Delezie J Mendoza

Sophie Reibel

Laurence Huck

Sophie Brangolo

Aline Grechez-Cassiau

Paule Felder

Daniel Clesse

Dominique Streicher

Jean-George Lorentz

Jevita, Slava, Edith, Stefanie, Ouafa, Tounès, Cristina, Mio Coralie Cathy

Daniela Lucas

Adrien Laurent

Alexis

Julien

Vy

Madah, Taole… David

Julien Delezie

Jorge Mendoza

Stéphanie Dumont

Cristina Sandu

Dominique Sage-Ciocca

Paul Pévet

Etienne Challet

Hugues Dardente

Hugues Oudart

Aline Gréchez-Cassiau

Paul Klosen

Michèle Teboul

Franck Delaunay

French review)

Keywords: food-entrainable oscillator, restricted feeding, clock gene, hypothalamus

que vous m'avez transmises en statistiques notamment.

« Le Soleil envie la Terre comme la Terre envie le Soleil.

L'un reçoit la lumière et l'autre brille de mille feux. » J.D.

Acknowledgments/Remerciements

A few words in Shakespeare's language… I am deeply grateful to the members of my thesis committee, Andries Kalsbeek, Franck Pfrieger and Urs Albrecht, for having accepted to evaluate this work.

Et un peu plus dans la langue de Voltaire… J'exprime toute ma reconnaissance à l'égard de mon directeur de thèse, Etienne Challet, pour m'avoir accueilli au sein de son équipe, encadré et soutenu pendant plus de trois années. J'ai énormément appris à tes côtés en pouvant notamment profiter de ton excellente culture en chronobiologie. Tu m'as donné beaucoup de liberté dans mon travail tout en sachant être présent quand il le fallaitet ce, même dans les moments les plus ardus.

Je tiens à remercier Jorge Mendoza, qui m'a pris comme stagiaire dès mes études en Master et qui m'a aidé tout au long de ces années. Tu m'as transmis le goût de l'effort. Sur les plans scientifique et technique, j'ai beaucoup appris avec toi.

Ma gratitude s'adresse également à Stéphanie Dumont. A tes côtés, j'ai profité d'une formation exceptionnelle sur le plan technique et d'une aide précieuse au quotidien. Sans toi, une partie de cette thèse n'aurait jamais vu le jour.

Un grand merci à Sylviane Gourmelen qui m'a souvent assisté dans le suivi des animaux durant cette thèse. Tu as toujours été spontanée et avenante.

Mes remerciements se destinent également à Franck Delaunay, Hugues

Dardente et Hugues Oudart. Avec de grandes qualités pédagogiques, vous m'avez chacun instruit sur différents domaines scientifiques qui m'étaient jusqu'alors quasi inconnusje pense en particulier au métabolisme ou à la génétique. Merci pour le temps que vous avez consacré à nos recherches.

De façon plus particulière, j'aimerais remercier des personnes qui ont influencé mon parcours scolaire et universitaire : Mme Hen, mon professeur de Biologie et Nutrition en BEP Carrières Sanitaires et Sociales, pour avoir un jour prononcé quelques mots qui ont eu une portée singulière et qui font encore écho en moi aujourd'hui ; Mme Max, mon professeur de Biologie lorsque j'entreprenais des études en Sciences Médico-Sociales, pour m'avoir donné goût à la biologie, peutêtre sans le savoir ; Mme Weber, professeur de Français, pour m'avoir mis plusieurs livres entre les mains dont le surprenant Candide, qui a depuis délivré tout son sens ;

et M Dalle Mese, professeur de Philosophie, pour m'avoir évalué comme un littéraire et fait comprendre que derrière les mots se cache souvent une imposture.

J'ai également une pensée chaleureuse pour mes (lointains) amis de « Psycho » : Amandine, Alexandra, Pierrela communauté. Nous avons partagé quelques années ensemble ; vous m'avez chacun marqué par vos personnalités, par nos échanges, par votre bienveillance.

« Et c'est à cet endroit […] » que je tiens également à t'ajouter. J'ai beaucoup écrit par le passé, tu m'as toujours lu avec un rare discernement. Merci Ewa.

Sans oublier Guillaume et Jérémy qui m'ont aussi inspiré. Les détours de la vie ne nous ont pas épargné, nous avons su cependant nous retrouver aux croisements de différents chemins, sans que le temps n'affecte nos amitiés.

Mes derniers mots s'adressent à « mon coeur ma mie mon âme », Caroline.

Alors peut-être devrais-je user de quelques citations, du père du Petit Prince par exemple, qui n'a jamais été très loin et qui a écrit de belles choses sur l'amour, ou bien tenter de déposer mes propres pensées pour te témoigner toute ma gratitude et mon affection ; il y a cependantpour faire dans ce qui nous ressemble -, ce fameux extrait d'une oeuvre cinématographique écrite par Tarantino dans laquelle deux personnages tombent éperdument amoureux l'un de l'autre et vivent une folle aventure, et avec du recul à présent, j'ai envie de le reprendre, de retranscrire à quelques mots près ce que nous dit Alabama en ouverture, tout simplement… Il a fallu que je fasse toute la route depuis mon p'tit bled perdu au fin fond de la [Lorraine], jusqu'à la grisaille de [Strasbourg], pour trouver l'amour de ma vie. Même en gambergeant pendant un million d'années, il me serait jamais venu à l'idée d'associer l'amour avec la capitale de l' [Europe]. Et aujourd'hui encore, ce qui a suivi ressemble à un rêve lointain. Pourtant ce rêve était bien vrai, et il a changé nos deux vies à tout jamais […]. True Romance

Abstract

Circadian oscillators rely on transcriptional mechanisms that regulate a wide array of biological activities. In mammals, the suprachiasmatic nucleus pacemaker is the chief conductor of the body that sets the tempo and dynamics through timekeeping signals to unify the timing of the different performers-the so-called secondary or peripheral oscillators. Each element of this biological orchestra shares common molecular mechanisms, in which clock genes such as Per1-3, Cry1-2, Clock, Bmal1 and Rev-erbα are essential.

In this large multi-oscillatory system, one putative performer, the foodentrainable oscillator, has been shown to respond to the restriction of food availability to a specific time of the day. As a result, beyond the reach of the main conductor, the food-entrainable oscillator is able to adjust the phase and amplitude of a plethora of behavioral and physiological events controlled by secondary oscillators. This is especially highlighted by the ability of animals to predict feeding time, as evidenced by food-seeking behaviors a few hours prior to the expected food access. In addition, many circadian oscillators are specifically involved in metabolic functions for a proper energy balance. The tight relationship between the metabolic states of the body and functioning of circadian oscillators is particularly illustrated by the genetic perturbations of the molecular clockwork.

The work performed during this PhD thesis aimed at investigating the role of the transcriptional silencer Rev-erbα in both the circadian clockwork of the foodentrainable oscillator and metabolic regulations. Firstly, by evaluating foodanticipatory components in animals fed once a day at the same time, we showed that mice lacking Rev-erbα display a reduction in locomotor activity prior to food access compared to littermate controls. Accordingly, the rises in body temperature and corticosterone that anticipate mealtime are also diminished. Interestingly, daily p-ERK expression in hypothalamic regions and daily PER2 expression in the cerebellum of Rev-erbα KO mice are not phase-adjusted to feeding time. These results indicate that Rev-erbα participates in the integration of feeding signals and in food-seeking behaviors. Secondly, by investigating energy balance in fasted, normal chow or highfat fed animals, we revealed that Rev-erbα KO mice exhibit greater reliance on lipid fuels as energy substrates, contributing to a mild hyperglycemic state. We also found that Lipoprotein lipase (Lpl) expression, important for fatty acid uptake and utilization, is strongly up-regulated in peripheral tissues of Rev-erbα KO mice, predisposing mice to obesity. In this regard, we uncovered a new molecular pathway that ties clock-driven Lpl expression to energy homeostasis. These findings highlight the significance of daily Rev-erbα oscillations to prevent the appearance of the metabolic syndrome.

In conclusion, we provide evidence that REV-ERBα may be a part of the foodentrainable oscillator clockwork that triggers food-anticipatory components, and represents a pivotal player to link the core clock machinery to metabolic pathways.

Résumé

Introduction

Les rythmes biologiques sont une propriété fondamentale du vivant. En effet, la plupart des espèces animales présentent une rythmicité journalière (p.ex. rythme d'activité-repos). Cette rythmicité est la conséquence d'une adaptation de ces espèces aux variations périodiques de leur environnement (p.ex. cycle lumièreobscurité). La rythmicité des fonctions biologiques repose sur l'existence d'un système endogène composé d'oscillateursou horlogesbiologiques capables d'imposer une rythmicité circadienne (circa : environ, dies : jour) à de nombreux paramètres physiologiques et comportementaux, tels que les métabolismes lipidique et glucidique, les sécrétions hormonales (p.ex. corticostérone), la prise alimentaire ou le cycle veille-sommeil.

Chez les mammifères, l'horloge principale est localisée dans les noyaux suprachiasmatiques (NSC) situés à la base de l'hypothalamus. Bien que ces noyaux présentent des oscillations auto-entretenues proches de 24 h, ces dernières sont ajustées principalement par le cycle d'alternance jour/nuit. Plus exactement, l'activité des NSC est synchronisée (c.-à-d. remise à l'heure) par la lumière du jour, qui leur est transmise directement par la rétine via le tractus rétino-hypothalamique. A partir des NSC, l'information va être relayée vers différentes structures centrales (p.ex. l'hypothalamus) ou périphériques (p.ex. le foie) dont les activités seront à leur tour synchronisées sur 24 h par l'intermédiaire de voies neuronales et neuroendocrines. D'autres oscillateurs, dits secondaires, sont localisés dans différentes parties du cerveau et dans les organes périphériques (Fig. 1). Afin d'explorer davantage les rouages moléculaires de cette horloge alimentaire, nous avons étudié des souris déficientes (ou knockout ; KO) pour le gène d'horloge Rev-erb. De manière intéressante, en condition de lumièreobscurité 12 h -12 h, l'activité locomotrice ainsi que l'augmentation de température en anticipation de l'heure du repas observées chez des souris témoins, étaient diminuées chez des souris KO pour Rev-erb (Fig. 3). Ce résultat a également été observé chez des souris KO placés en condition d'obscurité totale, ou porteuses de lésions des NSC. En parallèle à une diminution de l'activité locomotrice et de la température corporelle, le pic de libération de corticostérone en anticipation de l'heure du repas présent chez les souris sauvages, n'était pas observé chez les souris KO pour Rev-erb ; indiquant qu'une sortie physiologique additionnelle contrôlée par l'horloge alimentaire était altérée chez ces dernières. Toutefois, la synchronisation de l'oscillateur hépatique à l'heure du repas était conservée chez les souris KO (Fig. 4). De la même manière, les oscillations de la protéine d'horloge PER2 dans les structures hypothalamiques était modifiée sous l'effet de la restriction temporelle dans les deux génotypes. Autrement dit, la synchronisation des oscillateurs hypothalamiques et de l'oscillateur hépatique à l'heure des repas, était préservée en l'absence du gène d'horloge Rev-erb.

Cependant, ni PER2, ni p-ERK (un marqueur d'activation cellulaire) ne répondaient clairement à la restriction dans le cervelet des souris KO. L'ensemble de ces résultats suggèrent un rôle essentiel de Rev-erb dans le fonctionnement de cette horloge alimentaire qui pourrait, parmi les régions cérébrales étudiées, faire intervenir le cervelet dans la modulation de l'expression de l'activité anticipatrice. Néanmoins, du fait de la participation de Rev-erb aux régulations du métabolisme, nous avons évalué des paramètres métaboliques comme le niveau de glycémie ou de glycogène hépatique. De manière inattendue, nous avons découvert que les souris KO avaient des niveaux plus élevés de glucose et de glycogène hépatique en comparaison des souris témoins, indépendamment de la condition de nourrissage (Fig. 5). De plus, en période de jeûne induite par la restriction temporelle, les KO montraient des valeurs de température corporelle nettement supérieures à celles de souris témoins (Fig. 3). Ces observations rendaient compte de changements non négligeables dans l'homéostasie énergétique des souris déficientes en Rev-erb qui pourraient contribuer à l'altération des sorties comportementales et physiologiques de l'horloge alimentaire. En période de nourrissage ad libitum (gauche) ainsi qu'en période de restriction alimentaire (droit), les niveaux de glucose plasmatique et de glycogène hépatique sont plus élevés chez les souris KO (en rouge) en comparaison aux souris contrôles (en bleu).

Objectifs et résultatspartie 2

La seconde partie de mon travail de thèse a dès lors consisté à étudier le phénotype métabolique des souris KO pour Rev-erb. Par une association originale d'approches in vivo et in vitro pour l'exploration des métabolismes glucidique et lipidique, nous avons pu mettre en évidence que le gène Rev-erb joue un rôle essentiel dans la balance énergétique. De manière remarquable, les souris KO ont une plus grande adiposité et sont hyperglycémiques, même si elles mangent la même quantité de nourriture standard (ou normocalorique), au même moment de la journée et avec une activité physique similaire aux souris témoins. En outre, l'hyperglycémie a également été observée après un jeûne de 24 h (Fig. 6), et n'est pas la conséquence d'un défaut de sécrétion d'insuline ou d'une insulino-résistance comme le confirment notamment les résultats du clamp euglycémique hyperinsulinémique (Fig. 6). Les souris KO (en rouge) montrent une hyperglycémie même après une période de jeûne.

La différence majeure entre les souris sauvages et les souris déficientes en

Rev-erb réside dans la manière dont ces dernières métabolisent les nutriments : en effet, nos données de calorimétrie indirecte ont révélés que les KO synthétisent plus de lipides pendant la phase de prise alimentaire (c.-à-d. phase nocturne) contribuant à une surchage lipidique, et utilisent moins de glucides pendant la phase de repos (Fig. 7), contribuant au maintien d'une légère hyperglycémie sur le cycle de 24 h. De façon intéressante, cette augmentation de l'utilisation des lipides (ou des corps cétoniques) en l'absence de Rev-erb est également observée lors d'une période de jeûne de 24 h. En outre, nous avons montré que le phénotype « gras » est très accentué lorsque les KO sont sous nourrissage hypercalorique (c.-à-d. riche en graisses ; Fig. 8). Cette obésité induite par la nourriture va conduire à de multiples altérations métaboliques. Des modifications de la quantité et du rythme d'expression de la glycémie, de l'insuline, de la leptine (Fig. 8) et des acides gras libres sont également constatées de manière importante chez les souris KO. Les oscillateurs périphériques tels que le foie et le tissu adipeux, sont également touchés par une surcharge lipidique. Notamment, l'expression hépatique de gènes de la lipogenèse est augmentée chez les souris KO. Indépendamment de la condition de nourrissage (normo-ou hypercalorique), les souris KO ont une plus grande adiposité corrélée à de plus grandes concentrations de leptine.

Enfin, nous avons démontré que le phénotype « gras » des souris KO résultait également d'une perte de contrôle par les oscillateurs périphériques, en l'absence de REV-ERB, de l'expression d'un gène clé du métabolisme, la Lipoprotéine lipase (Lpl), intervenant dans l'hydrolyse des triglycérides et l'entrée des acides gras libres dans les cellules. En effet, le gène de la Lpl était surexprimé dans différents tissus périphériques tels que le foie, le muscle et le tissu adipeux (Fig. 9). Cette surexpression notamment dans le tissu adipeux et le muscle conduit respectivement à un meilleur stockage et à une utilisation préférentielle des acides gras.

Conclusion

L'ensemble de nos données indique que le répresseur transcriptionnel Rev-erb joue un rôle charnière dans les fonctions circadiennes ainsi que dans le métabolisme. En effet, d'un point de vue circadien, l'absence de Rev-erb altère la synchronisation à l'heure des repasdémontré par une réduction des sorties comportementales et physiologiques de l'horloge alimentaire, ainsi que par l'absence d'ajustement du rythme de PER2 dans l'oscillateur cérébelleux. Sur le plan métabolique, la délétion de ce gène modifie notamment le métabolisme des lipidesdémontré par une accumulation excessive de tissu adipeux, une utilisation préférentielle des acides gras, ainsi qu'une perte de contrôle de l'expression de la Lpl.

Néanmoins, la possibilité que la diminution de la capacité d'anticiper l'heure des repas soit essentiellement reliée aux modifications de la balance énergétique ne peut pas être exclue. Cette hypothèse qui donnerait dès lors un rôle moindre aux perturbations du coeur de la machinerie moléculaire circadienne induites par l'absence même du gène d'horloge Rev-erb, est soutenue par la synchronisation de l'oscillateur hépatique à l'heure des repas, ainsi que par l'ajustement dans divers oscillateurs hypothalamiques de l'expression de la protéine d'horloge PER2 en condition de restriction alimentaire.

En conclusion, au-delà de positionner Rev-erb à l'interface entre régulations circadiennes et métaboliques, ces travaux indiquent l'importance de prendre en considération les modifications du métabolisme induites par la délétion d'un gène d'horlogenotamment dans un organisme entier -pour l'étude des fonctions circadiennes telles que l'entraînement à l'heure des repas. En ce sens, l'utilisation d'outils génétiques permettant de désactiver spécifiquement l'expression d'un gène d'horloge dans un tissu donné ou à un stade précis du développement pourrait être une méthode de choix pour mieux explorer les rouages de cette mystérieuse horloge alimentaire.

Chapter 1 GENERAL INTRODUCTION

1. Beats, rhythms and life 11.1 Introduction Dynamic cyclic events and the steady recurrence of identical rhythm patterns are a common feature of both music and life. Various characteristic and repetitive pulses can be identified in different kinds of music, such as hip-hop, blues or classical music, just as we can observe various regular recurring events in nature. Some occur at a high frequency (e.g., human heartbeats) and others at a much slower frequency (e.g., annual migration). The idea that life is organized rhythmically, which has been originally hypothesized a long time ago (see part 1.2), paved the way for the development of a discipline of research studying biological rhythms, called chronobiology (from the greek: chrónos, meaning "time"; bios, "life" and -logia, "study of").

The historical emergence of this relatively new scientific research area radically changed our way of seeing living creatures, from basic organisms such as algae to more complex organic systems such as human beings. Since the last century, the study of biological rhythms is turning out to be one of the most fascinating interdisciplinary research fields in modern biology, receiving a vast amount of attention from other disciplines. I will present first the brilliant thoughts and discoveries that gave birth to chronobiology. Then, I will progressively introduce the different concepts and terminology used in our research area and show that biological rhythms can vary according to their duration and can have very complex and fascinating beats, the same as rhythmic patterns in music.

Horologium Florae

The observation of rhythmic events in living species comes first from description on leaf movements, which has been probably known over 2300 years ago (mentioned in Wolfgang Engelmann, 2002). Daily periodic movements of the leaves of the tamarind tree, Tamarindus indica, were initially and clearly reported by Androsthenes of Thasos, admiral under Alexander the Great, 400 years before Christ (mentioned in [START_REF] Bretzl | Botanische Forschungen des Alexanderzuges[END_REF]. Much later, in 1729, the French astronomer Jean Jacques Ortous de Mairan observed the daily opening and closing of the leaves of a sensitive heliotrope plant (probably Mimosa Pudica) 2 . de Mairan then performed an experiment to test whether this biological "behavior" was simply a response to the daily appearance of the sun. To do so, he confined the plant to the dark (i.e., a constant condition of darkness). He observed that in the absence of sunlight, the leaves of this plant opened during the usual daylight hours and closed at night. Even if de Mairan did not conclude that Mimosa Pudica has an internal clock, his experiment was the first scientific demonstration of the persistence of a rhythmic event in constant darkness.

It took 30 years before further scientific investigations on this biological mechanism in plants followed de Mairan's observation. Indeed, independent researches by Duhamel Du Monceau and Zinn in 1759 (for review, see [START_REF] Mcclung | Plant circadian rhythms[END_REF], showed that the rhythm of leaf movements in constant darkness was independent of fluctuations in environmental temperature. In other words, this "clock" mechanism appeared to be endogenous. In 1832, De Candolle gave more details on the period of this inner clock. He showed that in constant light conditions, opening and closing rhythm of the leaflets of a sensitive plant (e.g., Mimosa), was maintained with a period close to, but not exactly, 24 h. To make use of circadian terminology, this signifies that the endogenous period of Mimosa's clock runs free (or free-runs 3 ) with its own timing (Fig. 1). De Candolle also showed that the rhythm could be inverted by manipulating the light-dark cycle. Later on, Darwin tried to understand the selective advantage of the periodic leaves movements (mentioned in [START_REF] Bunning | Interference of moonlight with the photoperiodic measurement of time by plants, and their adaptive reaction[END_REF].

These pioneering observations paved the way for further studies focusing on biological rhythms, and thus the establishment of the discipline of chronobiology.

Chronobiology, modern times

In the 20 th century, chronobiology became its own discipline of research (in parallel to evolution, genetics and development). Observations of rhythmic events became numerous, particularly in animals. For example, the psychologist JS Szymanski reported in 1914 that goldfish swimming occurs with a 24-h activity rhythm, even in the absence of external cues such as light. In 1922, Curt Richter reported daily pattern of locomotor activity in rats. In 1937, researchers created an international organization for the study of biological rhythms (for review, see Koukarri) 4 . At the same time, brilliant and leading figures in chronobiology were influential in establishing many concepts and key criteria to define the biological clock.

Halberg, "Circadian" rhythm

In the 1950s, Franz Halberg-regarded as the father of chronobiologyobserved several kinds of differences among groups of mice in the counts of circulating blood eosinophils when they were sampled at different clock hours. He also noticed that after blinding, rectal temperature in mice showed an approximately 24-h periodicity. He created the term circadian, which comes from the Latin circa, meaning "around" and dies, "day" [START_REF] Halberg | Glossary of chronobiology (author's transl)][END_REF][START_REF] Halberg | Transdisciplinary unifying implications of circadian findings in the 1950s[END_REF]. As a result, biological phenomena observed with a roughly 24-h period (i.e., from 20 to 28 h) were so named. 4 Research on biological rhythms was strong and colorful in Germany and the rest of Europe in the 18 th and 19 th centuries. In the 20 th century, in spite of the prevalent skepticism of scientific establishment in the USA, chronobiology no longer hid, became popular and conceptual advances were flourishing. In the sense, the 1960 symposium at Cold Spring Harbor Laboratory laid the groundwork for the field of chronobiology.

Figure 1. Leaf movements of a representative species

(A) Sleep movements of Phaseolus coccineus. The position of the primary leaves of a seedling at night is at the left and during the day is at the right. (B) Circadian rhythm of leaf movements of P. coccineus entrained to light/dark cycles and monitored in continuous light. As can be inferred from the leaf positions in (A), the peaks of the curve represent the nightime leaf position. The vertical lines indicate 24-h intervals. The period for this trace is ∼27 h. From [START_REF] Mcclung | Plant circadian rhythms[END_REF] Circadian: relating to biologic variations or rhythms with a frequency of 1 cycle in 24 ± 4 h; circa (about, approximately) and dies (day or 24 h). Note: term describes rhythms with an about 24-h cycle length, whether they are frequency-synchronized with (acceptable) or are desynchronized or free-running from the local environmental time scale, with periods slightly yet consistently different from 24 h … [START_REF] Halberg | Glossary of chronobiology (author's transl)][END_REF] In spite of further extensive studies of circadian rhythms, many other important cycles were concurrently delineated and studied. This includes infradian rhythms, which show a period longer than 24-h and can occur at monthly, seasonal or yearly intervals (e.g., annual migration); and ultradian rhythms, which occur with a period shorter than 24-h (e.g., thermoregulation). It is important to mention that within each cycle, the time at which the peak of a rhythm occurs is called the "acrophase", whereas the time at which the trough of a rhythm occurs is the "bathyphase". The difference between the peak (or trough) and the mean value (mesor) of a wave is called the amplitude (Fig. 2) 5 . The period is defined as the time to complete one cycle. The mesor is the average value around which the variable measured oscillates. The amplitude of the rhythm is defined as the distance between the mesor and the acrophase (or half the peak-to-peak amplitude). Within each cycle, the acrophase and the bathyphase represent the time point where the parameter measured shows the highest or the lowest value, respectively.

Whether we measure, hour by hour, the number of dividing cells in any tissue, the volume of urine excreted, the reaction to a drug, or the accuracy and the speed with which arithmetical problems are solved, we usually find that there is a maximum value at one time of day and a minimum value at another. [START_REF] Aschoff | Circadian Rhythms in Man[END_REF]

Aschoff, light is a Zeitgeber

Jürgen Aschoff, one of the brilliant founders of chronobiology, used for the first time in 1954 the German term Zeitgeber ("time giver" or "synchronizer") to define an exogenous (external) cue that triggers some sort of change in an organism's endogenous clock. Light is the most prominent entraining agent for a wide variety of species, but other, so-called non-photic Zeitgebers such as food, which will be discussed further, can influence internal clocks. In 1960, Aschoff observed that an increase in the intensity of light in constant (light) conditions causes a lengthening of the free-running period for a nocturnal (dark-active) organism and a shortening of the period for a diurnal (light-active) organism6 [START_REF] Aschoff | Exogenous and endogenous components in circadian rhythms[END_REF]. He also contributed in unraveling the physiological mechanisms that regulate circadian rhythms in birds, mammals and especially in humans [START_REF] Chandrashekaran | Biological rhythms research: A personal account[END_REF]. Indeed, Aschoff & Wever from 1962 demonstrated that in the absence of environmental cues (e.g., light) human's endogenous clock free runs with a period slightly longer than 24 h [START_REF] Aschoff | Circadian Rhythms in Man[END_REF])-an observation also made by the French explorer and scientist, Michel Siffre (1964), when he isolated himself in a deep cave without time cues for several months.

Bünning, the heritability of circadian rhythms

In the early 1930s, Erwin Bünning, a great biologist and botanist, demonstrated the inheritance of circadian rhythms. In 1932, Bünning started to cross bean plants with different endogenous periods and demonstrated that the next generation had periods of intermediate duration, thus refuting the idea that daily rhythmicity is learned, as first argued earlier by Richard Semon (1904). In 1935, Bünning undeniably determined in plants the genetic origin of the "biological clock," a term he coined. However, it took several years until the existence of a genetic-basis for circadian rhythms was firmly admitted. Bünning also laid out the basis of photoperiodism. He suggested that plants display endogenous phases of light and dark sensitivity, important in the photoperiod response of flowering (mentioned in [START_REF] Koukkari | Introducing biological rhythms: a primer on the temporal organization of life, with implications for health, society, reproduction and the natural environment[END_REF].

Pittendrigh, temperature compensation

The biologist Colin Pittendrigh (1918Pittendrigh ( -1996) ) "brought to chronobiology a degree of experimental elegance and rigour which remained unsurpassed" [START_REF] Chandrashekaran | Biological rhythms research: A personal account[END_REF]. Work by Pittendrigh confirmed Bünning's hypothesis that a rhythmic process underlies the photoperiodic time-measurement. He defined several key criteria (highlighted below) a biological clock must have to be so called. His scientific research on Drosophila pseudoobscura in the early 1950s 7 , demonstrated that daily rhythm in eclosion persists in constant conditions and can be entrained by light cycles close to the flies' endogenous period; and more importantly, that the period of eclosion remains relatively constant when exposed to changes in environmental temperature. In other words, the eclosion rhythms of Drosophila were temperature compensated, unlike the rates of most chemical reactions [START_REF] Pittendrigh | On Temperature Independence in the Clock System Controlling Emergence Time in Drosophila[END_REF].

In 1958, Pittendrigh also developed the concept of the phase response curve (PRC), which predicts how the biological clock would be affected by a change in its light schedule [START_REF] Pittendrigh | On Temperature Independence in the Clock System Controlling Emergence Time in Drosophila[END_REF][START_REF] Menaker | Colin S. Pittendrigh (1918-96)[END_REF][START_REF] Mcclung | Plant circadian rhythms[END_REF]

Take-home message

In living creatures, most essential biological functions show a rhythmic pattern close to 24 h. These endogenous daily variations, called circadian rhythms, allow organisms to anticipate and prepare for periodic changes in the environment, such as light, temperature, food availability and other periodic phenomena. Circadian rhythms are generated by biological clocks, which are a temperature compensated system.

Circadian rhythms are mostly synchronized to the daily environmental light-dark cycle-ultimately determined by the Earth's rotation. When isolated from periodic environmental time cues or Zeitgebers, the endogenous rhythm persists (i.e., freeruns), with a period slightly longer or shorter than 24 h (Fig. 5).

Figure 5. Input and ouput pathways of a biological clock

Different Zeitgebers can entrain the internal biological clock in order to regulate circadian rhythms to a precise 24-hour period.

One clock to rule them all

In the recent past, scientific investigations were conducted in bacteria, fungi, plants, birds and mammals, as well as molecules and cells. The fact that circadian rhythms have been observed in a wide range of organisms, suggests that temporal organization result from an evolutionary process, which started when life consisted only of single cells.

The tree of life

As aforementioned, circadian rhythms-thus the presence of a biological clock-have been revealed in organisms ranging from prokaryotes to eukaryotes. As an aside, it is important to remember than until the mid 1980s, it was thought that only eukaryotes had circadian rhythms. However, several research groups in the 80's discovered that prokaryotes were also capable of expressing circadian rhythms [START_REF] Johnson | Circadian clocks in prokaryotes[END_REF]) that meet all the criteria of bona fide biological clocks. For instance, the prokaryote, Synechococcus8 , has been shown to possess a circadian clock regulating cell division9 that is entrained by a light-dark cycle and that is temperature-compensated (Fig. 6) [START_REF] Sweeney | A Circadian-Rhythm in Cell-Division in a Prokaryote, the Cyanobacterium Synechococcus Wh7803[END_REF][START_REF] Huang | Circadian Rhythm of the Prokaryote Synechococcus sp. RF-1[END_REF]). (Left) Rhythms of nitrogenase activity in several different light-dark (LD) cycles followed by constant light conditions (LL). The cultures were adapted to the LD conditions for one week before exposing them to LL. (Right) Endogenous rhythm of nitrogenase activity of Synechococcus sp. RF-1 at several different temperatures. The cultures were all initially grown at 28°C under a 12:12 LD regimen for 1 week. They were then transferred to LL at 22, 26, 29, and 33°C, respectively. The N2-fixing activity of these cultures incubated at various temperatures was assayed at 2 h interval for a period of 4 days. From [START_REF] Huang | Circadian Rhythm of the Prokaryote Synechococcus sp. RF-1[END_REF] Hence, the 20 th century has left us with an impressive list of living organisms that exhibit circadian rhythms (Fig. 7) (for review, see [START_REF] Dunlap | Molecular bases for circadian clocks[END_REF][START_REF] Vansteensel | Organization of cell and tissue circadian pacemakers: a comparison among species[END_REF]. At present, a common statement is that circadian rhythms are ubiquitous in living systems. Besides the observations of rhythmic behavioral/physiological outputs in many organisms supporting this thought, additional evidence comes from the identification of specific genes, named "clock genes", which are the genetic basis for circadian oscillations and which can be expressed in virtually all cells. The diagram, designed by Jay C. Dunlap, represents the range of organisms in which circadian systems/rhythms have been described. In blue are represented the phylogenic groups in which circadian rhythms have been investigated. In red are represented those species in which genetic and molecular analysis of clock mechanism has progressed significantly. Line lengths correspond to evolutionary distance between phylogenic groups. From [START_REF] Dunlap | Molecular bases for circadian clocks[END_REF] 2.2 Discovery of (the) clock gene In 1971, Konopka and Benzer isolated the first clock mutant Drosophila melanogaster. They showed that a gene mutation on a specific region of the X chromosome drastically alters the period length of the circadian clock regulating eclosion and locomotor activity rhythms [START_REF] Konopka | Clock mutants of Drosophila melanogaster[END_REF]. The gene was thus named Period, and its precise location discovered later [START_REF] Bargiello | Molecular genetics of a biological clock in Drosophila[END_REF]. This was the first description of a genetic component of a circadian clock.

From then on, "clock" mutation was also demonstrated in Neurospora and the green algae Chlamydomonas (mentioned in [START_REF] Ralph | A mutation of the circadian system in golden hamsters[END_REF].

Figure 8. Individual locomotor activity rhythms of normal or mutant flies kept in constant darkness

All flies were previously exposed to a 12 h-12 h light-dark cycle. Each line represents the start of a successive interval. Vertical black bars represent the level of activity. For visual continuity, data are double-plotted, so that two days are represented horizontally. From [START_REF] Konopka | Clock mutants of Drosophila melanogaster[END_REF] Later, Ralph and Menaker described for the first time a single-gene mutation that specifically affects the circadian clock of a vertebrate. This mutation, Tau, caused a dramatic shortening of the period (called Tau) of the circadian locomotor rhythm of golden hamsters (Fig. 8) [START_REF] Ralph | A mutation of the circadian system in golden hamsters[END_REF]). In the 1990s, Takahashi and colleagues succeed in identifying the first mammalian clock gene by inducing circadian clock mutations in mice with the N-ethyl-N-notrosourea (ENU)-induced mutagenesis strategy. As a result, a semidominant mutation in a key gene, named Clock, was found to dramatically affect circadian rhythms (Fig. 9). Clock mutant mice displayed an abnormally long period of daily activity and eventually became arrhythmic in constant darkness [START_REF] Vitaterna | Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior[END_REF][START_REF] King | Positional cloning of the mouse circadian clock gene[END_REF].

Figure 9. Effect of the Clock mutation in mice

Locomotor activity and DNA sequence data from wild-type and Clock/Clock mutant mice. On the actogram, the white and black bars represent the day and night periods, respectively. Note that activity records are double-plotted. Mice were kept in light-dark cycle for 8 days and then transferred to constant darkness (arrows). Below each actograms are represented the DNA sequence to illustrate the AT transversion that causes the mutation. Adapted from [START_REF] King | Positional cloning of the mouse circadian clock gene[END_REF] All these findings demonstrated that clock genes are fundamental components of a biological clock and are necessary for normal circadian function. Considerable progress has been now made in discovering many more genetic components of the biological clock 10 . In addition, the ongoing characterization of these genes at the transcriptional and translational levels contributes to precisely characterizing their role and notably, how they interact to generate circadian oscillations (see part 2.3.2).

Hereafter the focus will be especially on mammals.

Discovery of the first pacemaker in mammals

Since several physiological processes are controlled in a circadian way, the question of an anatomical substrate (in parallel to the identification of clock genes) at the basis of the temporal organization of dynamic events-like humans optimize their social/professional activities by referring to their wristwatch-has emerged decades ago. By referring to the results presented above, I will introduce what we currently know regarding the functioning of the circadian timing system in mammals. Is there only one main clock which drives all the circadian rhythms? Or are several clocks/oscillators11 involved? Where are they localized? Do they all contain the same "clock" genes?

The suprachiasmatic nucleus: a great conductor

Considering the fact that mammals showed daily rhythms of biological functions, synchronized to the 24-h light-dark cycle, it has been thought that an entrainment pathway must involved an organ that is sensitive to the periodic variations of illumination. In this context, a cerebral structure identified relatively early in the 20 th century became the subject of much attention.

In 1937, Pate demonstrated that enucleation in cats induced atrophy of a specific region of the hypothalamus-nucleus ovidens. Much later in 1972, a direct projection from the retina to the suprachiasmatic area of the hypothalamus was demonstrated in the rat [START_REF] Moore | A retinohypothalamic projection in the rat[END_REF]. This area was further described as a bilateral region containing two groups of neurons (10000-12000 total in population) dorsal to the optic chiasm and just lateral to the periventricular nucleus and third ventricle [START_REF] Moore | Suprachiasmatic nucleus organization[END_REF]. Of note, these nuclei are also subdivided in a ventral (core) area and a dorsal (shell) area based on their afferents and the neurochemical nature of cells in each area (Fig. [START_REF] Panda | Coordinated transcription of key pathways in the mouse by the circadian clock[END_REF][START_REF] Turek | Obesity and metabolic syndrome in circadian Clock mutant mice[END_REF]. Interestingly, Curt Richter at about the same time identified the anterior hypothalamus as a "biological pacemaker" involved in sleep-wake cycles [START_REF] Richter | Sleep and activity: their relation to the 24-hour clock[END_REF]. Two years later, the effect of a removal of the suprachiasmatic nucleus (SCN) by bilateral electrolytic lesions in rats was tested by [START_REF] Moore | Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat[END_REF] and [START_REF] Stephan | Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions[END_REF]. Surprisingly, the SCN ablation abolished the rhythm of adrenal corticosterone [START_REF] Moore | Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat[END_REF], as well as those of locomotor activity, food intake and drinking behavior [START_REF] Stephan | Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions[END_REF][START_REF] Nagai | Effect of bilateral lesions of the suprachiasmatic nuclei on the circadian rhythm of food-intake[END_REF]). Further observations were then made in other mammals (Fig. 11) [START_REF] Edgar | Effect of SCN lesions on sleep in squirrel monkeys: evidence for opponent processes in sleep-wake regulation[END_REF]DeCoursey et al. 1997). In 1979, a brilliant work by Inouye and Kawamura tested the autonomy of the rat SCN clock. They isolated a small hypothalamic "island" that contains the SCN, thus free of all neural inputs from other brain structures. Then, they recorded neural (multiple unit) activity from two electrodes simultaneously, one in or near the SCN and the other in different neural locations. In intact animals, SCN and all brain sites tested presented a circadian rhythmicity of spontaneous neural activity, while in animals (that were also blinded) with this hypothalamic island containing the SCN, rhythmic electrical activity was lost elsewhere but persisted in the island [START_REF] Inouye | Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus[END_REF]. In brief, the rhythmic activity of the island did not depend on inputs from other brain areas.

A few years later, it was demonstrated that brain slices containing the SCN maintained a circadian rhythm in electrical activity [START_REF] Green | Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice[END_REF]. It was also shown after isolation of the SCN that the peptide hormone vasopressin-which is synthesized in and secreted daily by the SCN (see Fig. 12, for the distribution of the different peptides in the SCN)-continue to be released in a circadian fashion for several days in vitro [START_REF] Moore | Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus[END_REF][START_REF] Earnest | Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro[END_REF]. Of interest, in the early 90's, a circadian rhythm in membrane conductance was revealed in isolated basal retinal neurons [START_REF] Michel | Circadian rhythm in membrane conductance expressed in isolated neurons[END_REF]. Afterward, isolated neurons from the rat SCN were shown to express independently phased circadian firing rhythms, indicative of the presence of single-cell circadian oscillators [START_REF] Welsh | Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms[END_REF]. To further investigate a role for the SCN as a main conductor of circadian rhythmicity, Ralph and colleagues in 1990 used wild-type and Tau mutant hamsters (the latter having shortened endogenous periods, see part 2.2). By transplanting the SCN from a donor to a host animal bearing SCN lesions (SCN-x), they were capable of restoring a circadian rhythmicity. Interestingly, the transplant restored a freerunning rhythm that was characteristic of the period of the donor. Indeed, if the donor was a Tau mutant hamster, the SCN-lesioned wild-type hamster behaved as a Tau mutant. SCN-lesioned Tau mutant in turn, could be converted to a wild-type hamster.

In other words, the restored rhythms always exhibited the period of the donor genotype [START_REF] Ralph | Transplanted suprachiasmatic nucleus determines circadian period[END_REF].

All these findings showed that the SCN is a driving oscillator in the circadian system. The SCN will therefore be referring as a circadian pacemaker, i.e., a selfsustained biological oscillator that can be entrained to external cues and distribute a rhythmic message to the rest of the body.

Light input to the pacemaker

SCN neurons form an endogenous pacemaker sensitive to the daily light-dark cycle which appears to orchestrate a wide range of physiological and behavioral circadian rhythms in mammals. Since terminals of retinal axons were discovered in that location (Moore et al. 1972), a mediating role for the mammalian visual system in photic entrainement has been thus extensively investigated.

The eye is the sole light-sensitive tissue in mammals 12 . The SCN, especially the core region, receives input from the retina through a monosynaptic projection from retinal ganglion cells, the retinohypothalamic tract, which uses glutamate as a main neurotransmitter and is the main carrier of photic information 13 . The retina contains many neurons interconnected by synapses. Three types of neurons, called photoreceptors, are sensitive to light: rods that are primarily used in situations of less intense light and provide black-and-white vision; cones that function best in bright daytime light and are responsible for colour vision; and the intrinsically-photosensitive retinal ganglion cells (ipRGCs), crucial for non-image forming functions, that are presented below.

In 1999, Freedman and colleagues observed that photoentrainment was maintained in mice lacking both rods and cones, thus demonstrating that synchronization of circadian wheel-running activity to the light-dark cycle depends on an additional photoreceptor [START_REF] Freedman | Regulation of mammalian circadian behavior by non-rod, non-cone, ocular photoreceptors[END_REF]. Meanwhile, a group of retinal ganglion cells were shown to contain a sensitive photopigment, called melanopsin that is excited in particular by blue-light. It was also revealed that the anatomical distribution of melanopsin-positive retinal cells was similar to the pattern of cells known to connect with the SCN [START_REF] Provencio | A novel human opsin in the inner retina[END_REF]. Interestingly, the thus named 12 In other vertebrates, light can be perceived by the pineal gland or deep brain photoreceptors. 13 Two addition neural pathways connect to the SCN: an indirect retinal projection from the ventral lateral geniculate nucleus of the thalamus, the geniculo-hypothalamic tract, which uses the neuropeptide Y; and a raphe-hypothalamic pathway that connects raphe nuclei to the SCN and uses 5-HT as neurotransmitter.

ipRGCs were capable of depolarization even when all synaptic inputs from rods and cones were pharmacologically blocked, supporting Freedman's observation. In addition, the spectral sensitivity of ipRGCs and the fact that these cells respond more sluggishly to light than cones and rods, suggested that they could be the primary photoreceptors for photic entrainment of the SCN pacemaker [START_REF] Berson | Phototransduction by retinal ganglion cells that set the circadian clock[END_REF].

This has been further confirmed by Hattar and his colleagues, who showed that axons of ipRGCs directly target the SCN [START_REF] Hattar | Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity[END_REF].

To formally investigate the role of melanopsin in light entrainment of the SCN pacemaker, Panda and colleagues generated mice lacking the melanopsin gene.

These mice entrained normally to a light-dark cycle and did not show altered endogenous period in constant darkness. However, they displayed altered lightinduced phase resetting of the SCN demonstrating that melanopsin is required for normal circadian photoentrainement (Panda et al. 2002). Of interest, mice lacking ipRGCs expressed more deficits in circadian light responses [START_REF] Guler | Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision[END_REF].

Moreover, mice deficient in both melanopsin and classical photoreceptors exhibited an entrainment phenotype comparable to that of enucleated mice [START_REF] Panda | Melanopsin is required for non-image-forming photic responses in blind mice[END_REF]. In short, partial functional redundancy may exist between ipRGCs, rods, and, to a lesser extent, cones to ensure normal photic entrainment (Fig. 13) [START_REF] Paul | The role of retinal photoreceptors in the regulation of circadian rhythms[END_REF][START_REF] Altimus | Rod photoreceptors drive circadian photoentrainment across a wide range of light intensities[END_REF][START_REF] Lall | Distinct contributions of rod, cone, and melanopsin photoreceptors to encoding irradiance[END_REF]. Among the multiple neurotransmitters that can affect SCN cells, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) release from the retinohypothalamic terminals play a crucial role for mediating the circadian actions of light [START_REF] Morse | Time after time: inputs to and outputs from the mammalian circadian oscillators[END_REF][START_REF] Meijer | In search of the pathways for light-induced pacemaker resetting in the suprachiasmatic nucleus[END_REF][START_REF] Hannibal | Roles of PACAP-containing retinal ganglion cells in circadian timing[END_REF]. SCN cells contain receptors sensitive to the peptides, NMDA, PAC2 and VPAC, leading to the activation of signaling pathways that will lead in turn to transcription-transduction and phosphorylation reactions that will influence the SCN's molecular machinery. In this context, neurons located in the ventrolateral part of the SCN have a strong capability of light-induced gene expression. The c-Fos gene, which has DNA-binding properties for transcriptional activation and whose transcription is regulated by phosphorylated CREB [START_REF] Ginty | Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock[END_REF], is proposed as a functional marker of the photic pathway.

Indeed, bright light pulses administrated during the subjective night have been shown

to induce FOS expression in the SCN (Fig. 14) [START_REF] Rea | Light increases Fos-related protein immunoreactivity in the rat suprachiasmatic nuclei[END_REF][START_REF] Kornhauser | Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus[END_REF]). To summarize, for a proper phase-adjustment of the mammalian pacemaker to the external environment, SCN neurons receive direct input from the retina in order to synchronize cellular activities to the 24-h light-dark cycle. In this context, the ipRGCs play an important role to mediate photic entrainment and activate (through glutamate and PACAP releases) multiple protein kinases, immediate early genes such as c-Fos and clock genes (Fig. 15), which will be further presented in the following part.

