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Fasting drives the metabolic, molecular and geroprotective effects of a calorie-restricted diet in mice

Nature Metabolism volume 3, pages 1327–1341 (2021)Cite this article

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Abstract

Calorie restriction (CR) promotes healthy ageing in diverse species. Recently, it has been shown that fasting for a portion of each day has metabolic benefits and promotes lifespan. These findings complicate the interpretation of rodent CR studies, in which animals typically eat only once per day and rapidly consume their food, which collaterally imposes fasting. Here we show that a prolonged fast is necessary for key metabolic, molecular and geroprotective effects of a CR diet. Using a series of feeding regimens, we dissect the effects of calories and fasting, and proceed to demonstrate that fasting alone recapitulates many of the physiological and molecular effects of CR. Our results shed new light on how both when and how much we eat regulate metabolic health and longevity, and demonstrate that daily prolonged fasting, and not solely reduced caloric intake, is likely responsible for the metabolic and geroprotective benefits of a CR diet.

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Fig. 1: Prolonged fasting is required for the CR-mediated increase in insulin sensitivity and alterations in fuel source selection in male C57BL/6J mice.
Fig. 2: Distinct physiological responses to diet in different strains and sexes of mice.
Fig. 3: Fasting alone recapitulates the metabolic effects of a CR diet.
Fig. 4: Fasting and calorie restriction result in highly similar transcriptomic signatures in liver and white adipose tissue.
Fig. 5: Prolonged fasting is required for the CR-mediated increase in insulin sensitivity and fuel selection in aged mice.
Fig. 6: Fasting is required for the geroprotective effects of a CR diet.
Fig. 7: Fasting plays a critical role in the response to a CR diet.

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Data availability

RNA-sequencing data have been deposited with the Gene Expression Omnibus and are available under accession number GSE168262. Source data are provided with this paper. Other data that support the plots and findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank all members of the laboratory of D.W.L., as well as J. Simcox and R. Jain, for their valuable insights and comments. We thank S. Simpson and S. Solon-Biet for advice regarding animal care. We thank T. Herfel (Envigo) for assistance with the formulation of the Diluted AL diet. We thank M. Schaid for critical reading of the manuscript. The laboratory of D.W.L. is supported in part by the National Institutes of Health (NIH)/NIA (AG050135, AG051974, AG056771, AG062328 and AG061635 to D.W.L.), NIH/National Institute of Diabetes and Digestive and Kidney Diseases (DK125859 to D.W.L and J.M.D.) and start-up funds from the UW School of Medicine and Public Health and Department of Medicine (to D.W.L.). Metabolomic and histone proteomic analysis was supported in part by a grant from the NIH (R37GM059785 to J.M.D.) and a UAB Nathan Shock Center of Excellence in the Basic Biology of Aging (P30AG050886) Core Services Pilot Award (to D.W.L.). Bomb calorimetry was supported by S10OD028739 (to C.-L.E.Y.), and gut integrity analysis was supported in part by DK124696 (to C.-L.E.Y.). H.H.P. is supported in part by a NIA F31 pre-doctoral fellowship (AG066311). C.L.G. is supported by a Glenn Foundation for Medical Research Postdoctoral Fellowship and was supported in part by a generous gift from D. Philanthropies. N.E.R. was supported in part by a training grant from the UW Institute on Aging (NIA T32 AG000213). S.A.H. was supported in part by a training grant from the UW Metabolism and Nutrition Training Program (T32 DK007665). Support for this research was provided by the UW Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation. This work was supported in part by the US Department of Veterans Affairs (I01-BX004031), and this work was supported using facilities and resources from the William S. Middleton Memorial Veterans Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work does not represent the views of the Department of Veterans Affairs or the US Government.

