Dietary restriction in senolysis and prevention and treatment of disease

Abstract Aging represents a key risk factor for a plethora of diseases. Targeting detrimental processes which occur during aging, especially before onset of age-related disease, could provide drastic improvements in healthspan. There is increasing evidence that dietary restriction (DR), including caloric restriction, fasting, or fasting-mimicking diets, extend both lifespan and healthspan. This has sparked interest in the use of dietary regimens as a non-pharmacological means to slow aging and prevent disease. Here, we review the current evidence on the molecular mechanisms underlying DR-induced health improvements, including removal of senescent cells, metabolic reprogramming, and epigenetic rejuvenation.

Box 1. tYPes oF dietarY restriCtion (dr) Caloric restriction (CR): limiting caloric intake to a certain amount per day without malnutrition Intermittent fasting (IF): limitation of energy intake to certain periods, such as 5:2 intermittent fasting (food intake for 5 days, fasting for 2 days), but also including other intermittent fasting schedules such as: • Time-restricted feeding (TRF): Food consumption limited to certain periods of the day, e.g.16:8 (feeding for 8 h a day), or 18:6 (feeding 6 h a day, fasting for the remaining 18).variations of this can define the time during which food is consumed, including eTRF (early trF, feeding restricted to a period during early hours of the day).
• Alternate day fasting (ADF): Fasting and normal food consumption in alternating day patterns Ketogenic diet (KD): High-fat, low-carb (or very low-carb) diet designed to simulate the state of fasting while supplying nutrients and calories; also sometimes referred to as fasting-mimicking diet (FMD).Kd and other FMds constitute special meal plans that do not involve fasting, but instead aim to elicit similar metabolic benefits by restricting access to certain food components.