A journey to the center of the clock

The process of SCN synchronization involves the transcriptional activation of different genes. As outlined above, light-induced cellular changes in calcium and kinase levels within the SCN [START_REF] Motzkus | The human PER1 gene is transcriptionally regulated by multiple signaling pathways[END_REF][START_REF] Hannibal | Roles of PACAP-containing retinal ganglion cells in circadian timing[END_REF]) lead to activation of immediate early genes, such as c-Fos, containing a cAMP responsive element (CRE) in their promoter region (Fig. 15). Interestingly, the Period (Per) clock gene, in particular its two isoforms Per1 and Per2 [START_REF] Albrecht | A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light[END_REF][START_REF] Albrecht | MPer1 and mper2 are essential for normal resetting of the circadian clock[END_REF],

have been shown to be light-responsive and to also possess CRE sequence in their promoter (Travnickova-Bendova et al. 2002). Thus the Per genes play a major role for the establishment of a new circadian phase following a change/perturbation in the light-dark schedule (Fig. 16). Coronal sections of the SCN of diurnal rodents exposed to a light-pulse (100 lux, 1 h) or not (control) during the subjective day (midday or Circadian Time 04) and the early (CT12) and late night period (CT20). Scale bar = 1 mm. Adapted from [START_REF] Caldelas | Circadian profile and photic regulation of clock genes in the suprachiasmatic nucleus of a diurnal mammal Arvicanthis ansorgei[END_REF] However, in the absence of environmental light variations, biological rhythms are still observed in the circadian range, from cellular (e.g. electrical activity of SCN neurons) to behavioral levels (e.g., locomotor activity of an animal). These selfsustained activities take their origin from a clockwork that orchestrates rhythmic gene expression, and thus periodic fluctuations in cellular functions. Indeed, the mammalian molecular basis of circadian oscillations relies on clock genes/proteins, which control their own transcription/translation. Among some of the discovered factors are the essential Period 1-3 (Per1, Per2, Per3), Cryptochrome 1,2 (Cry1,Cry2), circadian locomotor output cycles kaput (Clock) or its analogue neuronal PAS domain protein 2 (Npas2), brain and muscle Arnt-like 1 (Bmal1), reverse viral erythroblastis oncogene products (Rev-erbα and Rev-erbβ) and retinoic acid-related orphan receptors (Rorα, Rorβ, Rorγ) (Ko and Takahashi 2006;[START_REF] Guilding | Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain?[END_REF].

Clock genes are co-expressed (in virtually all tissues) and their products reciprocally interact at the transcriptional/translational levels to generate circadian oscillations. At least three auto-regulatory feedback loops are interconnected (Fig. Npas2, via retinoic acid-related orphan receptor response element (RORE 15 ) (Preitner et al. 2002;Guillaumond et al. 2005;[START_REF] Crumbley | Characterization of the core mammalian clock component, NPAS2, as a REV-ERBalpha/RORalpha target gene[END_REF]Crumbley and Burris 2011). This loop ensures the fine-tuning of circadian rhythms. In addition, posttranslational mechanisms such as protein phosphorylation, affect stabilization, degradation and subcellular localization of clock proteins, thus contributing to the molecular clockwork 16 (Lee et al. 2001; Ko et al. 2006). 14 The consensus sequence for the E-box element is CANNTG (where N = ANY base). 15 The 11pb consensus sequence for the RORE is WAWNTRGGTCA (where W = A or T, N = ANY, R = A or G). 16 This model is continuously completed. Overall, this highly sophisticated clock mechanism takes ~24 h to complete one full oscillation. Thus, each clock component has its acrophase at a specific time of the day 17 (Fig. 18) and mutation/deletion have profound consequences on circadian organization (see part 5.3).

17 Intriguingly, regardless of whether an animal is diurnal or nocturnal in terms of behavior, the circadian pattern of clock gene expression in the SCN is not markedly different. Not to mention the same aspect in SCN electrical activity that occurs during the day period in both diurnal and nocturnal animals.

It is now well-established that the circadian clockwork can function within a single cell. Besides, SCN neurons are circadian pacemakers that can show rhythms in electrical activity when isolated from other neurons (Fig. 19). However, SCN neurons in vivo are part of a network. Hence, in order to be in an exact synchrony, SCN neurons may communicate with each other through gap-junctions [START_REF] Jiang | Tracer and electrical coupling of rat suprachiasmatic nucleus neurons[END_REF][START_REF] Long | Electrical synapses coordinate activity in the suprachiasmatic nucleus[END_REF], synaptic interactions, GABAergic transmission [START_REF] Shirakawa | Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons[END_REF] and neuropeptide release [START_REF] Aton | Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons[END_REF][START_REF] Kim | Deletion of the secretory vesicle proteins IA-2 and IA-2beta disrupts circadian rhythms of cardiovascular and physical activity[END_REF]). As a result of synchronization, the precision of the period of the SCN pacemaker increases as a function of its cells interactions. In other words, the fluctuations in the period of electrical activity decrease as single-cells synchronize to a cellular network, which synchronize further to an entire living organism (Fig. 20). As an aside, it is important to mention that we currently have little knowledge regarding the inter-relationships within the SCN between rhythmic neural electrical activity and rhythmic gene From [START_REF] Herzog | Circadian rhythms in mouse suprachiasmatic nucleus explants on multimicroelectrode plates[END_REF] expression 18 . Despite this fact, it clearly appears that both ionic and molecular mechanisms are in perpetual reciprocal interaction (and can even be phased over time) to produce a functional pacemaker (for review, see Colwell 2011).

In the context of synchronization, the SCN pacemaker uses many output pathways to adjust biological functions to a 24-h schedule. The daily increase in SCN neuron firing rate itself has been assumed to be an output of the circadian system [START_REF] Colwell | Linking neural activity and molecular oscillations in the SCN[END_REF]). It will be show in the following part that the periodic synchronous fluctuations of thousand of neurons are interpreted outside the SCN by other brain regions and peripheral tissues at a specific time of the day.

The mammalian circadian timing system

Up to now, I focused on the SCN pacemaker to introduce the fundamental mechanisms at the basis of circadian oscillations. However, the SCN is not the sole biological entity that can exhibit self-sustained activity, rhythmic clock gene expression, and/or that can distribute a periodic message to the rest of the body. Indeed, the circadian timing system is multi-oscillatory by nature, particularly in 18 In the context of cell-autonomous oscillations and intercellular coupling, the participation of glial cells has to be taken into consideration. [START_REF] Honma | Diversity in the circadian periods of single neurons of the rat suprachiasmatic nucleus depends on nuclear structure and intrinsic period[END_REF] mammals. It is composed of several endogenous oscillators in anatomically discrete locations that form a highly sophisticated circadian network, regulating an amazing array of biological functions. In this context, major brain areas (e.g., hypothalamic nuclei) and peripheral organs (e.g., liver) possess their own clockwork to regulate the transcription of genes and hence local cellular events.

The brain oscillators

The output of the clock machinery consists of changes in neural activity and gene expression associated with neurotransmitter releases. Apart from generating self-sustained circadian clock gene expression, the interlocked positive and negative feedback loops within the SCN are essential for the rhythmic transcriptional control of circadian output genes or clock-controlled genes (CCGs) (Fig. 21).

GABA, glutamate, AVP, VIP, transforming growth factor α (TGFα), prokineticin 2 (PK2), cardiotrophin-like cytokine (CLC), are strong candidates for intrinsic timekeeping signals that travel from the SCN to the rest of the brain [START_REF] Guilding | Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain?[END_REF]). For instance, rhythmic immunoreactivity for the AVP CCGs has been demonstrated in efferent projections of the SCN (van Esseveldt et al. 2000).

Interestingly, as demonstrated by SCN grafts experiments, the humoral release of AVP is important for the restoration of circadian electrical activity in the paraventricular nucleus (PVN) of the hypothalamus in the absence of direct neural connections [START_REF] Tousson | Suprachiasmatic nuclei grafts restore the circadian rhythm in the paraventricular nucleus of the hypothalamus[END_REF]. In addition, AVP release from SCN terminals has been demonstrated to be crucial to control daily variations in corticosterone levels [START_REF] Kalsbeek | Vasopressincontaining neurons of the suprachiasmatic nuclei inhibit corticosterone release[END_REF][START_REF] Kalsbeek | A diurnal rhythm of stimulatory input to the hypothalamo-pituitary-adrenal system as revealed by timed intrahypothalamic administration of the vasopressin V1 antagonist[END_REF]. Another interesting candidate, PK2, has been shown to be expressed on a circadian basis and to be modulated by light exposure [START_REF] Cheng | Regulation of prokineticin 2 expression by light and the circadian clock[END_REF]. Furthermore, the intracerebroventricular injection of PK2 inhibits locomotor activity [START_REF] Cheng | Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus[END_REF].

Recent observations in mice lacking PK2 support its role in the circadian control of locomotor activity [START_REF] Li | Attenuated circadian rhythms in mice lacking the prokineticin 2 gene[END_REF]. Intriguingly, transplantation experiments of isolated SCN tissue have demonstrated that SCN projections are not required for the control of circadian locomotor rhythms, suggesting that a diffusible factor such as PK2 may sustain circadian activity [START_REF] Silver | A diffusible coupling signal from the transplanted suprachiasmatic nucleus controlling circadian locomotor rhythms[END_REF]. However, graft transplantation of SCN failed to restore endocrine rhythms [START_REF] Meyer-Bernstein | Effects of suprachiasmatic transplants on circadian rhythms of neuroendocrine function in golden hamsters[END_REF], showing that the daily SCN output signaling are either accomplished through synaptic connections or rely on nearby tissues that are direct targets of local diffusible factors.

Detailed topography of the SCN efferents has been revealed by studies using anterograde and retrograde tracing techniques (Watts and Swanson 1987;Watts et al. 1987;[START_REF] Kalsbeek | Efferent projections of the suprachiasmatic nucleus in the golden hamster (Mesocricetus auratus)[END_REF]. Within the hypothalamus, the SCN connects with the PVN, dorsomedial nucleus (DMH), ventromedial nucleus, lateral hypothalamic area (LH), arcuate nucleus (ARC) and retrochiasmatic area. SCN efferents can also terminate in the preoptic area, the bed nucleus of the stria terminalis and the lateral septum. The paraventricular nucleus of the thalamus (PVT) is also directly innervated. SCN projections to the habenula and amygdala (AMY) are also considered (Fig. 22) (for reviews, see [START_REF] Kalsbeek | Output pathways of the mammalian suprachiasmatic nucleus: coding circadian time by transmitter selection and specific targeting[END_REF][START_REF] Saper | The hypothalamic integrator for circadian rhythms[END_REF]Dibner et al. 2010). Furthermore, it has been revealed that SCN efferents can indirectly control crucial neuroendocrine rhythms for the distribution of rhythmicity to the entire body, such as those of melatonin. In short, a multisynaptic pathway involving the PVN, the preganglionic sympathetic neurons in the spinal cord and the superior cervical ganglia, allow the SCN to control the rhythmic synthesis of melatonin 19 by the pineal gland [START_REF] Moore | Visual pathways and the central neural control of a circadian rhythm in pineal serotonin N-acetyltransferase activity[END_REF][START_REF] Kalsbeek | SCN outputs and the hypothalamic balance of life[END_REF]).

Figure 22. Direct SCN projections

Efferent pathways from the SCN (red) to hypothalamic (yellow) and thalamic (green) brain regions. AMY, amygdala; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; DMH, dorsomedial hypothalamus; HB, habenula; IGL, intergeniculate leaflet; LS, lateral septum; POA, preoptic area; PVN, paraventricular nucleus of the hypothalamus; PVT, paraventricular nucleus of the thalamus; SCN, suprachiasmatic nuclei; sPVZ, subparaventricular zone. From (Dibner et al. 2010) The discovery of clock genes, amongst other things, uncovered in turn a substantial number of brain structures, outside the SCN, that exhibit circadian oscillations. The retina was the first autonomous oscillator-pacemaker-to be discovered outside the SCN. Cultured retina exhibited circadian rhythms of melatonin synthesis that can be entrained by light cycles; that was free-running in constant darkness; and that was temperature compensated 20 [START_REF] Tosini | Circadian rhythms in cultured mammalian retina[END_REF][START_REF] Tosini | The tau mutation affects temperature compensation of hamster retinal circadian oscillators[END_REF]. The olfactory bulb is one additional brain structure that can also oscillate in a self-sustained manner [START_REF] Granados-Fuentes | A circadian clock in the olfactory bulb controls olfactory responsivity[END_REF]). The habenula (Hb), especially the lateral Hb, may contain an autonomous oscillator as well [START_REF] Zhao | Circadian firing-rate rhythms and light responses of rat habenular nucleus neurons in vivo and in vitro[END_REF][START_REF] Guilding | Circadian oscillators in the epithalamus[END_REF]. A vast number of brain locations have now been identified as extra-SCN oscillators-thus oscillators that do not meet the criteria to be classified as pacemakers. Indeed, more than 20 extra-SCN tissues, such as the hippocampus and the AMY have shown at least one rhythmic cycle of clock components observed at the mRNA or protein level (for an exhaustive list, see [START_REF] Guilding | Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain?[END_REF]).

Circadian rhythms have also been observed by using transgenic Per1luciferase rats. Interestingly, 14 of the 27 brain areas examined expressed Per1 in a rhythmic fashion in vitro [START_REF] Abe | Circadian rhythms in isolated brain regions[END_REF]. Among these structures, the ARC nucleus, the pineal and pituitary glands all expressed rhythms that persisted for several days in vitro, although rhythmicity in some regions damped more rapidly over time than others (Fig. 23). All these findings indicate that the brain contains several circadian (strong or weak) oscillators that regulate multiple biological functions (Fig. 24). However, most oscillators depend on signals derived from the SCN for their cellular activities. In that respect, the SCN delivers circadian time information to the whole body through neuronal (e.g., autonomous nervous system) and humoral routes to fine-tune its control of many brain and peripheral tissues. Self-sustained circadian oscillators are shown shaded in red, semiautonomous oscillators in blue, and slave oscillators in green. AMY, amygdala; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; CB, cerebellum; CX, cortex; DG, dentate gyrus; DMH, dorsomedial hypothalamus; DRN, dorsal raphe nucleus; HB, habenula; Hip, hippocampus; LH, lateral hypothalamus; ME, median eminence; MRN, median raphe nucleus; NAc, nucleus accumbens; NTS, nucleus of the solitary tract; OB, olfactory bulb; OVLT, vascular organ of the lamina terminalis; Pi, piriform cortex; Pin, pineal gland; Pit, pituitary gland; PVN, paraventricular nucleus of the hypothalamus; PVT, paraventricular nucleus of the thalamus; Ret, retina; RVLM, rostral ventrolateral medulla; SCN, suprachiasmatic nuclei; SON, supraoptic nucleus; VLPO, ventrolateral preoptic area; VTA, ventral tegmental area. From (Dibner et al. 2010).

The peripheral oscillators

As aforementioned, several hypothalamic nuclei receive direct neuronal connections from the SCN (Watts et al. 1987;Dibner et al. 2010). Interestingly, these nuclei project in turn to peripheral tissues, such as the liver (la [START_REF] La Fleur | Polysynaptic neural pathways between the hypothalamus, including the suprachiasmatic nucleus, and the liver[END_REF][START_REF] Shibata | Neural regulation of the hepatic circadian rhythm[END_REF]), pancreas [START_REF] Buijs | Parasympathetic and sympathetic control of the pancreas: a role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake[END_REF]) and adipose tissue [START_REF] Kreier | Selective parasympathetic innervation of subcutaneous and intra-abdominal fat--functional implications[END_REF] via autonomic output pathways.

Indeed, the role of the autonomic nervous system is not only illustrated by the sympathetic innervations that permit the SCN to control melatonin release, but also by the multisynaptic pathway that connects the SCN to the adrenal gland. Autonomic projections of the SCN via the PVN to the intermedio-lateral column of the spinal cord to the adrenal contribute to the daily release of the adrenocorticotropic hormone (ACTH) [START_REF] Buijs | Anatomical and functional demonstration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway[END_REF]. This network is highlighted by the finding that light induces (clock) gene expression and corticosterone secretion in/by the adrenal gland via the SCN-sympathetic nervous system-since SCN lesions and transection of a sympathetic nerve route completely suppressed this effect [START_REF] Ishida | Light activates the adrenal gland: timing of gene expression and glucocorticoid release[END_REF].

Virtually all peripheral tissues contain a circadian oscillator, as many express clock genes and can, in culture, show 24-h rhythms in gene expression (see part 4.1.2 for more detailed) [START_REF] Tei | Circadian oscillation of a mammalian homologue of the Drosophila period gene[END_REF]Damiola et al. 2000;[START_REF] Abe | Circadian rhythms in isolated brain regions[END_REF]. As an example, the adrenal gland exhibits rhythmic clock gene expression that is disturbed in clock mutant mice (Oster et al. 2006). In addition, circadian oscillations are also observed in several peripheral tissues by using real-time visualization of circadian expression of mPER2::LUC reporter in vitro (Fig. 25) [START_REF] Yoo | PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues[END_REF]) and in vivo [START_REF] Tahara | In Vivo Monitoring of Peripheral Circadian Clocks in the Mouse[END_REF].

Surprisingly, some tissue explants can sustain circadian cycles that dampen over several days, even when they were derived from SCN-lesioned animals.

However, SCN lesions produced internal phase desynchrony between tissues but not between cells within a given tissue. These observations suggest that the SCN plays a role as a phase coordinator and that, organ-specific synchronizers at the cell and tissue levels may exist to ensure a coupling between cells and thus tissue oscillations for several cycles [START_REF] Yoo | PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues[END_REF]). Nevertheless, this conclusion was not supported by other studies which showed that the circadian oscillations observed in tissue explants did not necessarily imply that cells were synchronized in vivo [START_REF] Yamazaki | Resetting central and peripheral circadian oscillators in transgenic rats[END_REF][START_REF] Stratmann | Properties, entrainment, and physiological functions of mammalian peripheral oscillators[END_REF], and that the SCN central pacemaker is essential to impose phase coherence between oscillating cells within a peripheral Tissues were taken 1 h before light-offset (see arrow and light and dark periods in the bottom left plot). Note that all tissues show circadian oscillations for several days that dampened differently over time. From [START_REF] Yoo | PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues[END_REF] organ [START_REF] Guo | Suprachiasmatic regulation of circadian rhythms of gene expression in hamster peripheral organs: effects of transplanting the pacemaker[END_REF]). In addition, contrary to peripheral oscillators, the coupling between SCN neurons also renders them more resilient to clock gene mutations (Liu et al. 2007).

Taken together, these findings indicate that the SCN, in coordination with the light-dark cycle, is essential to synchronize cell-autonomous oscillators in the brain and peripheral organs, via direct and indirect neural projections as well as releases of humoral factors. However, light is not the unique input or Zeitgeber that can orchestrate rhythmic activities in an oscillator. I will show that non-photic cues such as food can influence the brain (including the SCN) as well as peripheral oscillators (Fig. 26).

Figure 26. Organization of the circadian timing system

The master clock, located in the suprachiamatic nuclei (SCN) of the hypothalamus, adjusts the timing of many secondary clocks/oscillators in the brain and peripheral organs, in part via nervous pathways (dotted red lines). Light perceived by the retina is the most potent synchronizer of the SCN clock (dashed yellow arrow) while mealtime can synchronize peripheral clocks (blue arrows).

Adaptive value of circadian oscillations

Long before humans introduced the concept of time and invented a clock mechanism to better apprehend their own world and organize their diverse activities, probably the first and simplest living organisms on earth adapted to nature's motions by developing a sensitive internal measuring system to evaluate the approximate duration of external events, and the intervals between them, enabling them not to merely passively follow their environment, but on the contrary anticipate changes in an active manner and optimize their own metabolism.

In such a perspective, it is therefore not surprising to observe periodic fluctuations of life functions in a wide variety of species and to identify gene sequences in portions of multiple organisms' DNA that are at the base of a remarkable molecular clock mechanism. The likely ubiquitous nature of the latter can be viewed as the result of evolution. Indeed, organisms having a clock machinery (functional or not) probably share a common ancestry. Biological clocks could represent a vestigial mechanism, providing evidence for evolution. In that respect, chronobiology may in the future unlock some of the mysteries relating to development and evolution.

In any case, it is certain that either the inheritance of the characteristics of a well-adapted ancestral predecessor, or the fact that more than a few creatures on, in and above earth have evolved an internal time-keeping system in parallel, imparted a non-negligible survival advantage allowing them to deal with nature's fickleness, such as the daily variations of sunlight, temperature, water/food availability, as well as greater phenomena such as seasonality, severe cold conditions, etc. Furthermore, the presence of a clock timing system not only improves the ability of a living organism to adapt-metabolically speaking-in a rhythmic environment but considerably enhances its fitness under competitive conditions (e.g., feeding, mating and predation). In this context, the benefit of a clock system is also highlighted by its ability to respond to several stimuli, even in distinctive conditions.

For instance, cavefish have evolved for millions of years in the perpetual darkness of subterranean caves in Somalia and present complete eye degeneration.

Nevertheless, this fascinating animal still retains a circadian molecular clock that can be entrained by feeding time [START_REF] Cavallari | A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception[END_REF].

The benefits of having a clock therefore accrue to all organisms across their diverse kingdoms.

The mysterious food-entrainable oscillator

3.1 Food as a Zeitgeber, first evidence Foraging is fundamental for an animal to survive and can be influenced by several factors such as genetics and learning and memory capacities. Another element that may help animals in their search for food resources is the circadian timing system. Although light is the most prominent Zeitgeber, several non-photic stimuli, such as food, have been also shown to entrain circadian rhythms.

Interestingly, feeding time can act not only on the SCN and peripheral tissues, but also on an oscillator which is not yet well-defined, called the food-entrainable oscillator (FEO), which helps multiple animals in their search for food.

"A primary task of the foraging organism is to exploit spatial and temporal regularities of food availability in the environment." (Mistlberger 1994)

Honey bees

In the early 20 th century, it was already known from von Buttel-Reepen that honey bees were able to learn the time of day when flowers secrete nectar (mentioned in [START_REF] Moore | Honey bee circadian clocks: behavioral control from individual workers to wholecolony rhythms[END_REF]. Meanwhile, the Swiss physician August Forel, when he had breakfast with his family on a terrace each morning, noticed that some forager bees from a hive located not far from his summer home, started to visit his breakfast table and even arrived, after a few days, at the time when breakfast was served. He also noticed that bees still came even if no food was put on the table, suggesting that this daily visit was not related to odor or other stimuli from food (mentioned in [START_REF] Renner | The contribution of the honey bee to the study of time-sense and astronomical orientation[END_REF]. He suggested that "the bees remember the hours at which they had usually found sweets … they have a memory for time (Zeitgedächtnis)".

In 1929, lngeborg Beling, a student of the famous ethologist Karl von Frish, published the first study on the time-sense of bees. Beling observed that honey bees were able to associate the time of day with the presentation of a food reward. By training bees to collect a sugar solution at specific location and during specific periods of time on several consecutive days, she observed that bees continued to return to the sugar source with greatest frequency at 24-h intervals even if sugar was no longer present (Fig. 27); food presented at 19-h or 48-h intervals did not train the bees. In addition, Beling and her colleague Otto Wahl demonstrated a few years later that bees shielded from external factors such as light, temperature and humidity could still be trained to collect nectar and pollen at different times of day 21 . However, von Frish and colleagues did not conclude that time-sense in bees relies on an endogenous clock (mentioned in [START_REF] Moore | Diurnal Changes in the Accuracy of the Honeybee Foraging Rhythm[END_REF][START_REF] Moore | Honey bee circadian clocks: behavioral control from individual workers to wholecolony rhythms[END_REF].

Evidence for the presence of an endogenous biological clock was given a few decades later by Max Renner (1955), in a series of translocation experiments.

Renner trained bees in his laboratory in France to collect sugar water between 8:15 p.m. and 10:15 p.m., and then he transported the bees overnight to New York, where they were placed in similar laboratory conditions. He observed that the following day of departure, bees arrived at the feeding table at the same previous French fixed time, despite having been displaced [START_REF] Renner | The contribution of the honey bee to the study of time-sense and astronomical orientation[END_REF]). Renner's studies give strong evidence that bees are oriented in time with an endogenous clock.

Craving rats

In the early 1920s, Curt Richter would have first observed a phenomenon of circadian food anticipation in rats (mentioned in [START_REF] Davidson | Search for the feeding-entrainable circadian oscillator: a complex proposition[END_REF]. Later, Bolles and colleagues, as well as Edmonds, showed that when food 22 was restricted to a 1-h span, rats exhibited increases in locomotor activity preceding food presentation, even thought food was presented during the light period (when rats are resting) or two times per day [START_REF] Bolles | Rat's anticipation of diurnal and a-diurnal feeding[END_REF][START_REF] Bolles | The rat's anticipation of two meals a day[END_REF][START_REF] Edmonds | Food and light as entrainers of circadian running activity in the rat[END_REF]. In 21 Bees were apparently moved 180 m below the surface of earth, in a salt mine, to exclude cosmic radiation influence. 22 Normocaloric standard chow pellets The bees were trained to come to a sugar source at a fixed time (16 to 18 h) during several consecutive training days. As they visited the sugar they were marked individually. The left graph shows the results on the test day, when sugar was omitted. The bees (individually numbered, see bar graphs) continued to arrive at the feeding location at the same time of the day. From [START_REF] Saunders | Insect Clocks[END_REF] 1979, Friedrich K. Stephan demonstrated that timely controlled food-seeking behaviors did not depend on the SCN pacemaker. He showed that bilateral SCN lesioned rats-thus arrhythmic in locomotor and feeding/drinking behaviors-that were previously fed on an ad libitum (AL) basis and then exposed to a restricted feeding (RF) schedule (i.e., rats received one meal every 12-h intervals) in constant darkness 23 , still displayed food-anticipatory locomotor activity (Fig. 28). Stephan concluded that food-anticipatory activity (FAA) was controlled by an oscillator outside the SCN (Stephan et al. 1979;Stephan et al. 1979;[START_REF] Stephan | Circadian-Rhythm Dissociation Induced by Periodic Feeding in Rats with Suprachiasmatic Lesions[END_REF]).

Food anticipation in nature

Of note, other species such as fishes [START_REF] Lopez-Olmeda | Feeding entrainment of food-anticipatory activity and per1 expression in the brain and liver of zebrafish under different lighting and feeding conditions[END_REF], birds, mice, hamsters, rabbits 24 [START_REF] Caba | The rabbit pup, a natural model of nursing-anticipatory activity[END_REF], golden shiners [START_REF] Lagud | Food-anticipatory activity of groups of golden shiners during both day and night[END_REF], marsupials and primates [START_REF] Boulos | Effects of restricted feeding schedules on circadian organization in squirrel monkeys[END_REF]) can all show abrupt foodseeking behaviors prior to feeding time (for a review, see Mistlberger 1994). As aforementioned, even cavefish that have evolved in dark subterranean caves can show FAA in response to periodic food availability (Fig. 29) [START_REF] Cavallari | A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception[END_REF]).

The circadian characteristics of the food-entrainable oscillator

As demonstrated above, there is much evidence of an extra-SCN oscillator sensitive to feeding time that can control circadian behavioral activity. This oscillator was the object of much attention in the early 80s, particularly in rats. Numerous studies characterized the properties of the so-called FEO.

Transient cycles and persistence

Stephan in the early 80s observed that SCN-lesioned rats 25 exposed to a 24-h interval RF schedule exhibited after phase shifts of mealtime, transient cycles that are characteristics of photic entrainment (Fig. 30) [START_REF] Stephan | Phase shifts of circadian rhythms in activity entrained to food access[END_REF]. Meanwhile, other groups demonstrated that rats previously fed at a fixed time of the day, could show after being in AL conditions a more rapid entrainment when reexposed to RF, and could also exhibit bursts of activity associated with the former mealtime when food deprived. In other words, a previous experience of a scheduled RF facilitates the (re)appearance of FAA in response to food deprivation (Fig. 31) [START_REF] Coleman | Evidence for a separate mealassociated oscillator in the rat[END_REF][START_REF] Stephan | Resetting of a feeding-entrainable circadian clock in the rat[END_REF]Mistlberger 1994). Of note, FAA can persist at its usual phase 25 As an aside, the study of FAA is facilitated in SCN-X rats, since the circadian control of locomotor activity by the SCN pacemaker is eliminated, making it easier to assess the FEO output under restricted feeding challenge.

Figure 29. Food-anticipatory activity in fishes

Representative actograms of zebrafish (A) and cavefish (B) maintained under constant darkness and fed once a day at a fixed time (ZT = 0). The mean waveforms of zebrafish and cavefish are represented below the actograms.

Each point in the mean waveform has been calculated as the mean 6 SEM from 10 min binned data across all the experimental days (n = 30) shown on each actogram and all experimental aquaria (n = 5 for zebrafish and n = 3 for cavefish).

From [START_REF] Cavallari | A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception[END_REF] for a few days in some food-restricted animals that are placed back in AL conditions. However, in most cases, persistent activity is not observed following a day of AL access. On the other hand, FAA can reappear at its usual period even after repeated periods of AL access interrupted by food deprivation, or even 50 days after the last RF schedule [START_REF] Clarke | Persistent meal-associated rhythms in SCN-lesioned rats[END_REF]Mistlberger 1994). Thus, this suggests that the FEO rapidly uncouple from (circadian) effectors that control (at least) locomotor activity, rather than dampen over time (Mistlberger 1994). Note that strong FAA appeared ~2-3 h prior to mealtime immediately few days after RF. Following phase delays of food access (e.g., -4 h or -8 h), clear transient cycles in activity are observed. Following phase advances, however, this rat did not show clear transients in activity. From [START_REF] Stephan | Phase shifts of circadian rhythms in activity entrained to food access[END_REF])

Feeding behavior and entrainment limits

Interestingly, rats both with and without SCN lesions are not capable of anticipating feedings at 18-h intervals (Stephan et al. 1979). Further experiments demonstrated that FAA is expressed only if food access is given under a circadian range. As a comparison, anticipatory wheel-running activity in SCN-ablated rats could not be reliably observed at 22-h intervals or shorter, and at 31-h intervals and longer (Fig. 32) [START_REF] Boulos | Feeding schedules and the circadian organization of behavior in the rat[END_REF][START_REF] Stephan | Limits of Entrainment to Periodic Feeding in Rats with Suprachiasmatic Lesions[END_REF].

Figure 32. Limits of food entrainment in SCN-X rats

Rats bearing SCN lesions were maintained in constant darkness and exposed to RF at short interval periods (i.e., 21, 20, 22 and 23-h intervals between food access; left figures) and long interval periods (i.e., 27, 29, 31, 33-h intervals; right figures). Note that rats entrained to the 24-h feeding schedule and can show consistent entrainment to schedules between 23 and 29 h. Black lines indicate food access and number on the left indicate the period of the feeding schedule. From [START_REF] Stephan | Limits of Entrainment to Periodic Feeding in Rats with Suprachiasmatic Lesions[END_REF] The circadian limit of entrainment and the fact that FAA onset occurs regularly with a positive phase angle (i.e., a few hours before mealtime, see Fig. 31), especially for long intervals, may suggest that the mechanism at the base of the FEO is relatively supple and have a memory of phase displacement. The latter can be also supported by the post-feeding activity that can be seen in some animals (Fig. 32, left actograms, 24-h and 21-h intervals). The study of the free-running capacity of this putative FEO is obviously limited by the deleterious effect of prolonged food deprivation. These experiments are nevertheless informative of the circadian nature of the FEO.

Conclusion on FEO's properties

The demonstration of a circadian range of entrainment, split transients and strong phase-specific persistence suggests that FAA is not simply the result of associative learning, classical conditioning or hourglass mechanism 26 . On the contrary, the above FAA characteristics are indicative of an oscillator that possesses a flexible intrinsic mechanism and that probably has the ability to retain or "remember" a circadian phase-likely associated with the memory of a specific location, as observation of food anticipation in bees supports this thought 27 .

All these findings thus confirm the existence of one (or multiple) oscillator(s) that drive FAA under restricted food access and that is anatomically independent of the SCN.

Food anticipation, outputs and inputs

The multiple aspects of the food-entrained physiology

In addition to general and wheel-running lomocotor activities, other behavioral/physiological parameters appeared to be controlled by the FEO as well.

Not surprisingly, drinking behavior can be synchronized to the periodic food access, increasing (i.e., number of licks) a few hours before mealtime or being maintained during food deprivation [START_REF] Boulos | Feeding schedules and the circadian organization of behavior in the rat[END_REF][START_REF] Clarke | Persistent meal-associated rhythms in SCN-lesioned rats[END_REF]. FAA can also be observed by assessing lever pressing behavior [START_REF] Boulos | Feeding schedules and the circadian organization of behavior in the rat[END_REF] or by evaluating food-bin approaches (Mistlberger 1994).

Concomitant to an increase of locomotor activity, a rise of body temperature can also be measured in normal or SCN-X rats prior to feeding time. In addition to this food-anticipatory thermogenesis (FAT), a diet-induced thermogenesis (DIT) is observed as well (Fig. 33) [START_REF] Krieger | Food and water restriction shifts corticosterone, temperature, activity and brain amine periodicity[END_REF]Nelson et al. 1975;[START_REF] Challet | Phase-advanced daily rhythms of melatonin, body temperature, and locomotor activity in food-restricted rats fed during daytime[END_REF]). The anticipatory rise in temperature, regardless of its association with physical activity, can be viewed as an adaptive mechanism that prepares an organism to ingest food efficiently. Moreover, DIT is related to the cost of digestion, absorption and nutrient processing and is as well a consequence of enhanced brown-adipose tissue (BAT) thermogenesis due to excessive food consumption during a short time window. On the other hand, the decrease in body temperature before FAT can reflect the economizing strategy to save energy during food deprivation and to optimize foodseeking behaviors at a specific time of the day (Fig. 33).

Figure 33. Rise in body temperature before mealtime in control rats

Mean core body temperature during 2 days of restricted feeding. The dashed rectangle indicates food access time. FAT: food-anticipatory thermogenesis; DIT: diet-induced thermogenesis. Adapted from [START_REF] Davidson | Feeding-entrained circadian rhythms are attenuated by lesions of the parabrachial region in rats[END_REF] Corticosterone is another physiological parameter that shows a drastic elevation in anticipation to food arrival [START_REF] Krieger | Food and water restriction shifts corticosterone, temperature, activity and brain amine periodicity[END_REF]Nelson et al. 1975). In nocturnal animals fed on AL basis, a regular peak of corticosterone occurs at the day-night transition and is controlled by the SCN pacemaker (Fig. 43). However, if food is restricted to a temporal window during the day, a second peak of corticosterone is observable before feeding time (Fig. 34). Of note, this RF-induced peak is not related to a possible stress induced by a food deprivation, since neither 24-h ACTH levels nor 24-h adrenal and plasma corticosterone levels were higher in food-restricted animals [START_REF] Wilkinson | Daily rhythms in adrenal responsiveness to adrenocorticotropin are determined primarily by the time of feeding in the rat[END_REF]). In addition, rats or mice that were stressed by tube restraint for 2 h at the same time for several days do not develop an anticipatory corticosterone peak prior to stress-time [START_REF] Ottenweller | Repeated stress at the same time of day does not mimic timed feeding in its effects on the plasma corticosterone rhythm in rats[END_REF] nor show a phase advance of circadian activity rhythm [START_REF] Challet | Entrainment in calorie-restricted mice: conflicting zeitgebers and free-running conditions[END_REF]). Thus, these findings suggest that the anticipatory acrophase of corticosterone induced by feeding time is likely controlled by a FEO.

Finally, other metabolic variables, such as leptin and ghrelin rhythms can be phase-shifted in response to restricted day feeding (Fig. 35) [START_REF] Bodosi | Rhythms of ghrelin, leptin, and sleep in rats: effects of the normal diurnal cycle, restricted feeding, and sleep deprivation[END_REF][START_REF] Martinez-Merlos | Dissociation between adipose tissue signals, behavior and the foodentrained oscillator[END_REF]). Both leptin and free fatty acids are entrained by a 2-h RF schedule. In particular, leptin and free fatty acids levels increase after and before mealtime, respectively, and can persist during food deprivation. However, i.c.v. administration of leptin failed to elicit FAA, suggesting that leptin rise is likely a direct response to food intake. On the other hand, the elevation of free fatty acids before mealtime could reflect mobilization of energy substrates to sustain FAA [START_REF] Martinez-Merlos | Dissociation between adipose tissue signals, behavior and the foodentrained oscillator[END_REF]). Parameters such as glucagon, insulin, and cholesterol, can also be affected by RF [START_REF] Velasco | Entrainment of Circadian-Rhythms of Cholesterol-Ldl, Corticosterone and Motor-Activity by Feeding Schedules in Rats[END_REF][START_REF] Diaz-Munoz | Anticipatory changes in liver metabolism and entrainment of insulin, glucagon, and corticosterone in food-restricted rats[END_REF][START_REF] Martinez-Merlos | Dissociation between adipose tissue signals, behavior and the foodentrained oscillator[END_REF]). However, further investigations are needed to ensure that these metabolic actors are controlled by the FEO in the absence of a circadian control from the SCN pacemaker, and are not only a response to a low metabolic state.

Figure 34. Rise in plasma corticosterone before mealtime in control rats

Mean plasma corticosterone levels in male and female rats under RF schedule. Food access is indicated by white rectangles along the X-axis. Note that both male and female rats showed a peak of corticosterone before food access. Adapted from [START_REF] Krieger | Food and water restriction shifts corticosterone, temperature, activity and brain amine periodicity[END_REF])

Figure 35. Effects of feeding restricted to the light cycle on the diurnal rhythms of leptin and ghrelin in rats

In rats with feeding restricted to the 12-h light period, phase-shift of plasma leptin (Pl Lep) and plasma ghrelin (Pl Ghre) can be observed. Hypothalamic ghrelin contents (HY Ghre) was not significantly affected. Filled symbols, RF rats; open symbols, freefeeding rats. Gray column, dark period. Adapted from [START_REF] Bodosi | Rhythms of ghrelin, leptin, and sleep in rats: effects of the normal diurnal cycle, restricted feeding, and sleep deprivation[END_REF]

Will run for almost anything

As outlined above, standard food provided only for a few hours per day can elicit behavioral and physiological activation, especially in rats. A temporal restriction protocol can be replaced by a hypocaloric feeding protocol, in which a limited amount of food is given at a fixed time point (i.e., usually 50% of the AL daily intake). In this procedure as well, food anticipation is observed in SCN intact rats [START_REF] Challet | Phase-advanced daily rhythms of melatonin, body temperature, and locomotor activity in food-restricted rats fed during daytime[END_REF]. In addition, it has been mentioned that the anticipation of two mealtimes can be observed in rats [START_REF] Mistlberger | Circadian clocks for all meal-times: anticipation of 2 daily meals in rats[END_REF]. Of note, if three meals are given for instance at 8-h intervals (thus three times per day), FAA is only observed for two out of the three meals and sometimes FAA jumps or shifts (with transient cycles) to another feeding time (Mistlberger 1994). This observation gives support to the control of FAA by the circadian system and not (only) by a memory/learning system.

Besides normocaloric food, access to salt solutions or salty food for 2 h each day failed to induce FAA [START_REF] Rosenwasser | Circadian wheel-running activity of rats under schedules of limited daily access to salt[END_REF]. Similar results have been shown in rats that had access to a daily meal of protein, carbohydrate or fat, plus a free access to a complementary diet. Conversely, rats limited to two daily singlemacronutrient meals (i.e., associated protein-fat, or protein-carbohydrate meals) can exhibit FAA [START_REF] Mistlberger | Food-anticipatory rhythms under 24hour schedules of limited access to single macronutrients[END_REF]). Thus, the energetic content of a meal appears to be crucial to entrain the FEO. This view is partially confirmed by a study demonstrating that glucose is sufficient to affect the circadian properties (i.e., elicit transients after phase-shift of feeding time) of the FEO (Stephan and Davidson 1998). Of note, gastric distention does not seem necessary to synchronize the FEO [START_REF] Stephan | Calories affect zeitgeber properties of the feeding entrained circadian oscillator[END_REF].

Palatable but nonnutritive meals, such as saccharin, do not arouse the FEO, excluding taste as a main factor of entrainment [START_REF] Mistlberger | Palatable daily meals entrain anticipatory activity rhythms in free-feeding rats: dependence on meal size and nutrient content[END_REF]. Brief access to sucrose solution produces behavioral anticipation only after food deprivation in rats [START_REF] Pecoraro | Brief access to sucrose engages foodentrainable rhythms in food-deprived rats[END_REF][START_REF] Waddington Lamont | Restricted access to food, but not sucrose, saccharine, or salt, synchronizes the expression of Period2 protein in the limbic forebrain[END_REF]). Other attractive snacks, such as chocolate provided once a day at the same time without food deprivation, can induce FAA (Fig. 36) (Mendoza et al. 2005). However, this is not observed if the chocolate has no nutritional value, again confirming the importance of food energy content [START_REF] Mistlberger | Palatable daily meals entrain anticipatory activity rhythms in free-feeding rats: dependence on meal size and nutrient content[END_REF]. On the other hand, in hamsters, temporally limited daily access to chocolate can elicit FAA only if the SCN is ablated or if the animals have limited access (i.e., 70%) to their standard food [START_REF] Abe | Anticipatory activity and entrainment of circadian rhythms in Syrian hamsters exposed to restricted palatable diets[END_REF]. In free-fed mice, however, a palatable chocolate snack failed to induce food anticipation, even if mice avidly consumed the snack and even if some related food-seeking behaviors can be detected [START_REF] Hsu | Palatable meal anticipation in mice[END_REF]. Thus, the ability of the circadian system, in particular the FEO, to respond to palatable food, may depend on the motivational and metabolic states specific to each species, in addition to the nutritive aspects of the food.

From all these findings, we have seen that FAA can be induced by temporal restriction, hypocaloric feeding and to some extent by a daily palatable meal. Hence, the role of brain reward pathways in anticipatory behavior in rats can be highly considered. In addition, some addictive substances such as methamphetamine (MAP) have been demonstrated to act on the circadian timing system. In particular, daily injections of MAP have been shown to induce behavioral activation a few hours before the time of injection that can persist on the day of withdrawal [START_REF] Shibata | Aging impairs methamphetamineinduced free-running and anticipatory locomotor activity rhythms in rats[END_REF][START_REF] Iijima | Methamphetamine-induced, suprachiasmatic nucleus-independent circadian rhythms of activity and mPer gene expression in the striatum of the mouse[END_REF]. In addition, in SCN-X rats and mice, continuous MAP exposure can restore a locomotor rhythm in the circadian range [START_REF] Honma | Activity rhythms in the circadian domain appear in suprachiasmatic nuclei lesioned rats given methamphetamine[END_REF], while in intact animals exposed to constant dark conditions, MAP lengthens the freerunning period [START_REF] Tataroglu | The methamphetaminesensitive circadian oscillator (MASCO) in mice[END_REF]. Of interest, the free-running activity induced by MAP can be entrained by a RF schedule in SCN-x rats. However, no FAA is detectable in this experimental design (Fig. 37) [START_REF] Honma | Methamphetamine Induced Locomotor Rhythm Entrains to Restricted Daily Feeding in Scn Lesioned Rats[END_REF]. The intriguing effect of MAP suggests that a methamphetamine-sensitive oscillator (MASCO), also independent of the SCN, can control behavioral rhythmicity. However, it is not excluded that both MAP and food can act on the same circadian system, a unique oscillator (originally the FEO), or more likely, a multiple oscillator system (at least FEO + MASCO). As an aside, this system could be tightly associated with brain regions that form reward pathways and could rely on close molecular feedback loops that generate circadian oscillations in the SCN and extra-SCN tissues as will be further discussed.

When restricted feeding affects the circadian timing system

Usually, locomotor activity, body temperature, fatty acid synthesis [START_REF] Hems | Fatty acid synthesis in liver and adipose tissue of normal and genetically obese (ob/ob) mice during the 24-hour cycle[END_REF], or expression of specific genes (see below), start to increase at the beginning of the light-dark transition, provided that free-fed nocturnal animals are synchronized to a 12:12 light-dark cycle. Conversely, after having been acclimatized to a circadian feeding schedule, many animals' body functions adapt to be in phase with feeding time. Triple plotted actographs of SCN lesioned rats before, during and after the RF schedule. Note that rats were also blinded during the procedure (black arrow heads). Parallel vertical lines in the actograph indicate (left) methamphetamine treatment and (right) RF schedule: RF 10-14 means the food presentation from 10 to 14 h and RF 10-16 from 10 to 16 h.

Note that no FAA can be detected during the RF protocol. Note also that after termination of methamphetamine treatment, animals were de novo arrhythmic.

