Melatonin in energy control: Circadian time-giver and homeostatic monitor.

Melatonin is a neurohormone synthesized from dietary tryptophan in various organs, including the pineal gland and the retina. In the pineal gland, melatonin is produced at night under the control of the master clock located in the suprachiasmatic nuclei of the hypothalamus. Under physiological conditions, the pineal gland seems to constitute the unique source of circulating melatonin. Melatonin is involved in cellular metabolism in different ways. First, the circadian rhythm of melatonin helps the maintenance of proper internal timing, the disruption of which has deleterious effects on metabolic health. Second, melatonin modulates lipid metabolism, notably through diminished lipogenesis, and it has an antidiabetic effect, at least in several animal models. Third, pharmacological doses of melatonin have antioxidative, free radical-scavenging, and anti-inflammatory properties in various in vitro cellular models. As a result, melatonin can be considered both a circadian time-giver and a homeostatic monitor of cellular metabolism, via multiple mechanisms of action that are not all fully characterized. Aging, circadian disruption, and artificial light at night are conditions combining increased metabolic risks with diminished circulating levels of melatonin. Accordingly, melatonin supplementation could be of potential therapeutic value in the treatment or prevention of metabolic disorders. More clinical trials in controlled conditions are needed, notably taking greater account of circadian rhythmicity.

Deletion of the Clock Gene Bmal2 Leads to Alterations in Hypothalamic Clocks, Circadian Regulation of Feeding, and Energy Balance.

BMAL2 (ARNTL2) is a paralog of BMAL1 that can form heterodimers with the other circadian factors CLOCK and NPAS2 to activate transcription of clock and clock-controlled genes. To assess a possible role of Bmal2 in the circadian regulation of metabolism, we investigated daily variations of energy metabolism, feeding behavior, and locomotor behavior, as well as ability to anticipate restricted food access in male mice knock-out for Bmal2 (B2KO). While their amount of food intake and locomotor activity were normal compared with wild-type mice, B2KO mice displayed increased adiposity (1.5-fold higher) and fasted hyperinsulinemia (fourfold higher) and tended to have lower energy expenditure at night. Impairment of the master clock in the suprachiasmatic nuclei was evidenced by the shorter free-running period (-14 min/cycle) of B2KO mice compared with wild-type controls and by a loss of daily rhythmicity in expression of intracellular metabolic regulators (e.g., Lipoprotein lipase and Uncoupling protein 2). The circadian window of eating was longer in B2KO mice. The circadian patterns of food intake and meal numbers were bimodal in control mice but not in B2KO mice. In response to restricted feeding, food-anticipatory activity was almost prevented in B2KO mice, suggesting altered food clock that controls anticipation of food availability. In the mediobasal hypothalamus of B2KO mice, expression of genes coding orexigenic neuropeptides (including Neuropeptide y and Agouti-Related Peptide) was downregulated, while Lipoprotein lipase expression lost its rhythmicity. Together, these data highlight that BMAL2 has major impacts on brain regulation of metabolic rhythms, sleep-wake cycle, and food anticipation.

Mice with humanized livers reveal the role of hepatocyte clocks in rhythmic behavior.

The synchronization of circadian clock depends on a central pacemaker located in the suprachiasmatic nuclei. However, the potential feedback of peripheral signals on the central clock remains poorly characterized. To explore whether peripheral organ circadian clocks may affect the central pacemaker, we used a chimeric model in which mouse hepatocytes were replaced by human hepatocytes. Liver humanization led to reprogrammed diurnal gene expression and advanced the phase of the liver circadian clock that extended to muscle and the entire rhythmic physiology. Similar to clock-deficient mice, liver-humanized mice shifted their rhythmic physiology more rapidly to the light phase under day feeding. Our results indicate that hepatocyte clocks can affect the central pacemaker and offer potential perspectives to apprehend pathologies associated with altered circadian physiology.

Reciprocal Interactions between Circadian Clocks, Food Intake, and Energy Metabolism.