Box 2. tHe ConCePt oF senolYsis
Composite of the words senescere, to grow old, and lytic, to loosen, unfasten, or destroy, the term senolysis was coined to describe the process of selective elimination of senescent and aged cells. in recent years, senolysis has become a central focus of aging research.it is hypothesized that removal of senescent cells may aid tissue function, delay age-related symptoms, and even increase the healthspan by removing nonfunctional or actively harmful (e.g., via release of cytokines) cells.a variety of studies are ongoing to identify successful senolytic strategies, including small molecules such as dasatinib and quercetin (Kirkland and tchkonia 2020;robbins et al. 2021;Zhu et al. 2015;Chaib, tchkonia, and Kirkland 2022), and lifestyle changes, such as described in this review.The accumulation of senescent cells in tissues occurs in response to many different endogenous triggers, such as age-related dysfunction of cell-mediated clearance, mitochondrial signaling, epigenome and chromatin organization, cytokines and chemokines, (Yousefzadeh et al. 2021;Ovadya et al. 2018), or exogenous triggers such as obesity (Wang et al. 2009;Shirakawa et al. 2016;Palmer et al. 2019) and cigarette smoking (Nyunoya et al. 2006;Paschalaki, Starke, Mercado, et al. 2013;Paschalaki, Starke, Hu, et al. 2013;Baskara et al. 2020).A well-functioning immune system is critical for healthy aging, and senescent cells may impair healthy aging and cause disease via the immune axis (Hashimoto et al. 2019).In line with the wider field of aging research, senescent cell burden has become relevant over the last decade due to an increase in human lifespan -prior to this, the majority of individuals did not live long enough to accumulate a detrimental critical mass of senescent cells.
Senescence is traditionally thought of as a tumor-suppressive mechanism, rendering cells that have encountered genotoxic stresses as non-proliferative (Sikora, Bielak-Zmijewska, and Mosieniak 2019).Moreover, cellular senescence has vital roles during wound healing and development, both of which are two molecular processes that must be tightly spatially and temporally coordinated.Senescence is a programmed but self-limiting response during optimal wound repair, ensuring generation of "appropriate" amounts of fibrosis and remodeling of the extracellular matrix (Jun and Lau 2010a), for instance via release of platelet-derived growth factor AA (Demaria et al. 2014).Notably, dysregulation of the timing or amount of senescence may cause loss of tissue function and impaired wound healing.Programmed senescence also plays a vital role in morphogenesis during vertebrate development, modulating tissue remodeling and patterning (Muñoz-Espín et al. 2013;Storer et al. 2013;Davaapil, Brockes, and Yun 2016;Villiard et al. 2017).Yet, the role of senescent cells is not exclusively beneficial, particularly with increasing age.While senescent cells can protect from malignancies modulators, growth factors, and proteases into the tissue microenvironment, have been shown to elicit bystander senescence and cause stem cell exhaustion (Acosta et al. 2013;Hubackova et al. 2012;Chang et al. 2016;Molofsky et al. 2006), contribute to expansion of preneoplastic cells (Krtolica et al. 2001;Ohanna et al. 2011;Lasry and Ben-Neriah 2015;Coppé et al. 2008;Gonzalez-Meljem et al. 2017), drive tumorigenesis (Gonzalez-Meljem et al. 2018;Ruhland et al. 2016), and increase cancer invasiveness (Ghosh et al. 2020;Benítez et al. 2021).Krtolica et al. have termed this duality of senescent cell function -both tumor-suppressive and tumor-driving -as an example of evolutionary antagonistic pleiotropy (Krtolica et al. 2001).
The diverse roles of senescent cells and SASP in tumorigenesis were recently summarized by Wang et al. (Wang, Lankhorst, et al. 2022).
Age-related diseases, potentially caused by accumulation of senescent cells, may exacerbate age-related dysfunction, and accrete further disease.For example, recently it was shown that Alzheimer's disease led to impaired mitochondrial function and increased inflammation and resulted in decreased cardiac contractility in mice (Murphy et al. 2022).Controlling senescence-associated inflammation and clearance of senescent cells have been proposed as means for both prevention and treatment of cancer and other age-related diseases (Lasry and Ben-Neriah 2015;Kirkland and Tchkonia 2017;Wang, Lankhorst, et al. 2022).Depletion of senescent cells has been shown to exert anti-aging effects on stem cells in mice (Chang et al. 2016;Xu et al. 2015).Furthermore, depletion of genes or proteins involved in cell cycle regulation and senescence, such as CDKN2A (thereafter referred to as p16INK4a [gene] or p16INK4a [protein]), can rejuvenate aged muscle stem cells (Sousa-Victor et al. 2014; S. R. Kim et al. 2021), indicating a potential for reversal of senescence features.Consistent with this, removal of senescent cells has been shown to increase lifespan and delay onset of age-associated diseases, including cancer, in various mouse models (Baker et al. 2011;Baker et al. 2016;Demaria et al. 2014).DR has been proposed to achieve its beneficial effects on lifespan, health, and cancer reduction, at least in part, by reducing or removing senescent cells (Fontana, Nehme, and Demaria 2018;Longo and Cortellino 2020;Cheng et al. 2014) and may thus provide an attractive strategy for non-pharmacological disease prevention.
Pharmacological agents targeting senescent cells in the context of human disease are also of increasing interest.Conceptually, there is a distinction between agents that target senescence via removal of senescent cells (senolytics) or via modulation of the senescence-associated phenotype (senomorphics).Common senolytics include dasatinib (D) and quercetin (Q), both of which are BCL-2 family inhibitors, and fisetin, a naturally occurring flavonoid (Yousefzadeh et al. 2018).Senomorphics include agents such as rapamycin, metformin, or resveratrol, a natural compound found in red grape skins and other food sources (Zhang, Pitcher, Prahalad, et al. 2022).A key difference in senolytics versus senomorphics is that senolytics could be used for intermittent dosing regimens as they remove the presumed underlying cause (accumulation of senescent cells), whereas senomorphics do not directly remove the underlying senescent cell burden and their effect is likely dependent on continuous presence of the senomorphic agent.There is a number of ongoing human studies investigating senolytics and senomorphics (Zhang, Pitcher, Yousefzadeh, et al. 2022;Gasek et al. 2021) tin]).The above list of trials is non-exhaustive, and a more detailed description of ongoing trials and agents is provided by Zhang, Pitcher, Yousefzadeh, et al. (2022) and Gasek et al. (2021).
In Table 3, we have compiled information on a number of recent murine studies addressing the role of senescent cells and their elimination in health, longevity, and disease.A comprehensive list of additional preclinical studies utilizing specifically pharmacological/small molecule agents to target senescent cells is provided by two recent publications by Zhang, Pitcher, Yousefzadeh, et al. 2022 andZhang, Pitcher, Prahalad, et al. 2022).

