Translational Medicine of Aging 3 (2019) 70e89

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Translational Medicine of Aging

journal homepage: www.keaipublishing.com/TMA

Amino acids in the regulation of aging and aging-related diseases

Clare-Ann Canfield a, Patrick C. Bradshaw b, *
a
Department of Biomedical Sciences, Keiser University Tampa, Tampa, FL, USA
b
Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA

a r t i c l e i n f o a b s t r a c t

Article history: Amino acids are the building blocks of protein, but also play important cellular signaling roles. The
Received 8 July 2019 mechanisms through which altered levels of many amino acids are sensed and the signals transmitted
Received in revised form are still largely unknown. Increasing evidence is showing that these signals may influence the aging
30 August 2019
process. In this regard, methionine restriction appears to be an evolutionary conserved mechanism to
Accepted 3 September 2019
Available online 4 September 2019
delay aging and supplementation with glycine can mimic methionine restriction to extend lifespan in
rodents. Tryptophan restriction may also activate specific anti-aging pathways, but it could interfere with
cognitive function. With exercise the consumption of dietary branched chain amino acids (BCAAs) may
Keywords:
Amino acids
be beneficial in building muscle mass, but high levels of BCAAs as well as tyrosine and phenylalanine in
Aging the bloodstream are associated with metabolic disease such as insulin resistance. Individual supple-
Lifespan mentation or restriction of several different amino acids has shown promise in the treatment of insulin
Yeast resistance in rodents. Much progress regarding the effects of amino acids on longevity has been made
C. elegans using yeast, nematodes, and fruit flies. Clearly, much more research is needed to understand the
signaling pathways activated by amino acid imbalance before efficacious and well-tolerated dietary in-
terventions can be developed for human aging and aging-related disorders. In this review the mecha-
nisms through which altered dietary and cellular levels of the twenty proteogenic amino acids affect
aging or aging-related disorders are discussed.
© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communication Co., Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

1. Introduction
There are 20 proteogenic amino acids used as the building
blocks of protein synthesis. Plasma amino acid imbalance can result
from liver or kidney disease and may lead to some of the pheno-
types of those diseases [1]. Roughly 40 years ago amino acid solu-
tions were formulated to treat such imbalances [1]. However, an
imbalance of amino acids can also lead to the activation of stress
response pathways and lifespan extension in model organisms
[2e4]. Dietary intake of large amounts of a single amino acid can be
toxic [5,6]. Single amino acids have been administered to humans
without severe side effects in the 3e40 g per dose range with
cysteine and methionine being the most toxic [1]. However, rodents
showed a slowed rate of growth when large amounts of a single
amino acid were given with a low protein diet [1]. Rodents can
sense the lack of a single amino acid in their food using the anterior
* Corresponding author.
E-mail addresses: cedwardscanfield@keiseruniversity.edu (C.-A.
bradshawp@etsu.edu (P.C. Bradshaw).
piriform cortex region of the brain and will become anorexic on
such a diet [1].
There are mechanisms present to detect uncharged tRNAs and
activate the eukaryotic initiation factor 2a (eIF2a) kinase general
amino acid control non-derepressible 2 (GCN2) to signal the
decreased level of a specific amino acid so the cell can decrease the
rate of protein synthesis accordingly [7]. The other major pathway
for sensing the level of single amino acids and adjusting the rate of
protein synthesis is the target of rapamycin (TOR) pathway [8].
Modulation of either of these pathways in C. elegans can extend
lifespan in part due to increases in proteostasis [9], as decreased
protein synthesis was found to correlate with increased lifespan
under several experimental conditions [10,11]. But other mecha-
nisms such as activation of the PHA-4/FoxA transcriptional regu-
lator [12] and the prevention of aging-induced mitochondrial
fission [13] to maintain energy levels [14] also likely play an
important role in lifespan regulation by TOR and GCN2. Since
rapamycin, a TOR inhibitor, can extend the lifespan of mice [15],
these pathways are also important targets for the regulation of
Canfield), mammalian longevity.

https://doi.org/10.1016/j.tma.2019.09.001
2468-5011/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communication Co., Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89
When amino acid intake exceeds the requirements for protein
synthesis they are deaminated and catabolized into pyruvate,
acetyl-CoA, or mitochondrial citric acid cycle intermediates for
energy generation [16]. In the nematode C. elegans, individual
supplementation at an optimal concentration with the vast ma-
jority of amino acids or citric acid cycle intermediates extended
lifespan [3,17]. Higher concentrations frequently decreased lifespan
suggesting a hormetic dose response. Supplementation with
peptone, a balanced source of amino acids decreased lifespan when
C. elegans was cultured in liquid media [3,18], but not when
cultured on agar plates [19], suggesting that the effects of high
amino acid levels depend upon the culture conditions.
Like mammals, C. elegans evolved to utilize several D-amino
acids as signaling molecules and can metabolize a subset of these D-
amino acids for energy generation [20,21]. Individual supplemen-
tation to C. elegans with each of the three D-amino acids, D-alanine,
D-aspartate, or D-glutamate, which are metabolized by the worms
and present in the E. coli that the C. elegans standardly consume as
the major portion of their diet [21], also extended lifespan [3], while
supplementation with D-proline or D-serine, which are not readily
metabolized by the four characterized C. elegans D-amino acid
oxidizing enzymes [20,21], did not extend lifespan [3]. This data
suggests metabolism as an important component of the longevity
effects of supplemented amino acids. However, other data suggest
that metabolism is not always required for amino acid-mediated
longevity in C. elegans as increasing the intracellular levels of
tryptophan [22] or branched chain amino acids (BCAAs) (isoleu-
cine, leucine, and valine) [23] by preventing their catabolism
increased lifespan. So, clearly different signaling pathways are
activated by different amino acids in the regulation of lifespan.
Whether or not individual amino acid supplementation at an
optimal dose can influence the lifespan of other multicellular or-
ganisms such as fruit flies, rodents, or humans is mostly unknown.
But recently it was shown that glycine supplementation increased
the lifespan of mice [24] and the molecular mechanism is likely
similar to that induced by methionine restriction [25].
Calorie restriction (CR) or dietary restriction (DR) is an evolu-
tionary conserved mechanism to delay aging resulting in lifespan
extension. In fruit flies the lifespan extending effects of DR have
been shown to be regulated by the protein to carbohydrate ratio.
Adding back essential amino acids to dietary restricted flies pre-
vented lifespan extension [4]. This area has been extensively
reviewed [26] and will not be covered here in detail. Decreased
levels of single amino acids in the culture media also extended
replicative [27] or chronological (Table 1) lifespan in yeast. Repli-
cative aging in yeast appears to be due to a loss of vacuole acidifi-
cation that leads to the vacuolar release of neutral amino acids that
reduce mitochondrial membrane potential. This aging-induced loss
of vacuolar acidification is delayed by CR [28]. One large study using
a Geometric Framework of Nutrition approach in mice also impli-
cated decreased protein levels as having an important role in the
longevity effects of CR [29]. But these results could be due to the
unique method (using cellulose to fill the void of restricted carbo-
hydrate, lipid, or protein food mass) of implementing the restricted
diets, as many older studies using rodents have shown that protein
restriction is not responsible for the large (~30%) increase in me-
dian lifespan of the typical 30%e40% CR diet [30]. But protein re-
striction of 50%e85% can also increase lifespan up to roughly 15%,
apparently independently of the effects of CR. Many beneficial
disease-delaying effects of protein restriction have been found [31].
Since amino acids signal through distinct pathways to modulate
lifespan in model organisms, consuming an optimized ratio of
amino acids in the diet may be a viable approach for delaying aging
in humans as well. Along these lines, research using fruit flies and
mice has shown that consuming a diet where the amino acid
71
content matches the in silico translated codon usage of the
expressed genome (the exome) enhances the rate of growth and
development without affecting lifespan [32]. Therefore, the levels
of amino acids in the diet may also one day be optimized for other
purposes including increased stress resistance or a decreased rate
of aging. This could be particularly useful for the treatment of
diabetes where the plasma levels of all three BCAAs and phenyl-
alanine and tyrosine positively associate with the disease [33]. This
may somehow be related to the finding that the level of these same
five amino acids plus glutamate increased in brain when rats were
fed diets of altered protein content, while the levels of other amino
acids changed little to none [34]. More research is needed on the
ability of amino acid supplementation to alter levels of free amino
acids in mammalian brain regions.
When examining the fibroblasts taken from 15 primate species,
33 bird species, and 13 rodent species, free levels of 11 of the 20
amino acids including arginine, glutamate, histidine, leucine,
lysine, methionine, phenylalanine, proline, tryptophan, tyrosine,
and valine were found to have a positive association with species
lifespan [35]. Individual supplementation with 18 of the 20 amino
acids extended lifespan in C. elegans (Table 1). These data suggest
that free amino acids have an important role in the regulation of
aging. Interestingly 8 of the 20 amino acids, including 5 of the 11
listed above, were able to protect Caco-2 intestinal epithelial cells
from oxidative stress including alanine, cysteine, histidine, isoleu-
cine, leucine, lysine, tryptophan, and valine [36]. Five of the amino
acids where the abundance associates with longevity that did not
show an antioxidant effect in Caco-2 cells including arginine [37],
glutamate [38], methionine [39,40], proline [41,42], and tyrosine
[43,44] have shown antioxidant effects using in vitro assays or have
been shown to stimulate the expression of antioxidant enzymes in
other cell types or tissues. These data suggest that decreased
oxidative stress may play a role in the positive association between
free amino acid levels and longevity. In the sections below we
outline the current state of knowledge regarding how altered levels
of each of the 20 proteogenic amino acids affect aging, aging-
related diseases, and the associated signaling pathways.
2. Amino acids
2.1. Alanine
Alanine is a non-essential amino acid and also one of 13
exclusively glucogenic amino acids [45]. Following its uptake and
entry into the hepatic circulation, alanine is taken up by the liver,
where much of the alanine pool is converted to pyruvate, which
enters into gluconeogenesis. In a fasted state, skeletal muscle re-
leases large amounts of alanine and glutamine into the circulation
where they are taken up by the liver and kidney and used for
gluconeogenesis [46]. Increased alanine consumption may be
beneficial for metabolic disorders such as type 2 diabetes as its
supplementation to a cultured pancreatic beta cell line increased
both non-oxidative and oxidative glucose metabolism and the
secretion of insulin [47]. Consistent with this, alanine infusion into
hyperglycemic dogs increased insulin secretion without much
change in glucagon levels, but in the fasting state when glucose
levels were low, alanine infusion stimulated glucagon secretion,
while having little effect on insulin levels [48]. In mice, dietary
alanine was shown to prevent obesity due to a high fat diet [49].
Alanine supplementation has also been shown to stimulate the
proliferation of lymphocytes [50] and thymocytes [51], which may
decrease the aging-related loss of immune system function. A diet
containing 10% alanine administered to rats was also able to pre-
vent the formation or dissolve preexisting kidney stones [52].
Alanine levels have been shown to decline with aging in mouse
72 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89

