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Daytime-restricted feeding enhances running endurance without prior exercise in mice

Nature Metabolism volume 5, pages 1236–1251 (2023)Cite this article

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Abstract

Physical endurance and energy conservation are essential for survival in the wild. However, it remains unknown whether and how meal timing regulates physical endurance and muscle diurnal rhythms. Here, we show that day/sleep time-restricted feeding (DRF) enhances running endurance by 100% throughout the circadian cycle in both male and female mice, compared to either ad libitum feeding or night/wake time-restricted feeding. Ablation of the circadian clock in the whole body or the muscle abolished the exercise regulatory effect of DRF. Multi-omics analysis revealed that DRF robustly entrains diurnal rhythms of a mitochondrial oxidative metabolism-centric network, compared to night/wake time-restricted feeding. Remarkably, muscle-specific knockdown of the myocyte lipid droplet protein perilipin-5 completely mimics DRF in enhancing endurance, enhancing oxidative bioenergetics and outputting rhythmicity to circulating energy substrates, including acylcarnitine. Together, our work identifies a potent dietary regimen to enhance running endurance without prior exercise, as well as providing a multi-omics atlas of muscle circadian biology regulated by meal timing.

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Fig. 1: Day/sleep time-restricted feeding enhances running endurance in sedentary and trained female mice.
Fig. 2: DRF enhances running endurance through the myocyte clock.
Fig. 3: Day/sleep time-restricted feeding entrains diurnal rhythm of mitochondrial lipid metabolism genes.
Fig. 4: Day/sleep time-restricted feeding entrains diurnal oscillation of energy substrates in the muscle and serum.
Fig. 5: Day/sleep time-restricted feeding entrains diurnal rhythms of mitochondrial functions and dynamics in muscle tissues.
Fig. 6: Day/sleep time-restricted feeding induces diurnal rhythm of muscle Plin5 in a clock-dependent manner.
Fig. 7: Day/sleep time-restricted feeding enhances running endurance via myocyte Plin5.
Fig. 8: Myocyte Plin5 knockdown mimics day/sleep time-restricted feeding in outputting rhythmicity to muscle mitochondrial lipid metabolism and circulating energy substrates.

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Data availability

RNA-seq data in this paper have been deposited into the Gene Expression Omnibus under accession number GSE194106. The published human transcriptome data are available at GSE28422. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the iProX59 partner repository under dataset identifier PXD039355. Graphs of these multi-omics data can be found at http://www.circametdb.org.cn/. Raw quantification data are deposited to the Science Data Bank (https://www.scidb.cn/en/). Source data are provided with this paper. Any other relevant data are available from the corresponding author upon request.

Code availability

Custom code is deposited to the Science Data Bank (https://doi.org/10.57760/sciencedb.08081).

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Acknowledgements

Per1/Per2-DKO mice were gifted from E. E. Zhang, and bred by J. R. He. Bmal1fl/fl mice were gifted from W. He. We thank D. Zhu, Z. Gan, Z. Meng and L. Xie for suggestions. This study was supported by National Natural Science Foundation of China Grants (32271208 to M.-D.L., 92057109 to M.-D.L., 81900776 to M.-D.L. and 32150005 to C.C.L.W.), Chongqing Science Fund for Distinguished Young Scholars (CSTB2022NSCQ-JQX0009 to M.-D.L.), National Key Research and Development Programme of China (2022YFA0806001 to C.C.L.W.), CAMS Innovation Fund for Medical Sciences (2022-I2M-1-004 to C.C.L.W.).

Author information

Author notes

  1. These authors contributed equally: Haoran Xin, Rongfeng Huang, Meiyu Zhou, Jianghui Chen.

Authors and Affiliations

  1. Department of Cardiovascular Medicine, Center for Circadian Metabolism and Cardiovascular Disease, Southwest Hospital, Army Medical University, Chongqing, China

    Haoran Xin, Rongfeng Huang, Meiyu Zhou, Jianghui Chen, Jianxin Zhang, Xinyu Bao, Lihua Li, Zhihui Zhang & Min-Dian Li

  2. Department of Cardiology, Third Affiliated Hospital of Chongqing Medical University, Chongqing, China

    Jianghui Chen & Shifei Tong

  3. Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China

    Tingting Zhou & Catherine C. L. Wong

  4. Department of Bioinformatics, GFK Biotech, Shanghai, China

    Shushen Ji & Xiao Liu

  5. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    He Tian, Sin Man Lam & Guanghou Shui

