaging of brain was done later in the animals’ lives. Early effects of CR included loss of body weight and body fat, reduced insulin levels and increased insulin sensitivity, and improved lipid profiles. Loss of skeletal muscle occurred during middle age for males, but the decline was slower for R than for C. CR decreased sleeping metabolic rate, but R displayed increased physical activity with lower cost of movement than for C. CR dramatically reduced the incidence of cancer, cardiovascular disease, and type 2 diabetes mellitus. CR also preserved brain cortical and subcortical grey matter volume and attenuated astrogliosis but did not affect amyloid plaque burden or the decline in integrity of the corpus callosum with advancing age. CR increased both healthspan and lifespan in these nonhuman primates.
- Robert Koza, Maine Medical Center Research Institute
Rea Anunciado-Koza, Justin Manuel, Robert A Koza
Heterogeneity in high fat diet (HFD)-induced obesity within a population of inbred mice has been shown to be associated with changes of gene expression in adipose tissue. A gene with a large degree of variation among mice, mesoderm specific transcript (Mest), has also been shown to be highly inducible after short-term exposure to dietary fat, and its expression in adipose tissue prior to HFD-feeding is predictive of individual susceptibility to the development of obesity. To gain insight on the relationship of Mest with phenotypic changes in body composition within a population of inbred mice after a short exposure to dietary fat, 96 individually housed 8-week-old C57BL/6J mice were fed a diet containing 58% kcal fat for a period of only 2 weeks. Measurements of Mest mRNA after the dietary regimen in visceral epididymal (EPI) and subcutaneous inguinal (ING) fat shows a range of 12-fold and 90-fold respectively and was highly and positively associated with changes in fat mass. Surprisingly, there was only a slight association of adipose Mest expression with food intake normalized to either bodyweight or lean mass as measured via NMR. In addition, adipose Mest expression coincides (ING; R=0.91; EPI; R=0.62) with the expression of transcription factor Kruppel-like factor 14 (KLF14), an imprinted gene that is thought to play a major role in the regulation of gene expression in adipose tissue. Other genes shown to be predictive for the development of adiposity (Bmp3, Sfrp5, and Nkd1) identified in previous studies were also highly associated with variation in fat mass as expected, whereas Pparg2 showed no association with any indices of fat mass accumulation. Our data suggest that KLF14 transcriptional activity may at least partially mediate adipose tissue Mest to promote fat mass accumulation in mice following exposure to an obesogenic diet.
- Warren Kruger, Fox Chase Cancer Center
The effect of dietary modulation of sulfur amino acids on cystathionine beta synthase deficient mice
Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, PA
Cystathionine beta synthase (CBS) is a key enzyme in the methionine and cysteine metabolic pathway. It catalyzes the irreversible conversion of homocysteine to cystathionine, which is subsequently converted to cysteine. Thus, CBS acts as a metabolic “gate keeper”, regulating the flow of fixed sulfur to the cysteine metabolic pathway. Mutations in the CBS gene cause clinical CBS deficiency, a disease characterized by elevated plasma total homocysteine (tHcy) and methionine and reduced plasma cysteine. The treatment goal of CBS-deficient patients is to normalize the metabolic values of these three metabolites using a combination of vitamin therapy and dietary manipulation.
In order to better understand the effectiveness of nutritional treatment strategies, we have performed a series of long-term dietary manipulation studies using our previously developed Tg-I278T Cbs-/- mouse model or CBS deficiency. These mice have undetectable levels of CBS activity, extremely elevated levels of plasma tHcy, modestly elevated plasma methionine, and low plasma cysteine. They exhibit several easily discernible phenotypes, including osteoporosis, loss of fat mass, reduced life-span, and facial alopecia. In these mice, we have tested three different dietary manipulations: 1) supplementation with N-acetylcysteine; 2) restriction of dietary methionine; and 3) supplementation with betaine. We found that methionine restriction is the most effective approach at reversing Cbs-/- phenoytpes, although some beneficial effect is also observed with betaine supplementation. Our studies suggest that dietary methionine restriction is the most effective treatment for CBS deficiency, and that elevated tHcy is the key pathogenic factor.
- Jason Locasale, Cornell University
Dynamics of histone methylation mediated by the status of methionine metabolism
S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) link metabolism to methylation status. However, it is unknown whether fluctuations in SAM and SAH can be sensed to alter the kinetics of key histone methylation marks such as H3K4me3. We provide evidence that methionine metabolism is sufficient to directly determine the levels of histone methylation through its ability to modulate SAM and SAH. This dynamic interaction occurred rapidly and methionine insufficiency leading to a depletion of H3K4me3 could be fully recovered upon restoration of methionine levels. Modulation of methionine in diet led to changes in both metabolism and histone methylation in liver. In humans, methionine variability in fasting serum was found to be commensurate with concentrations needed for these dynamics and could be explained in part by diet. Together these findings demonstrate that flux through methionine metabolism and the sensing of methionine availability may be configured to allow for direct communication to the chromatin state in cells.
