Beyond the Calorie: The regulation of health and longevity by specific dietary amino acids

Dudley Lamming

Mechanisms of yeast chronological lifespan extension by amino acid supplementation

Elisa Enriquez-Hesles¹, Christopher Letai¹, James R. Bain², Michael J. Muehlbauer², and Jeffrey S. Smith^1
1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Va, USA
²Department of Medicine, Duke University Physiology Institute, Duke University, Durham, N.C., USA

Caloric restriction (CR) in the form of glucose limitation extends both replicative and chronological lifespan (CLS) of the budding yeast Saccharomyces cerevisiae. CLS is defined as the number days that cells in stationary phase remain viable and is typically measured by tracking the fraction of cells able re-enter the cell cycle to form a colony provided with fresh nutrients. We previously reported that the conditioned media from CR stationary phase cultures extends CLS when supplemented into the expired media of non-restricted stationary phase cultures. Active longevity molecules in the CR conditioned media turned out to be specific amino acids, with L-glutamine, L-arginine, L-isoleucine, and L-serine having the strongest effects on CLS extension. L-serine supplementation occurred independent of amino acid auxotrophies in a common lab strain (BY4741) utilized in the study. Serine also extended CLS via the one-carbon metabolism pathway, although additional uncharacterized mechanisms were likely involved. On the other hand, CLS extension by increasing concentrations of L-arginine supplementation was specific to strains with an auxotrophic met15∆ mutation, which genetically mimics methionine restriction (MetR). Metabolomics analysis of BY4741 (met15∆) vs SY1183 (MET15⁺) strains revealed significantly upregulated flux through the polyamine biosynthesis and methionine salvage pathways in the met15∆ strain, most likely due to the impaired methionine synthesis pathway. Methylthioadenosine (MTA) produced during polyamine synthesis is salvaged back to methionine. Since arginine is a major precursor of polyamine (putrescine, spermidine, spermine) biosynthesis, it makes sense that arginine would extend CLS of BY4741 (met15∆), rescuing the limited methionine condition. In the MET15⁺ strain, however, we observed the opposite effect, with arginine supplementation actually shortening CLS, presumably through excessive methionine generation. Eliminating spermidine, spermine, and MTA production by deleting SPE2 (SAM decarboxylase) prevented arginine from shortening CLS in the MET15⁺ strain, thus supporting the methionine toxicity model. Methionine is an essential amino acid in mammals, so we are continuing investigation of arginine mechanisms of CLS extension in the met15∆ context where methionine is also essential.

Dr. Smith is currently a tenured Professor in the UVA Department of Biochemistry and Molecular Genetics and is Director of Graduate Studies for the Ph.D. program. He is also a recipient of the Robert Kadner Award for Graduate Student Mentoring and Teaching from the School of Medicine. In the aging field, Dr. Smith is an Editorial Board member for the new journal Aging Biology and is a member of the Cellular Mechanisms of Aging and Development Study Section at the NIA. His current research program is focused on caloric restriction mechanisms in yeast chronological aging and genomic instability and chromosome architecture in the context of replicative aging.

Molecular mechanisms of lifespan extension and the struggle to bring them to the clinic

Christian Sell
Drexel University College of Medicine, Philadelphia, Pa., USA

Inhibitors of the mTOR pathway are among the most promising interventions to target age-related dysfunction; however, there is a critical need to further define the pro-longevity effects to facilitate clinical development of mTOR inhibitors. The overarching goal of our research program is to develop a mechanistic understanding of novel downstream targets of rapamycin, in order to facilitate safer and more effective strategies to promote healthy aging. Cellular senescence occurs in both somatic and stem cell populations and contributes to age-related dysfunction, and our laboratory has shown that mTOR inhibition using rapamycin can prevent entry into the senescent state. The mTOR pathway also regulates senescence and pluripotency in a variety of stem cell populations. Our recent results suggest that mTOR inhibition by rapamycin prevents senescence and enhances pluripotency by increasing the levels of the non-coding RNA (lncRNA) H19. The rationale for this hypothesis is our observation that rapamycin increases lncRNA H19. We find that levels of H19 decrease during senescence and in pluripotent cells. H19 plays a central role during development and differentiation and maintenance of adult stem cell populations. Rapamycin increases H19 levels, prevents senescence, and maintains pluripotency. The results suggest that increasing H19 levels in response to mTOR inhibition may play a dual role, inhibiting senescence while simultaneously increasing pluripotency in adult stem cell populations. The proposed work will provide transformative data regarding a novel mechanism for lifespan extension and improvement of late-life function in multiple tissues.

