The regulation of healthspan and lifespan by dietary amino acids

Abstract

As a key macronutrient and source of essential macromolecules, dietary protein plays a significant role in health. For many years, protein-rich diets have been recommended as healthy due to the satiety-inducing and muscle-building effects of protein, as well as the ability of protein calories to displace allegedly unhealthy calories from fats and carbohydrates. However, clinical studies find that consumption of dietary protein is associated with an increased risk of multiple diseases, especially diabetes, while studies in rodents have demonstrated that protein restriction can promote metabolic health and even lifespan. Emerging evidence suggests that the effects of dietary protein on health and longevity are not mediated simply by protein quantity but are instead mediated by protein quality – the specific amino acid composition of the diet. Here, we discuss how dietary protein and specific amino acids including methionine, the branched chain amino acids (leucine, isoleucine, and valine), tryptophan and glycine regulate metabolic health, healthspan, and aging, with attention to the specific molecular mechanisms that may participate in these effects. Finally, we discuss the potential applicability of these findings to promoting healthy aging in humans.

Introduction

We live in a rapidly graying society beset with an epidemic of obesity; over 70% of adults in US, and 30% (2.1 billion) of the world population are overweight or obese. Obesity is becoming increasingly prevalent for those over 65, and is a key risk factor for the development of diabetes, which affects 29.1 million Americans, with health care costs of over $176 billion per year in the US alone (1, 2). Obesity and diabetes are risk factors for other serious diseases of aging, including cardiovascular diseases (CVD), cancer, and Alzheimer’s disease, amplifying the impact of obesity considerably (3–6).

While traditional dieting – essentially, reducing calorie intake – can reverse obesity, adherence to a low calorie diet is unsustainable for most. The three major macronutrients, compounds we ingest that are used as fuel, are carbohydrates, fats, and protein. While the prevailing view has long been that calories from one of these sources is equivalent to calories from another, recent studies have demonstrated that a calorie is “not just a calorie” – and that dietary macronutrients have metabolic impacts beyond their simple caloric value (7–9). Diets that alter the level of specific macronutrients, but which do not restrict calories, may therefore be able to promote metabolic health and leanness in a more sustainable way than traditional dieting (10).

Dietary protein is composed of amino acids, twenty of which – those directly encoded by the genome – are considered common. Amino acids are essential building blocks for proteins; but in addition to this function, amino acids also have roles as signaling molecules, and can be catabolized for use as fuel or as building blocks for a range of other macromolecules. Here, we summarize the current knowledge on how the level of protein in the diet, as well as protein quality – the precise amino acid composition of the protein – impacts both healthspan and longevity, with an emphasis on the role of specific dietary amino acids.

Dietary protein regulates the health and longevity of animals

Evidence for the effect of dietary protein on lifespan dates to the 1920’s, when McCay et al. found that trout fed a low protein diet lived longer (11). Following this finding, a series of systematic studies examined the effect of diets containing varying amounts of protein (10%−26% of calories from protein) on growth and lifespan in rats, concluding that lower protein diets reduce growth but extend lifespan (12). In a later study, Sprague Dawley rats fed a 7.8% protein diet were shown to live longer than ones fed on a 20.8% protein diet (13). Despite these intriguing findings, interest in protein restriction (PR) was limited by a number of studies that found dietary PR or supplementation did not affect rat lifespan (14–16). Based on what we know now, the variation in these results were likely due to the highly variable protein sources and quantities used, but the confusion engendered delayed serious consideration of the effects of dietary protein on aging.

Interest in dietary protein as a regulator of longevity re-emerged in this century, stimulated in part by studies in Drosophila which found that the level of dietary protein regulates lifespan (17). Subsequent research using a nutritional geometry approach to study how multiple different diets influence fitness and lifespan found that the ratio of protein to carbohydrate in the diet strongly influenced both the longevity and fecundity of flies. The lifespan of flies was maximized at a very low ratio (1:16) of dietary protein to carbohydrate (18). A similar nutritional geometry approach was undertaken in mice; mice were fed 25 different diets with varying ratios of dietary macronutrients as well as energy density. In agreement with the results found in flies, mice fed a low protein diet, and in particular those animals fed a low protein: high carbohydrate diet, lived longer (19).

Numerous beneficial effects of protein restricted (PR) diets on health in various target organs have now been identified in rodents (Fig. 1). PR has strong effects on the metabolic health of mice and rats, promoting glucose tolerance, insulin sensitivity, and energy expenditure (20–23). A low protein diet prevents age related declines in motor coordination and cognition in female mice (24). In a mouse model of Alzheimer’s disease (AD), periodic protein restriction reduced cognitive deficits as well as phosphorylation of the tau protein (25). PR also decreases the production of reactive oxygen species by mitochondria, and reduced oxidative damage to lipids and endogenous DNA in the livers of rats (26); and in rodent cancer xenograft models, PR inhibits tumor growth (27).

Figure 1: Benefecial effects of protein restricted (PR) diet on health in various target organs.

As depicted in this figure, a low protein diet exerts its beneficial effects on metabolic status through actions in multiple tissues, including liver, skeletal muscle and adipose tissue. PR has also been shown to have cardioprotective effects and has positive influence on memory and cognition function. Figure created with Biorender (https://biorender.com).

