In 2013, Carlos López-Otín and colleagues published a review titled The Hallmarks of Aging in Cell, describing nine common features of aging across species. Among them, alongside mechanisms already targeted by anti-aging enthusiasts—such as stem cell exhaustion, telomere attrition, cellular senescence, and blunted IGF/mTOR/AMPK/sirtuins signaling—they highlighted "mitochondrial dysfunction" as a key hallmark of aging.
Mitochondria are often called "cellular powerhouses" because nutrients enter mitochondria to undergo tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) under aerobic conditions, ultimately breaking down into carbon dioxide, water, and the universal energy molecule adenosine triphosphate (ATP). This oxygen-dependent process is known as cellular "oxidative respiration."
Mitochondrial dysfunction, as the name suggests, refers to failures in these nutrient catabolic steps, leading to insufficient ATP production and abnormal levels of intermediate metabolites.
Before The Hallmarks of Aging was published, improving mitochondrial function was primarily a therapeutic focus for clinical mitochondrial myopathies like MELAS, MERRF, KSS, and CPEO syndromes. After the review, scientists in the mitochondrial field shifted their focus: "If myopathies are hard to treat, anti-aging should be easier!" Gradually, "restoring mitochondrial function to a youthful state" became a mainstream direction in anti-aging research. Anti-aging substances such as NAD+ precursors, α-ketoglutarate (α-KG), α-lipoic acid, and even the "old standby" CoQ10 all target this pathway.
A common trait of these versatile anti-aging substances is that they are intermediate products of mitochondrial oxidative respiration. Components of OXPHOS—NAD+, CoQ10, FAD, etc.—have been thoroughly studied. The TCA cycle, however, is a "treasure trove" of over a dozen substrates. Our "old friend" Brian Kennedy, a leading anti-aging researcher from Singapore, was the first to identify α-KG as a hidden gem from this cycle. This raises the question: how many more anti-aging "treasures" lie along the oxidative respiration pathway?
Research shows there are more than just α-KG—several TCA cycle substrates can regulate cellular function, senescence, and even lifespan.
What Are the Substrates of the TCA Cycle?
Even science graduates may need a refresher on the TCA cycle. Here’s a recap: Nutrients are converted into pyruvate in the body, which then synthesizes oxaloacetate and acetyl-CoA. These enter the TCA cycle, sequentially transforming into citrate, isocitrate, aconitate, α-ketoglutarate (α-KG), succinate, and more, ultimately regenerating oxaloacetate while releasing chemical energy. This energy feeds into OXPHOS to produce "biological energy" (ATP). Oxaloacetate continues to react with incoming acetyl-CoA, sustaining the cycle and continuous ATP production.
Evolutionary biology research reveals that mitochondria existed before life evolved to use oxygen, indicating they have functions beyond ATP production—likely mediated by TCA cycle substrates. In short: Some TCA substrates escape mitochondrial consumption for ATP synthesis, moving to various cellular compartments to exert diverse biological effects. This process, as constant as ATP synthesis, is the core mechanism behind these substrates’ longevity-promoting effects.
Reunderstanding the Biological Functions of TCA Cycle Substrates
Beyond affecting ATP production, TCA substrates influence cell fate. Below is a summary of the well-studied biological functions of key substrates in recent years, including synthesizing lipids/proteins, regulating epigenetics, supporting stem/immune cell function, adapting to oxygen changes, modulating inflammation, and even "burning fat," "raising blood pressure," or "inhibiting bacteria":
| TCA Substrate | Biological Functions |
|---|---|
| Pyruvate | a) Main carbon source for TCA flux, regulating total cycle activity (affecting both ATP-synthesizing and cell-regulating substrates). b) In aging, less pyruvate enters TCA for oxidative respiration; more undergoes anaerobic glycolysis to lactate, reducing ATP production efficiency—a hallmark of cellular senescence. |
| Acetyl-CoA | a) In cytoplasm: Precursor for fatty acids, cholesterol, ketones, and amino acids. b) In nucleus: Acyl donor for histone acetylation, altering gene expression. c) In immune cells: High levels enhance antigen presentation and interferon secretion against infections [5,6]. d) In stem cells: Deficiency halts differentiation into specialized cells. |