Table 1
Changes in lifespan caused by increased or decreased levels of specific amino acids.

Amino acid C. elegans % change in mean lifespan [3] Yeasta Fruit flies Rodents

1 mM in media 5 mM in media 10 mM in media Incr. amino acid Decr. Incr. amino acid Decr. amino acid Incr. amino acid Decr. amino acid
Amino acid

Ala þ8 ns þ11 Y [56] ↑[56]

Arg þ8 þ11 þ15 [[83]
Asn ns þ5 25 [[93]
Asp ns ns 6 [[106]
Cys þ9 þ16 ns
Gln þ15 þ16 6 [[93]
Glu þ14 þ11 8 [[106] [[93]
Gly þ10 ns ns [[24]
His ns þ9 þ12 ns[200]
Ile ns ns þ3 [[209] ns[273]
Leu þ16 þ6 þ7 [[209] [[238]
Lys þ7 þ8 þ6 [[93] ns[273]
Met ns þ8 þ14 [[273] [[4] [[245]
[[106]
Phe ns 8 12
Pro þ17 þ19 þ18
Ser þ8 þ18 þ22 [[321]
[[322]
Thr ns ns þ8 [[209] ns[273]
[[322]
Trp þ14 þ6 ns [[356] ns[4] [[337]
[[379]
Tyr þ10 þ5 þ2
Val þ13 þ8 ns [[209] ns[273][[322]

ns ¼ not significantly different than the control. A plus sign (þ) or up arrow ([) indicates increased lifespan, while a minus sign () or down arrow (Y) refers to decreased
lifespan. Incr. ¼ Increased and Decr. ¼ Decreased.
a
Data indicates yeast chronological lifespan, except for that for arginine and tryptophan, where it indicates replicative lifespan.