Epigenetics of aging
Aging is associated with distinct epigenetic changes, such as alterations in DNA methylation (DNAme), histone modification, and chromatin remodeling.Age-dependent altered methylation of cytosine (5-methyl-C) in the CpG context, which includes a global decrease in methylation (hypomethylation) in repetitive genomic regions and interspersed elements (Bollati et al. 2009;Jintaridth and Mutirangura 2010) and increased methylation (hypermethylation) in promoter regions, is one of the most striking hallmarks of aging and may be a useful biomarker of aging and healthspan (Horvath and Raj 2018).Age-associated hypermethylated promoter regions are frequently found at tumor suppressor genes (Siegmund et al. 2007) and genes involved in cellular fate and differentiation (e.g., polycomb group target genes, PCGTs) (Maegawa et al. 2010;Teschendorff et al. 2010;Rakyan et al. 2010).Deregulation of epigenetic control with age is associated with progressive diseases such as cancer and diabetes (Egger et al. 2004).
While causality is not yet proved, several lines of evidence point to a significant contribution of epigenetic alterations to the aging process: 1) methylation alterations cause chromosomal instability (Esteller and Herman 2002) and contribute to gene expression alterations, transcriptional 'noise' , and increased transcriptional cell-to-cell variability associated with age and/or cancer (Hernando-Herraez et al. 2019); 2) the aging process in offspring can be modulated by epigenetic alterations accumulated in a parent (e.g., the offspring of younger paternal mice develop aging phenotypes later and live longer than the offspring of aged paternal mice (Xie et al. 2018)); and 3) during cellular reprogramming, amelioration of age-associated phenotypes is observed, highlighting a role for epigenetic remodeling as a driver for aging (Ocampo et al. 2016).Global hypomethylation has been suggested to result in chromatin instability (Esteller and Herman 2002) and has been correlated with frailty in elderly patients (Bellizzi et al. 2012) and increased tumor incidence in mice (Howard et al. 2008).Lastly, the finding that cancers exhibit methylation changes in genes associated with age and stem cell signatures (e.g., PCGTs) (Widschwendter et al. 2007) has led to the progenitor model: epigenetic deregulation, inter alia through age or senescence, can render cells more likely to develop cancer.
Although epigenetic alterations are associated with, and possibly causal for, aging and cancer, DNAme exhibits remarkable plasticity.Age-and senescence-associated DNAme changes can be reversed in vitro by reprogramming to pluripotent (Koch et al. 2013;Frobel et al. 2014;Horvath 2013;Weidner et al. 2014) or multipotent stem cells (Sheng et al. 2018), and DNAme rejuvenation in vivo has been achieved by environmental enrichment or partial reprogramming in mice (Browder et al. 2022) and dietary interventions in humans (Gensous et al. 2020).
Above, we primarily discuss age-associated changes relating to DNAme, yet many other forms of epigenetic changes with age have been described.For instance, aging changes histone levels and nucleosome occupancy, distribution and utilization of histone variants, and histone modification (e.g.acetylation and methylation) (Yi and Kim 2020;Pal and Tyler 2016).An altered chromatin structure and loss of heterochromatin with aging can moreover lead to activation of transposable elements (Pal and Tyler 2016; Andrenacci, Cavaliere, and Lattanzi 2020; Cecco et al. 2013).A detailed discussion of all age-associated epigenetic changes is beyond the scope of the current review, but excellent articles (including, but not limited to, the abovementioned) are available as further reading (Zhang et al. 2020;Wang et al. 2022).

Molecular mechanisms of DR and healthspan extension
DR without malnourishment can produce beneficial metabolic effects such as reduction of plasma glucose levels and induction of ketosis.The mechanisms by which DR delays or inhibits aging are not fully understood, but DR appears to simultaneously influence multiple cellular pathways.
Growing evidence indicates that ketone bodies, especially β-hydroxybutyrate (βHB) as the predominant ketone body in blood, are major mediators of the benefits of CR, fasting, and KD, and are involved in several "anti-aging" mechanisms.In the following section, we highlight potential molecular mechanisms underlying the anti-senescent effects of DR and provide further details from preclinical and clinical studies in Tables 1 and 2.

Lysosomes and autophagy
Lysosomes are specialized organelles involved in the breakdown of macromolecules via the process of autophagy and undergo prominent senescence-related changes (White, Mehnert, and

Mitochondrial dysfunction and reactive oxygen species
Senescent cells accumulate damaged mitochondria (Park et al. 2018;Vernier and Giguere 2021).Peroxisome proliferator-activated receptors (PPARs), in coordination with coactivators such as PPARγ coactivator 1α (PGC-1α), regulate mitochondrial function and biogenesis (Duszka et al. 2020), and aberrant PPAR/PGC-1α activity is considered to be the main reason for impaired mitochondrial bioenergetics and function in senescent cells (Vernier and Giguere 2021;Duszka et al. 2020).Mitochondrial dysfunction induces the generation of reactive oxygen species (ROS) and can lead to oxidative stress, contributing to aging as well as a variety of pathologies such as diabetes, cancer, and cardiovascular and neurodegenerative disease (Duszka et al. 2020).While free radicals may somewhat contribute to disease, the free radical theory of aging (later oxidative stress theory of aging (Lin and Beal 2003)) has been cast into doubt by results from several animal studies: overexpression of antioxidant genes has been found to have little influence on lifespan (Pérez et al. 2009).One exception to this were findings in an animal model with overexpression of catalase targeted to mitochondria (mCAT) (Schriner et al. 2005), which suggested that specific targeting of antioxidants to mitochondria may be beneficial for healthspan.Further work to dissect the role mitochondria-targeted interventions in the framework of a new "mitochondrial free radical theory" in aging (Dai et al. 2014) is required.βHB treatment of myoblasts and cardiomyocytes in vitro results in improvements in mitochondrial function and alleviation of oxidative stress (Parker et al. 2018;Deng et al. 2021;Liu et al. 2020).More generally, DR has been shown to restore mitochondrial function and thereby ameliorate signs and symptoms of aging (Yu et al. 2020;López-Lluch et al. 2006;Zhou et al. 2021;Duszka et al. 2020;Hegab et al. 2019;Redman et al. 2018), part of which are mediated by upregulation of PPAR and PGC-1α (Hasan-Olive et al. 2019; Duszka et al. 2020).DR may also alleviate oxidative stress via upregulation of nuclear factor-erythroid 2-related factor (NRF2), a primary sensor of cellular stress and regulator of the expression of a range of enzymes with important detoxification and antioxidant functions (Lettieri-Barbato et al. 2020;Vasconcelos et al. 2019;Martin-Montalvo et al. 2011).Nonetheless, Xu et al. recently reported that long-term KD and βHB may in fact reduce mitochondrial biogenesis and increase cardiac fibrosis in human heart tissue (Xu et al. 2021), so further research is needed to untangle the effects of DR on mitochondrial function.