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Authors and Affiliations

  1. Department of Medicine, University of Wisconsin–Madison, Madison, WI, USA

    Heidi H. Pak, Cara L. Green, Mikaela Koller, Nicole E. Richardson, Shany E. Yang, Sabrina N. Dumas, Michelle Sonsalla, Lindsey Bray & Dudley W. Lamming

  2. William S. Middleton Memorial Veterans Hospital, Madison, WI, USA

    Heidi H. Pak, Cara L. Green, Mikaela Koller, Nicole E. Richardson, Shany E. Yang, Sabrina N. Dumas, Michelle Sonsalla, Lindsey Bray & Dudley W. Lamming

  3. Interdisciplinary Graduate Program in Nutritional Sciences, University of Wisconsin–Madison, Madison, WI, USA

    Heidi H. Pak, Spencer A. Haws, Mitchell T. Lavarias, Chi-Liang Eric Yen, John M. Denu & Dudley W. Lamming

  4. Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, WI, USA

    Spencer A. Haws & John M. Denu

  5. Wisconsin Institute for Discovery, Madison, WI, USA

    Spencer A. Haws & John M. Denu

  6. Department of Nutritional Sciences, University of Wisconsin–Madison, Madison, WI, USA

    Mitchell T. Lavarias & Chi-Liang Eric Yen

  7. Endocrinology and Reproductive Physiology Graduate Training Program, University of Wisconsin–Madison, Madison, WI, USA

    Nicole E. Richardson & Dudley W. Lamming

  8. Nathan Shock Center of Excellence in the Basic Biology of Aging, Department of Pathology, University of Alabama, Birmingham, Birmingham, AL, USA

    Michelle Johnson, Victor Darley-Usmar & Jianhua Zhang

  9. Department of Pharmacology, University of Alabama, Birmingham, Birmingham, AL, USA

    Stephen Barnes

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Contributions

Experiments were performed in the laboratories of D.W.L., J.M.D. and C.-L.E.Y. at UW-Madison and in the UAB Nathan Shock Center Mitometabolism Core. All authors participated in the performance of the experiments and/or analysed the data. H.H.P., S.A.H., J.Z., J.M.D. and D.W.L. prepared the manuscript.

Corresponding author

Correspondence to Dudley W. Lamming.

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Competing interests

D.W.L. has received funding from, and is a scientific advisory board member of, Aeovian Pharmaceuticals, which seeks to develop novel, selective mTOR inhibitors for the treatment of various diseases. J.M.D. is a consultant for FORGE Life Sciences and co-founder of Galilei Bio-Sciences. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Metabolism thanks Leonie Heilbronn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt.

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Extended data

Extended Data Fig. 1 Additional measures of glucose homeostasis in male C57BL/6J mice.

(a) Food Consumption (b) Glucose (AL, n = 12; Diluted AL, n = 10; MF.cr, n = 11; CR, n = 12 biologically independent mice) (c) and insulin (AL, n = 12; Diluted AL, n = 11; MF.cr, n = 10; CR, n = 8 biologically independent mice) tolerance tests after 13 or 14 weeks on the indicated diets. * symbol represents a significant difference versus AL-fed mice (p = 0.0009); # symbol represents a significant difference versus Diluted AL-fed mice (p = 0.0021); * symbol represents a significant difference versus MF.cr-fed mice (p = 0.0013) based on Tukey’s test post one-way ANOVA. (D-F) Fasting blood glucose (d), fasting and glucose-stimulated insulin secretion (15 minutes) (e), and calculated HOMA-IR (F). (d-f) AL, n = 9; Diluted AL, n = 8; MF.cr, n = 9; CR, n = 9 biologically independent mice; * symbol represents a significant difference versus AL-fed mice (Diluted AL, p = 0.0256; CR, p = 0.0127); insulin levels in fasted and glucose-stimulated states were analyzed separately. All data are represented as mean ± SEM.

Source data

Extended Data Fig. 2 Fasting is required for CR-mediated reprogramming of the hepatic metabolome.