Dietary protein is negatively associated with metabolic health and lifespan of humans

A number of popular diet plans are based on the widely-held idea that high protein, low carbohydrate diets promote weight loss (28). The efficacy of high protein diets in clinical studies have been mixed, with weight loss observed in some trials (29–31), particularly in highly compliant subjects (32). At least part of the success of high protein diets comes through promoting satiety (33), but one study suggested that high protein diets may also increase thermogenesis, thereby driving weight loss through increased energy expenditure (34). Dietary protein supplementation has also been pursued in the elderly as a means of treating or preventing sarcopenia (35, 36). A limitation to these studies is that they were generally short-term.

In stark contrast to these short-term results, long-term retrospective and prospective cohort several studies have found that high protein consumption is associated with increased insulin resistance, diabetes, cancer, and overall mortality (37, 38). A retrospective analysis of data from The National Health and Nutrition Examination Survey (NHANES III), found that dietary protein consumption was also correlated with mortality in individuals under the age of 65 (38). High protein, low carbohydrate diets were associated with cardiovascular mortality as well as overall mortality in a cohort of over 40,000 Swedish women followed for over a decade (39). In line with these results, a recent population-based study from Finland showed that higher protein intake was associated with an increased risk of heart failure in middle aged men (40) and an overall increase in mortality among those with a history of cancer, CVD, or diabetes (41).

Supporting the epidemiological link between dietary protein consumption and the risk of developing diabetes, a recent short-term randomized clinical trial of PR found that reducing dietary protein reduced weight, fat mass, fasting blood glucose levels, and lowered plasma triglycerides in overweight middle-aged males (21, 42). PR also alters biomarkers associated with insulin and leptin signaling in plasma extracellular vesicles (43). However, no long-term clinical trial of PR has yet been undertaken.

Molecular mechanisms by which dietary protein impacts health and longevity

While the precise physiological and molecular mechanisms by which PR promotes metabolic health and longevity are unknown, there are several molecular pathways which likely play important roles in this response (Fig 2). Here we briefly discuss some of the key pathways that likely mediate the beneficial effects of a PR diet, and on metabolic health and lifespan.

Figure 2: Overview of nutrient signaling pathways altered by methionine restriction (MR).

The benefical effects of MR are likely mediated through multiple metabolic pathways and molecular mechanisms. These include the integrated stress response pathway (PERK/GCN2-ATF4-FGF21), decreased GH/IGF-1 signaling, and decreased mTORC1 signaling as a result of decreased IGF-1 signaling, the activation of GNMT and reduced levels of the methionine metabolite SAM. The suppression of mTORC1 activity leads to increased autophagy and decreased protein translation, which act to promote health and longevity. Abbreviations: eIF2α -eukaryotic transcription factor 2α, FGF21 -fibroblast growth factor 21, GH/IGF-1- Growth hormone/insulin-like growth factor 1, mTORC1- mechanistic target of rapamycin complex 1, UCP1 - uncoupling protein 1, PERK- protein kinase R (PKR)-like endoplasmic reticulum kinase, GCN2 -general control nonderepressible 2, GNMT- glycine‐N‐methyl transferase, ATF4- Activating Transcription Factor 4, SAM-S-Adenosyl methionine, AKT- Protein kinase B, Foxo- forkhead box transcription factors.

mTOR

The mechanistic target of Rapamycin (mTOR) is a serine/threonine protein kinase which regulates and coordinates numerous cellular processes by integrating nutrient sensing and growth factor signals. Highly conserved in eukaryotic cells as a major growth regulator, mTOR exerts its function through two different protein complexes mTOR complex 1 (mTORC1) and complex 2 (mTORC2) which are composed of distinct protein subunits and phosphorylate different substrates. The defining components of mTORC1 are mTOR itself, Raptor (regulatory protein associated with mTOR), and mLST8 (mammalian lethal with Sec13 protein 8); while the defining components of mTORC2 are mTOR, Rictor, mLST8, and mSin1; but both complexes have been shown to associate with numerous other proteins in a variety of cell types (reviewed in (44)).

mTORC1 activity is directly regulated by the availability of nutrients, most notably amino acids but also including glucose and cholesterol and requires that growth factor signaling must be permissive for growth. Pharmacological or genetic inhibition of mTORC1 extends life span in numerous diverse species, ranging from yeast to mice (45–52). In contrast to mTORC1, which integrates information about many different environmental and hormonal cues, mTORC2 primarily acts as an effector of insulin/PI3K signaling. While mTORC2 regulates lifespan in worms, flies, and mice (53–59), it does not appear to be key in the response to dietary protein, and thus we will not discuss it in detail.