The protein paradox—resolving the roles of dietary protein in obesity, aging, and age-related disease

Stephen J. Simpson
Charles Perkins Centre, The University of Sydney, Sydney, Australia

Reducing protein intake (and that of key amino acids) extends lifespan, especially during mid-life and early late-life. Yet, due to a powerful protein appetite, reducing protein in the diet leads to increased food intake, promoting obesity—which shortens lifespan. That is the protein paradox. In the talk I will first explore both sides of the paradox, introducing nutrient-specific appetites, protein leverage, FGF-21, and macronutrient interactions on metabolism and ageing. I will then attempt to resolve the paradox by considering age-specific effects and the quality of dietary protein (amino acid balance) and carbohydrate. I will conclude by showing how these pieces fit together and play out in the modern industrialized food environment to result in the global pandemic of unhealthy aging.

After graduating as a biologist from the University of Queensland, Steve undertook his Ph.D. at the University of London, then spent 22 years at Oxford before returning to Australia in 2005 as an Australian Research Council Federation Fellow.

Steve and colleague David Raubenheimer developed an integrative modelling framework for nutritional biology (the Geometric Framework), which was devised and tested using insects and has since been applied to a wide range of organisms—from slime molds to humans—and problems—from aquaculture and conservation biology to the dietary causes of human obesity and aging.

Dr. Simpson has also pioneered understanding of swarming in locusts, with research spanning neurochemical events within the brains of individual locusts to continental-scale mass migration.

In 2007 Steve was elected a Fellow of the Australian Academy of Science, in 2013 he was elected a Fellow of the Royal Society of London, in 2015 was made a Companion of the Order of Australia, and in 2022 he was awarded the Macfarlane Burnet Medal of the Australian Academy of Sciences.

Heterogeneity in the impact of dietary protein on metabolic health highlights the importance of precision dietetics

Cara Green^1,2, Michaela Murphy^1,2, Isaac Grunow^1,2, Yang Liu^1,2, Reji Babygirija^1,2, Mariah Calubag^1,2, Shelly Sonsalla^1,2, Astrid Martin^1,2, Yang Yeh^1,2, Dudley Lamming^1,2
1Department of Medicine, University of Wisconsin-Madison, Madison, Wis., USA
²William S. Middleton Memorial Veterans Hospital, Madison, Wis., USA

Low protein (LP) diets can improve metabolic health without caloric restriction and may be effective to promote healthy aging and combat diabetes and obesity. Many dietary recommendations exist at the population level, but individual information about the metabolic response to diet is lacking. In mice, an LP diet can promote weight loss, improve glycemic control, and increase lifespan; however, this is sex and strain dependent. It is unknown which key genes may be responsible for determining the individual response to dietary protein. To identify genetic markers that may determine how dietary protein impacts metabolism, we characterized 40 recombinant inbred strains of male and female BXD mice. We found huge variation across strains and sexes in the response to protein restriction (PR), including in weight loss, adiposity, and fasting blood glucose. PR promoted positive and negative responses depending on strain and sex; male mice could lose 4g or gain 6g after 8 weeks on PR depending on strain. One of the phenotypes almost universally improved by PR was fasting blood glucose; this was reflected in correlation analyses, in which 11 strains of mice showed a strong positive correlation (R > 0.4) between protein intake and fasting blood glucose, relative to only 5 strains with total calorie intake. Interestingly, there was very little correlation of protein with final lean mass, with 4 strains showing a positive (R > 0.4) correlation with protein intake. However, 21 BXD strains had a positive correlation between calorie intake and final lean mass, suggesting that calories, and not protein, in the diet is a modulator of fat-free mass in mice. Quantitative trait locus (QTL) analysis to discover links between these complex phenotypes and chromosome regions indicated there were no significant regions of interest conserved between males and females for any of the phenotypes we investigated. QTL analyses found genomic regions significantly associated with changes in lean mass and glucose tolerance with PR in males and females. In females, fasting blood glucose and fat mass were significantly associated with different genomic regions. These data show that the metabolic health effects of dietary protein are highly individualized based on sex and genetic background. This demonstrates the importance of precision dietetics to maximize metabolic health and the potential significance of personalized dietary strategies if a similar response exists in humans. In the future, this may help us to promote healthy aging and improve metabolic health on an individual basis.

Cara Green completed her undergraduate degree in Molecular Biology and Biochemistry at the University of Durham (UK) in 2013, where she first became interested in aging, before travelling north to the University of Aberdeen in Scotland for her Ph.D. in Biological Sciences with Professor John Speakman. There, she specialized in calorie restriction and its impact on whole body metabolomics, focusing on the linear relationship in mice between calories and lifespan, and embarked on a tumultuous but rewarding journey with bioinformatics. In 2018, Ph.D. completed, she moved across the pond to the University of Wisconsin-Madison for a postdoctoral position with Dr. Dudley Lamming to further investigate the relationship between nutrition and age-associated diseases. She is particularly interested in the metabolic response to dietary protein and branched chain amino acids and the impact of sex and genetic background on such responses and what may govern them.