Metabolic hormones
Age-related disorders are often associated with abnormal secretion and signaling of various metabolic hormones, including insulin and insulin-like growth factor (IGF-1) (Kolb et al. 2020;Ferreira 2021;Rose and Vona-Davis 2012).Insulin and IGF-1 act on glucose homeostasis by promoting cellular glucose uptake, regulate the carbohydrate, lipid, and protein metabolism (Clemmons 2012;Petersen and Shulman 2018), and enhance cellular proliferation via insulin/IGF1 receptor signaling (Hakuno and Takahashi 2018;Petersen and Shulman 2018).Numerous preclinical and clinical studies have identified a link between insulin secretion deficiency, insulin resistance, and hyperinsulinemia, which is a major cause of age-related diseases like cancer and diabetes (Bartke 2019;Shou, Chen, and Xiao 2020;Pak et al. 2021).DR lowers the secretion of both insulin and IGF-1 by limiting glucose intake and restraining the glucose metabolism (Urbain et al. 2017;Newman et al. 2017;Hopkins et al. 2018;Stubbs et al. 2018).DR can therefore improve insulin resistance (Cho et al. 2019;Forsythe et al. 2008;Sutton et al. 2018;Albosta and Bakke 2021).A recent study found while both CR and KD reduce blood glucose and insulin levels, only CR was able inhibit the growth of pancreatic tumor allografts in mice (Lien et al. 2021): the authors propose that a metabolic shift in the lipid metabolism and the reduced availability of lipids in the diet may drive the anti-tumor effects of CR.While KD also induced this metabolic shift, the increased availability of dietary fats abrogated the tumor growth-inhibiting effects.In contrast to the study by Lien et al., findings from other pancreatic mouse models showed that KD in combination with chemotherapy suppressed tumor growth by lowering insulin and glucose and increasing βHB levels (Yang et al. 2022).In addition to the latter study, several other studies also reported that KD can exert an anti-tumor effect even in the absence of an effect on plasma glucose levels (Weber et al. 2020).This raises the question of whether the composition of lipids in the KD may suppress tumor growth or whether other factors such as the type of animal model or tumor type could lead to different responses to the KD.
Ames (Prop df ) and Snell (Pit1(dw)) dwarf mice carry recessive mutations in pituitary genes resulting in a lack of growth hormone (GH) and have been found to exhibit an increased lifespan, indicating a disruption of the growth hormone axis could delay aging and/or promote longevity (Brown-Borg et al. 1996;Flurkey et al. 2001).Interestingly, the lifespan of Ames mice can be further extended by CR (Bartke et al. 2001), but supplementation of GH abrogates the beneficial effects of CR in both Ames and wild type mice (Gesing et al. 2014;Bonkowski et al. 2006).GH treatment during the early postnatal period in Ames mice can reduce lifespan, suggesting that early-life priming may result in metabolic or molecular memories that can, in part, explain developmental origins of aging phenotypes and disease (Sun et al. 2017).
The ratio of the adipose tissue hormones adiponectin and leptin has been proposed as a biomarker of adipose tissue dysfunction.High leptin and low adiponectin plasma levels are associated with aging and obesity (Filippi and Lam 2014;Balasko et al. 2014;Goktas et al. 2005) as well as a poor prognosis for various malignancies such as breast, colon, and prostate cancer (Garofalo and Surmacz 2006;Artac and Altundag 2012).IF, ADF, and CR reduce circulating leptin (Balasko et al. 2014;Cho et al. 2019;Varady et al. 2013) and increase adiponectin levels (Varady et al. 2013;Cui et al. 2020;Wan et al. 2010;Varady et al. 2010), although some studies have found no effect on adiponectin levels (Varady et al. 2007;Gavrila et al. 2003;Rogozina et al. 2011).KD alongside regular exercise increased the ratio of adiponectin to leptin in adults (Cipryan et al. 2021).While long-term KD alone altered the levels of leptin in children and adolescents (Amicis et al. 2019), short-term KD increased the levels of adiponectin in obese adults (Monda et al. 2020).
The effects of DR on metabolic hormone levels appear to be strongly dependent on the type of DR, and further research will be required to pinpoint the exact roles hormones have in mediating the beneficial effects of DR.

