Age-related circadian disorganization caused by sympathetic dysfunction in peripheral clock regulation

Yu Tahara, Yuta Takatsu, Takuya Shiraishi, Yosuke Kikuchi, Mayu Yamazaki, Hiroaki Motohashi, Aya Muto, Hiroyuki Sasaki, Atsushi Haraguchi, Daisuke Kuriki, Takahiro J Nakamura, Shigenobu Shibata, Yu Tahara, Yuta Takatsu, Takuya Shiraishi, Yosuke Kikuchi, Mayu Yamazaki, Hiroaki Motohashi, Aya Muto, Hiroyuki Sasaki, Atsushi Haraguchi, Daisuke Kuriki, Takahiro J Nakamura, Shigenobu Shibata

Abstract

The ability of the circadian clock to adapt to environmental changes is critical for maintaining homeostasis, preventing disease, and limiting the detrimental effects of aging. To date, little is known about age-related changes in the entrainment of peripheral clocks to external cues. We therefore evaluated the ability of the peripheral clocks of the kidney, liver, and submandibular gland to be entrained by external stimuli including light, food, stress, and exercise in young versus aged mice using in vivo bioluminescence monitoring. Despite a decline in locomotor activity, peripheral clocks in aged mice exhibited normal oscillation amplitudes under light-dark, constant darkness, and simulated jet lag conditions, with some abnormal phase alterations. However, age-related impairments were observed in peripheral clock entrainment to stress and exercise stimuli. Conversely, age-related enhancements were observed in peripheral clock entrainment to food stimuli and in the display of food anticipatory behaviors. Finally, we evaluated the hypothesis that deficits in sympathetic input from the central clock located in the suprachiasmatic nucleus of the hypothalamus were in part responsible for age-related differences in the entrainment. Aged animals showed an attenuated entrainment response to noradrenergic stimulation as well as decreased adrenergic receptor mRNA expression in target peripheral organs. Taken together, the present findings indicate that age-related circadian disorganization in entrainment to light, stress, and exercise is due to sympathetic dysfunctions in peripheral organs, while meal timing produces effective entrainment of aged peripheral circadian clocks.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Locomotor activity and peripheral PER2::LUC rhythms under light–dark and constant darkness conditions. (a) Representative double-plotted actograms of locomotor activity measured by an infrared sensor in young (n=1) and aged (n=2) mice maintained under normal light–dark (LD) or constant darkness (DD) conditions for 1 month. The dark shadow indicates the dark (active) period. Arrowheads on the right indicate the timing of in vivo monitoring of peripheral PER2::LUC bioluminescence. (b–d) Amplitude (b) and period (c) of the rhythmicity of locomotor activity analyzed by χ2 periodogram, and total daily activity (d) in the DD condition (n=4–5). (e–h) Representative photo images (e), analyzed wave forms (f), peak phases (g), and amplitudes (h) of PER2::LUC bioluminescence in the kidney, liver, and submandibular gland (sub gla) of young and aged mice in each condition. ZT, zeitgeber time; pZT, projected ZT in constant darkness (pZT0 and pZT12 representing the same times as ZT0 and ZT12, respectively). Values are expressed as the mean±s.e.m. The number of mice used is indicated in Supplementary Table S1. *P<0.05, **P<0.01, ***P<0.001 versus the young group or the LD group (Student’s t test or two-way analysis of variance (ANOVA) with Tukey’s or Sidak’s post hoc tests). The P value on the lower right side of each figure (f) indicates the result of two-way ANOVA between the young and aged groups.
Figure 2
Figure 2
Locomotor activity and peripheral PER2::LUC rhythms under the experimental jet lag condition. (a) Representative double-plotted actograms of locomotor activity as measured by an infrared sensor in young and aged mice. The dark shadow indicates the dark period. (b) Averaged time of activity onset during the shifting phase of the light–dark cycle (n=8). (c–e) Analyzed wave forms (c), peak phases (d), phase advance values (e), and amplitudes (f) of PER2::LUC bioluminescence in the kidney, liver, and submandibular gland (sub gla) of young and aged mice 5 or 9 days after the light–dark cycle phase shift (different mouse cohorts were used on day 5 and day 9). Phase advance values were calculated as the difference between the peak times before and after the light–dark cycle phase shift. Values are expressed as the mean±s.e.m. The number of mice used is indicated in Supplementary Table S1. *P<0.05, **P<0.01, ***P<0.001 versus the young group or the pre group (Student’s t test, 1-way analysis of variance (ANOVA) with Dunnett post hoc tests, or two-way ANOVA with Sidak’s post hoc tests). The P value on the lower right side of each figure (c) indicates the result of a two-way ANOVA between the young and aged groups.
Figure 3
Figure 3
