Molecular insights into human daily behavior

Abstract

Human beings exhibit wide variation in their timing of daily behavior. We and others have suggested previously that such differences might arise because of alterations in the period length of the endogenous human circadian oscillator. Using dermal fibroblast cells from skin biopsies of 28 subjects of early and late chronotype (11 "larks" and 17 "owls"), we have studied the circadian period lengths of these two groups, as well as their ability to phase-shift and entrain to environmental and chemical signals. We find not only period length differences between the two classes, but also significant changes in the amplitude and phase-shifting properties of the circadian oscillator among individuals with identical "normal" period lengths. Mathematical modeling shows that these alterations could also account for the extreme behavioral phenotypes of these subjects. We conclude that human chronotype may be influenced not only by the period length of the circadian oscillator, but also by cellular components that affect its amplitude and phase. In many instances, these changes can be studied at the molecular level in primary dermal cells.

Fig. 1.

Comparison of subject chronotype with fibroblast period length. (A) Schematic diagram of the experimental procedure. (B) Representative raw data (un-normalized, with background, smoothened by means of a running average over 10-minute intervals) from four subjects. Period lengths were, from Top to Bottom, 23.1 h, 24.2 h, 24.7 h, 25.6 h. (C) Graph showing average period length versus Horne–Ostberg score for all subjects. Period values shown are average ± SEM from two different biopsies, each measured twice. The Wilcoxon test suggests that the difference between lark and owl period lengths taken collectively is highly significant (P = 0.000027). Nevertheless, a Spearman rank test to compare Horne–Ostberg Questionnaire score and fibroblast period length shows only moderate correlation (ρ = −0.36, P = 0.04), implying the existence of factors additional to period length that affect Horne–Ostberg score.

Fig. 2.

Comparison of fibroblast period length with phase of reporter expression. (A) Expression of an E-box-luciferase reporter transgene in NIH 3T3 cells maintained under conditions of temperature entrainment. Days 0–4, 16 h 37°C and 8 h 33°C per day. Days 5–10, constant 37°C. (B) Sample human data from two different biopsies of three subjects (one each of early, intermediate, and late phase). Cells were phase-entrained for 6 days in a cycle identical to A, and then transferred to a multichannel measurement device at constant 37°C for 1.5 days of measurement. Graphed bioluminescence levels were normalized for ease of viewing to compensate for different degrees of infection. (C) Graph showing average period length versus entrained phase of reporter expression for fibroblasts from all subjects. Values are average ± SEM from two biopsies, each measured twice. x axis, period length in hours. y axis, phase in hours, relative to the mean of all subjects. Pearson Correlation Coefficient R = 0.6133 suggests moderate correlation, again indicating that factors in addition to period can influence phase. (t test for coefficient significance = 4.53, P = 0.0001.)

Fig. 3.

Theoretical modeling of the effects of clock properties upon entrained phase. (A) Graph of the circadian oscillations of mRNA in a 12 h:12 h light:dark cycle for two hypothetical oscillators in which the amplitude of transcription varies 2-fold. Dashed line, high-amplitude oscillator; solid line, low-amplitude oscillator. The resultant difference in entrained phase (dotted lines) is 1 h. In this scenario, the high-amplitude oscillator was created by setting h = 14, and the low-amplitude by setting h = 11 (see SI Materials and Methods for a description of mathematical methodology and variables used). (B) Similar graph for two hypothetical oscillators in which the amplitude of perceived phase shifting (i.e., light) varies 2-fold. Dashed line, lesser perceived light; solid line, more perceived light. The resultant difference in entrained phase (dotted lines) is 1.7 h. The curve from lesser perceived light was obtained with a = 0.015, and that from more perceived light used a = 0.03.

Fig. 4.

Comparison of fibroblast clock transcriptional amplitude with phase-shifting behavior. (A) Eight identical plates of fibroblasts from four subjects, two morning types and two evening types of identical period, were synchronized with dexamethasone, then harvested every 3 h. Graphs show the relative amount of Rev-Erbα RNA determined by qPCR. x axis, time of harvest relative to dexamethasone synchronization. y axis, relative RNA levels in arbitrary units (see Materials and Methods). (B) Schematic of phase-shifting protocol, with resulting Bmal1-luc bioluminescence levels from one subject. (C) Phase response curves in response to a stimulus with 0.3 μM forskolin. x axis, time of stimulation relative to dexamethasone synchronization. y axis, phase shift in hours.