How Dietary Restriction Changes Metabolism Differently with Age in Mice

Aging and aging‐related diseases have been widely introduced from molecular mechanisms to interventions and treatments (Guo et al. 2022). Healthy aging likely delays aging and reduces aging‐related impairments, thus extending lifespan. Many studies suggest that healthy aging is attributed to regular exercise, a healthy diet, regular doctor visits, and mental health care, even though genetics are not in our control. From a biological point of view, it is noteworthy that the most reliable, robust, and long‐known interventions for healthy aging target energy metabolism. For example, the attenuation of the growth hormone signaling induces the shift of energy resources from growth toward repair activities and thus extends lifespan (Brown‐Borg et al. 2014). Indeed, dietary restriction (DR) such as intermittent fasting (IF), time‐restricted feeding (TRF), and calorie restriction (CR) act by increasing metabolic plasticity. Metabolic plasticity is known as the metabolic resilience of the cells and their ability to swiftly alternate between metabolic substrates for energy production and decline with age (Chaudhari and Ermolaeva 2024; Longo and Anderson 2022). Therefore, since energy metabolism mainly occurs in mitochondria and aging significantly alters energy metabolism, modulation of mitochondrial resilience plays a crucial role in improving metabolic plasticity and thus extending lifespan. Chaudhari and Ermolaeva (2024) have demonstrated that the key variable for an effective metabolic intervention for healthy aging such as exercise and metformin, which elicit moderate mitochondrial and metabolic stress, as well as NAD+ boosters, rapamycin, and polymerase I inhibitors, which help reduce metabolic stress and molecular damage accumulation, is age. However, the effectiveness of these interventions may diminish over time due to the body's adaptive resilience and failure of mitochondrial function and plasticity mechanisms later in life. Therefore, these authors underline that the age‐adjustment concept of intervention for healthy aging may be an important step toward personalizing longevity treatments. Metabolic responses to DR were not associated with increased lifespan, indicating that improving health and extending lifespan are not synonymous and raising questions about which endpoints are the most relevant for evaluating aging interventions (Di Francesco et al. 2024). Indeed, mice subjected to 40% CR exhibited overall health benefits; however, they also experienced some adverse effects, including a lifelong loss of lean mass, lower body temperature, and alterations in their immune profile (Di Francesco et al. 2024). Therefore, it has been suggested that the health benefits of CR may vary significantly among individuals, depending on their genetic background (Di Francesco et al. 2024). Pak et al. (2021) have shed new light on how both when and how much we eat regulate metabolic health and longevity, and they demonstrated that daily prolonged fasting, following reduced caloric intake, is likely responsible for the metabolic and geroprotective benefits of the CR diet. Therefore, optimizing these benefits may require a tailored approach that considers factors such as age, the degree of CR, and the implementation of daily fasting alongside CR.

Metabolomics, the study of comprehensive small molecules in plants, microbial, animal and mammalian cells, tissues, and biofluids, is increasingly incorporated into aging research. With technological advancements with large datasets, metabolomics has enabled the population‐scale profiling of metabolites in humans, the findings of associations between multiple health conditions and metabolites, and thus prediction of aging, common incident diseases, and mortality risk (Cheng et al. 2015; Wang et al. 2011; Zhang et al. 2024). Taken together, age‐dependent metabolites in biofluids of humans and their associations with age‐related diseases have been widely explored (Lau et al. 2024; van den Akker et al. 2020; Yu et al. 2012). Over the last decade, a significant amount of metabolomic data has been collected, leading to the development of machine learning algorithms that create a metabolomic aging clock known as MileAge (Mutz et al. 2024). These algorithms can predict health and lifespan and are useful in health assessments, risk stratification, and proactive health monitoring, and thus promote healthy aging and longevity. In metabolomics studies on aging in animals (Houtkooper et al. 2011; Mitchell et al. 2016), CR is recognized as the most effective intervention for delaying aging and promoting healthy longevity. For example, metabolomics studies have revealed that CR‐induced Sirt3 regulates ornithine transcarbamoylase (OTC) activity and promotes the urea cycle and fatty acid oxidation, demonstrating improved mitochondrial function in the liver during CR (Hallows et al. 2011). Also, Aon et al. (2020) have found that fasting time and total calorie intake were determinants of increased survival in mice, rather than diet composition. DR is a broad term encompassing various feeding patterns such as CR, IF, TRF, specific nutrient restriction, and reduction in food intake. While CR focuses on an overall reduction of calories. these dietary patterns all retard the aging process, even though not in all mammals (Sohal and Forster 2014).

