Aging and circadian clock gene expression in peripheral tissues in rats

Aging is associated with alterations of the circadian rhythms (shortened amplitude and phase-advance). We studied by quantitative RT-PCR the influence of aging on the expression of circadian clock genes (Clock, Bmal1, Cry1,2, Per1-3) in peripheral tissues (liver and heart) of middle-aged (13 months) and old (27 months) rats of the Wag/Rij strain exposed to a 12 hours light/12 hours dark cycle. Rats were killed at the light-dark transition (8 am and 8 pm). In the liver, Per, Cry et Bmal1 genes showed a morning/evening difference of expression; in addition, old rats exhibited a significant decrease of Per gene expression in the evening vs middle-aged rats. The heart showed similar profiles with only a tendency toward a decrease of Per expression and an increased Bmal1 expression in the evening in old rats. These results show that aging is associated with circadian gene expression changes.     

Mistimed wheel running interferes with re‐entrainment of circadian Per1 rhythms in the mouse skeletal muscle and lung

Previously, we showed the acceleration of re‐entrainment to 8‐h phase‐advanced light/dark cycles (LD) in the circadian Per1 expression rhythms of the mouse lung and skeletal muscle by 3‐h wheel running (WR) at the beginning of shifted dark phase. In the present study, the effects of WR at the end of shifted dark phase were examined on the re‐entrainment in mice. LD was advanced by shortening and was delayed by lengthening the first light period in the phase‐advance and phase‐delay protocol, respectively. Shifted LD was continued for 4 days, which was followed by constant darkness (DD). Per1 expression was measured in the cultured tissues obtained on the first day of DD from mice carrying a bioluminescence reporter of Per1 expression. In the phase‐advance protocol, re‐entrainment was not influenced by WR in any circadian rhythm examined. In the phase‐delay protocol, re‐entrainment of the circadian locomotor rhythm was not affected by WR. However, re‐entrainment of circadian Per1 rhythm was significantly decelerated in the skeletal muscle and lung. These findings indicate that the effects of WR on re‐entrainment depend on the time of day and the peripheral tissues. Mistimed WR interferes with re‐entrainment of circadian rhythms in the lung and skeletal muscle.     

Circadian clock and cell cycle gene expression in mouse mammary epithelial cells and in the developing mouse mammary gland.

Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC-11 cells and mammary glands, elevated Per2 expression was positively correlated with c-Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with beta-casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation.     

The suprachiasmatic nuclei are involved in determining circadian rhythms during restricted feeding

The circadian clock in the suprachiasmatic nuclei (SCN) responds to light and regulates peripheral circadian rhythms. Feeding regimens also reset the clock, so that time-restricted feeding (RF) dictates rhythms in peripheral tissues, whereas calorie restriction (CR) affects the SCN clock. To better understand the influence of RF vs. CR on circadian rhythms, we took advantage of the transgenic alphaMUPA mice that exhibit spontaneously reduced eating, and can serve as a model for CR under ad libitum feeding, and a model for temporal CR under RF compared with wild type (WT) mice. Our results show that RF advanced and generally increased the amplitude of clock gene expression in the liver under LD in both mouse types. However, under disruptive light conditions, RF resulted in a different clock gene phase in WT mice compared with alphaMUPA mice, suggesting a role for the reduced calories in resetting the SCN that led to the change of phase in alphaMUPA mice. Comparison of the RF regimen in the two lighting conditions in WT mice revealed that mPer1, mClock, and mBmal1 increased, whereas mPer2 decreased in amplitude under ultradian light in WT mice, suggesting a role for the SCN in determining clock gene expression in the periphery during RF. In summary, herein we reinforce a role for calorie restriction in resetting the SCN clock, and unravel a role for the SCN in determining peripheral rhythms under RF.     

Circadian profile and photic regulation of clock genes in the suprachiasmatic nucleus of a diurnal mammal Arvicanthis ansorgei