Like other biological functions, food intake and energy metabolism display daily rhythms controlled by the circadian timing system that comprises a main circadian clock and numerous secondary clocks in the brain and peripheral tissues. Each secondary circadian clock delivers local temporal cues based on intracellular transcriptional and translational feedback loops that are tightly interconnected to intracellular nutrient-sensing pathways. Genetic impairment of molecular clocks and alteration in the rhythmic synchronizing cues, such as ambient light at night or mistimed meals, lead to circadian disruption that, in turn, negatively impacts metabolic health. Not all circadian clocks are sensitive to the same synchronizing signals. The master clock in the suprachiasmatic nuclei of the hypothalamus is mostly synchronized by ambient light and, to a lesser extent, by behavioral cues coupled to arousal and exercise. Secondary clocks are generally phase-shifted by timed metabolic cues associated with feeding, exercise, and changes in temperature. Furthermore, both the master and secondary clocks are modulated by calorie restriction and high-fat feeding. Taking into account the regularity of daily meals, the duration of eating periods, chronotype, and sex, chrononutritional strategies may be useful for improving the robustness of daily rhythmicity and maintaining or even restoring the appropriate energy balance.

Circadian functioning of Locus Cœruleus of the nocturnal rat and diurnal rodent Arvicanthis.

The noradrenergic Locus Cœruleus is one of the major arousal structures involved in inducing wakefulness. While brain noradrenaline (NA) amounts display 24-h variations, the origin of NA rhythm is currently unknown. In this study, we tested the hypothesis that NA rhythm could result from its rhythmic synthesis. Therefore, we investigated the 24-h expression profile of NA rate-limiting enzyme, tyrosine hydroxylase (th), in the Locus Cœruleus (LC) of the nocturnal rat and the diurnal rodent Arvicanthis, under 12 h:12 h light/dark (LD) and constant darkness (DD) conditions. In both species, th mRNA levels vary significantly over 24-h. In nocturnal rats, th mRNA profiles show a unimodal rhythm, with peak values in late day in LD, and in the middle of the subjective day in DD. In contrast, th mRNA rhythm in Arvicanthis is characterized by a bimodal profile, with higher levels at the beginning of the day and of the night in LD, and in the middle of the subjective day and night in DD. The rhythmic pattern of th expression may be dependent on a LC clock machinery. Therefore, we investigated the expression of three clock genes, namely bmal1, per1, and per2, and found that their mRNAs display significant variations between day and nighttime points in both species, but in opposite directions. These data show that NA rhythm may be related to circadian expression of th gene in both species, but differs between nocturnal and diurnal rodents. Furthermore, the phase opposition of clock gene expression in the rat compared to Arvicanthis suggests that the clock machinery might be one of the mechanisms involved in th rhythmic expression.

Comparison of Locomotor and Feeding Rhythms between Indoor and Outdoor Cats Living in Captivity.

The plastic nature of cat behaviour allows this "friendly symbiont" of humans to adapt to various housing conditions. Beyond daylight, one could wonder if other environmental factors affect its patterns. Yet, how its activity and feeding rhythms are impacted by its environment is rarely studied in standardised conditions between populations. We compared the behaviour of cats living in a 29 m2 indoor room and cats living in a 1145 m2 outdoor enclosure, tracking them simultaneously in summer for 21 days, with advanced technologies. Both populations received daylight but weather fluctuations only occurred outdoors. Bimodality was detected in the activity and feeding rhythms of both groups, while twilight triggered crepuscular peaks. Daily, the outdoor population covered more distance (4.29 ± 0.27 km; p < 0.001) and consumed more food (67.44 ± 2.65 g; p < 0.05) than the indoor population (2.33 ± 0.17 km, 57.75 ± 2.85 g, respectively), but displayed less rhythmic behaviours, assumedly because of rhythm disruptors met only in outdoor conditions. Finally, outdoor housing seemed to promote the exploratory behaviour of the cats at night, while indoor housing increased both meal frequency (p = 0.063) and the impact of human interactions on the feeding rhythms of the cats.

Sleep pattern in the dromedary camel: a behavioral and polysomnography study.

STUDY OBJECTIVES: To investigate sleep patterns in the camel by combining behavioral and polysomnography (PSG) methods. METHODS: A noninvasive PSG study was conducted over four nights on four animals. Additionally, video recordings were used to monitor the sleep behaviors associated with different vigilance states. RESULTS: During the night, short periods of sporadic sleep-like behavior corresponding to a specific posture, sternal recumbency (SR) with the head lying down on the ground, were observed. The PSG results showed rapid shifts between five vigilance states, including wakefulness, drowsiness, rapid eye movement (REM) sleep, non-REM (NREM) sleep, and rumination. The camels typically slept only 1.7 hours per night, subdivided into 0.5 hours of REM sleep and 1.2 hours of NREM sleep. Camels spent most of the night being awake (2.3 hours), ruminating (2.4 hours), or drowsing (1.9 hours). Various combinations of transitions between the different vigilance states were observed, with a notable transition into REM sleep directly from drowsiness (9%) or wakefulness (4%). Behavioral postures were found to correlate with PSG vigilance states, thereby allowing a reliable prediction of the sleep stage based on SR and the head position (erected, motionless, or lying down on the ground). Notably, 100% of REM sleep occurred during the Head Lying Down-SR posture. CONCLUSIONS: The camel is a diurnal species with a polyphasic sleep pattern at night. The best correlation between PSG and ethogram data indicates that sleep duration can be predicted by the behavioral method, provided that drowsiness is considered a part of sleep.