plasma [53] and muscle [54].
Alanine, leucine, and glutamine levels were shown to decline in
aged parasitoid wasps [55]. In yeast, deletion of the Alt1 alanine
transaminase gene increased alanine levels and decreased chro-
nological lifespan, while longevity extending CR decreased alanine
levels [56]. In C. elegans supplementation with 1 mM or 10 mM
alanine extended lifespan [3], while supplementation with 5 mM
did not [3,23] (Table 1). The metabolism of alanine to pyruvate may
play a role in the extended longevity of nematodes as pyruvate
supplementation also extended lifespan [57].
2.2. Arginine
Arginine is a semi-essential amino acid and the anti-aging ef-
fects of arginine supplementation have been reviewed [58]. Argi-
nine catabolism is altered in learning and memory centers in the
aged brain [59]. Arginine has been shown to stimulate the prolif-
eration of young human T lymphocytes to aid in wound healing
[60,61], but it did not stimulate the proliferation of young or aged
rat lymphocytes [62]. Increased arginine levels resulted in a switch
from glycolysis to oxidative phosphorylation in activated human T
lymphocytes and an upregulated anti-tumor response directly
associated with their enhanced survival [63]. Arginine was shown
to reverse the aging-related loss of acetylcholine-induced vasodi-
lation of coronary endothelial cells in humans [64] and to improve
depressed macrophage and wound immune function following
hemorrhage in rats [65,66]. Long term arginine supplementation
prevented the aging-related decrease in renal function [67]. The
protective effects of arginine may be due in part to its metabolism
to polyamines that are known to have anti-aging effects [68].
Consistent with this mechanism, anti-aging DR has been shown to
increase the levels of polyamine synthesis enzymes through a post-
transcriptional mechanism [69].
Arginine is used for nitric oxide synthesis [70] and decreased
nitric oxide synthesis contributes to the aging of the cardiovascular
system [71]. Arginine supplementation has been shown to increase
the secretion of insulin-like growth factor-1 (IGF-1) [72] and
growth hormone [73], both of which have been shown to have pro-
aging functions in mice [74,75]. Arginine is hydrolyzed by the
enzyme arginase to form ornithine and urea in the liver during urea
cycle function [76] and in the liver and other tissues for polyamine
synthesis from ornithine. The role of arginase in aging has been
reviewed [77].
Arginine stimulates mechanistic target of rapamycin (mTORC1)
kinase activity by two different mechanisms [78,79]. First cyto-
plasmic arginine binds the CASTOR1 (cytosolic arginine sensor for
mTORC1 subunit 1) protein to stimulate mTORC1. Second the
SLC38A9 lysosomal amino acid transporter (that transports leucine,
phenylalanine, isoleucine, tryptophan, methionine, valine, and
tyrosine [80]) interacts with the Ragulator protein complex that
signals to mTORC1 dependent upon high (likely lysosomal) argi-
nine levels [81]. The activation of mTORC1 leads to increased pro-
tein synthesis and inhibition of autophagy that may exert a pro-
aging effect in many cellular contexts.
Increased arginine levels were found in aged Drosophila [82]. In
yeast deletion of the arginine permease CAN1 extended replicative
lifespan depending upon the transcriptional regulator GCN4 and
translational activation of stress response genes [83]. Arginine
supplementation potently increased the lifespan of C. elegans
(Table 1) [3]. Although C. elegans lacks a CASTOR1 homolog, its
genome does contain homologs to the lysosomal amino acid
transporter SLC38A9 and the Ragulator complex protein Lamtor2
that may play a role in signaling cellular arginine levels.
2.3. Asparagine
Asparagine is a non-essential amino acid except when gluta-
mine levels are very low [84]. Asparagine synthetase catalyzes the
cytoplasmic synthesis of asparagine from aspartate and ammonia.
Older subjects showing physical frailty and sarcopenia were shown
 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89
to possess increased plasma levels of asparagine, aspartate, and
glutamate [85]. Asparagine plays a role in the proliferation of
cancer cells via activation of mTOR during tumor development [86].
Consistent with this role, a diet low in asparagine has been shown
to be protective against breast cancer metastasis [87]. Asparagine
and glutamine metabolism play essential roles in blood vessel
formation [88] and tumor growth [89]. Asparagine and glutamine
also play anti-inflammatory roles to protect intestinal integrity and
stimulate intestinal epithelial cell division to protect against lipo-
polysaccharide (LPS) treatment [90]. Treatment with asparagine
and aspartate together decreased fatigue and increased time to
exhaustion during moderate or intense exercise [91,92]. In yeast
removal of asparagine from the media extended the chronological
lifespan by inhibiting TOR kinase [93]. The asparagine catabolizing
enzyme asparaginase was shown to be selectively retained in yeast
mother cells following the budding of daughter cells [94], largely
explaining the decrease in asparagine levels in replicatively aged
yeast [95]. Asparagine supplementation only slightly extended
mean lifespan in C. elegans at a 5 mM dose and decreased lifespan
to the greatest extent of any amino acid at a 10 mM dose (Table 1)
[3].
2.4. Aspartate
The non-essential amino acid, aspartate, participates in a range
of important cellular functions such as the urea cycle and the
malate-aspartate shuttle, which transports nicotinamide adenine
dinucleotide (NADH) reducing equivalents between the cytoplasm
and mitochondrial matrix. The shuttle plays an essential role in CR-
mediated longevity in yeast by increasing the nucleocytoplasmic
NADþ/NADH ratio to activate sirtuin deacetylases [96]. Aspartate is
synthesized from oxaloacetate by aspartate aminotransferase.
Mitochondrial electron transport chain function declines with ag-
ing [97] and is required for aspartate and pyrimidine synthesis [98].
Metals such as Mn2þ, Cu2þ, Zn2þ, and Mg2þ bind to aspartate to
form complexes with antioxidant function [99]. Aspartate (as well
as proline and serine) supplementation was effective at decreasing
reactive oxygen species (ROS) production and increasing ATP levels
in M17 neuroblastoma cells expressing alpha-synuclein treated
with ferrous sulfate [100]. Aspartate or glutamate was effective in
promoting blood antioxidant levels when male pigs [101] or
weaned piglets [38] were challenged with hydrogen peroxide.
However, glutamate, but not aspartate was effective at reducing
diquat-mediated oxidative stress in piglets [102]. Aspartate sup-
plementation also decreased ethanol-induced hepatotoxicity and
oxidative stress in the testes of rats [103].
Within the brain, aspartate acts as an agonist of N-methyl-D-
aspartate (NMDA) receptors to regulate neurotransmission [104].
Therefore increased amounts of aspartate may contribute to aging-
related cognitive impairment by facilitating excitotoxicity [105].
Restriction of aspartate was shown to increase the chronological
lifespan of yeast [106], likely by inhibiting TOR kinase activity [107].
However, the replicative lifespan of many yeast gene deletion
strains positively correlated with increased levels of aspartate,
methionine, proline, threonine, isoleucine, histidine, and glutamine
[108]. So some amino acids, such as aspartate, may show opposite
effects on the chronological and replicative lifespan of yeast.
Aspartate was one of two amino acids that did not extend lifespan
when supplemented in the 1e10 mM range to C. elegans (Table 1)
[3].
2.5. Cysteine
Cysteine is a non-essential amino acid. Total cysteine (including
reduced cysteine and oxidized cystine) and the cystine to cysteine
73
ratio in human plasma were shown to increase with aging [109],
while the levels of some amino acids were shown to decline [110].
The plasma cystine/cysteine oxidation state is an important
determinant of systemic inflammation [111], which likely contrib-
utes to aging [112]. A reduced extracellular cystine/cysteine
oxidation state was also shown to restore free mitochondrial NADH
levels in isolated neurons from aged mice and the 3xTg-AD Alz-
heimer's disease mouse model [113]. Mitochondrial cysteine
oxidation may play a role in the aging process as long-lived animals
have evolved to encode less cysteines in the genomes of their
mitochondria, where ROS production is the highest [114]. Cysteine
supplementation has shown promise as an adjuvant for type 2
diabetes [115]. When rats were fed a low protein diet cysteine
supplementation was shown to decrease the plasma levels of ho-
mocysteine [116], a risk factor for neurodegenerative and cardio-
vascular diseases [117]. However, high levels of plasma cysteine
have been associated with many disorders including coronary heart
disease, Alzheimer's and Parkinson's diseases [118], rheumatoid
arthritis [119], and systemic lupus erythematosus [120]. Systems
biology experiments in yeast identified that high cysteine levels
inhibit protein synthesis to cause toxicity and that supplementa-
tion with leucine and pyruvate prevented this toxicity. The pro-
tective effect of leucine relied upon its transamination to alpha-
ketoisocaproate (KIC), which depended upon the function of the
tRNA methyltransferase NCL1 [121].
The antioxidant glutathione is composed of cysteine, glutamate,
and glycine [122]. The oxidized glutathione disulfide (GSSG) to
reduced glutathione (GSH) ratio increases with aging, but is not in
strict equilibrium with the cystine/cysteine ratio [123]. When GSH
is consumed orally by humans it is hydrolyzed in the intestine by g-
glutamyltransferase and plasma GSH was not altered [124]. Sup-
plementing with cysteine and glycine together has shown more
promise and led to enhanced plasma GSH levels in aged subjects
[125]. This elevation corresponded with a marked decrease in
oxidative damage. N-acetyl-L-cysteine (NAC) is a membrane
permeable prodrug form of cysteine that releases cysteine through
the action of intracellular esterases. NAC supplementation has been
shown to extend the lifespan of mice [126], fruit flies [127], and
nematodes [128,129]. But the effects were gender-specific and not
concentration dependent suggesting hormesis [127]. The negative
effects at higher doses may also suggest reductive stress [130].
Studies using NAC have been reviewed [131]. Consistent with the
effects of NAC, cysteine supplementation potently increased life-
span in C. elegans at two lower doses, but not at the highest 10 mM
dose administered (Table 1) [3].
2.6. Glutamate
Glutamate is a non-essential amino acid most well-known for
its role as an excitatory neurotransmitter in the brain. Therefore,
glutamate plays an essential role in learning and cognition, but at
high levels glutamate induces neuronal excitotoxicity, contributing
to neuronal injury in neurodegenerative disorders including Alz-
heimer's disease (AD) [132] and amyotrophic lateral sclerosis (ALS)
[133]. Glutamate levels have been shown to decline in human brain
during young adulthood [134]. Glutamate is only very slightly
permeable through the blood brain barrier (BBB), so relatively high
levels of dietary glutamate are non-toxic in healthy subjects. But it
is not recommended to consume membrane permeable glutamate
esters that may permeate the BBB. Patients with neurodegenerative
disorders or who have had a stroke may also choose to limit their
glutamate consumption as their BBB may be more permeable [135]
and lower plasma glutamate levels may stimulate limited release of
glutamate from the brain to decrease excitotoxicity [136,137]. The
crucial balance in the brain between low extracellular glutamate
74 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89
concentrations and high intracellular glutamate concentrations is
kept in check by plasma membrane glutamate transporters. As-
trocytes take up glutamate, convert it to glutamine, and release the
glutamine to the extracellular space where it can be taken up by
neurons that convert it back to glutamate for release at synaptic
terminals [138].
One potential therapy for lowering systemic glutamate levels in
neurodegenerative disorders and stroke is the infusion of oxalo-
acetate and the aspartate transaminase enzyme [136,137]. How-
ever, glutamate plays important roles in the body such as binding
free ammonia, which is especially toxic to neurons [139], and it
serves as an important anaplerotic source of the anti-aging citric
acid cycle intermediate alpha-ketoglutarate [140]. Higher levels of
glutamate were found in the plasma of long-lived naked mole rats
compared to mice, while lower levels of many other amino acids
including glycine, methionine, leucine, tyrosine, phenylalanine, and
tryptophan were found [141]. Another group found glutamate,
alanine, aspartate, isoleucine, leucine, serine, and threonine levels
to be higher in the plasma of naked mole rats than mice [142]. One
study found that high glutamate levels increased the chronological
lifespan of yeast [106]. However, another group using different
experimental conditions found removal of glutamate from the
media to extend chronological lifespan [93]. In C. elegans studies,
glutamate was one of the most potent lifespan extending amino
acids when supplemented at the 1 and 5 mM doses, but it
decreased lifespan when supplemented at the 10 mM dose
(Table 1) [3]. Knockout of the glutamate transporter glt-4 decreased
nematode lifespan, while knockout of the glutamate transporter
glt-5 increased lifespan [143].
2.7. Glutamine
Glutamine is an essential amino acid and the most abundant
amino acid in the serum, CSF, and muscle. Its anti-aging roles have
been reviewed [144]. Glutamine is an important energy source,
especially in rapidly dividing cells such as epithelial cells and im-
mune cells, due to its ability to be readily broken down and feed
carbon into alpha-ketoglutarate in the citric acid cycle [145].
Increased glutamine levels can inhibit fatty acid oxidation, decrease
plasma glucose, and attenuate weight gain in mice prone to obesity
[146]. A mitochondrial glutamine transport activity has been
characterized, but its molecular identity is still unknown [147].
Following prolonged exercise by athletes supplemented with
glutamine, an increased T-helper/T-suppressor cell ratio was
measured, which corresponded with a decrease in the rate of
infection [148]. Glutamine regulates the expression of genes
involved with cellular proliferation and survival, as well as de-
creases the expression of pro-inflammatory genes such as NFҠb
[149]. Supplementation with glutamine increased the levels of anti-
inflammatory monocytes and regulatory T lymphocytes in the
serum of diabetic mice contributing to enhanced muscle regener-
ation following limb ischemia [150]. Alanine facilitates glutamine
metabolism. The combination of glutamine and alanine was shown
to reduce the glutathione couple (GSSG/GSH) [151] to increase
antioxidant function and had anti-inflammatory and cytoprotective
effects in rats undergoing resistance exercise [152]. Long-term
supplementation with glutamine was shown to be beneficial for
maintaining intestinal immunity in the elderly [153].
Glutamine synthetase is highly expressed in the mammalian
brain [154] where it may play a neuroprotective role in healthy
young animals [155]. Glutamine levels and glutamine synthetase
activity are decreased in Alzheimer's disease brain, but glutamine
synthetase was increased in a fraction of hippocampal neurons
where it can play a maladaptive, cytotoxic role when glutamine
levels are low [156]. Consistent with this, glutamine
supplementation was shown to protect against Alzheimer's
amyloid-beta toxicity in cultured neurons [157]. The role of amino
acids in Alzheimer's disease brain has been reviewed [158,159]. In
yeast inhibition of glutamine synthesis decreased glutamine levels
[160] and increased chronological longevity [93]. During chrono-
logical aging the intracellular levels of 16 of the 20 amino acids
were found to decrease with glutamine levels declining to the
greatest extent followed by glutamate, lysine, histine, methionine,
and cysteine [161]. Supplementation of glutamine to C. elegans had
an almost identical effect on lifespan as glutamate at all three doses
administered (Table 1) suggesting that the glutamine synthetase
and glutaminase enzymes readily interconvert these amino acids
when they are consumed in the diet [3].
2.8. Glycine
The amino acid glycine is considered conditionally essential as it
can be synthesized from serine, but not always at the levels needed.
The known effects of dietary glycine supplementation have been
reviewed [162]. As mentioned earlier glycine is the one amino acid
studied thus far in which supplementation has been shown to
extend the lifespan of mice, albeit moderately [24]. Dietary glycine
supplementation also extended the lifespan of Fisher 344 rats
through a mechanism mimicking methionine restriction to in-
crease the hepatic clearance of methionine [163]. Glycine supple-
mentation has been show to restore T cell activation, T cell one-
carbon metabolism, and mitochondrial function in aged mice
[164]. Administration of glycine was also shown to decrease
oxidative stress and inflammation by decreasing the levels of
advanced glycation endproducts (AGEs) by inducing the expression
of glyoxalase 1 [165].
By comparing young and old human fibroblasts it was found
that epigenetic downregulation of the glycine-C-acetyltransferase
(GCAT) and serine hydroxymethyltransferase 2 (SHMT2) genes
involved in mitochondrial glycine synthesis (see Fig. 1) are involved
in the aging-related loss of cellular respiration. Adding glycine to
the culture media restored the phenotype of the aged cells back to
that of the young cells [166]. Supplemental glycine can be trans-
ported across the mitochondrial inner membrane by the mito-
chondrial carrier family member SLC25A38 [167]. Another study
did not find much change in human whole body glycine meta-
bolism with aging, but found large changes following altered pro-
tein consumption [168]. Glycine is an inhibitory neurotransmitter
that plays especially important roles in the spinal cord, brainstem,
and retina [169]. Increased glycine levels can induce hypothermia
and sleep by binding NMDA receptors in the suprachiasmatic nu-
cleus of the brain [170]. Glycine supplementation protected the
colon wall during radiotherapy [171]. Glycine infusion led to
vasodilation in the rat kidney, but this effect was greatly reduced in
aged animals [172]. N-arachidonoyl glycine and other N-acyl amino
acids play important anti-inflammatory and analgesic functions
[173,174]. Therefore, therapies using these compounds could be
tested for improving inflammation in aging-related disorders.
Mild mitochondrial uncoupling is associated with increased
longevity in nematodes [175] and fruit flies [176]. Overexpression of
uncoupling protein 1 (UCP1) in skeletal muscle most greatly
induced the expression of genes involved in glycine, serine, and
asparagine synthesis, and one-carbon metabolism [177], while the
expression of pro-longevity cytoplasmic phosphoenolpyruvate
carboxykinase (PEPCKc) [178] was also induced. Overexpression of
glycine-N-methyltransferase (gnmt), which converts glycine and S-
adenosylmethionine to sarcosine and S-adenosylhomocysteine
extended lifespan in Drosophila and knockdown of gnmt prevented
DR-mediated longevity [179]. In mice plasma sarcosine levels
declined with age and increased with anti-aging CR [180].
 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89 75