The molecular mechanisms of the mammalian circadian clock located in the suprachiasmatic nucleus have been essentially studied in nocturnal species. Currently, it is not clear if the clockwork and the synchronizing mechanisms are similar between diurnal and nocturnal species. Here we investigated in a day-active rodent Arvicanthis ansorgei, some of the molecular mechanisms that participate in the generation of circadian rhythmicity and processing of photic signals. In situ hybridization was used to characterize circadian profiles of expression of Per1, Per2, Cry2 and Bmal1 in the suprachiasmatic nucleus of A. ansorgei housed in constant dim red light. All the clock genes studied showed a circadian expression. Per1 and Per2 mRNA increased during the subjective day and decreased during the subjective night. Also, Bmal1 exhibited a circadian expression, but in anti-phase to that of Per1. The expression of Cry2 displayed a circadian pattern, increasing during the late subjective day and decreasing during the late subjective night. We also obtained the phase responses to light for wheel-running rhythm and clock gene expression. At a behavioral level, light was able to induce phase shifts only during the subjective night, like in other diurnal and nocturnal species. At a molecular level, light pulse exposure during the night led to an up-regulation of Per1 and Per2 concomitant with a down-regulation of Cry2 in the suprachiasmatic nucleus of A. ansorgei. In contrast, Bmal1 expression was not affected by light pulses at the circadian times investigated. This study demonstrates that light exposure during the subjective night has opposite effects on the expression of the clock genes Per1 and Per2 compared with that of Cry2. These differential effects can participate in photic resetting of the circadian clock. Our data also indicate that the molecular mechanisms underlying circadian rhythmicity and photic synchronization share clear similarities between diurnal and nocturnal mammals.     

Effect of lipopolysaccharide on circadian clock genes <Emphasis Type="Italic">Per2</Emphasis> and <Emphasis Type="Italic">Bmal1</Emphasis> in mouse ovary

In mammals, circadian rhythms are associated with multiple physiological events. The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on circadian systems in the ovary. Immature female mice were received an intra-peritoneal injection of equine chorionic gonadotropin (eCG) and LPS. Total RNA was collected from the ovary at 6-h intervals throughout a 48 h of experimental period. The expression of the circadian genes period 2 (Per2) and brain and muscle ARNT-like 1 (Bmal1) such as circadian genes was measured by quantitative PCR. Although expression of Per2 and Bmal1 in the ovary did not display clear diurnal oscillation, LPS suppressed the amplitude of Per2 expression. Additionally, LPS inhibited the expression of cytochrome P450 aromatase (CYP19) and luteinizing hormone receptor (LHr) genes in the ovary of eCG-treated mice. Our data suggest that Per2 may be associated with the inhibition of CYP19 and LHr expression by LPS in the ovaries of immature mice.     

Scheduled exposures to a novel environment with a running-wheel differentially accelerate re-entrainment of mice peripheral clocks to new light-dark cycles

Effects of scheduled exposures to novel environment with a running-wheel were examined on re-entrainment to 8 h shifted light–dark (LD) cycles of mouse circadian rhythms in locomotor activity and clock gene, Per1 , expression in the suprachiasmatic nucleus (SCN) and peripheral tissues. Per1 expression was monitored by a bioluminescence reporter introduced into mice. The animals were exposed to the novel environment for 3 h from the shifted dark onset for four cycles and released into constant darkness. In the phase-advance shift, the circadian rhythm in locomotor activity fully re-entrained in the exposed group, whereas it was in transients in the control. On the other hand, the circadian rhythm of Per1 expression in the SCN almost completely re-entrained in both the control and exposed groups. In the skeletal muscle and lung, the circadian rhythm fully re-entrained in the exposed group, whereas the rhythms in the control did not. In the phase-delay shift, the circadian rhythms in locomotor activity and Per1 expression almost completely re-entrained in both groups. These findings indicate that the scheduled exposures to novel environment with a running-wheel differentially accelerate the re-entrainment of the mouse peripheral clocks to 8 h phase-advanced LD cycles.     

Potential role of the pancreatic hormone insulin in resetting human peripheral clocks

Mammalian circadian rhythms are phase‐adjusted and amplified by external cues such as light and food. While the light input pathway via the central clock, the suprachiasmatic nucleus, has been well defined, the mechanism of feeding‐induced circadian resetting remains undefined, particularly in humans. Animal studies have indicated that insulin, a pancreatic hormone that is secreted rapidly in response to feeding, is an input factor for a few peripheral clocks, such as liver and adipose tissue. In this study, using plucked and cultured hair follicles as a representative human peripheral clock, we examined the effect of insulin on circadian characteristics of clock gene expression. Our results demonstrate that insulin phase‐shifts or amplifies the clock gene expression rhythms of ex vivo cultured hair follicles in a phase‐responsive manner. To reduce the possibility that differences in species, genetic or environmental background, and experimental methods affected experimental outcomes, we also treated surgically extracted whisker follicles of Period2::Luciferase (Per2^Luc) mice with insulin and found that the effect of insulin on clock gene expression was reproducible. These results suggest the possibility that feeding‐induced insulin resets peripheral circadian clocks in humans.     