Cellular integrators of energy, nutrients, and growth factor signals: AMPK and mTOR
Various studies have shown that the AMPK-mTOR pathway is associated with longevity and senescence (Weichhart 2018).In response to bioenergetic stress, AMPK upregulates numerous catabolic pathways (e.g., fatty acid oxidation) to restore cellular ATP levels and modulates the activity of mTOR, an intracellular nutrient sensor that regulates protein synthesis, cell growth, metabolism, and inflammation (Weichhart 2018).Interestingly, Jordan et al. reported that AMPK activation not only coordinated the metabolic adaptation to fasting, but also regulated the pool of circulating inflammatory cells (Jordan et al. 2019).
During periods of nutrient deficiency, AMPK activation results in inhibition of mTOR (Xu, Ji, and Yan 2012).As DR induces a state of metabolic stress, it has been hypothesized that DR results in mTOR inhibition, but recent studies have painted a more complex picture, revealing that KD-induced downregulation of mTOR is independent of AMPK (Genzer et al. 2015).KD reduces the levels of essential amino acids (Aminzadeh-Gohari et al. 2017;Douris et al. 2015;Roberts et al. 2016;Weber et al. 2022), a process which may be involved in mTOR inhibition, in particular via downregulation of leucine and arginine (Weichhart 2018;Sheen et al. 2011).The effect of DR on the AMPK-mTOR pathway may vary by tissue.Murine studies showed that KD-induced bioenergetic stress activated AMPK in neuroblastoma or liver cells, reduced AMPK activation in retinal cells, and had no effect on muscle or brain cells (Aminzadeh-Gohari et al. 2017;Harun-Or-Rashid and Inman 2018;McDaniel et al. 2011).Similarly, mTOR activity in murine muscle tissue is increased by KD (Roberts et al. 2017;You et al. 2020), while it is decreased in the liver (Roberts et al. 2017;Genzer et al. 2015;McDaniel et al. 2011;Newman et al. 2017) and brain (Singh et al. 2018;Genzer et al. 2015;McDaniel et al. 2011).

Sirtuins: NAD + -sensitive metabolic sensors
The sirtuin family (SIRT1-7) consists of evolutionarily conserved nicotinamide adenine dinucleotide (NAD + )-dependent lysine deacetylases involved in a variety of biological processes, including aging, cell survival and proliferation, apoptosis, DNA repair, and metabolism (Kratz et al. 2021).NAD + is a substrate for all sirtuins.Reduced levels of both are observed in aging (Covarrubias et al. 2021;Akter et al. 2021).Several studies have indicated that the anti-aging effects of DR may be associated with an induction of sirtuins (Tang et al. 2021;Ma et al. 2020;Lilja et al. 2021;Palacios et al. 2009).Both in yeast and mammals, homologues of sirtuin SIRT2 mediate the lifespan-extending effects of CR (Imai et al. 2000;Mercken et al. 2014).Overexpression of Sirt1 mimics CR and delays aging in mice (Satoh et al. 2013;Bordone et al. 2007).Deficiency of SIRT6 leads to a shortened lifespan in mice and non-human primates, whereas SIRT6 overexpression and CR-induced SIRT6 activation both delay aging phenotypes (Kanfi et al. 2012).Similarly, a decrease in SIRT7 significantly attenuated the anti-tumor effects of IF (Tang et al. 2021).
Upregulation of NAD + and sirtuins has been a topic of substantial interest for the prevention of age-related diseases (Braidy and Liu 2020;Elamin et al. 2017;Qin et al. 2006;Kane and Sinclair 2018;Akter et al. 2021;Yoshino et al. 2021).Following promising preclinical results, boosting NAD + levels (and, subsequently, sirtuins) via supplementation has become a topical strategy to combat aging.Several clinical studies are currently ongoing to investigating safety and tolerability of NAD + supplements, such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) (Reiten et al. 2021).DR may provide an alternative path to induce sirtuin levels: for example, SIRT1 and SIRT3 were found to be increased in blood after five days of periodic fasting in humans (Lilja et al. 2021).