Over the last decade, major advances have been made in understanding the regulation of mTORC1 by amino acids, which occurs at the lysosomal surface; as this has been thoroughly reviewed elsewhere (60), we will only touch upon it briefly. Amino acid sensing by mTORC1 is controlled by the Rag family of GTPases, which recruits mTORC1 to the lysosomal surface in the presence of amino acids (61). Amino acids regulate the GTP/GDP-bound status of the Rag GTPases through controlling the activity of the Ragulator complex, which has guanine nucleotide exchange factor (GEF) activity for two of the Rag proteins, RagA and RagB; the GATOR1 complex, which has GTPase-activating protein (GAP) activity and is controlled in turn by the GATOR2 complex; and a folliculin (FLCN) and folliculin-interacting proteins 1 and 2 (FNIP1/2) complex that acts as a GAP for Rag C and Rag D heterodimers (61–65). Sensing of specific amino acids occurs via specific sensors, including Sestrin2, CASTOR1, and SAMTOR, which modulate the activity of GATOR 1/2 upon binding to leucine, arginine, and SAM, respectively (66–69). Additional sensing of amino acid availability is mediated by Ragulator via SLC38A9, a lysosomal arginine sensor, as well as a mechanism that requires the vacuolar ATPase (70–72).

After localizing to the lysosome, mTORC1 needs to interact with Rheb-GTP, which binds to mTORC1 allosterically and realigns active-site residues (73). In the absence of insulin/IGF-1 or other growth factor signaling, Rheb is found bound to GDP due to the action of the tuberous sclerosis complex (TSC), which acts as a GAP for Rheb (74). In the presence of insulin/IGF-1 signaling, Akt phosphorylates TSC which leads to its departure from the lysosome, allowing Rheb-GTP to bind to and activate mTORC1 (75).

As amino acids, which function as the building blocks of protein, are known to stimulate mTORC1 activity, it is logical to assume that a low protein diet results in reduced mTORC1 activity. Drosophila fed a high sugar low protein diet have decreased TOR signaling (76). In mice, as the protein: carbohydrate ratio decreased, there was a decrease in hepatic mTOR activation (19). Similarly, tumor-bearing mice fed a PR diet have decreased mTORC1 activity relative to ad libitum fed controls in multiple somatic tissues (77), as well as in mouse models of obesity (78), and mTORC1 is repressed by in a mouse model of ischemia reperfusion injury (79). Given the profound beneficial effects of reduced mTORC1 signaling on healthspan and longevity, decreased mTORC1 activity likely contributes to or mediates the ability of PR to promote health and longevity.

Gcn2-ATF4-FGF21

A second major evolutionarily conserved amino acid sensitive kinase is general control nonderepressible 2 (GCN2), one of four kinases that can activate the integrated stress response pathway (ISR). GCN2 is canonically activated by binding to uncharged transfer ribonucleic acids (tRNAs); following activation, GCN2 phosphorylates eukaryotic initiation factor 2-α (eIF2α), leading to the inhibition of protein translation (80). More recently, it has been recognized that ribosome stalling can also lead to GCN2 activation independently of an increase in uncharged tRNAs; this may be one of the principle mechanism of GCN2 activation under many physiological conditions (81, 82).

Phosphorylation of eIF2α leads to a global decrease in translation, but increases translation of specific stress-responsive transcripts. One of the best characterized of these and a key effector of the ISR is activating transcription factor 4 (ATF4) (83–86). ATF4 is a basic leucine zipper (bZIP) transcription factor that plays a key role in both basal metabolism and stress response, binding to amino acid response elements (AARE), primarily activating as a transcriptional activator, and upregulating transcription of genes involved in amino acid uptake and biosynthesis among other stress response genes (reviewed in (87)). Interestingly, ATF4 is a key factor in coordinating GCN2 and mTORC1 activity; ATF4 induces expression of Sestrin2, and indeed GCN2 and phosphorylation of eIF2α are required to prevent mTORC1 activation by depletion of specific amino acids (88, 89).

Another key gene regulated by ATF4 is fibroblast growth factor 21 (FGF21), a peptide hormone which plays an important role in adaptive responses to starvation (90). FGF21 can be produced by multiple tissues, including the liver, muscle, white adipose tissue, and pancreas (91, 92). FGF21 can be induced by a variety of stresses including PR, which increases levels of FGF21 in both rodents and humans (21, 22). FGF21 is believed to be a critical regulator of many of the effects of a PR diet, as experiments using mice lacking Fgf21 have found that FGF21 is required for PR-mediated changes in food intake, energy expenditure, body weight, and glucose tolerance (21, 22, 93, 94). Loss of Gcn2 does not entirely block the ability of a PR diet to induce Fgf21 transcription, but it does delay it by about 2 weeks (95), highlighting a key role for GCN2 in the response to PR.

In mice, FGF21 induces hepatic insulin sensitivity, in part via inhibition of mTORC1 activity (96). However, the most robust mechanism by which FGF21 promotes metabolic health is by activating UCP1 in white and brown adipose tissue, promoting energy expenditure as well as food intake (93). This physiological response involves signaling through the brain to the adipose tissue, as mice lacking a brain FGF21 receptor are unable to respond to low protein diets (94). Thus, it seems likely that the GCN2-FGF21-UCP1 axis is a key mediator in the response to low protein diets; and as transgenic expression of FGF21 increases the lifespan of mice (97), this hormone may also play a role in the ability of a low protein diet to extend lifespan.