  6. LipidALL Technologies, Changzhou, China

    Sin Man Lam

  7. Department of Pathophysiology, College of High Altitude Military Medicine, Army Medical University, Chongqing, China

    Fang Deng

  8. Tsinghua University-Peking University Joint Center for Life Sciences, Tsinghua University, Beijing, China

    Catherine C. L. Wong

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Contributions

Conceptualization: M.-D.L.; Methodology: H.X., R.H., M.Z., J.C., G.S., C.C.L.W. and M.-D.L.; Investigation: M.-D.L., H.X., R.H., M.Z., J.C., T.Z., S.J., X.L., H.T., S.M.L., X.B., J.Z. and L.L.; Funding acquisition: M.-D.L. and C.C.L.W.; Project administration: M.-D.L.; Resources: M.-D.L., C.C.L.W., G.S., S.T., F.D. and Z.Z.; Supervision: M.-D.L., C.C.L.W. and G.S.; Writing—original draft: M.-D.L., H.X., R.H., M.Z. and J.C.; Writing—review and editing: M.-D.L., C.C.L.W. and G.S.

Corresponding authors

Correspondence to Zhihui Zhang, Catherine C. L. Wong or Min-Dian Li.

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Nature Metabolism thanks Chih-Hao Lee, Kristin Stanford and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Exercise phenotyping in time-restricted fed mice.

a, Running power in 3-week TRF female mice, as measured by time to exhaustion, distance, cumulative stimulations, and maximum speed (n = 14 NRF or 16 DRF mice). b, Running endurance in the early night (ZT14, n = 15 NRF or 16 DRF mice), as measured by time to exhaustion and distance. c, Running endurance in 3-week DRF mice (Male: n = 15 mice; Female: n = 16 mice), as measured by time to exhaustion, distance, and blood glucose. d, Running power in 3-week DRF mice (Male: n = 15 AL or 16 DRF mice; Female: n = 16 mice), as measured by time to exhaustion, distance, and maximum speed. e, Immunofluorescence (IF) of slow- (type 1)/fast- (type 2B, 2 A, 2X) myofibers and histochemical staining of bioenergetic enzyme activity in soleus muscle after 3 weeks of TRF (n = 8 mice, one image per mouse). MyHC, myosin heavy chain. SDH, succinate dehydrogenase. GPDH, alpha-glycerophosphate dehydrogenase. Arrows indicate positively stained fibers. Scale bars, 200 µm. f, Total locomotor activity in 3-week TRF female mice (n = 9 AL or 8 DRF/NRF mice). g, Respiratory exchange ratio (RER) in 1-week TRF female mice (n = 4 mice). h, The scheme for testing endurance in trained female mice (n = 12 mice, slope: 15o). i, Myofiber typing in GA muscle from ad libitum (AL)-fed exercised (Exe) mice or TRF-fed sedentary (Sed) mice, as measured by IF and bioenergetic enzyme activity (n = 11 Sed-NRF, 12 Sed-DRF (IF/GPDH), 11 Sed-DRF (SDH) or 16 Exe-AL mice). TRF, time-restricted feeding; NRF, night/wake time-restricted feeding; DRF, day/sleep time-restricted feeding (food available from ZT0 to ZT12); AL, ad libitum feeding. Data were represented as the mean ± s.e.m. Statistical tests: ANOVA, post-hoc Sidak/Tukey test, Kruskal-Wallis test, or two-sided unpaired Student’s/Wilcoxon test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Source data

Extended Data Fig. 2 Striated muscle-specific knockdown of Per2 gene by systemic administration of MyoAAV does not alter running endurance under DRF in female mice.