From [START_REF] Honma | Methamphetamine Induced Locomotor Rhythm Entrains to Restricted Daily Feeding in Scn Lesioned Rats[END_REF]

Peripheral tissues

In response to RF, the stomach has been shown to increase its capacity for distension [START_REF] Martinez-Merlos | Dissociation between adipose tissue signals, behavior and the foodentrained oscillator[END_REF]). Disaccharidase and even mitotic activities showed rhythmic changes in the gastro-intestinal system with higher levels around the feeding time [START_REF] Nishida | Parallel between Circadian-Rhythms of Intestinal Disaccharidases and Food-Intake of Rats under Constant Lighting Conditions[END_REF][START_REF] Scheving | Chronobiology of the Intestinal-Tract of the Mouse[END_REF]. Moreover, clock gene oscillations in peripheral tissues can be shifted by feeding time and thus be uncoupled from the circadian control of the central SCN pacemaker (Damiola et al. 2000;Stokkan et al. 2001;Feillet et al. 2006). In particular, the liver (Fig. 38), kidney, heart and pancreas, showed gradual phase resetting of rhythmic gene expression in response to daytime feeding, similar to a change in photoperiod, indicative of the involvement of a clock-dependent mechanism (Damiola et al. 2000). Moreover, the liver oscillator, important for glucose and lipid homeostasis, has been demonstrated to exhibit significant metabolic phase adjustments and increased levels of metabolic genes expression in response to food restriction [START_REF] Lima | Metabolic Consequences of Food Restriction in Rats[END_REF][START_REF] Baez-Ruiz | The food entrainable oscillator studied by DNA microarrays: What is the liver doing during food anticipatory activity?[END_REF]. The fact that many physiological, metabolic and endocrine events (e.g., temperature, corticosterone changes as outlined above) are affected by a RF schedule suggests that some peripheral signals may serve to regulate, or even entrain the circadian functioning of other organs and tissues that communicate with (or represent) the FEO.

Figure 38. Circadian accumulation of clock and clock-controlled genes in the liver

Daytime feeding changes the phase of circadian gene expression in the liver. Mice, kept under a lightdark regimen (lights on 6 a.m., lights off 6 p.m.), were fed exclusively during the light phase (6 a.m. to 6 p.m.) or during the dark phase (6 p.m. to 6 a.m.). From (Damiola et al. 2000) In this context, glucocorticoids are good candidates. Indeed, the glucocorticoid hormone analog dexamethasone has been shown to induce circadian gene expression in cultured fibroblasts and to transiently change the phase of clock gene expression in the liver, kidney, and heart [START_REF] Balsalobre | Resetting of circadian time in peripheral tissues by glucocorticoid signaling[END_REF]. Besides, glucocorticoids can feed back to the central nervous system to modulate brain oscillators [START_REF] Sage | Influence of the corticosterone rhythm on photic entrainment of locomotor activity in rats[END_REF][START_REF] Malek | Daily rhythm of tryptophan hydroxylase-2 messenger ribonucleic acid within raphe neurons is induced by corticoid daily surge and modulated by enhanced locomotor activity[END_REF]). The nutrient-responsive adenosine monophosphate-activated protein kinase (AMPK) is another candidate to inform the core clock machinery of metabolic changes (see part 4.4.2) [START_REF] Lamia | AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation[END_REF]).

Thus, feeding schedule has strong Zeitgeber properties on peripheral tissues, as evidenced by the uncoupling of peripheral oscillators from the SCN. Interestingly, peripheral organs connect directly through nervous pathways to the brain and can release multiple metabolic signals that can affect the core clock machinery.

Brain nuclei

Contrary to peripheral oscillators, the SCN pacemaker is relatively impervious to the synchronizing effect of mealtime, provided that the animals are exposed to a light-dark cycle and ingest enough daily energy. Under severe food restriction (i.e., caloric restriction), however, the phase of the SCN and its synchronization to light are modified [START_REF] Challet | Interactions between light, mealtime and calorie restriction to control daily timing in mammals[END_REF]. Different studies have measured c-FOS expression, as a marker of neuronal activity [START_REF] Dragunow | The Use of C-Fos as a Metabolic Marker in Neuronal Pathway Tracing[END_REF] in different brain areas, to assess their responses to a scheduled feeding. In food-entrained rats, increased c-FOS immunoreactivity before and after mealtimes have been observed in the DMH, LH, perifornical area, and only after feeding in the PVN (Angeles-Castellanos et al. 2004;Gooley et al. 2006). In hypothalamic regions such as the LH, DMH and perifornical area, c-FOS expression has even been shown to persist for a few days when food was not provided at the expected time (Angeles-Castellanos et al. 2004).

Several additional structures such as the BNST, lateral septum, nucleus accumbens (NAc), AMY, prefrontal cortex and PVT, showed up-regulated c-FOS expression in anticipation to food access (Fig. 39) [START_REF] Angeles-Castellanos | Restricted feeding schedules phase shift daily rhythms of c-Fos and protein Per1 immunoreactivity in corticolimbic regions in rats[END_REF]).

Besides, dopamine release is up-regulated in advance of a scheduled mealtime in the NAc [START_REF] Hsu | Palatable meal anticipation in mice[END_REF]). In addition, the neuropeptide Y (NPY) and agouti-related peptide (AgRP) has been shown to increase prior to mealtime in, for instance, the ARC nucleus, the PVN but not the DMH [START_REF] Yoshihara | Effects of restricted daily feeding on neuropeptide Y release in the rat paraventricular nucleus[END_REF][START_REF] Crowley | Differential effects of methamphetamine on expression of neuropeptide Y mRNA in hypothalamus and on serum leptin and ghrelin concentrations in ad libitum-fed and schedule-fed rats[END_REF]. PER2 rhythms are also sensitive to scheduled feeding in limbic forebrain areas [START_REF] Waddington Lamont | Restricted access to food, but not sucrose, saccharine, or salt, synchronizes the expression of Period2 protein in the limbic forebrain[END_REF]. Moreover, a phase-shift of mPer1 and mPer2 expression is observed in mice fed only for 4 h during the daytime in the cerebral cortex and hippocampus, as well as moderately in the striatum, piriform cortex and PVN, and without effect in the SCN [START_REF] Wakamatsu | Restrictedfeeding-induced anticipatory activity rhythm is associated with a phase-shift of the expression of mPer1 and mPer2 mRNA in the cerebral cortex and hippocampus but not in the suprachiasmatic nucleus of mice[END_REF]. Hypocaloric feeding has also been shown to affect PER1 and PER2 expression in multiple brain nuclei (e.g., DMH and ARC) (Feillet et al. 2008). These studies support the view that the FEO is probably not a unique structure, but involves several hypothalamic and corticolimbic brain regions.

Figure 39. Temporal patterns of c-FOS immunoreactivity in corticolimbic structures

Food-entrained rats (○) and their ad libitum controls AL (•). Significant cellular activation can be observed in all structures during the time when rats are anticipating food access and after mealtime. The hippocampus (F) is the only structure that does not show cellular activation associated with FAA and feeding. The horizontal bar on the abscissa represents food access. Values are mean±S.E. * Significant difference between AL and RF group obtained with the Tukey post hoc test (P<0.01), α for the AL group between peak and low time points, and δ for RF. From [START_REF] Angeles-Castellanos | Restricted feeding schedules phase shift daily rhythms of c-Fos and protein Per1 immunoreactivity in corticolimbic regions in rats[END_REF] It is worth noting that subdivisions of specific brain nuclei, such as the compact and ventral parts of the DMH, do not exhibit the same degree of response to RF in mice. In particular, mPer1 mRNA expression was strongly induced after feeding in the compact part of the DMH, while no significant rhythmicity was detected in AL conditions [START_REF] Mieda | The dorsomedial hypothalamic nucleus as a putative food-entrainable circadian pacemaker[END_REF]). In addition, the PER2 rhythm has been shown in some limbic forebrain structures to be dependent on adrenal integrity [START_REF] Lamont | The central and basolateral nuclei of the amygdala exhibit opposite diurnal rhythms of expression of the clock protein Period2[END_REF], suggesting that corticosterone levels during a RF-schedule can act downstream of the SCN to synchronize the activity of structures, probably important to anticipate food access.

In addition to the synchronizing properties of RF, palatable meals also have an entraining effect on extra-SCN oscillators. Indeed, in rats with unrestricted standard food access, c-FOS and PER1 expression were shown to be in phase with a daily palatable meal in the PVT and limbic structures (Mendoza et al. 2005;Mendoza et al. 2005;[START_REF] Angeles-Castellanos | Expectancy for food or expectancy for chocolate reveals timing systems for metabolism and reward[END_REF]). However, no effects are observed on PER2 levels in the limbic system in response to a daily access of sucrose solution in freely fed rats [START_REF] Waddington Lamont | Restricted access to food, but not sucrose, saccharine, or salt, synchronizes the expression of Period2 protein in the limbic forebrain[END_REF]). This was observed despite the efficiency of a sucrose solution in eliciting dopamine release in the NAc [START_REF] Bassareo | Differential responsiveness of dopamine transmission to foodstimuli in nucleus accumbens shell/core compartments[END_REF][START_REF] Rada | Daily bingeing on sugar repeatedly releases dopamine in the accumbens shell[END_REF]. Taken together, these results show that some "hedonic" feeding signals can affect gene expression. However, the effects appear rather limited since the expression pattern of additional clock components is lacking.

Therefore, the accurate synchronizing properties of a palatable meal on the core clock machinery remains to be further investigated.

To summarize, the results cited above give evidence that feeding synchronization-and the negative metabolic state associated with food restrictionimplies phase-adjustment of metabolic processes and gene expression in peripheral and brain tissues. As a result, many organs could likely play an essential role in food anticipation. Thus, the mammalian body is being carefully explored with a "fine-tooth comb" to delineate the FEO substrate(s) and its intrinsic core clock mechanism.

The food-entrainable oscillator, a needle in a haystack

As indicated above, the relevance of peripheral oscillators as well as brain nuclei for the generation of food-entrainable oscillations seems undeniable.

Nonetheless, very little is known as of yet regarding the location and the exact molecular basis on which the FEO relies. The identification of the neural substrate and the molecular basis of the so-called FEO have indeed proved to be a real challenge these last 30 years. Different approaches have been used, but neither anatomical nor molecular approaches have really succeeded in elucidating the neural substrate or the precise molecular functioning of this mysterious oscillator.

Lesional and pharmacological approaches

Subdiaphragmatic vagotomy was used in one study to remove afferents from the periphery to the brain and severely impact the normal function of the gastrointestinal tract. In this study, there was no change in the amount of FAA [START_REF] Comperatore | Effects of vagotomy on entrainment of activity rhythms to food access[END_REF]. Notably, the vagus nerve does not seem necessary for the ghrelin reponse to nutrients [START_REF] Williams | Vagotomy dissociates short-and long-term controls of circulating ghrelin[END_REF]. To further investigate neural routes of communication between the periphery and the central nervous system, capsaicin-induced visceral deafferentation was performed in rats bearing SCN lesions [START_REF] Davidson | Circadian food anticipation persists in capsaicin deafferented rats[END_REF]. However, as for vagal transection, capsaicin deafferentation did not prevent FAA, thus questioning the implication of neural pathways. The role of the liver oscillator in response to food restriction has also been investigated in rats rendered cirrhotic with carbon tetrachloride. These rats displayed, however, similar FAA and RF-entrained free fatty acids levels compared to normal rats [START_REF] Escobar | Rats made cirrhotic by chronic CCl(4) treatment still exhibit anticipatory activity to a restricted feeding schedule[END_REF][START_REF] Escobar | Metabolic parameters are food-entrained in rats made cirrhotic by chronic CCl4 treatment[END_REF].

Nonetheless, abnormalities in liver functioning were not described in these studies.

Of note, adrenalectomy did not block FAA either (Stephan et al. 1979). This result opposes to the highlighted role of corticosterone in the resetting of the circadian system, and can suggest that corticosterone signaling is somewhat dispensable for food entrainment. Of great interest, a study has investigated the role of the digestive system in the food-entrained oscillations that underlie FAA. The authors entrained rats to dual RF (i.e., two meals per day) and observed that FAA occurred for both meals. However, PER1-LUC rhythmicity in the digestive system (including liver, esophagus, stomach and colon) showed entrainment only to one-meal, dispelling the hypothesis that FAA arises as a unique output of rhythms in the gastrointestinal system. Thus food anticipation is likely an output of a circadian FEO, likely located in the brain (Davidson et al. 2003).

On the other hand, brain regions can detect blood-borne feedback signals that can be indicative of perturbations in energy balance, such as diminished circulating metabolic fuels (e.g., glucose) or high food intake in a short time-period. In that context, lesion or induced-degeneration of brain structures essential for relaying circulating signal from the periphery, such as the area postrema 28 and the ARC nucleus 29 , failed to affect FAA [START_REF] Mistlberger | Neonatal monosodium glutamate alters circadian organization of feeding, food anticipatory activity and photic masking in the rat[END_REF][START_REF] Davidson | Persistence of meal-entrained circadian rhythms following area postrema lesions in the rat[END_REF]). On the contrary, lesion of the parabrachial nuclei, which receive information from visceral and gustatory afferents and direct inputs from the area postrema and the nucleus of the solitary tract (NTS), altered food-anticipatory bin approaches and slightly attenuated FAT [START_REF] Davidson | Feeding-entrained circadian rhythms are attenuated by lesions of the parabrachial region in rats[END_REF]. In one study, destruction of the ventromedial nucleus have also been found to affect FAA [START_REF] Inouye | Ventromedial Hypothalamic-Lesions Eliminate Anticipatory Activities of Restricted Daily Feeding Schedules in the Rat[END_REF], but another study points to the importance of a sufficient time for recovery after lesion and the evaluation of food entrainment [START_REF] Mistlberger | Recovery of Anticipatory Activity to Restricted Feeding in Rats with Ventromedial Hypothalamic-Lesions[END_REF]. A lesional approach focusing on the paraventricular and lateral hypothalamic areas also failed to abolish FAA or mealtime associated food-bin approaches [START_REF] Mistlberger | Food-Anticipatory Circadian-Rhythms in Rats with Paraventricular and Lateral Hypothalamic Ablations[END_REF]. The pharmacological depletion of orexin neurons, essential for regulation of arousal and appetite, induced hypophagia and weight loss but did not affect anticipation of a daily meal [START_REF] Mistlberger | Food-and light-entrained circadian rhythms in rats with hypocretin-2-saporin ablations of the lateral hypothalamus[END_REF]. Rats bearing large lesions of the AMY, hippocampus or NAc were able to anticipate meal timing [START_REF] Mistlberger | The Limbic System and Food-Anticipatory Circadian-Rhythms in the Rat -Ablation and Dopamine Blocking Studies[END_REF]. Of note, specific ablation of the shell part of the NAc had a positive effect on FAA (Mendoza et al. 2005). Treatment with the dopamine antagonist haloperidol did not affected foodentrained rhythms in intact rats either [START_REF] Mistlberger | The Limbic System and Food-Anticipatory Circadian-Rhythms in the Rat -Ablation and Dopamine Blocking Studies[END_REF]). However, 6hydroxydopamine, a neurotoxin that selectively kills dopaminergic and noradrenergic neurons, injected into the PVN did eliminate the anticipatory rise of corticosterone [START_REF] Honma | Roles of Paraventricular Catecholamines in Feeding-Associated Corticosterone Rhythm in Rats[END_REF]. Lesion of the PVT appeared to change the phase of the foodanticipatory corticosterone peak [START_REF] Nakahara | Involvement of thalamic paraventricular nucleus in the anticipatory reaction under food restriction in the rat[END_REF]), without affecting FAA (Landry et al. 2007). Finally, zinc-induced anosmia in rats did not affect anticipation during scheduled feeding, indicating that olfaction does not play an exclusive role-Forel's assumption [START_REF] Coleman | Anticipatory Wheel-Running in Behaviorally Anosmic Rats[END_REF].

Of great interest, the DMH has been shown to be crucial for producing circadian rhythms such as those of sleep and waking, locomotor activity, feeding and corticosteroid production. In addition, the DMH connects with several hypothalamic nuclei including the lateral area and the PVN [START_REF] Chou | Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms[END_REF][START_REF] Saper | The hypothalamic integrator for circadian rhythms[END_REF].

The DMH has thus been tested as a putative FEO. In that context, a first study demonstrated that cell-specific lesions of the DMH in rats caused a drastic reduction in the preprandial rise in locomotor activity, body temperature and wakefulness (Fig. 40). The authors additionally showed that the degree of FAA correlated with the number of remaining DMH neurons (Gooley et al. 2006). After 3 weeks of restricted feeding, rats were deprived of food for 2 d so that the underlying behavioral and physiologic rhythms could be observed in the absence of the daily feeding-time cue. Black, rhythms during ad lib feeding; gray, rhythms during food deprivation period. (a-f) Unlesioned rats showed a marked shift induced by restricted feeding in the rhythms of locomotor activity (a), body temperature (c) and wakefulness (e). However, in DMH-lesioned rats, rhythms of locomotion (b), body temperature (d) and wakefulness (f) remained in phase with the light-dark cycle rather than with the daily meal. Data are reported as hourly mean s.e.m. White-black bars at top, light-dark cycle; vertical gray bars, timing of food availability during restricted feeding. Rhythms during ad lib feeding and food deprivation are plotted twice to emphasize daily rhythmicity. From (Gooley et al. 2006) Nevertheless, further studies focusing on the DMH showed, by using similar and complementary methods to induce lesion and assess food anticipation, that food entrainment in DMH-x rats was normal and even persisted during a food deprivation [START_REF] Landry | Persistence of a behavioral foodanticipatory circadian rhythm following dorsomedial hypothalamic ablation in rats[END_REF]Landry et al. 2007). Clock gene expression, temperature and activity rhythms could be RF-entrained in DMH-ablated mice as well [START_REF] Moriya | The dorsomedial hypothalamic nucleus is not necessary for food-anticipatory circadian rhythms of behavior, temperature or clock gene expression in mice[END_REF]). Conversely, two additional studies gave evidence that the DMH may still play a role in the control of food anticipation. The first in mice showed that after widespread destruction of the mediobasal hypothalamus, comprising the DMH and parts of the VMH and ARC, a reduction of FAA to a similar extent than the initial study of Gooley et al. 2006 was observed but without affecting the entrainment of Per2 gene expression to mealtime in the liver [START_REF] Tahara | Effects of medial hypothalamic lesions on feeding-induced entrainment of locomotor activity and liver Per2 expression in Per2::luc mice[END_REF]. The second study explored the tight connection between the SCN pacemaker and the putative DMH FEO in rats. The authors first revealed the presence of DMH GABA-projections to the SCN that can inhibit its intrinsic neuronal activity. Then they reported that DMHlesion, which results in a diminution of FAA, leads simultaneously to increased neuronal activity in the SCN (at the level of c-FOS expression). Intriguingly, the concomitant lesion of the SCN in the same animals (i.e., previously DMH-lesioned) restored FAA. They thus proposed that in intact animals, the DMH may play a role in food entrainment by inhibiting the activity of the SCN (Acosta-Galvan et al. 2011).

However, the SCN lesions appeared rather incomplete in the actograms provided in Acosta-Galvan et al. 2011, and the metabolic state of the animal, especially after deep surgery, has not been communicated. Therefore, further studies are needed to clarify the exact role of the DMH. In addition, it is important to note that, in both this last study and those cited above, further behavioral, physiological or gene expression measures, as well as their evaluation in different lighting conditions, are truly lacking for the study of food entrainment.

From all these results, it appears likely that the FEO is not confined to only one (peripheral or central) structure. In the contrary, the FEO may be a network of distributed circadian oscillators (weak or strong, see Fig. 24) that allow the integration of multiple messages and that permit sustained food-entrainable rhyhms of behavior and physiology even in the absence of one component (Fig. 41).

Gene deletion strategy

Genetic strategies allowing modifications of the genome, especially in mice, have been widely used to study the FEO clockwork. Clock mutants were the first models phenotyped in the context of food entrainment. In spite of having a strong circadian SCN defect [START_REF] Vitaterna | Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior[END_REF]), ClockΔ19 mutant mice exhibit higher and longer FAA than wild-type mice when challenged with RF, both in light-dark or constant dark conditions (Pitts et al. 2003). This result was also confirmed with the Clock-null mutant mice that also showed normal food-entrained clock gene expression to RF (Horikawa et al. 2005). It is interesting to note that both mutations did not affect the SCN functioning in the same manner and neither affected food entrainment. This could in part be explained by functional redundancy between clock genes, since NPAS2, a paralog of the CLOCK protein, can also heterodimerize with BMAL1 [START_REF] Reick | NPAS2: an analog of clock operative in the mammalian forebrain[END_REF]. In this context, deletion of Npas2 has been shown to have effect on Per2 mRNA oscillations in extra-SCN tissues [START_REF] Reick | NPAS2: an analog of clock operative in the mammalian forebrain[END_REF].

However, in response to temporal RF, mice knockout for Npas2 only displayed a Circles without clock hands represent processes directly driven by other clocks or external stimuli. Sine waves represent overt rhythms. Light-dark cycles entrain a circadian pacemaker in the suprachiasmatic nucleus (SCN). SCN outputs via polysynaptic pathways drive daily rhythms of activity and feeding, autonomic efferents, hormones and body temperature, all of which contribute to phase control of circadian clocks and driven processes in peripheral organs. If food is temporally restricted, locomotor activity comes under control of a timing mechanism (most likely neural) with circadian properties that generates food anticipatory activity. There may be a central food-entrainable oscillator (FEO) coordinating behavioral and physiological rhythms to mealtime, or there may be a distributed system of central and peripheral FEOs, entrained in parallel by feeding related stimuli. From (Mistlberger 2011) slightly delayed FAA (Dudley et al. 2003). The SCN functioning is also severely affected in mice double-mutant for Cry1 and Cry2 (van der Horst et al. 1999). These double-mutant mice expressed delayed and unstable FAA in light-dark or constant dark conditions, and disrupted FAA was also noticed when the SCN pacemaker was lesioned (Iijima et al. 2005).

A role in food anticipation for the main clock components Per1 and Per2 has also been investigated. Mice with a mutated Per1 gene (Per1 Brdm1 , considered a nullmutant allele) exhibited FAA at levels close to their wild-type littermates (Feillet et al. 2006). On the contrary, Per2 Brdm1 -mutant mice (that carry a deletion in the PAS domain) expressed reduced food anticipation of wheel-running and general activities as well as body temperature in response to temporal RF or hypocaloric feeding, regardless of the lighting conditions (Fig. 42). Per2 mutation, however, did not interfere with hepatic clock gene entrainment to RF (Feillet et al. 2006). On the contrary, in Per2 ldc -mutant mice (a null-mutant allele) normal FAA both in a light-dark cycle and in constant darkness was observed. In addition, double Per1 ldc ; Per2 ldc mutant mice exhibited normal FAA (Storch and Weitz 2009). The different consequence relative to complete or partial loss-of-function mutation strategies (i.e., creating partially or completely non-functional proteins) on the circadian timing system may explain these discrepancies (for review, see Challet et al. 2009). Gray shading indicates lights off. Animals were fed ad libitum, submitted to hypocaloric feeding, and subsequently released into constant darkness with food ad libitum with the exception of the last day (arrow) when no food was accessible. The gray line indicates the time when hypocaloric food was provided. The arrow shows FAA at the expected time in both WT and Per1 mutant mice fed ad libitum in constant darkness. The bottom graph represents the mean daily-activity profile during the last 8 days of hypocaloric feeding period (n = 6 in WT and Per1-/-mice and n = 4 in Per2

Brdm1 mice; mean ± SEM). The gray line on the X axis indicates time of feeding. Note the lack of food-anticipatory activity in Per2 mutant mice (C). From (Feillet et al. 2006) As studies that explored a role for the DMH in food anticipation gave diverging conclusions, investigations on the involvement of the Bmal1 clock gene in the FEO (and DMH) is controversial as well. Bmal1 -/-mice (null-allele) are arrhythmic in constant darkness and strong alterations of their SCN clock machinery are observed [START_REF] Bunger | Mop3 is an essential component of the master circadian pacemaker in mammals[END_REF]. In addition, when challenged with RF, Bmal1 -/-mice did not show significant increase of locomotor activity and body temperature prior to mealtime [START_REF] Fuller | Differential rescue of light-and food-entrainable circadian rhythms[END_REF]. By using viral vector containing the Bmal1 gene, Fuller and colleagues were able to demonstrate that the injection of this vector in the SCN of the Bmal1 -/-mice can restore light-entrainable circadian rhythms (i.e., those of locomotor activity and body temperature) without positively affecting food-entrained rhythms. Moreover, injection of the viral vector containing the Bmal1 gene in the DMH can restore the ability of Bmal1 -/-mice to entrain to RF without affecting light entrainment. The authors thus concluded that the DMH harbors a Bmal1-based FEO [START_REF] Fuller | Differential rescue of light-and food-entrainable circadian rhythms[END_REF]). However, these results have been challenged by further studies

showing that some FAA can be observed in the Bmal1 -/-mice [START_REF] Mistlberger | Comment on "Differential Rescue of Light-and Food-Entrainable Circadian Rhythms[END_REF][START_REF] Pendergast | Robust Food Anticipatory Activity in BMAL1-Deficient Mice[END_REF]Storch et al. 2009). Besides, methodological and data issues have been raised regarding Fuller and colleagues' findings [START_REF] Fuller | Standards of evidence in chronobiology: A response[END_REF]Mistlberger et al. 2009;Mistlberger et al. 2009). These caveats aside, it is important to mention that Bmal1 -/-mice have profound developmental and metabolic defects (see part 4.3.1). To avoid these problems, a recent study has investigated foodentrainable activity in mice lacking Bmal1 only in the nervous system. They showed that the emergence of FAA was severely delayed in these mice, supporting an essential role of the Bmal1 clock gene (in the nervous system) for a rapid adaptation to periodic feeding (Mieda and Sakurai 2011).

Mice lacking specific feeding-related genes have also been studied.

Genetically obese Zucker ( fa/fa) rats that carry a mutation in the leptin receptor (see part 4.2.1 for a detailed description) displayed anticipatory rhythms greater than that of control rats, that were maintained during food deprivation. This can suggest that leptin action is not an essential entrainment cue for the FEO [START_REF] Mistlberger | Enhanced food-anticipatory circadian rhythms in the genetically obese Zucker rat[END_REF]). On the other hand, mice carrying a mutation in the ghrelin receptor (GHSR knockout (KO) mice), exhibited diminished FAA and c-FOS immunoreactivity in several hypothalamic nuclei in response to RF [START_REF] Blum | Reduced anticipatory locomotor responses to scheduled meals in ghrelin receptor deficient mice[END_REF][START_REF] Lesauter | Stomach ghrelin-secreting cells as food-entrainable circadian clocks[END_REF]). Moreover, hypothalamic c-FOS expression patterns in unrestricted wild-type (WT) mice receiving daily i.p. injections of ghrelin can mimic those of mice under restricted feeding schedules [START_REF] Blum | Reduced anticipatory locomotor responses to scheduled meals in ghrelin receptor deficient mice[END_REF]). These results indicate that ghrelin can play a strong role in food entrainment. The role of ghrelin is highlighted by two additional studies. The first demonstrated that injections of ghrelin in free-fed animals (that were food-deprived following the injection) can increase general locomotor activity and subsequent food intake. In addition, the authors of the first study showed that the stomach oxyntic gland cells contained a putative FEO, since ghrelin and clock gene expression (i.e., PER1 and PER2) were controlled by the time of food availability and rhythmic expression of oxyntic cell ghrelin was abolished in Per1-Per2 (Brdm1) double-mutant mice [START_REF] Lesauter | Stomach ghrelin-secreting cells as food-entrainable circadian clocks[END_REF]). The second study gives evidence that the injection of ghrelin or an antagonist of the ghrelin receptor can increase or decrease, respectively, FAA prior to access to a palatable meal [START_REF] Merkestein | Ghrelin Mediates Anticipation to a Palatable Meal in Rats[END_REF]. Ghrelin thus seems to be a strong humoral signal to modulate food anticipation and motivational associated-behaviors such as feeding.

Genetic perturbations of several additional genes also led to interesting phenotypes regarding food entrainment. As outlined above, orexin is essential to regulate feeding and arousal. Orexin KO mice display reduced wakefulness and FAA but normal FAT before feeding time [START_REF] Akiyama | Reduced food anticipatory activity in genetically orexin (hypocretin) neuron-ablated mice[END_REF][START_REF] Mieda | Orexin neurons function in an efferent pathway of a food-entrainable circadian oscillator in eliciting food-anticipatory activity and wakefulness[END_REF][START_REF] Kaur | Entrainment of temperature and activity rhythms to restricted feeding in orexin knock out mice[END_REF]). Nevertheless, chemical destruction of hypothalamic orexin-producing neurons did not affect FAA [START_REF] Mistlberger | Food-and light-entrained circadian rhythms in rats with hypocretin-2-saporin ablations of the lateral hypothalamus[END_REF], thus not supporting an indispensable role for orexin. The role of the melanin-concentrating hormone (MCH, a neuropeptide that stimulates feeding) in food anticipation has been explored in mice deficient for the MCH1 receptor, with no effect [START_REF] Zhou | Enhanced running wheel activity of both Mch1r-and Pmch-deficient mice[END_REF]. On the other hand, melanocortins, crucial for the regulation of energy homeostasis, may influence food entrainment. Indeed, mice deficient for the melanocortin receptors MCR3 exhibit attenuated wakefulness and locomotor activity prior to food presentation concomitant with impaired clock gene expression in AL and RF conditions [START_REF] Sutton | The melanocortin-3 receptor is required for entrainment to meal intake[END_REF].

Deletion of the orphan nuclear receptor Ear2 in mice, essential for the development of the locus coeruleus, reduced FAA in response to a RF challenge. Of note, Ear2 -/- mice have impaired Per1 and Per2 expression in the frontal cortex as well as diminished NA levels in this brain region [START_REF] Warnecke | Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception[END_REF]. Investigations focusing on the role of mu-opiod receptors in adapted-reward behaviors are also informative. During or prior to the activation of anticipatory behavior to a coming reward, the mesolimbic opioid system is activated [START_REF] Spruijt | A concept of welfare based on reward evaluating mechanisms in the brain: anticipatory behaviour as an indicator for the state of reward systems[END_REF]. Of interest, mu-Opioid receptor KO mice do not show evidence of ethanol self-administration, or display attenuated MAP-induced behavioral sensitization and cocaine conditioned place preference-or reward cocaine effect-compared to wild-type littermates [START_REF] Roberts | mu-Opioid receptor knockout mice do not self-administer alcohol[END_REF][START_REF] Hall | mu-opioid receptor knockout mice display reduced cocaine conditioned place preference but enhanced sensitization of cocaine-induced locomotion[END_REF][START_REF] Shen | mu-Opioid receptor knockout mice are insensitive to methamphetamine-induced behavioral sensitization[END_REF]). In the context of food entrainment, mu-KO mice showed reduced locomotor activity in anticipation of food access [START_REF] Kas | Mu-opioid receptor knockout mice show diminished food-anticipatory activity[END_REF]). This may suggest that a functional opioid system is essential to adapt behavior to the limited temporal access of a daily reward such as food. Finally, mutation in mice of the ionotropic glutamate receptor δ2 gene (Grid2), selectively expressed in Purkinje cells, led to an impairment of motor coordination and to a severe reduction of food-anticipatory components (Mendoza et al. 2010).

Hence, the FEO location does not appear to be confined to a unique structure.

Multiple genes involved in clock regulation pathways, metabolic and feeding pathways, or even reward pathways, have emerged concurrently as important actors to drive food entrainment.

General conclusion

The necessary effort to feed life has favoured the evolution of a foodentrainable system to track temporal regularities and anticipate predictable changes of food resources in nature. Indeed, animals that occupy different time niches can forage at the most appropriate time of the day to ensure their survival. The increased expression of anticipatory behavior and physiology during artificially (i.e., in laboratory conditions) reduced food availability can thus reflect the triggering of circadian, motivational and metabolic processes to ensure that foraging takes place in the most efficient manner.

From the studies presented above, the molecular (circadian) control of food anticipation is still elusive in mammals-both at the tissue and cellular levels-, but two working hypotheses emerge. The first is an assumption that food entrainment is dependent on at least a few clock genes (i.e., Per2, Bmal1, Npas2 and Cry1-2) and thus a food-entrainable clockwork model can be shaped and served to further integrate the role of other clock-related genes yet to be tested (for a review, see Challet et al. 2009). The second hypothesis opts for the involvement of other (circadian) mechanisms for food entrainment to be determined and supporters based their hypothesis on the observations that canonical clock genes are not involved for the functioning of the FEO nor for the MASCO30 [START_REF] Mohawk | The methamphetamine-sensitive circadian oscillator does not employ canonical clock genes[END_REF]Storch et al. 2009).

Incidentally, most of the studies that investigate the functions of genes in food entrainment used germline KO mice, which make the interpretation of results a bit challenging. Indeed, on one hand, the circadian system has been demonstrated to be relatively impervious to genetic perturbations in vitro [START_REF] Baggs | Network Features of the Mammalian Circadian Clock[END_REF], which is supported in vivo by the few strong effects of clock gene deletion on the SCN pacemaker. Thus, significant impairment of the FEO clockwork could be hard to detect. On the other hand, clock gene deletion (as well as lesion of brain nuclei) can have broad consequences, in particular, on energy homeostasis (see part 4.3.1), rendering the evaluation of circadian food entrainment more difficult.

Finally, beyond the need to also (re)consider the participation of reward and learning processes in food entrainment [START_REF] Silver | Food anticipation depends on oscillators and memories in both body and brain[END_REF], study of the simplest organisms (e.g., bee) could probably be informative for further investigations of the food-entrainable system. In parallel, the use in the future of inducible and reversible knockout, or even, optogenetic tools for targeted neural inhibition/activation may help to find the "needle(s) in the haystack".

The circadian metabolism

Circadian control of metabolic functions

In the previous part, we have seen that food restriction triggers direct consequences on the circadian system and energy balance. The following part will show that metabolic regulations are tightly regulated within the brain and peripheral tissues and that clock genes play a crucial role in this context.

The hypothalamus

Considerable experimental work has highlighted the important role of hypothalamic nuclei in the control of feeding and energy metabolism. The ventromedial hypothalamic nucleus, LH and ARC nucleus contain glucose-sensing neurons and receive nutritional information from blood-borne signals and neuronal messages from brainstem nuclei, including parabrachial nucleus and NTS. Among the key hormones that modulate feeding via hypothalamic activity, leptin synthesized by adipocytes acts in particular on ARC to inhibit appetite and stimulate energy expenditure [START_REF] Ahima | Adipokines and the peripheral and neural control of energy balance[END_REF]. Insulin, released from the β cells of the pancreas, also has anorexigenic effects on the metabolic hypothalamus [START_REF] Gerozissis | Brain insulin, energy and glucose homeostasis; genes, environment and metabolic pathologies[END_REF]). On the other hand, ghrelin, released by the stomach (that play a strong role in food entrainment), activates NPY/AgRP containing neurons in the ARC to increase appetite and decrease energy expenditure [START_REF] Nogueiras | Central nervous system regulation of energy metabolism: ghrelin versus leptin[END_REF]).

As outlined before, an important role of the SCN is to orchestrate the internal ticking of different central and peripheral tissues. Therefore, 24-h rhythmic patterns can be observed for a plethora of metabolic substrates and hormones, such as glucose [START_REF] Yamamoto | Role of SCN in daily rhythms of plasma glucose, FFA, insulin and glucagon[END_REF][START_REF] Fleur | A suprachiasmatic nucleus generated rhythm in basal glucose concentrations[END_REF], non-esterified fatty acids (NEFA), insulin [START_REF] Yamamoto | Role of SCN in daily rhythms of plasma glucose, FFA, insulin and glucagon[END_REF]) and leptin levels [START_REF] Kalsbeek | The suprachiasmatic nucleus generates the diurnal changes in plasma leptin levels[END_REF], and are abolished in the absence of the SCN (Fig. 43). In addition, expression in the SCN of receptors to metabolic hormones (i.e., insulin, ghrelin and leptin) raises the possibility that peripheral hormonal signals can feedback to the SCN [START_REF] Unger | Distribution of insulin receptor-like immunoreactivity in the rat forebrain[END_REF][START_REF] Hakansson | Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus[END_REF][START_REF] Zigman | Expression of ghrelin receptor mRNA in the rat and the mouse brain[END_REF].

Circadian transcriptome

Microarray analysis of the mouse liver transcriptome showed that 9% of more than 2000 genes studied oscillate in a circadian manner and may be under the control of the SCN [START_REF] Akhtar | Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus[END_REF]. Thus, the SCN modulates both behavior and metabolism by altering the phase of peripheral oscillators provided that food is freely available. Recent genome-wide transcriptome analyses performed in the SCN, liver, adrenal gland of mice, revealed that around 10% of transcripts are regulated in a circadian manner [START_REF] Ueda | A transcription factor response element for gene expression during circadian night[END_REF][START_REF] Oishi | Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes[END_REF][START_REF] Lowrey | Mammalian circadian biology: elucidating genome-wide levels of temporal organization[END_REF]Oishi et al. 2005;[START_REF] Miller | Circadian and CLOCKcontrolled regulation of the mouse transcriptome and cell proliferation[END_REF]Hughes et al. 2009). Of note, among these transcripts are found key regulators of glucose and lipid metabolism, and components of the xenobiotic detoxification pathway. Of the 49 nuclear receptors, that are key actors of metabolic regulations (see below), approximately 40% were cyclic in the liver or white adipose tissue (WAT) [START_REF] Yang | Nuclear receptor expression links the circadian clock to metabolism[END_REF]. Liver posttranscriptional and translational mechanisms contribute as well to circadian coordination [START_REF] Reddy | Circadian orchestration of the hepatic proteome[END_REF].

Therefore, the whole circadian system participates in the daily variation of metabolism. Several biological approaches and genetic models have given further insight into the maintenance of metabolic homeostasis and the crosstalk between the circadian system and metabolism, as will be further discussed below.

4.2 Metabolic diseases are associated with circadian disturbances

Genetic obesity and diabetes

Monogenic causes of obesity and diabetes are relatively rare, especially in humans. For instance, early-onset obesity has been associated with a mutation in the leptin receptor in humans [START_REF] Clement | A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction[END_REF]. In rodents, however, genetic syndromes of obesity and diabetes mellitus offer models of choice to analyze circadian disturbances associated with metabolic physiopathology.

In obese Zucker rats, which carry a mutation (i.e., fa) in the leptin receptor gene, daily rhythms of locomotor activity, body temperature and feeding are phaseadvanced (i.e., the active phase starts in the afternoon in contrast to the nocturnal onset in fa/? littermates) and their day-night amplitude is generally reduced [START_REF] Fukagawa | Advance shift of feeding circadian rhythm induced by obesity progression in Zucker rats[END_REF][START_REF] Murakami | Circadian rhythms of temperature and activity in obese and lean Zucker rats[END_REF][START_REF] Mistlberger | Circadian rhythms in the Zucker obese rat: assessment and intervention[END_REF]). The molecular clockwork in peripheral tissues is consistently disrupted in genetically obese, diabetic

or not, mice. More precisely, in obese ob/ob and KK mice as well as in obese and diabetic KK-A Y mice, the amplitude of daily profiles of clock gene expression in the liver or white adipose tissue is generally reduced, if not barely sizeable [START_REF] Ando | Rhythmic messenger ribonucleic acid expression of clock genes and adipocytokines in mouse visceral adipose tissue[END_REF][START_REF] Ando | Impairment of peripheral circadian clocks precedes metabolic abnormalities in ob/ob mice[END_REF]. The results are more contrasted in obese and diabetic db/db mice because in their liver, expression of clock genes can be either decreased (i.e., Per2), up-regulated (i.e., Per1) or phase-advanced (i.e., Bmal1) [START_REF] Kudo | Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver[END_REF]).

Of interest, in ob/ob mice, alterations in peripheral clocks occur earlier than metabolic symptoms, such as morbid obesity and hyperinsulinemia, thus suggesting that metabolic disturbances are not the main cause of altered clockwork [START_REF] Ando | Impairment of peripheral circadian clocks precedes metabolic abnormalities in ob/ob mice[END_REF]). Within the central nervous system of ob/ob or KK-A Y mice, clock gene oscillations have been studied in the NTS. The most salient changes concern Bmal1

and Rev-erbα whose daily expression is up-and down-regulated, respectively [START_REF] Kaneko | Obesity alters circadian expressions of molecular clock genes in the brainstem[END_REF]). Moreover, the molecular clockwork of the SCN is not markedly affected in leptin-deficient, obese (i.e., ob/ob) mice [START_REF] Ando | Impairment of peripheral circadian clocks precedes metabolic abnormalities in ob/ob mice[END_REF]. Unexpectedly in view of the severe dampening of overt rhythmicity in db/db mice, the amplitude of SCN molecular oscillations is significantly increased in these mice compared to db/+ littermates [START_REF] Kudo | Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver[END_REF]). Finally, it is worth mentioning that ob/ob mice show increased photic resetting of the master clock (Sans-Fuentes et al. 2010).

Diet-induced obesity

In nature, species are not often confronted to food abundance. On the contrary, food can be rare for most animals and foraging is a key for survival (thus contributing to energy expenditure). Consequently, obese animals in the wild are uncommon, except for some species in which overweighting is important to support long periods of fasting (e.g., penguins, bears), and those species living in proximity to humans and consuming greasy food that is discarded by people. Most laboratorybased research on obesity is carried out in rodents [START_REF] West | Dietary fat, genetic predisposition, and obesity: lessons from animal models[END_REF]. Control animals usually have an ad libitum access to a normocaloric chow diet. Despite this free access, most rodents regulate their consumption and the time when they eat and do not become obese.

Obesity can be induced by feeding rodents on an ad libitum basis with a highfat diet (HFD, more than 50% of metabolizable energy derived from fat). Short-term high-fat feeding reduces circadian variations of leptin levels in rats [START_REF] Cha | High-fat diet feeding reduces the diurnal variation of plasma leptin concentration in rats[END_REF], and humans [START_REF] Havel | High-fat meals reduce 24-h circulating leptin concentrations in women[END_REF], thus suggesting that dampened circulating leptin could contribute to the development of obesity. In mice, high-fat feeding attenuates the daily pattern of food intake, with a higher consumption during the day and a concomitant decrease during the active period, before significant mass gain.

Moreover, changes in the concentration and temporal pattern of expression of glucose, insulin, leptin and NEFA are also observed (Fig. 44) (Kohsaka et al. 2007). High-fat feeding also leads to difficulties in maintaining wakefulness during the active period and increases non-rapid eye movement sleep in mice [START_REF] Jenkins | Sleep is increased in mice with obesity induced by high-fat food[END_REF], as well as postprandial sleepiness in humans [START_REF] Wells | Influences of fat and carbohydrate on postprandial sleepiness, mood, and hormones[END_REF], suggesting that metabolic state affects neural structures regulating sleep. Equally interesting, HFD seems to have a direct effect on the main circadian clock. In mice, HFD lengthens the free-running period (Kohsaka et al. 2007) and disrupts photic synchronization of the SCN to light, as shown by slower re-entrainment to shifted light-dark cycle and reduction in light-induced phase-shifts (Mendoza et al. 2008).

There is also a clear change in neuropeptide expression in the mediobasal hypothalamus, despite no major modification of the core clock machinery in that region (Kohsaka et al. 2007). In the brainstem, more precisely in the NTS, mice fed with HFD display altered daily patterns of clock gene expression [START_REF] Kaneko | Obesity alters circadian expressions of molecular clock genes in the brainstem[END_REF]. Taken together, these results suggest that central dysfunctions may contribute to the development of obesity.

Oscillations of clock genes have been found in human WAT [START_REF] Garaulet | An approximation to the temporal order in endogenous circadian rhythms of genes implicated in human adipose tissue metabolism[END_REF]), but no difference has been detected in the characteristics of these oscillations between lean, overweight and diabetic individuals [START_REF] Otway | Rhythmic diurnal gene expression in human adipose tissue from individuals who are lean, overweight, and type 2 diabetic[END_REF]. By contrast, the circadian timing of peripheral tissues is markedly modified by fat overload in rodents. HFD alters the diurnal variation in glucose tolerance and insulin sensitivity by influencing clock functioning [START_REF] Rudic | BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis[END_REF]). In the liver, changes are seen in the level as well as the rhythmic pattern of major components of lipid homeostasis and adiponectin metabolic pathway, with parallel modifications in clock gene expression (Kohsaka et al. 2007;[START_REF] Barnea | High-fat diet delays and fasting advances the circadian expression of adiponectin signaling components in mouse liver[END_REF]). In HFD-fed animals, expression of metabolic actors is asynchronous in liver and adipose tissue, suggesting the importance of temporal coordination among metabolic tissues.

Furthermore, HFD also attenuates the amplitude of Clock, Bmal1 and Per2 in adipose tissue (Kohsaka et al. 2007). Hence, altered circadian clock function within adipose tissue may promote excess fat storage (especially intra-abdominal fat).

In summary, impairment of clock gene oscillations and metabolic pathways may explain the altered coordination of metabolic functions and clock-controlled output signaling, contributing to obesity and associated disorders (e.g., diabetes, sleep disturbances). Recent evidence also demonstrates that disruption of the circadian timing system solely, has various consequences on metabolism.

Circadian disruption is associated with metabolic dysfunctions

Altered endogenous clockwork

As for the study of the FEO, one of the first clock genes studied was Clock.

Homozygous C57BL/6J Clock mutant mice are hyperphagic, show a dampened feeding pattern, with increased food intake during the rest period, and attenuated energy expenditure at night, thus contributing to fat excess. Clock mutant mice display severe metabolic alterations, including hypercholesteronemia, hypertriglyceridemia, hepatic steatosis and hyperglycemia. In addition, Clock mutation leads to day-night changes in the expression of hypothalamic neuropeptides, like CART (Cocaine and amphetamine regulated transcript) and orexin mRNA, which play a role in central circuits regulating feeding and arousal (Fig. 45) (Turek et al. 2005). Furthermore, Clock mutation induces profound changes in glucose homeostasis, such as increased insulin sensitivity and altered gluconeogenesis [START_REF] Rudic | BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis[END_REF]). Of interest, in the liver, Clock has been shown

to directly drive the expression of glycogen synthase 2 via E-boxes [START_REF] Doi | CLOCK regulates circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2[END_REF].

