Calorie restriction, without malnutrition, has been shown to increase lifespan and is associated with a shift away from glycolysis toward beta-oxidation. The objective of this study was to mimic this metabolic shift using low-carbohydrate diets and to determine the influence of these diets on longevity and healthspan in mice. C57BL/6 mice were assigned to a ketogenic, low-carbohydrate, or control diet at 12 months of age and were either allowed to live their natural lifespan or tested for physiological function after 1 or 14 months of dietary intervention. The ketogenic diet (KD) significantly increased median lifespan and survival compared to controls. In aged mice, only those consuming a KD displayed preservation of physiological function. The KD increased protein acetylation levels and regulated mTORC1 signaling in a tissue-dependent manner. This study demonstrates that a KD extends longevity and healthspan in mice.

In the current study, adult mice were fed isocaloric amounts of a control diet, LCD, or KD. The objective of this study was to determine the influence of an LCD or KD on longevity and healthspan in mice.

Low-carbohydrate diets (LCDs) also induce a shift in metabolism away from carbohydrates toward fatty acid oxidation. The most extreme LCD, the ketogenic diet (KD), has been shown to promote an anti-inflammatory metabolic state and to increase levels of ketone bodies in mice, resembling key features of CR (). Despite these similarities, there is a dearth of information on lifespan and healthspan outcomes of animals maintained long-term on these diets. To date, only one study has shown that mice fed a lifelong, ad libitum KD demonstrate no significant difference in longevity compared to mice fed a standard chow diet (). However, the control and KD C57BL/6 mice used by Douris and colleagues were short lived for this mouse strain and therefore the effect of a KD on aging is still equivocal, especially in animals that are not obese or fed ad libitum.

Caloric restriction (CR) extends longevity and delays age-related diseases across numerous animal models (). While the exact mechanisms contributing to increased longevity in CR animals are still subject to debate, CR induces a shift from carbohydrate to fat metabolism (). It remains to be determined whether the increased fatty acid oxidation and ketogenesis that occur with CR contribute to lifespan extension.

To further elucidate the mechanisms underlying the beneficial effects of a KD on longevity and healthspan, we examined the levels and activation state of several factors linked to the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway, which has been suggested to modulate aging in response to dietary interventions (). Total and phosphorylated levels of mTOR were not altered in liver by 1 month of LCD or KD ( Figures 3 A and S2 ). However, lower levels of phosphorylated 4E binding protein 1 (4E-BP1) and a similar trend for phosphorylated S6 ribosomal protein (rpS6) were detected, suggesting decreased mTORC1 signaling ( Figures 3 B and 3C) in liver. In contrast to liver, p-4E-BP1 levels were increased in the skeletal muscle of ketogenic mice after 1 month of diet ( Figure S3 ). We also analyzed several signaling cascades modulating hepatic nutrient sensing by mTORC1. No changes were detected in phosphorylated AMPK, p-Akt, or p-Erk1/2 between control and KD mice. Tuberous sclerosis complex 2 (TSC2) phosphorylation at S939 or S1387, as well as p-Raptor levels, was also unaltered by the KD ( Figure S2 ). In contrast, levels of DNA damage-inducible transcript protein 4 (DDIT4, also known as regulated in development and DNA damage or REDD1), a negative regulator of mTORC1, were significantly increased in the KD mice ( Figure 3 D).

βHB can inhibit histone deacetylase (HDAC) activity in vivo (). After 1 month on the KD, total acetyl-Lys levels were increased 5-fold in the liver of KD mice compared to control and LCD mice ( Figure 3 E). A 2.5-fold increase was detected in the skeletal muscle of the same mice ( Figure S3 ). The level of acetylated p53, a key tumor suppressor protein, was 10-fold higher in liver after 1 month on a KD ( Figure 3 F). Interestingly, acetylation levels of histone 3 at Lys9 (H3K9) were increased in the liver after 1 month on either a KD or an LCD ( Figure 3 G). HDAC inhibition and H3K9 acetylation by βHB have also been shown to upregulate gene expression of Foxo3a and some of its targets involved in antioxidant responses (), including manganese superoxide dismutase (MnSOD). FoxO3a and MnSOD protein levels were increased in liver after 1 month of either an LCD or KD compared to a control diet ( Figures 3 H and S2 ).

(I) Representative blots are shown for each of the quantified proteins. For total lysine acetylation, a fraction of the membrane is shown. A representative loading control is shown for those blots with n = 4, corresponding to the acetyl-p53 gel.

