Shin Yamazaki

Chronic jet-lag increases mortality in aged mice

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Effects of aging on central and peripheral mammalian clocks

Circadian organization changes with age, but we do not know the extent to which age-related changes are the result of alterations in the central pacemakers, the peripheral oscillators, or the coupling mechanisms that hold the system together. By using transgenic rats with a luciferase (luc) reporter, we assessed the effects of aging on the rhythm of expression of the Period 1 (Per1) gene in the suprachiasmatic nucleus (SCN) and in peripheral tissues. Young (2 months) and aged (24–26 months) Per1-luc transgenic rats, entrained to light–dark cycles, were killed, and tissues were removed and cultured. Per1-luc expression was measured from 10 tissues. In the SCN, the central mammalian pacemaker, Per1-luc expression was robustly rhythmic for more than 7 weeks in culture. The only difference between SCN rhythmicity in young and old rats was a small but significant age-related shortening of the free-running period. Circadian rhythmicity in some peripheral tissues was unaffected by aging, whereas rhythmicity in other tissues was either phase advanced relative to the light cycle or absent. Those tissues that were arrhythmic could be induced to oscillate by application of forskolin, suggesting that they retained the capacity to oscillate but were not being appropriately driven in vivo. Overall, the results provide new insights into the effects of aging on the mammalian circadian system. Aging seems to affect rhythms in some but not in all tissues and may act primarily on interactions among circadian oscillators, perhaps attenuating the ability of the SCN to drive damped oscillators in the periphery.

Age-Related Decline in Circadian Output

Disruptions in sleep/wake cycles, including decreased amplitude of rhythmic behaviors and fragmentation of the sleep episodes, are commonly associated with aging in humans and other mammals. While there are undoubtedly many factors contributing to these changes, a body of literature is emerging, suggesting that an age-related decline in the central circadian clock in the suprachiasmatic nucleus (SCN) may be a key element responsible. To explore age-related changes in the SCN, we have performed in vivo multiunit neural activity (MUA) recordings from the SCN of freely moving young (3–5 months) and middle-aged (13–18 months) mice. Importantly, the amplitude of day–night difference in MUA was significantly reduced in the older mice. We also found that the neural activity rhythms are clearly degraded in the subparaventricular zone, one of the main neural outputs of the SCN. Surprisingly, parallel studies indicate that the molecular clockwork in the SCN as measured by PER2 exhibited only minor deficits at this same age. Thus, the circadian output measured at the level of neural activity rhythms in the SCN is degraded by aging, and this decline occurs before the disruption of key components of the molecular clockwork.

Is the food-entrainable circadian oscillator in the digestive system?

Food‐anticipatory activity (FAA) is the increase in locomotion and core body temperature that precedes a daily scheduled meal. It is driven by a circadian oscillator but is independent of the suprachiasmatic nuclei. Recent results that reveal meal‐entrained clock gene expression in rat and mouse peripheral organs raise the intriguing possibility that the digestive system is the site of the feeding‐entrained oscillator (FEO) that underlies FAA. We tested this possibility by comparing FAA and Per1 rhythmicity in the digestive system of the Per 1‐luciferase transgenic rat. First, rats were entrained to daytime restricted feeding (RF, 10 days), then fed ad libitum (AL, 10 days), then food deprived (FD, 2 days). As expected FAA was evident during RF and disappeared during subsequent AL feeding, but returned at the correct phase during deprivation. The phase of Per1 in liver, stomach and colon shifted from a nocturnal to a diurnal peak during RF, but shifted back to nocturnal phase during the subsequent AL and remained nocturnal during food deprivation periods. Second, rats were entrained to two daily meals at zeitgeber time (ZT) 0400 and ZT 1600. FAA to both meals emerged after about 10 days of dual RF. However, all tissues studied (all five liver lobes, esophagus, antral stomach, body of stomach, colon) showed entrainment consistent with only the night‐time meal. These two results are inconsistent with the hypothesis that FAA arises as an output of rhythms in the gastrointestinal (GI) system. The results also highlight an interesting diversity among peripheral oscillators in their ability to entrain to meals and the direction of the phase shift after RF ends.

Activation of 5′-AMP-activated Kinase with Diabetes Drug Metformin Induces Casein Kinase Iϵ (CKIϵ)-dependent Degradation of Clock Protein mPer2

Metformin is one of the most commonly used first line drugs for type II diabetes. Metformin lowers serum glucose levels by activating 5′-AMP-activated kinase (AMPK), which maintains energy homeostasis by directly sensing the AMP/ATP ratio. AMPK plays a central role in food intake and energy metabolism through its activities in central nervous system and peripheral tissues. Since food intake and energy metabolism is synchronized to the light-dark (LD) cycle of the environment, we investigated the possibility that AMPK may affect circadian rhythm. We discovered that the circadian period of Rat-1 fibroblasts treated with metformin was shortened by 1 h. One of the regulators of the period length is casein kinase Iϵ (CKIϵ), which by phosphorylating and inducing the degradation of the circadian clock component, mPer2, shortens the period length. AMPK phosphorylates Ser-389 of CKIϵ, resulting in increased CKIϵ activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK α2 knock-out mice. We conclude that metformin and AMPK have a previously unrecognized role in regulating the circadian rhythm.

Real-time luminescence reporting of circadian gene expression in mammals.