Di Francesco et al. (2024) noted that the effectiveness of CR may vary with age. While studies with dietary interventions aiming at promoting longevity are mostly applicable in young organisms, their impact on the metabolic health of older individuals under CR has not been thoroughly investigated. To address this gap, we conducted a study examining the effectiveness of DR in young, middle‐aged, and old mice through metabolomic analysis of their plasma, liver tissues, and kidney tissues. Based on previous findings that a classic 30% DR is the most effective dietary intervention for metabolic and geroprotective benefits (Pak et al. 2021), we chose a 30% DR diet for the mice in the current study. The mice were fed once a day and subjected to daily inter‐meal fasting with reduced food intake.

To explore the dependences of the metabolic responses to DR on age, acute DR intervention for 5 days and chronic DR intervention for 30 days were applied to young (2‐month‐old, 5–6 weeks), middle‐aged (6‐month‐old, 26–28 weeks) and old (17‐month‐old, 73–75 weeks) mice (Figure 1a), in which control mice were fed ad libitum (AL) and 70% food was given for DR mice (30% DR). During both acute and chronic DR interventions, food intakes and body weight were monitored (Figure 1b,c), and ¹H NMR‐based metabolomics approach was involved in plasma, liver, and kidney to investigate the metabolic effect of DR at different ages (Figures 2, 3, 4). The representative 1D ¹H NMR spectra of plasma, liver, and kidney from control or AL young mice are shown in Figure S1. Metabolites identified in plasma, liver, and kidney included amino acid metabolism‐related metabolites (alanine, aspartate, betaine, creatine, glutamate, glutamine, glutathione, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, sarcosine, tyrosine, valine, and taurine), glycolysis metabolism‐related metabolites (citrate, fumarate, glucose, glycogen, lactate, pyruvate, succinate, and myo‐inositol), energy status‐related metabolites (AMP, ATP/ADP, niacinamide, and 3‐hydroxybutyrate), and lipid metabolism‐related metabolites (fatty acids, betaine, choline, phosphorylcholine, and glycerophosphocholine).

Aging is a natural process that occurs in all living organisms, but it increases susceptibility to various chronic diseases, physiological disorders, and cancer, which is considered a disease from certain perspectives. Therefore, modern society is increasingly focused on discovering breakthroughs to sustain health as we age, resulting in a wide range of interventions from lifestyle changes to therapeutic methods. Among these interventions, it is well established that DR or CR is the most effective strategy for delaying aging and extending lifespan. However, the benefits of DR can be limited if it is introduced later in life. The timing or age at which DR is initiated is crucial for realizing its benefits, as evidenced by nutritional memory effects observed in old mice (Hahn et al. 2019). In this study, we collected samples from the plasma, liver, and kidney of mice across three age groups: young, middle‐aged, and old. We examined metabolic alterations both during acute and chronic DR. The results revealed that metabolites in the plasma, liver, and kidney underwent distinct changes according to the age of mice both during a normal diet (AL) and DR intervention, demonstrating the complexity of metabolic adaptations at different life stages in mammals. During acute DR, we observed an increase in plasma fatty acid levels exclusively in young mice (Figure S12), indicating transient fatty acid mobilization from white adipose tissues (Selman et al. 2006). However, an increase in plasma 3‐hydroxybutyrate was observed in all age groups during acute DR, demonstrating an immediate energy requirement across ages.

The current study revealed not only metabolic changes during DR but also their dependence on age. Figure 4 presents the quantitative results for plasma, liver, and kidney metabolites that were found to be age‐dependent and uniquely altered during chronic DR. Previous research has widely reported age‐related variations in metabolites in plasma, muscle, and liver, highlighting their significant links to glucose and fat metabolism (Houtkooper et al. 2011). Our results confirmed that plasma glucose levels tend to increase with age (Figure 4), consistent with previous studies (Chen et al. 1985; Houtkooper et al. 2011; Reaven and Reaven 1980). Additionally, Pontzer et al. (2021) recently demonstrated that individuals over 60 experience reduced energy expenditure, as measured by total and basal energy levels across a large cohort ranging from 8 days to 95 years old. Therefore, it is common for healthy old mammals to show elevated plasma glucose levels, which may reflect a reduction in energy expenditure or a decline in glucose tolerance associated with aging.

In our study, we also observed that hepatic glucose levels gradually decreased with the age of mice fed AL (Figure 4). This finding contradicts results from a study on 23‐month‐old mice (Houtkooper et al. 2011), but aligns with results observed in 13‐month‐old mice (Atherton et al. 2009). Additionally, we noted reductions in hepatic glutathione levels and elevations in hepatic fatty acid levels with age, which are consistent with a previous report (Maher 2005). Consequently, the increases in plasma glucose and hepatic fatty acid levels, along with decreases in hepatic glucose, glycogen, lactate, and glutathione levels in aged AL‐fed mice, are likely related to declines in energy metabolism and antioxidant activity with age (Figure 4). These changes may serve as biomarkers for aging in our study.