Protein quality impacts health and longevity

For many years, there has been interest in understanding if protein source plays a role in health, with the greatest focus on understanding if there is a difference between the effect of plant protein and animal protein. Several studies have suggested that plant-based protein is healthier. One study found that consumption of a plant-based vegan diet decreased all-cause mortality, coronary heart disease and a decrease in risk of developing obesity in humans (98); a more recent study showed that a plant based diet significantly lowered the incidence of cardiovascular disease (CVD), CVD mortality, and all-cause mortality in a cohort of middle-aged adults (99). Vegan diets have also been implicated in reducing the risk of developing metabolic syndrome, lowering triglycerides, blood pressure, glucose, waist circumference and body mass index (100), and decreasing fat mass and insulin resistance (101).

One possibility that has been advanced to explain the beneficial effects of plant protein is that there is a difference in protein quality – the specific amino acid composition of the protein. Plant-based diets have a reduced level of methionine as compared to animal sources, and humans consuming a vegan diet have reduced plasma levels of methionine compared to humans who eat animal proteins (102, 103). As discussed below, significant data now suggests that the level of methionine – as well as of several other dietary amino acids – has a profound effect on health and longevity, not only in rodents, but also in humans. An overview of recent studies is provided in Table 1 and Table 2).

Table 1:

Summary of recent studies examining the effects of amino acid restriction or supplementation on the lifespan of rodents. Level of intake is the percent of the altered amino acids(s) in the experimental diet relative to the control diet. M -Male, F-Female, BCAAs = branched-chain amino acids (leucine, isoleucine, and valine).

Species/Strain/Sex	Altered Amino acid	Lifespan	Level of intake relative to control diet	Study
Methionine
Mice CB6F1 (F)	↓ Met	Increased	23–35%	Miller et al., 2005 (104)
Mice CB6F1 (M)	↓ Met	Increased	7%	Sun et al., 2009 (105)
Rats Fisher 344 (M)	↓ Met	Increased	20%	Orentreich et al., 1993 (106)
Rats Fisher 344 (M)	↓ Met	Increased	20%	Richie et al., 1994 (107)
Sprague Dawley Brown Norway Wistar	↓ Met	Increased	20%	Zimmerman et al., 2003 (108)
Tryptophan
Swiss Albino Mice (M)	↓ Trp	Increased	17%	(De Marte et al., 1986 (109)
Long Evans rats (F)	↓ Trp	Increased	30% or 40%	Ooka et al., 1988 (110)
Branched-chain amino acids
Mice C57BL/6J	↑ BCAAs	Decreased	200%	Solon-Biet et al., 2019 (111)
Mice C57BL/6J	↓ BCAAs	Increased (M only)	33%	Richardson et al., 2021 (112)
Glycine
Rats Fisher 344 (M)	↑ Gly	Increased	347% or 522%	Brind et al., 2011 (113)
Mice UM‐HET3	↑ Gly	Increased	772%	Miller et al., 2019 (114)

Table 2: Effect of altered dietary levels of methionine or the branched-chain amino acids on the metabolic health of rodents and humans.

M-Male, DIO – Diet Induced Obesity, T2D-Type 2 Diabetes, BCAAs - branched chain amino acids, FGF21-fibroblast growth factor 21, WAT-white adipose tissue. Table is adapted from Green et al., 2019 (128) with permission.

Species/Strain/Sex	Altered amino acid	Level of intake relative to control diet	Metabolic health	Length of intervention	Study
Methionine
Mice CB6F1 (F)	Met	23–35%	Decreased circulating IGF-1, insulin and glucose Increased resistance to liver stress	Lifespan study	Miller et al., 2005 (104)
Mice C57BL/6J (M)	Met	20%	Increased food intake Reduced body weight Improved hepatic insulin sensitivity Decreased hepatic lipogenic gene expression	8 weeks	Lees et al., 2014 (115)
Mice C57BL/6J (M)	Met	20%	Improved insulin sensitivity Reduced hepatic glucose production Increased FGF21	8 weeks	Stone et al., 2014 (116)
Mice C57BL/6J (M)	Met	15%	Increased food intake Increased energy expenditure Reduced accumulation of body weight and fat mass	10 weeks	Wanders et al., 2017 (117)
Mice C57BL/6 J (M)	Met	20%	Increased food intake Reduced body and fat mass Improved glycemic control Decreased fasting blood glucose and insulin Increased lipid cycling in WAT Decreased hepatic lipogenic gene expression Elevated FGF21	8 weeks	Lees et al., 2017 (118)
Mice C57BL/6J (M/F and M/F DIO)	Met	0%	Increased food intake Reduced weight and adiposity Improved glycemic control Increased energy expenditure Elevated FGF21 (males)	5 weeks	Yu et al., 2018 (119)
Mice C57BL/6J	Met	20%	Reduced Body Weight and Adiposity Increased food intake Increased expression of thermogenic markers Improved insulin sensitivity Decreased circulating lipid levels	7 weeks	Forney et al., 2020 (120)
Rats Fisher 344 (M)	Met	20%	Decreased Body weight Reduced visceral fat Decreased serum lipds and IGF-1	Lifespan study	Malloy et al., 2006 (121)
Humans (O)	Met	6%	Increased fat oxidation Decreased hepatic lipid metabolism	16 weeks	Plaisance et al., 2011 (122)
Branched-chain amino acids
Mice C57BL/6J (M)	Leu or Ile or Val	0%	Improved insulin sensitivity Improved glucose tolerance Increased hepatic insulin sensitivity (Leu) Decreased fasting blood glucose (Ile or Val)	1 or 7 days	Xiao et al., 2011 (123) and Xiao et al., 2014 (124)
Mice C57BL/6J (M)	Leu	33%	Increased adiposity	13 weeks	Fontana et al., 2016 (21)
Mice C57BL/6J (M)	BCAAs	33%	Increased food intake Reduced accumulation of body weight and fat mass Improved glycemic control Decreased fasting blood glucose	13 weeks	Fontana et al., 2016 (21)
Mice C57BL/6 J (M)	Leu	20%	Increased food intake Decreased body and fat mass Improved glycemic control Decreased fasting insulin Increased lipid cycling in WAT	8 weeks	Lees et al., 2017 (118)
Mice C57BL/6J (M, DIO)	BCAAs	33%	Rapid weight and fat mass loss Improved glycemic control Increased energy expenditure Transient increase in fasting FGF21	14 weeks	Cummings et al., 2018 (20)
Mice C57BL/6J	BCAAs	20%, 50%, or 200%	Increased food intake (200%) Decreased weight and fat mass Decreased leptin Decreased liver triglycerides Decreased fasting insulin (20%)	Lifespan study	Solon-Biet et al., 2019 (111)
Mice C57BL/6J	BCAAs	33%	Increased food intake Reduced weight and adiposity Improved glycemic control Increased energy expenditure	Lifespan study	Richardson et al., 2021 (112)
Mice C57BL/6J (M)	Ile	33%	Increased food intake Decreased weight and adiposity Improved glucose tolerance Increased hepatic insulin sensitivity Decreased fasting blood glucose Increased energy expenditure Elevated FGF21	12 weeks	Yu and Richardson et al., 2021 (125)
Rats Zucker fatty	BCAAs	55%	Improved skeletal muscle glucose disposal and insulin sensitivity	15 weeks	White et al., 2016 (126)
Humans (T2D)	BCAAs	40%	Decreased insulin secretion Induced FGF21 production, Improved oral glucose sensitivity Improves white adipose tissue metabolism	4 weeks	Karusheva et al., 2019 (127)