Methionine metabolism and growth hormone: Impact on aging

Holly M. Brown-Borg, Sharlene Rakoczy
Department of Biomedical Sciences, University of North Dakota School of Medicine & Health Sciences, Grand Forks, N.D., USA

Many factors affect metabolism, including the composition of the diet and hormone status. These factors, in turn, impact processes that lead to declines in function and aging. Reduced growth hormone (GH) signaling extends health and lifespan in part by altering metabolism, maintaining enhanced insulin sensitivity and defense mechanisms. Growth hormone also integrates nutrient signals, modulating metabolic responses that result in age-related disease susceptibility. GH appears to regulate oxidative defense and the methionine metabolic pathway via enzymes that affect S-adenosylmethionine, glutathione, DNA methylation, and detoxification activities as indicated in studies using model systems with low or elevated somatotropic signaling. Together, our work and that of others indicates that GH plays a significant role in an organism’s ability to respond to nutrients and cellular stressors by regulating factors that counter stress, modulating metabolic responsiveness to nutrients, and detoxification of endogenous and exogenous compounds.

She is a member of the Boards of FASEB and American Aging Association (AGE), and recently completed several years on the board of the Gerontological Society of America (GSA). She is a past-President of AGE and past-Chair of the Biological Sciences section of the GSA. She is a Fellow of the American Aging Association, GSA, and American Association for the Advancement of Science. She serves on the editorial boards of several journals and regularly reviews grant proposals for federal and foundation agencies that support aging research. She has organized and chaired several scientific meetings including AGE, GSA (Biological Sciences), Biology of Aging Gordon Research Conference, a Keystone Conference on aging and currently organizes the International Symposium on Neurobiology and Neuroendocrinology of Aging held in Bregenz, Austria biennially.

Nath Nichenametla received his Ph.D. in Integrative Biosciences from Penn State University (Hershey, Pa.) and DVM from Sri Venkateswara Veterinary University (Tirupati, India). He has a longstanding interest in understanding the effects of diet on health and disease susceptibility. Since 2013 he has primarily focused on deciphering the mechanisms by which sulfur amino acid restriction (SAAR) extends lifespan in laboratory models and aims to leverage these mechanisms to improve human health. A significant portion of his research portfolio includes preclinical studies that span nutritional biochemistry, aging, and metabolic diseases. To investigate the relevance of his preclinical findings, he conducts epidemiological and human dietary interventions. His recent work at the Orentreich Foundation for the Advancement of Science demonstrates that methionine restriction and cysteine restriction are two distinct components of SAAR and that they induce specific pathways in rodents. Through these studies, he identified serinogenesis as a potential targetable mechanism to ameliorate lipid metabolism.

Novel amino acid-related dietary interventions that improve the healthspan of mice

Jason D. Plummer¹, Mark C. Horowitz², Jay E. Johnson^1
1Orentreich Foundation for the Advancement of Science, Cold Spring, N.Y., USA
²Yale University School of Medicine, New Haven, Conn., USA

Continuous methionine restriction (MR) is one of only a few dietary manipulations known to dramatically extend mammalian healthspan. For example, continuously methionine-restricted rodents show less age-related pathology and are up to 45% longer-lived than controls. Despite the fact that a methionine-restricted diet is technically feasible for humans, long-term adherence to such a regimen is likely to be challenging (or even undesirable) for many individuals. Consequently, our lab has sought to develop more facile dietary interventions that may provide MR-like healthspan benefits.

We previously reported that an intermittent version of MR (IMR) confers to mice nearly identical metabolic health benefits as the continuous intervention. Not only does IMR have a much shorter interventional period than continuous MR (i.e., only 3 days per week of reduced methionine intake), but it also produces additional health benefits. In the current study, we demonstrate several such benefits, including 1) that mice undergoing IMR show a preservation of bone mass, as well as 2) a dramatic reduction in the accumulation of marrow fat. Additionally, we find that 3) the bones of intermittently methionine-restricted mice are significantly stronger than those of their continuously methionine-restricted counterparts.  Interestingly, our analyses suggest that IMR likely results in a higher bone density than that observed for continuously methionine-restricted mice by increasing the number of bone-producing osteoblasts.

In a complementary approach, we also attempted to identify compounds that produce MR-like healthspan benefits, but in a normal, methionine-replete context.  Here, we show that supplementation with any one of four different amino acids is sufficient to produce the same metabolic health benefits typically observed for MR. Specifically, animals supplemented with these amino acids are completely protected against diet-induced obesity and demonstrate similar beneficial plasma hormone changes as continuously methionine-restricted mice. Furthermore, subsequent experiments suggest that elevated levels of these amino acids produce a signal of underfeeding, which in turn beneficially alters central carbon metabolism.