| α-Ketoglutarate (α-KG) | a) In nucleus: High levels activate histone and DNA demethylation, altering gene expression [7]. b) In immune cells: Aids macrophages in fighting inflammation [8]. c) Under hypoxia or α-KG deficiency, HIF-1α/2α activate, promoting immunity, angiogenesis, and erythropoiesis. |
| 2-Hydroxyglutarate (2-HG) | a) Derivative of α-KG in cytoplasm; maintains T lymphocyte differentiation [9]. b) Mutations in 2-HG-synthesizing enzymes (IDH1/2) in cancer lead to 2-HG accumulation, making it a cancer biomarker [10-13]. |
| Succinate | a) In nucleus: High levels inhibit histone/DNA demethylases, causing hypermethylation and promoting tumor growth. b) In mitochondria: Promotes excessive ROS production, enhancing cellular inflammation [14]. c) Free succinate binds SUCNR1, stimulating renin-angiotensin secretion to raise blood pressure. d) Activates brown adipose tissue UCP-1, boosting fat burning [15]. |
| Fumarate | Mitochondrial fumarate is poorly studied, but dimethyl fumarate (DMF) is well-investigated: DMF modulates immunity via cAMP, treating multiple sclerosis and psoriasis [16]. |
| Itaconate | a) Derivative of cis-aconitate decarboxylation; inhibits bacterial/fungal isocitrate lyase (ICL) for antimicrobial effects. b) Derivatives treat immune diseases like lupus. |
These functions underpin how TCA substrates regulate cellular senescence and lifespan. Among them, α-KG—dubbed the "rising star" after NAD+ and championed by Professor Kennedy—has dozens of studies in the past five years supporting its anti-aging role:
| Metabolite | Model | Intervention | Results |
|---|---|---|---|
| α-KG | Mice, pig follicles | α-KG supplementation | Increased telomerase activity, telomere length, AMPK signaling; decreased mTOR signaling; improved fertility in aged mice; promoted in vitro maturation of pig follicles [17]. |
| α-KG | Mice | α-KG supplementation | Improved bone mass, reduced age-related bone loss, and ameliorated senescence-associated secretory phenotype (SASP) in aged mesenchymal stem cells [18]. |
| α-KG | Mice | α-KG calcium salt supplementation | Extended maximum lifespan in middle-aged female mice, prolonged healthspan in both sexes, reduced age-related morbidity, and alleviated chronic inflammation [19]. |
| α-KG | C. elegans | α-KG added to culture | Extended maximum lifespan; α-KG octyl ester precursor reduced ATP synthesis in nematodes [20]. |
Other TCA substrates, though less studied than α-KG, also extend lifespan (mostly in nematode models): Supplementing pyruvate, succinate, fumarate, malate, or oxaloacetate in C. elegans food prolonged their lifespan [21-24].
Acetyl-CoA, as the primary acyl donor in epigenetics [2-4], acts as a "double-edged sword" in aging regulation, with cell compartment-specific effects:
- In mitochondria: Acetyl-CoA maintains TCA flux and mitochondrial homeostasis. Supplementing acetyl-CoA in SAMP8 mice (an accelerated aging model) improved mitochondrial stability and restored cognitive function [25]. Compounds like CMS121 and J147, which boost acetyl-CoA, are candidates for Alzheimer’s treatment.
- In nucleus/cytoplasm: "More is not better":
a) In the nucleus: Acetyl-CoA availability determines histone acetylation. High levels upregulate glucose metabolism, cell cycle, and pro-aging gene expression.
b) In cytoplasm: As a precursor for lipids/proteins and a modifier of cytoplasmic proteins, excess acetyl-CoA causes anabolic overload, suppresses catabolism (including autophagy), and accelerates aging [16,26].
In summary, we reviewed anti- and pro-aging mechanisms of TCA substrates, including α-KG. Due to limited evidence (needing validation in higher organisms), the full picture remains unclear. Key observations include:
- Increased pyruvate influences anti-aging pathways via sirtuins, NAD+, FOXO homolog DAF-16, and AMPK.
- Malate and fumarate promote anti-aging via NAD+/sirtuins.
- α-KG’s benefits relate to inhibiting TOR signaling.
- Cytoplasmic citrate converts to acetyl-CoA; excess accelerates aging.
The network linking TCA cycle and aging continues to expand. Even α-KG’s longevity mechanisms remain debated. Scientists argue: "All roads lead to mitochondria." Age-related TCA cycle abnormalities not only reduce ATP but also disrupt aging-associated signaling outside mitochondria, causing metabolic dysfunction. As research advances, the TCA cycle will increasingly be recognized as a key target for aging intervention [27].