Methionine

Orentreich and colleagues first tested the hypothesis that a methionine restricted (MR) diet could extend lifespan in the 1990’s using Fischer 344 rats, finding that MR extended lifespan by about 30% (106). This effect was not strain specific, with a MR diet also able to extend the lifespan of Brown Norway, Sprague Dawley and Wistar rats (108). Subsequent research has demonstrated that restricting dietary methionine can extend the lifespan of many diverse species, including yeast, flies, and mice (104, 129–134).

In addition to these beneficial effects on lifespan, MR has many beneficial effects on metabolic health. Rodents fed a MR diet are leaner, with reduced adiposity; have improvements in glucose homeostasis with lower blood glucose and insulin levels and improved glucose tolerance and insulin sensitivity, and have decreased serum and hepatic triglyceride levels (121, 135–137). These metabolic benefits have led to the idea that MR diets might be effective in the treatment of diabetes and obesity, and indeed MR and methionine depleted dietary regimens promote weight loss and reduced adiposity in obese mice, decreases or reverses liver lipid accumulation, and normalizes glucose homeostasis (119, 138, 139). Beneficial metabolic effects, in particular increased fat oxidation, have also been seen in humans during a clinical trial of MR (122).

MR promotes metabolic health and longevity via multiple pathways and molecular mechanisms (Fig 2). As highlighted in Fig 2., decreased GH/IGF-1 signaling can suppress mTORC1 activity by activating glycine-N-methyl transferase (GNMT) (140, 141), which reduces levels of the key methionine metabolite S-Adenosyl methionine (SAM), which normally activates mTORC1 via binding to SAMTOR (68). In yeast, MR extends chronological lifespan through an autophagy/mitophagy dependent pathway that involves alterations in central carbon metabolism (133, 142). Rodents fed a MR diet have reduced levels of IGF-1, a key effector of the growth hormone signaling pathway that well-characterized as a regulator of lifespan (104, 143). Dietary supplementation with the amino acid selenium, which similarly reduces IGF-1 levels, mimics many of the healthspan benefits of a MR diet, protecting mice against weight gain and fat accumulation, and protecting against diet-induced obesity (144). Many of the metabolic effects of a MR diet are likely mediated by FGF21, which is induced by a MR diet (115). While it was initially believed that MR acted to induce FGF21 via the GCN2-ATF4-FGF21 axis discussed above, it was recently shown that GCN2 is dispensable for the effects of MR (145). Instead, the induction of FGF21 by MR is dependent upon the kinase PKR-like endoplasmic reticulum kinase (PERK), which is activated by MR as a result of oxidative stress, and similarly phosphorylates eIF2α and induces translation of ATF4 (Fig 2.). However, some of the metabolic effects of MR may not require a global increase in FGF21 levels, as female C57BL/6J mice have a robust metabolic response to a methionine depleted diet without an increase in FGF21 levels (119).

