Introduction

Immortality and longevity have fascinated people for millennia. Like in many other fields of science, one of the major discoveries in the field of longevity was a coincidence. Clive Maine McCay was a professor of animal husbandry. The initial aim of his studies was to investigate factors affecting farm animals' growth to increase yield. As one might expect, his studies showed that reducing calorie intake retards the growth of animals. However, the big surprise was that consuming fewer calories makes animals live longer¹.

Since McCay's discovery in 1930', scientists have confirmed that calorie restriction slows down ageing in almost all species ranging from tiny worms² or flies³ to rats⁴, dogs⁵ and even non-human primates⁶. Human studies showed that calorie restriction dramatically decreases the risk of major age-related diseases like diabetes, cancer or cardiovascular disease⁷. While the positive effect of calorie restriction on health is impressive, there is a catch. People can't adhere to prolonged periods of calorie restriction, and calorie restriction's benefits remain inaccessible.

Thousands of published studies investigate molecular mechanisms by which calorie restriction affects ageing. When we know which molecular processes are activated during calorie restriction, we can develop methods that mimic the effects of calorie restriction without going through the hardship of actually being in a calorie deficit. These methods are called calorie restriction mimetics⁸.

Ketones have a great potential to act as calorie restriction mimetics as they are evolutionarily linked to states of starvation⁹. As we covered in our blog DeltaG and Cognition, ketones serve as an alternative brain fuel to provide efficient brain fuel in times of foraging¹⁰. While the role of ketones as fuel is vital, it is not the only job ketones have. In the last decade, research confirmed that β-hydroxybutyrate (BHB), the ketone body elevated by drinking deltaG, is also a signalling molecule, a molecule which tells cells what to do¹¹.

The signals transmitted by ketones help the body activate molecular processes necessary to survive starvation. These processes spare energy or produce energy more efficiently. Importantly, these processes not only help us survive starvation but also make us more resilient and stand behind the longevity benefits of calorie restriction. In this blog, we will describe how ketones activate many of these molecular pathways and why they make us age slower.

Ketones Promote Stress Resilience

Genetic studies showed that our genes do not predetermine our lifespan. Genes account for about 25% of our lifespan¹², and some studies show even numbers as low as 7%¹³. This is good news as it shows us that a healthier lifestyle matters. However, studies investigating the link between genes and longevity still reveal interesting observations.

Out of ~20000 human genes, only two were shown to correlate with longevity¹⁴. The first, APOE4, dramatically increases the risk of Alzheimer's disease. The second gene is called the FOXO3. The lucky ones among us with the more active variant of the FOXO3 gene tend to live longer. This is an exciting observation because FOXO3 activity is also increased during calorie restriction.

Considering what we know about FOXO3, it makes sense that a more active variant of this gene can slow down ageing. FOXO3 regulates a wide scale of biological processes required for stress response. It stimulates the production of new mitochondria, which are very useful when we starve because they produce energy. However, getting more mitochondria is also beneficial as we age because mitochondrial dysfunction is one of the nine hallmarks of ageing¹⁵.

Age-related mitochondrial dysfunction also leads to greater oxidative stress16. One of the most serious consequences of oxidative stress is oxidative damage of the DNA17. FOXO3 not only activates internal antioxidants but also triggers mechanisms which repair our DNA18.

Now, we have reached the exciting part. A 2013 study¹⁹ showed that treating cells with BHB increases levels of the FOXO3 protein. BHB activates FOXO3 by changing the conformation of proteins called histones which our DNA winds around. BHB unwinds the part of DNA which codes for the FOXO3 gene, and the gene becomes more active. The ability of BHB to activate FOXO3 was shown in model organisms as well²⁰.

BHB likely triggers the activation of FOXO3 during starvation. Moreover, a study with genetically modified mice showed that FOXO3 is required for the lifespan extension induced by calorie restriction because mice lacking the FOXO3 gene did not live longer21 when exposed to calorie restriction. This is clear evidence that FOXO3 is an integral part of the longevity benefits of calorie restriction. Therefore, activating FOXO3 by drinking deltaG can help you unlock many of the benefits mediated by calorie restriction.