In total, our findings reveal multiple novel amino acid-related dietary interventions that improve the healthspan of mice, but are considerably more practicable (and less deleterious) than continuous MR. Should future studies find that these interventions also produce MR-like benefits for humans, then they would represent attractive alternatives to maintaining a continuously methionine-restricted diet.

New insights into the translational potential of rapamycin to target biological aging

Matt Kaeberlein
Optispan, Inc. and University of Washington, Seattle, Wash., USA

The mechanistic Target of Rapamycin (mTOR) is an evolutionarily conserved regulator of longevity that plays a central role linking environmental cues to aging biology. The mTOR inhibitor rapamycin is currently the most effective and reproducible pharmacological approach to extending lifespan in animals. Several groups have independently shown that short-term treatment with rapamycin in mice can prevent age-related decline or rejuvenate functional measures of health in various organs and tissues, including brain, heart, kidney, muscle, oral cavity, immune system, and ovary. Clinical trials have been initiated with the goal to determine whether rapamycin can positively impact age-related endpoints in humans and companion dogs, and several hundred “biohackers” are proactively using rapamycin off-label in hopes that it will increase healthspan and lifespan.

Dietary sulfur amino acid restriction in men and women with overweight and obesity: Evidence from a double-blind, randomized controlled dietary intervention study

Thomas Olsen¹, Emma Stolt¹, Bente Øvrebø², Amany Elshorbagy^3,4, Helga Refsum^1,4, Sindre Lee-Ødegård⁵, Kristýna Barvíková⁶, Marleen van Greevenbroek⁷, Tamàs Ditrói⁸, Peter Nagy⁸, Viktor Kožich⁶, Kjetil Retterstøl^1,9, Kathrine J. Vinknes^1
1Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
²Department of Food Safety, Norwegian Institute of Public Health, Oslo, Norway
³Department of Physiology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt
⁴Department of Pharmacology, University of Oxford, UK
⁵Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
⁶Department of Pediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic
⁷Department of Internal Medicine and CARIM School of Cardiovascular Diseases, Maastricht University, Maastricht, The Netherlands
⁸Department of Molecular Immunology and Toxicology and the National Tumor Biology Laboratory, National Institute of Oncology, Budapest, Hungary
⁹The Lipid Clinic, Department of Endocrinology, Morbid Obesity and Preventive Medicine, Oslo University Hospital, Oslo, Norway

Dietary restriction of the sulfur amino acids methionine and cysteine (sulfur amino acid restriction, SAAR) promotes longevity and metabolic health in experimental animal models. Among the demonstrated metabolic benefits in such studies are decreased body weight and improved body composition, increased energy expenditure, improved insulin sensitivity, and an improved plasma risk profile. Animal studies demonstrated that such effects are in part due to altered transcriptomic responses in adipose tissue and may further involve alterations in downstream metabolism of methionine and cysteine. In an 8-week double-blind dietary intervention study, we randomized men (n = 16) and women (n = 43) with overweight or obesity to receive SAAR (SAA content 2g/d, ~19 mg/kg body weight/d) or a control diet (SAA content (5.6 g/d, ~60 mg/kg body weight/d). The base diets in both groups were vegan and based on a whole-food approach. Sulfur amino acid contents were regulated with capsules, ensuring that both investigators and participants were blinded to the intervention allocation. All foods were provided in full to the participants on a weekly basis with recipes and meal plans. Eight weeks of SAAR led to ~20% greater weight loss compared to controls (p = 0.013), with no significant between-group differences in resting energy expenditure. SAAR further led to increased circulating ketone bodies (all p < 0.03) and decreased serum leptin concentrations (p = 0.03). Differential gene expression analyses showed that the SAAR group had decreased expression of genes involved in lipogenesis and fat storage in subcutaneous white adipose tissue compared to controls (FDR < 0.05). Marked reductions in intermediates and end-products of sulfur amino acid metabolism such as plasma and urinary cystathionine (‒50% and ‒5% vs. controls, respectively), plasma H₂S (‒25% vs. controls), and urinary sulfate (‒75% vs. controls) were also observed (all p < 0.01). No significant differences between groups were observed for fasting glucose and insulin, plasma lipids, or body fat compartments. In conclusion, some benefits of SAAR initially demonstrated in animals can be translated to humans using a whole-food based approach. Underlying mechanisms may involve altered adipose tissue lipid metabolism and changes to sulfur amino acid metabolism, but future studies are needed to address such potential mechanisms in greater detail.

He now works as a post-doc (Department of Nutrition, University of Oslo, Norway) in an international consortium focusing on establishing the relevance of sulfur amino acids and intermediates of their metabolism in human metabolic health. The project utilizes state-of-the art methodology and data from an epidemiological cohort study and a double-blind dietary intervention study with SAAR to achieve this objective.