Elevations in the levels of plasma 3‐hydroxybutyrate were observed in all mice during acute and chronic DR, likely demonstrating energy demand or improved energy metabolism. Taken together, the findings of elevated glucose levels alongside reduced lactate, pyruvate, and fatty acid levels in plasma exhibited classical indicators of systemic gluconeogenesis. This systemic gluconeogenesis was observed only in young mice during chronic DR (Figure 5), consistent with results reported during fasting or DR (Kolb et al. 2021; Rui 2014). These results suggest that there are distinct systemic responses to DR between young and aged mice, reflecting different metabolic adaptations.

Due to energy deficiency during DR, increases in fasting blood glucose levels occur commonly, which likely result from gluconeogenesis (Rui 2014). Lactate, pyruvate, glycerol, and amino acids serve as substrates for gluconeogenesis mainly in the liver and kidney (Rui 2014). In the current study, increased fasting plasma glucose levels during chronic DR were observed only in young mice, alongside decreased levels of plasma pyruvate and lactate (Figures 3 and 4), highlighting a reflection of a systematic increase in gluconeogenesis among young mice during chronic DR. Additionally, local gluconeogenesis in the liver during chronic DR was evidenced by decreased hepatic lactate and glucogenic amino acid (alanine, isoleucine, valine, phenylalanine, and tyrosine) levels in young mice, even as hepatic glucose levels trended upward (Figure 5a).

In AL mice, hepatic glucose levels decreased with age (Figure 4), which is consistent with findings from previous studies (Houtkooper et al. 2011). Following chronic DR, hepatic glucose levels in middle‐aged and old mice increased to levels comparable to those in the young mice. However, there was no evidence of gluconeogenesis in the liver of middle‐aged and old mice during chronic DR, as indicated by the lack of significant changes in the levels of hepatic pyruvate, lactate, and amino acids, unlike in young mice. To date, glycerol, which is derived from triglycerides through lipolysis and serves as a gluconeogenic precursor, contributes to 22% of total glucose production during prolonged fasting in humans, such as after 62–86 h of starvation (Baba et al. 1995; Bortz et al. 1972; Landau et al. 1996; Shah and Wondisford 2020). Additionally, free fatty acids are well known to enhance hepatic gluconeogenesis through the production of acetyl‐CoA, NADH, and ATP, and by providing a source for gluconeogenesis in the glyoxylate and the xylulose 5‐phosphate pathways (Lam et al. 2003). Therefore, large reductions in hepatic fatty acid levels and marked elevations in hepatic glucose levels in both middle‐aged and old mice may reflect fatty acid‐attributed gluconeogenesis during chronic DR in the current study (Figure 5b,c). In fact, hepatic fatty acids accumulate with age due to reduced β‐oxidation of free fatty acids (Son et al. 2012). Only the increased fatty acid levels in aged mice were shared with obese mice, as reported (Serkova et al. 2006), consistent with the current study. This result demonstrates that other metabolites perturbed as mice aged were dependent on age in the current study, rather than obesity. These findings highlight that the gluconeogenesis observed in the liver during chronic DR differs between young and aged mice and contributes distinctly to glucose production on energy demand or energy deficiencies induced by a reduced diet. The liver provides 90%–95% of circulating glucose during the postabsorptive state (Chia et al. 2018). However, increases in hepatic glucose levels in middle‐aged and old mice during chronic DR did not affect their plasma glucose levels in the current study. This might be because hepatic glucose is involved in other metabolism during chronic DR, such as the accumulation of hepatic glycogen.