- James Mitchell, Harvard T. H. Chan School of Public Health
Dietary sulfur amino acid control of endogenous hydrogen sulfide production
Harvard T. H. Chan School of Public Health, Department of Genetics and Complex Diseases, Boston, MA
Hydrogen sulfide (H2S) is a gas easily identified by its distinctive odor, and although toxic at high concentrations, it has recently gained recognition for its numerous beneficial health effects. Many experiments documenting such benefits, ranging from extended longevity in lower organisms to protection from ischemic injury in mammals, are based on exposure to exogenous sources of H2S. However, there is a growing appreciation for the importance of endogenously produced H2S in a variety of health outcomes. H2S is generated by enzymes of the evolutionary conserved transsulfuration pathway (TSP), including cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CGL), responsible for the biosynthesis of cysteine from methionine.
We recently linked functional benefits of dietary restriction (DR), or reduced food intake without malnutrition, on stress resistance and longevity in model organisms to increased TSP activity and endogenous H2S production. DR includes various regimens aimed at either reducing overall calorie intake (calorie restriction, intermittent/every-other-day fasting) or reducing particular nutrients such as protein or sulfur amino acids, methionine and cysteine (methionine restriction), with overlapping functional benefits on stress resistance, metabolic fitness, and lifespan. We found that multiple DR regimens increased CGL expression and H2S production, and that this was blocked by selective re-addition of dietary cysteine with a concomitant loss of DR benefits. We will discuss the small but growing body of literature linking the TSP to the functional benefits of DR in part through the production of endogenous H2S, with an emphasis on regulation of the TSP and H2S production by diet and molecular mechanisms of beneficial H2S action.
- James Mullin, Lankenau Institute for Medical Research
Improvement of epithelial barrier function by methionine restriction
1Lankenau Institute for Medical Research, Wynnewood, PA
2National Institute on Aging, NIH Biomedical Research Center, Baltimore, MD
3Division of Gastroenterology, Lankenau Medical Center, Wynnewood, PA
The singular importance of epithelial barrier function in both systemic physiology and prevention of inflammation induced us to examine the possible effects of methionine restriction (MR) on epithelial tight junction (TJ) structure and permeability. We used two very different but well described epithelial models, the LLC-PK1 renal epithelial cell line and rat distal colon. In LLC-PK1 epithelia, MR did not affect cell growth or differentiation. However, the TJ proteins claudin-3 and claudin-7 were significantly decreased in abundance, whereas claudin-4 and claudin-5 were markedly increased in abundance. The functional result of these structural changes was improved epithelial barrier function, measured as increased transepithelial electrical resistance (Rt) and decreased transepithelial (paracellular) diffusion of 14C-D-mannitol (Jm). In our animal model study, rats were maintained on a MR diet (0.17% L-methionine [w/w] vs. the normal 0.86%) for 28 days prior to removal of their distal colon. Animals on MR diet showed small but significant reductions in the plasma and (intracellular) colonocyte levels of methionine. Colon mucosal sheets from rats on MR diet showed increased Rt with simultaneous decrease in Jm, both indicating improved colon epithelial barrier function in MR. Western blot analyses and RT-PCR showed an increase in claudin-3 and a change in the post-translational modification of occludin, data reinforcing a paracellular barrier alteration. Improvement of epithelial barrier function at the level of the TJ may be a general benefit of MR.
- Arlan Richardson, University of Oklahoma Health Sciences Center
The role of DNA methylation in the anti-aging mechanism of dietary restriction
Reynolds Oklahoma Center on Aging and the Department of Geriatric Medicine,
the University of Oklahoma Health Sciences Center,
and the OKC VA-Medical Center, Oklahoma City, OK
Research conducted over the past 60 years has shown that dietary restriction (DR) extends the mean and maximum lifespan of a wide variety of organisms. In addition, DR has been shown to delay the onset and progression of most age-related diseases as well as improve most physiological processes that decline with age. Therefore, DR is believed to retard aging and has become the gold standard by which other manipulations that increase lifespan are compared. An important facet of DR that has been largely overlooked by the research community is that DR can have early effects that create a cellular memory, which persists even when DR is discontinued. Therefore, DR could be increasing lifespan and retarding aging through a novel mechanism that involves a molecular signal(s) that arises shortly after the implementation of DR and has an impact on the animal over its lifespan, even if DR is discontinued.
