Role of the microbiota in circadian rhythms of the host

ABSTRACT

Life for meta-organisms is based on a strong relationship between gut bacteria and body cells. This review summarizes to what extent the microbiota can influence host circadian rhythms via a literature review on the topic. The results show that microbiota can influence the host’s circadian gene expression through direct interactions via immunoreceptors and microbiota-derived metabolites, especially in peripheral tissues. Noteworthy metabolites that are only attributable to the microbiota are short-chain fatty acids and unconjugated bile acids. The microbiota also serves as a mediator for the interplay between the host’s diet and circadian rhythmicity. This work furthermore displays that the microbiota is subject to diurnal variations in terms of structure and function and that the host and the host’s diet influence these fluctuations. As most of these results originate in mouse models, we hope this work stimulates further research in human derived tissue to verify these conclusions.     

Gonadectomy reveals sex differences in circadian rhythms and suprachiasmatic nucleus androgen receptors in mice

In mammals, it is well established that circadian rhythms in physiology and behavior, including the rhythmic secretion of hormones, are regulated by a brain clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. While SCN regulation of gonadal hormone secretion has been amply studied, the mechanisms whereby steroid hormones affect circadian functions are less well known. This is surprising considering substantial evidence that sex hormones affect many aspects of circadian responses, and that there are significant sex differences in rhythmicity. Our previous finding that "core" and "shell" regions of the SCN differ in their expression of clock genes prompted us to examine the possibility that steroid receptors are localized to a specific compartment of the brain clock, with the discovery that the androgen receptor (AR) is concentrated in the SCN core in male mice. In the present study, we compare AR expression in female and male mice using Western blots and immunochemistry. Both of these methods indicate that ARs are more highly expressed in males than in females; gonadectomy eliminates and androgen treatment restores these sex differences. At the behavioral level, gonadectomy produces a dramatic loss of the evening activity onset bout in males, but has no such effect in females. Treatment with testosterone, or with the non-aromatizable androgen dihydrotestosterone, restores male locomotor activity and eliminates sex differences in the behavioral response. The results indicate that androgenic hormones regulate circadian responses, and suggest an SCN site of action.     

Embryonic development and maternal regulation of murine circadian clock function

The importance of circadian clocks in the regulation of adult physiology in mammals is well established. In contrast, the ontogenesis of the circadian system and its role in embryonic development are still poorly understood. Although there is experimental evidence that the clock machinery is present prior to birth, data on gestational clock functionality are inconsistent. Moreover, little is known about the dependence of embryonic rhythms on maternal and environmental time cues and the role of circadian oscillations for embryonic development. The aim of this study was to test if fetal mouse tissues from early embryonic stages are capable of expressing endogenous, self-sustained circadian rhythms and their contribution to embryogenesis. Starting on embryonic day 13, we collected precursor tissues for suprachiasmatic nucleus (SCN), liver and kidney from embryos carrying the circadian reporter gene Per2::Luc and investigated rhythmicity and circadian traits of these tissues ex vivo. We found that even before the respective organs were fully developed, embryonic tissues were capable of expressing circadian rhythms. Period and amplitude of which were determined very early during development and phases of liver and kidney explants are not influenced by tissue preparation, whereas SCN explants phasing is strongly dependent on preparation time. Embryonic circadian rhythms also developed in the absence of maternal and environmental time signals. Morphological and histological comparison of offspring from matings of Clock-Δ19 mutant and wild-type mice revealed that both fetal and maternal clocks have distinct roles in embryogenesis. While genetic disruptions of maternal and embryonic clock function leads to increased fetal fat depots, abnormal ossification and organ development, Clock gene mutant newborns from mothers with a functional clock showed a larger body size compared to wild-type littermates. These data may contribute to the understanding of the ontogenesis of circadian clocks and the risk of disturbed maternal or embryonic circadian rhythms for embryonic development.     