(A–H) Quantification of (A) p-mTOR, (B) p-4E-BP1, (C) p-rpS6, (D) DDIT4, (E) total acetyl-lysine, (F) p-53, (G) acetyl-H3K9, and (H) MnSOD protein levels in liver after analysis by western blot (n = 4–8).

Alterations in Protein Acetylation and mTOR Signaling in the Liver of Male Mice after 1 Month of Diet

Figure 3 Alterations in Protein Acetylation and mTOR Signaling in the Liver of Male Mice after 1 Month of Diet

To further characterize the metabolic shift on these diets, we analyzed protein content of several enzymes linked to fatty acid metabolism in the liver of these mice. A KD decreased hepatic levels of phosphorylated and total acetyl-CoA carboxylase (ACC), while increasing those of carnitine palmitoyltransferase 2 (CPT2) and medium-chain acyl-CoA dehydrogenase (MCAD) ( Figures 2 G–2L). Phosphorylated and total pyruvate dehydrogenase (PDH) protein levels were also reduced by both LCD and KD ( Figures 2 H and 2K).

Glucose and insulin tolerance tests (GTT and ITT) were conducted in mice after 1 month of dietary intervention. Mice on a KD displayed impaired glucose tolerance compared to those on a control diet ( Figure 2 E). The LCD group did not differ in glucose disposal compared to either the control or KD groups. Although no differences were observed between the control mice and the other groups, insulin sensitivity after a 4 hr fast was enhanced by a KD if compared to the LCD ( Figure 2 F), indicating that insulin signaling is functioning normally in mice fed a KD. Interestingly, hepatic levels of phosphorylated AS-160 (Akt substrate of 160 kDa), a key mediator of insulin sensitivity, were increased in ketogenic mice compared to the other diets ( Figure S2 ). Circulating levels of fibroblast growth factor 21 (FGF21) were not significantly altered by any of the dietary interventions ( Table S2 ).

Respiratory quotient (RQ), energy expenditure (EE), and physical activity were measured in mice at 13 and 26 months of age. At both ages, average RQ was decreased by an LCD or a KD compared to a control diet ( Figure 2 D; Table S3 ). No significant differences between diets were observed in 24 hr EE; however, aging decreased EE, unadjusted or adjusted for either BW or lean mass, in all diet groups ( Table S3 ). Overall, spontaneous physical activity did not differ with age or between diet groups at either 13 or 26 months of age ( Table S3 ).

Circulating β-hydroxybutyrate (βHB) levels were measured 3 hr postprandial ( Figure 2 C). In both age groups, blood ketones were significantly elevated in KD mice compared to control or LCD mice. We also analyzed other serum biomarkers after 1 and 14 months of dietary intervention ( Table S2 ). No changes were detected between diets at 13 months of age. At 26 months of age, the only observed difference was that free fatty acid concentration was higher in the LCD compared to the other groups.

To investigate the physiologic and metabolic changes induced by these diets, we measured body weight (BW) and composition, a panel of serum biomarkers, energy expenditure, and physical activity. Despite being fed the same amount of calories, mice fed an LCD were heavier throughout the study than mice fed either a control or KD ( Figure 2 A). Body composition analysis showed that lean mass increased with age in the control and LCD mice, and was significantly lower in the KD mice at 26 months of age ( Figure S1 ). LCD mice had significantly more fat mass compared to control or ketogenic mice ( Figure 2 B). In all diet groups, total fat mass peaked at 17 months of age.

(M) Representative blots are shown for each protein. A representative loading control is shown for each case (n = 4–8).

(E) GTT after a 16 hr fast; the areas under the curve (AUCs) differ between the KD and the control (n = 6).

For the physiological tests (D–F) and protein levels in liver (G–K), animals were on the diets for 1 month.

(B) Fat mass from 1 to 14 months of dietary interventions (n = 15); +, fat mass is greater (p < 0.05) for the LCD versus the other groups.

To assess the effects of these diets on healthspan, a battery of physical and behavioral tests was conducted after either 1 or 14 months of the dietary intervention (13 or 26 months of age). The results of the novel object recognition test () ( Figure 1 B) indicate that memory was preserved in old mice fed a KD compared to those fed a control or LCD. Coordination, strength, and endurance were assessed with the hanging wire and grip strength tests. Male mice fed a KD for 14 months were more resistant to falling from the hanging wire ( Figure 1 C) and had greater forelimb grip strength ( Figure 1 D) compared to age-matched controls. Old ketogenic mice were also faster in the Locotronic speed test () ( Figure 1 E) and more active during the rearing test ( Figure 1 F) compared to controls, suggesting better preservation of motor coordination. In most cases, LCD group performance was intermediate to the control and ketogenic groups. Noteworthy, and consistent with increased motor function, the relative mass of gastrocnemius and other hind limb muscles was higher in the old KD mice ( Figures 1 G–1I and S1 ). Altogether, these tests suggest that the KD is able to extend both lifespan and healthspan in adult mice.