The most likely molecular process by which DR could increase lifespan and retard aging after being discontinued would be through an epigenetic mechanism, specifically DNA methylation. We are currently generating the first data on the effect of DR on DNA methylation using a novel next generation sequencing approach that detects cytosine methylation (5mC) in CpG islands, shores, and shelves, as well as gene promoters and intragenic regions in the hypothalamus genome because the hypothalamus is a major nutritional sensor that integrates and co-ordinates various signals (e.g., nutrient sensing, satiety, and changes in adiposity).
- John Richie, Penn State University College of Medicine
Dietary sulfur amino acid restriction in healthy adults
Diets restricted in sulfur amino acids (SAA) have been shown in pre-clinical models to have profound beneficial effects, including enhanced lifespan and reductions in a variety of aging-related diseases, disorders, and impairments. These effects may be driven, in part, by changes in key metabolic pathways, resulting in a variety of physiological and cellular changes including reductions in body weight, adiposity, and oxidative damage. Altogether, these findings suggest that dietary SAA restriction may have clinical implications as a potential anti-aging/disease prevention/healthspan promoting intervention. In order to determine the potential viability of such a strategy in humans, we previously conducted a short-term cross-over controlled feeding study of dietary methionine restriction (MR) in healthy adults. Results demonstrated the overall feasibility of MR feeding in a controlled environment. Further, we observed that MR resulted in changes in a number of blood parameters, including reductions in SAA and lipids and an increase in FGF-21 after 3 wk; however, no effects were observed for other MR-related markers, including adipokines, IGF-1, and glutathione. Since more recent findings in animal models highlighted the importance of restricting total SAA and not just methionine (Met), we initiated a controlled feeding study of total SAA in healthy adults. The study design consists of two randomized groups (n=10/grp) each consisting of three 4-wk feeding periods separated by 3-4 wk washout periods. Each group started with control diet [30.1 mg/kg/d each of Met and cysteine (Cys)] followed by test diet periods of 70% and 90% Met restriction for one group and 50% and 65% SAA (Met + Cys) restriction for the other. Preliminary results from this study will be presented. Altogether, studies done to date indicate that dietary SAA restriction may represent an important intervention strategy for prevention and treatment, particularly in aging individuals.
- George Roth, GeroScience
Manipulation of healthspan and function by dietary restriction mimetics
After nearly a century of rigorous investigation and testing, dietary caloric restriction (CR) remains the most robust and reproducible method for slowing aging and maintaining health, function, and vitality. This intervention has been applied to species across the evolutionary spectrum, but for a number of reasons, practical applicability to humans has been questioned. To overcome these issues, we initiated to field of CR mimetics in 1998 and have observed its development into a full-fledged “anti-aging” industry at the present time. Basically, strategies which enable individuals to obtain the biological benefits of CR, WITHOUT REDUCING ACTUAL FOOD INTAKE, can be considered CR mimetics, whether functional, pharmaceutical, nutraceutical, or other. Some of the best known candidates include resveratrol and related agents, the antidiabetic drug metformin, and rapamycin and other mTOR regulators. While the mechanisms of action vary, these and essentially all CR mimetic candidates work through at least some of the same pathways as actual CR.
However, we believe the most efficient strategy for mimicking CR is to act as close to the initial energy processing events as possible and have, therefore, focused on glycolytic inhibition. Proof of principle was initially obtained with 2-deoxyglucose, which exerted a number a similar metabolic sequellae to full CR, although this compound exhibited a narrow efficacy/toxicity range. The second generation of glycoltic-inhibiting CR mimetics was therefore spearheaded with mannoheptulose, a seven carbon sugar, derived primarily from unripe avocados, that inhibits hexokinase without apparent side effects. CR-like benefits include better insulin/glucoregulatory control, maintenance of youthful body composition, increased strength and agility, immune and stress/inflammation protection, weight control, and improved health/longevity.
While the entire field continues to evolve rapidly, the current status will be reviewed with particular focus on recent developments, most practical relevance and applicability for potential consumers, and new strategies for the future.
- Jacob Selhub, Tufts University
The atherogenic effect of methionine
The popularity of the homocysteine theory of arteriosclerosis can be traced to the study by Wilken and Wilken in 1976 showing that the concentrations of homocysteine-cysteine mixed disulfide after a methionine load were higher in patients with CVD than in healthy controls. What was typical of this and the subsequent studies showing associations between homocysteine and a variety of diseases (CAD, stroke, cognitive impairment, etc.) was that the differences in homocysteine concentrations were small, unlike the high concentrations seen among those with the rare congenital vitamin B12 or cystathionine beta synthase deficiencies.