The immune system and inflammation
With advancing age, the immune system undergoes devitalizing changes, resulting in greater susceptibility to infection, inflammation, and autoimmunity (Ferrucci and Fabbri 2018;Muller, Benedetto, and Pawelec 2019).Individuals with age-related diseases have significantly higher serum levels of proinflammatory cytokines and chemokines.DR has been shown to reduce inflammation associated with inflammatory and autoimmune diseases without weakening the immune system against infections.For instance, KD increased the survival of COVID-infected mice by increasing tissue-protective T cells, reducing inflammation, and decreasing the number of pathogenic monocytes in the lungs (Ryu et al. 2021).Interestingly, like DR, treatment with fisetin, a naturally occurring senolytic compound that reduces senescent cell burden, has been shown to improve survival following SARS-CoV-2 challenge in old mice, indicating that removal of senescent cells could improve age-associated inflammation (Camell et al. 2021).Fisetin is now being explored for the prevention of COVID-19 complications in human clinical trials (NCT04771611, NCT04537299, and NCT04476953).
DR elicits immune-supportive responses and ameliorates inflammatory and autoimmune diseases, suggesting it may act in part as an immune adjuvant.DR stimulates lymphocyte-dependent killing of cancer cells (Buono and Longo 2019) and several in vitro and murine studies indicate that DR modulates the inflammatory response by reducing the levels of circulating pro-inflammatory cytokines (Forsythe et al. 2008;Moro et al. 2016;Faris et al. 2012;Goldberg et al. 2017;Youm et al. 2015;Lu et al. 2018;Monda et al. 2020; Harun-Or-Rashid and Inman 2018) and monocytes (Jordan et al. 2019).Furthermore, DR inhibits NOD-like receptor protein 3 (NLRP3)-inflammasome activation, an essential cytosolic regulator of innate immunity, in peripheral macrophages, neutrophils (Goldberg et al. 2017) and monocytes (Youm et al. 2015).Again, βHB appears to be a key mediator of the effects of DR on the immune system: βHB has been shown to reduce systemic inflammation via suppression of NLRP3-inflammasome formation in human monocytes (Youm et al. 2015), neutrophils (Goldberg et al. 2017) and cardiomyocytes (Youm et al. 2015;Byrne et al. 2020) as well as in rodent models of spinal cord injury, chronic unpredictable stress, and progressive eye abnormalities (Qian et al. 2017;Yamanashi et al. 2017;Harun-Or-Rashid and Inman 2018).Murine studies demonstrated that DR and βHB induce T cell-dependent anti-cancer effects, which result in cancer immunosurveillance and synergize with immune checkpoint blockade (Ferrere et al. 2021;Lussier et al. 2016).

Epigenetic modulation by DR
While the exact mechanisms linking age, diet, DNA methylation and senescent cells are not yet clear, it is evident that DR increases lifespan, reduces the accumulation of senescent cells (Fontana, Nehme, and Demaria 2018;Messaoudi et al. 2006), and influences (age-related) methylation signatures (current literature summarized in (Ng et al. 2019)).Both DR and mTOR-interference, which influence similar pathways, delay age-related DNAme signatures or drift in mice and rats (Cole et al. 2017;Miyamura et al. 1993;Hadad et al. 2018;Kim et al. 2016;Hahn et al. 2017;Maegawa et al. 2017).CR in Daphnia magna leads to DNAme changes of genes involved in methylation, providing an epigenetic feed-forward mechanism (Hearn et al. 2019), and DR has been shown to epigenetically reprogram lipid metabolism in mice (Hahn et al. 2017).Recent studies provide insights into the mechanistic links between DR and epigenetic alterations: βHB acts as an endogenous histone deacetylase (HDAC) inhibitor (Shimazu et al. 2013) and modifies the expression of genes involved in DNAme, including sirtuins.Sirtuins modify DNAme to prevent premature activation of inflammatory genes in immune cells (Li et al. 2020), and regulate DNAme and differentiation potential in stem cells by antagonizing DNA methyltransferase (DNMT) (Heo et al. 2017).Sirtuins also influence DNAme at PCGT promoters (Wakeling et al. 2015;Furuyama et al. 2004), which are strongly implicated in aging and cancer (Widschwendter et al. 2007).Sirtuin expression therefore could provide a link between diet, the epigenome, and longevity/senescence, and ultimately health and disease.
Cellular metabolism and the epigenome are tightly linked (Finkel 2015), and the effects of DR interventions appear to result, at least in part, from the prevention or reversal of age-associated DNAme changes (Zhang et al. 2020).Glucose restriction in mice increases DNMT1 activity and triggers p16INK4a gene promoter hypermethylation, thereby reducing the expression of the senescence-associated protein p16INK4a (Li, Liu, and Tollefsbol 2010).Another strand of evidence for potential involvement of epigenetics in mediating the effects of DR comes from a recent study in transgenerational inheritance of longevity in Caenorhabditis elegans.Exposure of C. elegans to transient fasting (TF) influences mortality not only in the exposed generation (parental, P0), but also in at least four descendant generations (F1-F4) (Ivimey-Cook et al. 2021).Longevity can be inherited via epigenetic factors in C. elegans, which have been shown to exhibit a specialized type of DNAme (Greer et al. 2015;Greer et al. 2011).These data raise the possibility that epigenetic changes caused by DR could be inherited.Importantly, Ivimey-Cook et al. found that whereas TF reduced mortality in the P0 generation, it increased mortality in the F4 generation.While the mechanisms underlying this phenomenon have not yet been explored, the findings suggest a potential need to consider the health of offspring in the pursuit of longevity.
We may also gain insights into the molecular mechanisms contributing to DR-associated clearance of senescent cells and DNAme changes by observations of other 'geroprotective' therapeutics (i.e., therapeutics protecting from aging) that target similar pathways.For example, metformin, a widely prescribed antidiabetic drug, has been found to target several molecular mechanisms of aging and increase healthspan (Piskovatska et al. 2020), and epidemiological evidence suggests it can also reduce the incidence of cancers (Zhang et al. 2013;Kasznicki, Sliwinska, and Drzewoski 2014).Like DR (Lei and Lixian 2012;Weir et al. 2017), metformin treatment activates AMPK, and its antineoplastic effect may be mediated via modulation of the mTOR signaling pathway and DNAme (Pernicova and Korbonits 2014; Zhong et al. 2017;Yan et al. 2020).Metformin has been suggested to represent a metabolo-epigenetic regulator linking cellular metabolism to the DNA machinery (Cuyàs et al. 2018).Another geroprotective drug, rapamycin, which directly targets the mTOR pathway, extends lifespan in mice, even when animals are treated later in life (Harrison et al. 2009), and slows accumulation of epigenetic aging signatures in mouse hepatocytes similar to CR (Wang et al. 2017).Interestingly, however, two recent studies found distinct transcriptomic profiles between long-term CR and rapamycin treatment (Ham et al. 2022;Orenduff et al. 2022), as well as additive effects on counteracting muscle loss, potentially opening the option to parallel interventions counteracting age-related events.
In summary, DR may promote epigenetic rejuvenation and thus induce longevity and reduce cancer risk (Topart, Werner, and Arimondo 2020;Sen et al. 2016;Zhang et al. 2020).It is currently not clear whether DR reduces the proportion of aged and/or senescent cells, for instance by reduced formation of senescent cells, or a suppression of the senescent phenotype e.g.acting at an epigenetic level on individual cells to promote "epigenetic" rejuvenation.It is likely a combination of both: e.g., CR could trigger epigenetic rejuvenation of immune cells to accelerate clearance of senescent cells.An initial insight into the effects of CR and cellular aging on individual cells has been provided by Ma et al., who found that CR attenuates age-associated cell type-specific gene expression changes and relieves the accumulation of pro-inflammatory cells in various tissues (Ma et al. 2020).