Glycogen breakdown in the liver, known as glycogenolysis, occurs during fasting to provide glucose as an energy source, leading to elevated blood glucose levels (Rui 2014). Glycogen can be directly observed in intact liver tissues by ¹H MAS spectroscopy (Bollard et al. 2000). In the current study, marked accumulation of hepatic glycogen and increased hepatic glucose levels were observed in middle‐aged and old mice during chronic DR, with the effects being particularly pronounced in old mice (Figure 4). However, their blood glucose levels remained unchanged (Figure 4). Hepatic glycogen accumulation has rarely been reported in the condition of glucose deprivation or during dietary or calorie restriction. Nevertheless, Hu et al. (2022) demonstrated that active glycogen synthesis contributed to the elevated glycogen levels in the liver of mice undergoing a 40% calorie restriction (CR) to compensate for lipid insufficiency. Furthermore, hepatic glucose may have contributed to the accumulation of hepatic glycogen (excess glycogenesis), possibly because fatty acids were primarily used to compensate for energy deficiency during chronic DR (Galgani and Fernandez‐Verdejo 2021). This relationship was indicated by a strong inverse correlation between glucose and fatty acids in the liver tissues of middle‐aged (p < 0.001) and old (p = 0.008) mice in the current study (Figure S18). In contrast, the correlation observed in the liver of young mice was not significant (p = 0.117). Therefore, the increased glycogen levels in the livers of middle‐aged and old mice during chronic DR may reflect glycogenesis through the excess β‐oxidation of fatty acids in the liver (Figure 5b,c). Nevertheless, glycogen levels in the liver of middle‐aged mice subjected to chronic DR were found to be similar to those in AL young mice. The differences in glycogen levels between AL mice and chronic DR mice were measured at 1.85 ± 1.04, 1.80 ± 2.77, and 8.58 ± 3.55 for young, middle‐aged, and old mice, respectively. Also, the differences of hepatic fatty acids were −0.16 ± 1.04, −3.20 ± 3.96, and −5.45 ± 4.10 for young, middle‐aged, and old mice, respectively, after chronic DR when compared to AL mice. As a result, the large accumulation of hepatic glycogen in old mice during chronic DR suggests excess glycogenesis due to a corresponding increase in the breakdown of hepatic fatty acids. Furthermore, a study by de Toro‐Martin et al. (2020) indicated that early undernutrition leads to substantial hepatic glycogen accumulation in the liver and lack of glycogen mobilization after birth. These findings demonstrated increased glycogen synthesis and decreased endogenous glucose production in the livers of undernourished neonates, which is linked to reduced glucagon secretion [37]. Thus, large glycogen accumulation in the liver during food restriction may elevate the risk of developing metabolic diseases in the future.

Ketone bodies or ketones, produced in the body are used as an alternative energy source when glucose is scarce. In AL mice, plasma levels of 3‐hydroxybutyrate did not change with age. However, these levels increased in all DR mice during both acute and chronic DR (Figures 4 and S12), consistent with previous reports that ketone bodies are highly activated during CR or in conditions of glucose deprivation (Veech et al. 2017; Wijeyesekera et al. 2012). 3‐hydroxybutyrate, one of the most abundant ketone bodies in human circulation, is derived in the liver through the β‐oxidation of free fatty acids, which are produced by the lipolysis of adipose tissues during starvation, fasting, or when following a low carbohydrate diet (Veech et al. 2017). Mitochondrial β‐oxidation not only provides energy for hepatocytes but also generates ketone bodies. Therefore, ketone bodies inhibit gluconeogenesis in the liver, lipolysis in adipose tissue, glucose oxidation in the brain, and protein degradation in skeletal muscle (Fernandez‐Verdejo et al. 2023). In addition to CR, ketogenic diets also lead to an increase in circulating 3‐hydroxybutyrate, which contributes to extending lifespan (Roberts et al. 2017; Veech et al. 2017). It has also been reported that 3‐hydroxybutyrate supplementation can extend the lifespan of C. elegans (Edwards et al. 2014). Indeed, elevated levels of 3‐hydroxybutyrate have been shown to alleviate various age‐related diseases and improve health outcomes (Han et al. 2020). The increases in plasma 3‐hydroxybutyrate levels during DR indicate a shift from a state of energetic balance to one of energy deficit, which was observed in all mice during DR in the current study. This shift may contribute to the extension of a healthy lifespan. Specifically, increases in plasma 3‐hydroxybutyrate levels may reflect an increase in energy expenditure. Furthermore, fatty acids in the liver, which significantly increased with age, were found to be decreased in middle‐aged and old mice, suggesting a potential improvement in metabolic diseases. However, excess reductions of hepatic fatty acid levels in old mice during chronic DR could present issues.

