Etienne Challet

Lack of food anticipation in Per2 mutant mice.

Predicting time of food availability is key for survival in most animals. Under restricted feeding conditions, this prediction is manifested in anticipatory bouts of locomotor activity and body temperature. This process seems to be driven by a food-entrainable oscillator independent of the main, light-entrainable clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus . Although the SCN clockwork involves self-sustaining transcriptional and translational feedback loops based on rhythmic expression of mRNA and proteins of clock genes , the molecular mechanisms responsible for food anticipation are not well understood. Period genes Per1 and Per2 are crucial for the SCNs resetting to light . Here, we investigated the role of these genes in circadian anticipatory behavior by studying rest-activity and body-temperature rhythms of Per1 and Per2 mutant mice under restricted feeding conditions. We also monitored expression of clock genes in the SCN and peripheral tissues. Whereas wild-type and Per1 mutant mice expressed regular food-anticipatory activity, Per2 mutant mice did not show food anticipation. In peripheral tissues, however, phase shifts of clock-gene expression in response to timed food restriction were comparable in all genotypes. In conclusion, a mutation in Per2 abolishes anticipation of mealtime, without interfering with peripheral synchronization by feeding cycles.

Melatonin: Both master clock output and internal time-giver in the circadian clocks network

Daily rhythms in physiological and behavioral processes are controlled by a network of circadian clocks, reset by inputs and delivering circadian signals to the brain and peripheral organs. In mammals, at the top of the network is a master clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus, mainly reset by ambient light. The nocturnal synthesis and release of melatonin by the pineal gland are tightly controlled by the SCN clock and inhibited by light exposure. Several roles of melatonin in the circadian system have been identified. As a major hormonal output, melatonin distributes temporal cues generated by the SCN to the multitude of tissue targets expressing melatonin receptors. In some target structures, like the Pars tuberalis of the adenohypophysis, these melatonin signals can drive daily rhythmicity that would otherwise be lacking. In other target structures, melatonin signals are used for the synchronization (i.e., adjustment of the timing of existing oscillations) of peripheral oscillators, such as the fetal adrenal gland. Due to the expression of melatonin receptors in the SCN, endogenous melatonin is also able to feedback onto the master clock, although its physiological significance needs further characterization. Of note, pharmacological treatment with exogenous melatonin can synchronize the SCN clock. From a clinical point of view, provided that the subject is not exposed to light at night, the daily profile of circulating melatonin provides a reliable estimate of the timing of the human SCN. During the past decade, a number of melatonin agonists have been developed for treating circadian, psychiatric and sleep disorders. These drugs may target the SCN for improving circadian timing or act indirectly at some downstream level of the circadian network to restore proper internal synchronization.

Feeding Cues Alter Clock Gene Oscillations and Photic Responses in the Suprachiasmatic Nuclei of Mice Exposed to a Light/Dark Cycle

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master mammalian circadian clock, which is mainly reset by light. Temporal restricted feeding, a potent synchronizer of peripheral oscillators, has only weak influence on light-entrained rhythms via the SCN, unless restricted feeding is coupled with calorie restriction, thereby altering phase angle of photic synchronization. Effects of daytime restricted feeding were investigated on the mouse circadian system. Normocaloric feeding at midday led to a predominantly diurnal (60%) food intake and decreased blood glucose in the afternoon, but it did not affect the phase of locomotor activity rhythm or vasopressin expression in the SCN. In contrast, hypocaloric feeding at midday led to 2-4 h phase advances of three circadian outputs, locomotor activity rhythm, pineal melatonin, and vasopressin mRNA cycle in the SCN, and it decreased daily levels of blood glucose. Furthermore, Per1 and Cry2 oscillations in the SCN were phase advanced by 1 and 3 h, respectively, in hypocalorie- but not in normocalorie-fed mice. The phase of Per2 and Bmal1 expression remained unchanged regardless of feeding condition. Moreover, the shape of behavioral phase-response curve to light and light-induced expression of Per1 in the SCN were markedly modified in hypocalorie-fed mice compared with animals fed ad libitum. The present study shows that diurnal hypocaloric feeding affects not only the temporal organization of the SCN clockwork and circadian outputs in mice under light/dark cycle but also photic responses of the circadian system, thus indicating that energy metabolism modulates circadian rhythmicity and gating of photic inputs in mammals.