Targeted metabolomics were performed on the livers of male C57BL/6J mice fed AL, Diluted AL and CR diets (n = 6 biologically independent mice per diet). a) Heatmap of 59 targeted metabolites, represented as log2-fold change vs. AL-fed mice. b) sPLS-DA of liver metabolomics with CR mice sacrificed in the fasted state. c) sPLS-DA of liver metabolomics with CR sacrificed in the fed state. (d) Relative abundance of methionine and its metabolite S-Adenosyl-Homocysteine. * symbol represents a significant difference versus AL mice (p ≤ 0.0023); # symbol represents a significant difference versus Diluted AL mice (p ≤ 0.0024) based on Tukey’s test post one-way ANOVA. Overlaid box plots show center as median and 25th-75th percentiles; whiskers represent minima and maxima.

Extended Data Fig. 3 Additional hepatic metabolomic data.

(a-c) Hepatic metabolites that showed a statistically significant difference during the fasted or fed state (n = 6 biologically independent mice per diet). a) Relative abundance of nucleotide/nucleoside metabolites. b) Relative abundance of TCA cycle metabolites. c) Relative abundance of amino acid metabolites. (a-c) * symbol represents a significant difference versus AL mice (p ≤ 0.05); # symbol represents a significant difference versus Diluted AL mice (p ≤ 0.05) based on Tukey’s test post one-way ANOVA. Overlaid box plots show center as median and 25th-75th percentiles; whiskers represent minima and maxima.

Extended Data Fig. 4 Fasting is required for CR-mediated reprogramming of the hepatic epigenome.

Histone proteomics were performed on the livers of male C57BL/6J mice fed AL, Diluted AL and CR diets (n = 6 mice per group). a) Heatmap of histone H3 and H4 peptides represented as log2-fold change from AL. B) sPLS-DA of histone modifications. c) Statistically significant modified histones. (a-c) * symbol represents a significant difference versus AL mice (p ≤ 0.05); # symbol represents a significant difference versus Diluted AL mice (p ≤ 0.05) based on Tukey’s test post one-way ANOVA. Overlaid box plots show center as median and 25th-75th percentiles; whiskers represent minima and maxima.

Extended Data Fig. 5 Metabolomic profile of skeletal muscle from AL, Diluted AL and CR mice.

Targeted metabolomics were performed on skeletal muscle from male C57BL/6J mice fed AL, Diluted AL, and CR diets (n = 10 biologically independent mice per diet). a) Heatmap of 28 targeted metabolites, represented as log2-fold change vs. AL-fed mice. b) sPLS-DA of skeletal muscle metabolites. c) Relative abundance of amino acid metabolites. d) Relative abundance of TCA cycle metabolites. (a–d) * symbol represents a significant difference versus AL mice (p ≤ 0.05); # symbol represents a significant difference versus Diluted AL mice (p ≤ 0.05) based on Tukey’s test post one-way ANOVA. Overlaid box plots show center as median and 25th-75th percentiles; whiskers represent minima and maxima.

Extended Data Fig. 6 The effect of three calorie restriction regimens on female C57BL/6J mice.