Diurnal pattern of clock gene expression in the hypothalamus of the newborn rabbit

In the European rabbit (Oryctolagus cuniculus) nursing acts as a strong non-photic synchronizer of circadian rhythmicity in the newborn young. Rabbits only nurse for a few minutes once every 24 h and previous studies have shown that the pups, blind at birth, display endogenous circadian rhythms in behavior and physiology entrained by this regular daily event. As a further step toward understanding the neural organization of the rabbit's early circadian system, we investigated the expression of clock genes in the suprachiasmatic nucleus of the hypothalamus (SCN; the principal circadian pacemaker in adult mammals) across the pups' 24-h day. We used 43 pups from seven litters maintained in constant darkness and entrained non-photically by nursing at the same time each day until P7. After nursing on day 7, pups were killed in the dark at 3-h intervals so as to obtain eight groups (n=5-6 pups/group) distributed evenly across the 24 h before the next scheduled nursing. Profiles in the expression of the clock genes Per1, Per2, Cry1 and Bmal1 were determined using in situ hybridization in brain sections through the hypothalamus at the level of the SCN. We report for the first time: 1) that Per1, Per2, Cry1 and Bmal1 are all expressed in the SCN of the newborn rabbit, 2) that the expression of Per1, Per2 and Bmal1 but not Cry1 shows diurnal rhythmicity similar to that in adult mammals, and 3) that the expression of Per1, Per2 and Bmal1 is consistent with the strong entraining effect of nursing found in previous studies. Unexpectedly, and contrasting somewhat to the pattern in the SCN, we also found diurnal rhythmicity in the expression of Cry1 and Bmal1 but not of Per1 in the anterior ventromedial hypothalamic nucleus. Overall, our findings suggest that the SCN is a functional part of the newborn rabbit's circadian system and that it can be entrained by non-photic cues associated with the mother's daily nursing visit.     

Peripheral clock gene expression in CS mice with bimodal locomotor rhythms

CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting, but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression (mPer1, mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN.     

Simulated body temperature rhythms reveal the phase-shifting behavior and plasticity of mammalian circadian oscillators

The circadian pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus maintains phase coherence in peripheral cells through metabolic, neuronal, and humoral signaling pathways. Here, we investigated the role of daily body temperature fluctuations as possible systemic cues in the resetting of peripheral oscillators. Using precise temperature devices in conjunction with real-time monitoring of the bioluminescence produced by circadian luciferase reporter genes, we showed that simulated body temperature cycles of mice and even humans, with daily temperature differences of only 3°C and 1°C, respectively, could gradually synchronize circadian gene expression in cultured fibroblasts. The time required for establishing the new steady-state phase depended on the reporter gene, but after a few days, the expression of each gene oscillated with a precise phase relative to that of the temperature cycles. Smooth temperature oscillations with a very small amplitude could synchronize fibroblast clocks over a wide temperature range, and such temperature rhythms were also capable of entraining gene expression cycles to periods significantly longer or shorter than 24 h. As revealed by genetic loss-of-function experiments, heat-shock factor 1 (HSF1), but not HSF2, was required for the efficient synchronization of fibroblast oscillators to simulated body temperature cycles.     

BK calcium-activated potassium channels regulate circadian behavioral rhythms and pacemaker output

Spontaneous action potentials in the suprachiasmatic nucleus (SCN) are necessary for normal circadian timing of behavior in mammals. The SCN exhibits a daily oscillation in spontaneous firing rate (SFR), but the ionic conductances controlling SFR and the relationship of SFR to subsequent circadian behavioral rhythms are not understood. We show that daily expression of the large conductance Ca(2+)-activated K(+) channel (BK) in the SCN is controlled by the intrinsic circadian clock. BK channel-null mice (Kcnma1(-/-)) have increased SFRs in SCN neurons selectively at night and weak circadian amplitudes in multiple behaviors timed by the SCN. Kcnma1(-/-) mice show normal expression of clock genes such as Arntl (Bmal1), indicating a role for BK channels in SCN pacemaker output, rather than in intrinsic time-keeping. Our findings implicate BK channels as important regulators of the SFR and suggest that the SCN pacemaker governs the expression of circadian behavioral rhythms through SFR modulation.     

Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks

The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase.