Research ArticleCircadian Rhythms

Effects of caffeine on the human circadian clock in vivo and in vitro

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Science Translational Medicine  16 Sep 2015:
Vol. 7, Issue 305, pp. 305ra146
DOI: 10.1126/scitranslmed.aac5125
  • Fig. 1. Protocol for the human experiment.

    After about 7 days of ambulatory monitoring, participants remained in an environment free of external time cues (days 8 to 13) under dim light during scheduled wakefulness (~1.9 lux, ~0.6 W/m2) and darkness during scheduled sleep (black bars) in the laboratory. Examples of in-laboratory procedures are as follows: day 8 included an 8-hour sleep opportunity; days 9 and 10 consisted of a 40-hour constant routine (hashed dark gray bars). On day 11, participants received either caffeine or rice powder–filled placebo (pill symbol) 3 hours before habitual bedtime and a 3-hour exposure (☼) to bright light (~3000 lux, ~7 W/m2) or continued exposure to dim light (light gray bars; ~1.9 lux, ~0.6 W/m2) beginning at habitual bedtime. Days 12 to 13 consisted of a 30-hour constant routine. Laboratory procedures were repeated four times over ~49 days (D1 to D49; fig. S1). Relative clock hour shown with the 2400 hour assigned to bedtime; actual times were dependent on and relative to the participant’s habitual bedtime.

  • Fig. 2. Phase-shifting response for each condition.

    (A) Average phase shifts. Circadian phase delays are denoted as negative numbers and error bars represent SEM. Lines represent significant differences between conditions at endpoints of the line (Dunnett’s test: dim-light placebo versus dim-light caffeine, P = 0.011; dim-light placebo versus bright-light placebo, P = 0.0007; dim-light placebo versus bright-light caffeine, P = 0.0003). Data are mean ± SEM, n = 5. (B) Individual differences in the phase-shifting response controlling for phase change during the dim-light placebo control condition. Symbols represent individual subjects.

  • Fig 3. Association between phase shifts induced by different conditions.

    (A) Dim-light caffeine with bright-light caffeine. (B) Dim-light placebo and bright-light placebo. Symbols represent individual subjects, and solid line represents the best linear fit to the data.

  • Fig. 4. Caffeine increases the circadian period in cultured human cells in vitro in an adenosine receptor/cAMP–dependent fashion.

    (A) Representative examples of grouped raw bioluminescence data (mean ± SEM, n = 6) showing the effect of different concentrations and combinations of IBMX, CGS-15943, and caffeine on human U2OS cells stably expressing bmal1:luc. (B) Grouped quantification of circadian period (mean ± SEM, n = 6) showing dose-dependent lengthening of the circadian period in response to CGS-15943 (blue, left panels) or IBMX (red, right panels) ± a fixed concentration of another period-lengthening drug [either 0.25 mM IBMX (red), 5 μM CGS-15943 (blue), or 2.5 mM caffeine (brown)]. Solid line depicts the linear regression in each case (R2 ≥ 0.98), with broken lines representing the null hypothesis (null, simple additive drug action, that is, no change in the slope). In each subpanel, sum-of-squares F-test P values are reported, where P < 0.05 indicates rejection of the null hypothesis (same slope for both groups). The significance and drug additivity are summarized below. Red arrows indicate that caffeine acts synergistically with IBMX but less than additively with CGS-15943. (C) All three drug treatments significantly increase cAMP signaling reported by pGloSensor activity over 6 days, plotted as mean ± SEM (n = 4) relative to vehicle control. P < 0.0001 by one-way ANOVA and by Bonferroni’s multiple comparisons test for each drug versus vehicle. (D) ADORA1 knockdown attenuates the period-lengthening effect of 2.5 mM caffeine in U2OS cells; representative detrended group mean ± SEM is shown (n = 4). (E) Grouped quantification of period lengthening by caffeine after siRNA knockdown of each adenosine receptor isoform (n = 7 or 8); P = 0.0002, one-way ANOVA. By Bonferroni’s multiple comparisons test, P = 0.0043 for control versus ADORA1, with no significant difference versus any other group (n.s., P > 0.67). (F) A1R is rhythmically expressed in U2OS cells in phase with per2:luc. Upper panel, representative A1R immunoblot; lower panel, normalized grouped A1R abundance (mean ± SEM, n = 3; P < 0.0001 by one-way ANOVA; * indicates P < 0.0001 for each time point versus 16 hours by Bonferroni’s multiple comparisons test). Per2:luc rhythms, recorded in parallel (n = 4), are shown for reference. RLU, relative light units.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/305/305ra146/DC1

    Fig. S1. Forty-nine day protocol for human experiment.

    Fig. S2. Ryanodine does not lengthen the circadian period reported by per2:luc in cultured human cells in vitro.

    Fig. S3. Caffeine increases the circadian period reported by bmal1:luc in cultured human cells in vitro in an adenosine receptor/cAMP–dependent fashion.

    Fig. S4. Caffeine increases the circadian period reported by per2:luc in cultured human cells in vitro in an adenosine receptor/cAMP–dependent fashion.

    Fig. S5. Caffeine acts at the same site as 8-SPT to increase the circadian period in cultured human cells in vitro.

    Fig. S6. Quantification of adenosine receptor siRNA knockdown efficacy.

  • Supplementary Material for:

    Effects of caffeine on the human circadian clock in vivo and in vitro

    Tina M. Burke, Rachel R. Markwald, Andrew W. McHill, Evan D. Chinoy, Jesse A. Snider, Sara C. Bessman, Christopher M. Jung, John S. O'Neill,* Kenneth P. Wright Jr.*

    *Corresponding author. E-mail: kenneth.wright{at}colorado.edu (K.P.W.); oneillj{at}mrc-lmb.cam.ac.uk (J.S.O.)

    Published 16 September 2015, Sci. Transl. Med. 7, 305ra146 (2015)
    DOI: 10.1126/scitranslmed.aac5125

    This PDF file includes:

    • Fig. S1. Forty-nine day protocol for human experiment.
    • Fig. S2. Ryanodine does not lengthen the circadian period reported by per2:luc in cultured human cells in vitro.
    • Fig. S3. Caffeine increases the circadian period reported by bmal1:luc in cultured human cells in vitro in an adenosine receptor/cAMP–dependent fashion.
    • Fig. S4. Caffeine increases the circadian period reported by per2:luc in cultured human cells in vitro in an adenosine receptor/cAMP–dependent fashion.
    • Fig. S5. Caffeine acts at the same site as 8-SPT to increase the circadian period in cultured human cells in vitro.
    • Fig. S6. Quantification of adenosine receptor siRNA knockdown efficacy.

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