Ketones Slow Down Cellular Senescence

Cellular senescence is a stage when a cell's ability to replicate is halted. A cell becomes senescent as a result of accumulated damage²². While senescent cells can still perform their function, the inability to replicate severely affects the capacity of tissues to regenerate. The accumulation of senescent cells leads to overall functional decline associated with ageing²³.

The good news is that BHB was shown to interact with some of the proteins which regulate senescence. One of these proteins is p53²⁴. Activation of p53 induces cellular senescence. BHB can be attached to proteins in a process called β-hydroxybutyrylation and p53 is one of many proteins BHB can be attached to. A 2019 study²⁵ showed that BHB could attenuate the activity of p53. Therefore, BHB should prevent cells from becoming senescent.

The anti-senescence effect of BHB was confirmed in kidney cells²⁶ and vascular cells²⁷. The anti-senescence effect of BHB in vascular cells is especially important. The accumulation of senescent vascular cells compromises the function of our arteries. Senescence is considered a major contributor to atherosclerosis²⁸, the condition characterised by plaque formation in the artery, which can lead to a heart attack.

By delaying cellular senescence, BHB can help to preserve the function of ageing tissues as cells are still able to replicate and regenerate. Moreover, BHB does not only interact with the regulators of senescence; it can also prevent the damage which triggered senescence in the first place. A very common trigger of senescence is DNA damage caused by oxidative stress. As mentioned, BHB can stimulate antioxidant defence and DNA repair by activating FOXO3¹⁹. The decrease of cellular damage is also one of the mechanisms by which calorie restriction prevents cellular senescence²⁹.

Ketones Prevent Age-Associated Inflammation

The problem associated with cellular senescence is not only the functional decline due to cells' inability to replicate. Senescent cells also secrete pro-inflammatory cytokines, collectively called senescence-associated secretory phenotype (SASP)³⁰. When there are only a few senescent cells, these cytokines can be beneficial as they help our immune system to clear senescent cells³¹. However, as we age, the function of our immune system declines and the number of senescence cells increases. As a result, senescent cells secrete more SASP and contribute to age-associated inflammation, also called inflammaging³².

As covered in our blog about deltaG and Metabolic Health, acute inflammation is important for fighting infection or other damage. However, when inflammation becomes chronic, it starts to break havoc. Increased inflammation significantly contributes to major age-related diseases like heart disease, diabetes, cancer and Alzheimer's disease³³. The inflammation associated with senescence is especially dangerous as it can "poison" the cells around and convert them into senescent cells as well³⁴. The newly senescent cells will start to produce more inflammatory SASP, and a vicious cycle of senescence and inflammaging starts to spin quicker and quicker

Evidence suggests that BHB might stop this cycle. A 2018 study³⁵ reported that BHB prevents SASP production in vascular cells. While more work needs to be done in this area, BHB likely blocks the SASP production by inhibiting NLRP3 inflammasome³⁶ and NF-κB³⁷. Both of these proteins are key regulators of inflammation and were shown to promote the production of SASP^38,39. Inhibition of NLRP3 and NF-κB by BHB should alleviate the toxic effects of SASP and prevent the spread of senescent cells.

Ketones Stimulate Autophagy

Autophagy is a process of cellular recycling of proteins. It prevents aggregation of damaged proteins, which would compromise cellular function⁴⁰. Unfortunately, as we age, the process of autophagy becomes less efficient and damaged or misfolded proteins might accumulate in the cell⁴¹. This is a problem because protein aggregates can clutter up the cell and severely affect its function. Loss of proteostasis (the balance between protein synthesis and protein degradation) is recognised as one of the hallmarks of ageing¹⁵, and it is implicated in diseases like Alzheimer's or diabetes⁴².

Autophagy is not only useful as a cellular garbage disposal system. When a protein gets recycled, it is broken down into its basic building blocks – amino acids. These amino acids can be used for building up a new protein, but they can also be converted into glucose and used as a source of energy⁴³. This explains why autophagy is activated by calorie restriction. The cell kills two birds with one stone – autophagy clears up damaged protein, making the cell more efficient, and at the same autophagy generates a new source of energy.

