Amino acids in the regulation of aging and aging-related diseases 氨基酸在調節衰老和衰老相關疾病中的作用

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 releases 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 prevent the formation or dissolve preexisting kidney stones [52]. Alanine levels have been shown to decline with aging in mouse 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 chronological 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 effects of arginine supplementation have been reviewed [58]. Arginine catabolism is altered in learning and memory centers in the aged brain [59]. Arginine has been shown to stimulate the proliferation 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 vasodilation 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 cytoplasmic 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) arginine levels [81]. The activation of mTORC1 leads to increased protein 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 glutamine 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 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 lipopolysaccharide (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 NAD⁺/NADH ratio to activate sirtuin deacetylases [96]. Aspartate is synthesized from oxaloacetate by aspartate aminotransferase. Mitochondrial electron transport chain function declines with aging [97] and is required for aspartate and pyrimidine synthesis [98]. Metals such as Mn²⁺, Cu²⁺, Zn²⁺, and Mg²⁺ 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 supplementation 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 1–10 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 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 contributes 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 Alzheimer’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 homocysteine [116], a risk factor for neurodegenerative and cardiovascular 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 supplementation with leucine and pyruvate prevented this toxicity. The protective 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 γ-glutamyltransferase and plasma GSH was not altered [124]. Supplementing 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 lifespan 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 Alzheimer’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 concentrations and high intracellular glutamate concentrations is kept in check by plasma membrane glutamate transporters. Astrocytes 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 oxaloacetate and the aspartate transaminase enzyme [136,137]. However, 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 immune 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 decreases the expression of pro-inflammatory genes such as NF_Ҡβ [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 regeneration 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 chronological 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 increase the hepatic clearance of methionine [163]. Glycine supplementation 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 transported across the mitochondrial inner membrane by the mitochondrial carrier family member SLC25A38 [167]. Another study did not find much change in human whole body glycine metabolism with aging, but found large changes following altered protein 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 nucleus 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.

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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 NAD⁺ to NADH. THF, tetrahydrofolate; 5-meTHF, 5-methyltetrahydrofolate; 5,10-meTHF, 5,10-methylenetetrahydrofolate; coA, coenzyme A; NAD⁺, nicotinamide adenine dinucleotide (oxidized); NADP⁺, nicotinamide adenine dinucleotide phosphate (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); ATP, adenosine triphosphate; PP_i, inorganic pyrophosphate; CO₂, carbon dioxide; SHMT1, Serine Hydroxymethyltransferase 1; SHMT2, Serine Hydroxymethyltransferase 2; MTHFD2L, Methylenetetrahydrofolate Dehydrogenase (NADP⁺ Dependent) 2 Like; MTHFD1L, Methylenetetrahydrofolate Dehydrogenase (NADP⁺ 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, Adenosylhomocysteinase; 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.

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 (PEPCK_c) [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].

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 potential 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 supplementation 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 activity to decrease liver injury induced by chronic alcohol consumption [194]. Individual administration of histidine, cysteine, or glycine, but not alanine, inhibited NF-κB 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, histidine 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 pathways.

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)-dependent 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 spectrometry) levels were found in aged Drosophila [82]. Increased isoleucine levels in the culture media extended chronological 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 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 90–140 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 [[220], [221], [222]] and by binding leucyl-tRNA synthetase [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 knowledge leucine supplementation has yet to result in decreased lifespan 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-1α 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 NAD⁺ 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 activate the same molecular mechanisms. One group showed that individual 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 mitigated 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 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 intestine 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 gluconeogenesis 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 administered to diabetic rats. An equivalent amount of a mixed amino acid supplement was also beneficial for both of these disease phenotypes, 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 [[246], [247], [248], [249], [250], [251]]. 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 R–like 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 adipose tissue (WAT) and a browning of WAT [256], which results in increased energy expenditure. FGF21 has been shown to be transported into the brain and signal to alter metabolism during a low protein diet [257]. It also likely plays a similar role during 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-adenosylmethionine, which normally binds the SAMTOR protein to stimulate mTORC1 activity [258]. In yeast increased methionine and S-adenosylmethionine levels also inhibit autophagy by stimulating the S-adenosylmethionine-dependent methylation of the catalytic subunit of the PP2A phosphatase by the Ppm1p methyltransferase leading to the dephosphorylation of the Npr2p regulator of autophagy [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 expenditure, 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 restriction 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 supplementation 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 transamination pathway decreased blood sugar levels by inhibiting hepatic gluconeogenesis through GCN5 acetyltransferase and PGC-1α-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, phenylalanine levels decreased with age [281]. Phenylalanine binds hydroxyl 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, methionine, 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 1–10 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 hydroxylase 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 metabolomics 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 mitigated the early stage of liver injury in rats by decreasing oxidative stress. It also improved redox status to improve nitric oxide availability 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 mediating 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 lifespan 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) 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 [[296], [297], [298], [299]]. Although short term serine supplementation 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 supplementation increased Sirt1 protein deacetylase expression and decreased ROS levels in the hypothalamus, and decreased levels of phosphorylated NFκB 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 synthesis 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 occurs 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 fragmentation 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 phosphorylation 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 supplementation 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 ketogenic. Threonine supplementation in humans can greatly increase its levels in plasma [323]. Threonine is a precursor for glycine synthesis in most mammals and threonine supplementation 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 protein 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 proliferation [327,328]. Threonine deficiency in mammals has been associated with depression and neurological dysfunction [329]. Threonine supplementation to chickens led to an improved immune response [330] and protection from LPS-induced inflammation 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 restriction (as well as valine restriction) to increase yeast chronological 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 dehydrogenase 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 product 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 production of hydrogen peroxide and methylglyoxal, which then activated the SKN-1 and HSF-1 transcriptional regulators. So lifespan 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]. However, 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 nicotinamide adenine dinucleotide (NAD⁺) 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 intermediate kynurenic acid is neuroprotective because it is an antagonist 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 tryptophan 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.

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 [[342], [343], [344]], 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 exceptions. 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 supplementation 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 tryptophan and cysteine were the only two lifespan-promoting amino acids out of ten tested that did not activate a transcriptional reporter 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 (UPR^mt) and the ER stress response pathways in C. elegans [3]. The UPR^mt 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 phenylalanine hydroxylase enzyme. Tyrosine is the precursor for the neurotransmitters 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]. Authors 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]. Tyrosine supplementation has been shown to increase the vasoconstriction response to body cooling in older adults, so it may aid thermoregulatory function [365]. Tyrosine supplementation has been shown to enhance dopaminergic neurotransmission in Parkinson’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 increase in mitochondrial biogenesis induced by BCAAs [210], as isoleucine showed a similar, but smaller effect [100]. The mitochondrial 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 asparagine, 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].