Fig. 1. Serine, glycine, threonine, methionine, cysteine, and histidine metabolism influence one-carbon and NADPH redox metabolism. TDH and GLY1 enzymes are not present in
humans. Metabolites shown on the mitochondrial (inner) membrane can be transported into and out of the matrix space. Glycine catabolism by the mitochondrial glycine cleavage
system is not shown. This results in the conversion of THF to 5,10-meTHF for additional one-carbon metabolism and is accompanied by the reduction of NADþ to NADH. THF,
tetrahydrofolate; 5-meTHF, 5-methyltetrahydrofolate; 5,10-meTHF, 5,10-methylenetetrahydrofolate; coA, coenzyme A; NADþ, nicotinamide adenine dinucleotide (oxidized); NADPþ,
nicotinamide adenine dinucleotide phosphate (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADPH, nicotinamide adenine dinucleotide phosphate (reduced);
ATP, adenosine triphosphate; PPi, inorganic pyrophosphate; CO2, carbon dioxide; SHMT1, Serine Hydroxymethyltransferase 1; SHMT2, Serine Hydroxymethyltransferase 2;
MTHFD2L, Methylenetetrahydrofolate Dehydrogenase (NADPþ Dependent) 2 Like; MTHFD1L, Methylenetetrahydrofolate Dehydrogenase (NADPþ Dependent) 1 Like; MTHFD1,
Methylenetetrahydrofolate Dehydrogenase, Cyclohydrolase And Formyltetrahydrofolate Synthetase 1; TDH, Threonine Dehydrogenase; GLY1, Threonine Aldolase; GCAT, Glycine C-
Acetyltransferase; ALDH1L1, Aldehyde Dehydrogenase 1 Family Member L1; ALDH1L2, Aldehyde Dehydrogenase 1 Family Member L2; HAL, Histidine Ammonia-Lyase; UROC1,
Urocanate Hydratase 1; AMDHD1, Amidohydrolase Domain Containing 1; FTCD, Formimidoyltransferase Cyclodeaminase; MTHFR, Methylenetetrahydrofolate Reductase; MTR, 5-
Methyltetrahydrofolate-Homocysteine Methyltransferase; MAT1A, Methionine Adenosyltransferase 1A; MAT2A, Methionine Adenosyltransferase 2A; AHCY, Adenosylhomocys-
teinase; CBS, Cystathionine-Beta-Synthase; CTH, Cystathionine Gamma-Lyase; SDS, Serine Dehydratase; GCLC, Glutamate-Cysteine Ligase Catalytic Subunit; GSS, Glutathione
Synthetase; PHGDH, Phosphoglycerate Dehydrogenase; PSAT1, Phosphoserine Aminotransferase 1; PSPH, Phosphoserine Phosphatase; AGXT2, Alanine–Glyoxylate And Serine–
Pyruvate Aminotransferase 2; GRHPR, Glyoxylate And Hydroxypyruvate Reductase; GLYCTK, Glycerate Kinase.

Metabolomics studies of aging using C. elegans have yielded
fairly consistent data showing decreased levels of the vast majority
of amino acids with aging [181,182]. One group found glycine and
aspartate levels increased in C. elegans with aging, while the levels
of the other amino acids peaked in the late larval stages or first few
days of adulthood and declined thereafter [183]. Another group
found glycine and alanine as the only amino acids to increase in
abundance with aging [184], and a third group found glycine and
glutamine to be the only amino acids that increased in abundance
with aging (see Table 2) [184]. DR resulting from the reduced
pharyngeal pumping in eat-2 mutants prevented these increases in
glycine and glutamine as well as prevented the decreased levels of
arginine, valine, phenylalanine, and isoleucine, but not the
decreased levels of the ketogenic amino acids lysine and leucine
that occurred with aging [185]. Glycine and serine supplementation
have been shown to extend lifespan in C. elegans through stimu-
lating one carbon metabolism including the methionine cycle [186].
Glycine supplementation only extended lifespan at concentrations
of 1 mM and below (Table 1) [3,186]. Knockdown of mel-32, the
C. elegans homolog of serine hydroxymethyltransferase 1 and 2 (see
Fig. 1), increased glycine levels and extended lifespan by preventing
glycine conversion to serine and instead funneled glycine to the
glycine cleavage system for one-carbon metabolism including the
methionine cycle. Consistent with this, knockdown of one of the
four glycine cleavage system subunits, gcst-1, increased glycine
levels and decreased lifespan [186].
2.9. Histidine
Histidine is an essential amino acid that acts as an anti-glycating
agent [187], free radical scavenger [188], and metal chelator
[189,190]. The dipeptide carnosine contains histidine and beta-
alanine, is found at high levels in muscle and brain, and has
similar physiological properties as histidine. But carnosine was
shown to be less toxic in cell culture studies, so is a more promising
therapeutic [187]. Much data in model systems supports the po-
tential use of carnosine for the treatment of aging-related disorders
[191]. But we will focus on histidine as the anti-aging effects of
carnosine have been previously reviewed [192]. Histidine supple-
mentation increased insulin secretion and glycemic control,
increased glutathione peroxidase activity, and decreased pro-
inflammatory cytokine levels in diabetic mice [193]. Histidine
supplementation increased GSH levels and enhanced catalase ac-
tivity to decrease liver injury induced by chronic alcohol con-
sumption [194]. Individual administration of histidine, cysteine, or
glycine, but not alanine, inhibited NF-kB activation in cultured
coronary endothelial cells [195]. Fig. 1 shows that catabolism of
histidine, cysteine, and glycine influence one-carbon and redox
metabolism.
Histidine can scavenge singlet oxygen and hydroxyl radicals
[196]. Low plasma histidine levels have been associated with
inflammation, oxidative stress, and mortality in chronic kidney
disease patients [197]. A metabolomics study of human plasma
found histidine levels to decline with aging [198]. A metabolomics
study of 647 human subjects linked high histidine levels (and two
other amino acids) with longevity as defined by attaining at least 80
years [199]. In studies using Drosophila histidine supplementation
showed no effect on the mean lifespan, while an equivalent dose of
carnosine extended the lifespan of male flies [200]. However, his-
tidine administration increased the lifespan of C. elegans at the two
higher concentrations tested, but not at a lower 1 mM dose
(Table 1) [3]. The lifespan extension observed at the higher doses
may have been due to its activation of the pro-longevity hypoxia
inducible factor-1 (HIF-1) and SKN-1/Nrf2 stress response
76 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89

Table 2
Changes in endogenous amino acid levels with aging.