Preventing the age-associated decline of autophagy is an integral part of the longevity benefits induced by calorie restriction. BHB plays an important role in this process. A 2016 study⁴⁴ showed that BHB stimulates autophagy in neurons, and a similar effect was also shown in lung cells⁴⁵. These studies suggest that autophagy activation during calorie restriction might be mediated by BHB. It is also worth noting that FOXO3 is a potent activator of autophagy¹⁸.

Conclusion

The studies in this blog demonstrate that ketones are much more than fuel. BHB stimulates adaptations which help cells to deal with the challenge of energy deficits, such as mitochondrial biogenesis, protection against oxidative stress, DNA repair or autophagy. These adaptations also mediate the longevity benefits of caloric restriction. BHB also mitigates cellular senescence and senescence-associated inflammation, which significantly alleviates the risk of numerous age-related diseases linked to inflammation. The ability of BHB to mimic many of the longevity benefits of calorie restriction makes it a promising anti-ageing tool that slows down the pace of our ageing clocks.

References

McCay, C.M., Crowell, M.F. and Maynard, L.A., 1935. The effect of retarded growth upon the length of life span and upon the ultimate body size: one figure. The journal of Nutrition, 10(1), pp.63-79.
Lakowski, B. and Hekimi, S., 1998. The genetics of caloric restriction in Caenorhabditis elegans. Proceedings of the National Academy of Sciences, 95(22), pp.13091-13096.
Partridge, L., Piper, M.D. and Mair, W., 2005. Dietary restriction in Drosophila. Mechanisms of ageing and development, 126(9), pp.938-950.
Masoro, E.J., 2009. Caloric restriction-induced life extension of rats and mice: a critique of proposed mechanisms. Biochimica et Biophysica Acta (BBA)-General Subjects, 1790(10), pp.1040-1048.
Kealy, R.D., Lawler, D.F., Ballam, J.M., Mantz, S.L., Biery, D.N., Greeley, E.H., Lust, G., Segre, M., Smith, G.K. and Stowe, H.D., 2002. Effects of diet restriction on life span and age-related changes in dogs. Journal of the American Veterinary Medical Association, 220(9), pp.1315-1320.
Mattison, J.A., Colman, R.J., Beasley, T.M., Allison, D.B., Kemnitz, J.W., Roth, G.S., Ingram, D.K., Weindruch, R., De Cabo, R. and Anderson, R.M., 2017. Caloric restriction improves health and survival of rhesus monkeys. Nature communications, 8(1), pp.1-12.
Anderson, R.M. and Weindruch, R., 2012. The caloric restriction paradigm: implications for healthy human aging. American Journal of Human Biology, 24(2), pp.101-106.
Madeo, F., Pietrocola, F., Eisenberg, T. and Kroemer, G., 2014. Caloric restriction mimetics: towards a molecular definition. Nature reviews Drug discovery, 13(10), pp.727-740.
Cahill Jr, G.F., 2006. Fuel metabolism in starvation. Annu. Rev. Nutr., 26, pp.1-22.
Dilliraj, L.N., Schiuma, G., Lara, D., Strazzabosco, G., Clement, J., Giovannini, P., Trapella, C., Narducci, M. and Rizzo, R., 2022. The Evolution of Ketosis: Potential Impact on Clinical Conditions. Nutrients, 14(17), p.3613.
Rojas-Morales, P., Tapia, E. and Pedraza-Chaverri, J., 2016. β-Hydroxybutyrate: A signaling metabolite in starvation response?. Cellular signalling, 28(8), pp.917-923.
Passarino, G., De Rango, F. and Montesanto, A., 2016. Human longevity: Genetics or Lifestyle? It takes two to tango. Immunity & Ageing, 13(1), pp.1-6.