a, Daytime locomotor activity in Per1−/− Per2−/− (Per1/2 DKO) male mice before and after 3 weeks of DRF beginning at the age of 34 weeks (3 weeks AL feeding > 3 weeks DRF) (n = 8 mice), as measured by percentage of daytime activity and 24-h beam breaks. b, Daytime locomotor activity in Per2−/− female mice before and after 3 weeks of DRF starting at the age of 7 weeks (n = 10 mice). c, RT-qPCR analysis of muscle and liver tissues in MyoAAV-transduced (MKD) female mice under DRF (N = 5 Ctrl-MKD mice, N = 6 Per2-MKD mice). d, Representative immunofluorescence image of PER2 protein in TA muscle tissue. This experiment was repeated independently twice with similar results (n = 200 cells examined over two independent experiments). GA muscle from Per1/2 DKO mice, as well as mock primary antibody-treated Per2-MKD, serves as the negative control. e, Daytime locomotor activity in MKD female mice before and after 3 weeks of DRF starting at the age of 8 weeks (n = 5 Ctrl-MKD or 6 Per2-MKD mice). f, Running endurance of MKD female mice in the early day (ZT2, n = 16 mice), as measured by time to exhaustion, distance and cumulative stimulations. Mice received MyoAAV by tain vein injection at the age of 8 weeks, tested 3 weeks later (AL), and fed DRF for 3 weeks before the next running test. g, Tissue mass in Bmal1 or Per2 MKO mice under DRF (n = 8 control or 7 MKO mice). Per2-MKD, striated muscle-specific knockdown of Per2; Ctrl-MKD, control group. Data are presented as the mean ± s.e.m. or minima, maxima, median, 25th and 75th percentiles. Statistical tests: two-sided paired Student’s t-test, ANOVA, post-hoc Sidak’s test, or two-sided Wilcoxon test, *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 3 General features and circadian clock oscillation of the multi-omics dataset.

a, Exercise performance in 1-week TRF mice as measured by time to exhaustion (n = 16 mice). b, Workflow of the multi-omics study. c-d, Diurnal expression of the muscle clock genes in TRF mice (c, female: n = 4 mice; d, male: n = 4 mice). e, Phase distribution of oscillating muscle genes and phase-shift between dual-oscillating genes in TRF mice. f-h, Diurnal expression of dual-oscillating (f), DRF-only (g), NRF-only (g), and mitochondrial (h) rhythmic genes in skeletal muscle (n = 4 mice per time-point). Data were represented as the mean ± s.e.m. or minima, maxima, median, 25th and 75th percentiles. Statistical test: ANOVA, two-sided unpaired Student’s t-test with Bonferroni correction, MetaCycle: P-values from JTK and LS methods were adjusted by Fisher’s method.

Source data

Extended Data Fig. 4 Global profiling of muscle metabolites.

Heatmap showing cycling metabolites in GA muscle during TRF. Inset shows phase distribution of muscle cycling metabolites.

Source data

Extended Data Fig. 5 Global profiling of muscle lipids and serum metabolites.

a, Heatmap showing cycling lipid species in GA muscle. Inset shows phase distribution of muscle cycling lipids. Abbreviations see Data S4. b, Diurnal expression of cycling serum metabolites (n = 7 mice for 2 time points). c, Principal component analysis of the four groups. D00, DRF ZT0; D12, DRF ZT12; N00, NRF ZT0; N12, NRF ZT12.

Source data

Extended Data Fig. 6 Global profiling of the muscle proteome.

a, 48-h profile of cycling muscle proteins from 3-week DRF mice. Samples were collected every 2 h for 48 h containing 25 time points (n = 2 mice per time point). b, Representative cycling muscle proteins (n = 2 mice per time point). Data were represented as mean and points. c, Representative immunoblots of pSer637-DRP1 in anterior hypothalamus (aHyp) of the brain in ad libitum fed mice (n = 2 mice, repeated three times with similar results). ALP, alkaline phosphatase treatment. d, Immunoblot analysis of key regulators involved in mitochondrial dynamics by DRF in GA muscle. Data were represented as the mean ± s.e.m. MetaCycle: P-values from JTK and LS methods were adjusted by Fisher’s method.

Source data

Extended Data Fig. 7 Daily rhythms of acylcarnitine and amino acids persist in the muscle of 3-week DRF female mice.

a, Heatmap showing cycling metabolites in GA muscle from 3-week DRF female mice. b, Class of cycling metabolites in muscle. c, Heatmap showing cycling acylcarnitines, amino acids and fatty acids. d, Daily abundance levels of selected cycling muscle metabolites representing acylcarnitines and amino acids. Stearoylcarnitine, 18:0-carnitine; L-Palmitoylcarnitine, 16:0-carnitine. Samples were collected every 2 h for 48 h containing 25 time points (n = 2 mice per time point). Data were shown as the mean ± s.e.m. Statistical tests: RAIN and MetaCycle.