Amino acid C. elegans Yeasta Fruit flies Mice or rats Humans

Tissue Effect Tissue Effect

Ala Y [183] ns [95] muscle Y [54]

Y [185] plasma Y [53]
[ [184]
Arg Y [185] ns [95] [[82] plasma Y [380]
Asn Y [183] Y [95]
Asp [ [183] ns [95]
[ [182]
Cys
Gln [ [185] ns [95] muscle [ [54] plasma [ [359]
Y [183] brain [ [59]
Glu Y [183] Y [95]
Y [185]
Gly [ [183] Y [95] muscle Y [54]
[ [185]
[ [184]
Y [182]
His Y [95] plasma Y [198, 359]
Ile Y [183] Y [95] brain [ [205] plasma Y [202]
Y [185]
Y [184]
Leu Y [183] Y [95] plasma [ [381] plasma Y [359]
Y [185] plasma Y [202]
Lys Y [185] [ [95]
Met Y [183] Y [95] plasma Y [53]
Y [184]
Phe Y [183] Y [95] [[82] muscle [ [54]
Y [185]
Y [184]
Pro Y [183] ns [95] plasma Y [53]
Ser Y [183] ns [95] plasma Y [53] plasma Y [359]
plasma [ [380]
Thr Y [184] ns [95] plasma Y [205] plasma Y [359]
Trp Y [183] Y [95] plasma Y [198, 359]
Y [184]
Tyr Y [183] Y [95] plasma Y [53] plasma [ [359]
Y [185] brain Y [205] plasma [ [380]
Val Y [183] Y [95] plasma [ [381]
Y [185]
Y [184]

ns ¼ not significantly different than the control. An up arrow ([) refers to increased amino acid level with aging, while a down arrow (Y) refers to decreased amino acid level
with aging.
a
Data from yeast was taken from results of replicative lifespan experiments.