Insect circadian rhythms and photoperiodism

Two clock-controlled processes, overt circadian rhythmicity and the photoperiodic induction of diapause, are described in the blow fly,Calliphora vicina and the fruit fly,Drosophila melanogaster. Circadian locomotor rhythms of the adult flies reflect endogenous, self-sustained oscillations with a temperature compensated period. The free-running rhythms become synchronised (entrained) to daily light:dark cycles, but become arrhythmic in constant light above a certain intensity. Some flies show fragmented rhythms (internal desynchronisation) suggesting that overt rhythmicity is the product of a multioscillator (multicellular) system. Photoperiodic induction of larval diapause inC. vicina and of ovarian diapause inD. melanogaster is also based on the circadian system but seems, to involve a separate mechanism at both the molecular and neuronal levels. For both processes in both species, the compound eyes and ocelli are neither essential nor necessary for photic entrainment, and the circadian clock mechanism is not within the optic lobes. The central brain is the most likely site for both rhythm generation and extra-optic photoreception. InD. melanogaster, a group of lateral brain neurons has been identified as important circadian pacemaker cells, which are possibly also photo-sensitive. Similar lateral brain neurons, staining for arrestin, a protein in the phototransduction ‘cascade’ and a selective marker for photoreceptors in both vertebrates and invertebrates, have been identified inC. vicina. Much less is known about the cellular substrate of the photoperiodic mechanism, but this may involve thepars intercerebralis region of the mid-brain.     

Photoperiodic modulation of circadian rhythms in the cricket Gryllus bimaculatus

The waveform and the free-running period of circadian rhythms in constant conditions are often modulated by preceding lighting conditions. We have examined the modulatory effect of variable length of light phase of a 24h light cycle on the ratio of activity (alpha) and rest phase (rho) as well as on the free-running period of the locomotor rhythm in the cricket Gryllus bimaculatus. When experienced the longer light phases, the alpha/rho-ratio was smaller and the free-running period was shorter. The magnitude of changes in alpha/rho-ratio was dependent on the number of cycles exposed, while the free-running period was changed by a single exposure, suggesting that there are separate regulatory mechanisms for the waveform and the free-running period. The neuronal activity of the optic lobe showed the alpha/rho-ratio changing with the preceding photoperiod. When different photoperiodic conditions were given to each of the two optic lobe pacemakers, the alpha/rho-ratio of a single pacemaker was rather intermediate between those of animals treated with either of the two conditions. These results suggest that the storage of the photoperiodic information occurs at least in part in the optic lobe pacemaker, and that the mutual interaction between the bilateral optic lobe pacemakers is involved in the photoperiodic modulation.     

Automatic data recording of circadian rhythms

This paper illustrates a method for automatic data recording using the printer port of personal computer and software designed ad hoc. The system was tested by measuring circadian rhythms of activity in the subterranean rodent Ctenomys talarum. Data is recorded in a text-only comma-delimited file, and displayed on screen.     

An overview of natural variation studies in the Arabidopsis thaliana circadian clock

Circadian clocks are ubiquitous mechanisms that provide an adaptive advantage by predicting subsequent environmental changes. In the model plant Arabidopsis thaliana (Arabidopsis), our understanding of the complex genetic network among clock components has considerably increased during these past years. Modeling has predicted the possibility of additional component to systematically and functionally complete the clock system. Mutagenesis screens have in the past been successfully employed to detect such novel components. With the advancement in sequencing technologies and improvements in statistical approaches, the extensive natural variation present in Arabidopsis accessions has emerged as a powerful alternative in functional gene discovery. In this review article, we review the previous efforts in mapping natural alleles affecting various clock parameters and will discuss further potentials of such natural-variation studies in physiological and ecological contexts.     

Mammalian activity-rest rhythms in Arctic continuous daylight

Activity - rest (circadian) rhythms were studied in two species of Arctic mammals living in Arctic continuous daylight with all human-induced regular environmental cues (zeitgebers) removed. The two Arctic species (porcupine and ground squirrel) lived outdoors in large enclosures while the Arctic summer sun circled overhead for 82 days. Would local animals maintained under natural continuous daylight demonstrate the Aschoff effect described in previously published laboratory experiments using continuous light, in which rats' circadian activity patterns changed systematically to a longer period, expressing a 26-hour day of activity and rest? The outdoor experiments reported here, however, showed that under natural continuous daylight, both species (porcupine and ground squirrel) had specific times of activity and rest on a nearly 24-hour scale, and their activity peaks did not come later each day. The daily rhythms of the two species were recorded using implanted physiological radio capsules, and from direct observation.     