Locomotor activity and peripheral PER2::LUC rhythms in response to daytime scheduled feeding. (a) Representative double-plotted actograms of locomotor activity as measured by an infrared sensor in young and aged mice. The dark shadow indicates the feeding period. (b) Wave forms of locomotor activity analyzed during the last 3 days of each condition (n=8). FF, free feeding; SF, scheduled feeding. (c) Per cent activity during 3 h prior to the scheduled feeding time (Zeitgeber time (ZT) 1–3) or during the dark period (ZT12–24). (d) The experimental feeding schedule. White and black bars indicate the light and dark periods, respectively. Arrowheads indicate food timings. Food pellets (1 g for the first and second days, 1.5 g for the third day) were given to mice after overnight fasting. (e–h) Analyzed wave forms (e), peak phases (f), phase change values (g), and amplitudes (h) of PER2::LUC bioluminescence in the kidney, liver, and submandibular gland (sub gla) of young and aged mice in each condition. Values are expressed as the mean±s.e.m. The number of mice used is indicated in Supplementary Table S1. *P<0.05, **P<0.01, ***P<0.001 versus the young group or the control group (Student’s t test, 1-way analysis of variance (ANOVA) with Dunnett’s post hoc tests, or two-way ANOVA with Sidak’s post hoc tests). The P value on the lower right side of each figure (e) indicates the result of a two-way ANOVA between the young and aged groups.
Figure 4
Figure 4
Effect of constant routine feeding on peripheral PER2::LUC rhythms. (a) Experimental constant routine feeding schedule. Mice were fed the same amount of food (0.54 g) 6 times per day at the same interval for 4 weeks. White and black bars indicate the light and dark periods, respectively. Arrowheads indicate food timings. ZT, zeitgeber time; SF, scheduled feeding. (b–e) Analyzed wave forms (b), peak phases (c), phase change values (d), and amplitudes (e) of PER2::LUC bioluminescence in the kidney, liver, and submandibular gland (sub gla). Values are expressed as the mean±s.e.m. The number of mice used is indicated in Supplementary Table S1. In evaluation of the aged kidney, 1 outlier was excluded. *P<0.05, **P<0.01 versus the young group (Student’s t test, Mann–Whitney test, or two-way analysis of variance (ANOVA) with Sidak’s or Tukey’s post hoc tests). The P value on the lower right side of each figure (b) indicates the result of a two-way ANOVA between the control and SF 6 points groups.
Figure 5
Figure 5
Peripheral PER2::LUC rhythms in response to daily scheduled restraint stress or treadmill exercise. (a) Experimental restraint stress and treadmill exercise schedules. White and black bars indicate the light and dark periods, respectively. Arrowheads indicate stimulation timings. ZT, zeitgeber time. (b–g) Analyzed wave forms (b and e), peak phases (c and f), and phase change values (d, g) of PER2::LUC bioluminescence in the kidney, liver, and submandibular gland (sub gla) of young and aged mice in each condition. Phase advance values were calculated as the difference between the peak times of the restraint/treadmill group and the intact group. Values are expressed as the mean±s.e.m. The number of mice used is indicated in Supplementary Table S1. *P<0.05, **P<0.01, ***P<0.001 versus the young group or the intact group (Student’s t test or two-way analysis of variance (ANOVA) with Sidak’s or Tukey’s post hoc tests). The P value on the lower right side of each figure (b, e) indicates the result of a two-way ANOVA on the intact and restraint groups or the young and aged groups.
Figure 6
Figure 6
Age-related changes in sympathetic regulation of the submandibular gland. (a) Experimental schedule of adrenergic receptor stimulation. Saline (vehicle), norepinephrine (NE), an α-adrenergic receptor agonist (phenylephrine; PHE), and a β-adrenergic receptor agonist (isoproterenol; ISO) were administered intraperitoneally (i.p.) on a daily basis. White and black bars indicate the light and dark periods, respectively. ZT, zeitgeber time. (b–d) Peak phase (b, c) and phase change values (d) of PER2::LUC bioluminescence in the submandibular gland in each condition. Phase advance values were calculated as the difference between the peak times of each drug group and the saline group. The number of mice used is indicated in Supplementary Table S1. (e, f) mRNA expression rhythms (e) and total daily mRNA expression (f, average of 4 time points) for each adrenergic receptor subtype in the submandibular gland (n=6). (g) Tissue contents of NE and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), and MHPG/NE ratios in the submandibular gland at ZT0, 6, 12, and 18 (n=4). Values are expressed as the mean+/±s.e.m. *P<0.05, **P<0.01, ***P<0.001 versus the young group or the saline group (Student’s t-test, Mann–Whitney test, or one-way analysis of variance (ANOVA) with Dunnett’s post hoc tests). (h) Model of age-related changes in the regulation of peripheral circadian clocks.

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