Bimodal serotonin synthesis in the diurnal rodent, Arvicanthis ansorgei.

In mammals, behavioral activity is regulated both by the circadian system, orchestrated by the suprachiasmatic nucleus (SCN), and by arousal structures, including the serotonergic system. While the SCN is active at the same astronomical time in diurnal and nocturnal species, little data are available concerning the serotonergic (5HT) system in diurnal mammals. In this study, we investigated the functioning of the 5HT system, which is involved both in regulating the sleep/wake cycle and in synchronizing the SCN, in a diurnal rodent, Arvicanthis ansorgei. Using in situ hybridization, we characterized the anatomical extension of the raphe nuclei and we investigated 24 h mRNA levels of the serotonin rate-limiting enzyme, tryptophan hydroxylase 2 (tph2). Under both 12 h:12 h light/dark (LD) and constant darkness (DD) conditions, tph2 mRNA expression varies significantly over 24 h, displaying a bimodal profile with higher values around the (projected) light transitions. Furthermore, we considered several SCN outputs, namely melatonin, corticosterone, and locomotor activity. In both LD and DD, melatonin profiles display peak levels during the biological night. Corticosterone plasma levels show a bimodal rhythmic profile in both conditions, with higher levels preceding the two peaks of Arvicanthis locomotor activity, occurring at dawn and dusk. These data demonstrate that serotonin synthesis in Arvicanthis is rhythmic and reflects its bimodal behavioral phenotype, but differs from what has been previously described in nocturnal species.

Seasonal variations in locomotor activity rhythm and diurnal activity in the dromedary camel (Camelus dromedarius) under mesic semi-natural conditions.

The dromedary camel (Camelus dromedarius) is a large ungulate that copes well with the xeric environment of the desert. Its peculiar adaptation to heat and dehydration is well-known. However, its behavior and general activity is far from being completely understood. The present study was carried out to investigate the ecological effect of the various seasons on the locomotor activity (LA) rhythm and diurnal activity of this species. Six adult female camels were maintained under mesic semi-natural conditions of the environment during four periods of 10 days in each season: autumn, winter, spring and summer. In addition, three female camels were used to test the effect of rain on the LA rhythm during a period of 18 days during the winter. The animal's LA was recorded using the locomotion scoring method. Camels displayed a clear 24.0h LA rhythm throughout the four seasons. Activity was intense during Day-time (6-22 fold higher in comparison to night) and dropped or completely disappeared during nighttime. Mean daytime total activity was significantly higher in the summer as compared to winter. Regardless of the season, the active phase in camels coincided with the time of the photophase and thermophase. Furthermore, the daily duration of the time spent active was directly correlated to the seasonal changes of photoperiod. The diurnal activity remained unchanged over the four seasons. For each season, the start and the end of the active phase were synchronized with the onset of sunrise and sunset. At these time periods, temperature remained incredibly stable with a change ranging from 0.002 to 0.210°C; whereas, changes of light intensity were greater and faster with a change from 0.1 to 600 lux representing a variation of 3215-7192 fold in just 25-29 min. Rainfall affected the pattern of the LA rhythm with occurrence of abnormal nocturnal activity during nighttime disturbing nocturnal rest and sleep. Here we show that the dromedary camel exhibits significant seasonal changes of its activity within daylight hours. However, the diurnal pattern remains unchanged regardless of the season; whereas, abnormal nocturnal activity is observed during periods of rain. The activity onset and offset in this species seems to be primarily driven by the changes in light intensity at dusk and dawn.

Melatonin and the circadian system: Keys for health with a focus on sleep.

Melatonin (MLT), secreted during the night by the pineal gland, is an efferent hormonal signal of the master circadian clock located in the suprachiasmatic nucleus (SCN). Consequently, it is a reliable phase marker of the SCN clock. If one defines as "chronobiotic," a drug able to influence the phase and/or the period of the circadian clock, MLT is a very potent one. The most convincing data obtained so far come from studies on totally blind individuals. Exogenous MLT administered daily entrains the sleep-wake cycle of these individuals to a 24-h cycle. MLT, however, is not essential to sleep. In nocturnally, active mammals, MLT is released during the night concomitantly with the daily period of wakefulness. Therefore, MLT cannot be simply considered as a sleep hormone, but rather as a signal of darkness. Its role in the circadian system is to reinforce nighttime physiology, including timing of the sleep-wake cycle and other circadian rhythms. MLT exerts its effects on the sleep cycle especially by a direct action on the master circadian clock. The sleep-wake cycle is depending not only on the circadian clock but also on an orchestrated network of different centers in the brain. Thus, the control of sleep-wake rhythm might be explained by a parallel and concomitant action of MLT on the master clock (chronobiotic effect) and on sleep-related structures within the brain. MLT acts through two high-affinity membrane receptors (MT1 and MT2) with striking differences in their distribution pattern. MLT is a powerful synchronizer of human circadian rhythms, thus justifying the use of MLT and MLT agonists in clinical medicine as pharmacological tools to manipulate the sleep-wake cycle, and to treat sleep disorders and other circadian disorders. Available MLT analogs/drugs are all nonspecific MT1/MT2 agonists. The development of new ligands which are highly selectivity for each subtype is clearly a new challenge for the field and will be at the root of new therapeutic agents for curing specific pathologies, including sleep disorders.

Pinealectomy and gonadectomy modulate amplitude, but not photoperiodic modulation of Clock gene expression in the Syrian hamster suprachiasmatic nuclei.

The duration of daytime light phase (photoperiod) controls reproduction in seasonal mammals. Syrian hamsters are sexually active when exposed to long photoperiod, while gonadal atrophy is observed after exposure to short photoperiod. The photorefractory period, or photorefractoriness, is a particular state of spontaneous recrudescence of sexual activity that occurs after a long-term exposure to short photoperiod. Expression of core clock genes in the master circadian clock contained in the suprachiasmatic nuclei depends on photoperiodic conditions. Interestingly, the expression of the Clock gene is also modified in photorefractory Syrian hamsters. Since melatonin and testosterone levels in seasonal species are dependent on photoperiod, photoperiodic variations of Clock mRNA levels in the suprachiasmatic clock could be a consequence of these hormonal changes. To test this hypothesis, we analysed the effects of pinealectomy on Clock mRNA changes due to long to short photoperiod transition and of gonadectomy on Clock mRNA levels in photorefractory period. Our data show that the suprachiasmatic integration of the short photoperiod (assessed by a rhythmic expression profile of Clock) is independent of the presence of melatonin. Furthermore, constitutively low expression of Clock observed during the photorefractory period does not require the presence of either melatonin or testosterone. However, we show that both hormones provide positive feedback on average levels of Clock expression. Thus, our data support the hypothesis that daily variations of Clock levels in the suprachiasmatic nuclei are influenced by photoperiodic changes and the time spent in short photoperiod, independently of seasonal modifications of melatonin or testosterone levels.

Daytime Restricted Feeding Affects Day-Night Variations in Mouse Cerebellar Proteome.

The cerebellum harbors a circadian clock that can be shifted by scheduled mealtime and participates in behavioral anticipation of food access. Large-scale two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry was used to identify day-night variations in the cerebellar proteome of mice fed either during daytime or nighttime. Experimental conditions led to modified expression of 89 cerebellar proteins contained in 63 protein spots. Five and 33 spots were changed respectively by time-of-day or feeding conditions. Strikingly, several proteins of the heat-shock protein family (i.e., Hsp90aa1, 90ab1, 90b1, and Hspa2, 4, 5, 8, 9) were down-regulated in the cerebellum of daytime food-restricted mice. This was also the case for brain fatty acid protein (Fabp7) and enzymes involved in oxidative phosphorylation (Ndufs1) or folate metabolism (Aldh1l1). In contrast, aldolase C (Aldoc or zebrin II) and pyruvate carboxylase (Pc), two enzymes involved in carbohydrate metabolism, and vesicle-fusing ATPase (Nsf) were up-regulated during daytime restricted feeding, possibly reflecting increased neuronal activity. Significant feeding × time-of-day interactions were found for changes in the intensity of 20 spots. Guanine nucleotide-binding protein G(o) subunit alpha (Gnao1) was more expressed in the cerebellum before food access. Neuronal calcium-sensor proteins [i.e., parvalbumin (Pvalb) and visinin-like protein 1 (Vsnl1)] were inversely regulated in daytime food-restricted mice, compared to control mice fed at night. Furthermore, expression of three enzymes modulating the circadian clockwork, namely heterogeneous nuclear ribonucleoprotein K (Hnrnpk), serine/threonine-protein phosphatases 1 (Ppp1cc and Ppp1cb subunits) and 5 (Ppp5), was differentially altered by daytime restricted feeding. Besides cerebellar proteins affected only by feeding conditions or daily cues, specific changes in in protein abundance before food access may be related to behavioral anticipation of food access and/or feeding-induced shift of the cerebellar clockwork.