The induction of oxidative stress in the liver of MR-treated mice is due to the depletion of the anti-oxidant glutathione, which is generated from methionine via cysteine (145). MR may have many other effects that are mediated indirectly via its metabolites. In addition to the effects of the methionine metabolite SAM on mTORC1 mentioned above, SAM is also a key metabolite for methyltransferases; MR therefore has profound effects on histone methylation and gene expression (146). Finally, the transulfuration pathway has been implicated in longevity and stress resistance, and both PR and MR promote the generation of hydrogen sulfide (H₂S), a key longevity regulator, in multiple species (147–150).

Branched-chain amino acids (BCAAs)

The three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, are so named because they have an aliphatic side-chain with a branched carbon structure. It has long been realized that these amino acids may play an important role in health and metabolism; in 1969, it was observed that the BCAAs are elevated in the blood of obese humans (151). Over the last 15 years, it has become apparent that plasma levels of BCAAs are correlated with obesity and insulin resistance in both humans and rodents (152–154).

Conversely, numerous interventions that reduce obesity and improve metabolic health in humans, including calorie restriction, protein restriction, and gastric bypass surgery, lower plasma levels of BCAAs (21, 155, 156). Branched-chain amino acids are essential, and increased consumption of BCAAs correlates with both plasma BCAAs and the incidence of type 2 diabetes in humans (157, 158). Rodent studies have demonstrated that dietary intake of BCAAs directly regulate metabolic health. Supplementation of BCAAs to a Western diet promotes adiposity and insulin resistance in both mice and rats (20, 153).

Conversely, short-term complete deprivation of any single BCAA promotes hepatic insulin sensitivity (123, 124). As complete deprivation of a BCAA is not physiologically relevant, several groups have recently investigated the results of simply reducing dietary levels of BCAAs. We have shown that reducing dietary levels of BCAAs by 67% improves the metabolic health of lean mice, and rapidly restores metabolic health to diet-induced obese mice, dramatically reducing adiposity and glucose tolerance (20, 21). Similarly, reduced levels of dietary BCAAs slows the accumulation of visceral adipose tissue and preserves the insulin sensitivity of Zucker fatty rats (126).

Overall, these studies demonstrate that BCAAs directly regulate metabolic health, and that reducing dietary levels of the BCAAs may be a strategy to rapidly promote healthy body composition and blood glucose control in overweight or obese humans. A recent randomized controlled study has found that short-term dietary restriction of BCAAs decreases insulin secretion, induces FGF21, improves oral glucose sensitivity index, and improves white adipose tissue metabolism in humans with type 2 diabetes (127). The longer-term effects of reducing dietary BCAAs in humans with and without metabolic syndrome remains to be determined.

The potent metabolic benefits of reduced BCAA consumption strongly suggested that reducing dietary levels of BCAAs might promote mammalian healthspan and even increase lifespan. Such effects could be mediated in part by reducing mTORC1 signaling, as the BCAAs, particularly leucine, are strong agonists of mTORC1 and genetic and pharmacological interventions that inhibit mTORC1 signaling extend mammalian lifespan and healthspan (46, 159–162). In agreement with this hypothesis, circulating levels of the BCAA are associated with hepatic mTOR activity and negatively associated with lifespan (19).

Consistent with a negative effect of BCAAs on longevity, dietary supplementation with extra BCAAs leads not only to impaired metabolic health, but to decreased lifespan (20, 111, 153, 163). Solon-Biet and colleagues did not observe increased or decreased longevity on mice fed a 50% or 80% restricted BCAA diet from 12 weeks of age (111). Similarly, Richardson and colleagues found that 67% restriction of BCAAs improved metabolic health and reduced frailty of male and female mice without increasing lifespan when started at 16 months of age (112). However, lifelong restriction of BCAAs reduced frailty and extended the lifespan of male, but not female mice by over 30%, reducing mTORC1 signaling in multiple tissues specifically in males (112). It is clear that the precise level of restriction, time of diet initiation, and sex may play a role in determining if BCAA restriction will extend lifespan, but both studies support a model in which reduced dietary consumption of BCAAs promote healthspan.

BCAAs have been linked to multiple diseases of aging, including Alzheimer’s disease; and defects in BCAA catabolism induced by diabetes may drive the pathogenesis of Alzheimer’s disease by increasing brain levels of the BCAAs and activating mTORC1 (164). Dietary supplementation of BCAAs has been shown to increase neuropathology and decrease the survival of a mouse model of Alzheimer’s disease (165). BCAAs are also suggested to be critically important in cancer; there is an increased uptake and catabolism of BCAAs by some types of cancer that drives cancer progression (166, 167). Conversely, defective BCAA catabolism leading to an accumulation of BCAAs and mTORC1 hyperactivation have been shown to be important in other cancer types, and dietary BCAA intake has been shown to be correlated with cancer risk (168).

However, not all experiments in model organisms clearly support a model in which reducing dietary BCAAs improves healthspan and extends longevity. In the budding yeast S. cerevisiae, supplementation of leucine actually promotes chronological lifespan during CR (169). In C. elegans, supplementation with BCAAs or impaired expression of a major BCAA catabolic enzyme, branched-chain amino acid transferase 1 (BCAT-1) resulted in extended lifespan and healthy aging without affecting fecundity (170). In contrast, dietary restriction of BCAAs in D. melanogaster improves stress resistance, ameliorates age-related pathologies, and extends lifespan (171); this is likely mediated in part via the regulation of mTORC1 activity by the single fly Sestrin orthologue (172). In contrast to the results described above, consumption of an essential amino acid supplement with extra BCAAs is reported to extend the longevity of male mice (173, 174). However, the BCAA supplement and diet used in these experiments actually alter the levels of many essential and non-essential dietary amino acids, including methionine, making the role of the BCAAs in this response unclear.