pathways. isoleucine levels in the culture media extended chronological

2.10. Isoleucine
Isoleucine, an essential amino acid that is both glucogenic and
ketogenic, is one of three branched-chain amino acids (BCAAs),
with leucine and valine being the other two [201]. A metabolomics
study showed that isoleucine and leucine levels decreased in the
blood of aged human subjects [202]. Mice undergoing fasting,
which is associated with anti-aging and anti-cancer properties
[203], showed increased plasma levels of all three BCAAs [204]. In a
metabolomics study of young and aged rat brains, isoleucine levels
increased, while tyrosine levels declined with aging [205]. Much
conflicting data has been obtained regarding the role of BCAAs on
metabolic health, insulin resistance, and glucose homeostasis
[206]. In one study using rats, oral isoleucine was shown to
decrease plasma glucose levels and stimulate glucose uptake into
C2C12 myotubes in a phosphatidylinositol 3-kinase (PI3K)-depen-
dent manner, while equivalent doses of leucine or valine did not
[207]. Consistent with this, high blood levels of isoleucine have
been associated with hypoglycemia in rats [208].
Altered isoleucine/leucine (indistinguishable by mass spec-
trometry) levels were found in aged Drosophila [82]. Increased
longevity in yeast [209] and just slightly increased lifespan in
C. elegans [3,23]. In C. elegans, DAF-16, a homolog of mammalian
FOXO proteins, is a pro-longevity transcriptional regulator activated
when insulin signaling is blocked. Metabolomics analysis of worms
found that many amino acids, including isoleucine, glutamine, and
valine were increased in long-lived daf-2 insulin receptor mutant
worms, while glutamate levels declined [210]. Isoleucine and valine
levels returned to wild-type levels in daf-2; daf-16 double mutants
as did lifespan, suggesting a tight connection between these amino
acids and longevity. However, isoleucine supplementation to
C. elegans only increased lifespan at the highest 10 mM dose of
three doses tested and this effect was small (Table 1) [3].
2.11. Leucine
Like isoleucine, leucine is an essential BCAA. Leucine is one of
two exclusively ketogenic amino acids and so is metabolized into
acetyl-CoA and potentially into ketone bodies if blood glucose
levels are low. A diet high in BCAAs has been shown to be associated
with increased mean lifespan of male mice [210]. However, a diet
low in BCAAs [211,212], or only low in leucine [213], increased
glucose homeostasis and enhanced metabolic health in mice. High
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levels of the alpha-ketoacid of leucine, alpha-ketoisocaproic acid,
has been shown to cause mitochondrial dysfunction in neurons
[214]. A review of 34 reports in the literature has identified leucine
supplementation to mice in a narrow range of 90e140 mg per day
consistently protected metabolic health against diet-induced
obesity, while BCAA administration had no effect [215]. However,
BCAA administration was protective in models of type 1 diabetes
[215]. BCAA levels increased with aging in mouse skeletal muscle
[216]. Leucine levels were found to decrease by 14% in the urine
from aged rats and increase by 6% with exercise [217].
Leucine strongly activates TOR kinase [218,219] by binding
Sestrin1 and Sestrin2 [220e222] and by binding leucyl-tRNA syn-
thetase [223,224]. Since inhibition of TOR kinase extends lifespan
across eukaryotic species, one may expect leucine supplementation
to decrease lifespan due to the downstream effects of TOR kinase
activation including stimulation of protein synthesis and inhibition
of autophagy [225]. However, strikingly, to the best of our knowl-
edge leucine supplementation has yet to result in decreased life-
span in any animal model yet tested, although a mouse diet high in
BCAAs, and compensatorily lower in other amino acids, has been
shown to decrease lifespan by decreasing the ratio of threonine and
tryptophan to BCAAs, which led to decreased serotonin synthesis,
hyperphagia, and obesity [226].
Leucine is a major amino acid regulating the rate of protein
synthesis by the ribosome in many tissues such as skeletal muscle
[227,228]. Activation of TOR kinase increased levels of the YY1-
PGC-1a complex in skeletal muscle to stimulate mitochondrial
oxidative function to match energy supply with energy demand
[229]. Administration of leucine has been shown to decrease
muscle degeneration in vitro [230] and may increase overall muscle
anabolism in young to middle-aged healthy humans [231,232].
Leucine supplementation has been shown to promote fatty acid
oxidation, stimulate mitochondrial biogenesis, increase NADþ
levels, and upregulate SIRT1 and AMP kinase (AMPK) activity in
skeletal myotube cells [233]. Although leucine is commonly
consumed by young or middle aged athletes to facilitate an increase
in muscle mass, there is little evidence to support the long-term use
of leucine supplementation without exercise in the elderly for the
amelioration of sarcopenia [234,235].
A study in Drosophila showed that lowering BCAAs in the diet
extended lifespan, but not more than a diet lowering three other
essential amino acids threonine, histidine, and lysine [236]. So at
least in fruit flies, the BCAAs do not suppress longevity more than
other essential amino acids. Restriction of BCAAs did not further
extend lifespan of an already low amino acid diet suggesting that
BCAA restriction and general protein/amino acid restriction acti-
vate the same molecular mechanisms. One group showed that in-
dividual supplementation with leucine could extend chronological
longevity in yeast [209] and that leucine availability was important
for maximal lifespan extension during CR [237]. However, another
group under different conditions showed that blocking leucine
synthesis in yeast stimulated chronological longevity [238].
Leucine supplementation led to a prominent increase in lifespan
in C. elegans that was slightly greater than valine and much greater
than isoleucine (Table 1) [3,23]. The extended longevity could be
mimicked by knocking down the bcat-1 BCAA aminotransferase
[23]. Surprisingly, knockdown of bcat-1 in neurons also extended
lifespan that was reduced by concurrent neuronal knockdown of
TOR kinase (let-363) [23]. The lifespan extension resulting from
bcat-1 knockdown or leucine supplementation could also be miti-
gated by ablation of ASI neurons (perform some functions of the
mammalian hypothalamus). This data suggests that neuronal TOR
has a pro-longevity function. In contrast to these results, long-lived
global mTORC1 pathway-deficient worms lost their enhanced
longevity and became normal-lived when mTORC1 activity was
77
restored specifically in neurons, but not when mTORC1 activity was
specifically restored in the intestine [13]. This data indicates that
neuronal TOR has a pro-aging function. Perhaps decreased function
of both mTORC1 and mTORC2 by TOR kinas/let-363 knockdown in
the first example above explains the divergent results. It has been
shown that specific mTORC1 deficiency compared to concurrent
mTORC1 and mTORC2 deficiency led to the activation of different
stress response pathways [239]. The inability to restore normal
lifespan by restoring mTORC1 activity in the intestine at first glance
appears to be inconsistent with other data where specific inhibition
of mTORC1 in the intestine extended lifespan [239]. But it is
possible that specific knockdown of mTORC1 activity in the intes-
tine generates a signal inactivating mTORC1 activity in neurons that
leads to lifespan extension, which is consistent with the other
findings.
2.12. Lysine
Lysine is an essential ketogenic amino acid. Therefore both
leucine and lysine can be supplemented while a subject is on the
anti-aging ketogenic diet [240,241] without stimulating gluco-
neogenesis to raise glucose levels. A metabolomics study of 647
human subjects linked high lysine levels (and two other amino
acids) with longevity as defined by attaining at least 80 years [199].
In humans lysine supplementation was shown to mimic protein
supplementation to increase plasma insulin and glucagon levels
and when lysine was administered with glucose it decreased blood
glucose levels [242]. Lysine supplementation also decreased blood
glucose levels and prevented cataract formation when adminis-
tered to diabetic rats. An equivalent amount of a mixed amino acid
supplement was also beneficial for both of these disease pheno-
types, but not to the extent of lysine [243]. Lysine supplementation
induced an angiogenic response and increased the rate of wound
healing [244]. In yeast deletion of the lysine biosynthesis gene
Lys12 increased chronological lifespan [93]. Lysine was the only
amino acid to increase in abundance in replicatively aged yeast,
while the abundance of roughly half of the amino acids declined
[95]. In C. elegans lysine supplementation yielded small increases in
lifespan at all three doses tested (Table 1) [3].
2.13. Methionine
Reducing dietary levels of methionine, an essential amino, in
rodents was shown to partially mimic the effects of CR and induce
lifespan extension [245]. This topic has been extensively reviewed
[246e251]. Many of the protective effects of DR are blocked by
adding back methionine to the diet [252]. Some of the protective
effects of CR and methionine restriction are due to increased
transsulfuration pathway flux to increase the expression of the
cystathionine gamma lyase (CTH) gene and the production of
hydrogen sulfide (H2S) [252,253]. Not only can CTH catabolize
cystathionine to cysteine, but it can catabolize cysteine to H2S
[253]. Cysteine supplementation can inhibit some of the beneficial
effects of methionine restriction [254] perhaps through restoring
glutathione levels that are decreased during methionine restriction
[255]. The decreased glutathione levels led to the activation of
protein kinase Relike endoplasmic reticulum kinase (PERK) and the
integrated stress response (ISR) including transcription of the ATF4
transcriptional regulator [256]. Increased hepatic ATF4 levels
stimulated release of fibroblast growth factor 21 (FGF21) leading to
increased expression of uncoupling protein 1 (UCP1) in white adi-
pose tissue (WAT) and a browning of WAT [256], which results in
increased energy expenditure. FGF21 has been shown to be trans-
ported into the brain and signal to alter metabolism during a low
protein diet [257]. It also likely plays a similar role during
78 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89
methionine restriction. The Nrf2 transcriptional regulator was also
activated by methionine restriction, likely due to the decreased
glutathione levels. Nrf2 increases transcription from antioxidant
response elements (AREs) in gene promoters.
Dietary methionine restriction inhibits mTORC1 activity to
activate autophagy by lowering tissue levels of S-adenosylme-
thionine, which normally binds the SAMTOR protein to stimulate
mTORC1 activity [258]. In yeast increased methionine and S-ade-
nosylmethionine levels also inhibit autophagy by stimulating the S-
adenosylmethionine-dependent methylation of the catalytic sub-
unit of the PP2A phosphatase by the Ppm1p methyltransferase
leading to the dephosphorylation of the Npr2p regulator of auto-
phagy [259]. Therefore, decreased S-adenosylmethionine levels,
inhibition of mTORC1, and activation of autophagy likely explain
the lifespan extension observed following knockdown of the S-
adenosylmethionine synthetase-1 (sams-1) gene in C. elegans [260],
even though stimulation of the methionine cycle is also associated
with longevity under other conditions [186].
Methionine restriction can also occur during a vegan diet
[261,262] or a strict ketogenic diet [263] due to the low protein
intake prescribed to these diets, and the addition of methionine to a
ketogenic diet resulted in reversal of weight loss in mice [263]. A
vegan diet is also typically lower in lysine content [264]. A diet
deficient in methionine is associated with many positive effects
such as enhanced fatty acid oxidation, increased energy expendi-
ture, decreased ROS production, reduced oxidative damage [265],
and inhibition of tumorigenesis [266]. Most notably, methionine
restriction extended lifespan in mice by nearly 7% [267], rats by 44%
[268], and Drosophila by 36% [269]. Similar to CR, methionine re-
striction produced a lifelong reduction in fatty body mass [245]. A
diet that restricts methionine by 83% in humans, similar to the
extent that produces the positive longevity effect in rodents, has
been established [266]. Glycine supplementation to rats mimicked