What fueled this area further are the many studies with cell culture, animals, and humans showing adverse effects of “high homocysteine”, which was introduced either as a bolus at high non-physiological concentrations or as a methionine load that resulted in higher plasma homocysteine and at the same time there was a decrease of flow mediated brachial artery after stimulation, higher oxidative stress, increased coagulation, and circulating adhesion molecule levels and others. These studies neglected the possibility that methionine load is also associated with increased plasma methionine. To address this question, we fed APOE-4-deficient mice with experimental diets designed to achieve three conditions: (i) high methionine intake with normal blood homocysteine; (ii) high methionine intake with B vitamin deficiency and hyperhomocysteinemia; and (iii) normal methionine intake with B vitamin deficiency and hyperhomocysteinemia. Mice fed methionine-rich diets had significant atheromatous pathology in the aortic arch, even with normal plasma homocysteine levels. Mice fed B vitamin-deficient diets developed severe hyperhomocysteinemia without any increase in vascular pathology. Our findings suggest that moderate increases in methionine intake are atherogenic in susceptible mice while high plasma homocysteine was not. We propose that it was the high methionine that was responsible for the adverse effects after the methionine load.
- Martha Stipanuk, Cornell University
Blocking metabolism of cysteine to cysteinesulfinate: Consequences of taurine depletion and hydrogen sulfide overproduction
Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to sulfur amino acid intake. Knockout of the murine Cdo1 gene results in elevated cysteine levels, severe impairment in ability to synthesize taurine, and an increased catabolism of cysteine to H2S. In an effort to distinguish whether the Cdo1-null mouse phenotype is due to taurine depletion or to excess levels of cysteine and its greater catabolism via desulfhydration, mice were fed either a basal or taurine-supplemented semi-purified diet.
The lack of taurine was associated with a lack of taurine conjugation of bile acids, a dramatic increase in the total and unconjugated hepatic bile acid pools, and an increase in betaine and other molecules that serve as organic osmolytes. We identified cysteinesulfinic acid decarboxylase, betaine:homocysteine methytransferase (BHMT), organic solute and steroid transporter subunit beta and cholesterol 7α-hydroxylase as proteins whose hepatic expression is strongly regulated in response to taurine depletion in the Cdo1-null mouse. Taurine supplementation of Cdo1-null mice restored hepatic levels of these proteins to wild-type levels, whereas taurine supplementation had no effect on abundance of these proteins in wild-type mice. The observation of a strong effect of taurine on BHMT expression suggests that BHMT downregulation may allow betaine to play a greater role as a cellular osmolyte. Parameters that were altered in Cdo1-null mice but were not corrected by taurine supplementation include elevated tissue and urine thiosulfate levels, which are indicative of excess flux of cysteine through desulfhydration pathways. In addition, low serum leptin levels, high serum insulin levels, elevated serum triglyceride levels, elevated hepatic levels of long-chain acyl-carnitines, and elevated hepatic levels of stearoyl-CoA desaturase 1 and acetyl-CoA carboxylase were observed, indicative of alterations in hepatic lipid metabolism. More work is needed to understand the cause of these changes in lipid metabolism.
- Suresh Tyagi, University of Louisville
Mitochondrial division/mitophagy inhibitor (Mdivi) ameliorates pressure overload-induced heart failure
Background: We have previously reported the role of anti-angiogenic factors in inducing the transition from compensatory cardiac hypertrophy to heart failure and the significance of MMP-9 and TIMP-3 in promoting this process during pressure overload hemodynamic stress. Several studies reported the evidence of cardiac autophagy, involving removal of cellular organelles like mitochondria (mitophagy), peroxisomes, etc., in the pathogenesis of heart failure. However, little is known regarding the therapeutic role of mitochondrial division inhibitor (Mdivi) in pressure overload induced heart failure. We hypothesize that treatment with Mdivi inhibits abnormal mitophagy in a pressure overload heart and thus ameliorates heart failure condition.
Materials & Methods: To verify this, ascending aortic banding was done in wild type mice to create pressure overload induced heart failure; mice were then treated with Mdivi and compared with vehicle treated controls.
Results: Expression of MMP-2, vascular endothelial growth factor, CD31, was increased, while expression of anti angiogenic factors like endostatin and angiostatin along with MMP-9, TIMP-3 was reduced in Mdivi treated AB 8-week-old mice compared to vehicle treated controls. Expression of mitophagy markers like LC3 and p62 was decreased in Mdivi treated mice compared to controls. Cardiac functional status assessed by echocardiography showed improvement, and there is also a decrease in the deposition of fibrosis in Mdivi treated mice compared to controls.
Conclusion: Above results suggest that Mdivi inhibits the abnormal cardiac mitophagy response during sustained pressure overload stress and propose the novel therapeutic role of Mdivi in ameliorating heart failure.