Clock proteins and training modify exercise capacity in a daytime-dependent manner

Abstract

Exercise and circadian biology are closely intertwined with physiology and metabolism, yet the functional interaction between circadian clocks and exercise capacity is only partially characterized. Here, we tested different clock mutant mouse models to examine the effect of the circadian clock and clock proteins, namely PERIODs and BMAL1, on exercise capacity. We found that daytime variance in endurance exercise capacity is circadian clock controlled. Unlike wild-type mice, which outperform in the late compared with the early part of their active phase, PERIODs- and BMAL1-null mice do not show daytime variance in exercise capacity. It appears that BMAL1 impairs and PERIODs enhance exercise capacity in a daytime-dependent manner. An analysis of liver and muscle glycogen stores as well as muscle lipid utilization suggested that these daytime effects mostly relate to liver glycogen levels and correspond to the animals' feeding behavior. Furthermore, given that exercise capacity responds to training, we tested the effect of training at different times of the day and found that training in the late compared with the early part of the active phase improves exercise performance. Overall, our findings suggest that clock proteins shape exercise capacity in a daytime-dependent manner through changes in liver glycogen levels, likely due to their effect on animals' feeding behavior.

Keywords: circadian clocks; exercise; glycogen; metabolism; training.

Fig. 1.

Male and female mice show a daytime difference in their exercise capacity. Mice were housed under a 12-h light–12-h dark regimen, and a moderate-intensity treadmill test was performed either at the Early (ZT14) or Late (ZT22) part of the active phase. (A) Schematic representation of the experimental design. (B and C) Blood glucose profiles of individual WT mice during the test (thin lines) and mean ± SEM (thick line) for each group for males (Early, n = 5; Late, n = 4) and females (Early, n = 5; Late, n = 4). End point for each group, at which either blood glucose levels reached ≤70 mg/dL or the animal finished the 5.5-h test, is presented as average time ± SEM. (D) Food intake was monitored using metabolic cages. Data are presented as mean ± SEM of average food consumed during 12 h per body weight either before ZT14 or ZT22 from an average of two consecutive days of four male and four female mice. See also SI Appendix, Fig. S3A. (E and F) Liver and gastrocnemius (Gas) glycogen content (n = 4 per group) of Early (ZT14) and Late (ZT22) sedentary (Sed) mice or after a 90-min run (Run). Student’s t test (B and C); two-way ANOVA with Tukey’s post hoc test (D); three-way ANOVA with Tukey’s post hoc test (E and F; for panel E, test was done on log-transformed values); ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, nonsignificant (ns).

Fig. 2.

Time difference in exercise capacity is circadian clock controlled. Mice were housed under a 12-h light–12-h dark regimen, and lights were not turned on at ZT0 on the day of the test. Moderate-intensity treadmill test was performed in constant dark conditions either at the Early (CT14) or Late (CT22) part of the respective active phase. (A) Schematic representation of the experimental design. (B and C) Blood glucose profiles of individual mice during the test (thin lines) and mean ± SEM (thick line) for each group for WT (Early, n = 4; Late, n = 5) and Per1,2^−/− (Early, n = 5; Late, n = 5). End point for each group, at which either blood glucose levels reached ≤70 mg/dL or the animal finished the 5.5-h test, is presented as average time ± SEM. (D) Food intake was monitored using metabolic cages. Data are presented as mean ± SEM of average food consumed during 12 h per body weight either before CT14 or CT22 from an average of three WT and three Per1,2^−/− mice. See also SI Appendix, Fig. S3B. Reanalysis of data presented in ref. . (E and F) Liver and gastrocnemius (Gas) glycogen content (n = 3 to 4 per group). Student’s t test (B and C), two-way ANOVA (F) with Tukey’s post hoc test (D and E); ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, nonsignificant (ns).

Fig. 3.

The effect of clock proteins on daytime variance in exercise capacity. Mice were housed under a 12-h light–12-h dark regimen, and a moderate-intensity treadmill test was performed either at the Early (ZT14) or Late part (ZT22) of the active phase. Early and Late group blood glucose profiles of individual mice during a moderate-intensity run (thin lines) and mean ± SEM for each time point (thick line) of (A) Per1,2^−/− mice (Early [n = 4] and Late [n = 5]), (B) Per1^−/− mice (Early [n = 5] and Late [n = 5]), and (C) Per2^−/− mice (Early [n = 5] and Late [n = 5]). End point for each group, at which either blood glucose levels reached ≤70 mg/dL or the animal finished the 5.5-h test, is presented as average time ± SEM. (D) Food intake was monitored using metabolic cages. Data are presented as mean ± SEM of average food consumed during 12 h per body weight either before ZT14 or ZT22 from an average of two consecutive days of six to seven mice from each genotype. See also SI Appendix, Fig. S3C. (E and F) Liver and gastrocnemius (Gas) glycogen content (n = 4 to 5 per group). Early and Late group blood glucose profiles of individual mice during a moderate-intensity run (thin lines) and mean ± SEM for each time point (thick line) of (G) Bmal1^+/+ mice (Early [n = 5] and Late [n = 4]), (H) Bmal1^−/+ mice (Early [n = 3] and Late [n = 3]), and (I) Bmal1^−/− mice (Early [n = 4] and Late n = 4]). End point for each group, at which either blood glucose levels reached ≤70 mg/dL or the animal finished the 5.5-h test, is presented as average time ± SEM. (J) Food intake was monitored using metabolic cages. Data are presented as mean ± SEM of average food consumed during 12 h per body weight either before ZT14 or ZT22 from an average of two consecutive days of two to three mice from each genotype. See also SI Appendix, Fig. S3D. Reanalysis of data presented in ref. . (K and L) Liver and gastrocnemius (Gas) glycogen content (n = 3 to 5 per group). Student’s t test (A–C and G–I), two-way ANOVA (F) with Tukey’s post hoc test (D, E, J, K, and L); ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, nonsignificant (ns).