Research has shown that as we age, glutathione levels decline in various tissues, including the liver. Glutathione is crucial for protecting against free radical damage associated with aging (Maher 2005). Several studies have reported that reduced glutathione (GSH) levels decreased in cells from old animals, while oxidized glutathione (GSSG) levels tend to increase (Sastre et al. 1996). In contrast to the findings of Houtkooper et al. (2011), hepatic glutathione levels in old mice were different in the current study (Figure 4). In comparison of serum metabolic differences among young (3–8 months), adult (13–23 months), and old (27–36 months) mice, serum levels of glucose, 3‐hydroxybutyrate, and glycerol were lower in the old mice [45]. However, the hepatic glutathione levels were found to be increased with age (Petr et al. 2021). Notably, discrepancies in the hepatic glutathione levels reported in previous studies compared to the current study may be attributed to the misidentification of reduced glutathione (GSH) as oxidized glutathione (GSSG), since GSH, an abundant form of glutathione, was not identified in the reports (Houtkooper et al. 2011; Petr et al. 2021). In our study, we clearly identified hepatic glutathione as GSH, and its levels were found to decrease with age. Notably, GSH levels were elevated in all mice during chronic DR (Figure 4), while their levels started to increase at Day 5 of acute DR in young and middle‐aged mice (Figure S13). Additionally, levels of hepatic glutamine and glutamate, which are precursors of glutathione, were positively associated with hepatic glutathione levels during chronic DR. These results indicate that chronic DR enhances protection against oxidative stress by removing reactive oxygen species through GSH.

Indeed, the markedly increased levels of taurine, an antioxidant in biological systems (Z. Y. Zhang et al. 2014), in the livers of only middle‐aged mice during chronic DR likely reflect an enhanced protective function of the liver in these middle‐aged mice (Figure 4). In young mice, hepatic taurine levels tended to increase after chronic DR, although this change was not statistically significant, and never changed in the old mice. Sarcosine is capable of inducing autophagy that helps cells survive, function, and protect liver cells under stress. However, sarcosine levels decline with age across multiple species (Walters et al. 2018). Glycine N‐methyltransferase (GNMT), which converts glycine to sarcosine, is predominantly expressed in the liver and its activity is suppressed as age increases (Johnson and Cuellar 2023). Walters et al. (2018) found that hepatic sarcosine levels remain relatively unchanged with aging but decrease with DR. These authors reported that plasma sarcosine levels decrease with age and increase with DR in both rodents and humans, which is attributed to liver GNMT activity. They also demonstrated that sarcosine could activate autophagy in cultured cells and enhance autophagic flux in vivo. Therefore, the marked reduction in hepatic sarcosine levels observed in young mice during chronic DR—bringing their levels down to those comparable to middle‐aged and old mice—may negatively impact liver functions (Figure 4). Young mice subjected to chronic DR may experience potential liver dysfunction due to decreased sarcosine levels. This reduction could be associated with liver malnutrition, resulting from gluconeogenesis, which might lead to excessive degradation of various amino acids. In contrast, middle‐aged mice demonstrate a tendency for increased hepatic sarcosine levels during chronic DR. This increase may support enhanced liver health and function in conjunction with elevated taurine levels in the liver.

Various dietary interventions have been introduced to extend lifespan, including dietary restriction, calorie restriction, time‐restricted feeding, and intermittent fasting. Among these dietary regimes, daily prolonged fasting combined with reduced caloric intake (CR‐DPF) has been found to be mostly effective for enhancing longevity and providing metabolic and geroprotective benefits. Additionally, it has been demonstrated that metabolic longevity treatments, such as DR and DR mimetic drugs, are mostly applicable in young and middle‐aged organisms, highlighting age limitations for these treatments. In the current study, young, middle‐aged, and old mice on a 30% DR received one meal a day at 7:00 p.m. and completed allotment within two hours. This resulted in an involuntary CR regime, leading to prolonged daily fasting until the next meal, similar to CR‐DPF. The metabolomic analysis of plasma, liver, and kidney samples from these mice revealed that chronic DR induced both systemic metabolite perturbations and gluconeogenesis in young mice, utilizing lactate and amino acids. In contrast, middle‐aged and old mice primarily relied on fatty acid oxidation for gluconeogenesis. Also, chronic DR may weaken liver function in young mammals possibly due to the high protein degradation for gluconeogenesis. In particular, the excessive oxidation of fatty acids and large glycogen accumulation in the livers of old mice during chronic DR, along with irreversible loss of body weight, highlight the potential harmful effects of chronic DR in aging mammals. Therefore, a personalized strategy for dietary or calorie intake reduction based on age groups, particularly for older mammals, is strongly recommended to avoid adverse effects.

J.L. and Y.‐S.H. conceived and designed the study. J.L. and V.H.J.H.A. performed the experiments. J.L. and E.‐H.K. conducted the data analysis. J.L. and Y.‐S.H. wrote the manuscript. E.B. and Y.‐S.H. reviewed and revised the manuscript. All authors contributed to finalizing the manuscript.

This study was supported by the National Research Foundation (NRF) grant funded by the Korea government (MIST) (RS‐2021‐NR059295).

The authors declare no conflicts of interest.