In contrast to the generally deleterious effects of life-long consumption of high levels of the BCAAs, short-term supplementation with BCAAs has shown promise in preclinical and clinical studies in the aged. BCAA supplementation along with low resistance exercise increased muscle strength and physical function in sarcopenic older adults (175). A clinical prospective study on blood metabolites revealed an inverse relation between BCAA levels, dementia and AD. Reduced levels of BCAAs were associated with higher risk of AD (176). BCAA supplementation enhanced cognitive recovery in patients with severe traumatic brain injury (177).Supplementation of BCAAs has also been shown to extend the survival time, reduce complications, and reduce the recurrence rate of patients treated for hepatocellular carcinoma (178, 179)

While one potential explanation for the conflicting results of different studies may be age or disease status, another explanation for the conflicting results of different studies may be the fact that the BCAAs have usually been considered as a group due to their structural similarity and shared catabolic pathways. However, there is emerging evidence that the individual BCAAs have unique effects on signaling and metabolism. Leucine, for example, is a strong activator of mTORC1 in most cell types, binding to Sestrin2 and activating mTORC1 by promoting the recruitment of mTORC1 to the lysosomal surface via a Rag-GTPase dependent pathway (67, 69, 180–182). This crucial signaling pathway leads to the activation of many downstream pathways that regulate metabolism and aging (44). In contrast, isoleucine and valine do not strongly interact with Sestrin2 and may be less potent activators of mTORC1.

Distinct roles for the individual BCAAs have also been observed in vivo. We have observed thicker dermal white adipose tissue and heavier epididymal fat pads in mice fed a diet in which the levels of leucine were specifically reduced (21). An intermediate valine catabolite, 3-hydroxy-isobutyrate (3-HIB), is secreted from muscle cells and can activate trans-endothelial fatty acid transport, thereby causing lipid accumulation and insulin resistance (183). Finally, in a recent study we demonstrated that each of the BCAAs has distinct metabolic effects in mice, with restriction of dietary isoleucine being necessary and sufficient for the metabolic benefits of a low protein diet. As highlighted in Fig. 3, these effects were independent of hepatic mTORC1 and GCN2 signaling, and mediated in part through FGF21, which was strongly induced by isoleucine restriction and not by restriction of leucine or valine alone (125). In humans, we found that dietary levels of isoleucine are positively associated with BMI (125); and another recent study found that blood levels of isoleucine are correlated with increased mortality in humans (184). In contrast, blood levels of leucine and valine were associated with decreased human mortality (184).

Figure 3: Overview of major signaling pathways altered by restriction or supplementation of amino acids.

Restriction of methionine, tryptophan or the BCAAs extends lifespan and improves metabolic health. Restriction of methionine, tryptophan, or isoleucine, but not restriction of leucine or valine, induces FGF21 and increases energy expenditure through the FGF21-UCP1 axis. All three BCAAs, Trp, and methionine (via SAM) stimulate mTORC1 activity, and restriction reduces mTORC1 activity. Glycine supplementation promotes longevity through its regulation of methionine and SAM levels. Abbreviations:Trp-tryptophan, Ile-Isoleucine, Val-valine, Leu-leucine, Met-methionine, Gly-glycine, FGF21 -fibroblast growth factor 21, mTORC1- mechanistic target of rapamycin complex 1, UCP1 - uncoupling protein 1, SAM-S-Adenosyl methionine

Tryptophan

The essential amino acid tryptophan and its metabolites have been widely studied as key regulators of metabolic health. Segall and Timiras were the first to report that a tryptophan deficient diet (185) could delay aging in rats, finding that a chronic deficiency of tryptophan delayed pathological signs of aging, including the onset of tumors, and increased lifespan (186). Although this small study was not carried to completion, follow up work demonstrated that tryptophan restriction reduced age-related pathologies and extended the lifespan of both rats (110) and mice (109). Similar beneficial responses have been shown in yeast, worms, and flies, where ibuprofen has been shown to extend longevity by destabilizing tryptophan permeases and reducing tryptophan uptake (187).

The physiological and molecular mechanisms by which tryptophan restriction promotes health and longevity are still under investigation. It was recently shown that tryptophan restriction in the context of a casein-based diet induces FGF21, promoting metabolic health (Fig. 3) (188). The major catabolic pathway of tryptophan is the kynurenine pathway, which produces metabolites like kynurenic acid and nicotinamide adenine dinucleotide (NAD⁺) (189). This pathway is in fact the sole de novo biosynthetic pathway for NAD⁺, which has been implicated in many metabolic processes, including mitochondrial function, and NAD+ supplementation has been shown to be beneficial in many diseases of aging (reviewed in (185)). Numerous studies have also investigated links between tryptophan metabolism, immune cell function, and inflammation (190–193). Finally, a recent study found that the serum level of tryptophan was associated with onset of diabetes (194).

