Serum factors in older individuals change cellular clock properties

Abstract

Human aging is accompanied by dramatic changes in daily sleep-wake behavior: Activity shifts to an earlier phase, and the consolidation of sleep and wake is disturbed. Although this daily circadian rhythm is brain-controlled, its mechanism is encoded by cell-autonomous circadian clocks functioning in nearly every cell of the body. In fact, human clock properties measured in peripheral cells such as fibroblasts closely mimic those measured physiologically and behaviorally in the same subjects. To understand better the molecular mechanisms by which human aging affects circadian clocks, we characterized the clock properties of fibroblasts cultivated from dermal biopsies of young and older subjects. Fibroblast period length, amplitude, and phase were identical in the two groups even though behavior was not, thereby suggesting that basic clock properties of peripheral cells do not change during aging. Interestingly, measurement of the same cells in the presence of human serum from older donors shortened period length and advanced the phase of cellular circadian rhythms compared with treatment with serum from young subjects, indicating that a circulating factor might alter human chronotype. Further experiments demonstrated that this effect is caused by a thermolabile factor present in serum of older individuals. Thus, even though the molecular machinery of peripheral circadian clocks does not change with age, some age-related circadian dysfunction observed in vivo might be of hormonal origin and therefore might be pharmacologically remediable.

Fig. 1.

Influence of age on period length and chronotype. (A) Chronotype of young and old subjects, as measured by the MCTQ. The y axis shows subject MSF-Sc. This statistic (the output of the MCTQ) is widely used as a reliable measure of human chronotype (27). Dataset variation is shown as a standard boxplot (n = 18; unpaired t test; **P < 0.01). (B) Period length of the primary fibroblasts of each subject participating in this study. For ease of display, data are sorted on the basis of the period length. Data are mean of six independent measurements of the period length for every subject ± SEM. (Inset) Population average of period lengths of skin fibroblasts from young (Y) and older (O) subjects, shown as a standard boxplot. No statistical difference was observed (n = 18; unpaired t test; P > 0.05).

Fig. 2.

Length of circadian period of skin fibroblasts treated with media containing human serum. (A) Lengths of circadian period in one representative cell line taken from a young subject (Y) measured in medium containing FBS (white) and media containing human serum from eight young (YS; gray) and five older (OS; black) donors. Bars represent the mean of three independent measurements ± SEM. (B) Equivalent measurements from a representative cell line from an older subject (O). (C) Bar graph showing the average differences in period length in four Y cell lines treated with YS and OS. Results are expressed as average ± SEM. (D) Equivalent measurements for two O cell lines. In both cases, treatment with YS gave a highly different period length (unpaired t test; ***P < 0.001 compared with the treatment with OS in Y cell lines). (E) Average period length across experiments using human serum, as a function of the age of the serum donor (linear regression: P < 0.0002; r² = 0.6274).

Fig. 3.

Circadian phase of fibroblasts in the presence of serum from young and older subjects. (A) Phase of cell lines from young subjects (Y) was determined after temperature entrainment in the presence of serum from younger subjects (YS) or from older subjects (OS). Results are expressed as phase difference (in h) between the two treatments. Each bar results from the average of two different cell lines, each treated with two different sera, ± SEM. (B) Equivalent data using cell lines from older subjects. In both cases, there was a significant advance in the phase of cells treated with OS compared with the same cell lines treated with YS (unpaired t test; *P < 0.05). (C) Correlation between phase-shift differences seen in this figure and differences in period length seen in Fig. 2. Phase shifts are plotted relative to the average phase for all sera from young subjects (linear regression: P = 0.0265; r² = 0.6488).

Fig. 4.

Length of the circadian period of skin fibroblasts treated with media containing normal or heat-inactivated human serum. For ease of analysis, data for normal serum are replotted from Fig. 2. (A) Length of circadian period obtained from one representative cell line from a young subject (Y) measured in media containing normal (YS or OS) or heat-inactivated (YSHI or OSHI) human serum from four young and seven older donors. Every bar shows the mean of three independent measurements ± SEM. (B) (Upper) Graph showing the average period length from Y cell lines treated with normal and heat-inactivated serum from both young and old subjects. Each bar represents the average of two different cell lines, each treated with four different sera, ± SE. (Lower) Equivalent graph for cell lines from older subjects. In both cases, YSHI did not modify the period length compared with YS (one-way ANOVA Tukey's multiple comparison test: P > 0.05), whereas OSHI increased the period length compared with OS (one-way ANOVA Tukey's multiple comparison test: *P < 0.05; **P < 0.01; ***P < 0.001) to a length equivalent to that obtained with YS (one-way ANOVA Tukey's multiple comparison test: P > 0.05). (C) Under temperature-entrained conditions, comparison of phase shifts obtained with untreated and heat-inactivated sera tested on two cell lines from young subjects and two from older subjects. Results are expressed relative to phase obtained with young serum. No differences were observed in phase among YS, YSHI, and OSHI (one-way ANOVA Tukey's multiple comparison test: P > 0.05), but OS resulted in significantly earlier phase (one-way ANOVA Tukey's multiple comparison test: *P < 0.05). (D) Model demonstrating why older subjects show altered circadian behavior. Blue, younger subjects. Red, older subjects. (1) In elderly individuals, lower light levels caused by less light exposure and changed lens properties reduce the ability of light to entrain the central clock in SCN. (2) A weakened circadian drive from the SCN results in fragmented sleep–wake cycles, which in turn affect self-selected light preferences. (3) Altered hormonal balance in elderly individuals changes cellular clock properties and shifts phase earlier at sleep–wake centers but cannot entrain the SCN. In cells, only intrinsic and hormonal influences are operative, resulting in shorter period and earlier phase in the presence of serum from older individuals.