Behavioral changes are not directly related to striatal monoamine levels, number of nigral neurons, or dose of parkinsonian toxin MPTP in mice.

To study the effects of LCDs on longevity in adult male mice, we compared an LCD (70% kcal from fat) and a KD (89% kcal from fat) with a control diet (65% kcal from carbohydrate). Diets were fed in isocaloric amounts starting at 12 months of age. Lifespan was significantly increased in the KD compared to control group ( Figure 1 A), with the ketogenic group showing a 13.6% increase in median lifespan versus the control mice. The LCD group had a lifespan intermediate to the KD and control groups, and was not significantly different from either group. Median lifespans were 886, 943, and 1,003 days for the control, LCD, and KD groups, respectively. Maximum lifespan (90percentile) was 1,064, 1,123, and 1,175 days, respectively. Median, but not maximum (p = 0.16), lifespan was significantly increased in the KD versus control group. Of specific interest, incidence of tumors at time of death, particularly histiocytic sarcoma, was decreased with a KD ( Table S1 ).

Discussion

Douris et al., 2015 Douris N.

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et al. Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels. The objective of this study was to mimic the metabolic changes accompanying CR by manipulating dietary macronutrient composition and to determine whether these changes in diet composition increase longevity and healthspan in mice. The results clearly demonstrate that lifespan is increased in mice consuming a KD compared to a standard control diet. However, feeding strategies and husbandry issues may play a role in determining the influence of KDs on aging. This hypothesis is supported by the fact that a previous study reported that KDs do not alter survival curves in C57BL/6 mice (). It is important to note that the lifespan of the control group reported by Douris and colleagues was shorter than would be expected for this strain (). Level of energy intake and prevention of weight gain may be particularly important for positive lifespan effects with a KD, and the results of the present study suggest that longevity is increased when a feeding strategy is followed that mitigates weight gain in adult mice.

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Rebuffé-Scrive M. Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice. Similar to KDs, very little is known about the impact of LCDs on longevity in animals that are not allowed ad libitum access to food. It is often assumed that high-fat diets shorten lifespan since they have been shown to induce weight gain and obesity when fed ad libitum to C57BL/6 mice (). However, our results indicate that a calorie-controlled LCD started in middle-aged mice does not have a negative impact on aging.

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et al. The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. There has been considerable interest in the impact of dietary macronutrient composition on longevity, with several studies focusing on protein or methionine restriction as an approach to increase lifespan (). In our study, lifespan did not significantly differ between the LCD and KD groups despite higher protein intake in LCD animals compared to the KD animals. Furthermore, no increase in lifespan was observed in rats fed diets in which protein content was decreased to levels, and in a proportion, comparable to those of our study (). Another study found that survival was not increased in rats consuming a 12% versus 20% protein diet (). Thus, available evidence does not support the idea that level of protein is primarily responsible for the increased longevity in our KD mice. It has also been proposed that a low dietary protein to carbohydrate ratio drives longevity (). However, the results of the present study are not consistent with this hypothesis. Nonetheless, additional studies are needed to determine if dietary protein contributes to improved physiological function and longevity in KD mice. It is also possible that the optimal dietary macronutrient composition may differ between an animal that is fed ad libitum and one that is not.

Our results show that a KD slows cognitive decline and preserves motor function in aging mice. It should be noted that although the LCD did not significantly differ from the ketogenic group in longevity, the two diets differed in their ability to preserve physiological function with age. This suggests that ketones may be necessary to elicit an extension of healthspan.

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et al. A periodic diet that mimics fasting promotes multi-system regeneration, enhanced cognitive performance, and healthspan. The novel object recognition test has been previously used to study memory in models of aging and its associated diseases (). Our results support the notion that ketones may play an important role as neuro-protective signaling molecules (). Further support for this hypothesis comes from the fact that a diet that mimics fasting also increases ketone production and improves memory in mice (). The present study along with the literature supports the notion that a KD promotes long-term cognitive health.