Distinct feedback actions of behavioural arousal to the master circadian clock in nocturnal and diurnal mammals.

The master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus provides a temporal pattern of sleep and wake that - like many other behavioural and physiological rhythms - is oppositely phased in nocturnal and diurnal animals. The SCN primarily uses environmental light, perceived through the retina, to synchronize its endogenous circadian rhythms with the exact 24 h light/dark cycle of the outside world. The light responsiveness of the SCN is maximal during the night in both nocturnal and diurnal species. Behavioural arousal during the resting period not only perturbs sleep homeostasis, but also acts as a potent non-photic synchronizing cue. The feedback action of arousal on the SCN is mediated by processes involving several brain nuclei and neurotransmitters, which ultimately change the molecular functions of SCN pacemaker cells. Arousing stimuli during the sleeping period differentially affect the circadian system of nocturnal and diurnal species, as evidenced by the different circadian windows of sensitivity to behavioural arousal. In addition, arousing stimuli reduce and increase light resetting in nocturnal and diurnal species, respectively. It is important to address further question of circadian impairments associated with shift work and trans-meridian travel not only in the standard nocturnal laboratory animals but also in diurnal animal models.

Entrainment of circadian rhythms of locomotor activity by ambient temperature cycles in the dromedary camel.

In the dromedary camel, a well-adapted desert mammal, daily ambient temperature (Ta)-cycles have been shown to synchronize the central circadian clock. Such entrainment has been demonstrated by examining two circadian outputs, body temperature and melatonin rhythms. Locomotor activity (LA), another circadian output not yet investigated in the camel, may provide further information on such specific entrainment. To verify if daily LA is an endogenous rhythm and whether the desert Ta-cycle can entrain it, six dromedaries were first kept under total darkness and constant-Ta. Results showed that the LA rhythm free runs with a period of 24.8-24.9 h. After having verified that the light-dark cycle synchronizes LA, camels were subjected to a Ta-cycle with warmer temperatures during subjective days and cooler temperatures during subjective nights. Results showed that the free-running LA rhythm was entrained by the Ta-cycle with a period of exactly 24.0 h, while a 12 h Ta-cycle phase advance induced an inversion of the LA rhythm and advanced the acrophase by 9 h. Similarly, activity onset and offset were significantly advanced. All together, these results demonstrate that the Ta-cycle is a strong zeitgeber, able to entrain the camel LA rhythm, hence corroborating previous results concerning the Ta non-photic synchronization of the circadian master clock.

Transcriptional Changes Involved in Atrophying Muscles during Prolonged Fasting in Rats.

Food deprivation resulting in muscle atrophy may be detrimental to health. To better understand how muscle mass is regulated during such a nutritional challenge, the current study deciphered muscle responses during phase 2 (P2, protein sparing) and phase 3 (P3, protein mobilization) of prolonged fasting in rats. This was done using transcriptomics analysis and a series of biochemistry measurements. The main findings highlight changes for plasma catabolic and anabolic stimuli, as well as for muscle transcriptome, energy metabolism, and oxidative stress. Changes were generally consistent with the intense use of lipids as fuels during P2. They also reflected increased muscle protein degradation and repressed synthesis, in a more marked manner during P3 than P2 compared to the fed state. Nevertheless, several unexpected changes appeared to be in favor of muscle protein synthesis during fasting, notably at the level of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, transcription and translation processes, and the response to oxidative stress. Such mechanisms might promote protein sparing during P2 and prepare the restoration of the protein compartment during P3 in anticipation of food intake for optimizing the effects of an upcoming refeeding, thereby promoting body maintenance and survival. Future studies should examine relevance of such targets for improving nitrogen balance during catabolic diseases.