In Clock mutant mice generated on a CBA/6CaH background (which synthesize melatonin contrary to the C57BL/6J strain), impairments in glucose tolerance, insulin secretion, liver Pepck mRNA expression and increased insulin sensitivity are also observed, although these mice do not become spontaneously obese unlike C57BL/6J Clock mutants [START_REF] Kennaway | Metabolic homeostasis in mice with disrupted Clock gene expression in peripheral tissues[END_REF]). Moreover, Clock Clock mutant mice display altered circadian patterns and abundances of Per2 mRNA (A) and mRNAs encoding hypothalamic peptides involved in energy balance. 12:12 LD cycle is indicated by bar at the bottom. Values for WT (redline) and Clock mutant (black line) mice are displayed as relative abundance (RA; mean +/-SEM). Brains were collected at 4-h intervals across the 24-h cycle. Orx: orexin; Ghr: ghrelin; CART: cocaine-and amphetamine-regulated transcript. From (Turek et al. 2005) (Jcl:ICR) mutant mice display levels of triglycerides and NEFA lower than control mice, and cholesterol and glucose levels do not differ. Due to reduced lipid absorption and hepatic lipogenesis, these mice challenged with HFD show reduced body mass elevation, leptin and insulin levels (Oishi et al. 2006;[START_REF] Kudo | Attenuating effect of clock mutation on triglyceride contents in the ICR mouse liver under a high-fat diet[END_REF]). In spite of these discrepancies, due notably to strain-related differences, mutation of the Clock gene clearly has an impact on lipid metabolism, since Clock has been shown

to participate in liver cholesterol accumulation [START_REF] Kudo | Clock mutation facilitates accumulation of cholesterol in the liver of mice fed a cholesterol and/or cholic acid diet[END_REF]) and to amplify obesity in ob/ob mice (Oishi et al. 2006).

Bmal1, a close partner of Clock, has been shown to participate in adipocyte differentiation and lipogenesis [START_REF] Shimba | Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis[END_REF]. Sensitivity to exogenous insulin is increased by deletion of Bmal1, while gluconeogenesis is suppressed [START_REF] Rudic | BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis[END_REF]). Liver-specific deletion of Bmal1 in mice nicely confirmed its strong involvement in glucose metabolism. L-Bmal1 -/-mice have impaired expression of clock-related genes involved in hepatic glucose regulation such as glucose transporter 2, glucokinase or pyruvate kinase. L-Bmal1 -/-mice were hypoglycemic during the resting period, were more glucose tolerant, have normal insulin sensitivity and production, and normal total body fat content compared to wild-type mice [START_REF] Lamia | Physiological significance of a peripheral tissue circadian clock[END_REF]). Altogether, these results demonstrate that Clock and Bmal1 modulate hepatic function to regulate glucose and fatty acid homeostasis.

Recent elegant studies have explored the role of Clock and Bmal1 in the pancreatic islets, demonstrating that the pancreas harbors a functional circadian oscillator (Fig. 46) (Marcheva et al. 2010;[START_REF] Sadacca | An intrinsic circadian clock of the pancreas is required for normal insulin release and glucose homeostasis in mice[END_REF]. They showed that pancreas-specific Bmal1 mutant mice have higher blood glucose levels during the whole 24 h, impaired glucose tolerance and reduced insulin secretion. Moreover, pancreatic islets of these mutant mice have altered development and produced less insulin, suggesting that intact clock functioning in the pancreas is essential for normal insulin secretion (Marcheva et al. 2010). This study establishes clearly that circadian components can regulate local metabolism.

Mutation of the core clock gene Per2 can also lead to abnormal conditions. In Per2 -/-(Per2tm1Brd) mice, daily corticosterone rhythm is markedly attenuated. Of interest, when fed with HFD, these mice eat more during the rest period compared to wild-type mice and develop bigger obesity [START_REF] Yang | The role of mPer2 clock gene in glucocorticoid and feeding rhythms[END_REF]). Moreover, the rhythmic pattern in Per2 -/-mice of alpha-melanocyte-stimulating hormone (α-MSH), a powerful appetite suppressing peptide, is disrupted and peripheral injection of α-MSH induces weight loss [START_REF] Yang | The role of mPer2 clock gene in glucocorticoid and feeding rhythms[END_REF]. In mice in which Per2 is fully ablated, lipid metabolism was altered with decreases in both plasma NEFA and total TG, and TG contents in the WAT [START_REF] Grimaldi | PER2 controls lipid metabolism by direct regulation of PPARgamma[END_REF]. In Per2 -/-mice, and to a lesser extent in Per1 -/- mice, glucose tolerance was increased (i.e., improved) compared to wild-type animals [START_REF] Dallmann | Impaired daily glucocorticoid rhythm in Per1 ( Brd ) mice[END_REF]. By contrast, in Per1 -/-;Per2 -/-double-mutant mice (129/sv background) glucose tolerance and insulin sensitivity were both attenuated [START_REF] Lamia | Physiological significance of a peripheral tissue circadian clock[END_REF]. Triple mutant mice for Per1-Per2-Per3 gain more body mass on HFD than wild-type mice, similar to Per3 single mutant mice, thus indicating that the Per3 mutation alone accounts for the obese phenotype (Dallmann and Weaver 2010).

Along with core clock components, secondary actors of circadian oscillations take part in metabolic functions. Rorα, activator of Bmal1 transcription, has been shown to participate in the regulation of Apolipoprotein C-III (Apo C-III) gene involved in triglyceride metabolism [START_REF] Raspe | Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha[END_REF], and lipid homeostasis in skeletal muscle [START_REF] Lau | RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR[END_REF]). Rev-erbα, repressor of Bmal1, is involved in adipocyte differenciation [START_REF] Fontaine | The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation[END_REF]Wang and Lazar 2008) and rhythmic bile and lipid homeostasis (Le Martelot et al. 2009). Rev-erbα -/-mice displayed elevated serum Apo C-III and triglycerides levels (Raspe et al. 2002). Moreover, although the deletion of Rev-erbα alone has only a minor effect on hepatic glucose regulation (Le Martelot et al. 2009), the alterations are more pronounced with concomitant mutation of Per2 (Schmutz et al. 2010). Indeed, the expression of key enzymes of glucose metabolism, such as glucose-6-phosphatase or glucokinase, was more affected in the liver of Rev-erbα -/-/Per2 mutant mice.

Altered rhythmic environment

There are two main external causes of disruption in circadian rhythmicity. The first one concerns chronic changes in timing of light-dark cycles, such as shift-work or chronic jet-lag. The second way relates to behavioral activity (e.g., physical activity, feeding) occurring on a regular basis during the usual resting period, such as during night-work. As detailed below, both situations have deleterious consequences on circadian organization and metabolic health.

Housing mice to light-dark cycles too short (i.e., 20 h) for enabling daily synchronization of their master clock leads to larger body mass gain and increased insulin/glucose ratio, indicative in the fasted state of insulin resistance [START_REF] Karatsoreos | Disruption of circadian clocks has ramifications for metabolism, brain, and behavior[END_REF]. In human subjects exposed to controlled 28-h sleep-wake cycles under dim light (so-called 'forced desynchronization' protocol), circadian misalignment impairs glucose tolerance and reduces sensitivity to insulin [START_REF] Scheer | Adverse metabolic and cardiovascular consequences of circadian misalignment[END_REF]).

Additionally, constant exposure of mice to bright or dim light leads to increased body mass and reduced glucose tolerance compared to mice housed under a regular lightdark cycle (Fig. 47) [START_REF] Fonken | Light at night increases body mass by shifting the time of food intake[END_REF]. Repeated weekly shifts of 24-h light-dark cycle in rats fed with regular chow diet lead to circadian desynchronization and trigger higher body mass gain [START_REF] Tsai | Repeated light-dark shifts speed up body weight gain in male F344 rats[END_REF]. + P ≤ 0.05 when glucose level is higher in the DM group than in the LD group). (D) Body mass at the time of the GTT correlated positively with final blood glucose levels. [START_REF] Fonken | Light at night increases body mass by shifting the time of food intake[END_REF] Being nocturnal animals, laboratory rats and mice exposed to a light-dark cycle consume most of their daily food during the night period. Access to food restricted to a few hours during the light phase usually leads to mild body mass loss or no change at all [START_REF] Castillo | Entrainment of the master circadian clock by scheduled feeding[END_REF]Feillet et al. 2006;[START_REF] Sutton | The melanocortin-3 receptor is required for entrainment to meal intake[END_REF]. In more rare cases, food-restricted rats with chow diet available only for a few hours can increase their body mass [START_REF] Martinez-Merlos | Dissociation between adipose tissue signals, behavior and the foodentrained oscillator[END_REF]. A clear contribution of circadian timing of food intake in body mass gain has been shown in Zucker rats (Fig. 48) [START_REF] Mistlberger | Circadian rhythms in the Zucker obese rat: assessment and intervention[END_REF]). As mentioned above, fa/fa rats ingest a large proportion of food during the usual resting phase (i.e., light phase) in nocturnal rats. This study was the first to demonstrate that by limiting food access to the normal period of activity and feeding (i.e., nighttime in nocturnal rats), Zucker rats gained less body mass compared to free-fed animals, in spite of similar amounts of food intake between both groups [START_REF] Mistlberger | Circadian rhythms in the Zucker obese rat: assessment and intervention[END_REF]). More recent observations confirm nicely the metabolic consequences of unusual timing of feeding. Mice fed ad libitum with HFD display a rapid increase in daytime feeding that takes place weeks before the obese phenotype is detectable (Kohsaka et al. 2007). Furthermore, when HFD was restricted to the light phase, mice gained more body mass than those on the same diet but with access limited to the dark phase (Fig. 48) [START_REF] Arble | Circadian timing of food intake contributes to weight gain[END_REF]).

Figure 48. Importance of feeding time in genetic or diet-induced obesity

In Zucker rats (upper panels), obese individuals (i.e., fa/fa; blue lines and bars) eat more during daytime than control littermates (fa/?; red lines and bars). When food access is limited to nighttime (orange lines and bars), fa/fa rats gain less body mass in spite of similar whole energy intake compared to free-fed individuals (modified with permission from [START_REF] Mistlberger | Circadian rhythms in the Zucker obese rat: assessment and intervention[END_REF]). In C57BL6 mice (lower panels), free access to a high-fat diet is associated with an increase in daytime feeding (purple line), while chow feeding is mostly nocturnal (red line). If high-fat feeding is restricted to daytime or nighttime hours (light and dark purple bars, respectively), body mass gain is larger in the former, despite comparable energy intake (drawn from data in refs. [START_REF] Martinez-Merlos | Dissociation between adipose tissue signals, behavior and the foodentrained oscillator[END_REF]Kohsaka et al. 2007).

Another paradigm used to awaken rodents during their resting period (i.e., daytime) is forced activity that leads to internal desynchronization [START_REF] Salgado-Delgado | Internal desynchronization in a model of night-work by forced activity in rats[END_REF]. Keeping food intake to the normal feeding period (i.e., night) in spite of diurnal forced activity prevents body mass and metabolic changes [START_REF] Salgado-Delgado | Food intake during the normal activity phase prevents obesity and circadian desynchrony in a rat model of night work[END_REF]. Considering that exposure to bright light at night is an aggravating factor for the occurrence of pathologies in human night-workers [START_REF] Arendt | Shift work: coping with the biological clock[END_REF], it may be clinically relevant to develop shift-work models in day-active animals.

Transcriptional networks connecting molecular clockwork and metabolic pathways

From genetic strategies allowing modifications of the genome in mice, it has been discussed above that most, if not all, clock gene deletions lead to a broad range of metabolic diseases. In addition, the effects of unhealthy food and incorrect timing of food intake on metabolic regulatory centers and the core clock machinery have been introduced. In this last section, an overview of the emergent understanding of the communication within the circadian clock circuitry will be given. The different molecular pathways by which clock-related nuclear receptors are involved in metabolism and the effects of nutrient "sensors" on core clock components are illustrated below (Fig. 49).

Figure 49. A schematic illustration of the cross-talk between circadian components and metabolic regulators

The master clock housed in the SCN is connected to several brain and peripheral clocks to orchestrate rhythmic activities. All clocks share a common molecular mechanism in which the clock genes are coexpressed, and the generation of circadian oscillations is made from transcriptional/translational interactions (represented in the gray box; for more details, see Fig. 17).

Clock genes can influence, directly or indirectly, the rhythmic expression of many metabolic genes (listed in the blue box). Clock components can also control rate-limiting enzymes in the NAD + salvage pathway (i.e., Nampt) and heme biosynthesis (i.e., Alas1). Intracellular metabolism through the action of SIRT1, AMPK, and heme can impinge both metabolic genes and core clock machinery. In addition, epigenetic modifiers, such as HDAC3 recruited by REV-ERBα via NCoR1, modulate clock and metabolic gene transcription (orange box).

The nuclear receptors' dynamic network

Transcriptomal analyses have demonstrated that a large number of nuclear receptors are expressed in a circadian manner [START_REF] Yang | Nuclear receptor expression links the circadian clock to metabolism[END_REF]). Among them, Rors, Rev-erbs and peroxisome proliferator-activated receptor (Ppars) genes appear to be pivotal players at the interface between the circadian system and metabolism [START_REF] Teboul | The nuclear hormone receptor family round the clock[END_REF].

As introduced earlier, RORs and REV-ERBs compete for binding RORE in clock gene promoters (Preitner et al. 2002;Guillaumond et al. 2005;[START_REF] Crumbley | Characterization of the core mammalian clock component, NPAS2, as a REV-ERBalpha/RORalpha target gene[END_REF]Crumbley et al. 2011). Rev-erbα has also been shown to repress its own transcription through a Rev-erbα responsive element (RevRE) located in its promoter [START_REF] Adelmant | A functional Rev-erb alpha responsive element located in the human Rev-erb alpha promoter mediates a repressing activity[END_REF]. Interestingly, RORα binds to this site, thus controlling the transcription of Rev-erbα (Raspe et al. 2002). Hence, genes containing RORE/RevRE in their promoter are activated by RORα and repressed by REV-ERBα. This highlights the occurrence of dynamic interactions within the core clock machinery.

In skeletal muscle, Rorα directly regulates the mouse caveolin-3 (Cav-3) and carnitine palmitoyltransferase-1 (Cpt1) genes, involved in fatty acid metabolism [START_REF] Lau | RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR[END_REF]). Rorα also enhances activity of the human Apo C-III gene promoter [START_REF] Raspe | Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha[END_REF]) and binds to the rodent Apolipoprotein A-I (ApoA1) RORE [START_REF] Vu-Dac | Transcriptional regulation of apolipoprotein A-I gene expression by the nuclear receptor RORalpha[END_REF]. Additional evidence for the role of Rorα in metabolism is provided by the fact that cholesterol is a putative ligand of RORα [START_REF] Kallen | Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A[END_REF]).

Numerous studies have shown that REV-ERBs also influence lipid and energy homeostasis. The expression of a dominant negative version of Rev-erbβ in transfected cell lines attenuates expression of genes involved in lipid metabolism, such as fatty acid binding protein [START_REF] Asher | Crosstalk between components of circadian and metabolic cycles in mammals[END_REF][START_REF] Zvonic | Characterization of peripheral circadian clocks in adipose tissues[END_REF], cluster of differentiation 36 (Cd36), These findings demonstrate that the circadian control of epigenetic modifiers by clock components is critical for normal metabolic processing. Additional evidence of the involvement of Rev-erbs in metabolism is provided by studies on heme which has diverse biological functions, including oxygen sensing, cell respiration and metabolism [START_REF] Tsiftsoglou | Heme as key regulator of major mammalian cellular functions: molecular, cellular, and pharmacological aspects[END_REF]. Heme, whose expression occurs in a circadian manner [START_REF] Kaasik | Reciprocal regulation of haem biosynthesis and the circadian clock in mammals[END_REF]) can bind to both REV-ERB (Raghuram et al. 2007).

Heme binding to REV-ERBs facilitates recruitment of the nuclear corepressor NCoR [START_REF] Yin | The orphan nuclear receptor Rev-erbalpha recruits the N-CoR/histone deacetylase 3 corepressor to regulate the circadian Bmal1 gene[END_REF]Raghuram et al. 2007). The rate-limiting enzyme in heme biosynthesis Aminolevulinate synthase 1 (Alas1), is directly controlled by the clock [START_REF] Zheng | Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock[END_REF]. Heme biosynthesis is also influenced by the nutritional status through the regulation of Alas1 by the PPARγ coactivator 1α (PGC-1α) (Handschin et al. 2005). This nuclear receptor coactivator is induced by fasting [START_REF] Handschin | Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha[END_REF]) and participates in a variety of metabolic pathways such as glucose and lipid regulation [START_REF] Lin | Metabolic control through the PGC-1 family of transcription coactivators[END_REF]. Moreover, both PGC-1α and heme are able to modulate the expression of circadian genes [START_REF] Dioum | NPAS2: a gas-responsive transcription factor[END_REF][START_REF] Kaasik | Reciprocal regulation of haem biosynthesis and the circadian clock in mammals[END_REF]Liu et al. 2007). These results illustrate how REV-ERBs can sense dynamic metabolic changes and transmit them to the core clock machinery.

PPARs are members of the nuclear hormone receptor superfamily of ligandactivated transcription factors. The three PPARs (α, δ, γ) have been shown to regulate carbohydrate, lipid, lipoprotein and energy metabolism [START_REF] Gervois | Regulation of lipid and lipoprotein metabolism by PPAR activators[END_REF][START_REF] Yoon | The role of PPARalpha in lipid metabolism and obesity: focusing on the effects of estrogen on PPARalpha actions[END_REF]). PPARα is rhythmically expressed in tissues with high fatty acid catabolism rates such as the liver and adipose tissue. PPARγ is rhythmically expressed in liver and WAT, while PPARδ expression oscillates in liver and brown adipose tissue (BAT) [START_REF] Yang | Nuclear receptor expression links the circadian clock to metabolism[END_REF]. PPARα is likely to play a role in connecting circadian physiology to metabolism. Pparα-deficient mice have normal rhythmic behavior and clock gene expression in the SCN, but PPARα deficiency alters Bmal1

and Per3 expression in the liver. PPARα directly regulates hepatic Bmal1 expression through a response element located in its promoter [START_REF] Canaple | Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock[END_REF]). Besides, it has been shown that Pparα is a clock-controlled gene (Oishi et al. 2005) and daytime feeding inverts its circadian expression pattern in the liver [START_REF] Canaple | Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock[END_REF].

Interestingly, PPARα is also connected to the nuclear receptor Rev-erbα because the PPARα agonists (fibrates) induce liver expression of Rev-erbα. In addition, PPARα (as well as PPARγ) can directly transactivate Rev-erbα via RevRE located in its promoter [START_REF] Vu-Dac | The nuclear receptors peroxisome proliferator-activated receptor alpha and Rev-erbalpha mediate the species-specific regulation of apolipoprotein A-I expression by fibrates[END_REF][START_REF] Gervois | Fibrates increase human REV-ERBalpha expression in liver via a novel peroxisome proliferator-activated receptor response element[END_REF][START_REF] Fontaine | The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation[END_REF]. Clearly, all these data demonstrate that the metabolic sensor PPARα may play a prominent role Interestingly, PGC-1 (PPARγ coactivator 1, as introduced above), which is involved in thermogenesis and associated metabolic responses [START_REF] Puigserver | A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis[END_REF]Liu et al. 2007), is rhythmically expressed in phase with PPARγ, PPARδ and thermogenic genes such as Ucp1 and Thyroid receptor α (Trα), in BAT [START_REF] Yang | Nuclear receptor expression links the circadian clock to metabolism[END_REF]).

Furthermore, PGC-1 can specifically bind PPARγ [START_REF] Puigserver | A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis[END_REF]), whereas the latter can also interact with Ucp-1 promoter [START_REF] Sears | Differentiation-dependent expression of the brown adipocyte uncoupling protein gene: regulation by peroxisome proliferator-activated receptor gamma[END_REF]. All these findings suggest that PPARs, could transmit (diurnal) changes in heat dissipation to the circadian system.

Based on these data, nuclear receptors are controlling an amazing array of metabolic functions and are capable of communicating the body metabolic state to the core clock machinery. Incidentally, other nuclear receptor subgroups also participate in the interplay between metabolic and circadian physiology (e.g., Retinoid X receptor, Liver X receptor and Estrogen-related receptor) (for reviews, see [START_REF] Yang | Nuclear receptor expression links the circadian clock to metabolism[END_REF][START_REF] Teboul | The nuclear hormone receptor family round the clock[END_REF]Asher and Schibler 2011). In the oscillator, both PER proteins act as repressors of E-box-mediated circadian transcription via interaction with BMAL1 and CLOCK. In addition, PER2 can modulate NRE-mediated transcription via interaction with nuclear receptors. This affects expression of Bmal1 in the oscillator and target genes. These output targets can be modulated via E-boxes, NREs, or both. PER2-nuclear receptor interactions may be involved in this regulatory process (hatched arrows) to coordinate clock output processes. Modified from (Schmutz et al. 2010)

Nutrient sensors interact with clock components

The tight connection between the circadian timing system and metabolism is also underscored by the role of nutrient sensors. Clock adaptation to metabolic reactions is likely to be achieved also by direct input of nutrient sensing regulators to the intrinsic clock machinery.

Recent evidence has demonstrated that local changes in cellular energy, such as redox reactions (portmanteau for reduction-oxidation), can influence the circadian expression of core clock genes and clock-related nuclear receptors. Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH, are coenzymes found in all living cells. NAD+ also exists in a phosphorylated form NADP+ and can be reduced (Only unphosphorylated NAD electron carriers are shown.). From [START_REF] Schibler | Circadian rhythms. Chronobiology--reducing time[END_REF] These results raise the possibility that cell energy metabolism can influence circadian rhythmicity. For example, these clock protein sensors might participate in the rhythm imposed by the alternation of fasting-feeding. Indeed, NAD:NADH ratio is modified under fasting and feeding conditions since starvation lowers NADP:NADPH (i.e., shift toward a reduced state) in both rat liver mitochondria and cytoplasm [START_REF] Williamson | The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver[END_REF]. Thus, for feeding synchronization, it is plausible that the cellular redox status could transmit changes in energy metabolism directly to the clock architecture. In this context, it is tempting to think that irregular mealtimes could also contribute to circadian misalignment by subtle perturbations of local circadian oscillator functioning.

Sirtuin 1 (SIRT1), another energy sensor, has recently been found to link circadian to metabolic physiology. SIRT1 is a NAD-dependent histone deacetylase that contributes to epigenetic gene silencing and a plethora of biological processes ranging from gluconeogenesis, insulin secretion and sensitivity, lipid regulation, mitochondrial activity and thermogenesis, adipogenesis and adipocyte differentiation, apoptosis, to caloric restriction-dependent life span extension [START_REF] Blander | The Sir2 family of protein deacetylases[END_REF][START_REF] Dali-Youcef | Sirtuins: the 'magnificent seven', function, metabolism and longevity[END_REF][START_REF] Yu | The role of sirtuins in the control of metabolic homeostasis[END_REF]. SIRT1, whose expression occurs in a circadian manner, has been shown to influence circadian transcription of several clock genes (e.g., Per2, Cry1, Bmal1 and Rorα) [START_REF] Asher | SIRT1 regulates circadian clock gene expression through PER2 deacetylation[END_REF]).

Moreover, SIRT1 can directly bind CLOCK:BMAL1 heterodimers and promote deacetylation of PER2 [START_REF] Asher | SIRT1 regulates circadian clock gene expression through PER2 deacetylation[END_REF]), BMAL1 and histone H3 [START_REF] Nakahata | The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control[END_REF]). Interestingly, SIRT1 can interact with and deacetylate PGC-1α to control the expression of gluconeogenic and glycolytic genes [START_REF] Rodgers | Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1[END_REF]). In addition, hepatic SIRT1 is able to modulate the expression of PPARα [START_REF] Purushotham | Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation[END_REF]). Importantly, SIRT1 can also repress the fat regulator PPARγ and it is activated during fasting to promote fat mobilization in WAT, again demonstrating its clear implication in lipid homeostasis [START_REF] Picard | Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma[END_REF]). Furthermore, SIRT1 has been shown to be decreased in adipose tissue endothelial cells from obese human subjects [START_REF] Villaret | Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence[END_REF]. Of interest, since fat accumulation is associated with several adverse complications, such as diabetes and hypertension, caloric restriction (and consequent fat depletion) which has multiple biological and life-extending benefits [START_REF] Barzilai | The role of fat depletion in the biological benefits of caloric restriction[END_REF], may be used to prevent or treat metabolic disruptions.

In addition to NAD(P)+ and SIRT1, AMP-activated protein kinase (AMPK) is a further important nutrient sensor. AMPK is sensitive to fluctuations in the cellular AMP:ATP ratio and can be activated by various factors such as exercise, glucose deprivation or leptin treatment. The functions of AMPK cover the whole-body energy balance (e.g., food intake, body mass, lipid and glucose homeostasis, cholesterol and triglyceride synthesis, energy expenditure) [START_REF] Kahn | AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism[END_REF]. In mouse skeletal muscle and cultured myotubes, AMPK has been demonstrated to regulate genes involved in energy metabolism, by acting in coordination with SIRT1. Interestingly, AMPK activation increases NAD+ levels, which in turn enhance SIRT1 activity, resulting in the deacetylation and activation of the downstream SIRT1 target PGC-1α [START_REF] Canto | AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity[END_REF]). Deletion of the AMPKγ3 subunit in mice leads to impaired expression profiles of clock genes in skeletal muscle in response to the AMPK activator AICAR (5-amino-4-imidazole-carboxamide riboside), and attenuated daily variations of the respiratory exchange ratio [START_REF] Vieira | Relationship between AMPK and the transcriptional balance of clockrelated genes in skeletal muscle[END_REF]. AMPK also has direct actions on the clock machinery. AMPK phosphorylates and destabilizes CRY1 [START_REF] Lamia | AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation[END_REF]). Casein kinase Iε, an important regulator of PER proteins stability, is also phosphorylated by AMPK which induces subsequent degradation of PER2 and phase-shifts of peripheral oscillators [START_REF] Um | Activation of 5'-AMP-activated kinase with diabetes drug metformin induces casein kinase Iepsilon (CKIepsilon)-dependent degradation of clock protein mPer2[END_REF]). Recently, it has been demonstrated that mice deficient for either AMPKα1 or AMPKα2 have altered circadian feeding behavior and free-running period [START_REF] Um | AMPK regulates circadian rhythms in a tissue-and isoform-specific manner[END_REF]. Surprisingly, the rhythmic gene expression of leptin, PGC-1α and nicotinamide phosphoryl-transferase (NAMPT), a rate-limiting enzyme in the NAD+ salvage pathway regulated by CLOCK:BMAL1 [START_REF] Nakahata | Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1[END_REF][START_REF] Ramsey | Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis[END_REF], was abolished in AMPKdeficient mice [START_REF] Um | AMPK regulates circadian rhythms in a tissue-and isoform-specific manner[END_REF]). This study reveals that AMPK is to some extent involved in the cycling of NAMPT-(NAD+)-SIRT1-PGC-1α pathway. In addition, prolonged activation of SIRT1 by its agonist SRT1720 causes an indirect activation of AMPK, and both SIRT1 and AMPK respond to low-energy levels [START_REF] Bordone | Calorie restriction, SIRT1 and metabolism: understanding longevity[END_REF][START_REF] Kahn | AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism[END_REF]. Therefore, SIRT1 and AMPK may have overlapping functions to ensure the fine-tuning of metabolic and clock regulations.

Finally, it is important to mention that the mammalian molecular circadian clockwork (based on transcriptional-translational events) overviewed in this section, is not a unique cellular clock model. Indeed, circadian redox rhythms of peroxiredoxins, which are antioxidant enzymes, can occur independently of transcription, thus defining a metabolic non-transcriptional oscillator (O'Neill and Reddy 2011).

General conclusion

At the molecular/cellular levels, the recent findings cited above highlight multiple transcriptional crossroads between circadian and metabolic pathways in organs involved in metabolism, namely liver, adipose tissue, pancreas and muscle.

Nuclear receptors such as Rors and Rev-erbs are well-situated to receive metabolic signals and integrate them into the core clock architecture. In addition, fluctuations in cellular metabolism can directly influence the transcriptional activity of core clock components such as Cry1, Per2, Clock and Bmal1. This can be of importance to adapt behavior and physiology to situation where food availability is limited in time.

At the level of the organism, the circadian timing system provides internal temporal organization controlled locally by brain and peripheral oscillators-including the FEO network-that can be reset by feeding time, and supervised by the master SCN pacemaker mainly reset by ambient light. Impairment of this internal timing due to altered endogenous (local) clockwork or misalignment with external cues, light and/or mealtime, has deleterious impact on health.

[…} the hierarchically dominant position of the SCN is impressive, but limited. We like to think of it as an orchestra's conductor, using a large repertoire of gestures to set the timing of individual musicians in playing their instruments. However, the success of the symphony not only depends on the signs given by the director, but also on the capacity of the individual musicians to play their parts. [START_REF] Schibler | A web of circadian pacemakers[END_REF]

Aim of the thesis

Role of Rev-erbα in the clockwork of food anticipation

During my PhD thesis, which started in October 2008, I was involved in the study of the food-entrainable oscillator (FEO). In terms of molecular machinery, one of the current predictions is that food-entrainable oscillations are generated by transcriptional/translational feedback loops similar to those already described in the SCN. In this context, several mutant and KO mice models were used to test this hypothesis and Feillet and colleagues in 2006 gave strong evidence that Per2 is a circadian gene critical for behavioral food anticipation. This finding was a first step to better understand the functioning of the FEO. However, the other key components of the clockwork involved in food anticipation remained to be identified. Thus, we assessed the involvement of the nuclear receptor Rev-erbα as a putative circadian regulator of food-entrainable oscillations. As outlined in the introduction, Rev-erbα is a transcriptional repressor that is expressed in a circadian manner (mRNA acrophase ZT6-10, Fig. 53) in all tissues studied up to now. Furthermore, Rev-erbα has been demonstrated to regulate several clock components including, Clock, Npas2 and Bmal1. In addition, the depletion of Rev-erbα in vivo was shown to shorten the SCN period length and disrupt the shifting response to light. All these findings thus support a role for Rev-erbα as a modulator of circadiandependent functions and make it an ideal candidate for the integration of environmental changes into the core clock machinery during food synchronization. As a model to investigate further the FEO, we used mice KO for Rev-erbα (kindly provided by Pr. Ueli Schibler and raised in our facility). WT and KO mice, with or without a functional SCN, were exposed to schedules of restricted feeding in different lighting conditions. We analyzed multiple food-entrained rhythms such as general locomotor and wheel-running activities, body temperature and corticosterone.

We also studied the expression of PER2, considered as a component of the FEO clockwork, and p-ERK proteins in different brain areas, both in ad libitum (AL) and restricted feeding (RF) conditions. Since the liver oscillator plays a role in food synchronization, we evaluated the hepatic expression of clock and clock-controlled genes in response to RF challenge (Fig. 54).

Figure 54. Study of the food-entrainable oscillator network in Rev-erbα KO mice

We assessed the effect of scheduled feeding on central oscillators as well as the liver oscillator. Several behavioral and physiological food-entrained rhythms were evaluated.

Role of Rev-erbα in energy homeostasis

As highlighted in the introduction, the nuclear receptor Rev-erbα participates in several molecular pathways, especially in the cross-talk between the circadian system and metabolism. Indeed, Rev-erbα is highly expressed in peripheral tissues essential for energy homeostasis such as the liver, adipose tissue, skeletal muscle.

In addition, this gene was shown to regulate the transcription of metabolic actors involved in lipid metabolism.

Besides, REV-ERBα expression has been demonstrated to be stabilized by the glycogen synthase kinase 3 beta (GSK3β). Heme binding to REV-ERBs can facilitate the recruitment of the nuclear corepressor NCoR, which in turn increase the capacity of REV-ERBα to recruit the repressive chromatin modifier HDAC3 to the genome (Fig. 55). Of importance, GSK3β, heme and HDAC3 are all involved in particular in energy homeostasis.

Figure 55. The nuclear receptor Rev-erbα and its partners

Rev-erbα transcription is controlled in particular by the CLOCK-BMAL1 heterodimer. REV-ERBα is regulated at the post-translational level by glycogen synthase kinase (GSK 3β), thereby regulating its stability. REV-ERBα protein can mediate repression by recruiting the nuclear corepressor NCoR1, which then activates the histone deacetylase (HDAC) 3. Heme binding to REV-ERBα facilitates the recruitment of NCor1. As a result, genes that contain a RORE sequence in their promoter are repressed.

Hence, we started in the early 2009 to gain insight into the role of Rev-erbα in energy homeostasis in vivo, since there was no report at this time on the metabolic adaptation of the Rev-erbα-deficient mice to various feeding challenges. Our investigation on energy balance was also justified by the fact that the use of clock mutants to study food entrainment implies a necessary exploration of their metabolic phenotype before drawing robust conclusions.

In this context, we explored daily metabolism of carbohydrates and lipids in chow-fed, fasted or high-fat-fed Rev-erbα KO mice and their WT littermates. In particular, we assessed the adaptation of both genotypes to different nutritional challenges by evaluating behavioral, physiological and metabolic parameters. We have also evaluated the functioning of metabolically active tissues that are themselves peripheral oscillators, including the liver, adipose tissue and muscle, at the molecular levels (Fig. 56).

Figure 56. Study of energy homeostasis in Rev-erbα KO mice

We analyzed the physiological and molecular responses of multiple metabolic tissues in response to different feeding challenges (i.e., normocaloric versus high-fat feeding, fasting versus highcarbohydrate refeeding challenge).

Chapter 2 RESULTS

The Rev-erb clock gene participates in the circadian clockwork of food anticipation

INTRODUCTION

In mammals, the circadian timing system is composed of several endogenous oscillators. The suprachiasmatic nucleus (SCN), the central clock located in the anterior hypothalamus, is principally synchronized to the environmental light-dark cycle. The SCN in turn controls the daily rhythmicity of many aspects of physiology and behavior. The molecular machinery of the SCN clock is based on transcription/translation feedback loops of clock gene expression. Among them, Bmal1, Clock, Period 1-3 (Per1-3), Cryptochrome 1-2 (Cry1-2), Ror,β and Rev-erb,β are essential (Crumbley and Burris, 2011;Guillaumond et al., 2005;Preitner et al., 2002). Secondary oscillators, whose molecular mechanisms are close to the SCN, are also present in multiple brain regions and peripheral tissues (Dibner et al., 2010).

Besides the light-dark cycle, feeding is a potent synchronizer for the circadian system as well (Damiola et al., 2000;[START_REF] Hara | Restricted feeding entrains liver clock without participation of the suprachiasmatic nucleus[END_REF]Stokkan et al., 2001). When food availability is limited to a regular time window each day, rodents can readily adapt those schedules and predict the time of food access. They develop food-seeking behaviors a few hours in anticipation of meal access, concomitant with physiological and hormonal activations (Mistlberger, 1994). In addition, RF-induced adaptation is highlighted by the phaseadjustment of clock-related gene expression in several peripheral tissues (Damiola et al., 2000).

Food-anticipatory rhythms have the properties of a clock-controlled process: it occurs gradually (1 to 3 h before mealtime); it persists over days in the absence of food; it is expressed only if food access is given within the circadian range (22-31 h); and it shows transient cycles when food is presented at a new phase (Mistlberger, 1994). Besides, food anticipation survives the ablation of the SCN clock [START_REF] Marchant | Anticipation and entrainment to feeding time in intact and SCN-ablated C57BL/6j mice[END_REF]Stephan et al., 1979). Altogether, these data indicate that food anticipation is an output of a circadian food-entrainable oscillator (FEO), likely located in the brain (Davidson et al., 2003).

The identification of the neural substrate and the molecular basis of the so-called FEO proved to be a real challenge these last 30 years (for reviews, see Challet et al., 2009;Mistlberger, 2011). In particular, the SCN-like molecular mechanism hypothesis at the basis of food entrainment is currently not fully supported. Few studies gave evidence that clock components (i.e., Per2 and Bmal1) may be essential for the FEO molecular clockwork (Feillet et al., 2006;Mieda and Sakurai, 2011). However, the implication of other canonical clock genes (e.g., Per1, Clock, Npas2, Cry1-2) has been revealed mild (Dudley et al., 2003;Horikawa et al., 2005;Iijima et al., 2005;Mendoza et al., 2010a) or even non significant (Feillet et al., 2006;Pitts et al., 2003;Storch and Weitz, 2009) Therefore, in the current study we sought to evaluate the role of the nuclear receptor Rev-erbα in the molecular clockwork of the FEO. For that purpose, we used mice lacking the Rev-erbα clock gene (knockout, -/-) and their control littermates (wild-type, +/+). Both genotypes were maintained in a regular 12 h:12 h light-dark cycle (LD) or constant darkness (DD) with food available ad libitum (AL) and then challenged with scheduled RF. Wheelrunning behavior, general activity and body temperature were recorded. We also investigated the effect of the daily feeding schedules on energy balance and hepatic clock genes oscillations. The expression of PER2 and p-ERK proteins was also determined in brain regions such as the dorsomedial hypothalamus and cerebellum, assumed to play a role in food entrainment (Acosta-Galvan et al., 2011;Gooley et al., 2006;Mendoza et al., 2010b).

METHODS

Animals and housing conditions

The founder Rev-erbα +/-mice kindly provided by Prof. Ueli Schibler (University of Geneva, Switzerland) were rederived on a C57BL6/J background (Charles River Laboratories, France)

and backcrossed until N5 in our local animal care facilities (Chronobiotron UMS 3415, Strasbourg). The Rev-erbα deletion strategy is described in (Preitner et al., 2002)

Genotype determination

Genotyping was performed by PCR using tail biopsy. Briefly, tissue was digested with NaOH

Locomotor activity and body temperature recordings

Mice were implanted intraperitoneally (i.p.) under constant gaseous anesthesia (oxygennitrogen protoxide with a constant rate at 0.2 L/min and isoflurane at 2%) with a small transponder (G2 E-Mitter, Mini Mitter, Bend, Oregon, USA) allowing the continuous recordings of gross locomotor activity (general activity hereafter) and core body temperature. A PC-based acquisition system (VitalView, MiniMitter) recorded the aforementioned parameters plus the wheel-running activity 24 hours a day.

Experimental procedures

Independent experiments were conducted with distinct cohorts of mice (sex ratio about 1:1). Mice were 2-to 5-month old at the beginning of each experiment and body mass and food intake were determined weekly.

LD conditions: Rev-erbα +/+ and -/-mice (4-month old, n = 8-12 / genotype) fed on an AL basis for several weeks post-weaning were exposed to RF schedules in which food availability was reduced to 6 h per day, from ZT6 to ZT12 (ZT refers to Zeitgeber Time and ZT0 and ZT12 are defined as time of lights-on and lights-off, respectively) for up to 3 weeks. Note that this experiment was reproduced with another set of mice (n = 9 / genotype) and that additional age matched AL-fed animals (n = 20 / genotype) were kept in LD for further study of the rhythmic expression of metabolic parameters, clock gene expression in the liver and PER2

and p-ERK expression in the brain (see results).

DD conditions: Rev-erbα +/+ and -/-mice (5-months old, n = 6 / genotype) were placed in DD conditions and fed AL and then challenged with a RF paradigm in which food was given for 6 h from 02:00 p.m. to 08:00 p.m for 4 weeks.

DD conditions and SCN lesions: Rev-erbα +/+ and -/-mice (2 to 4-months old, n = 9-12 / genotype) maintained in LD conditions, were SCN-lesioned and immediately placed several weeks in DD for recovery. Mice were then exposed to temporal RF for which the food access was limited to 6 h per day for up to 3 weeks (from 02:00 p.m. to 08:00 p.m.).

SCN lesions

2 to 4-month old mice (n = 9-12 / genotype) were anesthetized with ketamine-xylazine (100 mg/kg-10 mg/kg, respectively) and placed in a stereotaxic apparatus. A lesioning electrode (0.25 mm shaft diameter, 0.1 mm lead/contact diameter; SNEX-300, Rhodes Medical Instruments) was inserted into the SCN (0.5 mm posterior to bregma, 0.2 mm lateral to midline, 5.7 mm ventral to dura, incisor bar 0 mm below ears bars). Bilateral electrolytic lesions were generated with constant current (1.5 mA, 20 s; Lesion making device 53500, Ugo Basile, Italy). During the surgery procedure, animals were placed on heating pad (Harvard Apparatus, France) and treated with antiseptics and lidocaine 5%. After recovery from anesthesia, mice were weighted and injected i.p. with Metacam (1 mg/kg) and transferred back into their cages. Arrhytmicity was later assessed by evaluating locomotor activity with X² periodogram. Animals were killed at the end of the RF paradigm, and brains were removed and immediately frozen on dry ice. Lesions were confirmed by cresyl violet staining and only mice with complete SCN lesions were included in the analysis.

Procedure for tissue and blood collections

Mice (6-month old) from LD experiments, either on AL or RF conditions, were sacrificed at ZT0, ZT6, ZT12 and ZT18. Briefly, mice were injected with a lethal dose of pentobarbital, a blood sample was taken intracardially and two pieces of liver were cut and immediately flash-frozen in liquid nitrogen. Then animals were perfused transcardially with 50 mL PBS 1X

(Phosphate-buffered saline, pH 7.2) and 50 mL PFA buffer (4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4). Brains were removed and post-fixed in 4% PFA for 24 h at 4°C and cryoprotected successively for 24 h at 4°C into 10, 20 and 30% sucrose solution in 0.1M PB.

Brains were then frozen in isopentane at -30 to -40°C and then stored at -80°C.

Metabolic measurements

The blood glucose rhythm was determined in mice from repeated tail blood microsamples (<1 µL) using a Glucotrend®Premium (Roche Diagnostics, Germany). Plasma glucose was evaluated with GOD-PAP Kit (BIOLABO, Maizy, FRANCE). Plasma corticosterone was measured using an EIA kit (AC-14F1, IDS, Paris, FRANCE). Plasma β-Hydroxybutyrate concentrations were determined with a Cyclic Enzymatic Method (Autokit 3-HB, Wako, Japan). Hepatic glycogen was determined following the methods of Murat and Serfaty (Murat and Serfaty, 1974).

Immunohistochemistry

Coronal frozen brain sections (30 µm thick) of the hypothalamus and the cerebellum were made using a cryostat. Free-floating sections were rinsed in PBS 1X and incubated in a [START_REF] Guillaumond | Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors[END_REF]. cDNAs were synthesized from 1 µg of total RNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems).

solution
Quantitative Real-time PCR was performed and analyzed using an Applied Biosystems 7300

Real-time PCR System with 1X TaqMan Gene Expression Master Mix (Applied Biosystems), 1X TaqMan Gene Expression Assay (Applied Biosystems, see references below) and 1 µL of cDNA in a total volume of 20 µL. PCR conditions were 10 min at 95°C followed by 40 cycles of 15 s at 95°C, 1 min at 60°C. PCR reactions were done in duplicate and negative controls (i.e., no-reverse transcription and no-template controls) were added to the reactions. Relative expression levels were determined using the comparative ΔC T method to normalize target gene mRNA to β-actin (reference below). A dilution curve of the pool of all cDNA samples was used to calculate the amplification efficiency for each assay (values were between 1.9 and 2). The following TaqMan Gene Expression Assays were used: β-actin

(Mm01205647_g1), Bmal1 (Arnt1, Mm00500226_m1), Clock (Mm00455950_m1), Dbp (Mm01194021_m1), Per1 (Mm00501813_m1) and Per2 (Mm00478113_m1).

Analysis of locomotor activity and temperature data

Locomotor activity data were double-plotted in actograms using a 5 min block size. Daily rhythms of general activity, wheel-running activity and body temperature were analyzed using a Clocklab software (Actimetrics, Evanston, IL, USA) associated to MatLab (MathWorks France). Mean activity profiles were quantified every 1 h during the last 10 days of the AL and RF conditions in the LD experiments, whereas for the DD experiment only a period of 5

days was taken into account to avoid overlap between the nocturnal activity controlled by the SCN and the food-anticipatory activity. To determine the free-running period in animals in DD conditions, with or without SCN, X² periodogram was used.

Statistical analysis

All values are expressed as mean ± SEM.

RESULTS

Food anticipation is reduced in LD conditions in Rev-erbα -/-mice

We first determined if Rev-erbα deletion led to an impaired ability of mice to anticipate mealtime in LD conditions. No significant differences were found between genotypes fed AL regarding body mass and food intake (Figure S1A), 24-h body temperature rhythm (Figure 1B) and daily general locomotor activity (data not shown). The amount of daily wheel-running revolutions was not significantly different in Rev-erbα +/+ vs. Rev-erbα -/- mice exposed to LD (11665 ± 2382 vs. 7536 ± 2784, respectively; P = 0.27). For comparison purpose, data points were expressed as a percentage of total daily activity. The normalized circadian pattern of activity was similar in both genotypes placed in LD and fed AL (Figure 1A,B). During the scheduled RF, both genotypes showed a similar body mass (Figure S1A).