Source data

Extended Data Fig. 8 Diurnal expression of rhythmic transcripts in DRF muscle.

a, Diurnal expression of Pdp2 and Hmgcs2 in GA muscle from TRF female mice, as measured by RNA sequencing (n = 4 mice). b, Diurnal gene expression in GA muscle from TRF female mice (n = 4 mice). c, Diurnal gene expression in GA muscle from TRF male mice (n = 4 mice). d, Diurnal gene expression in GA muscle from female mice subjected to DRF and constant lightness (n = 4 mice). e, Diurnal gene expression in GA muscle from Per1−/−Per2−/− mutant mice subjected to constant darkness and DRF (N = 14 mice, n = 2 mice per time point for 7 time points). CT, circadian time, or hours under constant darkness; τ, period length. f, Gene expression in vastus lateralis muscle biopsies before (T1, T3) and after (T2, T4) resistance exercise. Y (young, n = 8 males/8 females), O (old, n = 6 males/6 females); T1/T2 is from the sedentary state, T3/T4 is from the trained state (Source: GSE28422). Data are presented as mean ± s.e.m. or minima, maxima, median, 25th and 75th percentiles. Statistical tests: multiple t-tests with Bonferroni correction, MetaCycle (rhythmicity), Circacompare (phase shift), or ANOVA, two-sided Wilcoxon test; ns, not significant, AR, arrhythmia.

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Extended Data Fig. 9 The role of myocyte Plin5 in the regulation of exercise capacity under DRF or ad libitum.

a, Treadmill running scheme for studying the role of myocyte Plin5 in exercise capacity. KD, TA muscle-specific knockdown of Plin5 gene; WT, control group dosed with negative control miRNA. b, Representative immunoblots of PLIN5 protein in different muscle depots from the ipsilateral and contralateral sides of the AAV injection site (one technical replicate of two biological replicates). c, Running power in female mice transduced with AAV-pre-miR-Plin5 (KD) or AAV-pre-miR-Ctrl (WT) and fed ad libitum (AL) or DRF for 3 weeks (n = 16 mice), as measured by time to exhaustion, distance, maximum speed, and cumulative stimulations. d, Cumulative stimulations and body weight in the endurance test for KD and WT mice fed AL or DRF for 3 weeks (n = 16 mice). Data were represented as the mean ± s.e.m. 2-way ANOVA or two-sided unpaired Student’s t test, *P < 0.05.

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Extended Data Fig. 10 Muscle phenotyping in Plin5 KD mice.

a, Running endurance in DRF Plin5 KD mice resuming AL feeding for 1 to 3 weeks (n = 16 mice), as measured by time to exhaustion, distance, and cumulative number of stimulations. b, Running endurance and histochemical staining of bioenergetic enzyme activity in GA muscle from diet reversed (DRF > AL, return to AL for 3 weeks after 3 weeks of DRF) and DRF mice (n = 16 mice, one image per mouse). Scale bars, 100 μm. c, Representative immunoblots for PLIN5 in GA muscle and heart (HT) of MKD mice (n = 5 mice). d, Distribution of phase-shift among dual-oscillating muscle genes in KD and WT mice. KD, TA-specific Plin5 knockdown. e, Diurnal expression of circadian clock genes in the muscle (n = 2 mice for 8 time points). f, Pathway analysis of differentially expressed muscle genes from ZT21 to ZT3 between KD and WT mice (n = 6 mice). Data were represented as the mean ± s.e.m. 2-way ANOVA or two-sided unpaired Student’s t test, *P < 0.05, ****P < 0.0001.

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Supplementary information

Supplementary Information

Supplementary Methods and Supplementary Table 4

Reporting Summary

Supplementary Tables 1–3

Table 1: Rhythmicity results of genes, proteins, metabolites and lipids. Table 2: PSEA results of oscillating genes in skeletal muscle. Table 3: Rhythmicity results of metabolites in the serum.

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Xin, H., Huang, R., Zhou, M. et al. Daytime-restricted feeding enhances running endurance without prior exercise in mice. Nat Metab 5, 1236–1251 (2023). https://doi.org/10.1038/s42255-023-00826-7

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