Ruby, J.G., Wright, K.M., Rand, K.A., Kermany, A., Noto, K., Curtis, D., Varner, N., Garrigan, D., Slinkov, D., Dorfman, I. and Granka, J.M., 2018. Estimates of the heritability of human longevity are substantially inflated due to assortative mating. Genetics, 210(3), pp.1109-1124.
Broer, L., Buchman, A.S., Deelen, J., Evans, D.S., Faul, J.D., Lunetta, K.L., Sebastiani, P., Smith, J.A., Smith, A.V., Tanaka, T. and Yu, L., 2015. GWAS of longevity in CHARGE consortium confirms APOE and FOXO3 candidacy. Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences, 70(1), pp.110-118.
López-Otín, C., Blasco, M.A., Partridge, L., Serrano, M. and Kroemer, G., 2013. The hallmarks of aging. Cell, 153(6), pp.1194-1217.
Elfawy, H.A. and Das, B., 2019. Crosstalk between mitochondrial dysfunction, oxidative stress, and age related neurodegenerative disease: Etiologies and therapeutic strategies. Life sciences, 218, pp.165-184.
Hemnani, T.A.R.U.N.A. and Parihar, M.S., 1998. Reactive oxygen species and oxidative DNA damage. Indian journal of physiology and pharmacology, 42, pp.440-452.
Fasano, C., Disciglio, V., Bertora, S., Lepore Signorile, M. and Simone, C., 2019. FOXO3a from the nucleus to the mitochondria: a round trip in cellular stress response. Cells, 8(9), p.1110.
Shimazu, T., Hirschey, M.D., Newman, J., He, W., Shirakawa, K., Le Moan, N., Grueter, C.A., Lim, H., Saunders, L.R., Stevens, R.D. and Newgard, C.B., 2013. Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science, 339(6116), pp.211-214.
Kong, G., Huang, Z., Ji, W., Wang, X., Liu, J., Wu, X., Huang, Z., Li, R. and Zhu, Q., 2017. The ketone metabolite β-hydroxybutyrate attenuates oxidative stress in spinal cord injury by suppression of class I histone deacetylases. Journal of neurotrauma, 34(18), pp.2645-2655.
Shimokawa, I., Komatsu, T., Hayashi, N., Kim, S.E., Kawata, T., Park, S., Hayashi, H., Yamaza, H., Chiba, T. and Mori, R., 2015. The life‐extending effect of dietary restriction requires F oxo3 in mice. Aging cell, 14(4), pp.707-709.
Chen, J.H., Hales, C.N. and Ozanne, S.E., 2007. DNA damage, cellular senescence and organismal ageing: causal or correlative?. Nucleic acids research, 35(22), pp.7417-7428.
Antelo-Iglesias, L., Picallos-Rabina, P., Estévez-Souto, V., Da Silva-Álvarez, S. and Collado, M., 2021. The role of cellular senescence in tissue repair and regeneration. Mechanisms of Ageing and Development, 198, p.111528.
Mijit, M., Caracciolo, V., Melillo, A., Amicarelli, F. and Giordano, A., 2020. Role of p53 in the Regulation of Cellular Senescence. Biomolecules, 10(3), p.420.
Liu, K., Li, F., Sun, Q., Lin, N., Han, H., You, K., Tian, F., Mao, Z., Li, T., Tong, T. and Geng, M., 2019. p53 β-hydroxybutyrylation attenuates p53 activity. Cell death & disease, 10(3), pp.1-13.
Fang, Y., Chen, B., Gong, A.Y., Malhotra, D.K., Gupta, R., Dworkin, L.D. and Gong, R., 2021. The ketone body β-hydroxybutyrate mitigates the senescence response of glomerular podocytes to diabetic insults. Kidney International, 100(5), pp.1037-1053.
Han, Y.M., Bedarida, T., Ding, Y., Somba, B.K., Lu, Q., Wang, Q., Song, P. and Zou, M.H., 2018. β-Hydroxybutyrate prevents vascular senescence through hnRNP A1-mediated upregulation of Oct4. Molecular cell, 71(6), pp.1064-1078.
Minamino, T. and Komuro, I., 2007. Vascular cell senescence: contribution to atherosclerosis. Circulation research, 100(1), pp.15-26.
Fontana, L., Nehme, J. and Demaria, M., 2018. Caloric restriction and cellular senescence. Mechanisms of ageing and development, 176, pp.19-23.