Adaptive significance of circadian clocks

Circadian clocks are ubiquitous and are found in organisms ranging from bacteria to mammals. This ubiquity of occurrence implies adaptive significance, but to date there has been no rigorous empirical evidence to support this. It is believed that an organism possessing circadian clocks gains fitness advantage in two ways: (i) by synchronizing its behavioral and physiological processes to cyclic environmental factors (extrinsic adaptive value); (ii) by coordinating its internal metabolic processes (intrinsic adaptive value). There is preliminary circumstantial evidence to support both. Several studies using organisms living in constant environments have shown that these organisms possess functional circadian clocks, suggesting that circadian clocks may have some intrinsic adaptive value. Studies to assess the adaptive value of circadian clocks in periodic environments suggest that organisms may have a fitness advantage in those periodic environments, which closely match their own intrinsic periodicity. Furthermore, evidence from organisms living in the wild, selection studies, and studies on latitudinal clines suggest that circadian clocks may have an extrinsic adaptive value as well. In this paper, I have presented several hypotheses for the emergence of circadian clocks and have reviewed some major empirical studies suggesting adaptive significance of circadian clocks.     

Pharmacological effects of vinorelbine on body temperature and locomotor activity circadian rhythms in mice

The effects of vinorelbine (VRL) on the circadian rhythms in body temperature and locomotor activity were investigated in unrestrained B6D2F₁ mice implanted with radio-telemetry transmitters. A single intravenous VRL dose (24 or 12 mg/kg) was given at 7 h after light onset (HALO), a time of high VRL toxicity, and resulted in transient suppression of temperature and activity circadian rhythms in mice kept in light-dark (LD) 12h:12h. Such suppression was dose-dependent. It occurred within 1-5 d after VRL dosing. Recovery of both rhythms was partially complete within 5 d following the high dose and within 2 or 3 d after the low dose and was not influenced by suppression of photoperiodic synchronization by housing in continuous darkness. Moreover, VRL induced a dose-dependent relative decrease in amplitude and phase shift of the temperature circadian rhythm. The mesor and amplitude of the activity rhythm were markedly reduced following the VRL administration. The relevance of VRL dosing time was studied in mice housed in LD 12h:12h. Vinorelbine was injected weekly (20 mg/kg/injection) for 3 wk at 6 or 18 HALO. Vinorelbine treatment ablated the rest-activity and temperature rhythms 3-6 d after each dose, with fewer alterations after VRL dosing at 18 HALO compared to 6 HALO, especially for the body temperature rhythm. There was at least partial recovery 1 wk after dosing, suggesting the weekly schedule of drug treatment is acceptable for therapeutic purposes. Our findings demonstrate that VRL can transiently, yet profoundly, alter circadian clock function. Vinorelbine-induced circadian dysfunction may contribute to the toxicokinetics of this and possibly other anticancer drugs.     

From circadian rhythms to cancer chronotherapeutics

Mammalian circadian rhythms result from a complex organization involving molecular clocks within nearly all “normal” cells and a dedicated neuroanatomical system, which coordinates the so-called “peripheral oscillators.” The core of the central clock system is constituted by the suprachiasmatic nuclei that are located on the floor of the hypothalamus. Our understanding of the mechanisms of circadian rhythm generation and coordination processes has grown rapidly over the past few years. In parallel, we have learnt how to use the predictable changes in cellular metabolism or proliferation along the 24h time scale in order to improve treatment outcome for a variety of diseases, including cancer. The chronotherapeutics of malignant diseases has emerged as a result of a consistent development ranging from experimental, clinical, and technological prerequisites to multicenter clinical trials of chronomodulated delivery schedules. Indeed large dosing-time dependencies characterize the tolerability of anticancer agents in mice or rats, a better efficacy usually results from treatment administration near the least toxic circadian time in rodent tumor models. Programmable in time multichannel pumps have allowed to test the chronotherapy concepts in cancer patients and to implement chronomodulated delivery schedules in current practice. Clinical phase I and II trials have established the feasibility, the safety, and the activity of the chronotherapy schedules, so that this treatment method has undergone further evaluation in international multicenter phase III trials. Overall, more than 2000 patients with metastatic disease have been registered in chronotherapy trials. Improved tolerability and/or better antitumor activity have been demonstrated in randomized multicenter studies involving large patient cohorts. The relation between circadian rhythmicity and quality of life and even survival has also been a puzzling finding over the recent years. An essential step toward further developments of circadian-timed therapy has been the recent constitution of a Chronotherapy cooperative group within the European Organization for Research and Treatment of Cancer. This group now involves over 40 institutions in 12 countries. It is conducting currently six trials and preparing four new studies. The 19 contributions in this special issue reflect the current status and perspectives of the several components of cancer chronotherapeutics.     