Practical Challenges in TCA Substrate Anti-Aging
Readers may already be planning "morning A, evening B" or age-specific supplementation regimens, but lab evidence is insufficient to support scientific TCA substrate combinations.
Current research mostly stays at the "administer substrate → count lifespan" stage. Mechanisms of transport and metabolism remain unclear, making it hard to ensure oral bioavailability. For example, α-KG faces transport barriers, so oral intake may not act in its native form. Even with protected delivery, feedback regulation complicates supplementation: Abundant NADH slows TCA enzymes; high ATP inhibits pyruvate/isocitrate reactions; excess succinyl-CoA negatively regulates α-KG production; and high oxaloacetate inhibits fumarate production and OXPHOS. Determining which substrates to take and in what doses to restore youthful substrate levels remains elusive.
This "ripple effect" of TCA substrates is a double-edged sword: In youth, it coordinates energy metabolism; in aging, substrates collectively "drift" after long-term compensation. Reliable, unbiased data from more labs—spurred by the recent NAD+/α-KG anti-aging—will be needed to guide targeted TCA substrate interventions.
References
[1] Feng, Z., et al., Reprogramming of energy metabolism as a driver of aging. Oncotarget, 2016. 7(13): p. 15410-20.
[2] Sivanand, S., I. Viney, and K.E. Wellen, Spatiotemporal Control of Acetyl-CoA Metabolism in Chromatin Regulation. Trends Biochem Sci, 2018. 43(1): p. 61-74.
[3] Lee, J.V., et al., Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. Cell Metab, 2014. 20(2): p. 306-319.
[4] Moussaieff, A., et al., Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells. Cell Metab, 2015. 21(3): p. 392-402.
[5] Peng, M., et al., Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science, 2016. 354(6311): p. 481.
[6] Infantino, V., et al., ATP-citrate lyase is essential for macrophage inflammatory response. Biochemical & Biophysical Research Communications, 2013. 440(1): p. 105-111.
[7] Carey, B.W., et al., Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature, 2015. 518(7539): p. 413-6.
[8] Liu, P.S., et al., alpha-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat Immunol, 2017. 18(9): p. 985-994.
[9] Xu, T., et al., Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism. Nature, 2017. 548(7666): p. 228-233.
[10] Hao, H.X., et al., SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science, 2009. 325(5944): p. 1139-42.
[11] Niemann, S. and U. Muller, Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet, 2000. 26(3): p. 268-70.
[12] Baysal, B.E., et al., Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science, 2000. 287(5454): p. 848-51.
[13] Astuti, D., et al., Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet, 2001. 69(1): p. 49-54.
[14] Littlewood-Evans, A., et al., GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis. J Exp Med, 2016. 213(9): p. 1655-62.
[15] Mills, E.L., et al., Accumulation of succinate controls activation of adipose tissue thermogenesis. Nature, 2018. 560(7716): p. 102-106.
[16] Kotra, L.P. and J. Park, Therapeutic Approaches to MS and Other Neurodegenerative Diseases. 2016: Reference Module in Chemistry, Molecular Sciences and Chemical Engineering.
[17] Zhang, Z., et al., alpha-ketoglutarate delays age-related fertility decline in mammals. Aging Cell, 2021. 20(2): p. e13291.
[18] Wang, Y., et al., Alpha-ketoglutarate ameliorates age-related osteoporosis via regulating histone methylations. Nat Commun, 2020. 11(1): p. 5596.
[19] Asadi Shahmirzadi, A., et al., Alpha-Ketoglutarate, an Endogenous Metabolite, Extends Lifespan and Compresses Morbidity in Aging Mice. Cell Metab, 2020. 32(3): p. 447-456 e6.
[20] Partridge, L., M.D. Piper, and W. Mair, Dietary restriction in Drosophila. Mech Ageing Dev, 2005. 126(9): p. 938-50.
[21] Chuang, M.H., et al., The lifespan-promoting effect of acetic acid and Reishi polysaccharide. Bioorg Med Chem, 2009. 17(22): p. 7831-40.
[22] Edwards, C.B., et al., Malate and fumarate extend lifespan in Caenorhabditis elegans. PLoS One, 2013. 8(3): p. e58345.
[23] Edwards, C., et al., Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans. BMC Genet, 2015. 16: p. 8.