Young, A.R. and Narita, M., 2009. SASP reflects senescence. EMBO reports, 10(3), pp.228-230.
Prata, L.G.L., Ovsyannikova, I.G., Tchkonia, T. and Kirkland, J.L., 2018, December. Senescent cell clearance by the immune system: Emerging therapeutic opportunities. In Seminars in immunology (Vol. 40, p. 101275). Academic Press.
Olivieri, F., Prattichizzo, F., Grillari, J. and Balistreri, C.R., 2018. Cellular senescence and inflammaging in age-related diseases. Mediators of Inflammation, 2018.
Chung, H.Y., Kim, D.H., Lee, E.K., Chung, K.W., Chung, S., Lee, B., Seo, A.Y., Chung, J.H., Jung, Y.S., Im, E. and Lee, J., 2019. Redefining chronic inflammation in aging and age-related diseases: proposal of the senoinflammation concept. Aging and disease, 10(2), p.367.
Ohtani, N., 2022. The roles and mechanisms of senescence-associated secretory phenotype (SASP): can it be controlled by senolysis?. Inflammation and Regeneration, 42(1), pp.1-8.
Han, Y.M., Bedarida, T., Ding, Y., Somba, B.K., Lu, Q., Wang, Q., Song, P. and Zou, M.H., 2018. β-Hydroxybutyrate prevents vascular senescence through hnRNP A1-mediated upregulation of Oct4. Molecular cell, 71(6), pp.1064-1078.
Yamanashi, T., Iwata, M., Kamiya, N., Tsunetomi, K., Kajitani, N., Wada, N., Iitsuka, T., Yamauchi, T., Miura, A., Pu, S. and Shirayama, Y., 2017. Beta-hydroxybutyrate, an endogenic NLRP3 inflammasome inhibitor, attenuates stress-induced behavioral and inflammatory responses. Scientific reports, 7(1), pp.1-11.
Fu, S.P., Li, S.N., Wang, J.F., Li, Y., Xie, S.S., Xue, W.J., Liu, H.M., Huang, B.X., Lv, Q.K., Lei, L.C. and Liu, G.W., 2014. ΒHBA suppresses LPS-induced inflammation in BV-2 cells by inhibiting NF-κ B activation. Mediators of inflammation, 2014
Salminen, A., Kauppinen, A. and Kaarniranta, K., 2012. Emerging role of NF-κB signaling in the induction of senescence-associated secretory phenotype (SASP). Cellular signalling, 24(4), pp.835-845.
Acosta, J.C., Banito, A., Wuestefeld, T., Georgilis, A., Janich, P., Morton, J.P., Athineos, D., Kang, T.W., Lasitschka, F., Andrulis, M. and Pascual, G., 2013. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nature cell biology, 15(8), pp.978-990.
Mizushima, N., 2007. Autophagy: process and function. Genes & development, 21(22), pp.2861-2873.
Ichimiya, T., Yamakawa, T., Hirano, T., Yokoyama, Y., Hayashi, Y., Hirayama, D., Wagatsuma, K., Itoi, T. and Nakase, H., 2020. Autophagy and autophagy-related diseases: a review. International Journal of Molecular Sciences, 21(23), p.8974.
Saha, S., Panigrahi, D.P., Patil, S. and Bhutia, S.K., 2018. Autophagy in health and disease: A comprehensive review. Biomedicine & Pharmacotherapy, 104, pp.485-495.
Bujak, A.L., Crane, J.D., Lally, J.S., Ford, R.J., Kang, S.J., Rebalka, I.A., Green, A.E., Kemp, B.E., Hawke, T.J., Schertzer, J.D. and Steinberg, G.R., 2015. AMPK activation of muscle autophagy prevents fasting-induced hypoglycemia and myopathy during aging. Cell metabolism, 21(6), pp.883-890.
Camberos-Luna, L., Gerónimo-Olvera, C., Montiel, T., Rincon-Heredia, R. and Massieu, L., 2016. The ketone body, β-hydroxybutyrate stimulates the autophagic flux and prevents neuronal death induced by glucose deprivation in cortical cultured neurons. Neurochemical research, 41(3), pp.600-609.
Finn, P.F. and Dice, J.F., 2005. Ketone bodies stimulate chaperone-mediated autophagy. Journal of Biological Chemistry, 280(27), pp.25864-25870.