Moreover, body mass loss after one-week of RF was not different in both genotypes (+/+ = -8.09 ± 2.12% vs. -/-= -9.52 ± 3.07%; n.s.). As expected, Rev-erbα +/+ mice expressed a strong FAA 1 h (from ZT5 to ZT6) before meal access, illustrated by significant increases of wheelrunning and general activities. In contrast, Rev-erbα -/-mice showed a reduced, albeit non significant (P = 0.09), percent elevation of wheel-activity 1 h prior to mealtime (Figure 1C,D).

This result was further confirmed by the amount of general activity in Rev-erbα -/-mice, that did not vary significantly with time and was significantly decreased in anticipation, in comparison to Rev-erbα +/+ controls (Figure 1C,D). Moreover, a clear rise in body temperature a few hours before food arrival was observed in control animals, whereas in Rev-erbα -/-mice, thermogenesis in anticipation was nearly absent (Figure 1C,D). Of note, LD experiment was reproduced with an additional series of mice for the metabolic and mRNA/protein expression analyses presented thereafter. Their results as well as those of Rev-erbα +/-mice, which demonstrated similar increases of food-anticipatory components than Rev-erbα +/+ mice, are shown in Figure S2.

FAA is nearly absent in Rev-erbα -/-mice exposed to constant darkness

Under LD conditions, it was difficult to exclude a potential masking effect of light that could contribute to the reduced food anticipation in Rev-erbα -/-mice. We therefore evaluated food-anticipatory parameters in DD conditions. During AL access to food, body mass and food intake (Figure S1B), and total daily amount of general activity were similar between both genotypes (data not shown), while total wheel-running activity was significantly decreased in Rev-erbα -/-mice compared to Rev-erbα +/+ animals (5912 ± 2440 vs.

15886 ± 2753, respectively; P = 0.018). As in LD conditions, Rev-erbα +/+ mice displayed a significant elevation of both wheel-running and general activities 1 h prior to food access, whereas Rev-erbα -/-mice demonstrated a significant reduction of FAA (Figure 2A,B).

Moreover, the sharp preprandial rise in body temperature seen in control animals was not observed in Rev-erbα -/-mice, although the postprandial peak of body temperature occurred in both genotypes, thus indicating active food processing (Figure 2B). To further confirm the failure of Rev-erbα -/-mice to express a strong FAA, we performed SCN lesions (SCNx) in order to abolish the nocturnal activity component controlled by the SCN that could obscure food anticipation in DD. In control animals with successful SCN lesion, FAA was recognizable in some Rev-erbα +/+ animals (n = 4, Figure 2C), but not all (n = 4). However, none of SCNx Reverbα -/-mice displayed significant FAA in response to food restriction (n = 5, Figure 2C). Of note, food-anticipatory wheel-running activity was also diminished in Rev-erbα -/-mice exposed to a skeleton photoperiod (data not shown). Thus, it was clear that, regardless of the lighting conditions, the time of the day when food was provided and the presence of a functional SCN pacemaker, the expression of FAA in Rev-erbα -/-mice was markedly reduced.

Corticosterone and ketone body levels are decreased in anticipation in Rev-erbα -/-mice

We then decided to evaluate the energy balance in both genotypes fed AL or exposed to RF in LD conditions. 24-h plasma glucose levels were significantly higher in Rev-erbα -/- mice compared to their control littermates fed AL. This significant hyperglycemia was not detected when food access was restricted to 6 h during daytime, despite elevated plasma glucose levels at ZT6 and ZT18 in Rev-erbα -/-mice (Figure 3A). In addition, we observed that hepatic glycogen levels were also significantly higher in Rev-erbα -/-mice at ZT12, which correspond to the end of the resting period (Figure 3B). While RF induced a change in the daily rhythm of glycogen levels in both genotypes, significant higher values were also detected at ZT6 (i.e., prior to mealtime) in Rev-erbα -/-mice compared to Rev-erbα +/+ mice (Figure 3B). We then measured plasma ketone bodies, an important source of energy during starvation, which have been demonstrated to increase prior to food access in food-restricted rats [START_REF] Escobar | Persistence of metabolic rhythmicity during fasting and its entrainment by restricted feeding schedules in rats[END_REF]. In contrast to the absence of significant difference between both populations fed AL, however, plasma ketone bodies concentrations were significantly reduced in Rev-erbα -/-mice in RF conditions, in particular, at ZT6 (i.e., before food arrival) and at ZT18 (i.e., after robust nocturnal activity) (Figure 3C). Taken together, these results could suggest that Rev-erbα -/-mice do not mobilize energy to the same extent as their control littermates. Previous studies have shown that a second peak of corticosterone appears in anticipation of food access in wild-type animals (Nelson et al., 1975). There was no significant difference in the 24-h corticosterone profiles between both genotypes, in spite of lower rhythm amplitude in Rev-erbα -/-mice (Figure 3D). In RF conditions, a strong rise of plasma corticosterone concentration emerged prior to food access in Rev-erbα +/+ mice. On the opposite, there was no significant change in corticosterone values of Rev-erbα -/-mice during scheduled RF at ZT6 when both AL and RF conditions are compared. As a result, ZT6 corticosterone levels were significantly lower in Rev-erbα -/-mice when compared to control animals (Figure 3D). Since the anticipatory peak of corticosterone is considered as an output of the FEO, the absence of a rise in corticosterone in Rev-erbα -/-mice further substantiates our behavioral observations.

Hepatic clock gene expression is shifted by RF in both genotypes in LD conditions

Since daytime feeding changes the phase of circadian gene expression in peripheral tissues (Damiola et al., 2000), we also evaluated whether the synchronizing effects of RF on the liver oscillator involves Rev-erbα. For that purpose, we analyzed the mRNA expression of key actors of circadian molecular oscillations in both genotypes. In line with a previous report (Preitner et al., 2002), we found that Bmal1 and Clock mRNA levels were up-regulated in Rev-erbα -/-mice (Figure 4A). As a consequence, Clock mRNA 24-h rhythmicity was even abolished in the absence of Rev-erbα. However, the circadian temporal pattern of Per1, Per2

and Dbp mRNA were not different between genotypes in AL conditions (Preitner et al., 2002; Figure 4C-E), in accordance with previous results. As expected, feeding time induced a 6-h phase shift of clock-related gene expression in control animals (Damiola et al., 2000;Stokkan et al., 2001) (Figure 4A-E). Food synchronization of hepatic oscillations also occurred in Reverbα -/-mice, however, no phase-shift of mRNA expression could be detected for Bmal1

(Figure 4A-E). This can be explained by the fact that the mRNA level of this clock component is severely up-regulated in the absence of Rev-erbα, and could therefore mask a change in their temporal expression. On the other side, Clock and Bmal1 genes may not be essential for hepatic food entrainment. In that respect, Rev-erbα does not appear either essential for phase-adjustment of peripheral oscillations, since 6-h phase-shifts or the core clock components Per1, Per2, as well as the crucial clock output Dbp were observed when it is lacking (Figure 4A-E).

Feeding time differently affected brain PER2 and p-ERK expression in Rev-erbα +/+ and Reverbα -/-mice

A number of extra-SCN oscillators that could be part of the FEO network are sensitive to changes in the temporal organization of feeding behavior as well as those in energy balance (Dibner et al., 2010;Feillet et al., 2008;Mendoza et al., 2010b). We therefore investigated PER2 expression in key hypothalamic areas in mice fed AL and under RF paradigm. We also used as a marker of neuronal activation, the phosphorylated form of the Extracellular signal Regulated Kinases I/II (p-ERK), to assess its expression in both genotypes in response to a scheduled feeding. We found that all hypothalamic structures showed daily rhythmic expression of PER2 in both genotypes (Figure 5A). Of note, the amplitude rhythm of PER2 expression in the PVN and VMH was fairly diminished in Rev-erbα -/-mice, while PER2 expression was 6-h phase-advanced in the DMH and ARC of Rev-erbα -/-mice (Figure 5A). No change was detected in the SCN which serve as an internal control to evaluate the reliability of our immunohistochemistry experiments (Figure 5A). Unexpectedly, adaptation of hypothalamic PER2 oscillations to food restriction, in particular in the DMH, was observed in both genotypes, as evidenced by an elevation of PER2 expression at time of food access (Figure 5A). Hence, as for the liver oscillator, feeding synchronization of hypothalamic oscillators, involved in particular in feeding regulation, does not seem to require the clock gene Rev-erbα. On the other hand, we analyzed p-ERK expression to identify relevant structures that may directly respond to food restriction. In AL conditions, we could not detect significant rhythmic pattern of p-ERK expression in hypothalamic regions when both genotypes were considered in the analysis (Figure 5B). However, our results indicate that the acrophase of p-ERK expression occurred around ZT12 in all hypothalamic nuclei in control animals, while p-ERK peak expression was less evident in the hypothalamus of Rev-erbα -/- mice. Interestingly, in response to RF, p-ERK expression peaked at mealtime in Rev-erbα +/+ mice. On the opposite, temporal changes in the expression of p-ERK in the hypothalamus of Rev-erbα -/-mice were hardly detected. Indeed, daily expression patterns were quite similar to those of AL conditions, in spite of a noticeable acrophase of p-ERK at the night-day transition in the SCN, PVN, VMH and DMH of Rev-erbα -/-mice. Besides that, only the ARC nucleus seemed to adjust to food access in Rev-erbα -/-mice (Figure 5B). In the cerebellum, implicated in food anticipation (Mendoza et al., 2010b), the temporal pattern of PER2 and p-ERK expression was undistinguishable in the two genotypes fed AL (Figure 5A,B). In response to daytime RF, PER2 expression was severely phase-advanced in control mice. Conversely, the shift in PER2 expression was not observed in Rev-erbα -/-mice (Figure 5A). In addition, p-ERK expression was clearly up-regulated during food access in control animals, while the trough of p-ERK expression occurred prior to mealtime in Rev-erbα -/-mice (Figure 5B).

DISCUSSION

In the present study, we evaluated the role of an essential actor of the circadian timing system, the nuclear receptor Rev-erbα, which has not been considered in the context of food anticipation. We show that Rev-erbα-deficient mice have disrupted food-entrainable circadian rhythms, in particular those of locomotor activity, body temperature and plasma corticosterone.

Our results indicate that Rev-erbα -/-mice display a significant reduction of FAA in LD conditions compared to control animals. This is further confirmed by our investigations in DD conditions, in animals bearing or not SCN lesions. The lack of food-seeking behaviors in Reverbα -/-mice was not linked to larger body mass loss, reduced food intake or delayed food intake during the 6-h daily access (data not shown). In accordance with behavioral results, a sharp rise in body temperature before food access occurred in Rev-erbα +/+ mice, whereas this anticipatory thermogenesis was nearly absent in the Rev-erbα -/-mice. Indeed, while control mice decreased their body temperature during food withdrawal, Rev-erbα -/- maintained body temperature values close to their respective values in AL feeding (Figure 1B,C). The absence of phase-and amplitude-adjustment of the body temperature rhythm in Rev-erbα -/-mice could reflect altered FEO influence on thermoregulation. However, relative normothermia was not observed in Per2 mutant mice that showed a drastic reduction of FAA and preprandial thermogenesis. On the contrary, Per2 mutant mice showed similar RFinduced drop of body temperature in late night hours than that in control littermates (Feillet et al., 2006). Hence, relative normothermia in Rev-erbα -/-mice could, instead, indicate that RF does not trigger to the same degree adaptive mechanisms to reduce energy expenditure as in wild-type littermates.

To further substantiate altered food entrainment in the absence of Rev-erbα, we measured hormonal and metabolic parameters, important for energy balance and that have been shown to increase prior to mealtime. In AL conditions, the 24-h plasma corticosterone profiles were not different between genotypes, suggesting that the adrenal peripheral oscillator (Oster et al., 2006) is likely unaltered in the absence of Rev-erbα. Interestingly, scheduled feeding affects the 24-h plasma corticosterone rhythm, with a shift of the acrophase of corticosterone at the night-day transition in Rev-erbα -/-mice, without changing the peak at the day-night transition in control animals. However, the food-anticipatory peak of corticosterone, considered as a strong output of the FEO (Nelson et al., 1975) was absent in Rev-erbα -/-mice. Plasma glucose and hepatic glycogen levels were higher in Rev-erbα -/- mice in LD conditions. Of note, REV-ERBα has been suggested to play a role in hepatic gluconeogenesis in vitro (Yin et al., 2007). Hence, altered expression of gluconeogenesic actors could contribute to the elevated plasma glucose levels in Rev-erbα -/-mice. Mild hyperglycemia and increased hepatic glycogen levels in Rev-erbα -/-mice were also observed in RF conditions prior to food access. Of interest, dietary glucose can affect the circadian properties of the FEO (Stephan and Davidson, 1998). Thus, abnormal circulating blood glucose levels could alter the FEO functioning. However, rats rendered diabetic by streptozotocine injection exhibit normal FAA [START_REF] Davidson | Food-anticipatory activity and liver per1-luc activity in diabetic transgenic rats[END_REF]. Therefore, the higher blood glucose levels and hepatic glycogen stores in Rev-erbα -/-mice could rather reflect decreased utilization of energy substrates to feed FAA-since the latter is clearly reduced in these mice. Nonetheless, we could not detect an elevation of ketone bodies levels in Reverbα -/-mice prior to mealtime in RF conditions. Since Rev-erbα -/-mice are not hypoactive indicated by measurements of general activity and maintain relative normothermia in RF conditions, whether the absence of a rise in ketone bodies results from an increased utilization or a decreased mobilization of this energy source has to be further investigated.

Our findings also confirm that the molecular functioning of the liver oscillator is not impaired in LD conditions with food provided on an AL basis in Rev-erbα -/-mice, as demonstrated by undistinguishable Per1-2, Cry1-2 and Dbp circadian pattern (present results; Preitner et al., 2002). In addition, we demonstrate that central clock components can be phase-shifted in response to limited food access in the absence of Rev-erbα, despite its suggested role in the acute response of the liver clockwork to feeding [START_REF] Tahara | Refeeding after fasting elicits insulin-dependent regulation of Per2 and Rev-erbalpha with shifts in the liver clock[END_REF].

Indeed, synchronization of the liver oscillator to feeding time was preserved in Rev-erbα -/- mice as demonstrated by a shift in Per1-2 and Dbp expression, suggesting that the disrupted FAA is not the consequence of impaired entrainment of the liver to scheduled feeding. Of note, the expression of clock-related genes in the liver of Per2 mutant mice was also shifted in response to daytime feeding (Feillet et al., 2006). Furthermore, mice with streptozotocininduced diabetes which demonstrate drastic elevation and reduction of serum glucose and insulin levels, respectively, displayed a similar phase shift of clock gene expression in peripheral tissues such as liver in response to RF [START_REF] Oishi | Gene-and tissue-specific alterations of circadian clock gene expression in streptozotocin-induced diabetic mice under restricted feeding[END_REF]. Thus, if liver dysfunctions contribute to altered energy status (i.e., high plasma glucose and low ketone bodies levels) in RF conditions, it is unlikely due to altered circadian oscillations.

We then investigated the expression of the core clock protein PER2 in key hypothalamic regions, involved in particular in feeding behavior and that exhibit circadian oscillations (Dibner et al., 2010;[START_REF] Horvath | The floating blueprint of hypothalamic feeding circuits[END_REF]. Indeed, on one hand, PER2

expression has been previously demonstrated to strongly respond to feeding time (Feillet et al., 2008). On the other hand, the DMH oscillator has been proposed as important for the expression of food-entrainable oscillations (Acosta-Galvan et al., 2011;Gooley et al., 2006).

Our results show that PER2 expression in all hypothalamic nuclei studied (i.e., SCN, PVN, VMH, DMH, ARC) was not significantly different between both genotypes either fed AL or challenged with RF. Therefore, the diminished capability of food entrainment in Rev-erbα -/- mice does not appear to be correlated with altered molecular functioning of central hypothalamic areas. Conversely, increased expression of a marker of neuronal activation such as c-FOS, before and after mealtime, has been observed in several hypothalamic nuclei, including the DMH and the VMH (Angeles-Castellanos et al., 2004;Gooley et al., 2006;Ribeiro et al., 2007). Of interest, our data on p-ERK expression, which is a marker of neuronal activation, indicate that all hypothalamic nuclei, including the SCN, demonstrated a phaseadjustment of p-ERK oscillations in control animals. On the contrary, the circadian pattern of p-ERK was only in phase with mealtime in the ARC of Rev-erbα -/-mice. Taken together, these results on p-ERK expression can suggest that hypothalamic regions are not activated to the same extent in Rev-erbα -/-mice compared to control animals during temporal food restriction.

It was reported recently that the cerebellum harbors a circadian oscillator sensitive to feeding schedules and that genetic and pharmacological impairment of the cerebellar function led to reduced or lacking FAA (Mendoza et al., 2010b). In this context, we also determined the expression pattern of PER2 and p-ERK proteins in the cerebellum of Reverbα -/-mice, since the latter exhibited reduced food-anticipatory components. Interestingly, we found no phase-adjustment of PER2 oscillations as well as a slight diminution of p-ERK expression in anticipation of eating in Rev-erbα -/-mice. Of interest, Per2 mRNA was not increased in anticipation to mealtime in Grid2 ho/ho mice that have genetic cerebellar deficits, and which demonstrated a lack of FAA (Mendoza et al., 2010b). Therefore, these results on PER2 and p-ERK expression in the cerebellum of Rev-erbα -/-mice could be a direct correlate to their defect to display strong food-anticipatory components.

The present findings indicate that mice lacking the Rev-erbα clock gene exhibit altered behavioral and physiological food anticipation, in addition to impaired p-ERK response to RF, implying that Rev-erbα may be a key component of the FEO. Our results could thus support the hypothesis that the basis of the FEO molecular mechanism is likely similar to those of well-known circadian oscillators, in line with previous studies (Dudley et al., 2003;Feillet et al., 2006;Iijima et al., 2005;Mieda and Sakurai, 2011). However, considering the strong involvement of Rev-erbα in metabolic regulations (Duez et al., 2008;Raspe et al., 2002;Yin et al., 2007), our results do not fully exclude that the decrease of FAA in Rev-erbα -/-mice could be linked to a metabolic defect at central or peripheral levels beyond preserved core clock gene oscillations. Therefore, the implication of Rev-erbα in metabolic processes in vivo has to be further explored. Supp. Fig. 1 Supp. Fig. 2 2

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

Altered circadian rhythmicity is a newly identified determinant of metabolic disorders in humans [START_REF] Froy | Metabolism and circadian rhythms-implications for obesity[END_REF]. Most aspects of behavior and metabolism display daily rhythms, including sleep-wake and feeding-nonfeeding cycles [START_REF] Kalsbeek | The hypothalamic clock and its control of glucose homeostasis[END_REF]. These daily variations are controlled by a circadian timing system made of interconnected clocks and oscillators. In mammals, the master circadian clock, located in the suprachiasmatic nuclei of the hypothalamus, is mainly reset by ambient light and synchronizes peripheral oscillations to 24 h. Secondary oscillators are present in many brain regions and peripheral organs (e.g., liver), and can be shifted by feeding-related cues [START_REF] Asher | Crosstalk between components of circadian and metabolic cycles in mammals[END_REF][START_REF] Zvonic | Characterization of peripheral circadian clocks in adipose tissues[END_REF][START_REF] Shibata | The adjustment and manipulation of biological rhythms by light, nutrition, and abused drugs[END_REF].

The molecular clockwork is based on autoregulatory transcriptional and translational feedback loops involving clock genes and proteins that generate a rhythmic transcriptional activity with a ϳ24 h period. In this clock network, two transcriptional activators, CLOCK and BMAL1, stimulate the expression of Period (Per1-3) and Cryptochrome (Cry1, 2) genes, whose proteins in turn repress the CLOCK-BMAL1 transactivation [START_REF] Ko | Molecular components of the mammalian circadian clock[END_REF]. In addition to these main components, the nuclear receptors ROR(␣,␤,␥) and REV-ERB(␣,␤) compete to activate and repress, respectively, the transcription of the Bmal1 and Clock genes, thereby reinforcing the robustness of circadian oscillations [START_REF] Guillaumond | Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors[END_REF][START_REF] Preitner | The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator[END_REF][START_REF] Crumbley | Direct regulation of CLOCK expression by REV-ERB[END_REF].

Approximately 10% of the mammalian transcriptome is under circadian regulation [START_REF] Panda | Coordinated transcription of key pathways in the mouse by the circadian clock[END_REF]. In particular, 1 Correspondence: Department of Neurobiology of Rhythms, Institute of Cellular and Integrative Neurosciences, CNRS UPR3212, University of Strasbourg, 5 rue Blaise Pascal, F-67084 France. E-mail: challet@inci-cnrs.unistra.fr doi: 10.1096/fj.12-208751 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

Abbreviations: AUC, area under the curve; DMEM, Dulbecco's modified Eagle's medium; GST, glucagon stimulation test; HDL, high-density lipoprotein; HFD, high-fat diet; IPIST, intraperitoneal insulin sensitivity test; LDL, low-density lipoprotein; MyHC, myosin heavy chain; NEFA, nonesterified fatty acid; OGTT, oral glucose tolerance test; PTT, pyruvate tolerance test; RLU, relative luminescence unit; RQ, respiratory quotient; VLDL, very low density lipoprotein; WAT, white adipose tissue; ZT, zeitgeber time metabolic processes are closely linked to circadian oscillations, as evidenced by the transcriptional control of genes involved in lipid and glucose metabolism by clock-related factors [START_REF] Asher | Crosstalk between components of circadian and metabolic cycles in mammals[END_REF][START_REF] Delezie | Interactions between metabolism and circadian clocks: reciprocal disturbances[END_REF]. Consequently, disruption of the circadian clockwork leads to profound changes in energy balance. Mutation of Clock leads to obesity [START_REF] Turek | Obesity and metabolic syndrome in circadian Clock mutant mice[END_REF], while pancreas-specific Bmal1-mutant mice develop diabetes [START_REF] Marcheva | Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes[END_REF]. On the other hand, diet-induced obesity affects the master clock as well as peripheral oscillations in wild-type mice [START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF]15) and exacerbates metabolic phenotypes in mice with deficient clocks [START_REF] Turek | Obesity and metabolic syndrome in circadian Clock mutant mice[END_REF][START_REF] Dallmann | Altered body mass regulation in male mPeriod mutant mice on high-fat diet[END_REF].

As outlined above, REV-ERB␣ is a component of the circadian clockwork that is expressed in the brain and peripheral tissues, such as liver, pancreas, adipose tissue, and muscle [START_REF] Preitner | The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator[END_REF][START_REF] Lazar | A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit[END_REF][START_REF] Vieira | The clock gene Rev-erbalpha regulates pancreatic betacell function: modulation by leptin and high-fat diet[END_REF]. Furthermore, REV-ERB␣ participates in the regulation of diverse metabolic pathways, including adipocyte differentiation, gluconeogenesis, bile acid synthesis, and cholesterol homeostasis (19 -22). Besides, REV-ERB␣ can modulate the expression of its own ligand, heme [START_REF] Wu | Negative feedback maintenance of heme homeostasis by its receptor, Rev-erbalpha[END_REF][START_REF] Raghuram | Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-erbbeta[END_REF], implicated in cellular metabolism. Daily expression of REV-ERB␣ has also been shown to control the circadian transcription of various lipid metabolism genes by recruiting the repressive chromatin modifier histone deacetylase 3 in the liver [START_REF] Feng | A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism[END_REF]. Taken together, these data suggest that REV-ERB␣ may tightly connect the circadian system to energy metabolism on a daily basis.

Here we show that mice lacking Rev-erb␣ display changes in daily energy homeostasis, leading to enhanced lipid fuel utilization and production during daytime and nighttime, respectively, and predisposing to diet-induced obesity. This occurs with constitutive elevation of the Lpl gene in peripheral tissues, likely due to the defective clock regulation of this gene.

MATERIALS AND METHODS

Animals and housing conditions

Rev-erb␣ ϩ/Ϫ mice, kindly provided by Prof. Ueli Schibler (University of Geneva, Geneva, Switzerland) were rederived on a C57BL6/J background (Charles River Laboratories, L'Arbresle, France) and backcrossed until N5 in our local animal care facilities (Chronobiotron, Strasbourg, France). Characterization of the genetic background of the strain by Charles River Laboratories indicated that mice were Ͼ95% C57BL6/J. The Rev-erb␣-deletion strategy is described in ref. [START_REF] Preitner | The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator[END_REF]. All mice were maintained under a 12-h light-dark cycle in a temperaturecontrolled room (22 Ϯ 1°C). Normocaloric chow diet (2.89 kcal/g, 14% kcal from fat, 27% kcal from protein and 59% from carbohydrates; SAFE 105; SAFE, Augy, France) and water were provided ad libitum unless specified otherwise. All experiments were performed in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996), the French National Law (implementing the European Communities Council Directive 86/609/EEC) and approved by the Regional Ethical Committee of Strasbourg for Animal Experimentation (CREMEAS).

Animal experiments

Behavior, physiology, and glucose homeostasis Mice (4 mo old, nϭ10/genotype, sex ratio 1:1) were used for measuring general locomotor activity and body temperature after intraperitoneal implantation with a small transponder (G2 E-Mitter; MiniMitter, Sunriver, OR, USA) under gaseous anesthesia (2% isoflurane in 50:50 O 2 /N 2 O). Locomotor activity and temperature data were recorded every 5 min, 24 h/d, with a PC-based acquisition system (VitalView; Mini-Mitter) and analyzed with Clocklab (Actimetrics, Evanston, IL, USA). Indirect calorimetry was performed on other mice (nϭ10/genotype, sex ratio 1:1) as described previously [START_REF] Fergani | A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1[END_REF] as well as the hepatocyte study (see procedure below). For glucose, insulin measurements and pancreas studies, mice (nϭ12/genotype, sex ratio 1:1) were sampled at zeitgeber time 0 (ZT0; lights on, onset of the resting/nonfeeding period) and ZT12 (lights off, onset of the active/feeding period).

Food withdrawal and refeeding experiments

Mice (4 mo old) were divided into 2 groups (4 males and 3 females/group/genotype); one group was unfed for 24 h starting at ZT0, and the other was unfed for 24 h and then refed for the next 24 h with a high-carbohydrate diet (3.63 kcal/g, 7.4% kcal from fat, 14.1% kcal from protein, and 78.5% kcal from carbohydrate; SAFE U8960v2). Both groups were killed by lethal injection of pentobarbital at ZT0. Blood samples were collected with 4% EDTA and centrifuged for 10 min at 5000 rpm at 4°C. Liver samples were flash-frozen in liquid nitrogen. Locomotor activity and body temperature were recorded as above.

High-fat-diet (HFD) challenge

Mice (4 wk old, sex ratio 1:1) were fed either with a chow diet (as described above; nϭ12/genotype) or an HFD (4.65 kcal/g, 53.2% kcal from fat, 14.6% kcal from protein and 32.2% kcal from carbohydrate, SAFE U8955v3; n ϭ 12/ genotype) for up to 12 wk. Then, animals were sacrificed at ZT0 and ZT12. Mice were unfed for 1 h before injection of a lethal dose of pentobarbital. Blood samples were collected as above. Liver, white adipose tissue (WAT; retroperitoneal and perigonadal), and rectus femoris muscle were taken and immediately flash-frozen in liquid nitrogen.

In vivo glucose homeostasis

The blood glucose rhythm was determined in mice fed ad libitum from repeated tail-blood microsamples (Ͻ0.5 l) using an Accu-Check glucometer (Roche Diagnostics, Meylan, France). For all metabolic tests, mice were unfed for 14 h (from ZT12 to ZT2) and injected at ZT2. For the oral glucose tolerance test (OGTT), mice received a glucose load via gavage (2 g/kg; d-glucose; Sigma, Saint Quentin Fallavier, France). For the glucagon stimulation test (GST) and pyruvate tolerance test (PTT), glucagon (1 mg/kg; GlucaGen; Novo Nordisk, Bagsvaerd, Denmark) and pyruvate (1.5 g/kg; P5280; Sigma) were administered intraperitoneally. Note that metabolic tests were separated by several days. The euglycemic clamp study was done by the Mouse Clinical Institute (MCI; Strasbourg, France) from ZT6 in animals previously unfed from ZT0 to ZT6 (see method in ref. [START_REF] Heikkinen | Evaluation of glucose homeostasis[END_REF].

Pancreatic histology and immunohistochemistry

Pancreases were fixed in Bouin's solution for 24 h and dehydrated successively in 70% ethanol, 2-ethoxyethanol, and butanol. The tissue was embedded in paraffin, and 8-m-thick serial sections were cut on a microtome and collected on gelatin-coated slides. For determination of islet area and circularity, sections were stained with Carrazzi's hematoxylin. To assess insulin and glucagon content, sections were washed with Tris buffered saline and incubated overnight with either mouse anti-insulin (1:50,000; clone HUI018; Novo Nordisk) or mouse anti-glucagon (1:60,000; clone GlU001; Novo Nordisk) antibodies. The sections were then rinsed and incubated for 1 h with a biotinylated secondary antibody (1:2000; Jackson ImmunoResearch, West Grove, PA, USA). Finally, streptavidin-peroxidase conjugate (1:2000; Roche) was added for 1 h after washing. Peroxidase activity was visualized with 0.5 mg/ml 3,3=-diaminobenzidine (Sigma) in the presence of 0.003% H 2 O 2 in 50 mM Tris buffer containing 10 mM imidazole (pH 7.6). Micrographs were taken on a Leica DMRB microscope (Leica Microsystems, Nanterre, France) with an Olympus DP50 digital camera (Olympus, Rungis, France) and analyzed with ImageJ software (W. S. Rasband, U. S. National Institutes of Health, Bethesda MD, USA).

Plasma metabolic parameters

Total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol levels were determined by a direct colorimetric method (Biolabo, Maizy, France). Plasma leptin was assayed with an ELISA kit (Leptin EZML-82K; Millipore, Molsheim, France). Plasma glucose was evaluated with a GOD-PAP kit (Biolabo). Plasma insulin was determined with an ultrasensitive mouse insulin ELISA kit (Crystal Chem, Downers Grove, IL, USA). Plasma glucagon was assayed with a glucagon RIA (GL-32K, Millipore) after adding aprotinin (250 kIU/ml) when blood samples were taken. Triglyceride concentration was determined by a triglyceride determination kit (TR-0100; Sigma). The ACS-ACOD method (NEFA-HR2; Wako, Osaka, Japan) was used for assaying plasma nonesterified fatty acids (NEFAs). Plasma ␤-hydroxybutyrate concentrations were determined with a cyclic enzymatic method (Autokit 3-HB; Wako).

Hepatic glycogen and triglycerides

Hepatic glycogen and triglycerides were assayed following the methods of Murat and Serfaty (28) and Miao et al. (29), respectively.

Primary hepatocyte culture

Livers sampled from adult mice at ZT2 to ZT4 were immediately perfused at the flow rate of 3 ml/min via the portal vein for 5 min with a calcium-free perfusion buffer (10 mM HEPES, 140 mM NaCl, and 6.7 mM KCl, pH 7.65), supplemented by 0.6 mM EGTA, followed by an additional 2 min with the Ca 2ϩ -free buffer, then 5 min with the same buffer containing 5 mM CaCl 2 and 15 mg/ml thermolysin (Liberase, medium research grade; Roche). All solutions were kept at 37°C. The livers were then excised, and hepatocytes were released by mechanical disruption of the liver capsule into 20 ml of Leibovitz L-15 medium (Invitrogen, Cergy-Pontoise, France) supplemented with 100 IU penicillin and 100 mg/ml streptomycin. The cells were filtered through a 70-m nylon mesh and centrifuged at 800 rpm for 2 min. The supernatant and cell debris were aspirated, and the cell pellet was washed 2 times into 10 ml of the same medium. The resulting cell pellet was finally resuspended in William's medium E with Glutamax (Invitrogen) containing 10% fetal bovine serum (Invitrogen), 100 IU penicillin, 100 mg/ml streptomycin, and 0.2 M insulin. Hepatocyte viability was assessed by trypan blue exclusion. Hepatocytes were placed at 2.5 ϫ 10 4 cells/ cm 2 in collagen-coated 35-mm plates and cultured for up to 6 h in the resuspended medium at 37°C in 5% CO 2 chamber. After cell attachment, the medium was renewed without fetal calf serum and supplemented with 10 nM dexamethasone and 20 nM insulin for 12-16 h. Glucose production was measured 2 and 24 h after incubation of hepatocytes in glucose-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 16 mM lactate, 4 mM pyruvate, 10 nM dexamethasone, and 20 nM insulin. Glucose release in the medium was determined by the GOD-POD colorimetric method (Sclavo Diagnostics International, Sovicille, Italy) and normalized to the protein concentration determined by the Bradford method.

mRNA extraction and quantitative real-time PCR

Samples of frozen livers were homogenized in lysis buffer supplemented with ␤-mercaptoethanol, and total RNA was extracted (Absolutely RNA Miniprep Kit; Stratagene; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocol. The RNA samples were further purified by precipitation with sodium acetate and isopropyl alcohol. For retroperitoneal WAT and rectus femoris muscle samples, homogenization was done with QIAzol Lysis Reagent (Qiagen, Courtaboeuf, France), and total RNA was extracted using an RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's protocol. RNA quality was evaluated with the Bioanalyzer 2100 (Agilent Technologies; RNA integrity number for all samples was Ͼ7). RNA quantity was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA; A 260 /A 280 and A 260 / A 230 values were Ͼ1.8). cDNAs were synthesized from 1 g of total RNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Courtaboeuf, France). Quantitative Realtime PCR was performed and analyzed using a 7300 Real-Time PCR System with 1ϫ SYBR Green PCR Master Mix (Applied Biosystems), 0.9 M primers (Invitrogen), and 1 l of cDNA in a total volume of 20 l. PCR conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. PCR reactions were done in duplicate and negative controls (i.e., no reverse transcription and no template controls) were added to the reactions. Relative expression levels were determined using the comparative ⌬C T method to normalize target gene mRNA to 36b4. Primers were designed and optimized for an annealing temperature of 60°C. To assess primer specificity and product uniformity, sequences were tested with the Basic Local Alignment Search Tool (BLAST; U.S. National Center for Biotechnology Information; http://blast.ncbi.nlm.nih.gov/), and postamplification dissociation curves were determined. A dilution curve of the pool of all cDNA samples was used to calculate the amplification efficiency for each assay (values were between 1.85 and 2). Primer sequences are summarized in Supplemental Table S1.

Cell culture, transfection, and luciferase assay COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc.; Invitrogen), 1% penicillin/streptomycin (Life Technologies) mix and sodium pyruvate in a humidified atmosphere with 5% CO 2 at 37°C. Cells were plated in 24-well plates and transfected with GeneJuice (Novagen; Merck, Darmstadt, Germany). Depending on the experiment, hROR␣ and hREV-ERB␣ expression vectors [START_REF] Guillaumond | Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors[END_REF] were used at either 100 or 200 and 200 ng/well, respectively. The oBmal1-luc and mPer1-luc reporter construct and mClock/mBmal1 expression vectors have been used pre-viously [START_REF] Dardente | Transcriptional feedback loops in the ovine circadian clock[END_REF][START_REF] Travnickova-Bendova | Bimodal regulation of mPeriod promoters by CREB-dependent signaling and CLOCK/BMAL1 activity[END_REF]. The proximal promoter region of mLpl (Ϫ143 to ϩ186) was cloned in pGL3 basic (see Supplemental Data for further information). All reporter constructs and ␤-galactosidase reporter construct were used at 50 and 100 ng/well, respectively. Total transfected DNA amount was set to an equal amount between all conditions by addition of the corresponding empty vector. Luciferase assay was performed 48 h after transfection. Briefly, cells were rinsed twice in cold PBS and lysed for 15 min in lysis buffer (25 mM Tris, 2 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 1% TritonX-100). The luciferase assay was performed using a luciferase assay system kit (Promega, Charbonnieres, France) and a PolarStar Optima luminometer (BMG Labtech, Ortenberg, Germany). Results were normalized to ␤-galactosidase activity. Data [in relative luminescence units (RLU)] represent fold induction once normalized to ␤-galactosidase. Experiments were repeated 3 times, each condition in triplicate.

Statistical analysis

All values are presented as means Ϯ se. Area under the curve (AUC) was determined as incremental area (i.e., above baseline; see ref. 27). The 48-h respiratory quotient (RQ) data were fitted to a cosinor function [f ϭ a ϩ (b ء cos (2 ء ء (x Ϫ c)/ 24))], where x indicates the time, a the mean value, b the amplitude, and c the acrophase. Statistical difference between genotypes for a given parameter (e.g., amplitude) was tested in an analysis of covariance. Normality and homogeneity of variance were assessed with Kolmogorov-Smirnov and Lilliefors test and Levene's test, respectively. Data that were non-normal and/or heteroscedastic were subjected to logarithmic transformation before analysis. Value of ␣ was set at 0.05. Unpaired Student's t test was used to compare 2 groups. Luciferase assay data were analyzed using 1-way analyses of variance (ANOVA) followed by Tukey HSD post hoc analysis. Data collected successively at different time points (e.g., locomotor activity) within each group were compared with ANOVA for repeated measures. Two-way ANOVA was performed to assess the effects of genotype and feeding condition and the interaction between these factors. Threeway ANOVA design was employed to test the aforementioned factors plus the effect of time. Simple main effects approach was conducted to examine each factor separately if the interaction term was significant. Statistical analyses were performed with Statistica 10 (StatSoft,France).

RESULTS

Rev-erb␣ ؊/؊ mice are slightly hyperglycemic without insulin resistance

We first evaluated whether Rev-erb␣ deficiency affects general physiology and behavior. No significant difference was found between Rev-erb␣ Ϫ/Ϫ mice and their wild-type (ϩ/ϩ) littermates regarding the amount and timing of chow intake (Fig. 1A). Body mass was not significantly different between 4-mo-old Rev-erb␣ ϩ/ϩ and Rev-erb␣ Ϫ/Ϫ mice (24.02 Ϯ 1.11 vs. 25.58 Ϯ 1.20 g, respectively). The daily patterns of general locomotor activity and body temperature were very close in the two genotypes (Fig. 1B,C). In sharp contrast, 24-h blood glucose rhythm showed higher values in Rev-erb␣ Ϫ/Ϫ mice compared to Rev-erb␣ ϩ/ϩ mice (Fig. 1D), suggesting altered glucose homeostasis. Of note, mild hyperglycemia at the day-night transition has been previously observed in Rev-erb␣ Ϫ/Ϫ mice [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF]. To understand the origin of this hyperglycemia in Rev-erb␣ Ϫ/Ϫ mice, we determined the day-night levels of insulin, glucagon, and hepatic glycogen in both genotypes. Plasma insulin was increased at ZT0 (lights on, onset of the resting period; Fig. 2A), while hepatic glycogen content was higher at ZT12 (lights off, onset of the active period; Fig. 2B). These higher insulin and glycogen levels in Rev-erb␣ Ϫ/Ϫ mice are consistent with previous results [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF][START_REF] Schmutz | The mammalian clock component PERIOD2 coordinates circadian output by interaction with nuclear receptors[END_REF]. Plasma glucagon levels showed no difference between genotypes (Fig. 2C). According to standardized guidelines [START_REF] Heikkinen | Evaluation of glucose homeostasis[END_REF], we then compared the responses Figure 1. Normal food intake, locomotor activity, and body temperature (but higher blood glucose) in Rev-erb␣ Ϫ/Ϫ mice. A) Rhythm of 24 h chow intake in Rev-erb␣ ϩ/ϩ and Ϫ/Ϫ mice. B) Rhythm of general locomotor activity. C) Rhythm of core body temperature. D) Blood glucose profiles. Values are expressed as means Ϯ se (nϭ10/group). Shaded area indicates the dark (i.e., active) period. Data from ZT0 and ZT24 are double plotted in panels B-D. Open circles, Rev-erb␣ ϩ/ϩ mice; solid circles, Rev-erb␣ Ϫ/Ϫ mice. ϳ P Ͻ 0.05 for main effect of time; *P Ͻ 0.05 for main effect of genotype.

of both genotypes to metabolic challenges, by comparing their respective AUC values, calculated as incremental area (above baseline, to exclude the effect of differences in fasting glucose levels between animals). The OGTT (performed at the same ZTs as in ref. [START_REF] Marcheva | Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes[END_REF] showed no significant difference between genotypes either at ZT2 (Fig. 2D) or at ZT14 (Supplemental Fig. S1C). Insulin sensitivity, assessed by the intraperitoneal insulin sensitivity test (IPIST), was not significantly modified in Rev-erb␣ Ϫ/Ϫ mice compared to wild-type mice, either at ZT2 (despite a trend toward increased sensitivity; Pϭ0.07 for AUC; Supplemental Fig. S1B), or at ZT14 (Supplemental Fig. S1D). When Rev-erb␣ Ϫ/Ϫ mice were challenged with an OGTT after overnight food withdrawal, their glucose-stimulated insulin secretion was also similar to that of control mice (Supplemental Fig. S1A). Similarly, a hyperinsulinemic-euglycemic clamp study performed from ZT6 in animals previously unfed for 6 h indicated that the glucose infusion rate, averaged over the last 60 min of the clamp, was similar between genotypes (Fig. 2E), ruling out any major alteration in whole-body insulin sensitivity. In addition, pancreatic islets from both genotypes were identical in area, circularity, and insulin content (Fig. 2F, G and Supplemental Fig. S2A). Altogether, these observations suggest that the high blood glucose seen in chow-fed Rev-erb␣ Ϫ/Ϫ mice is not the result of either insulin resistance or a defect in insulin secretion.

Gluconeogenesis is not enhanced in Rev-erb␣ ؊/؊ mice

The following experiments were conducted to determine whether the hyperglycemia in Rev-erb␣ Ϫ/Ϫ mice was of hepatic origin. For that purpose, we investigated glycogenolysis by challenging mice with a GST. Rev-erb␣ Ϫ/Ϫ mice did not show a larger hyperglycemic response compared to that in controls (Fig. 2H), excluding hypersensitivity to glucagon as a potential cause of hyperglycemia. We then challenged mice with a PTT at ZT2 to investigate a potentially greater contribution , from ZT12 to ZT2) and the resulting incremental AUC (nϭ12/group). E) Hyperinsulinemic-euglycemic clamp and the resulting glucose infusion rates averaged over the last 60 min of the clamp (nϭ6/group). F) Insulin immunostaining from pancreas sections of ϩ/ϩ and Ϫ/Ϫ animals and their respective quantification (nϭ6/group). G) Pancreas sections of a Rev-erb␣ ϩ/ϩ (left panel) and a Rev-erb␣ Ϫ/Ϫ mouse (right panel) stained with insulin antibody. Scale bar ϭ 50 m. H, I) GST (H) and PTT (I) in mice unfed overnight and the resulting AUCs (nϭ12/group). J) Glucose production in Rev-erb␣ ϩ/ϩ and Rev-erb␣ Ϫ/Ϫ primary hepatocytes incubated with (ϩ) or without (Ϫ) lactate-pyruvate (LP) for 2 or 24 h (nϭ7/group). Data are means Ϯ se. Open circles, Rev-erb␣ ϩ/ϩ mice; solid circles, Rev-erb␣ Ϫ/Ϫ mice. ϳ P Ͻ 0.05 for main effect of time (top left corner) or vs. corresponding ZT0 group of same genotype (above bar); *P Ͻ 0.05 vs. ϩ/ϩ genotype at same ZT; # P Ͻ 0.05 vs. 24 h LPϪ treatment.

of gluconeogenesis in Rev-erb␣ Ϫ/Ϫ mice. No significant difference was detected between the genotypes (Fig. 2I). Metformin, known to decrease hepatic glucose production by inhibiting gluconeogenesis and increasing insulin sensitivity, improved glucose tolerance to the same degree in both genotypes (data not shown). To confirm these in vivo results, we compared glucose production in vitro in primary hepatocytes from Rev-erb␣ Ϫ/Ϫ and control animals. Following 24 h incubation in the presence of lactate-pyruvate, Rev-erb␣ Ϫ/Ϫ hepatocytes did not produce more glucose than wild-type cells (Fig. 2J). We conclude that the chronic hyperglycemic phenotype of Rev-erb␣ Ϫ/Ϫ mice cannot be explained by increased gluconeogenesis. As REV-ERB␣ is a potential link between the circadian timing system and metabolism, we hypothesized that these mice may exhibit a circadian misalignment of energy utilization.