Fig. 4.

Clock proteins modify exercise capacity in a time-dependent manner. (A and B) Time until reaching blood glucose levels ≤70 mg/dL (time to fatigue) upon moderate-intensity treadmill test for Per and Bmal1 mutant mice, respectively. Red dots represent mice that finished the 5.5-h run without reaching blood glucose levels ≤70 mg/dL. (C and D) Peak blood glucose levels upon moderate-intensity treadmill test for Per and Bmal1 mutant mice, respectively. (E and F) Representative linear fits for glucose consumption during the test for the average profiles of WT, Per, and Bmal1 mutant mice. (G and H) Calculated GCRs (slopes) based on the linear fits for individual WT (R² > 0.90), Per (R² > 0.78), and Bmal1 (R² > 0.73) mutant mice. (I and J) Calculated time to fatigue (intercept with y = 70) based on the linear fits for individual WT, Per, and Bmal1 mutant mice. The analysis is based on data presented in Figs. 1B and 3 A–C and G–I. One-way ANOVA for each Late and Early groups (nonsignificant [ns] for this test is marked on the graphs) with Tukey’s post hoc test (A–D and G–J); ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; multiple comparisons that were nonsignificant are not marked.

Fig. 5.

Muscle lipid composition in sedentary and exercised Per1,2^−/− and Bmal1^−/− mice. Mice were housed under a 12-h light–12-h dark regimen and performed the moderate-intensity treadmill test at ZT22 for 1 h. Sedentary (Sed) and Exercised (Ex) mice were killed, and the gastrocnemius muscle was harvested and analyzed for its lipids content. Fold induction of mean total triglycerides identified in WT (n = 6) and Per1,2^−/− (n = 6) mice (A) and in Bmal1^−/+ (n = 8) and Bmal1^−/− (n = 10) (E). Heatmap representation of triglyceride levels for WT and Per1,2^−/− (B) and for Bmal1^−/+ and Bmal1^−/− (F). Fold induction of mean total acylcarnitines identified in WT and Per1,2^−/− mice (C) and in Bmal1^−/+ and Bmal1^−/− (G). Heatmap representation of acylcarnitine levels for WT and Per1,2^−/− (D) and for Bmal1^−/+ and Bmal1^−/− (H). Triglycerides and acylcarnitines with positive and negative Z scores are depicted in purple and orange, respectively. Clustering was performed using Python’s Seaborn clustermap function on log2-transformed data. Two-way ANOVA (A, E, and G) with Bonferroni’s post hoc test (C). *P < 0.05, nonsignificant (ns).

Fig. 6.

The effect of daytime scheduled training on endurance capacity. (A) An image of the TRW. (B) A schematic representation of the experimental design. (C) Representative actograms of Early- and Late-trained mice throughout their scheduled training period. (D) The daily running pattern and the mean daily distance covered by the Early- and Late-trained mice throughout the scheduled training period (n = 5 to 7 in each group). (E) Blood glucose profiles of individual mice (thin lines) during an extended moderate-intensity treadmill test performed at ZT14 of Untrained (n = 5), Early- (n = 6), or Late-trained mice (n = 7) and mean ± SEM (thick line) for each time point. End point for each group, at which either blood glucose levels reached ≤70 mg/dL or the animal finished the 8-h test, is presented as average time ± SEM. (F and G) Running wheels of Early- and Late-trained mice were locked for a washout period of 24 h, after which at ZT0 the wheels were freely opened to all groups, and mice were kept in constant dark. Onset of activity of the first day and period lengths were analyzed from the recorded actograms. (H) Polar plot of the phase distribution of gastrocnemius muscle based on gastrocnemius organotypic slice from PER2::LUC mice that were subjected to the training protocol in B (n = 6 for each group). Each point represents the CT value of the first peak of the second day of recordings from a single mouse (mean of 3 to 5 technical replicates). The line’s angle represents the circular mean of each condition, and line’s radius anticorrelates with the circular variance (n = 6; P < 0.05, Watson–Williams test). (I) Period length based on gastrocnemius muscle PER2::LUC bioluminescence recordings (n = 6). One-way ANOVA (G and I), with Tukey’s post hoc test (D, E, and F); ****P < 0.0001, ***P < 0.001, **P < 0.01, nonsignificant (ns).