(a) Outline of feeding regimens: AL, Diluted AL, CR and MF.cr. (b-e) Body composition measurement over 16 weeks on diet (AL, n = 12; Diluted AL, n = 10; MF.cr, n = 12; CR, n = 12 biologically independent mice); total body weight (b), lean mass (c), fat mass (d) and adiposity (e). (f-g) Glucose (n = 12 biologically independent mice per diet) (f) and insulin (AL, n = 12; Diluted AL, n = 12; MF.cr, n = 11; CR, n = 10 biologically independent mice) (g) tolerance tests after 9 or 10 weeks on the indicated diets. * symbol represents a significant difference versus AL-fed mice (Diluted AL, p < 0.0001; MF.cr, p ≤ 0.0012, CR, p < 0.0001); # symbol represents a significant difference versus Diluted AL-fed mice (MF.cr, p = 0.0019; CR, p ≤ 0.0001); @ symbol represents a significant difference versus MF.cr-fed mice (CR, p = 0.0043) based on Tukey’s test post one-way ANOVA. (h-j) Metabolic chamber analysis of mice fed the indicated diets. (h) Respiratory exchange ratio vs. time (n = 12 biologically independent mice per diet) (i) Fuel utilization was calculated for the 24-hour period following the indicated (arrow) refeeding time (n = 12 biologically independent mice per diet). * symbol represents a significant difference versus AL (Diluted AL, p = 0.0255); # symbol represents a significant difference versus Diluted AL (CR, p = 0.0274) based on Tukey’s test post one-way ANOVA performed separately for FAO and C/PO). (j) Energy expenditure as a function of lean mass was calculated for the 24-hour period following the indicated (arrow) refeeding time (n = 12 biologically independent mice per diet, data for each individual mouse is plotted; slopes and intercepts were calculated using ANCOVA). k) Food consumption (AL, n = 12; Diluted AL, n = 12; MF.cr, n = 12; CR, n = 11-12). All data are represented as mean ± SEM.

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Extended Data Fig. 7 The effect of three calorie restriction regimens on male DBA/2J mice.

(a) Outline of feeding regimens: AL, Diluted AL, CR and MF.cr. (b–e) Body composition measurement over 16 weeks on diet (AL, n = 11; Diluted AL, n = 11; MF.cr, n = 12; CR, n = 12 biologically independent mice); total body weight (b), lean mass (c), fat mass (d) and adiposity (e). (f-g) Glucose (AL, n = 12; Diluted AL, n = 12; MF.cr, n = 12; CR, n = 11 biologically independent mice) (f) and insulin (AL, n = 12; Diluted AL, n = 12; MF.cr, n = 12; CR, n = 11 biologically independent mice) (g) tolerance tests after 9 or 10 weeks on the indicated diets. * symbol represents a significant difference versus AL-fed mice (Diluted AL, p = 0.0059; MF.cr, p = 0.0052; CR, p < 0.0001) based on Tukey’s test post one-way ANOVA. (H-J) Metabolic chamber analysis of mice fed the indicated diets. (h) Respiratory exchange ratio vs. time (AL, n = 12; Diluted AL, n = 11; MF.cr, n = 11; CR, n = 11 biologically independent mice) (i) Fuel utilization was calculated for the 24-hour period following the indicated (arrow) refeeding time (AL, n = 12; Diluted AL, n = 11; MF.cr, n = 11; CR, n = 11 biologically independent mice). * symbol represents a significant difference versus AL (Diluted AL, p < 0.0001; MF.cr, p < 0.0001; CR, p < 0.0001); # symbol represents a significant difference versus Diluted AL (MF.cr, p ≤ 0.0002; CR, p ≤ 0.0001) based on Tukey’s test post one-way ANOVA performed separately for FAO and C/PO). (j) Energy expenditure as a function of lean mass was calculated for the 24-hour period following the indicated (arrow) refeeding time (AL, n = 12; Diluted AL, n = 11; MF.cr, n = 11; CR, n = 11 biologically independent mice, data for each individual mouse is plotted; slopes and intercepts were calculated using ANCOVA). K) Food consumption (n = 12 biologically independent mice per diet). All data are represented as mean ± SEM.

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Extended Data Fig. 8 The effect of three calorie restriction regimens on female DBA/2J mice.