Daily balance in energy stores utilization is exacerbated in Rev-erb␣ ؊/؊ mice

To test whether the daily cycle of lipid and glucose utilization is modified in Rev-erb␣ Ϫ/Ϫ mice, we assessed the 24-h variations of energy metabolism in vivo by using indirect calorimetry. Basal metabolism, determined from the 5 lowest O 2 consumption values during the second 24-h cycle, was not significantly different between genotypes (Supplemental Fig. S3). The RQ (VCO 2 /VO 2 ) tracks the type of fuel oxidized. High (i.e., close to 1) and low (i.e., close to 0.7) RQ values indicate preferential utilization of carbohydrates and lipids, respectively. Of interest, Rev-erb␣ Ϫ/Ϫ mice showed altered daily variations in RQ values compared to control mice, with lower and higher values during the day and the night, respectively (Fig. 3A,B). Cosinor fitting followed by analysis of covariance of 48 h RQ data revealed that the acrophase of RQ occurred 1.4 h later (i.e., was phase delayed) in Rev-erb␣ Ϫ/Ϫ compared to control mice (ZT 17.4 Ϯ 0.1 vs. 16.0 Ϯ 0.2, respectively; P Ͻ 0.001). Furthermore, daily changes of RQ in Rev-erb␣ Ϫ/Ϫ mice displayed larger amplitude than in control littermates (0.11 Ϯ 0.003 vs. 0.06 Ϯ 0.003, respectively; P Ͻ 0.001), whereas the daily mean RQ, which reflects energy homeostasis, did not differ between genotypes (ϩ/ϩ vs. Ϫ/Ϫ: 0.85 Ϯ 0.002 vs. 0.85 Ϯ 0.002, respectively).

These data demonstrate that Rev-erb␣ Ϫ/Ϫ mice have increased fatty acid utilization during the resting period compared to littermate controls, while their glucose utilization (i.e., glucose supply to active organs and/or conversion of dietary carbohydrates to fat) is delayed and increased during the active period.

Increased utilization of lipid fuels in unfed

Rev-erb␣ ؊/؊ mice To test a preferential use of fatty acids over glucose in Rev-erb␣ Ϫ/Ϫ mice, we analyzed the physiological and hepatic molecular responses of both genotypes after 24 h of food withdrawal (i.e., to trigger mobilization of energy stores) and 24 h of food withdrawal followed by 24 h refeeding with high-carbohydrate diet (i.e., to trigger lipogenesis). Changes in body mass associated with food withdrawal and refeeding did not differ significantly between the genotypes (Fig. 4A). Compared to controls, Rev-erb␣ Ϫ/Ϫ mice exhibited a significantly lower hypothermia response to 24 h of food deprivation, resulting in higher body temperature at the end of the nonfeeding period (Fig. 4B). This lack of hypothermia was not due to changes in the level of locomotor activity (Supplemental Fig. S4A). In addition, fasting blood glucose levels were still significantly higher in Rev-erb␣ Ϫ/Ϫ mice compared to Rev-erb␣ ϩ/ϩ animals (Fig. 4C). This confirms the mild hyperglycemia after overnight food withdrawal before metabolic tests. However, there was no difference between the genotypes in the refeeding condition (Fig. 4C). The absence of hyperglycemia in refed Rev-erb␣ Ϫ/Ϫ mice was not linked to differential changes in insulin levels (Fig. 4D) or food intake (ϩ/ϩ vs. Ϫ/Ϫ: 6.09 Ϯ 0.16 vs. 6.33 Ϯ 0.17 g, respectively). One possible explanation is that previously unfed Rev-erb␣ ϩ/ϩ mice are more sensitive than Rev-erb␣ Ϫ/Ϫ animals to the hyperglycemic effect of high-carbohydrate diet. In other words, Rev-erb␣ Ϫ/Ϫ mice may be more efficient to process highcarbohydrate intake for fatty acid synthesis. While most metabolic parameters studied did not differ between the two genotypes in the refed condition (Fig. 4D-I), many of them were differentially affected by 24 h of food withdrawal in Rev-erb␣ ϩ/ϩ vs. Ϫ/Ϫ animals. In particular, hepatic glycogen was significantly more elevated in Rev-erb␣ Ϫ/Ϫ mice (Fig. 4E), indicating that blood glucose is not used as a primary source of energy in these mice. Interestingly, plasma

Figure 3. Changes in daily fuel utilization in

Rev-erb␣ Ϫ/Ϫ mice. A) RQ (48 h; i.e., VCO 2 /VO 2 values) of Rev-erb␣ ϩ/ϩ and Ϫ/Ϫ mice, determined by indirect calorimetry. Shaded and solid traces show fitting of the 48-h RQ data of Rev-erb␣ ϩ/ϩ and Rev-erb␣ Ϫ/Ϫ mice, respectively, to the cosine function. Note that data were collected every 15 min. Shaded area indicates the dark (i.e., active/feeding) period. Open circles, Rev-erb␣ ϩ/ϩ mice; solid circles, Rev-erb␣ Ϫ/Ϫ mice. B) Day and night averages of RQ. Values are expressed as means Ϯ se (nϭ10/ group). ϳ P Ͻ 0.05 for main effect of time.

NEFA and ketone body levels were significantly decreased in Rev-erb␣ Ϫ/Ϫ mice compared to control mice (Fig. 4H,I). This suggests that fatty acids are used as the main source of energy during 24 h of food withdrawal in Rev-erb␣-deficient animals.

To substantiate this possibility, we analyzed the mRNA expression of peroxisome proliferator-activated receptor ␣ (Ppar␣) and Ppar␣-target genes known to be involved in ketogenesis: carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2). Food withdrawal similarly enhanced the mRNA levels of Ppar␣, Cpt1a, and Hmgcs2 in both genotypes (Fig. 4J), ruling out an abnormal ketogenesis in Rev-erb␣ Ϫ/Ϫ mice, which could have explained the reduction of their ketone body levels. In addition, the expression of phosphoenolpyruvate carboxykinase 2 (Pepck) and glucose-6-phosphatase (G6pc), two key actors of gluconeogenesis, was reduced in unfed Rev-erb␣ Ϫ/Ϫ mice (Fig. 4K). Furthermore, the mRNA levels of glucose transporter 2 (Glut2) and glucokinase (Gk), involved in glucose transport and both glycogen synthesis and glycolysis, respectively, were higher after food withdrawal in Rev-erb␣ Ϫ/Ϫ mice than in wild-type mice (Fig. 4K). In contrast, loss of Rev-erb␣ did not block the refeeding-induced increase of lipogenic genes (Fig. 4L and Supplemental Fig. S4C). The mRNA levels of other regulators of glucose and lipid metabolism were similar between the genotypes (Supplemental Fig. S4B,C). To summarize, unfed Rev-erb␣ Ϫ/Ϫ mice maintain relative hyperglycemia, concomitant with available glycogen stores and reduced gluconeogenesis. In agreement with these findings, the lower RQ values in chow-fed Rev-erb␣ Ϫ/Ϫ mice during daytime demonstrate that Rev-erb␣ deletion in vivo leads to a greater mobilization and oxidation of fatty acids as well as a greater ketolysis during the resting period.

Rev-erb␣ ؊/؊ mice are more prone to HFD-induced obesity

The experiments reported above indicate that the absence of Rev-erb␣ leads to a shift from glucose to fatty I) levels in unfed (fasted) and refed animals. J-L) mRNA expression levels of ketogenic genes (J), glucose metabolism genes (K), and lipogenic genes (L). Animals were sampled at ZT0. Values are expressed as means Ϯ se (nϭ7/group). See Supplemental Table S1 for gene names. # P Ͻ 0.05 for main effect of feeding condition (midpanel) or vs. corresponding unfed (fasted) group of same genotype (above bar); *P Ͻ 0.05 vs. ϩ/ϩ genotype of same feeding condition.

acid utilization for energy supply, at least during the resting period and 24 h of food withdrawal. During nighttime (i.e., period of dietary carbohydrate intake), however, mild hyperglycemia, normal insulin sensitivity, and high RQ values in chow-fed Rev-erb␣ Ϫ/Ϫ mice indicate an active absorption of carbohydrates. We wondered whether de novo lipogenesis from dietary carbohydrates could be increased at night in Rev-erb␣ Ϫ/Ϫ mice. We first assessed day-night parameters related to lipid metabolism in 4-wk-old Rev-erb␣ ϩ/ϩ and Ϫ/Ϫ mice fed chow or HFD for up to 12 wk. Rev-erb␣ Ϫ/Ϫ mice fed HFD gained much more body mass than HFD-fed control animals (Fig. 5A). This cannot be attributed to biased (i.e., different) values of baseline body mass, because they were similar between the genotypes at 4 wk of age. Moreover, all groups had a similar body size at the end of the experiments (data not shown). Mean calorie intake did not significantly differ between the genotypes, despite a trend for increased calorie consumption in the Rev-erb␣ Ϫ/Ϫ group (Pϭ0.08; Supplemental Fig. S5A). Rev-erb␣ Ϫ/Ϫ mice had a significantly higher body mass index, especially in the HFD group (Supplemental Fig. S5B). In accordance with this observation, the amount of WAT (i.e., perigonadal and retroperitoneal) was also significantly greater in Rev-erb␣ Ϫ/Ϫ mice compared to controls, regardless of the feeding conditions (Fig. 5B,C). As expected [START_REF] Gallou-Kabani | C57BL/6J and A/J mice fed a high-fat diet delineate components of metabolic syndrome[END_REF], HFD-fed control animals had significantly higher body mass and adiposity than chow-fed control animals, and in addition, they reached a similar adiposity as in chow-fed Rev-erb␣ Ϫ/Ϫ mice (Fig. 5B,C).

When fed HFD, both genotypes expressed an attenuated diurnal feeding rhythm (data not shown) and showed significant hyperleptinemia (Fig. 5D), increased hepatic triglycerides (Fig. 5F), decreased plasma triglycerides (Fig. 5G), increased plasma LDL and plasma NEFAs (Fig. 5H,I), hypercholesterolemia (Supplemental Fig. S5D) and hyperinsulinemia (Supplemental Fig. S5G), compared to their chow-fed controls. Interestingly, irrespective of the feeding conditions, Rev-erb␣ Ϫ/Ϫ mice had significantly increased plasma leptin, glucose, NEFA, LDL, ketone body, cholesterol, insulin, and adiponectin levels compared to Rev-erb␣ ϩ/ϩ mice (Fig. 5D,E, H-J and Supplemental Fig. S5D,G,H). Incidentally, HFD-induced hyperglycemia was only detected with blood sampling by tail incision (Supplemental Fig. S5K,L). The high levels of adiponectin and leptin in chow-and HFD-fed Rev-erb␣ Ϫ/Ϫ mice could have prevented the development of insulin resistance [START_REF] Yamauchi | The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity[END_REF]. Corticosterone levels, important for carbohydrate metabolism, were not significantly enhanced in chow-fed Rev-erb Ϫ/Ϫ mice (Supplemental Fig. S5I). Hepatic triglycerides in chow-fed Rev-erb␣ Ϫ/Ϫ mice were found to be increased at ZT10 [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF] and reduced at ZT12 [START_REF] Feng | A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism[END_REF]. However, we noticed a trend toward elevated hepatic triglycerides at ZT12 in Rev-erb␣ Ϫ/Ϫ mice, regardless of the feeding conditions (Pϭ0.06; Fig. 5F). Of interest, plasma glycerol in Rev-erb␣ Ϫ/Ϫ mice was enhanced at ZT12 in chow-fed conditions and at both time points in HFD-fed conditions (Supplemental Fig. S5F), which may indicate enhanced hydrolysis of triglycerides. Taken together, our data indicate that Rev-erb␣ deletion leads to a fat phenotype in chow-fed conditions, which is severely amplified in HFD-fed conditions. Rev-erb␣ Ϫ/Ϫ mice exhibit a fat phenotype amplified on prolonged high-fat feeding. A) Body mass change over time in chow-fed and HFD-fed ϩ/ϩ and Ϫ/Ϫ animals (nϭ12/group). Sac., sacrifice. Note that statistical analysis was performed on the last week of feeding. Open circles, chow-fed Rev-erb␣ ϩ/ϩ mice; solid circles, chow-fed Rev-erb␣ Ϫ/Ϫ mice; light shaded triangles, HFD-fed Rev-erb␣ ϩ/ϩ mice; dark shaded triangles, HFD-fed Rev-erb␣ Ϫ/Ϫ mice. B) Total adiposity, expressed as a percentage of body mass (nϭ12/group). C) Amount of perigonadal and retroperitoneal WAT (WATp and WATr, respectively; nϭ12/group). D, E) Plasma leptin (D) and glucose (E) levels (nϭ6/group). F, G) Hepatic triglyceride (TG; F) and plasma TG (G) levels (nϭ6/group). H-J) Plasma LDL (H), NEFA (I), and ketone body (KB; J) levels (nϭ6/group). Values are expressed as means Ϯ se. ϳ P Ͻ 0.05 for main effect of time; *P Ͻ 0.05 for main effect of genotype; # P Ͻ 0.05 for main effect of feeding condition.

Expression of metabolic genes is disrupted in chow-and HFD-fed Rev-erb␣ ؊/؊ mice To gain further insight into the role of REV-ERB␣ as transcriptional repressor for the daily control of energy balance, we examined the day-night levels of glucose and lipid-related genes in the liver, WAT, and skeletal muscle of chow-and HFD-fed Rev-erb␣ ϩ/ϩ and Ϫ/Ϫ mice. Of note, day-night expression patterns of most metabolic genes studied in Rev-erb␣ ϩ/ϩ mice were in accordance with expression profiling from transcriptome data [START_REF] Panda | Coordinated transcription of key pathways in the mouse by the circadian clock[END_REF][START_REF] Hughes | Harmonics of circadian gene transcription in mammals[END_REF]. We first determined whether the defective energy homeostasis in Rev-erb␣ Ϫ/Ϫ mice was the consequence of altered hepatic gene expression. Notably, hepatic glycogen synthase 2 (Gys2) and Glut2 expression was significantly diminished at ZT12 in Rev-erb␣ Ϫ/Ϫ mice in both feeding conditions (Fig. 6A), while no significant differences were found for Gk, Pepck, and G6pc (Supplemental Fig. S6A), in keeping with a previous report [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF]. Hepatic expression of lipogenic genes [e.g., sterol regulatory element binding transcription factor 1 (Srebp1c) and fatty acid synthase (Fas)] showed decreased levels at ZT12 in chow-fed Rev-erb␣ Ϫ/Ϫ mice (Fig. 6A). However, while HFD did not markedly change the expression of Srebp1c, the hepatic mRNA levels of Fas, acetyl-coenzyme A carboxylase (Acc), and ELOVL fatty acid elongase 6 (Elovl6) in the Rev-erb␣ Ϫ/Ϫ group were ϳ2-fold higher than in control animals at both time points (Fig. 6A). Similarly, we found that the fatty acid transporter cluster of differentiation 36 (Cd36) was significantly induced by HFD exclusively in Rev-erb␣ Ϫ/Ϫ animals (Fig. 6A). Of special interest, Lpl mRNA, which encodes the rate-limiting enzyme in triglyceride hydrolysis (36), was significantly increased at ZT12 in the liver of Rev-erb␣ Ϫ/Ϫ mice Figure 6. Lipid metabolism gene expression is modified in the absence of Rev-erb␣. mRNA expression of glucose and lipid metabolism genes in liver (A), retroperitoneal WAT (B), and rectus femoris skeletal muscle (C). Data are means Ϯ se (nϭ6/group). See Supplemental Table S1 for gene names. ϳ P Ͻ 0.05 for main effect of time (top left corner) or vs. corresponding ZT0 group of same genotype (above bar); *P Ͻ 0.05) for main effect of genotype (top left corner) or vs. corresponding ϩ/ϩ genotype group at same ZT (above bar); # P Ͻ 0.05 for main effect of feeding condition (top left corner) or vs. corresponding chow-fed group at same ZT (above bar).

compared to controls (Fig. 6A). Taken together, the elevated expression of these metabolic actors may contribute to obesity.

To further specify the obesity phenotype of Rev-erb␣ Ϫ/Ϫ mice, we analyzed the mRNA levels of metabolic actors in WAT and skeletal muscle. Glut4, Fas, Acc, and Srebp1c were significantly down-regulated by HFD in both genotypes (Fig. 6B and Supplemental Fig. S6B). Incidentally, this HFD-dependent regulation of the lipogenic genes Acc and Fas has also been observed in obese humans and rodents [START_REF] Ortega | The gene expression of the main lipogenic enzymes is downregulated in visceral adipose tissue of obese subjects[END_REF]. While there was no difference in Cd36 expression between the two genotypes, the deletion of Rev-erb␣ also increased Lpl mRNA expression in WAT (Fig. 6B). This suggests that in Rev-erb␣ Ϫ/Ϫ mice, WAT Lpl enhanced uptake of NEFA for fat storage, thus contributing to fat overload. In skeletal muscle, an overall tendency to down-regulate Glut4 was seen in Rev-erb␣ Ϫ/Ϫ mice, which only reached statistical significance in HFD-fed Rev-erb␣ Ϫ/Ϫ animals at ZT12 (Fig. 6C), when massive body fat overload started to impede physical activity (data not shown). Expression of Fas and Acc in the muscle was also down-regulated in response to HFD in both genotypes, whereas the expression of Cd36 was increased by HFD in both genotypes (Fig. 6C, Supplemental S6C). Of note, Cd36 and Lpl expression was significantly increased in Rev-erb␣ Ϫ/Ϫ mice, irrespective of the feeding conditions (Fig. 6C). This further supports our hypothesis of an enhanced utilization of fatty acids as energy substrate for the muscle.

Altogether, these results clearly indicate that Rev-erb␣ Ϫ/Ϫ mice display a defective molecular control of energy homeostasis which contributes to HFD-induced obesity. Both liver and skeletal muscle appear to play a role in the production and preferential use of fatty acids at the expense of peripheral utilization of glucose, thus leading to a chronic mild hyperglycemia.

CLOCK drives transcriptional activation of Lpl

The overexpression of Lpl mRNA that we found in 3 peripheral tissues of Rev-erb␣ Ϫ/Ϫ mice led us to test whether this gene could be clock controlled. Since several putative RORE motifs were found in the proximal promoter region of Lpl (see Supplemental Data), we first decided to check whether the latter would be responsive to REV-ERB␣ and ROR␣. Neither ROR␣ nor REV-ERB␣ had any effect toward this reporter construct (Fig. 7A). In contrast, ROR␣ consistently activated Bmal1-luc, an effect that could be partially overcome by the addition of REV-ERB␣ (Fig. 7B).

However, the transcriptions of Rev-erb␣ and Clock/ Bmal1 are interlocked: The heterodimer CLOCK/ BMAL1 drives Rev-erb␣ transcription [START_REF] Triqueneaux | The orphan receptor Rev-erbalpha gene is a target of the circadian clock pacemaker[END_REF], and REV-ERB␣ inhibits both Bmal1 and Clock transcriptions, as previously mentioned. This explains why the mRNA levels of both Bmal1 and Clock are higher in the Rev-erb␣ Ϫ/Ϫ mice than in their wild-type littermates (Supplemental Fig. S7). Therefore, it seems plausible that at least a subset of CLOCK/BMAL1 targets will display enhanced expression levels in these Rev-erb␣ Ϫ/Ϫ mice. We thus tested the potential implication of a putative E-box, which is located in the most proximal promoter region of mLpl. This reporter construct significantly responded to CLOCK/BMAL1, although the amplitude of the response was not different from that elicited by CLOCK alone (Fig. 7C). These results demonstrate that the circadian clockwork is somehow involved in the transcriptional activation of the Lpl gene.

DISCUSSION

Energy metabolism can be affected by circadian disturbance, as evidenced by epidemiologic studies in shift workers and genetic disruption of clock components in mice [START_REF] Froy | Metabolism and circadian rhythms-implications for obesity[END_REF][START_REF] Delezie | Interactions between metabolism and circadian clocks: reciprocal disturbances[END_REF]. REV-ERB␣ is a transcription factor involved both in the molecular clockwork and in several metabolic pathways. Therefore, dissecting REV-ERB␣ contribution to the daily balance between glucose and lipid metabolism is an important step toward understanding the functional crosstalk between the circadian and metabolic systems. Here we show that the absence of REV-ERB␣ in vivo leads to a preferential use of fatty acids as energy substrate during daytime, in association with non-insulin-dependent hyperglycemia.

During the daily resting period, liver and muscle derive most of their energy from fatty acids released from WAT. Chow-fed Rev-erb␣ Ϫ/Ϫ mice rely more on lipid fuels during the inactive period than do control mice, as evidenced by low (i.e., close to 0.7) RQ values. Accordingly, hepatic glycogen is less mobilized at the end of the resting period, while the hyperglycemia persists. A defect of glucose entry due to tissue-specific impaired insulin signaling would have explained the increased fatty acid utilization in Rev-erb␣ Ϫ/Ϫ mice. Deficiency in Glut4 expression has been associated with insulin resistance [START_REF] Stenbit | GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes[END_REF]. However, chow-fed Rev-erb␣ Ϫ/Ϫ mice have normal whole-body insulin sensitivity without obvious changes in Glut4 mRNA levels in peripheral tissues.

Another explanation for the increased fatty acid utilization stems from the implication of REV-ERB␣ in the regulation of myosin heavy chain (MyHc) isoform expression in the skeletal muscle, based in particular on a significant fast-to-slow MyHC isoform transformation in skeletal muscle of Rev-erb␣ Ϫ/Ϫ mice [START_REF] Pircher | Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the Rev-erbAalpha orphan receptor gene[END_REF]. Thus, the preponderance of this slow myosin may have increased aerobic lipid metabolism, sparing glucose use in the muscles of Rev-erb␣-deficient mice. In agreement with this hypothesis, the membrane protein Cd36 involved in the binding and transport of fatty acids ( 36) is up-regulated in Rev-erb␣ Ϫ/Ϫ mice in the rectus femoris muscle, which has both oxidative and glycolytic capabilities [START_REF] Manttari | Comparative analysis of mouse skeletal muscle fibre type composition and contractile responses to calcium channel blocker[END_REF]. Muscle-specific overexpression of Cd36 in mice promotes clearance of circulating fatty acids and increases plasma glucose and insulin [START_REF] Ibrahimi | Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin[END_REF]. Furthermore, Lpl mRNA in the muscle of Rev-erb␣-deficient animals is up-regulated both in the morning and the evening. Of note, LPL activity is higher in muscles composed predominantly of high-oxidative slow-twitch fibers [START_REF] Ferraro | Relationship between skeletal muscle lipoprotein lipase activity and 24-hour macronutrient oxidation[END_REF] and is inversely phased to daily variation of RQ [START_REF] Tsutsumi | The relationship between lipoprotein lipase activity and respiratory quotient of rats in circadian rhythms[END_REF]. Moreover, Rev-erb␣ Ϫ/Ϫ mice are resistant to cold-induced hypothermia (Supplemental Fig. S8), as are transgenic mice that overexpress Lpl in skeletal muscles [START_REF] Jensen | Increased thermoregulation in cold-exposed transgenic mice overexpressing lipoprotein lipase in skeletal muscle: an avian phenotype?[END_REF]. This improved cold tolerance could be linked to changes in muscle physiology (i.e., enhanced oxidative capacity) due to LPL overexpression [START_REF] Jensen | Increased thermoregulation in cold-exposed transgenic mice overexpressing lipoprotein lipase in skeletal muscle: an avian phenotype?[END_REF]. Taken together, these findings show that increased Cd36 and Lpl expression reflects increased muscular uptake of fatty acids in Rev-erb␣ Ϫ/Ϫ mice and can contribute not only to hyperglycemia but also to the absence of food withdrawal-induced drops in blood glucose and body temperature.

The physiological responses to acute food withdrawal include depletion of glycogen stores, hepatic gluconeogenesis, and mobilization of triglyceride in adipose tissues, which together provide energy supply. After 24 h of food withdrawal, glycogen depletion in Rev-erb␣ Ϫ/Ϫ mice remains incomplete and the hepatic expression of gluconeogenic genes much less increased in their liver. Strikingly, blood glucose levels in unfed Rev-erb␣ Ϫ/Ϫ mice are not decreased as in control animals, but instead show similar values to those of fed Rev-erb␣ Ϫ/Ϫ mice. In sharp contrast, plasma NEFA and ketone bodies levels in unfed Rev-erb␣ Ϫ/Ϫ mice are lower than in unfed control mice, reflecting acute utilization of fatty acids. This conclusion is substanti-ated by the facts that plasma NEFA and ketone body levels are either similar or greater in chow-fed Rev-erb␣ Ϫ/Ϫ mice compared to their controls, and that ketogenesis at the molecular level is induced by food withdrawal. Thus, it is unlikely that reduced NEFA and ketone bodies levels are due to decreased lipolysis and ketogenesis. Hence, the relative hyperglycemia and normothermia in starved Rev-erb␣ Ϫ/Ϫ mice result from a combination of lower activation of gluconeogenesis, diminished glucose utilization, and increased production and utilization of lipid fuels.

During the daily activity period, feeding allows the restoration of energy stores depleted over the previous daytime food withdrawal. Nocturnal RQ values in Rev-erb␣ Ϫ/Ϫ mice are higher than in control mice, suggesting a greater utilization of glucose. This feature, however, cannot be explained by increased Glut4 mRNA levels, fast glycolytic fibers [START_REF] Pircher | Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the Rev-erbAalpha orphan receptor gene[END_REF], or physical activity. Alternatively, higher nocturnal RQ values in Rev-erb␣ Ϫ/Ϫ mice can result from increased de novo lipogenesis, leading to enhanced production of lipids. Indeed, when fatty acids are synthesized from glucose, for instance during the absorption of dietary carbohydrates, the average RQ remains close to or below 1 [START_REF] Schutz | Concept of fat balance in human obesity revisited with particular reference to de novo lipogenesis[END_REF]. Rev-erb␣ Ϫ/Ϫ mice exhibit increased adiposity even when fed chow. Of note, body fat can derive from dietary fat and de novo lipogenesis, mainly from carbohydrates [START_REF] Schutz | Concept of fat balance in human obesity revisited with particular reference to de novo lipogenesis[END_REF]. Fas mRNA expression shows a 2-fold increase at ZT12 in the muscle and WAT of Rev-erb␣ Ϫ/Ϫ mice, indicating elevated fat synthesis in these tissues. However, no increased expression of lipogenic genes has been detected in the liver of chow-fed Rev-erb␣ Ϫ/Ϫ mice. This is explained by our sampling times, because expression of Srebp1c, Fas, and Elovl6 is phase shifted in the liver of Rev-erb␣-deficient mice [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF], suggesting a delay in the occurrence of fat synthesis. This was confirmed by Feng et al. (25), who demonstrated that REV-ERB␣ controls the expression of lipid metabolism genes by recruiting the repressive chromatin modifier histone deacetylase 3, ensuring a temporal control of lipogenesis. In line with this study, we also show a trend toward elevated hepatic triglycerides, an important source of lipids for very low density lipoprotein (VLDL) assembly at the end of the day. Hepatic de novo lipogenesis contributes to VLDL production, the latter being increased in Rev-erb␣-deficient mice [START_REF] Raspe | The FASEB Journal ⅐ www.fasebj.org tion of Rev-erbalpha as a physiological repressor of apoC-III gene transcription[END_REF]. In addition, our calorimetry data indicating a possible nocturnal elevation of fat synthesis from dietary glucose in chow-fed Rev-erb␣ Ϫ/Ϫ mice are in accordance with an increased de novo lipogenesis found in these mice following the administration of deuterated water [START_REF] Feng | A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism[END_REF]. The improved ability to convert dietary glucose to fat in the absence of Rev-erb␣ may explained the diminished differences in blood glucose levels between both genotypes fed high-carbohydrate diet or HFD.

In addition, our study demonstrates that the altered circadian control of lipid homeostasis due to the absence of REV-ERB␣ in vivo facilitates HFD-induced obesity. The metabolic phenotype in Rev-erb␣ Ϫ/Ϫ mice is robust, as increased adiposity occurs even on chow diet without significant hyperphagia. Interestingly, an opposite phenotype (i.e., resistance to HFD) has been described in Ror␣-deficient mice [START_REF] Lau | The orphan nuclear receptor, RORalpha, regulates gene expression that controls lipid metabolism: staggerer (SG/SG) mice are resistant to diet-induced obesity[END_REF]. While Rev-erb␣ ϩ/ϩ and Rev-erb␣ Ϫ/Ϫ mice challenged with HFD become overweight and develop hyperleptinemia, hyperlipidemia, hypercholesterolemia, hyperinsulinemia, and fatty liver, these metabolic responses are all significantly greater in Rev-erb␣ Ϫ/Ϫ mice. Besides, high-fatfed Rev-erb␣ Ϫ/Ϫ mice display increased ketonemia, which is likely a response to an overload of the citric acid cycle, blood glucose being converted to acetyl-CoA in parallel to the production of acetyl-CoA from fat breakdown. This indicates that the enhancement of the fat oxidation capacity of the skeletal muscle of Rev-erb␣ Ϫ/Ϫ mice is not sufficient to cope with the adverse effects of prolonged HFD feeding. In addition, hepatic expression of Acc, Fas, and Elovl6 increases only in Rev-erb␣ Ϫ/Ϫ mice at both time points on HFD, in keeping with increased de novo lipogenesis.

The overexpression of Lpl observed in Rev-erb␣ Ϫ/Ϫ mice is not restricted to the skeletal muscle. Indeed, expression of Lpl, which is barely detectable in the adult liver [START_REF] Staels | Perturbation of developmental gene expression in rat liver by fibric acid derivatives: lipoprotein lipase and alpha-fetoprotein as models[END_REF], is significantly up-regulated at ZT12 in the liver of Rev-erb␣ Ϫ/Ϫ mice. This is in line with the 24-h up-regulation of the Lpl transcript found in these mice by Le Martelot et al. (20). Of note, hepatic endothelial lipase mRNA levels are decreased in the liver [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF] while hepatic lipase mRNA expression is similar in Rev-erb␣ Ϫ/Ϫ mice (Supplemental Fig. S6). Interestingly, the metabolic phenotype of chow-fed Rev-erb␣ Ϫ/Ϫ animals is quite similar to that of liver-only Lpl-expressing mice, which show increased liver triglyceride content and ketone body levels [START_REF] Merkel | Lipoprotein lipase expression exclusively in liver. A mouse model for metabolism in the neonatal period and during cachexia[END_REF][START_REF] Kim | Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance[END_REF]. However, Rev-erb␣ Ϫ/Ϫ mice do not exhibit insulin resistance like mice overexpressing Lpl in liver or skeletal muscle [START_REF] Kim | Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance[END_REF]. Of importance, conflicting results have been reported regarding insulin resistance induced by Lpl overexpression [START_REF] Voshol | In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake[END_REF]. In addition, our results show that the overexpression of Figure 8. Hypothetical model illustrating the consequence of altered Lpl expression in Rev-erb␣ Ϫ/Ϫ mice. In addition to altered daily hepatic expression of lipogenic genes [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF][START_REF] Feng | A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism[END_REF], Rev-erb␣ deletion impaired circadian control of Lpl, leading to its overexpression in peripheral tissues. As a result, skeletal muscle may adapt by preferentially utilizing fatty acids (FA), thus sparing glucose. The surplus of glucose is therefore converted to acetyl-CoA through de novo lipogenesis in parallel to the generation of acetyl-CoA from fat breakdown. Acetyl-CoA production exceeding the cellular energy needs, ketone bodies (KB) are synthesized to make use of the energy available (e.g., by muscle). At the same time, fat storage is increased as triglycerides (TG) in both liver and adipose tissue. This leads to a frail energy balance in which greater uptake of fatty acids by the muscle may relatively protect from severe hepatic steatosis and body fat overload. CHYL, chylomicrons; CHYL R, chylomicron remnants.

Lpl also occurs in WAT of Rev-erb␣ Ϫ/Ϫ mice. Specific overexpression of Lpl in adipose tissue does not lead to increased adiposity, an effect that could be due to down-regulation of LPL in other tissues or increase in energy expenditure [START_REF] Hensley | Transgenic mice expressing lipoprotein lipase in adipose tissue. Absence of the proximal 3=-untranslated region causes translational upregulation[END_REF]. Interestingly, Rev-erb␣ Ϫ/Ϫ mice do not show increased body temperature, basal metabolism, or thermogenic response to noradrenaline (data not shown). Moreover, a direct correlation between adipocyte-derived LPL expression and lipid storage has been proposed [START_REF] Gonzales | Role of adipocytederived lipoprotein lipase in adipocyte hypertrophy[END_REF]. Therefore, it is likely that increased Lpl transcription in WAT of Rev-erb␣ Ϫ/Ϫ mice reflects increased activity of LPL, leading to enhanced hydrolysis of circulating triglycerides and facilitated NEFA uptake for storage.

The potential implication of REV-ERB␣ in the transcriptional control of Lpl was assessed by luciferase assay using the proximal promoter region of this gene. In contrast to a Bmal1-luc reporter, mLpl-luc was unresponsive to either REV-ERB␣ or ROR␣. Of interest, an E-box has been found in the mouse Lpl promoter [START_REF] Bey | Cloning, sequencing and structural analysis of 976 base pairs of the promoter sequence for the rat lipoprotein lipase gene. Comparison with the mouse and human sequences[END_REF]. Instead of a direct repression by REV-ERB␣, activation by CLOCK/BMAL1 could drive the temporal expression of the Lpl gene. Indeed, REV-ERB␣ expression is crucial on a daily basis for the transcriptional control of Clock and Bmal1. In the absence of Rev-erb␣, Clock and Bmal1 mRNA are overexpressed in peripheral tissues, which can affect the expression of CLOCK/BMAL1 target genes. Our results show that Lpl expression can be elicited by CLOCK alone. Whether this effect depends on the acetyltransferase activity of CLOCK [START_REF] Doi | Circadian regulator CLOCK is a histone acetyltransferase[END_REF][START_REF] Nader | Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications[END_REF] remains to be established. Such a positive regulation of Lpl transcription by CLOCK/BMAL1 has been previously mentioned (J. M. Gimble and Z. E. Floyd, Pennington Biomedical Research Center, Baton Rouge, LA, USA; unpublished results mentioned in ref. [START_REF] Gimble | Fat circadian biology[END_REF]. Thus, the overexpression of Lpl appears to be the result of a defective clockwork, keeping in mind that the regulation of Lpl transcription can also be achieved by other pathways [START_REF] Gachon | Proline-and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPARalpha) activity[END_REF][START_REF] Schoonjans | PPARalpha and PPARgamma activators direct a distinct tissuespecific transcriptional response via a PPRE in the lipoprotein lipase gene[END_REF].

This study provides strong insight into the role of REV-ERB␣ in the regulation of in vivo energy balance. Our findings do not fully exclude the possibility that impaired development of skeletal muscles [START_REF] Pircher | Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the Rev-erbAalpha orphan receptor gene[END_REF] and adipocytes [START_REF] Wang | Bifunctional role of Reverbalpha in adipocyte differentiation[END_REF] in Rev-erb␣-deficient mice may participate in the observed metabolic phenotype. Alternatively, altered clock functioning in the absence of REV-ERB␣ could account for impaired temporal coordination of developmental processes related to muscle and adipose physiology [START_REF] Gimble | Fat circadian biology[END_REF][START_REF] Andrews | CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function[END_REF]. We propose that altered circadian control of metabolic pathways across peripheral tissues is likely the main cause of the metabolic phenotype of the Rev-erb␣ Ϫ/Ϫ mice. Our data are in agreement with the involvement of REV-ERB␣ in the timing of hepatic lipid metabolism genes [START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF][START_REF] Feng | A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism[END_REF]. Furthermore, we show that altered clock machinery leads to up-regulated transcription of Lpl. In line with a clock-regulated timing of Lpl mRNA expression, we could not detect day-night variation of Lpl in the liver of Rev-erb␣ Ϫ/Ϫ mice. Circadian variations of Lpl mRNA and LPL activity have been observed in various tissues including the liver, with hepatic mRNA acrophase in the early morning [START_REF] Zvonic | Characterization of peripheral circadian clocks in adipose tissues[END_REF][START_REF] Panda | Coordinated transcription of key pathways in the mouse by the circadian clock[END_REF][START_REF] Martelot | REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis[END_REF][START_REF] Tsutsumi | The relationship between lipoprotein lipase activity and respiratory quotient of rats in circadian rhythms[END_REF][START_REF] Gachon | Proline-and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPARalpha) activity[END_REF]. Therefore, in the context of glucose and fat regulation, circadian control of Lpl appears to be essential for a balanced energy metabolism, as suggested by Gimble and Floyd (58). If Lpl is expressed continuously around the clock, fat overload can ensue, while at the same time muscles adapt toward a more oxidative metabolism (Fig. 8).

In summary, the present study uncovers a molecular pathway that ties clock-driven Lpl expression to energy homeostasis and highlights circadian disruption as a potential cause for the etiology of the metabolic syndrome.

SUPPLEMENTAL DATA

Animal experiments: For the intra-peritoneal insulin sensitivity test (IPIST), mice (n = 10 / genotype, sex ratio 1:1) were fasted for 14 h (either from ZT12 to ZT2 or from ZT22 to ZT14) and injected i.p. with 0.5 IU/kg insulin (Umuline®NPH, Lilly, France). For the oral glucose tolerance test (OGTT), mice were fasted for 14 h (from ZT22 to ZT14) and received a glucose load via gavage (2 g/kg; D-Glucose Sigma). Adaptive thermogenesis was assessed in 4-month-old mice (n = 6 / genotype) exposed to 4°C for 6 h from ZT2 (see Argmann et al. (2006) Curr Protoc Mol Biol) and recorded with rectal thermometer (Harvard apparatus, MA, USA). Note that the animals were transferred from their housing room to a 4°C room.

Plasma metabolic parameters: Plasma adiponectin was assayed with ELISA Kits (Adiponectin EZMADP-60K). Plasma corticosterone was measured using an EIA kit (AC-14F1, IDS EURL).

Cell Culture, Transfection, and Luciferase Assay: The entire locus of the Lpl gene (including 10kb upstream of the transcription start site and 10kb downstream of the last exon) was searched for the presence of consensus ROREs (WAWNTRGGTCA, where W is A/T, R is A/G and N corresponds to any nucleotide). A single consensus RORE was found in the Lpl gene within the 46.4kb region examined (NC_000074, nt 71394454..71440831); this corresponds to an intergenic region, located 3.4kb downstream of the last Lpl exon (minus strand, position 39788-39778 of the above-mentioned genomic fragment). Due to its location, the functional relevance of this element is difficult to evaluate and was consequently not investigated. Proximal promoter regions often bear functionally important response elements and most transcription factors can bind sequences slightly divergent from their consensus DNA binding motif. Indeed, several potential non-consensus ROREs were found in the proximal promoter regions of Lpl. We therefore decided to check whether this proximal promoter region was responsive to REV-ERBα/RORα (see results). S1 Chapter 3 DISCUSSION

Supplemental Table

The FEO in Rev-erbα knockout mice

Numerous studies have explored the role of canonical clock genes in the functioning of the FEO. Some have suggested that the latter is a true circadian oscillator and that clock genes such as Per2 or Bmal1 are essential for its clockwork.

Others have indicated that the molecular mechanism of the FEO could either involve non-classical clockwork yet to be discovered or that clock gene deletion fails to deeply affect food entrainment due to the involvement of metabolic and cognitive cues.

Key findings discussed

In 12:12 light-dark conditions, Rev-erbα -/-mice exhibit reduced FAA prior to food access. This result is further confirmed in DD conditions in animals with or without SCN. However, contrary to Per2 mutant mice (Feillet et al. 2006;Mendoza et al. 2010), Rev-erbα deletion does not lead to a total disappearance of FAA 31 .

In contrast to slight reductions of behavioral components, thermogenesis in anticipation is almost abolished in Rev-erbα -/-mice. The anticipatory corticosterone peak and the rise in plasma ketone bodies are not observed either. However, 24-h hepatic clock mRNA oscillations and 24-h hypothalamic PER2 oscillations are RFentrained in Rev-erbα -/-mice as in their control littermates. Nevertheless, we found that the daily PER2 oscillations in the cerebellum are not similarly changed between

Rev-erb +/+ and Rev-erbα -/-mice in response to RF.

At first glance, our results could support the circadian nature of the FEO functioning. Some matters, however, remain unsettled.

Rev-erbα deletion and locomotor behaviors

By evaluating Rev-erbα contribution to food entrainment, we give a new evidence that clock genes are important for the ability of mice to properly anticipate mealtime, in line with previous reports (Dudley et al. 2003;Horikawa et al. 2005;Iijima et al. 2005;Feillet et al. 2006;Mendoza et al. 2010;Mieda et al. 2011). Indeed, the expression of general and wheel-running locomotor activities is significantly impacted prior to food access in Rev-erbα -/-mice. Nevertheless, in our study, the absence of Rev-erbα leads to an overall reduction of wheel-running activity.

Interestingly, this has been observed in some [START_REF] Lopezmolina | The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior[END_REF][START_REF] Bunger | Mop3 is an essential component of the master circadian pacemaker in mammals[END_REF], but not all circadian mutants [START_REF] Vitaterna | Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior[END_REF][START_REF] Zheng | The mPer2 gene encodes a functional component of the mammalian circadian clock[END_REF]). Thus, it is not excluded that mice that do not behave similarly in the presence of a running wheel can eventually influence the accurate evaluation of food entrainment.

As previously mentioned, not all studies agree on the involvement of clock genes for the expression of FAA or for a proper MASCO functioning [START_REF] Mohawk | The methamphetamine-sensitive circadian oscillator does not employ canonical clock genes[END_REF]Storch et al. 2009), disputing the reliance of the putative FEO and MASCO oscillators on a SCN-like molecular mechanism. Most often, however, these studies did not investigate other parameters than wheel-running behaviors. In addition, FAA is rarely evaluated: in different lighting conditions 32 ; following mealtime jet-lag test; or in T-cycle experiments (period of feeding different from 24 h, but in the circadian range). We have evaluated FAA in constant dark conditions, in a skeleton photoperiod (i.e., 1 h light-pulse at the beginning of the resting period and 1 h lightpulse at the end of the resting period) as well as following mealtime jet-lag test, without revealing more FAA than in LD conditions (unpublished data; see appendix 1). Incidentally, considering the fact that the FEO and MASCO may be a unique entity, the use of MAP injections could be informative to further substantiate the involvement of Rev-erbα in circadian pathways.

Rev-erbα deletion and clock gene oscillations

As outlined above, the SCN functioning is altered in the absence of Rev-erbα (Preitner et al. 2002). Regarding the FEO, since its location/network has not been yet

clearly revealed, we have to search rather blindly for altered molecular oscillations. At least, we found neither major changes of core clock mRNA expression (i.e., Per1-2)

in liver nor of PER2 expression in key hypothalamic nuclei known to be circadian oscillators, in both AL and RF conditions. It is thus plausible that the circadian network (probably including the FEO) could still work in spite of genetic perturbations induced by the depletion of Rev-erbα-thus contributing to the expression of some FAA. For instance, active compensatory mechanisms in the clock network have been recently demonstrated in vitro [START_REF] Baggs | Network Features of the Mammalian Circadian Clock[END_REF]. In that study, knockdown of Reverbβ was shown to increase expression of its paralog Rev-erbα, ensuring a "genetic backup". In this regard, more recently, Rev-erbβ has been proposed to compensate for the absence of Rev-erbα in mouse embryonic fibroblasts and to protect circadian clock function [START_REF] Bugge | Rev-erbalpha and Rev-erbbeta coordinately protect the circadian clock and normal metabolic function[END_REF]) 33 . At least in the liver (an organ which demonstrates significant metabolic changes in the absence of Rev-erbα, see below), 24-h Rev-erbβ levels are unchanged in adult Rev-erbα -/-mice (Schmutz et al. 2010).

On the other hand, without strong modifications in the core clock functioningapart from altered Bmal1 and Clock levels-of hypothalamic and peripheral oscillators, it is possible that downstream targets of the FEO and of the Rev-erbα gene are differentially impacted. This could therefore explained why some physiological and metabolic parameters, considered as FEO outputs, such as thermogenesis (mainly controlled by the preoptic area), corticosterone release (controlled by the adrenal), ketone bodies production (by the liver), are severely impaired in Rev-erbα -/-mice under scheduled food restriction, although we did not investigate clock and metabolic gene expression in the adrenal gland and the preoptic area (for investigations on the liver, see part 2.1).

As for the CRB oscillator, our data on PER2 expression in Rev-erbα -/-mice under RF are intriguing in the light of a recent study demonstrating that a functional cerebellar oscillator is essential to ensure proper FAA-but not for the development of the food-anticipatory peak of corticosterone and the phase-adjustment of hypothalamic Per1-2 expression to mealtime (Mendoza et al. 2010). However, it seems premature to correlate the altered PER2 oscillations in the cerebellum of Reverbα -/-mice to their defect to show robust food anticipation. Our view of the oscillator properties of the CRB of Rev-erbα -/-mice is limited since we did not evaluate the daily pattern of expression of additional clock proteins. Indeed, mutation of clockrelated factors such as Overtime or Cry1 can significantly affect the circadian expression of some, but not all, clock transcripts/proteins in the CRB [START_REF] Miyamoto | Circadian regulation of cryptochrome genes in the mouse[END_REF][START_REF] Siepka | Circadian mutant overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression[END_REF]. Moreover, the role of the CRB in circadian timing processes such as food-entrainable oscillations is not yet defined. There is evidence of clock mRNA/protein oscillations in this brain region [START_REF] Siepka | Circadian mutant overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression[END_REF][START_REF] Farnell | Neonatal alcohol exposure differentially alters clock gene oscillations within the suprachiasmatic nucleus, cerebellum, and liver of adult rats[END_REF]) that can be entrained by RF (present results and Mendoza et al. 2010).