High‐fat feeding alters the clock synchronization to light

High‐fat feeding in rodents leads to metabolic abnormalities mimicking the human metabolic syndrome, including obesity and insulin resistance. These metabolic diseases are associated with altered temporal organization of many physiological functions. The master circadian clock located in the suprachiasmatic nuclei controls most physiological functions and metabolic processes. Furthermore, under certain conditions of feeding (hypocaloric diet), metabolic cues are capable of altering the suprachiasmatic clocks responses to light. To determine whether high‐fat feeding (hypercaloric diet) can also affect resetting properties of the suprachiasmatic clock, we investigated photic synchronization in mice fed a high‐fat or chow (low‐fat) diet for 3 months, using wheel‐running activity and body temperature rhythms as daily phase markers (i.e. suprachiasmatic clocks hands). Compared with the control diet, mice fed with the high‐fat diet exhibited increased body mass index, hyperleptinaemia, higher blood glucose, and increased insulinaemia. Concomitantly, high‐fat feeding led to impaired adjustment to local time by photic resetting. At the behavioural and physiological levels, these alterations include slower rate of re‐entrainment of behavioural and body temperature rhythms after ‘jet‐lag’ test (6 h advanced light–dark cycle) and reduced phase‐advancing responses to light. At a molecular level, light‐induced phase shifts have been correlated, within suprachiasmatic cells, with a high induction of c‐FOS, the protein product of immediate early gene c‐fos, and phosphorylation of the extracellular signal‐regulated kinases I/II (P‐ERK). In mice fed a high‐fat diet, photic induction of both c‐FOS and P‐ERK in the suprachiasmatic nuclei was markedly reduced. Taken together, the present data demonstrate that high‐fat feeding modifies circadian synchronization to light.

Forebrain oscillators ticking with different clock hands

Clock proteins like PER1 and PER2 are expressed in the brain, but little is known about their functionality outside the main suprachiasmatic clock. Here we show that PER1 and PER2 were neither uniformly present nor identically phased in forebrain structures of mice fed ad libitum. Altered expression of the clock gene Cry1 was observed in respective Per1 or Per2 mutants. In response to hypocaloric feeding, PERs timing was not markedly affected in few forebrain structures (hippocampus). In most other forebrain oscillators, including those expressing only PER1 (e.g., dorsomedial hypothalamus), PER2 (e.g., paraventricular hypothalamus) or both (e.g., paraventricular thalamus), PER1 was up-regulated and PER2 largely phase-advanced. Cry1 expression was selectively modified in the forebrain of Per mutants challenged with hypocaloric feeding. Our results suggest that there is not one single cerebral clock, but a system of multiple brain oscillators ticking with different clock hands and differentially sensitive to nutritional cues.

Phase-advanced daily rhythms of melatonin, body temperature, and locomotor activity in food-restricted rats fed during daytime.

This study was performed to investigate possible effects of a timed caloric restriction on the light-dark (LD) synchronization of four biological rhythms pair-studied in the same animals. In Experiment 1, food-restricted rats kept under a photoperiod of 12 h light:12 h dark received 50% of previous ad libitum food 2 h after the onset of light. Their daily rhythm of pineal melatonin and rhythms of plasma melatonin and corticosterone were examined and com pared to those of ad libitum control rats after 1 or 2 months of food restriction. A significant phase advance (about 2 h) was found for the pineal melatonin rhythm and for the daily onset of plasma melatonin. Timing of nocturnal peak of circulating corticosterone was unchanged, and a diurnal peak anticipated food presentation by about 2 h. In Experiment 2, effects of a timed caloric restriction under 12L:12D were studied on the expression of daily rhythms of body tem perature and locomotor activity. To discriminate between the effects of timed meal feeding and those of the added caloric restriction, these rhythms were analyzed in food-restricted rats, as in Experiment 1, and were compared to those in sham-restricted rats, concomitantly fed twice more than food-restricted rats (i.e., a timed meal feeding without caloric restriction). Acrophase of the nocturnal peak of body temperature rhythm reached the greatest phase advance (7 h) in food-restricted rats, in which it was close to LD transition. The nocturnal com ponent of locomotor activity rhythm also was markedly phase advanced (6 h) by caloric restriction, as indicated by wheel-running and general activity occur ring from early afternoon to midnight. A smaller 4-h phase advance of the nocturnal peak of body temperature also was observed in sham-restricted rats, although the onset of locomotor activity rhythm apparently was unaffected by meal feeding and the end of activity rhythm was phase advanced by 2 h. These results indicate that timed caloric restriction is a potent phase-shifting agent that interacts with the LD cycle zeitgeber. This nonphotic stimulus phase advances melatonin, corticosterone, body temperature, and activity rhythms to different extents and thus suggests a change in the internal synchronization of the cir cadian system.

The nuclear receptor REV-ERBα is required for the daily balance of carbohydrate and lipid metabolism