Melatonin rhythm and other outputs of the master circadian clock in the desert goat (Capra hircus) are entrained by daily cycles of ambient temperature.

In desert areas, mammals such as camel and goat are exposed to harsh environmental conditions. The ambient temperature (Ta) cycles have been shown to entrain the circadian clock in the camel. In the present work, we assumed that, in the goat living in a desert biotope, Ta cycles would have the same synchronizing effect on the central clock. Therefore, the effects of Ta cycles on body temperature (Tb), locomotor activity (LA) and melatonin (Mel) rhythms as outputs of the master circadian clock have been studied. The study was performed on bucks kept first under constant conditions of total darkness (DD) and constant Ta, then maintained under DD conditions but exposed to Ta cycles with heat period during subjective day and cold period during subjective night. Finally, the Ta cycles were reversed with highest temperatures during the subjective night and the lowest temperatures during the subjective day. Under constant conditions, the circadian rhythms of Tb and LA were free running with an endogenous period of 25.3 and 25.0 hours, respectively. Ta cycles entrained the rhythms of Tb and LA to a period of exactly 24.0 hours; while when reversed, the Ta cycles led to an inversion of Tb and LA rhythms. Similarly, Ta cycles were also able to entrain Mel rhythm, by adjusting its secretion to the cooling phase before and after Ta cycles inversion. All together, these results show that the Ta cycles entrain the master circadian clock in the goat.

The circadian regulation of food intake.

Feeding, which is essential for all animals, is regulated by homeostatic mechanisms. In addition, food consumption is temporally coordinated by the brain over the circadian (~24 h) cycle. A network of circadian clocks set daily windows during which food consumption can occur. These daily windows mostly overlap with the active phase. Brain clocks that ensure the circadian control of food intake include a master light-entrainable clock in the suprachiasmatic nuclei of the hypothalamus and secondary clocks in hypothalamic and brainstem regions. Metabolic hormones, circulating nutrients and visceral neural inputs transmit rhythmic cues that permit (via close and reciprocal molecular interactions that link metabolic processes and circadian clockwork) brain and peripheral organs to be synchronized to feeding time. As a consequence of these complex interactions, growing evidence shows that chronodisruption and mistimed eating have deleterious effects on metabolic health. Conversely, eating, even eating an unbalanced diet, during the normal active phase reduces metabolic disturbances. Therefore, in addition to energy intake and dietary composition, appropriately timed meal patterns are critical to prevent circadian desynchronization and limit metabolic risks. This Review provides insight into the dual modulation of food intake by homeostatic and circadian processes, describes the mechanisms regulating feeding time and highlights the beneficial effects of correctly timed eating, as opposed to the negative metabolic consequences of mistimed eating.

Smartphone and a freely available application as a new tool to record locomotor activity rhythm in large mammals and humans.

Daily pattern of locomotor activity (LA), one of the most studied rhythms in humans and rodents, has not been widely investigated in large mammals. This is partly due to the high cost and breakability of used automatic devices. Since last decade, smartphones are becoming ubiquitous. Meanwhile, several applications detecting activity by using internal sensors were made available. In this study, we assumed that this device could be a cheaper and easier way to measure the LA rhythm in humans and large mammals, like camel and goat. A smartphone application (Nokia Mate Health), normally used to quantify physical activities in humans, was chosen for the study. To validate the rhythm data obtained from the smartphone, LA rhythm was simultaneously recorded using an automatic device, the Actiwatch-Mini®. Results showed that the smartphone provided a clear and significant daily rhythm of LA. The visual assessment of the superimposed LA rhythm's curves in all three species showed that the smartphone application displayed similar rhythms as those recorded by the Actiwatch-Mini. Highly significant positive correlation (p≤ 0.0001) exists between the two recording rhythms. The daily periods were both the same at 24.0 h. Acrophases were also significantly similar and occurring around mid-day: 11:40 ± 0.35 h vs 11:41 ± 0.35 h for the camel, 11:25 ± 0.19 h vs 11:37 ± 0.25 h for the goat and 13:04 ± 0.11 h vs 13:51 ± 0.28 h for humans using smartphone and Actiwatch, respectively. The related mesor and amplitude were also close between the two recording devices. Results indicate clearly that using smartphones constitutes a reliable cheap tool to study LA rhythm for chronobiology studies, especially in laboratories facing lack of funding.

Circadian Analysis of the Mouse Cerebellum Proteome.

The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.