Stoichiometric relationship among clock proteins determines robustness of circadian rhythms

The mammalian circadian oscillator is primarily driven by an essential negative feedback loop comprising a positive component, the CLOCK-BMAL1 complex, and a negative component, the PER-CRY complex. Numerous studies suggest that feedback inhibition of CLOCK-BMAL1 is mediated by time-dependent physical interaction with its direct target gene products PER and CRY, suggesting that the ratio between the negative and positive complexes must be important for the molecular oscillator and rhythm generation. We explored this idea by altering expression of clock components in fibroblasts derived from Per2(Luc) and Per mutant mice, a cell system extensively used to study in vivo clock mechanisms. Our data demonstrate that the stoichiometric relationship between clock components is critical for the robustness of circadian rhythms and provide insights into the mechanistic organization of the negative feedback loop. Our findings may explain why certain mutant mice or cells are arrhythmic, whereas others are rhythmic, and suggest that robustness of circadian rhythms can be increased even in wild-type cells by modulating the stoichiometry.     

Non-stationary time series and the robustness of circadian rhythms

Circadian rhythms are regular oscillations in the value of behavioral and physiological variables of organisms that recur on a daily basis. The purpose of this study was to evaluate the extent of non-stationarity of circadian rhythms over several days, to determine how damaging is the violation of the assumption of stationarity in the analysis of circadian rhythms, and to formalize the concept of "rhythm robustness" as an index of oscillatory ("weak") stationarity. Simulated (computer-generated) and experimental data sets (rhythms of body temperature and running-wheel activity in several rodent species) were analysed. Tests of stationarity based on the variance of the daily means and the variance of the daily variances revealed that most experimental data sets are not stationary. Analysis of linear trends indicated that significant trends are rare in experimental data sets. Although the non-stationarity of the experimental data sets reduced the spectral energy of the Enright periodogram used to assess rhythmicity, detection of circadian rhythmicity was not prevented in any of the rhythmic data sets. The results of the various analyses allow the inference that, after high-frequency noise is filtered out, the value of the periodogram's Q(P) statistic reflects the extent of stationarity of the time series. Thus, the "robustness" of a circadian rhythm (i.e. the magnitude of the empirical Q(P) value as compared to the Q(P) value associated with a perfectly rhythmic time series) can serve as an index of the stationarity of the rhythm.     

Differential effect of lithium on the circadian oscillator in young and old hamsters

Lithium is one of the most commonly used drugs in the prophylaxis and treatment of bipolar disorder. It is also known to lengthen circadian period in several organisms. Previously, we reported that there was the association between lengthening circadian period by lithium and GSK-3 protein and its enzyme activity in the mouse suprachiasmatic nucleus (SCN). In this study, we show that lithium affects the circadian oscillator in young and old hamster SCN, in an age-dependent manner. We found that basal levels of phosphorylated GSK-3 (pGSK-3) protein expression in old hamsters are much lower than that in young hamsters. Furthermore, in the old hamsters, lithium did not affect the period of the locomotor activity rhythm or pGSK-3 expression, while changing period and pGSK-3 in the younger animals. These results indicate that the content of pGSK-3 in the SCN has an important role in age-dependent effects of lithium on the circadian oscillator.     

Exogenous corticosteroid and shifts of circadian rhythms in hamsters

We tested the hypothesis that glucocorticoid stimulation mediates the effect of exercise on circadian clock resetting in hamsters. We injected animals with 1 and 5 mg dexamethasone—a potent glucocorticoid agonist—at zeitgeber time (ZT) 4 and ZT6, circadian phases at which vigorous exercise induces maximal phase advances of about 3h. Neither dose of dexamethasone induced phase shifts that were significantly larger than those induced by injections of saline vehicle at either of the phases tested. Some animals, however, showed quite large and consistent phase shifts to repeated injections whether with saline or dexamethasone, such that there was a statistically significant correlation between individuals' responses to the two treatments. The data indicate no role for increased glucocorticoid activity in mediating the effects of exercise on circadian phase shifting, but suggest a modest role for nonspecific stimulation, independent of exercise, in inducing phase shifts at ZT4-ZT6. (Chronobiology International, 18(2), 203-213, 2001)     

Sleep and circadian rhythms: key components in the regulation of energy metabolism

In this review, we present evidence from human and animal studies to evaluate the hypothesis that sleep and circadian rhythms have direct impacts on energy metabolism, and represent important mechanisms underlying the major health epidemics of obesity and diabetes. The first part of this review will focus on studies that support the idea that sleep loss and obesity are "interacting epidemics." The second part will discuss recent evidence that the circadian clock system plays a fundamental role in energy metabolism at both the behavioral and molecular levels. These lines of research must be seen as in their infancy, but nevertheless, have provided a conceptual and experimental framework that potentially has great importance for understanding metabolic health and disease.