As with protein and the BCAAs, while some studies suggest that tryptophan restriction is beneficial, several studies have found that instead dietary supplementation with tryptophan may be beneficial for aging and specific age-related diseases. Tryptophan supplementation extends the lifespan of C. elegans (195), while low serum tryptophan levels and alterations in tryptophan catabolism are associated with reduced life expectancy in people with coronary artery disease (196, 197). Dietary supplementation of tryptophan suppresses blood glucose level and thereby delays diabetes progression in diabetic rats (198).

Tryptophan is a well-known precursor of serotonin, which has been implicated in many neurodegenerative disorders. Tryptophan-rich diets can prevent age-associated cognitive decline in rats (199), and in humans there is a negative correlation between tryptophan levels and cognitive functioning during aging (200). Humans with Alzheimer’s disease have been shown to have increased tryptophan catabolism and altered kynurenine levels, which may be linked to impaired cognitive function (200). However, tryptophan does not simply have beneficial effects on the aging brain; a study in young adults recently showed that consumption of a tryptophan-rich diet has a positive effect on anxiety and depression (201).

Glycine

Multiple studies have demonstrated that dietary supplementation of glycine can increase longevity and promote metabolic health. Dietary glycine increases lifespan in C. elegans, suppressing many genes involved in aging processes (202). This effect requires methionine synthase and S-adenosyl methionine synthetase, suggesting that glycine promotes longevity through alterations in methionine metabolism (Fig. 3). Dietary glycine regulates the action of GNMT, an enzyme that removes the methyl group from methionine (203, 204).

Glycine supplementation also increases the lifespan of rodents, including a small study conducted in Fisher 344 rats (113), and a large study conducted in UM-HET3 mice (114) by the NIA Interventions Testing Program. Although the overall effect of glycine supplementation on longevity was small, numerous rodent and human studies have found benefits from glycine supplementation. Glycine supplementation in aged mice enhanced activation of T cells as well as mitochondrial biogenesis (205), and has anti-inflammatory properties via inhibition of pro-inflammatory cytokines (206). Glycine intake also promotes metabolic health, reducing the accumulation of abdominal fat, plasma triglyceride levels and blood pressure induced by a high sucrose diet in rats (207).

In humans, glycine supplementation has been shown to be protective against chronic inflammation, oxidative stress and immune responses caused by type 2 diabetes (208). Cell culture studies suggest that glycine rescues age-related mitochondrial defects in human cells (209). Glycine is essential for the proliferation of muscle progenitor cells in cell culture and conditionally essential in vivo in mice (210), and humans with a genetic variant that raises blood levels of glycine have a reduced risk of cardiovascular disease (211). In humans, blood levels of glycine are positively associated with insulin sensitivity (212), and higher blood levels of glycine are associated with a reduced risk of diabetes (213).

Conclusions

Promoting healthy aging is becoming of increasing importance around the world as the population grays. Many people, particularly the elderly, are also threatened by the increasing prevalence of obesity and diabetes, which are both deleterious in themselves and increase the risk of developing many other diseases of aging. Here, we have discussed how instead of cutting calories, changing the composition of the diet – in particular, altering the levels of dietary protein or of specific dietary amino acids – may be a translatable and sustainable method to promote healthy aging.

An emerging consensus of animal and human data suggests that, contrary to long-held popular beliefs, lower protein consumption is more beneficial for health and longevity than high protein consumption. Recent human studies have found that lower protein intake is correlated with improved metabolic health as well as increased longevity, while a high protein intake correlates with an increased risk of diabetes and cardiovascular disease. This is not to say that there may not be risks of reducing protein consumption; in older people, lower protein intake has been associated with frailty and sarcopenia, and increased protein intake has been suggested as an intervention to preserve muscle mass in this population (214–216). Long-term clinical trials of PR will be critical to determining if PR can promote healthy aging and longevity in humans, as well as the time periods when PR may be beneficial and identifying any portions of the life where PR may be detrimental.

It is now becoming clear that dietary protein quality – the specific amino acid composition of the dietary protein – has a profound effect on metabolic health and longevity in mammals. In this review, we have discussed current knowledge on how dietary amino acids can affect metabolic health and longevity. These include studies demonstrating that restriction of methionine, BCAAs or tryptophan can improve healthspan and lifespan in rodents. While only a few human studies on these types of diets have been conducted, preliminary evidence suggests that restriction of methionine or BCAAs may also have metabolic benefits in humans. Data from rodents already suggests that the age of initiation of methionine or BCAA restriction, as well as the degree of restriction, will influence the ultimate effect of these diets (111, 112, 217). As with PR, long-term clinical trials will be critical to determining if restriction of specific dietary amino acids – and which ones, and when – can promote healthy aging in humans.

As discussed here, multiple molecular mechanism – including mTORC1, eIF2α, ATF4, and FGF21 – engaged by PR or restriction of specific amino acids may contribute to the beneficial effects of protein and amino acids restricted diets. While long-term adherence to amino acid restricted diets in humans is likely to be low, an expanding range of chemicals to modulate signaling through these pathways is being developed (218–223). It therefore seems likely that as we achieve a deeper understanding of the shared and distinctive molecular mechanisms engaged by restriction of different amino acids, pharmaceuticals can be developed that mimic the benefits of these diets to promote health and longevity.