However, the CRB fine-tunes motor activity, but does not initiate it; thus, decreased food-anticipatory activities cannot be solely imputed to impaired clockwork in this structure. Besides that, the CRB represents rather a weak oscillator compared to the SCN, since the slight PER1 oscillations in tissue explants damped out relatively quickly (Mendoza et al. 2010). In this regard, it is not known if the coordinated daily clock gene expression in the CRB requires extrinsic factors from the brain (e.g., indirect coupling with the SCN pacemaker) and/or the periphery (e.g., feedback from feeding-related signals). Furthermore, since daytime RF challenge leads to the appearance of locomotor activity prior to mealtime and can shorten the main bout of nocturnal activity [START_REF] Challet | Entrainment in calorie-restricted mice: conflicting zeitgebers and free-running conditions[END_REF][START_REF] Holmes | Food anticipatory activity and photic entrainment in food-restricted BALB/c mice[END_REF], to what level these changes are interpreted by the CRB oscillator? This is especially important considering that locomotor activity, particularly the access to a running wheel, can feedback to the circadian system [START_REF] Turek | Effects of stimulated physical activity on the circadian pacemaker of vertebrates[END_REF][START_REF] Lax | Coupling effect of locomotor activity on the rat's circadian system[END_REF][START_REF] Campuzano | The role of wheel running in the coupling of two simultaneous circadian rhythms of motor activity in the rat[END_REF][START_REF] Cambras | Entrainment of the rat motor activity rhythm: effects of the light-dark cycle and physical exercise[END_REF][START_REF] Van Der Veen | Behavioral Responses to Combinations of Timed Light, Food Availability, and Ultradian Rhythms in the Common Vole (Microtus arvalis)[END_REF]. Since Rev-erbα -/-mice show decreased wheel-running activity, this could render difficult the establishment and expression of food-entrained circadian rhythms34 . To summarize, beyond the need to evaluate the property of CRB cells (i.e., the Purkinje cells) to exhibit for instance rhythmic neuronal activity as a putative circadian electrical output signal to modulate food anticipation, it would be informative to study the degree of sensitivity of clock gene oscillations to diverse manipulations, such as following SCN removal or in the absence of a feedback from the periphery 35 . Likewise, instead of pharmacological/genetic impairments of the cerebellar architecture, the use of inducible circadian mutant to only affect the oscillator properties of the CRB could be enlightening.

Rev-erbα deletion and noncircadian effects

It is also important not to exclude impaired additional biological functions in the observed phenotype. Circadian oscillators are considered ubiquitous and the nuclear receptor Rev-erbα is no exception to the rule.

In this context, oligophrenin-1, important for dendritic spine morphogenesis and hippocampus-dependent spatial memory [START_REF] Khelfaoui | Loss of X-linked mental retardation gene oligophrenin1 in mice impairs spatial memory and leads to ventricular enlargement and dendritic spine immaturity[END_REF], has been recently shown to interact with Rev-erbα [START_REF] Valnegri | A circadian clock in hippocampus is regulated by interaction between oligophrenin-1 and Rev-erbalpha[END_REF]. Lack of FAA in Reverbα -/-mice could hence result from disrupted higher order cognitive processes and associative learning-and their interactions with the circadian system 36 . This view is supported by studies on Cry1 -/-;Cry2 -/-mice, which show altered onset and robustness of FAA (Iijima et al. 2005) concomitant to a lack of their ability to achieve time-place learning (Van der [START_REF] Van Der Zee | Circadian time-place learning in mice depends on Cry genes[END_REF]. This is also supported by reports on Npas2 -/-mice that demonstrate deficits in contextual and cues memory [START_REF] Garcia | Impaired cued and contextual memory in NPAS2-deficient mice[END_REF] as well as delayed development of FAA in response to RF (unpublished data and Dudley et al. 2003). We have not investigated yet, however, cognitive defects (such as the ability to discriminate and remember a circadian phase) as a potential confounding effect of the disrupted FAA in Rev-erbα -/-mice 37 .

Another element that could account for the reduced FAA in Rev-erbα -/-mice concerns mood and anxiety disorders. Mice harboring mutations in clock-related genes have interesting phenotypes [START_REF] Easton | The circadian Clock mutation increases exploratory activity and escape-seeking behavior[END_REF][START_REF] Roybal | Mania-like behavior induced by disruption of CLOCK[END_REF][START_REF] Li | Disruption of the Circadian Output Molecule Prokineticin 2 Results in Anxiolytic and Antidepressant-like Effects in Mice[END_REF]. In particular, mice overexpressing GSK3β are considered as a good model of hyperactivity and mania [START_REF] Prickaerts | Transgenic mice overexpressing glycogen synthase kinase 3 beta: A putative model of hyperactivity and mania[END_REF]. At least, what we have noticed is that Rev-erbα deficient mice do not suffer from a loss of motivation to eat after a period of food withdrawal or an aversion to consume a palatable snack (personal observations). However, we still have to assess more specifically motivation-oriented behaviors. On the other hand, we have evaluated anxiety and depressive-like behaviors in Rev-erbα -/-mice and found no noteworthy difference in comparison to wild-type littermates (unpublished data, see appendix 2).

36 And vice-versa; indeed, the recourse to higher order cognitive processes and associative learning could contribute to the apparition of residual FAA in the presence of a dysfunctional FEO (network). 37 As an aside, Per2 Brdm1 mutant mice that exhibit abolished FAA (Feillet et al. 2006), show normal spatial and contextual learning (Zueger et al. 2006). However, the time-of-day regulation of memory has not yet been studied in Per2 mutant mice.

Rev-erbα deletion and energy balance

As aforementioned in the introduction, studies on the circadian nature of the FEO can also be inconclusive due to the strong interactions between the circadian network and metabolism.

In our study Rev-erbα-deficient mice were healthy as evidenced by similar body mass, water/food intake, and general locomotor activity. Nevertheless, we revealed significant changes in bodily functions such as glucose metabolism, ketone bodies utilization and heat production, either on AL or on RF conditions. Moreover, the responses of several hypothalamic nuclei to food access were not clearly observed at the level of p-ERK expression.

These elements could contribute to altered food entrainment as well, and are discussed in the following part.

The energy homeostasis of Rev-erbα knockout mice

In the last decades, tight connections between clock and metabolic cycles have been revealed. Body's functions and body's energy levels cycle along the day.

The SCN pacemaker and slave oscillators timely coordinate metabolic processes to ensure their occurrence at the right time of the day. Rev-erbα, a gear of the circadian clockwork, holds a prominent role in this regard.

Key findings discussed

Rev-erbα -/-mice show higher blood glucose values than those of control mice across the whole 24-h cycle and after a fasting period as well. However, this is not due to decreased sensitivity to exogenous insulin, decreased production of endogenous insulin or increased hepatic glucose production in the absence of Reverbα 38 . Instead, indirect calorimetric measurements of resting energy expenditure in AL conditions and a 24-h fast challenge indicate that Rev-erbα -/-mice rely heavily on fatty acids as a source of energy.

In addition, Rev-erbα -/-mice fed with a standard normocaloric diet exhibit increased adiposity that can be severely enhanced under high-fat diet regimen. They also develop hyperlipidemia and hyperleptinemia, regardless of the feeding conditions. Their defective energy homeostasis was a consequence of abnormal timing in the liver clockwork, as evidenced by a slight delayed in lipogenesis during the night period and altered expression of lipogenic genes. Moreover, lipoprotein lipase (Lpl) gene expression is significantly enhanced in peripheral oscillators following Rev-erbα depletion, thus contributing to altered lipid homeostasis (i.e., lipid utilization and storage).

These results highlight the significance of daily variations in clock gene expression for the control of metabolic gene expression, essential for fuel balance.

Rev-erbα and glucose metabolism

Depletion of Rev-erbα by using small interfering RNA (siRNA) molecules has been shown to increase the expression of G6pc and Pepck mRNA in a human hepatoma cell line (Yin et al. 2007). Of note, Rev-erbα overexpression has the opposite effect. The human G6pc promoter contains a RORE site, important for the repressive action of Rev-erbα. Moreover, hemin treatment also diminishes gluconeogenic gene expression and glucose production in vitro, demonstrating the metabolic relevance of heme binding to Rev-erbα. These results suggest that Reverbα may play a role in the daily variation of gluconeogenesis (Yin et al. 2007). In that regard, we observed that Rev-erbα deletion affects glucose levels in vivo, in animals fed AL 39 , fasted overnight, fasted for 4 h (from ZT16 to ZT0; unpublished data) or for 24 h. However, this is not due to increased gluconeogenesis as first hypothesized.

Indeed, neither Pepck nor G6pc mRNA are significantly elevated in the liver of Reverbα -/-mice (our results and Le Martelot et al. 2009) or decreased in the liver of Reverbα overexpressing mice (Le Martelot et al. 2009). Likewise, in vitro hepatic glucose production from Rev-erbα -/-hepatocytes is not enhanced in the presence of lactate and pyruvate.

Recently, the pancreatic islets have been shown to exhibit robust oscillations of clock gene, and deletion of Clock or Bmal1 has deleterious consequence on βcells function (Marcheva et al. 2010). Interestingly, Rev-erbα depletion by siRNA treatment in islets cells impairs glucose-induced insulin secretion and β-cell proliferation [START_REF] Vieira | The Clock Gene Rev-erbalpha Regulates Pancreatic beta-Cell Function: Modulation by Leptin and High-Fat Diet[END_REF]). However, we did not detect alterations in pancreas physiology that could explain the mild hyperglycemia in germline KO mice. It is thus not improbable that metabolic regulations (in relation with the circadian system) are reorganized to some extent during development (i.e., compensatory mechanisms as discussed above) in the absence of Rev-erbα.

Since we have done metabolic tests around the clock to evaluate glucose homeostasis and have explored the hormonal system (e.g., insulin, glucagon and glucocorticoïds) that regulate glucose balance, without being able to find strong 39 Review of the literature and our data indicate strong inter-study variations of the blood glucose acrophase in AL-fed C57BL6/J wild-type mice under 12:12 LD cycle, making difficult to assess alterations in the daily pattern of blood glucose. We did not investigate the daily/circadian blood glucose rhythm in fasted animals.

defects that could account for the recurring mild hyperglycemia seen in Rev-erbα -/- mice, we favor the presence of altered timing in energy substrate utilization.

Rev-erbα and lipid metabolism

As mentioned in the introduction, the nuclear receptor Rev-erbα has been shown to play a role in bile acid, cholesterol and lipoprotein metabolism, particularly by controlling the expression of key components of metabolic pathways.

Regarding lipoprotein metabolism, liver ApoA1 expression 40 can be regulated by Rev-erbα in vitro [START_REF] Vu-Dac | The nuclear receptors peroxisome proliferator-activated receptor alpha and Rev-erbalpha mediate the species-specific regulation of apolipoprotein A-I expression by fibrates[END_REF] and in vivo (Le Martelot et al. 2009). Apoc3 transcript 41 has also been demonstrated to be controlled by Rev-erbα in vitro and to be up-regulated in the liver of Rev-erbα -/-mice (Raspe et al. 2002) 42 , an observation, however, not confirmed by others (our results and Le Martelot et al. 2009). Further in the lipoprotein metabolism, we found that the Lpl transcript is consistently elevated in three peripheral tissues of Rev-erbα -/-mice. The up-regulation of Lpl is not directly a consequence of abolished repressive action of Rev-erbα on Lpl transcription, but involves enhanced CLOCK-BMAL1 transactivation on the Lpl promoter. Intriguingly, the expression of Lpl-luc reporter could be elicited by CLOCK alone. Whether CLOCK acts directly (as an E-Box binding partner for a protein endogenously present in COS7 cells) or indirectly (e.g. through its acetyltransferase activity; see Doi et al. 2006;Nader et al. 2009) remains to be established. At least, co-transfection with CLOCK-BMAL2 does not induce more luciferase activity than transfection with CLOCK alone (unpublished data, Dardente Hugues). Conversely, the role of CLOCK alone in the modulation of clock and metabolic gene transcription has been previously observed (Oishi et al. 2005;[START_REF] Cai | Expression levels of estrogen receptor beta are modulated by components of the molecular clock[END_REF]Nader et al. 2009;[START_REF] Doi | CLOCK regulates circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2[END_REF][START_REF] Shi | Circadian Clock Gene Bmal1 Is Not Essential; Functional Replacement with its Paralog, Bmal2[END_REF]. It is also important to mention that a recent study demonstrate that small hairpin RNA-mediated knockdown of Rev-erbβ potentiates the upregulation of Lpl in the liver of Rev-erbα -/-mice [START_REF] Bugge | Rev-erbalpha and Rev-erbbeta coordinately protect the circadian clock and normal metabolic function[END_REF]-although Clock mRNA level has not been evaluated in this study. 40 APOA1 is a major protein component of high density lipoprotein in plasma. 41 APOC3 is a protein component of very low density lipoprotein. It is has been shown to inhibit lipoprotein lipase and hepatic lipase in vitro, however, APOC3 could have rather little effect in vivo (Mendivil et al. 2010). 42 It is important to mention that genetic variations between mouse stains can contribute to different metabolic phenotype in mutant mice.

Worth to mention, as well, is the induction of Lpl in other circadian mutants [START_REF] Shimba | Deficient of a Clock Gene, Brain and Muscle Arnt-Like Protein-1 (BMAL1), Induces Dyslipidemia and Ectopic Fat Formation[END_REF]. Indeed, Bmal1 -/-and liver-specific Bmal1 -/-mice display a startling up-regulation of hepatic Lpl mRNA expression 43 , while muscle Lpl expression remains unchanged in both mutant mice-however, again, without data on Clock mRNA expression. In addition, in primary hepatocytes from control animals, a two-fold reduction of Bmal1 expression via siRNA knockdown, which is associated with a two-fold decrease of Rev-erbα mRNA levels without affecting Clock expression, has a slight effect on Lpl transcript level. None mechanistic explanation has been proposed in that study [START_REF] Shimba | Deficient of a Clock Gene, Brain and Muscle Arnt-Like Protein-1 (BMAL1), Induces Dyslipidemia and Ectopic Fat Formation[END_REF]. Additional studies show: 1) that BMAL1 overexpression in 3T3-L1 adipocytes has no consequence on the expression of the Lpl gene [START_REF] Shimba | Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis[END_REF]; 2) that Rev-erbα and Rev-erbβ are nearly undetectable at all time points in Bmal1 -/-animals (Kondratov et al. 2006;[START_REF] Grechez-Cassiau | The circadian clock component BMAL1 is a critical regulator of p21WAF1/CIP1 expression and hepatocyte proliferation[END_REF]) and 3) that the circadian oscillations of the nuclear accumulation and degradation of CLOCK have been suggested to depend on BMAL1 [START_REF] Kondratov | BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system[END_REF][START_REF] Kwon | BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer[END_REF]. Thus, further studies are needed to shed more light on the importance of the molecular clockwork for the daily control of Lpl expression with regard to the possible contribution of other pathways (Schoonjans et al. 1996;[START_REF] Schoonjans | Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes[END_REF]Gachon et al. 2011).

"Once we succeed in finding the clock gene … boy, we'd lick the problem of mechanisms …" C. Pittendrigh to M.K. Chandrashekaran in Berkeley, 1968(mentioned in Chandrashekaran 1998). 43 In the study of Shimba et al. 2011 -Figure 7, the normalized Lpl mRNA levels to the 36b4 levels are quite similar among peripheral tissues. In addition, the acrophase of Lpl mRNA (see the daily and circadian patterns of Lpl expression) are 6-h phase-delayed compared to our results, those of Le Martelot et al. 2009 andGachon et al. 2011. Besides, Lpl levels are similar to those of Bmal1 in control animals, while when L-Bmal -/-and control mice are compared, Lpl levels are just above 0 in control animals. All these elements are hard to understand and raise concerns about the normalization method and the Lpl primer sequences. Furthermore, regardless of Lpl overexpression, the body mass of Bmal1 -/-mice was much lower than that of control littermates, both in chow-fed and high-fat-fed conditions, which deeply contrasts with independent studies [START_REF] Lamia | Physiological significance of a peripheral tissue circadian clock[END_REF][START_REF] Guo | The clock gene, brain and muscle Arnt-like 1, regulates adipogenesis via Wnt signaling pathway[END_REF]. Discrepancies between studies that specifically evaluate adjustment to environmental changes and energy metabolism can result from the development of diseases due to pathogen exposition. All our experiments were done with mice raised in a pathogen-free facility.

As evidenced by the evaluation of energy expenditure in chow-fed mice and their circulating energy substrates following a 24-h challenge, Lpl overexpression leads to increased utilization of fatty acids as energy source in Rev-erbα -/-mice, thus sparing glucose use as shown by higher blood glucose levels and glycogen stores in chow-fed, RF and fasting conditions 44 . Since the resting muscle is a major contributor of fatty acid oxidation, we analyzed metabolic gene expression in this tissue. We showed no significant decrease in the capacity of the muscle of Rev-erbα -/-mice to oxidize fatty acids, regarding Cpt1b mRNA expression. Along with overexpressed Lpl mRNA, we found that Cd36 transcript is also up-regulated in the muscle of Rev-erbα - /-mice. These findings thus support enhanced fatty acids entry into muscular cells, although we did not evaluate LPL activity in muscle tissues of Rev-erbα -/-mice or in vivo metabolic flux. Our results are consistent with the previously described shift towards an oxidative muscle phenotype in Rev-erbα -/-mice. Indeed, the type I MyHc isoform in the slow-twitch soleus muscle (mainly oxidative) and the type I fiber area in the fast-twitch extensor digitorum longus muscle (oxidative and highly glycolytic) were found to be increased in Rev-erbα -/-mice (Pircher et al. 2005). Incidentally, the importance of the circadian nuclear receptors in skeletal muscle cells is also highlighted by the role of Rorα in lipid homeostasis 45 [START_REF] Lau | RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR[END_REF]) and the control of genes important for lipid absorption (e.g., Cd36) by Rev-erbβ [START_REF] Ramakrishnan | Rev-erbbeta regulates the expression of genes involved in lipid absorption in skeletal muscle cells: evidence for crosstalk between orphan nuclear receptors and myokines[END_REF].

The overexpression of Lpl also favors fat accumulation. We found that chowfed Rev-erbα -/-mice suffer from dyslipidemia, in line with previous reports (Raspe et al. 2002;Le Martelot et al. 2009). In addition, Rev-erbα -/-mice have severe adipose accumulation in chow-fed conditions and are more prone to HFD-induced obesity compared to control mice 46 , likely due to enhanced LPL-mediated fatty acids uptake.

The level of hepatic triglycerides at the day-night transition is also elevated in these mice (our results and Feng et al. 2011;[START_REF] Bugge | Rev-erbalpha and Rev-erbbeta coordinately protect the circadian clock and normal metabolic function[END_REF]. Whether increased hepatic TG is due to increased Lpl mRNA expression remains unclear, considering that the capacity of the normal adult liver to synthesize LPL and its role are still a 44 We did not, however, evaluate energy expenditure in fasted Rev-erbα -/-animals. 45 Of note, Rorα -/-mice expressed similar levels of Lpl mRNA in skeletal muscle in comparison to wild-type animals. 46 Interestingly, 3 mo. old Rev-erbα -/-mice high-fat-fed for 12 w also developed an obesity phenotype and severe dyslipedemia in contrast to their controls that did not (i.e., high-fed Rev-erbα +/+ mice and chow-fed Reverbα -/-mice; unpublished data).

subject of debate [START_REF] Vilaro | Intralipid Administration Induces a Lipoprotein Lipase-Like Activity in the Livers of Starved Adult-Rats[END_REF][START_REF] Vilaro | Lipoprotein-Lipase Uptake by the Liver -Localization, Turnover, and Metabolic Role[END_REF][START_REF] Benavides | Circadian rhythms of lipoprotein lipase and hepatic lipase activities in intermediate metabolism of adult rat[END_REF]Merkel et al. 1998;[START_REF] Merkel | Lipoprotein lipase: genetics, lipid uptake, and regulation[END_REF]. However, several works have shown that hepatic Lpl up-regulation can contribute to hepatic steatosis in humans and rodents (Kim et al. 2001;[START_REF] Westerbacka | Genes involved in fatty acid partitioning and binding, lipolysis, monocyte/macrophage recruitment, and inflammation are overexpressed in the human fatty liver of insulin-resistant subjects[END_REF][START_REF] Pardina | Lipoprotein Lipase Expression in Livers of Morbidly Obese Patients Could be Responsible for Liver Steatosis[END_REF][START_REF] Barclay | GH-Dependent STAT5 Signaling Plays an Important Role in Hepatic Lipid Metabolism[END_REF]. Of note, the fatty acid transporter Fabp5 is up-regulated in the liver of Rev-erbα -/-mice (Le Martelot et al. 2009). Moreover, we showed that HFD induces an up-regulation of Cd36 mRNA only in Rev-erbα -/-mice. Hence, it is clear that hepatic fatty acid transport is altered in the absence of Rev-erbα.

Emblematic of significant changes in lipid homeostasis in the liver of Rev-erbα-/-mice, its capacity of lipogenesis is also modified. We demonstrate in vivo that Reverbα depletion results in a change in RQ values during the active/feeding period-in line with the described phase-shifted expression of lipogenic genes in the late night (Le Martelot et al. 2009) and the importance of genomic recruitment of HDAC3 by

Rev-erbα on a daily basis to ensure fatty acid synthesis at the most appropriate time of the day (Feng et al. 2011). Indeed, the acrophase of RQ values is slightly delayed in Rev-erbα -/-mice and RQ values are higher compared to control littermates during nighttime, indicative of enhanced hepatic de novo lipogenesis 47 . Interestingly, the importance of Rev-erbα in the control of lipogenesis is highlighted by several studies.

The circadian transcription activity of Elovl3, which plays a role in fatty acid chain elongation, has been suggested to be mediated by Rev-erbα [START_REF] Anzulovich | Elovl3: a model gene to dissect homeostatic links between the circadian clock and nutritional status[END_REF]Le Martelot et al. 2009). Additionally, Elovl6 mRNA expression is phase-shifted in the liver of Rev-erbα -/-mice (Le Martelot et al. 2009). These results support a modulating role for Rev-erbα in the synthesis of very long chain fatty acids. In this context, we showed that Elovl6 mRNA is severely increased in the liver of high-fat fed Rev-erbα -/- mice. Additional lipogenic genes (i.e., Fas, Acc) are up-regulated as well. Incidentally, this elevated expression of lipogenic genes in Rev-erbα -/-mice could also be an adaptive response to energy surplus [START_REF] Tepperman | Adaptive hyperlipogenesis--late 1964 model[END_REF]) and reflect a remodeling of hepatic fatty acids [START_REF] Oosterveer | High fat feeding induces hepatic fatty acid elongation in mice[END_REF]).

Rev-erbα and developmental defects

We have demonstrated that the consistent overexpression of Lpl in liver, adipose tissue and skeletal muscle, results from a defective clock control of this gene. However, beyond clock impairment, we do not entirely exclude growth and developmental defects that could contribute to the metabolic phenotype of Rev-erbα -/- mice.

As previously introduced, both REV-ERBs are crucial during developmental stages. In particular, REV-ERBα is essential for the appropriate balance of transcriptional activators and repressors during postnatal cerebellar development [START_REF] Chomez | Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor[END_REF]. REV-ERBβ has been suggested to be important for latter aspects of neuronal development, such as axon guidance, and at later stages of muscle differentiation [START_REF] Bonnelye | Rev-erb beta, a new member of the nuclear receptor superfamily, is expressed in the nervous system during chicken development[END_REF]. REV-ERBα expression was linked to muscle fiber composition (Pircher et al. 2005) and both Rev-erbα and Rev-erbβ have been shown to play a role in skeletal muscle growth and differentiation, the former notably by regulating the MyoD and Myogenin genes [START_REF] Downes | Constitutive expression of the orphan receptor, Rev-erbA alpha, inhibits muscle differentiation and abrogates the expression of the myoD gene family[END_REF][START_REF] Burke | Transcriptional repression by the orphan steroid receptor RVR/Rev-erb beta is dependent on the signature motif and helix 5 in the E region: functional evidence for a biological role of RVR in myogenesis[END_REF]. Besides, Rev-erbα mRNA has been shown to increase during differentiation of 3T3-L1 cells into adipocytes [START_REF] Chawla | Induction of Rev-ErbA alpha, an orphan receptor encoded on the opposite strand of the alpha-thyroid hormone receptor gene, during adipocyte differentiation[END_REF][START_REF] Fontaine | The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation[END_REF], although this is more likely the proteasomal degradation of the REV-ERBα protein that is critical for late stages of adipocyte differentiation (Wang et al. 2008).

Regarding muscle development, additional studies give evidence that interactions between clocks component and actors of muscle physiology are crucial for proper muscle function. Indeed, MyoD, a regulator of myogenesis, is regulated by CLOCK and BMAL1 (Andrews et al. 2010). Moreover, Bmal1-deficient mice develop sarcopenia (Kondratov et al. 2006). Clock Δ19 and Bmal1 -/-mice exhibit reduced skeletal muscle specific tension concomitant to altered myofilament architecture (Andrews et al. 2010). Muscle performance and metabolism are also affected in mPer2 -/-mice [START_REF] Bae | Differential effects of two period genes on the physiology and proteomic profiles of mouse anterior tibialis muscles[END_REF]). On the other hand, the importance of circadian genes and adipose functions are also highlighted by several studies. High-fat-fed Clock Δ19

mice develop adipocyte hypertrophy relative to wild-type controls (Turek et al. 2005).

Lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts and increased TG contents in WAT in mice [START_REF] Grimaldi | PER2 controls lipid metabolism by direct regulation of PPARgamma[END_REF]. Disruption of the Bmal1 gene also leads to increased total body fat content [START_REF] Lamia | Physiological significance of a peripheral tissue circadian clock[END_REF][START_REF] Shi | Circadian Clock Gene Bmal1 Is Not Essential; Functional Replacement with its Paralog, Bmal2[END_REF]) that is amplified under HFD due to up-regulation of adipogenic factors and down-regulation of genes of the canonical Wnt signaling pathway, important to suppress adipogenesis [START_REF] Ross | Inhibition of adipogenesis by Wnt signaling[END_REF][START_REF] Guo | The clock gene, brain and muscle Arnt-like 1, regulates adipogenesis via Wnt signaling pathway[END_REF].

The implication of Rev-erbα in specific developmental pathways and its capacity to modulate adipogenesis and muscle development were first considered with no overt effects on WAT expansion and the organization of aerobic, intermediate and anaerobic muscle fibers in adult Rev-erbα -/-mice [START_REF] Chomez | Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor[END_REF].

However, these mice do display increased adiposity, and altered MyHC isoform expression in skeletal muscle 48 (Pircher et al. 2005), that contribute to enhanced oxidative metabolism. To what extent the involvement of Rev-erbα in clockindependent functions has contributed to the phenotype that we and other groups have observed remains to be further investigated. Indeed, while we privilege altered circadian molecular oscillations per se, we could not fully exclude that aberration in the early stages of adipose tissue development, such as hyperplastic growth, could have favored lipid storage, beyond Lpl overexpression. Besides, it is plausible that the fast-to-slow transition during muscle development in the absence of Rev-erbα could underlie Lpl up-regulation. In that perspective, Lpl overexpression could rather appear as a consequence of developmental defects and the role of Clock transactivation on the Lpl promoter that we demonstrate in vitro could rather be negligible in vivo. However, why does Lpl overexpression also occur in the liver oscillator? Indeed, in the presence of an enhanced muscle oxidative metabolism, the liver would have just likely compensated for the unused glucose by eliciting the expression of lipogenic genes, but not Lpl. In this context, the evaluation of Lpl expression in other tissues could be informative to confirm defective clock control of Lpl.

Lpl in central and peripheral tissues

"[…] the whole is something besides the parts" Aristotle, Metaphysics

The etiology for the mild hyperglycemia seen in chow fed, high-fat fed and fasted Rev-erbα -/-mice is believed to be caused by a greater reliance on fatty acids as energy source, particularly by the muscle. But it remains uncertain why liver or muscle Lpl overexpression has not induced insulin resistance (IR) in chow-fed Reverbα -/-mice, as in previous reports [START_REF] Ferreira | Overexpressing human lipoprotein lipase in mouse skeletal muscle is associated with insulin resistance[END_REF]Kim et al. 2001) 49 . This is all the more surprising because insulin sensitivity (assessed by the intraperitoneal insulin sensitivity test) was either similar or greater in high-fat-fed Rev-erbα -/-mice compared to high-fat-fed control littermates or chow-fed Rev-erbα -/-mice (unpublished data), thus excluding an evolution toward diabetes. However, not all studies show that Lpl overexpression induces a state of IR in mice. For instance, muscle TG content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake in muscle-specific Lpl-overexpressing mice. In addition, these mice exhibited a strong decrease in whole-body glucose oxidation, whereas nonoxidative glucose disposal (i.e., for glycogen and lipid synthesis) was increased during hyperinsulinemic-euglycemic clamp conditions (Voshol et al. 2001).

In this study, the liver was suggested to have compensated by increasing insulin sensitivity. This is intriguing since we show in Rev-erbα -/-mice that whole-glucose insulin sensitivity is normal and that the liver may have enhanced its capacity to process glucose for fat synthesis, which is supported by a recent study (Feng et al. 2011).

Pancreatic islet cells can express Lpl transcript and show enzyme LPL activity (i.e., delivery of fatty acids to β-cells), which are regulated by glucose and insulin [START_REF] Cruz | Glucose and insulin stimulate heparin-releasable lipoprotein lipase activity in mouse islets and INS-1 cells. A potential link between insulin resistance and beta-cell dysfunction[END_REF]. However, higher LPL activity in β-cell has been predicted to impair cell function such as insulin release and to promote lipotoxicity and apoptosis [START_REF] Cruz | Glucose and insulin stimulate heparin-releasable lipoprotein lipase activity in mouse islets and INS-1 cells. A potential link between insulin resistance and beta-cell dysfunction[END_REF]). More recently, the effect of Lpl depletion and overexpression in pancreatic islets has been investigated. β-cell-specific Lpl-overexpressing mice had normal glucose tolerance until 5 months of age, while βLPL-KO mice display 49 Of note, in Kim et al. 2001, no effect of liver-Lpl overexpression was observed on total body insulinstimulated glucose uptake. However, the steady-state glucose infusion rate (average rate from 90 to 120 min) required to maintain euglycemia was significantly decreased in the liver-LPL mice during hyperinsulinemiceuglycemic clamp.

defective glucose tolerance from 1 month of age. Additionally, the increased LPL activity has been shown to reduce glucose oxidation in islets [START_REF] Pappan | Pancreatic beta-cell lipoprotein lipase independently regulates islet glucose metabolism and normal insulin secretion[END_REF]. At least, our observations of unaltered glucose-induced insulin secretion, plasma insulin levels, as well as number, morphology and insulin content of pancreatic islets in Reverbα -/-mice, do not support a strong effect of a putative Lpl overexpression in pancreas. Incidentally, in rabbits, systemic overexpression of human LPL transgene (under the control of the actin promoter) has been shown to increase whole body insulin sensitivity [START_REF] Liu | High lipoprotein lipase activity increases insulin sensitivity in transgenic rabbits[END_REF]) and protect from HFD-induced IR [START_REF] Kitajima | Overexpression of lipoprotein lipase improves insulin resistance induced by a high-fat diet in transgenic rabbits[END_REF]). It has been also suggested that LPL could modulate insulin signal pathway (e.g., in muscle), via a direct action on the insulin receptor substrate 1 [START_REF] Liu | High lipoprotein lipase activity increases insulin sensitivity in transgenic rabbits[END_REF]). However, we did not evaluate total plasma LPL or the intracellular handling of glucose in Rev-erbα -/-mice (e.g., in the absence of altered insulin-mediated glucose transport, a disrupted glycolytic pathway could have spared glucose in favor of lipid utilization 50 ). Besides, we have to consider that the adverse effect of Lpl/LPL upregulation may be species-dependent-especially considering that systemic Lpl overexpression can have beneficial effects by correcting hyperlipidemia and reducing body fat accumulation in high-fat-fed rabbits [START_REF] Koike | Overexpression of lipoprotein lipase in transgenic Watanabe heritable hyperlipidemic rabbits improves hyperlipidemia and obesity[END_REF]).

The role of Lpl in the nervous system has been particularly highlighted recently. Mice with neuron-specific deletion of Lpl have reduced Lpl mRNA and LPL enzyme activity in both the hypothalamus and hippocampus and display altered energy balance, as reflected by increased fat mass and lower daily average metabolic rate-correlated with a substantial reduction in physical activity.

Interestingly, LPL deficiency promotes the expression of the AgRP orexigenic peptide in the hypothalamus. From their findings, the authors proposed that TG-rich lipoproteins are sensed in the brain by a LPL-dependent mechanism [START_REF] Wang | Deficiency of lipoprotein lipase in neurons modifies the regulation of energy balance and leads to obesity[END_REF]. What would happen in the hypothalamus of Rev-erbα -/-mice if Lpl was also up-regulated? Does hypothalamic sensing of fatty acids would have been affected?

This is interesting since central administration of oleic acid has been shown to inhibit food intake [START_REF] Obici | Central administration of oleic acid inhibits glucose production and food intake[END_REF]. Regarding the hypothalamus, we observed that food restriction does not elicit the same metabolic responses in Rev-erbα -/-mice as in control littermates. Indeed, daily p-ERK expression was shown to be slightly altered in several hypothalamic areas in both AL and RF conditions, which can suggest that circulating signals are not integrated similarly or are different in Rev-erbα -/-mice. In this context, we observed that plasma leptin levels are not severely decreased following 24-h fast in Rev-erbα -/-mice (unpublished data). Of note, this absence of fasting-induced reduction of leptin levels has been suggested to be associated with low corticosterone levels [START_REF] Jeong | Impaired leptin expression and abnormal response to fasting in corticotropin-releasing hormone-deficient mice[END_REF])-which are observed in Rev-erbα -/-mice under RF schedules. Considering that low leptin levels signal low energy stores and facilitate entry into torpor [START_REF] Ahima | Role of leptin in the neuroendocrine response to fasting[END_REF][START_REF] Gavrilova | Torpor in mice is induced by both leptin-dependent and -independent mechanisms[END_REF], the relative normothermia in food-deprived Rev-erbα -/-mice finds a first explanation 51 . Besides, the loss of hunger due to an altered starvation-sensing system may explain their defect to develop strong food-seeking behaviors-without altered food consumption after food deprivation. It would have been interesting to assess the time course of energy substrate utilization during starvation. Additionally, the characterization of hypothalamic neurons implicated in feeding and energy balance (e.g., in the ARC neurons, which are particularly sensitive to feeding-related signals such as leptin and ghrelin) could be informative, more especially as clock mutation can modify the levels and temporal expression of neuropeptides (see part 4.3.1). In that regard, AgRP is a good candidate considering that it is activated by ghrelin [START_REF] Nogueiras | Central nervous system regulation of energy metabolism: ghrelin versus leptin[END_REF] and that its expression is increased prior to mealtime [START_REF] Yoshihara | Effects of restricted daily feeding on neuropeptide Y release in the rat paraventricular nucleus[END_REF][START_REF] Crowley | Differential effects of methamphetamine on expression of neuropeptide Y mRNA in hypothalamus and on serum leptin and ghrelin concentrations in ad libitum-fed and schedule-fed rats[END_REF]. Moreover, AgRP neurons are sufficient to orchestrate feeding behavior [START_REF] Aponte | AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training[END_REF][START_REF] Krashes | Rapid, reversible activation of AgRP neurons drives feeding behavior in mice[END_REF]. Besides AgRP neuronal activity has been shown to adapt in response to signals from hormones and metabolites such as ghrelin, leptin, glucose and fatty acids (mentioned in Sternson 2011). 51 Of note, we cannot exclude either an up-regulation of Lpl transcript in the BAT of Rev-erbα -/-mice to explain their greater capacity to sustain normal body temperature levels during a period of food deprivation or during cold exposition. Indeed, LPL supplies BAT with fatty acids to sustain nonshivering thermogenesis (Klinegenspor et al. 1996).

Conclusion and perspectives

The involvement of the nuclear receptor Rev-erbα in circadian functions is undeniable. Indeed, its absence in vivo leads to a reduction of the SCN period length (Preitner et al. 2002;[START_REF] Cho | Regulation of circadian behaviour and metabolism by REV-ERB-alpha and REV-ERB-beta[END_REF] and to an enhanced shifting response to light exposure, along with disrupted SCN molecular clockwork due to blunted Bmal1 expression (Preitner et al. 2002). Rev-erbα expression has been shown to be differentially sensitive, in regard to the peripheral tissue considered, to jet-lag [START_REF] Kiessling | Adrenal glucocorticoids have a key role in circadian resynchronization in a mouse model of jet lag[END_REF]. Moreover, food schedules differentially affect Rev-erbα expression in normal mice (Damiola et al. 2000;[START_REF] Filipski | Effects of light and food schedules on liver and tumor molecular clocks in mice[END_REF]Feillet et al. 2006).

In this circadian context, we further evaluate the involvement of well-known canonical clock genes in the molecular mechanisms of the FEO. We showed that Rev-erbα deletion in vivo leads to altered food-entrainable physiology, as evidenced by decreased locomotor activities as well as an absence of a rise in body temperature, corticosterone and ketone bodies prior to mealtime. These findings, however, were not correlated with disrupted molecular clockwork in the liver or DMH, the latter being likely a candidate for the development and expression of food anticipation (Gooley et al. 2006;Acosta-Galvan et al. 2011;[START_REF] Landry | Evidence for Time-of-Day Dependent Effect of Neurotoxic Dorsomedial Hypothalamic Lesions on Food Anticipatory Circadian Rhythms in Rats[END_REF][START_REF] Verhagen | Anticipation of meals during restricted feeding increases activity in the hypothalamus in rats[END_REF]. Incidentally, the actual view privileges rather a spatially distributed FEO particularly at the central level. In that respect, our evaluation of PER2 oscillations in other hypothalamic nuclei essential to regulate energy balance and feeding behavior, did not allow us to detect altered phase-adjustment of PER2 expression in response to RF. Activation of hypothalamic neurons as measured through c-FOS expression in phase with feeding time has been described in several studies (Angeles-Castellanos et al. 2004;Gooley et al. 2006;Ribeiro et al. 2007). Our data on p-ERK activation in control mice, for the first time used to assess the effect of RF, further suggest that the FEO is integrated into a network of oscillators. Of interest, in Rev-erbα -/-mice the peak of p-ERK in phase with mealtime is not clearly observed as in their control littermates. In addition, we uncover a potential role for Rev-erbα in the food synchronization of the cerebellar clockwork. Yet, the relative importance of clock genes as well as the CRB structure for food entrainment remains to be further explored. Hence, an approach could be to selectively disrupt the Rev-erbα clock gene in the Purkinje cells of the CRB, to evaluate the exact participation of the CRB oscillator to food entrainment and its capacity to generate an output signal to synchronize the activity of other components of the FEO network. In addition, since Rev-erbα depletion leads only to a mild reduction of FAA, and considering the recently revealed cooperative role of both Rev-erbs isoforms in circadian clock function [START_REF] Cho | Regulation of circadian behaviour and metabolism by REV-ERB-alpha and REV-ERB-beta[END_REF], it would be enlightening to evaluate the phenotype of double KO mice (i.e., for both Rev-erbα and Rev-erbβ) regarding food entrainment.

The involvement of the nuclear receptor Rev-erbα in metabolism is also patent. Indeed, Rev-erbα acts to fine-tune metabolic pathways, by modulating SREBP target genes and by recruiting HDAC3 to the genome on a daily basis, both being important for the control of lipid metabolism (Le Martelot et al. 2009;Feng et al. 2011). Our results demonstrate that in vivo deletion of the circadian component Reverbα results in profound metabolic changes. Chow-fed Rev-erbα -/-mice display increased adiposity, mild hyperglycemia concomitant to a greater reliance on fatty acids as energy source with non-significant daily changes in energy intake or energy expenditure. We uncover a molecular pathway that ties together CLOCK expression to the 24-h rhythmic accumulation of the Lpl transcript. Disruption of this CLOCK-Lpl interaction due to elevated CLOCK expression in the absence of Rev-erbα is likely to have caused higher oxidative muscle metabolism and enhanced fat storage in the WAT and the liver of Rev-erbα -/-mice. Furthermore, this imbalance in fuel utilization predisposes Rev-erbα -/-mice to HFD-induced obesity, as supported by enhanced hepatic de novo lipogenesis and adipose fat storage in these mice. These results should be considered in the light of the recent study demonstrating that in vivo administration of synthetic REV-ERB agonists have positive effects on lipid and glucose oxidation. Moreover, these drugs are effective to reduce lipogenesis and to protect mice from the detrimental effects of HFD [START_REF] Solt | Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists[END_REF]). Collectively, these findings and ours demonstrate that Rev-erbα is a key gene for the regulation of energy balance. In the future, it would be attractive to target Rev-erbα to limit circadian disorders and metabolic defects. In the same context, the relevance of clock-dependent and -independent pathways for the modulation of (daily) Lpl expression-and fuel balance-have to be strengthened by further investigations.

To summarize, our two studies give novel evidence that Rev-erbα is fundamental for an accurate crosstalk between the circadian system and metabolism.

The use of germline KO for our investigations of food entrainment cannot rule out that the significant changes in the internal energy status of our mice may be a confounding effect for the observed reduction of food-anticipatory components. Indeed, we cannot fully argue that Rev-erbα is essential to shape the mice's 24-hour pattern of activity in limited food access conditions, since these mice do not display decreased hepatic glycogen reserves, plasma glucose and leptin levels, and body temperature values during periods of food deprivation. These body signals are likely essential to convey the energy status of an organism for instance to the hypothalamus, where nutrient-dependent signals are integrated and transduced into efferent signals-including food-seeking behaviors. Thus, it seems necessary to make use of brain-specific and inducible knockout, to solve the long-lasting and unresolved enigma regarding the (circadian) nature of the FEO (Fig. 57)-a strategy that we have already adopted. We will evaluate several behavioral and physiological food-entrained rhythms in mice lacking Reverbα in neurons. The effect of scheduled feeding on central oscillators will be evaluated as well.

b) Results

Data were analyzed with analyses of variance. We did not find any significant differences between genotypes regarding the time spent inside or the number of entries in the open arms (Fig. 2). Thus, the absence of a reduction in both the time spent and the number of entries in Rev-erbα -/-mice does not reveal any anxiety-like behavior. The light-dark test box is a method to evaluate anxiety responses in rodents (i.e, conflict between their aversion to light and their motivation to explore a novel environment). For more information, see Bourin and Hascoët 2003. Briefly, mice were moved from their housing room in their individual cage to an adjacent behavioral testing room under dim-light. They were then placed in the box for a 5 min session (Fig. 3). The entry latency to the lit compartment as well the number of entries and the time spent in the light box were evaluated. Data were analyzed with analyses of variance, followed by the Unequal N HSD test, as appropriate (i.e., when P-value of main effect of genotypes was less than 0.05). Rev-erbα -/-mice displayed a significant decrease in the time spent in the lit compartment compared to the other groups. However, the latency to enter the lit compartment and the total number of entries were not significantly different between genotypes (Fig. 4). Thus, Rev-erbα -/-mice demonstrated an aversion to a brightly illuminated area; however, in view of our results in the Elevated plus maze, this aversion may be related to other causes than enhanced "anxiety".

Forced swimming test a) Protocol

The forced swimming test is a method for screening antidepressants drugs. For more information, see Petit-Demoulire et al. 2004. In a first attempt to evaluate a depression-like behavior consecutive to Rev-erbα deletion, we measured baseline levels of immobility (i.e. without any antidepressant drug injection) in each genotype (as in [START_REF] Roybal | Mania-like behavior induced by disruption of CLOCK[END_REF]Hampp et al. 2008).

Briefly, mice were placed in a transparent cylinder filled with water (25 ± 0.5°C) to a depth of 10 cm for 6 min (Fig. 5). The water was changed between each mouse. A video camera recorded the testing session for later analysis. Immobility was defined as the absence of active behaviors such as swimming, limb movements, etc. The first two minutes were excluded from the analysis.

b) Results

Data were analyzed with analyses of variance. Immobility time did not differ between genotypes (Fig. 6).

Résumé

La première partie de mon travail de thèse a été de définir le rôle joué par le récepteur nucléaire Rev-erb dans les mécanismes de synchronisation par la nourriture d'une horloge circadienne putative, non encore localisée, appelée « horloge alimentaire ». La seconde partie de mon travail a consisté à étudier la participation de Rev-erb dans les régulations des métabolismes glucidique et lipidique.

L'ensemble de nos données indique que le répresseur transcriptionnel Rev-erb joue un rôle charnière dans les fonctions circadiennes ainsi que dans le métabolisme. En effet, d'un point de vue circadien, l'absence de Rev-erb altère la synchronisation à l'heure des repasdémontré par une réduction des sorties comportementales et physiologiques de l'horloge alimentaire, ainsi que par l'absence d'ajustement du rythme de la protéine d'horloge PER2 dans l'oscillateur cérébelleux. Sur le plan métabolique, la délétion de ce gène modifie notamment le métabolisme des lipidesdémontré par une accumulation excessive de tissu adipeux, une utilisation préférentielle des acides gras, ainsi qu'une perte de contrôle de l'expression de la Lipoprotéine lipase.