Mutations of clock genes can lead to diabetes and obesity. REV‐ERBα, a nuclear receptor involved in the circadian clockwork, has been shown to control lipid metabolism. To gain insight into the role of REV‐ERBα in energy homeostasis in vivo, we explored daily metabolism of carbohydrates and lipids in chow‐fed, unfed, or high‐fat‐fed Rev‐erbα−/− mice and their wild‐type littermates. Chow‐fed Rev‐erbα−/− mice displayed increased adiposity (2.5‐fold) and mild hyperglycemia (∼10%) without insulin resistance. Indirect calorimetry indicates that chow‐fed Rev‐erbα−/− mice utilize more fatty acids during daytime. A 24‐h nonfeeding period in Rev‐erbα−/− animals favors further fatty acid mobilization at the expense of glycogen utilization and gluconeogenesis, without triggering hypoglycemia and hypothermia. High‐fat feeding in Rev‐erbα−/− mice amplified metabolic disturbances, including expression of lipogenic factors. Lipoprotein lipase (Lpl) gene, critical in lipid utilization/storage, is triggered in liver at night and constitutively up‐regulated (∼ 2‐fold) in muscle and adipose tissue of Rev‐erbα−/− mice. We show that CLOCK, up‐regulated (2‐fold) at night in Rev‐erbα−/− mice, can transactivate Lpl. Thus, overexpression of Lpl facilitates muscle fatty acid utilization and contributes to fat overload. This study demonstrates the importance of clock‐driven Lpl expression in energy balance and highlights circadian disruption as a potential cause for the metabolic syndrome.—Delezie, J., Dumont, S., Dardente, H., Oudart, H., Gréchez‐Cassiau, A., Klosen, P., Teboul, M., Delaunay, F., Pévet, P., Challet, E. The nuclear receptor REV‐ERBα is required for the daily balance of carbohydrate and lipid metabolism. FASEB J. 26, 3321–3335 (2012). www.fasebj.org

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.

Entrainment in calorie-restricted mice: conflicting zeitgebers and free-running conditions

Phase-shifting effects of timed calorie restriction were investigated in mice during exposure to a 12:12-h light-dark cycle. Food-anticipatory activity (FAA), the output of a food-entrainable pacemaker, was expressed before the time of feeding whether mice received daily hypocaloric food (3.3 g of chow/day) or normocaloric food (5 g of chow/day) at zeitgeber time (ZT) 2 (ZT12 = lights off). Subsequently, mice were placed in constant darkness and fed ad libitum. The onset of the nocturnal period of locomotor activity was phase advanced by 1 h in calorie-restricted mice compared with normocalorie-fed controls. The phase advance still occurred when FAA was prevented by restraining calorie-restricted mice. Giving hypocaloric food at ZT2, ZT10, ZT14, or ZT22 phase advanced the nocturnal pattern of activity by 1, 3, 1, and 1 h, respectively. After transfer to constant darkness, FAA free ran in parallel with the normal nocturnal period of locomotor activity. A light pulse during the early subjective night phase delayed both components. These results indicate that 1) timed calorie restriction under a light-dark cycle can phase advance the light-entrainable pacemaker with a phase-dependent magnitude, 2) FAA feedback is not crucial for the observed phase advance, and 3) the light-entrainable pacemaker may control the period of the food-entrainable pacemaker in mice fed ad libitum.Phase-shifting effects of timed calorie restriction were investigated in mice during exposure to a 12:12-h light-dark cycle. Food-anticipatory activity (FAA), the output of a food-entrainable pacemaker, was expressed before the time of feeding whether mice received daily hypocaloric food (3.3 g of chow/day) or normocaloric food (5 g of chow/day) at zeitgeber time (ZT) 2 (ZT12 = lights off). Subsequently, mice were placed in constant darkness and fed ad libitum. The onset of the nocturnal period of locomotor activity was phase advanced by 1 h in calorie-restricted mice compared with normocalorie-fed controls. The phase advance still occurred when FAA was prevented by restraining calorie-restricted mice. Giving hypocaloric food at ZT2, ZT10, ZT14, or ZT22 phase advanced the nocturnal pattern of activity by 1, 3, 1, and 1 h, respectively. After transfer to constant darkness, FAA free ran in parallel with the normal nocturnal period of locomotor activity. A light pulse during the early subjective night phase delayed both components. These results indicate that 1) timed calorie restriction under a light-dark cycle can phase advance the light-entrainable pacemaker with a phase-dependent magnitude, 2) FAA feedback is not crucial for the observed phase advance, and 3) the light-entrainable pacemaker may control the period of the food-entrainable pacemaker in mice fed ad libitum.

Involvement of corticosterone in the fasting-induced rise in protein utilization and locomotor activity

During fasting, most of the energy is derived from lipids whereas proteins are efficiently spared. However, there is a late rise in net protein utilization. Fasting is also associated with an increase in locomotor activity. Because the plasma corticosterone level increases concomitantly with these metabolic and behavioral changes, the involvement of corticosterone has been hypothesized. To test this, the net protein utilization and locomotor activity were investigated in fasted adrenalectomized (Adx) rats, with or without replacement with corticosterone, and in fasted intact rats treated with RU486, an antagonist of type II glucocorticoid receptors. During the phase of fasting characterized by protein sparing, urine nitrogen loss was further reduced in Adx rats and in RU486-treated controls compared with intact rats and with Adx rats with corticosterone replacement: this indicates a catabolic effect of corticosterone through type II receptors. In the last phase of fasting, the rise in net protein breakdown was suppressed in Adx rats and restored by corticosterone replacement. The increase in locomotor activity induced by fasting in controls was suppressed in Adx and restored by corticosterone replacement. This rise in running activity was still present in RU486-treated rats. In conclusion, this study shows that corticosterone plays a critical role in the changes of both protein catabolism and locomotor activity during prolonged fasting.