[24] Williams, D.S., et al., Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathway. Aging Cell, 2009. 8(6): p. 765-8.
[25] Currais, A., et al., Elevating acetyl-CoA levels reduces aspects of brain aging. Elife, 2019. 8.
[26] Li, X., et al., Regulation of chromatin and gene expression by metabolic enzymes and metabolites. Nat Rev Mol Cell Biol, 2018. 19(9): p. 563-578.
[27] Sharma, R. and A. Ramanathan, The Aging Metabolome-Biomarkers to Hub Metabolites. Proteomics, 2020. 20(5-6): p. e1800407.
[1] Feng, Z., et al., Reprogramming of energy metabolism as a driver of aging. Oncotarget, 2016. 7(13): p. 15410-20.
[2] Sivanand, S., I. Viney, and K.E. Wellen, Spatiotemporal Control of Acetyl-CoA Metabolism in Chromatin Regulation. Trends Biochem Sci, 2018. 43(1): p. 61-74.
[3] Lee, J.V., et al., Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. Cell Metab, 2014. 20(2): p. 306-319.
[4] Moussaieff, A., et al., Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells. Cell Metab, 2015. 21(3): p. 392-402.
[5] Peng, M., et al., Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science, 2016. 354(6311): p. 481.
[6] Infantino, V., et al., ATP-citrate lyase is essential for macrophage inflammatory response. Biochemical & Biophysical Research Communications, 2013. 440(1): p. 105-111.
[7] Carey, B.W., et al., Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature, 2015. 518(7539): p. 413-6.
[8] Liu, P.S., et al., alpha-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat Immunol, 2017. 18(9): p. 985-994.
[9] Xu, T., et al., Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism. Nature, 2017. 548(7666): p. 228-233.
[10] Hao, H.X., et al., SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science, 2009. 325(5944): p. 1139-42.
[11] Niemann, S. and U. Muller, Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet, 2000. 26(3): p. 268-70.
[12] Baysal, B.E., et al., Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science, 2000. 287(5454): p. 848-51.
[13] Astuti, D., et al., Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet, 2001. 69(1): p. 49-54.
[14] Littlewood-Evans, A., et al., GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis. J Exp Med, 2016. 213(9): p. 1655-62.
[15] Mills, E.L., et al., Accumulation of succinate controls activation of adipose tissue thermogenesis. Nature, 2018. 560(7716): p. 102-106.
[16] Kotra, L.P. and J. Park, Therapeutic Approaches to MS and Other Neurodegenerative Diseases. 2016: Reference Module in Chemistry, Molecular Sciences and Chemical Engineering.
[17] Zhang, Z., et al., alpha-ketoglutarate delays age-related fertility decline in mammals. Aging Cell, 2021. 20(2): p. e13291.
[18] Wang, Y., et al., Alpha-ketoglutarate ameliorates age-related osteoporosis via regulating histone methylations. Nat Commun, 2020. 11(1): p. 5596.
[19] Asadi Shahmirzadi, A., et al., Alpha-Ketoglutarate, an Endogenous Metabolite, Extends Lifespan and Compresses Morbidity in Aging Mice. Cell Metab, 2020. 32(3): p. 447-456 e6.
[20] Partridge, L., M.D. Piper, and W. Mair, Dietary restriction in Drosophila. Mech Ageing Dev, 2005. 126(9): p. 938-50.
[21] Chuang, M.H., et al., The lifespan-promoting effect of acetic acid and Reishi polysaccharide. Bioorg Med Chem, 2009. 17(22): p. 7831-40.
[22] Edwards, C.B., et al., Malate and fumarate extend lifespan in Caenorhabditis elegans. PLoS One, 2013. 8(3): p. e58345.
[23] Edwards, C., et al., Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans. BMC Genet, 2015. 16: p. 8.
[24] Williams, D.S., et al., Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathway. Aging Cell, 2009. 8(6): p. 765-8.
[25] Currais, A., et al., Elevating acetyl-CoA levels reduces aspects of brain aging. Elife, 2019. 8.
[26] Li, X., et al., Regulation of chromatin and gene expression by metabolic enzymes and metabolites. Nat Rev Mol Cell Biol, 2018. 19(9): p. 563-578.
[27] Sharma, R. and A. Ramanathan, The Aging Metabolome-Biomarkers to Hub Metabolites. Proteomics, 2020. 20(5-6): p. e1800407.