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Zhu D. Acetoacetate accelerates muscle regeneration and ameliorates muscular dystrophy in mice. Motor function was evaluated with approaches previously used to detect age-related deficits in muscle strength and function (). The KD mice did not show the age-related decrease in grip strength observed in the control mice, and 26-month-old KD mice outperformed the other diet groups in the hanging wire test. This suggests that the KD maximizes and preserves forelimb grip strength with age. There is evidence that the ketone body acetoacetate plays an important role as a signaling molecule in muscle cells independent of its metabolic effects (). Acetoacetate has been shown to improve muscular dystrophy outcomes and accelerate muscle regeneration, suggesting that it may be an important player in attenuating age-related decline in muscle function. The results of our tests and the work of others suggest that ketones positively impact muscle homeostasis. However, more work is needed to elucidate the exact mechanisms underlying this protective effect.

According to our results, extension of both longevity and healthspan appears to be unique to the KD. Interestingly, there is evidence of interaction between ketone bodies and pathways proposed to modulate aging.

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et al. Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor. The ketone body βHB is a direct inhibitor of HDACs, a process that has been shown to occur in vivo (). HDAC inhibitors extend lifespan in models from yeast to flies, through mechanisms not yet elucidated, but associated with hyperacetylation of histones and a large number of other proteins. In our study, a KD resulted in a dramatic increase in total levels of acetylated lysine. Interestingly, in both the LCD and the KD mice, we observed an increase in acetyl-H3K9, concomitant with increased FoxO3a and MnSOD in liver, an effect previously described in the literature as a contribution of ketone bodies to stress response pathways and potentially longevity (). However, since the effect on acetyl-H3K9, FoxO3a, and MnSOD was similar in the LCD and KD groups, our data suggest that these changes do not underlie the lifespan and healthspan of the diet.

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et al. Low protein intake is associated with a major reduction in IGF-1, cancer, and overall mortality in the 65 and younger but not older population. Many dietary interventions known to extend or modulate lifespan have been shown to be mediated, at least partially, by decreased mTORC1 activity (), an effect that has been previously reported to occur with KDs (). The decreased protein content in the KD likely contributes to lower mTORC1 activity, eliciting a response analogous to that of protein or methionine restriction (). Our experimental design attempted to minimize this effect by using a higher protein content (10% of calories) than previous laboratory rodent studies of KDs and protein restriction ().

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Bost F. Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. We detected a tissue-dependent modulation of mTORC1 signaling. In skeletal muscle, the KD increased p-4E-BP1 levels. Similarly, a recent study in rats fed a relatively high-protein (∼20% kcal) KD detected no change in p-rpS6 and a trend toward an increase in p-4E-BP1 (). Thus, protein level may have a major influence on mTORC1 signal in response to a KD. There is a lack of sufficient understanding of the trade-off between pro-longevity mTOR modulation and skeletal muscle homeostasis (), and further research is needed to fully understand the mechanisms responsible for the preservation of muscle mass with aging in our KD mice. In liver, however, we have shown that mTORC1 signaling is inhibited by the KD. Interestingly, it has been reported that p53 hyperacetylation inhibits mTORC1 in response to fasting by increasing the expression of Ddit4, a negative regulator of mTORC1 (). Moreover, this same pathway seems to mediate the effects of metformin (). Our results, including increased p53 acetylation and DDIT4 levels and decreased mTORC1 downstream signaling, only in the ketogenic group are in accordance with this model. Of note, p53 hyperacetylation and stabilization may also be contributing to the marked decrease in cancer incidence in the KD mice. Crosstalk between HDAC inhibition and liver mTORC1 signaling is therefore a potential mechanism contributing to the longevity extension with a KD.

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Hamilton K.L. Calorie restriction does not increase short-term or long-term protein synthesis. A goal of the present study was to determine if a KD could mimic the changes in healthspan and longevity induced by CR. The KD did induce some of the same changes reported with CR. In particular, overall lifespan is increased by both CR () and the KD. Fatty acid β-oxidation and βHB production are stimulated (), and protein acetylation is increased as described in previous CR studies (). Signaling downstream of mTORC1 is also downregulated in liver with both CR () and a KD.

Douris et al., 2015 Douris N.

Melman T.

Pecherer J.M.

Pissios P.

Flier J.S.

Cantley L.C.

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Maratos-Flier E. Adaptive changes in amino acid metabolism permit normal longevity in mice consuming a low-carbohydrate ketogenic diet. The KD, however, also showed several differences from CR. Unlike CR, the KD mice in the present work were glucose intolerant compared to controls, in contrast with previous reports of enhanced glucose tolerance in ad libitum-fed KD (). Additionally, the level of intake of the KD in the present study did not produce the decrease in body weight observed with a CR diet.

This study demonstrates that energy-controlled high-fat LCDs are not detrimental to health, but rather a KD extends lifespan and slows age-related decline in physiological function in mice. Future studies are warranted to further investigate the mechanisms through which this diet works and to optimize diet composition and feeding approaches to further extend healthspan.