Not only what we eat, but when: merging roles of timing on the effects of DR
Recent studies dissecting the effects of calorie intake, fasting, and type of diet on DR have shed further light on what factors may mediate the beneficial effects on survival.In CR, the beneficial and anti-tumor effects have been proposed to be a result of reduced calorie intake, but a recent study by Pak et al. has overturned this long-held belief (Pak et al. 2021).CR in rodents is typically carried out using once-a-day feeding, resulting in fasting periods of up to 22 hours.The authors found that the same number of calories delivered by feeding three times a day rather than once (removing the effect of fasting) abrogated the beneficial effects of CR on metabolic health and longevity, suggesting fasting is required.Fasting alone also recapitulated many of the beneficial metabolic effects of CR (Pak et al. 2021).Timing of feeding and fasting also appears to be crucial.CR has been shown to be particularly successful in extending lifespan in mice when the animals fasted for at least 12 h during rest phases and consumed food during the active phase, "aligning" the feeding and fasting patterns to circadian rhythms (Acosta-Rodríguez et al. 2022).A recent study in humans investigating the effects of restricting feeding to 8 h either in the morning or the middle of the day found that only "early", but not "mid-day", time-restricted feeding produced beneficial effects on insulin sensitivity (Xie et al. 2022).In line with these findings, results from a 2-year CR study in humans (CALERIE trial) indicated that food consumption occurring earlier in the day (as opposed to later in the day) and smaller windows (i.e.longer fasting periods) were associated with bigger weight loss (Fleischer et al. 2022).
While both daily CR and a FMD regimen using caloric cycling (4 days severe CR, 10 days ad libitum feeding) reduced breast cancers in a mouse model, the protective effect was significantly higher in the daily CR group (Pomatto-Watson et al. 2021).A study in Drosophila melanogaster showed that distinct TRF schedules prolonged life and highlighted the need for further in-depth study of different DR mechanisms: the authors defined a life-and healthspan-extending schedule of TRF, but also identified that other schedules either reduced lifespan or did not alter lifespan at all (Ulgherait et al. 2021).Importantly, the lifespan-extending effects of TRF were dependent on an intact circadian clock, in line with other studies that suggest timing of feeding is crucial for the beneficial effects of DR.Thus, evidence from humans, mice, and Drosophila suggests that DR-induced anti-aging effects are linked to circadian rhythms (Singh et al. 2020;Pak et al. 2021;Jamshed et al. 2019).