Luminescence reporters have been used successfully in studies of circadian rhythms. Real-time measurements of circadian variations in gene expression were made in living cells, cultured tissues, and whole organisms. Because this technique is relatively easy and continuous noninvasive measurement from tissue cultures allows for a drastic reduction in the number of experimental animals, we believe this method will become a common technique for studying circadian rhythms. Using a multichannel recording apparatus, it may also become a powerful tool for the discovery of new drugs. In the past, measurements were done using hand-made apparatuses or by modifying commercially available equipment. We, along with other investigators, have developed user-friendly equipment for performing circadian rhythms experiments, and these systems are now available commercially. This article describes the use of luminescence reporters in circadian research and provides detailed methods used in these experiments. One of our goals in this article is to reduce experimental variability in different laboratories by proposing standard protocols.

Circadian gene expression in mammalian fibroblasts revealed by real-time luminescence reporting: Temperature compensation and damping

Mammalian cells such as rat-1 fibroblasts have been shown to exhibit daily oscillations in the expression of several gene transcripts in culture. After induction, these oscillations persist with a period of ≈24 h for several days. This characteristic suggests that the oscillations are controlled by a circadian clock, but the crucial criterion of temperature compensation has not been demonstrated for rat-1 fibroblasts. We have developed an automated assay of circadian expression of the mPer1 promoter in rat-1 fibroblasts that have been stably transfected with a luciferase reporter. Using this cell culture-based in vitro luminescent reporter assay, we found that the daily oscillation of mPer1 promoter activity in rat-1 cells is temperature compensated over the range of 28.5-36.5°C. This finding means that these oscillations are bona fide circadian rhythms. Moreover, the circadian clock of these homeothermic mammalian cells not only is temperature compensated but also is overcompensated such that it runs faster at cooler temperatures (Q10 of 0.85-0.88). The oscillations in rat-1 fibroblasts damp more rapidly at cooler temperatures, and damping is not due to cells becoming unhealthy because a second stimulus will reinitiate a robust rhythm. These data show that rat-1 cell cultures that are stably transfected with luminescence reporters are an excellent model system for studying circadian clocks at the cellular level in mammals.

An autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA.

The influence of the mammalian retinal circadian clock on retinal physiology and function is widely recognized, yet the cellular elements and neural regulation of retinal circadian pacemaking remain unclear due to the challenge of long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal circadian clock. In the current study, we developed a protocol for long-term culture of intact mouse retinas, which allows retinal circadian rhythms to be monitored in real time as luminescence rhythms from a PERIOD2::LUCIFERASE (PER2::LUC) clock gene reporter. With this in vitro assay, we studied the characteristics and location within the retina of circadian PER2::LUC rhythms, the influence of major retinal neurotransmitters, and the resetting of the retinal circadian clock by light. Retinal PER2::LUC rhythms were routinely measured from whole-mount retinal explants for 10 d and for up to 30 d. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the inner nuclear layer. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate) and the inner nuclear layer (dopamine, acetylcholine, GABA, glycine, and glutamate) did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36 (cx36)-containing gap junctions, indicating that PER2::LUC rhythms generation in the inner nuclear layer is likely cell autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization and casein kinase, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Light pulses reset the phase of the in vitro retinal oscillator and dopamine D1 receptor antagonists attenuated these phase shifts. Thus, dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock.

Robust Food Anticipatory Activity in BMAL1-Deficient Mice

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Even though nocturnal rodents prefer to forage at night, daytime food anticipatory activity (FAA) is observed prior to short meals presented at a scheduled time of day. Under this restricted feeding regimen, rodents exhibit two distinct bouts of activity, a nocturnal activity rhythm that is entrained to the light-dark cycle and controlled by the master clock in the suprachiasmatic nuclei (SCN) and a daytime bout of activity that is phase-locked to mealtime. FAA also occurs during food deprivation, suggesting that a food-entrainable oscillator (FEO) keeps time in the absence of scheduled feeding. Previous studies have demonstrated that the FEO is anatomically distinct from the SCN and that FAA is observed in mice lacking some circadian genes essential for timekeeping in the SCN. In the current study, we optimized the conditions for examining FAA during restricted feeding and food deprivation in mice lacking functional BMAL1, which is critical for circadian rhythm generation in the SCN. We found that BMAL1-deficient mice displayed FAA during restricted feeding in 12hr light:12hr dark (12L:12D) and 18L:6D lighting cycles, but distinct activity during food deprivation was observed only in 18L:6D. While BMAL1-deficient mice also exhibited robust FAA during restricted feeding in constant darkness, mice were hyperactive during food deprivation so it was not clear that FAA consistently occurred at the time of previously scheduled food availability. Taken together, our findings suggest that optimization of experimental conditions such as photoperiod may be necessary to visualize FAA in genetically modified mice. Furthermore, the expression of FAA may be possible without a circadian oscillator that depends on BMAL1.

Differential Response of Period 1 Expression within the Suprachiasmatic Nucleus

The suprachiasmatic nuclei (SCNs) of the hypothalamus contain a circadian clock that exerts profound control over rhythmic physiology and behavior. The clock consists of multiple autonomous cellular pacemakers distributed throughout the rat SCN. In response to a shift in the light schedule, the SCN rapidly changes phase to achieve the appropriate phase relationship with the shifted light schedule. Through use of a transgenic rat in which rhythmicity in transcription of the Period 1 gene was measured with a luciferase reporter (Per1-luc), we have been successful in tracking the time course of molecular rhythm phase readjustments in different regions of the SCN that occur in response to a shift in the light schedule. We find that different regions of the SCN phase adjust at different rates, leading to transient internal desynchrony in Per1-luc expression among SCN regions. This desynchrony among regions is most pronounced and prolonged when the light schedule is advanced compared with light schedule delays. A similar asymmetry in the speed of phase resetting is observed with locomotor behavior, suggesting that phase shifting kinetics within the SCN may underlay the differences observed in behavioral resetting to advances or delays in the light schedule.