Adrenal glucocorticoids have a key role in circadian resynchronization in a mouse model of jet lag

Abstract

Jet lag encompasses a range of psycho- and physiopathological symptoms that arise from temporal misalignment of the endogenous circadian clock with external time. Repeated jet lag exposure, encountered by business travelers and airline personnel as well as shift workers, has been correlated with immune deficiency, mood disorders, elevated cancer risk, and anatomical anomalies of the forebrain. Here, we have characterized the molecular response of the mouse circadian system in an established experimental paradigm for jet lag whereby mice entrained to a 12-hour light/12-hour dark cycle undergo light phase advancement by 6 hours. Unexpectedly, strong heterogeneity of entrainment kinetics was found not only between different organs, but also within the molecular clockwork of each tissue. Manipulation of the adrenal circadian clock, in particular phase-shifting of adrenal glucocorticoid rhythms, regulated the speed of behavioral reentrainment. Blocking adrenal corticosterone either prolonged or shortened jet lag, depending on the time of administration. This key role of adrenal glucocorticoid phasing for resetting of the circadian system provides what we believe to be a novel mechanism-based approach for possible therapies for jet lag and jet lag-associated diseases.

Figure 1. Behavioral entrainment during jet lag.

(A) Representative double-plotted actogram of a mouse before and after 6-hour LD phase advance applied at day 1. Tick marks represent wheel running activity; gray shading denotes dark lighting conditions. (B) Average onset of mice (n = 9) during jet lag. PS₅₀ (4.0 ± 0.1 days) was defined as the time at which half the phase shift was completed. All values are average ± SEM.

Figure 2. Resetting of clock genes during jet lag in the SCN.

(A) Diurnal mRNA profiles (average ± SEM) of 5 different clock genes at days 0, 2, 3, 4, and 12 after the LD shift, superimposed with sine wave fits (black). Dark phases are marked by gray shading. 3 animals were used per time point. (B) Shifts of gene expression peak times obtained from the ISH data in A showed that adaptation to the new light schedule varied for the 5 clock genes (average ± SEM). Colors are as indicated in A and C. (C) PS₅₀ values (average ± SEM) of clock genes in the SCN (from B) and of activity reentrainment (from Figure 1B). **P ≤ 0.01, ***P < 0.001 versus Per2. See Supplemental Table 2 for the results of statistical analysis.

Figure 3. Clock gene resetting kinetics in different tissues following 6-hour LD phase advance.

Resetting is represented by PS₅₀ values (average ± SEM). *P ≤ 0.05, **P < 0.01, ***P < 0.001 versus Per2. Determination of PS₅₀ values from expression data (Supplemental Figure 1; n = 3 animals per time point) was done as described in Figure 2. See Supplemental Table 2 for statistical analysis. (A) In the somatosensory cortex, similar and rapid adaptation of the Per1 and Per2 was followed by slower adaptation of Dbp, Arntl, and Nr1d1. (B) In the adrenal, Per1 and Per2 both showed comparable and fast adaptation, whereas Dbp and Nr1d1 rhythms shifted at a similar, but slower, rate. Arntl showed the slowest adaptation, with a PS₅₀ value of 3.5 ± 0.2 days. (C) A similar hierarchy was observed for kidney, with fast adaptation for Per, Dbp, and Nr1d1, and a slow one for Arntl. (D) In liver, Per2 expression shifted significantly faster than that of the other clock genes except Nr1d1. Per1 and Dbp followed at comparable speed, while Arntl adapted slowest (4.1 ± 0.2 days). (E) In the pancreas, Per1 and Per2 shifting was slow, with PS₅₀ values of 4.8 ± 0.7 and 4.8 ± 0.7 days, respectively, followed by Dbp and Arntl. Nr1d1 adaptation was fastest in this tissue, with a PS₅₀ value of 3.5 ± 0.4 days.

Figure 4. Influence of adrenal clock function on activity reentrainment after 6-hour phase advance of the LD cycle.

(A) Representative double-plot actograms of h^WT/a^WT and h^WT/aP2/C1 animals. Dark phases are denoted by gray shading. (B) Resetting kinetics of activity onsets (average ± SEM). The curves differed significantly between days 3 and 8 (0.003 ≤ P ≤ 0.016). On average, PS₅₀ values of activity resetting were reduced by 28.4% for h^WT/aP2/C1. n = 9 (h^WT/a^WT); 8 (h^WT/aP2/C1). (C) Resetting kinetics of corticosterone excretion maxima peak times (average ± SEM). The curves differed significantly between days 2 and 5 (0.030 ≤ P ≤ 0.004). On average, PS₅₀ values of corticosterone resetting were reduced by 36.5% in h^WT/aP2/C1. n = 5 (h^WT/a^WT); 6 (h^WT/aP2/C1). (D) Corticosterone maxima and activity onset phase shifts are plotted against each other for individual UT, h^WT/a^WT, and h^WT/aP2/C1 mice. Because nearly all individual experimental values were located above the normal (y = x; dashed line), cortico­sterone concentration rhythms shifted more rapidly than did locomotor behavior at all times. A strong correlation between both factors for all groups was found (UT, r² = 0.65; h^WT/a^WT, r² = 0.79; h^WT/aP2/C1, r² = 0.87). UT activity is as shown in Figure 1.

Figure 5. Shifting corticosterone rhythms prior to jet lag affects behavioral resetting kinetics in a phase-advance paradigms.

(A and B) Advanced and delayed corticosterone excretion rhythms of WT MET_D (A) and MET_N (B) mice after 16 days of MET treatment. The direction of the shift of the corticosterone peak time prior to jet lag in treated mice in comparison to the peak time in SAL_D, SAL_N, and UT control mice (n = 5) is indicated (ΔZT_max). (C and D) Representative double-plotted actograms of SAL_D and MET_D mice (C) and SAL_N and MET_N mice (D) 2 weeks before and 2 weeks after a 6-hour phase advance of the LD cycle. Time and duration of MET treatment is shown by red bars. Dark phases are denoted by gray shading. (E and F) Resetting kinetics of activity onsets of MET_D and SAL_D mice (E), MET_N and SAL_N mice (F), and UT controls. Resetting kinetics of injected animals differed significantly from that of saline-treated animals (P < 0.0001, MET_D vs. SAL_D; P = 0.0003, MET_N vs. SAL_N; n = 6 per group). All values are average ± SEM.

Figure 6. MET injection prior to jet lag affects behavioral resetting kinetics in a phase-delay paradigm.

After injection of MET or saline for 16 days, animals were released into an 8-hour phase delay paradigm. (A and B) Representative double-plotted actograms of SAL_D and MET_D mice (A) and SAL_N and MET_N mice (B) 2 weeks before and 2 weeks after 8-hour phase delay of the LD cycle. Time and duration of MET treatment is shown by red bars. Dark phases are denoted by gray shading. (C and D) Resetting kinetics of activity onsets of MET_D and SAL_D mice (C) and MET_N and SAL_N mice (D). The curves of injected animals differed significantly from that of saline-treated control animals (P < 0.0001, MET_D vs. SAL_D and MET_N vs. SAL_N; n = 6 per group). Differences between MET- and saline-injected animals were still significant (P = 0.0105) when the shift was shortened to 7 hours, caused by delayed onset after MET injection at ZT12. All values are average ± SEM.