Limitations of current animal models and experimental approaches to study aging, and translation to human health
Many studies investigating aging have traditionally investigated lifespan or specific pathologies associated with age, yet it is important to distinguish the (albeit) fine line between age-associated specific pathologies or the general decline of physiological systems.For instance, neoplasms account for up to 90% of natural age-related deaths in laboratory mouse strains (Xie, Fuchs, et al. 2022) and hence "anti-aging" therapies that target neoplasms may therefore influence lifespan without influencing the aging process itself.Lifespan may therefore not always be a reliable proxy for aging.By comparing effect sizes of putative anti-aging interventions on a variety of biological parameters in both young and aged mice, a recent study indicated that many of these "anti-aging" interventions may in fact not target aging itself but other molecular pathways linked with pathology: many of the "anti-aging" effects of IF already manifested in young mice, at a time when no aging phenotypes were observed (Xie, Fuchs, et al. 2022).The authors concluded that IF may therefore target pathology-associated pathways rather than aging.These results indicate that stronger markers for aging and a dissection of underlying molecular and phenotypic changes is required for future studies.
Another limitation of many current studies investigating various DR is the common use of ad libitum feeding in controls.Compared to wild animals, ad libitum feeding in laboratory animals essentially constitutes a form of overfeeding, and hence is likely not comparable to a "normal feeding" state.It may overestimate the effects of DR on lifespan and pathology.Future studies including controls that more closely mimic normal feeding states are warranted (Feige-Diller et al. 2020).
Lastly, whether observations on changes in lifespan and disease following DR in various animal models can be translated to humans remains to be seen.In Table 4, we provide an overview of molecular findings from animal models that have -or have not -been confirmed in humans.As for longer-term and/or disease outcomes, a recent meta-analysis suggested that evidence for a beneficial effect of intermittent fasting is moderate to strong for weight loss but limited for other outcomes such as cardiovascular disease (Patikorn et al. 2021).Due to the much longer lifespan and many individual lifestyle factors of humans compared to laboratory animals that are kept in a tightly controlled environment, human studies investigating mortality outcomes are extremely challenging to virtually impossible, and only few studies so far have sufficient duration, participants, or compliance with the intervention to provide deep insights into key phenotypes such as cardiovascular, cancer, or mortality outcomes.Future studies investigating either disease outcomes or reliable "surrogate" markers of disease in humans are warranted.

Conclusion and future questions
There is increasing evidence that DR reduces the accumulation of senescent cells and improves healthspan and lifespan.We propose that DR-associated senolysis may be achieved in part via metabolic reprogramming and epigenetic rejuvenation (summary and graphical overview in Figure 1).While we welcome the increasing interest in dietary, non-pharmacological approaches in longevity and prevention research, we emphasize the urgent requirement for further research to fully understand both the beneficial and potential harmful effects, including to future generations of offspring, and to further dissect the underlying molecular mechanisms.In particular, many preclinical studies to date have been carried out on male animals only (see Table 1), and more needs to be done to understand the effects of DR in females in relation to sex hormones and their fluctuation.
A hindrance in the interpretation of current studies is the lack of comparability between highly variable DR regimens and their effects in different species.More research is required to identify robust markers of the beneficial effects of DR, for instance on clearance of senescent cells, which could provide universal markers of DR efficacy.Circadian rhythms are likely to play a major role and timing of sampling may also influence results.Additionally, new research is required to understand the effects of DR on individual cells.Undoubtedly, future studies will assess senescence using single-cell technologies to gain a better understanding of the role of cellular heterogeneity and its relationship to dietary interventions.Cellular and molecular profiling of the effects of DR may enable the development of novel non-pharmacological strategies for longevity and disease prevention.

Figure 1 .
Figure 1.Schematic overview of involvement of senescent cells in aging and how DR may aid disease prevention or treatment and increase healthspan.abbreviations: sasP, senescence-associated secretory phenotype; dname: dna methylation; ros: reactive oxygen species.

Table 1 .
diet and impact on lifespan and disease in selected preclinical studies.

Table 2 .
diet and impact on lifespan and disease in selected human studies.

Table 3 .
selected studies on elimination of pathological effects caused by senescent cells, alleviating age-related diseases.
rhythms involved in autJamshed et al. 2019)al for the beneficial effects of DR(Ulgherait et al. 2021;Jamshed et al. 2019), but more research is required to understand the underlying molecular mechanisms.
(Galluzzi et al. 2014)uzzi et al. 2014;Mu et al. 2021).DR moreover reduces the cellular access to nutrients such as glucose and amino acids in the extracellular fluids, which triggers autophagy(Galluzzi et al. 2014).βHB is a key factor mediating DR-induced autophagy during glucose deprivation, as it stimulates the autophagic flux and prevents autophagosome accumulation (Camberos-Luna et al. 2016; Torres-Esquivel et al. 2020).Recent studies suggest that circadian . IF or CR have been reported to elicit increased ghrelin secretion (Amitani et al. 2017; Bayliss and Andrews 2016; Al-Rawi et al. 2020; Amicis et al. 2019), although serum levels of ghrelin, melatonin, and leptin have been reported to decrease after diurnal IF (Al-Rawi et al. 2020).KD or ketone esters reduced ghrelin secretion in children with refractory epilepsy

Table 4 .
Comparison of animal models and human studies on the effects of dr or senolysis.