the longevity effects of methionine restriction though stimulating
the clearance of hepatic methionine [270]. Methionine restriction
had greater benefits to the metabolic health of mice than equivalent
leucine restriction [271].
When fruit flies were placed on a protein-deficient diet that
extended lifespan, the addition of methionine could restore
fecundity without decreasing lifespan, suggesting that it is possible
to design a modified amino acid diet that offers the positive effects
of DR without the negative effects [4]. Methionine restriction also
extended lifespan in Drosophila [4], but only on a low amino acid
diet [272]. In one study in yeast, chronological lifespan was
extended by methionine restriction, but not by lysine, isoleucine,
threonine, or valine restriction [273], and this increased longevity
required autophagy-dependent vacuolar acidification [274].
Methionine restriction extended the chronological lifespan of yeast
even when H2S production was inhibited [275]. Methionine sup-
plementation increased mitochondrial oxidative metabolism in
yeast [276] and also increased oxidative stress resistance by
increasing pentose phosphate pathway flux and cellular reduced
nicotinamide adenine dinucleotide phosphate (NADPH) levels
[277]. Methionine supplementation to fasted mice and catabolism
of the methionine through the little studied methionine trans-
amination pathway decreased blood sugar levels by inhibiting he-
patic gluconeogenesis through GCN5 acetyltransferase and PGC-
1a-dependent mechanisms [278]. Methionine supplementation
was shown to extend the lifespan of C. elegans at the two highest
doses administered, but not at the lowest dose (Table 1) [3].
2.14. Phenylalanine
Phenylalanine is an essential amino acid that is both glucogenic
and ketogenic and is converted to tyrosine by phenylalanine
hydroxylase. Phenylalanine and glutamine levels were found to be
higher in the plasma of centenarians compared to other elderly
individuals [279]. Using a longitudinal approach phenylalanine/
tyrosine metabolism, tryptophan metabolism, and methionine
metabolism were shown to be altered with aging in the plasma of
marmosets, a primate aging model [280,281]. Specifically, phenyl-
alanine levels decreased with age [281]. Phenylalanine binds hy-
droxyl radicals and prevented hydroxyl radical-mediated inhibition
of acetylcholinesterase activity in brain homogenates [282].
Elevated serum phenylalanine has been linked to telomere loss
in men [283], inflammatory disease [284], and type 2 diabetes [33].
Dietary protein restriction in rats increased lifespan and decreased
phenylalanine levels in liver [285]. Increased phenylalanine levels
were found in aged Drosophila [82]. Increased dietary protein as
well as individual supplementation with phenylalanine, methio-
nine, serine, or threonine greatly decreased the lifespan of the
Argentine ant, while individual supplementation with most other
amino acids slightly decreased lifespan and supplementation with
glutamate, tyrosine, or tryptophan did not affect lifespan [286].
Phenylalanine was one of two amino acids that did not extend the
lifespan of C. elegans when added in the 1e10 mM range and was
the only amino acid that decreased lifespan at the 5 mM dose
(Table 1) [3]. Phenylalanine can be oxidized to the toxic metabolite
meta-tyrosine (m-tyrosine), which decreases C. elegans lifespan. So
during oxidative stress C. elegans upregulates the expression of
tyrosine aminotransferase (tatn-1) to deaminate excess tyrosine,
which likely decreases product inhibition of phenylalanine hy-
droxylase to stimulate the conversion of phenylalanine to tyrosine
and decrease phenylalanine and m-tyrosine levels [287].
2.15. Proline
Proline is a non-essential amino acid that is synthesized from
glutamate in a two-step NADPH-requiring pathway. A metab-
olomics study of aging mice found proline, alanine, serine, tyrosine
and methionine to decrease in plasma with aging [53]. Increased
plasma proline concentrations have also been associated with
sarcopenia in the elderly [288]. Dietary proline supplementation
had an immunostimulatory effect to protect mice from Pasteurella
multocida infection [289]. The topical application of proline to
cutaneous wounds led to an acceleration of healing in rats [290].
Proline acts as a metal chelator, supports osmotic balance, and
prevents oxidative damage [291]. Proline supplementation miti-
gated the early stage of liver injury in rats by decreasing oxidative
stress. It also improved redox status to improve nitric oxide avail-
ability and prevent a pathological increase in blood pressure [292].
Proline was the second most potent amino acid at extending
lifespan in C. elegans (Table 1) and the most potent for increasing
thermotolerance [3] suggesting that it stimulates pathways medi-
ating proteostasis. It has been shown to extend the lifespan by
transiently increasing ROS levels, which then stimulate stress
response pathways that induce antioxidant gene expression [293].
Proline is hydroxylated to form hydroxyproline, a prominent amino
acid in collagen, a major protein of the C. elegans cuticle [294].
Collagen remodeling was shown to be required for C. elegans life-
span extension due to daf-2 insulin receptor mutation [295].
2.16. Serine
Serine is a non-essential amino acid that is synthesized in a 3
step pathway from the glycolytic intermediate 3-phosphoglycerate
(Fig. 1). Serine supplementation showed many protective effects on
the phenotypes induced by a high fat diet in mice including
increasing glucose tolerance and insulin sensitivity, decreasing
hepatic lipid levels, and preserving reduced glutathione (GSH)
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levels by preventing the hypermethylation of promoters of GSH
synthesis genes [296]. Serine supplementation also activated AMPK
in palmitic acid-treated primary hepatocytes to decrease ROS
production [296e299]. Although short term serine supplementa-
tion did not reduce weight gain on a high fat diet [296], long term
serine supplementation to a normal chow diet was shown to
decrease food consumption and weight gain [300]. Serine supple-
mentation increased Sirt1 protein deacetylase expression and
decreased ROS levels in the hypothalamus, and decreased levels of
phosphorylated NFkB to decrease the levels of pro-inflammatory
cytokines [300]. Serine has been shown to be neuroprotective
[301,302] in part through upregulation of the expression of ER
protein disulfide isomerase to increase proteostasis [303,304]. With
aging, microglia synthesize and release increased amounts of D-
serine, synthesized from L-serine. D-serine is a co-agonist for NMDA
receptors [305] and therefore this increased extracellular D-serine
may contribute to the excitotoxicity induced by neuronal NMDA
receptor activation in aged animals [306]. A phase I clinical trial has
been conducted testing the effects of serine supplementation on
amyotrophic lateral sclerosis (ALS) patients [307,308]. A 15 g dose
of serine twice a day was well tolerated with only mild intestinal
discomfort in a few subjects. It has been postulated that the long
life of Ogimi village Okinawans may be due in part to the high
content of serine in their diet [309].
Serine metabolism can increase cellular antioxidant function
through several different mechanisms. In studies with human
endothelial cells, serine supplementation increased antioxidant
gene expression through upregulation of Nrf2 and its downstream
effector heme oxygenase-1 [310]. Serine is a direct precursor for the
synthesis of glycine and cysteine, which are limiting for the syn-
thesis of GSH. See Fig. 1. Once GSH is oxidized, the GSSG is reduced
by NADPH and recycled for use. One-carbon units derived from
serine can also be added to homocysteine for methionine synthesis.
Serine is transported into the mitochondrial matrix by the SFXN1
transporter [311]. Catabolism in mitochondria, initiated by the
enzyme SHMT2, increased mitochondrial NADPH levels to increase
antioxidant function [312]. Data has emerged that mitochondrial
serine can also be catabolized in a three step pathway to 2-
phosphoglycerate (Fig. 1), which can then be exported to the
cytoplasm for gluconeogenic or glycolytic metabolism [313,314],
while synthesis of serine from glycolytic 3-phosphoglycerate oc-
curs in the cytoplasm [315,316]. Serine is also an essential building
block of lipids such as phosphatidylserine [317], sphingolipids, and
ceramides. Serine deficiency results in mitochondrial fragmenta-
tion and dysfunction [318].
Anti-aging CR was shown to increase serine dehydratase levels
in the liver of aged mice to increase the catabolism of serine to
pyruvate to fuel gluconeogenesis [319]. Subjects with fibromyalgia
showed lower plasma levels of serine and histidine [320]. In yeast
inhibition of serine synthesis from 3-phosphoglycerate (see Fig. 1)
increased chronological lifespan by stimulating glyoxylate shunt
activity and trehalose synthesis [321]. In other studies with yeast,
serine was further shown to promote aging by activating a phos-
phorylation signaling cascade using the Pkh1/2, Sch9 (homolog of
mammalian S6 kinase), and Rim15 protein kinases [322]. In
contrast to the results with yeast, in C. elegans serine supplemen-
tation increased lifespan to the greatest extent of any amino acid
(Table 1) [3]. Serine supplementation activated the HIF-1, DAF-16,
and SKN-1 stress response pathways and lifespan extension was
dependent upon the function of AAK-2/AMPK and sirtuin SIR-2.1
[3].
2.17. Threonine
Threonine is an essential amino acid that is both glucogenic and
79
ketogenic. Threonine supplementation in humans can greatly in-
crease its levels in plasma [323]. Threonine is a precursor for
glycine synthesis in most mammals and threonine supplementa-
tion has been shown to increase plasma glycine levels in pigs [324]
and increase glycine neurotransmitter levels in the brain of rats
[325]. An excess or deficiency in dietary threonine decreased pro-
tein synthesis rates in pigs [324]. Threonine levels were shown to
decline in the plasma of aged rats [205]. A metabolomics study of
647 human subjects linked high threonine levels (and two other
amino acids) with longevity as defined by attaining at least 80 years
[199]. In human [326] and mouse embryonic stem cells, threonine
levels can regulate the cell cycle G1/S phase transition and prolif-
eration [327,328]. Threonine deficiency in mammals has been
associated with depression and neurological dysfunction [329].
Threonine supplementation to chickens led to an improved im-
mune response [330] and protection from LPS-induced inflamma-
tion and intestinal barrier damage [331]. Threonine
supplementation to pigs led to increased protection against a viral
challenge [332]. Supplementation with threonine has been shown
to extend chronological longevity in yeast [209]. However, a
different group using different conditions found threonine re-
striction (as well as valine restriction) to increase yeast chrono-
logical lifespan [322,333,334] by inhibiting TOR kinase [322].
Threonine supplementation only extended C. elegans lifespan
slightly at the highest (10 mM) dose of three doses applied
(Table 1). Threonine can be converted to glycine in in one step by
threonine aldolase and in two steps by the threonine dehydroge-
nase and GCAT enzymes (Fig. 1). Therefore threonine could extend
lifespan by a similar stimulation of the methionine cycle as shown
for glycine or serine supplementation [186]. However, another
mechanism could be involved. The 2-amino-3-ketobutyrate prod-
uct of the threonine dehydrogenase reaction is unstable and
spontaneously decarboxylates into aminoacetone, which is further
broken down producing hydrogen peroxide and methylglyoxal.
Knockdown of the C. elegans GCAT homolog T25B9.1 extended
lifespan through increasing 2-amino-3-ketobutyrate and the pro-
duction of hydrogen peroxide and methylglyoxal, which then
activated the SKN-1 and HSF-1 transcriptional regulators. So life-
span was extended by a hormetic effect [335]. Therefore, increased
production of these reactive products could also contribute to the
lifespan extension that occurs during threonine supplementation.
2.18. Tryptophan
Tryptophan is an essential amino acid that is both glucogenic
and ketogenic. Dietary tryptophan restriction in rats delayed
reproductive aging [336] and extended lifespan [337,338]. How-
ever, the rats were switched to a normal diet later in life, so more
experiments need to be done to confirm these lifespan findings
[339]. Tryptophan is a precursor for the neurotransmitter serotonin