(a) Outline of feeding regimens: AL, Diluted AL, CR and MF.cr. (b-e) Body composition measurement over 16 weeks on diet (AL, n = 12; Diluted AL, n = 7; MF.cr, n = 11; CR, n = 12 biologically independent mice); total body weight (b), lean mass (c), fat mass (d) and adiposity (e). (f-g) Glucose (AL, n = 12; Diluted AL, n = 11; MF.cr, n = 12; CR, n = 12 biologically independent mice) (f) and insulin (AL, n = 12; Diluted AL, n = 11; MF.cr, n = 12; CR, n = 12 biologically independent mice) (g) tolerance tests after 9 or 10 weeks on the indicated diets. * symbol represents a significant difference versus AL-fed mice (Diluted AL, p = 0.0033; MF.cr, p = 0.0003; CR, p < 0.0001) based on Tukey’s test post one-way ANOVA. (H-J) Metabolic chamber analysis of mice fed the indicated diets. (h) Respiratory exchange ratio vs. time (AL, n = 11; Diluted AL, n = 11; MF.cr, n = 11; CR, n = 10 biologically independent mice) (i) Fuel utilization was calculated for the 24-hour period following the indicated (arrow) refeeding time (AL, n = 11; Diluted AL, n = 11; MF.cr, n = 11; CR, n = 10 biologically independent mice). * symbol represents a significant difference versus AL (Diluted AL, p < 0.0001; MF.cr, p < 0.0001; CR, p < 0.0001); # symbol represents a significant difference versus Diluted AL (MF.cr, p ≤ 0.0128; CR, p < 0.0001); @ symbol represents a significant difference versus MF.cr (CR, p ≤ 0.0074) based on Tukey’s test post one-way ANOVA performed separately for FAO and C/PO). (J) Energy expenditure as a function of lean mass was calculated for the 24-hour period following the indicated (arrow) refeeding time (AL, n = 11; Diluted AL, n = 11; MF.cr, n = 11; CR, n = 10 biologically independent mice, data for each individual mouse is plotted; slopes and intercepts were calculated using ANCOVA). K) Food consumption (AL, n = 12; Diluted AL, n = 7-12; MF.cr, n = 12; CR, n = 12 biologically independent mice). All data are represented as mean ± SEM.

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Extended Data Fig. 9 Additional data for C57BL/6J male mice fed CR or TR.al diets.

a) Food consumption (n = 12 biologically independent mice per diet). b-e) Body composition (body weight, lean mass, fat mass and adiposity) of C57BL/6J male mice fed the indicated diets for 4 months (n = 12 biologically independent mice per diet; statistics on supplementary table 7). F-G) Glucose (n = 12 biologically independent mice per diet) (f) and insulin (AL, n = 12; TR.al, n = 12; CR, n = 11 biologically independent mice) (g) tolerance tests were performed after 13-14 weeks, respectively on the indicated diets. (h-j) Fasting blood glucose (h), fasting and glucose-stimulated insulin secretion (15 minutes) (i), and calculated HOMA2-IR (j) (AL, n = 12; TR.al, n = 11; CR, n = 12 biologically independent mice). * symbol represents a significant difference versus AL (TR.al, p ≤ 0.0018; CR, p ≤ 0.0477); # symbol represents a significant difference versus TR.al (MF.cr, p ≤ 0.0187; CR, p = 0.0478) based on Tukey’s test post one-way ANOVA. All data are represented as mean ± SEM.

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Extended Data Fig. 10 Food consumption, absorption and gut integrity.

a) Food consumption (AL, n = 27-33; Diluted AL, n = 8-33; CR, n = 30-33 biologically independent mice). b) Food absorption calculation by bomb calorimetry of 19-month-old C57BL/6J male mice fed the indicated diets for 13 months (n = 6 biologically independent mice per diet) c) Gut integrity calculation by FITC-dextran of 20-month-old C57BL/6J male mice (n = 6 biologically independent mice per diet). * symbol represents a significant difference versus AL (CR, p = 0.0160); # symbol represents a significant difference versus Diluted AL (CR, p ≤ 0.0281) based on Tukey’s test post two-way ANOVA) Data are represented as mean ± SEM.

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Supplementary Information

Supplementary Figs. 1–6

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Supplemental Tables 1–11

Supplementary Data

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Pak, H.H., Haws, S.A., Green, C.L. et al. Fasting drives the metabolic, molecular and geroprotective effects of a calorie-restricted diet in mice. Nat Metab 3, 1327–1341 (2021). https://doi.org/10.1038/s42255-021-00466-9

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