and for the hormone melatonin that regulates circadian rhythms.
Tryptophan is degraded by the kynurenine pathway to nicotin-
amide adenine dinucleotide (NADþ) and other end products with
several of the intermediates being toxic such as quinolinic acid, due
to it being an agonist of the neuronal NMDA receptor. The inter-
mediate kynurenic acid is neuroprotective because it is an antag-
onist of the NMDA receptor. Tryptophan levels were shown to
decrease in human serum with aging [198], while toxic tryptophan
catabolites were shown to increase [340]. This increase in trypto-
phan catabolism with aging is likely due to increased levels of the
enzyme indoleamine-2,3-dioxygenase (IDO) [340]. IDO breaks
down tryptophan to kynurenine in tissues outside the liver, while
tryptophan-2,3-dioxygenase performs this task in the liver. IDO is
induced by pro-inflammatory cytokines and superoxide [340],
which increase with aging.
80 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89
Tryptophan plasma levels have been measured or tryptophan
supplementation studies have been performed for many disorders
as recently reviewed [341]. In brief, studies have been performed
focusing on tryptophan in the following aging-related disorders:
cardiovascular disease [342e344], chronic kidney disease [345],
diabetes [346], depression [347,348], inflammatory bowel disease
[349], and multiple sclerosis [350]. Tryptophan levels were most
frequently lower in the disease state and supplemental tryptophan
most often decreased disease phenotypes. There were two excep-
tions. Limiting tryptophan was beneficial for chronic kidney disease
and increased plasma tryptophan levels were found to predict
future type 2 diabetes in a Chinese population [346]. However, this
association with type 2 diabetes has yet to be found in other ethnic
groups. In fact, several tryptophan catabolites have been found to
be increased in the plasma of subjects with type 2 diabetes [351]
suggesting increased tryptophan catabolism. In rats tryptophan
supplementation was shown to lower blood glucose levels, and
delay disease progression in hereditary diabetic rats [352]. A
decrease in plasma tryptophan levels with aging was associated
with a loss of cognitive function in non-demented women [340].
Supplemental tryptophan was also shown to improve the memory
of aged rats [353], while tryptophan-enriched cereals were shown
to improve the sleep/wake cycle of elderly humans [354].
Altered tryptophan levels were found in aged Drosophila [82]
and long-lived flies showed higher tryptophan levels [355].
Decreasing tryptophan uptake by deleting the tryptophan
permease Tat2p increased replicative lifespan in yeast [356].
Blocking tryptophan catabolism extended lifespan and decreased
aging-related proteotoxicity in C. elegans [22]. Tryptophan supple-
mentation increased the lifespan of C. elegans at two lower doses,
but not at the highest 10 mM dose tested (Table 1) [3]. Tryptophan
was the only lifespan-extending amino acid tested out of ten tested
that extended the lifespan in daf-16 mutant worms. Also trypto-
phan and cysteine were the only two lifespan-promoting amino
acids out of ten tested that did not activate a transcriptional re-
porter for SKN-1/Nrf2 activity, suggesting novel mechanisms of
lifespan extension [3]. Tryptophan and a few of its catabolites were
shown to activate the mitochondrial unfolded protein response
(UPRmt) and the ER stress response pathways in C. elegans [3]. The
UPRmt is frequently, but not always associated with nematode
longevity [357,358].
2.19. Tyrosine
Tyrosine, a non-essential amino acid, is both glucogenic and
ketogenic and is synthesized from phenylalanine by the phenylal-
anine hydroxylase enzyme. Tyrosine is the precursor for the neu-
rotransmitters dopamine and norepinephrine. In a metabolomics
study of human plasma, tyrosine and glutamine levels increased
with age, while histidine, threonine, tryptophan, leucine, and
serine decreased with age [359]. Supplementation with tyrosine in
both hypertensive humans and rats led to a decrease in blood
pressure [360]. In one study of human subjects, increased dietary
tyrosine levels were linked with increased cognitive performance
[361]. However, another study found that supplementation of
moderate tyrosine levels had no effect, while high dietary tyrosine
levels decreased cognitive performance in older adults [362]. Au-
thors of a recent review suggest that tyrosine supplementation
likely only increases cognition during times of stress when either
dopamine or norepinephrine levels are depleted [363]. There is no
consistent data that tyrosine supplementation can decrease the
effects of stress on cognitive or physical performance [364]. Tyro-
sine supplementation has been shown to increase the vasocon-
striction response to body cooling in older adults, so it may aid
thermoregulatory function [365]. Tyrosine supplementation has
been shown to enhance dopaminergic neurotransmission in Par-
kinson's disease patients [366]. However, high levels of tyrosine in
the plasma increase the risk of type 2 diabetes to a similar extent as
increased levels of each of the three BCAAs [33]. In C. elegans
tyrosine supplementation increased lifespan moderately at the
lowest 1 mM dose, but only slightly at the two higher doses
(Table 1) [3].
2.20. Valine
Valine is an essential glucogenic BCAA. A breakdown product of
valine, 3-hydroxyisobutyrate (3-HIB) was shown to drive vascular
fatty acid transport and insulin resistance [367]. However, valine
supplementation to rats reduced fatigue during swim exercise and
partly prevented the drop in blood glucose and liver glycogen
following exercise, while leucine or isoleucine supplementation did
not [368]. Valine supplementation also protected against paraquat-
induced toxicity in rats [369]. Valine supplementation, more than
the supplementation of any other amino acid, increased ATP levels
and protected against oxidative stress in M17 neuroblastoma cells
overexpressing alpha-synuclein [100]. This could be due to an in-
crease in mitochondrial biogenesis induced by BCAAs [210], as
isoleucine showed a similar, but smaller effect [100]. The mito-
chondrial carrier family member SLC25A44 has recently been
identified as a mitochondrial BCAA transporter that plays an
especially important role in brown adipose tissue metabolism
[370]. Using yeast, one group showed that supplemental valine
extended chronological longevity [209]. However, another group
using different conditions found valine restriction to increase yeast
chronological lifespan by inhibiting TOR kinase [322,333,334].
Replicative aging in yeast has been shown to lead to an increase in
the expression of amino acid catabolism genes, a decrease in the
expression of amino acid biosynthesis genes, and a decrease in
most amino acid levels, especially all three BCAAs but also aspar-
agine, glutamate, glycine, histidine, and methionine (see Table 2)
[95]. In C. elegans valine supplementation increased lifespan
moderately at the two lower 1 mM and 5 mM doses, but not at the
highest dose (Table 1) [3].
3. Conclusions
In the last decade major strides have been made in the under-
standing that amino acids are signaling molecules and can be used
as a relatively non-toxic treatments to alter the rate of aging in
experimental models. Important molecular details of how specific
amino acids such as leucine and arginine activate TOR to decrease
lifespan have been elucidated. But the molecular mechanisms
through which increased levels of many of the amino acids provide
health benefits remain largely unknown. The ability of glycine to
extend longevity in rodents is an important breakthrough as amino
acid-restricted diets, such as methionine-restricted diets, are
expensive and can be relatively unpalatable resulting in difficulties
in subject retention in clinical trials. Another important goal should
be the determination of the effects of serine and threonine on the
lifespan of mice, since these amino acids can be metabolized to
glycine but also have their own unique properties. In support of
these unique properties, serine or glycine administration to rats
altered brain amino acid levels in different ways [371]. Women
have been shown to have higher plasma levels of serine and glycine
than men, while 13 of the other 18 amino acids were lower [359].
Whether these amino acid level differences contribute to the
extended longevity of women compared to men remains to be
determined. Serine supplementation in humans has been shown to
lower plasma homocysteine levels, a marker of metabolic and
cardiovascular disease [372]. This may occur through stimulation of
 C.-A. Canfield, P.C. Bradshaw / Translational Medicine of Aging 3 (2019) 70e89
cystathionine-beta synthase (CBS) activity (See Fig. 1). Serine
metabolism in mitochondria leads to NADPH production that pro-
tects mitochondria from ROS-mediated damage, but at the expense
of NADPH oxidation in the cytoplasm [373]. However, some of these
lost cytoplasmic reducing equivalents can be restored if increased
serine levels stimulate CBS activity of the transsulfuration pathway
to increase cysteine levels, which decreases the plasma membrane
import of oxidized cystine and its subsequent reduction to cysteine,
which is indirectly coupled to cytoplasmic NADPH oxidation [374].
The human threonine dehydrogenase [375] and threonine
aldolase [376] genes have mutated so functional proteins are not
expressed, while the expressed proteins are functional in rodents
for the conversion of threonine to glycine (Fig. 1) [373]. So threo-
nine supplementation to humans would not likely yield similar
effects to those of glycine. Several amino acids from the diet are
catabolized to a large extent by first pass splanchnic [377] and
hepatic [378] metabolism and amino acid supplementation stra-
tegies may fail to raise amino acid levels to a large extent in pe-
ripheral tissues due to this issue. Since the hypothalamus is a major
regulator of the aging process, amino acids may need to cross the
BBB to exert their anti-aging effects and in doing so they must
compete with other amino acids for transport [158]. So it may be
difficult to raise the level of many amino acids in the brain. To
address these issues, studies could be performed with membrane
permeable forms of amino acids such as N-acetylated forms and
ethyl ester forms that may more readily permeate throughout the
body.
Another challenge that needs to be addressed is determining
the correct amount of an amino acid to supplement to obtain a
therapeutic effect as a hormetic dose response is often observed
[3]. The presence of transaminases and alpha-ketoacids in the
blood and many tissues including the liver lead to the relatively
quick transamination of one amino acid and synthesis of another.
Many of the common transaminases such as aspartate trans-
aminase and alanine transaminase use alpha-ketoglutarate as an
acceptor of an amino group to form glutamate. Therefore, the ef-
fects of supplementation with certain amino acids may be a result
of increased glutamate or decreased alpha-ketoglutarate levels
instead of from a direct effect of the supplemented amino acid. In
this regard, alpha-ketoglutarate has anti-aging properties [140]
even at low doses [3] and so supplementation with high levels of
certain amino acids may shorten lifespan through alpha-
ketoglutarate depletion. Amino acid transamination likely ex-
plains why 18 of the 20 amino acids extended lifespan in C. elegans
and why many of the amino acids demonstrated a similar hormetic
dose response [3]. At high doses of supplementation a common
mechanism of toxicity may be occurring even though a different
amino acid was supplemented. Heavy isotope amino acid tracer
studies will be useful to follow the metabolism of the supple-
mented amino acids to determine if common downstream pro-
longevity and pro-aging catabolites can be identified as causative
longevity modulators. Therefore, much work remains to harness
the great potential of altering amino acid levels for the treatment
of aging and aging-related diseases.
Conflict of interest
The authors declare there is no conflict of interest.
Declarations of interest
Declarations of interest: none. The funders had no role in the
writing of the manuscript or in the decision to publish.
81
Funding
This research was funded by The National Institutes of Health
grant numbers AG059096 and AG046769 awarded to PB.
Acknowledgements
We would like to thank John Canfield, Neil Copes, Vedad Delic,
and Jeddidiah Griffin for helpful discussion.
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