Mots-clés : Rev-erb alpha, gène d'horloge, horloge alimentaire, métabolisme lipidique et glucidique, lipoprotéine lipase, obésité, anticipation de l'heure des repas, rythme circadien

Résumé en anglais

The work performed during this PhD thesis aimed at investigating the role of the transcriptional silencer Rev-erbα in both the circadian clockwork of the food-entrainable oscillator and metabolic regulations. Firstly, by evaluating food-anticipatory components in animals fed once a day at the same time, we showed that mice lacking Rev-erbα display a reduction in locomotor activity prior to food access compared to littermate controls. Accordingly, the rises in body temperature and corticosterone that anticipate mealtime are also diminished. Interestingly, daily p-ERK expression in hypothalamic regions and daily PER2 expression in the cerebellum of Rev-erbα KO mice are not phase-adjusted to feeding time. These results indicate that Rev-erbα participates in the integration of feeding signals and in food-seeking behaviors. Secondly, by investigating energy balance in fasted, normal chow or high-fat fed animals, we revealed that Rev-erbα KO mice exhibit greater reliance on lipid fuels as energy substrates, contributing to a mild hyperglycemic state. We also found that Lipoprotein lipase (Lpl) expression, is strongly up-regulated in peripheral tissues of Rev-erbα KO mice, predisposing mice to obesity. In this regard, we uncovered a new molecular pathway that ties clock-driven Lpl expression to energy homeostasis. These findings highlight the significance of daily Rev-erbα oscillations to prevent the appearance of the metabolic syndrome.

In conclusion, we provide evidence that REV-ERBα may be a part of the food-entrainable oscillator clockwork that triggers food-anticipatory components, and represents a pivotal player to link the core clock machinery to metabolic pathways. Keywords: Rev-erb alpha; Food-entrainable oscillator; Lipid metabolism; Obesity; Lipoprotein lipase; Respiratory quotient; Clock gene; Food-anticipatory activity, Circadian rhythm

Figure 1 .

 1 Figure 1. Les oscillateurs centraux et périphériques L'activité cellulaire des noyaux suprachiasmatiques (NSC), situés à la base de l'hypothalamus, est synchronisée sur 24 h par l'alternance rythmique du jour et de la nuit. Ce message rythmique est ensuite distribué par les NSC à différents oscillateurs centraux (c.-à-d. dans le cerveau) ou périphériques (p.ex. le foie).

Figure 2 .

 2 Figure 2. Modèle cellulaire simplifié de la rythmicité circadienne Les oscillations moléculaires des gènes d'horloges se déroulent sur 24 h grâce à un jeu réciproque d'activation (flèches bleues) et d'inhibition (flèches noires) de la transcription.

Figure 3 .

 3 Figure 3. Activité locomotrice de roue (à gauche) et température corporelle (à droite) en condition de restriction alimentaire Quelques heures précédant l'accès à la nourriture (rectangle orange), les souris témoins (en bleu) présentent une forte augmentation de l'activité locomotrice, en parallèle à une élévation de leur température corporelle. Ces composantes d'anticipation sont diminuées chez les souris KO (en rouge).

Figure 4 .

 4 Figure 4. Expression de l'ARNm Per2 dans le foie La restriction alimentaire temporelle induit un changement de phase du rythme de Per2 dans les deux génotypes.

Figure 5 .

 5 Figure 5. Rythme de la glycémie (haut) et du glycogène hépatique (bas)

Figure 6 .

 6 Figure 6. Concentration de glucose plasmatique après une période de jeûne de 24 h (à gauche) et clamp euglycémique hyperinsulinémique (à droite)

Figure 7 .

 7 Figure 7. Calorimétrie indirecte Les souris KO (en rouge) métabolisent davantage les glucides de nuit et les lipides de jour, en comparaison aux souris contrôles (en bleu).

Figure 8 .

 8 Figure 8. Adiposité totale (à gauche) et concentration de leptine (à droite)

Figure 9 .

 9 Figure 9. Expression du gène de la Lipoprotéine lipase dans différents tissus L'ARNm de la Lpl est surexprimé à différents temps dans les organes périphériques des souris KO pour Rev-erbα.

Figure 10 .

 10 Figure 10. Dosage de l'activité luciférase L'analyse des séquences promotrices du gène de la Lpl a mis en évidence une E-box fonctionnelle qui peut interagir avec le facteur CLOCK.

Figure 2 .

 2 Figure 2. Terms used to describe circadian rhythms.

  . It was also in 1960 that Patricia DeCoursey demonstrated on a time graph a daily light sensitivity rhythm of locomotor activity in rodent by testing the effects of light pulse at different times of the day (De Coursey 1960). The PRC is now a powerful and precise tool for the study of circadian rhythms (Fig. 3-4).

Figure 3 .

 3 Figure 3. Light-pulse phase response-curve of a nocturnal animalAnimals are kept in constant darkness. The free-running activity can be phase shifted by light-pulses in a phase-dependent manner. (A) When light-pulse is given in the middle of the subjective day (light/rest-phase for a nocturnal animal), there is no strong effect on the onset of an animal activity. Whereas, light-pulses administrated at the beginning of the subjective night (i.e., dark/active phase for a nocturnal animal), phase-delayed the activity rhythm, while light-pulse delivered at the end of the active period caused phase-advance. Note that "subjective" day/night are defined by the segments of inactive and active period, respectively, during entrainment by a light-dark cycle.

Figure 4 .

 4 Figure 4. Light-pulse phase response-curve of a flying squirrel rodent (Left) 24-h activity record, called actogram, of a flying squirrel. The first yellow arrow indicated the first light-pulse, which was given 1 min after the onset of the daily activity period and which caused a phase-delay. The second yellow arrow points to a ligh-pulse that was given 9 h after the onset of activity, which caused a phase-advance of the locomotor activity the following day. (Right) Lightreponse curves for two flying squirrels, A and B. Adapted from (De Coursey 1960).

Figure 6 .

 6 Figure 6. Rhythms of nitrogenase activity of Synechococcus sp. RF-1

Figure 7 .

 7 Figure 7. Circadian systems in the universal tree of life

Figure 10 .

 10 Figure 10. SCN localization in mice Coronal section of a mouse brain showing the localization of the SCN, and below cresyl violet staining demonstrating the dense cellular packing in the ventromedial and dorsolateral part of the SCN. See text for description. OC: optic chiasma; 3V: third ventricule. Adapted from the Franklin-Paxinos atlas.

Figure 11 .

 11 Figure 11. Effect of SCN lesions in the diurnal rodent, Ammospermophilus leucurus Two actograms representing the locomotor activity of an intact SCN animal (Left) and an SCNlesioned animal (Right). Recordings were performed using microchip transponder scanner for a period of 12 days. Each line corresponds to one day. Vertical black data line represents the number of bouts of activity per 10-min bin. Above the actograms, the schedule bars indicate the light and the dark conditions. Adapted from (DeCoursey et al. 1997)

Figure 12 .

 12 Figure 12. Drawing representing neuropeptide distribution in the SCN neurons Many of the neurons within the core SCN express the neuropeptides VIP, GRP and the neurotransmitter GABA (not represented here). Neurons of the dorsal shell mostly express the neuropeptide AVP and GABA as well. AVP: arginine vasopressin; VIP: vasoactive intestinal polypeptide; GRP: gastrin-releasing peptide.

Figure 13 .

 13 Figure 13. Schematic view of retinal circuitry and light entrainment Photic signals are conveyed to the SCN via photoreceptor cells, especially the melanopsin-containing cells, which project directly to the SCN along the retinohypothalamic tract. Signal transduction to the molecular oscillator likely involves VIP, PACAP and glutamate releases at terminals. Changes in intracellular Ca2+ concentration lead to activation of several kinase pathways (e.g., MAPK, PKA) that in turn activate the transcription of different genes that are part of the SCN molecular architecture. Adapted from (Morse and Sassone-Corsi 2002).

Figure 14 .

 14 Figure 14. Photomicrographs of SCN coronal sections stained for c-Fos immunoreactivity.(A) Control rat, and (B) a rat which received 15 min of bright (2200 lux) white light exposure. SON, supraoptic nucleus; OC, optic chiasm. Magnification = 30x. From[START_REF] Rea | Light increases Fos-related protein immunoreactivity in the rat suprachiasmatic nuclei[END_REF]

Figure 15 .

 15 Figure 15. Signal transduction of photic messages in the mammalian SCN

Figure 16 .

 16 Figure 16. Photic modulation of Per genes in a diurnal rodent

17

 17

  ): one positive in which CLOCK and BMAL1 dimerize to activate the E-box 14 mediated transcription of Per and Cry genes; one negative whereby upon reaching a critical concentration, PER and CRY proteins enter into the nucleus to inhibit the transactivation mediated by CLOCK:BMAL1, therefore inhibiting their own transcription; and an interconnecting loop, in which Rors can activate the transcription of Bmal1 and Npas2, whereas Rev-erbs can repress Bmal1, Clock and

Figure 17 .

 17 Figure 17. Simplified cellular model of the mammalian molecular circadian clockwork Circadian rhythms are generated by transcriptional/translational feedback regulatory loops of core clock genes. CLOCK (or NPAS2) and BMAL1 can dimerize to rhythmically transactivate genes containing a specific DNA sequence (E-box) in their promoter region. As a result, the transcriptional activity of Pers, Crys, Rors, and Rev-erbs is enhanced, and their products from translational activity are cyclically released in the cytoplasm. Then, when PERs and CRYs proteins reach a critical concentration, they form heterodimers that translocate into the nucleus to repress transcriptional activity induced by CLOCK (NPAS2):BMAL1, leading to their own repression. An additional loop involves the nuclear receptors Rev-erbs and Rors, which can translocate into the nucleus to modulate Bmal1, Clock, and Npas2 transcription via opposite action on a RORE sequence located in their promoter.

Figure 18 .

 18 Figure 18. Circadian variations of clock genes mRNA in the SCNRev-erb α and Per1 mRNA levels peak early in the subjective day, Per2 and Cry1 levels peak later in the subjective day, while Bmal1 peaks in antiphase to these, in the subjective night. Expression of clock proteins are delayed by 4-6 h with regard to mRNA. Subjective night is indicated by the black bar along the time axis. Note that Clock (not represented) is continuously expressed. From[START_REF] Guilding | Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain?[END_REF]

Figure 20 .

 20 Figure 20. Distribution of circadian periods in two types of SCN culture and at the animal level (A, D) Electrical activity in dispersed cell culture. Note the strong fluctuations in the intrinsic periods between single cells. (B) Electrical activity of single-cell recorded in SCN slice culture. Note that the periods vary less between single-cells. (C) Period of locomotor activity in individual rats, depicting slight variations from one animal to another. (D, E) Period variations within a dish and a slice. (F) Distribution range of behavioral rhythm. From[START_REF] Honma | Diversity in the circadian periods of single neurons of the rat suprachiasmatic nucleus depends on nuclear structure and intrinsic period[END_REF]

  Figure 21.Clock-controlled genes linked to the circadian molecular mechanism.Both positive and negative limbs reciprocally interact to govern the transcription of CCGs.

Figure 23 .

 23 Figure 23. Real-time recording of bioluminescence from Per1-luciferase transgenic rats Tissues were explanted 1 h before light-offset (see arrow and light and dark periods in the top left plot). Note that the SCN differs from other tissues in the phase and amplitude of PER1-luc circadian expression. AN: arcuate nucleus; Pit: pituitary gland; Pin: pineal gland. Adapted from (Abe et al. 2002).

Figure 24 .

 24 Figure 24. Potential circadian oscillators in the mammalian brain

Figure 25 .

 25 Figure 25. Circadian oscillations of PER2-luc in the SCN and peripheral tissues

Figure 27 .

 27 Figure 27. The time-memory of bees

Figure 28 .

 28 Figure 28. Food-anticipatory activity in SCN-x ratsDouble-plotted actograms (48 h time scale) showing locomotor activity for 2 rats (SCN6-7) in constant darkness. The arrow indicates the time when rats were SCN lesioned. Black line indicates the beginning of 1 h restricted feeding (food was given every 12-h intervals, thus twice a day). Note the increase of activity prior to the two mealtimes. From[START_REF] Stephan | Circadian-Rhythm Dissociation Induced by Periodic Feeding in Rats with Suprachiasmatic Lesions[END_REF]

Figure 30 .

 30 Figure 30. Food-anticipatory activity in an SCN-X rat followed phase shifts of mealtime Double-plotted actogram of daily locomotor activity. Rectangles indicate 2 h food access during RF. DD or constant dark conditions. Phase shifts of food access are shown on the left. * indicates 2 days power failure and loss of data.

Figure 31 .

 31 Figure 31. Food-anticipatory activity in a normal rat under light-dark conditions (A) Single-plotted actogram of wheel-running activity of a rat during AL food and RF access. The white rectangle during the light period indicates time of food access. Triangles represent the beginning (pointing down) or end (pointing up) of total food deprivation. (B, C, D) Waveforms of wheel running during AL (7 day average), RF (7 day average), and total food deprivation (3 day average), respectively. From(Mistlberger 1994)

Figure 36 .

 36 Figure 36. Daily fixed access to a palatable meal entrained locomotor activity in rats.Mean activity waves for locomotor activity in restricted feeding scheduile (RFS; white circles) and in palatable meal entrainment (PME; black circles) groups during AL (A, D), during entrainment (B, E) and for the 3 days in food deprivation (C) or 4 days following interruption of palatable meal (F). Horizontal black bar indicates mealtime and vertical line indicates palatable mealtime (B, E). Horizontal white bar and dotted line indicate time of expected mealtime (C, F). Values represent mean±S.E. * Post hoc Tukey P<0.05. From(Mendoza et al. 2005)

Figure 37 .

 37 Figure 37. Methamphetamine-induced locomotor rhythm entrains to restricted daily feeding in SCN lesioned rats.

Figure 40 .

 40 Figure 40. DMH lesions attenuate food entrainment.

Figure 41 .

 41 Figure 41. Cartoon of the mammalian circadian system, illustrating known (solid arrows and circles) and hypothesized (dashed arrows and circles) components and pathways

Figure 42 .

 42 Figure 42. Daily wheel-running activity in wild-type (WT) (A), Per1 (B), and Per2 (C) mutant mice with hypocaloric feeding under light-dark conditions

Figure 43 .

 43 Figure 43. Daily rhythms in basal plasma glucose concentrations and a number of glucoregulatory hormones during different experimental conditionsPlasma glucose, corticosterone, insulin and glucagon concentrations across the 24-h light/dark cycle show clear day/night rhythms in intact rats under conditions of ad libitum (Ad lib) feeding but are abolished by an SCN lesion (SCN-x). Adapted from(Kalsbeek et al. 2010)

Figure 44 .

 44 Figure 44. High-Fat Diet Alters Diurnal Patterns of Metabolic Markers.Mice were maintained on a 12:12 LD cycle and fed either RC (black lines) or HF diet (gray dotted lines) ad libitum for 6 weeks (n = 6-8 per group per time point).(A) Diurnal variation in serum leptin, glucose, insulin, free fatty acid (FFA), and corticosterone levels. Adapted from(Kohsaka et al. 2007)

Figure 45 .

 45 Figure 45. Clock mutation alters the diurnal rhythms of clock and metabolic genes.

Figure 46 .

 46 Figure 46. Cell autonomous oscillator in pancreasIslets from Per2Luc mice were imaged, and the orange trace at the right represents the bioluminescence rhythm collected from the islet in the orange square (left). Traces from other islets are shown below. Adapted from(Marcheva et al. 2010)

Figure 47 .

 47 Figure 47. Body mass, fat pad mass, and glucose tolerance were altered in mice exposed to bright or dim light at night. (A) Weekly body mass for mice throughout the study (*P ≤ 0.05 when ligh-dark (LD) differs from both ligh-light (LL) and dim-light (DM) groups; & P ≤ 0.05 between all groups). Mice exposed to light at night had elevated body mass beginning 1 wk after placement in experimental light conditions and continuing throughout the remainder of the study. (B) Body mass gain and epididymal fat pad mass differed among groups at the conclusion of the study, suggesting increases in body mass may be caused by changes in body fat composition. (C) Mice exposed to either DM or LL had reduced glucose tolerance, and DM and LL mice failed to recover blood glucose as rapidly as LD mice (*P ≤ 0.05 when LD differs from both LL and DM groups;+ P ≤ 0.05 when glucose level is higher in the DM group than

  stearoyl-Coenzyme A desaturase 1, and cellular energy balance (e.g., uncoupling protein[START_REF] Asher | Crosstalk between components of circadian and metabolic cycles in mammals[END_REF] Ucp3). Similarly to Rorα, Rev-erbα can bind directly to the human Apo C-III promoter(Raspe et al. 2002) and the rat Apoa-I promoter RevRE[START_REF] Vu-Dac | The nuclear receptors peroxisome proliferator-activated receptor alpha and Rev-erbalpha mediate the species-specific regulation of apolipoprotein A-I expression by fibrates[END_REF]. For the repressive action on Bmal1 transcription, Rev-erbα has been shown to recruit the nuclear receptor corepressor (NCoR)/histone deacetylase 3 (HDAC3) complex[START_REF] Yin | The orphan nuclear receptor Rev-erbalpha recruits the N-CoR/histone deacetylase 3 corepressor to regulate the circadian Bmal1 gene[END_REF]. Of interest, genetic disruption of NCoR1-HDAC3 interaction in mice induces changes in Bmal1 expression and circadian behavior with concomitant effects on metabolism (e.g., increased insulin sensitivity, altered expression of metabolic genes). Surprisingly, loss of a functional NCoR1-HDAC3 complex also protects mice from diet-induced obesity[START_REF] Alenghat | Nuclear receptor corepressor and histone deacetylase 3 govern circadian metabolic physiology[END_REF]). On the other hand, a recent study has explored the role of the circadian genomic recruitment of HDAC3 in the mouse liver. They showed that REV-ERBα controls the circadian expression of lipid metabolism genes by recruiting the repressive chromatin modifier HDAC3 (and NCoR) to the genome during the light period, therefore preventing lipogenesis at a time when animals are resting. In addition, deletion of either Hdac3 or Rev-erbα results in hepatic steatosis indicating steady lipogenesis (Fig.50)(Feng et al. 2011).

Figure 50 .

 50 Figure 50. Regulation of hepatic lipid homeostasis by HDAC3 (Left) Hepatic TG levels in livers from 9-week-old WT and Rev-erbα KO. Values are mean ± SEM (n = 4). *P < 0.05 by Student's t test. (Right) Model depicting the mechanistic links between the daily cycles of Rev-erbα expression (orange oval), HDAC3 genomic recruitment, epigenomic status, and hepatic lipogenesis. Adapted from(Feng et al. 2011)

  in circadian functioning. Other results indicate that PER2 has the capacity to recruit PPARα or REV-ERBα for the modulation of Bmal1 expression (Fig. 51) (Schmutz et al. 2010), again showing intimate interactions between clock and metabolic components in the clock circuitry.PPARγ and PPARδ are both involved in energy homeostasis. A recent study reveals that PER2 can interact directly with PPARγ to repress its transcriptional activity. Ablation of Per2 in cell culture increased induction of adipogenic genes, while deletion of Per2 in mice results in slight changes in circadian expression of PPARγ target genes in WAT[START_REF] Grimaldi | PER2 controls lipid metabolism by direct regulation of PPARgamma[END_REF]. These results demonstrate that the control of PPARγ by a circadian actor is essential for normal lipid metabolism.

Figure 51

 51 Figure 51. Model of how PER2 may couple E-box-driven and nuclear receptor-regulated gene expression.

  to NADPH. NAD(P)+ and NAD(P)H are involved in cellular redox reactions.Interestingly, CLOCK:BMAL1 and NPAS2:BMAL1 heterodimers can sense intracellular redox status. The DNA-binding activities of these dimers are influenced by both reduced and oxidized forms of NAD(H) and NADP(H) in an opposing manner. The reduced forms (NADH and NADPH) activate DNA binding of CLOCK:BMAL1 and NPAS2:BMAL1, whereas the oxidized forms (NAD+ and NADP+) inhibit it, consistent with a role as redox sensors (Fig.52)[START_REF] Rutter | Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors[END_REF]).

Figure 52 .

 52 Figure 52. Metabolic states and the mammalian circadian feedback loop BMAL1 can bind to Clock or NPAS2, and these heterodimers activate transcription. Alternatively, BMAL1 can form homodimers with itself that do not activate transcription. The formation of the Clock: BMAL1 and NPAS2:BMAL1 heterodimers and their binding to DNA is stimulated by reduced NADH and inhibited by oxidized NAD. These heterodimers enhance the expression of the clock genes Cry and Per (not shown) and the clock output gene Ldh. CRY proteins repress Clock: NPAS2-mediated gene activation, possibly by oxidizing the NAD + cofactors associated with these proteins. Conceivably, the negative action of CRY proteins on Clock-NPAS2 could be reinforced by lactate dehydrogenase (LDH), which may increase the cellular concentration of NAD + .(Only unphosphorylated NAD electron carriers are shown.). From[START_REF] Schibler | Circadian rhythms. Chronobiology--reducing time[END_REF]

Figure 53 .

 53 Figure 53. Daily expression of Rev-erbα mRNA Note that the acrophase of Rev-erbα expression occurs in the middle of the light period. From http://bioinf.itmat.upenn.edu/circa

(

  25 mM) in the presence of EDTA (0.2 mM) at 95 °C for 25 min. 2µL of digested tissue was added to a DNA amplification reaction containing (in final concentration): 1X Taq Reaction buffer (Fermentas), MgCl2 2.8 mM (Fermentas), DMSO (Hybri-Max, Sigma D2650), dNTP 0.39 mM and Taq DNA polymerase 1.25 U (Fermentas). Three primers were added to the mixture: a forward primer located in exon 2 (5'-CCAGGAAGTCTACAAGTGGCCATGGAAGA-3', 0.79 µM) and a reverse primer located in exon 3 (5'-CACCTTACACAGTAGCACCATGCCATTC-3', 0.55 µM) amplified a 340 bp band corresponding to the wild-type allele; and a forward primer located in the LacZ cassette (5' AAACCAGGCAAAGCGCCATTCGCCATTCA-3', > 0.55 µM) amplified a band about 200 bp from the targeted allele. The PCR protocol consisted of 5 min at 95°, 35 cycles of amplification (each cycle consisting of 1 min 15 s at 95°C, 1 min at 62°C and 1 min 30 s at 72°C) and 10 min at 72°C. Products were separated by electrophoresis on 2% agarose gels stained with ethidium bromide and visualized under UV light.

Figure 1 .

 1 Figure 1. Food-anticipatory components are reduced in Rev-erbα -/-mice exposed to a 12:12 light-dark cycle.(A) Representative double-plotted actograms (left, middle) and thermograms (right) of Reverbα +/+ (upper panel) and -/-mice (lower panel) under LD conditions with food provided AL and then restricted to 6 h per day (grey box, food was provided from ZT6 to ZT12). (B-C) Normalized activity profiles and body temperature raw data representing the average of 10 days during AL conditions (B) and RF conditions (C). Daytime and nighttime are indicated by white and black bars, respectively. The period of food access during RF is represented by the grey rectangle. (D) Percent of FAA over total daily activity from 4 h to 1 h before mealtime (left, middle) and rise in body temperature in anticipation (right). * significantly different from control animals (P < 0.05). Note that the main effect of time was not represented (see text).

Figure 2 .

 2 Figure 2. Food-anticipatory components are reduced in Rev-erbα -/-mice exposed to constant darkness.(A) Representative double-plotted actograms (left, middle) and thermograms (right) of Reverbα +/+ (upper panel) and -/-mice (lower panel) under DD conditions with food provided AL and then restricted to 6 h per day (grey box, food was provided from 02:00 p.m. to 08:00 p.m.). Constant darkness is indicated by a black bar. (B) Percent of FAA over total daily activity (average of 5 days) from 4 h before mealtime and 2 h after mealtime (left, middle) and rise in body temperature in anticipation (right). (C) Representative double-plotted actograms (left, middle) and corresponding χ 2 periodograms (right) from SCN-X Rev-erbα +/+ (upper panel) and -/-mice (lower panel) under LD conditions and DD conditions (DD onset indicated by the black arrow) with food provided AL and then restricted to 6 h per day (grey box, food was provided from 02:00 p.m. to 08:00 p.m.). * significantly different from control animals (P < 0.05). Note that the main effect of time was not represented (see text).

Figure 3 .

 3 Figure 3. Plasma ketone bodies and corticosterone peaks in Rev-erbα -/-mice are decreased in anticipation of feeding time.24-h (A) plasma glucose, (B) hepatic glycogen, (C) plasma ketone bodies and (D) plasma corticosterone levels in both AL (left) and RF conditions (right). Daytime and nighttime are indicated by white and black bars, respectively, on the X axis. Food access during RF schedules is depicted by a grey rectangle. Data for ZT0 are double-plotted. * significantly different from control animals (P < 0.05). ~ for main effect of time (top left corner) or vs. control animals (above data point) (P < 0.05).

Figure 4 .

 4 Figure 4. mRNA expression of clock-related genes in the liver oscillator are synchronized by feeding time in both genotypes. Expression of Bmal1 (A), Clock (B), Per1 (C), Per2 (D), Dbp (E) under AL conditions (left panel) and RF conditions (right panel). Daytime and nighttime are indicated by white and black rectangles, respectively, on the X axis. Food access during RF schedules is depicted by a grey rectangle. Data for ZT0 are double-plotted. * significantly different from control animals (P <

Fig. 4

 4 Fig.4

Fig. 5

 5 Fig.5

Figure 2 .

 2 Figure 2. Lack of whole insulin resistance or increased gluconeogenesis in Rev-erb␣ Ϫ/Ϫ mice. A-C) Plasma insulin levels (A), hepatic glycogen concentration (B), and plasma glucagon levels (C); n ϭ 6/group. D) OGTT in mice unfed overnight (i.e., from ZT12 to ZT2) and the resulting incremental AUC (nϭ12/group). E) Hyperinsulinemic-euglycemic clamp and the resulting glucose infusion rates averaged over the last 60 min of the clamp (nϭ6/group). F) Insulin immunostaining from pancreas sections of ϩ/ϩ and Ϫ/Ϫ animals and their respective quantification (nϭ6/group). G) Pancreas sections of a Rev-erb␣ ϩ/ϩ (left panel) and a Rev-erb␣ Ϫ/Ϫ mouse (right panel) stained with insulin antibody. Scale bar ϭ 50 m. H, I) GST (H) and PTT (I) in mice unfed overnight and the resulting AUCs (nϭ12/group).J) Glucose production in Rev-erb␣ ϩ/ϩ and Rev-erb␣ Ϫ/Ϫ primary hepatocytes incubated with (ϩ) or without (Ϫ) lactate-pyruvate (LP) for 2 or 24 h (nϭ7/group). Data are means Ϯ se. Open circles, Rev-erb␣ ϩ/ϩ mice; solid circles, Rev-erb␣ Ϫ/Ϫ mice. ϳ P Ͻ 0.05 for main effect of time (top left corner) or vs. corresponding ZT0 group of same genotype (above bar); *P Ͻ 0.05 vs. ϩ/ϩ genotype at same ZT; # P Ͻ 0.05 vs. 24 h LPϪ treatment.

Figure 4 .

 4 Figure 4. Shift from glucose to fatty acid utilization in unfed Rev-erb␣ Ϫ/Ϫ mice. A) Body mass changes determined before (left, upper values) and after a 24 h fast (left, lower values), and before (right, upper values) and after 24 h of food withdrawal ϩ 24 h refeeding (right, lower values) in Rev-erb␣ ϩ/ϩ and Rev-erb␣ Ϫ/Ϫ mice. B) Body temperature during 24 h of food withdrawal and the adjacent ZT22-24 average. Open circles, Rev-erb␣ ϩ/ϩ mice; solid circles, Rev-erb␣ Ϫ/Ϫ mice. C-I) Blood glucose (C), plasma insulin (D), hepatic glycogen (E), hepatic triglyceride (TG; F), plasma TG (G), plasma NEFA (H), and plasma ketone body (KB;I) levels in unfed (fasted) and refed animals. J-L) mRNA expression levels of ketogenic genes (J), glucose metabolism genes (K), and lipogenic genes (L). Animals were sampled at ZT0. Values are expressed as means Ϯ se (nϭ7/group). See Supplemental TableS1for gene names. # P Ͻ 0.05 for main effect of feeding condition (midpanel) or vs. corresponding unfed (fasted) group of same genotype (above bar); *P Ͻ 0.05 vs. ϩ/ϩ genotype of same feeding condition.

Figure 5 .

 5 Figure5. Rev-erb␣ Ϫ/Ϫ mice exhibit a fat phenotype amplified on prolonged high-fat feeding. A) Body mass change over time in chow-fed and HFD-fed ϩ/ϩ and Ϫ/Ϫ animals (nϭ12/group). Sac., sacrifice. Note that statistical analysis was performed on the last week of feeding. Open circles, chow-fed Rev-erb␣ ϩ/ϩ mice; solid circles, chow-fed Rev-erb␣ Ϫ/Ϫ mice; light shaded triangles, HFD-fed Rev-erb␣ ϩ/ϩ mice; dark shaded triangles, HFD-fed Rev-erb␣ Ϫ/Ϫ mice. B) Total adiposity, expressed as a percentage of body mass (nϭ12/group). C) Amount of perigonadal and retroperitoneal WAT (WATp and WATr, respectively; nϭ12/group). D, E) Plasma leptin (D) and glucose (E) levels (nϭ6/group). F, G) Hepatic triglyceride (TG; F) and plasma TG (G) levels (nϭ6/group). H-J) Plasma LDL (H), NEFA (I), and ketone body (KB; J) levels (nϭ6/group). Values are expressed as means Ϯ se. ϳ P Ͻ 0.05 for main effect of time; *P Ͻ 0.05 for main effect of genotype; # P Ͻ 0.05 for main effect of feeding condition.

Figure 7 .

 7 Figure 7. Transactivation by CLOCK of the Lpl promoter. A, B) Effects of ROR␣ and REV-ERB␣ on the transcription of Lpl-luc (A) and Bmal1-luc (B) promoter reporter constructs. C, D) Effects of CLOCK and BMAL1 on the transcription of Lpl-luc (C) and Per1-luc (D) promoter reporter constructs. Luciferase assay was performed on COS-7 cells as indicated in Materials and Methods. Data are means Ϯ se of a representative experiment performed in triplicate wells. RLU, relative luminescence units. *P Ͻ 0.05 vs. control (empty vector).

Figure 57 .

 57 Figure 57. Study of the food-entrainable oscillator network in brain-specific Rev-erbα KO mice

Figure 2 .

 2 Figure 2. Evaluation of anxiety responses in Rev-erbα +/-and Rev-erbα -/-mice

Figure 3 .

 3 Figure 3. Light-dark box test

Figure 4 .

 4 Figure 4. Evaluation of anxiety responses in Rev-erbα +/-and Rev-erbα -/-mice Rev-erbα-/-mice spent significantly less time in the light side of the light-dark box (P < 0.05) compared to wild-type and Rev-erbα+/-mice.

Figure 5 .Figure 6 .

 56 Figure 5. Forced swimming test

  NanoDrop Technologies; A 260 /A 280 and A 260 /A 230 values were >1.

	of 3% H 2 O 2 (Sigma-Aldrich) in PBS for 30 min at room temperature. Sections were
	then rinsed in PBS, and incubated for 2 h in a blocking solution containing 10% goat serum in
	PBS with 0.3% Tween 20 in PBS. Then sections were incubated in the primary antibody
	solution (in PBS + 0.3% Tween 20 + 10% goat serum) for 24 h with gentle agitation at 4°C.
	We used a rabbit polyclonal anti-PER2 (1:2000; Alpha Diagnostic International, Cat. PER21-A;
	#869900A1) and a rabbit anti-p44/42 MAPK (1:20000; Cell Signaling #4370). Sections were
	then rinsed in PBS with 0.05% Tween 20 and incubated for 2 h at 4°C with a biotinylated
	anti-rabbit IgG made in goat (Vectastain ABC peroxydase kit PK6101), diluted 1:500 with
	0.3% Tween 20 in PBS on plate agitation at 4°C. Thereafter, sections were rinsed in PBS +
	0.05% Tween 20 and incubated for 1 h at room temperature with an avidin-biotin-
	mRNA extraction and quantitative Real-time PCR
	Pieces of frozen livers were homogenized in lysis buffer supplemented with β-
	mercaptoethanol and total RNA was extracted according to the manufacturer's protocol
	(Absolutely RNA Miniprep Kit, Stratagene, Agilent technologies). The RNA samples were

peroxidase complex (1:250; Vectastain Kit, PK6101; Vector Laboratories) in PBS + 0.05% Tween 20. Next, sections were rinsed in PBS, and incubated with 3,3′-diaminobenzidine (0.5 mg/mL; Sigma) with 0.015% H 2 O 2 in H 2 O. Thereafter, sections were rinsed with PBS, wetmounted onto gel-coated slides, dehydrated through a series of alcohol, soaked in toluene, and coverslipped with EUKITT. Photomicrographs were taken on Leica DMRB microscope (Leica Microsystems) with an Olympus DP50 digital camera (Olympus France). The intensity and number of immunoreactive cells in hypothalamic nuclei and the Purkinje layer of the cerebellum, respectively, were determined using NIH ImageJ software (Rasband, W.S., U. S. National Institutes of Health, Bethesda MD, USA). The average intensity or cell numbers was determined, as far as possible, from three brain sections per animal. The number of animals / genotype / ZT was comprised between 2 to 6. further purified by precipitation with sodium acetate and isopropyl alcohol. RNA quality was evaluated with the Bioanalyseur 2100 (Agilent Technolgies; RNA integrity number > 6 for all samples). RNA quantity was measured using NanoDrop ND-1000 Spectrophotometer (

. Sequences of primers used for Quantitative Real-Time PCR

	Genes	Forward	Reverse
	36b4	GCTGATGGGCAAGAACACCA	CCCAAAGCCTGGAAGAAGGA
	Acc (Acetyl-Coenzyme A carboxylase alpha)	GCCTCTTCCTGACAAACGAG	TGACTGCCGAAACATCTCTG
	Acly (ATP citrate lyase)	ATCAACCCCCTTGTGGTGA	GCTTCAAGCTTGCTCCACTT
	ApocIII (Apolipoprotein CIII)	ACATGGAACAAGCCTCCAAG	TGGTTGGTCCTCAGGGTTAG
	Bmal1 (Brain and muscle aryl hydrocarbon receptor nuclear	CTCATTGATGCCAAGACTGG	GGTGGCCAGCTTTTCAAATA
	translocator (ARNT)-like)
	Cd36 (Cluster of Differentiation 36)	AAGAGGTCCTTACACATACAGAGTTC	AGCTGCTACAGCCAGATTCA
	Clock* (Circadian Locomotor Output Cycles Kaput)	ACCACAGCAACAGCAACAAC	GGCTGCTGAACTGAAGGAAG
	Cpt1a (Carnitine palmitoyltransferase 1A (liver))	TACTACGCCATGGAGATGCT	TGACGTGTTGGATGGTGTCT
	Cpt1b (Carnitine palmitoyltransferase 1B (muscle))	GTCGCTTCTTCAAGGTCTGG	AAGAAAGCAGCACGTTCGAT
	Elovl6 (ELOVL fatty acid elongase 6)	GAGCAGAGGCGCAGAGAAC	ATGCCGACCACCAAAGATAA
	Fas (Fatty acid synthase)	TCGACTTCAAAGGACCAAGC	TTCATGAACTGCACAGAGGTG
	Fgf21 (Fibroblast growth factor 21)	AGATCAGGGAGGATGGAACA	TCAAAGTGAGGCGATCCATA
	G6pc (Glucose-6-phosphatase, catalytic subunit)	CTCGTCTTCAAGTGGATTCTGT	TGGCTTTTTCTTTCCTCGAA
	Gk (Glucokinase)	CCAGAAGGCTCAGAAGTTGG	TGCTTGTCCAGGAAGTCAGA
	Glut2 (Glucose transporter 2)	GGACAAACTTGGAAGGATCA	CAGTCCTGAAATTAGCCCACA
	Glut4 (Glucose transporter 4)	GATTCTGCTGCCCTTCTGTC	ATTGGACGCTCTCTCTCCAA
	Gys2 (Glycogen synthase 2)	CCAGCTTGACAAGTTCGACA	CCTCTTCAGCATGTGCTCTG
	Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthase 2	AAGACCAAGGCCTCCCTTTA	TAAGCCTGAGCCGTAGGAGA
	(mitochondrial))
	Insig2a (Insulin induced gene 2a)	CACGCCAGTGCTAAAGTAGAC	GGGTGACAACGGTTGCTAAG
	L-Pk (Liver-pyruvate kinase)	CGGAAAATTGGCCCAGA	CACCACATCACTGGCTTTTC
	Lipc (Hepatic lipase)	GCTGCTGGGAACAAAAGAAG	AATGAGGCCAGAGTGGTGAG
	Lpl (Lipoprotein lipase)	AGGGCTCTGCCTGAGTTGTA	CCATCCTCAGTCCCAGAAAA
	Pepck (Phosphoenolpyruvate carboxykinase 2	TTTGATGCCCAAGGCAACTT	ATCGATGCCTTCCCAGTAAA
	(mitochondrial))
	Ppara (Peroxisome proliferator-activated receptor alpha)	GGATGTCACACAATGCAATTC	GGCCTTGACCTTGTTCATGT
	Pparbp (Ppar binding protein)	GAGAATCCTGTGAGCTGTCC	CGTTGGTTGCCTTCCAGTA
	Pparg (Peroxisome proliferator-activated receptor gamma)	AGACCACTCGCATTCCTTTGACAT	TCCCCACAGACTCGGCACTCAATG
	Srebp1c (Sterol regulatory element binding transcription	GGCACTGAAGCAAAGCTGAA	TCATGCCCTCCATAGACACA
	factor 1)

* Primers from

Kohsaka et al. (2007)

Cell Metab

This title is freely inspired from "Beats, Rhymes and Life", the fourth album of the famous hip-hop groug "A Tribe Called Quest".

The botanical observations of de Mairan would have passed into obscurity, if his colleague, M. Marchand, had not published the results for him.

"Free running" being a term used to describe a circadian rhythm that is not entrained to external time cues.

Acrophase and other parameters of a rhythm are defined by a mathematical model, usually a cosine function.

A phenomenon extended and called now "Aschoff's Rule".

As an aside, it is important to mention that it was also in the 50's that Gustav Kramer and Klaus Hoffman demonstrated that starlings, which use the sun to navigate, possess an internal clock, important to compensate for the sun's movements throughout the day, and thus essential for migration.

Synechococcus is a unicellular cyanobacterium that is very widespread in the marine environment.

Cell division cycle in cyanobacteria can be a much more rapid process than circadian cycle. However, it has been demonstrated that the circadian clock mechanism is independent of cell division cycle, while the later depends on the phase of the circadian clock such that cell division is gated to occur only in specific circadian phases.

The cross-species approach, in particular between fruit flies and mice, was a driving force in chronobiology to identify clock gene by homology of DNA sequence.

I am adding the term "oscillator" to define a biological entity capable of generating circadian rhythms (e.g., of gene expression), but, amongst other things, that does not impose its rhythmicity to other entities.

Melatonin, secreted during the night, is a key hormone for seasonal and circadian variations in biological functions.

Of note, the Tau mutation shortened the free-running rhythm of melatonin synthesis by 4 h, suggesting that common mechanisms may be shared between the SCN and the retina pacemakers.

To avoid a masking effect (i.e., inhibiting effect of light) on locomotor activity, such experiments are conducted in constant dark conditions.

Rabbit pups are normally fed by nursing only once a day and show a clear locomotor anticipatory behavior to the arrival of the nursing mother (see[START_REF] Caba | The rabbit pup, a natural model of nursing-anticipatory activity[END_REF].

Not excluding, however, the participation of memory, conditioning, etc.

As starling birds give evidence of interactions between compass navigational system, memory and circadian clock systems.

A region that lacks the blood-brain barrier (BBB).

The presence of a BBB is controversial for the ARC since some vessels in the ventromedial part lacks some BBB markers. At least, the close relationship of the ARC with the median eminence makes it important to integrate metabolic changes in the periphery.

The close nature between the MASCO and the FEO (a sole entity or two different systems) was mentioned earlier.

Vol. 26 August 2012 DELEZIE ET AL. The FASEB Journal ⅐ www.fasebj.org

ALTERED ENERGY HOMEOSTASIS IN REV-ERB␣ Ϫ/Ϫ MICE

Vol. 26 August 2012 DELEZIE ET AL. The FASEB Journal ⅐ www.fasebj.org

ALTERED ENERGY HOMEOSTASIS IN REV-ERB␣ Ϫ/Ϫ MICE

Vol. 26 August 2012 DELEZIE ET AL. The FASEB Journal ⅐ www.fasebj.org

ALTERED ENERGY HOMEOSTASIS IN REV-ERB␣ Ϫ/Ϫ MICE

Vol. 26 August 2012 DELEZIE ET AL. The FASEB Journal ⅐ www.fasebj.org

ALTERED ENERGY HOMEOSTASIS IN REV-ERB␣ Ϫ/Ϫ MICE

Vol. 26 August 2012 DELEZIE ET AL. The FASEB Journal ⅐ www.fasebj.org

ALTERED ENERGY HOMEOSTASIS IN REV-ERB␣ Ϫ/Ϫ MICE

Vol. 26 August 2012 DELEZIE ET AL. The FASEB Journal ⅐ www.fasebj.org

On the other hand, not all control mice show strong FAA in our studies, in line with previous results[START_REF] Pendergast | Robust Food Anticipatory Activity in BMAL1-Deficient Mice[END_REF]).

It has been recently shown that FAA in Bmal1 -/-mice could be observed in 18L:6D lighting cycles or in constant darkness, while it was nearly absent in 12L:12D conditions[START_REF] Pendergast | Robust Food Anticipatory Activity in BMAL1-Deficient Mice[END_REF]

This study reveals a close identity of the Rev-erbα and Rev-erbβ cistromes.

Of note, the absence of Rev-erbα leads to several defects during the postnatal development of the cerebellar cortex (e.g., delayed differentiation of Purkinje cells), yet with no reported deficits in motor coordination in adult mice[START_REF] Chomez | Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor[END_REF]-a phenotype which contrasts to Rorα -/-mice that suffer from cerebellar ataxia(Hamilton et al. 1996).

Interestingly, PER1 expression in the DMH has been shown to increase in response to food that was presented at variable and unpredictable times each day, in the absence of entrained FAA. In addition, peak of PER2 expression is observed in the DMH after mealtime. These data suggest that oscillations in the DMH are sensitive to feeding-related signals rather than synchronized by mealtime (Verwey and Amir 2009).

Of note, no sex differences were observed regarding our in vivo glucose homeostasis studies, apart from higher blood glucose values in Rev-erbα -/-males than in Rev-erbα -/-females, which was also observed between wild-type males and females. Moreover, physiological and molecular data following the 24-h fasting and refeeding experiments were analyzed with no sex differences, apart from Pepck mRNA expression that was stronger in Rev-erbα -/-females than Rev-erbα -/-males (unpublished data).

The liver is the main organ that removes the glucose from the blood. It is the principle site of de novo lipogenesis.

It is not excluded that the skeletal muscle phenotype or the delayed maturation of Purkinje cells in Rev-erbα-/-mice may have, to some extent, contributed to their reduced wheel-running activity levels (e.g., altered energy-mobilization or altered fine motor coordination).

In addition, disrupted brain control of daily glucose homeostasis may also have contributed to steady hyperglycemia in Rev-erbα -/-mice.

Acknowledgments

We are indebted to Prof. Ueli Schibler for kindly providing the founder Rev-erbα+/-mice (University of Geneva, Switzerland). We would like to thank Magaly Mayer-Schmitt for taking part in the immunohistochemistry experiments as well as Sylviane Gourmelen, Laurence Huck and Anita Wulgué for animal care. J.D. is supported by a doctoral fellowship from the French Ministry of National Education and Research, and E.C. is supported by Centre National de Recherche Scientifique, University of Strasbourg and Agence Nationale pour la Recherche "Jeunes chercheurs/jeunes chercheuses" (ANR-07-JCJC-0111).

In preparation…

The authors thank Dr. André Malan for statistical advice. The authors are grateful to Laurence Huck and Dr. Sophie Reibel-Foisset for animal care and Sophie Brangolo, Marion Grandjean, and Sylviane Gourmelen for helpful technical assistance. The authors are indebted to the Mouse Clinical Institute (MCI; Strasbourg, France) for the clamp study. The authors also thank Prof. Ueli Schibler (University of Geneva, Geneva, Switzerland) for kindly providing the founder Rev-erb␣ ϩ/Ϫ mice. J.D. is supported by a doctoral fellowship from the French Ministry of National Education and Research, and E.C. is supported by Centre National de la Recherche Scientifique, CNRS), University of Strasbourg, and Agence Nationale pour la Recherche Jeunes Chercheurs/Jeunes Chercheuses (ANR-07-JCJC-0111). A.G.C., M.T., and F.D. are supported by the University of Nice Sophia-Antipolis, CNRS, and the European Commission (integrated project CRESCENDO LSHM-CT-2005-018652).

SUMMARY

Limiting food access to a time window during the day (termed restricted feeding, RF) has profound synchronizing effects on circadian rhythms. A few hours before mealtime, rodents display food-anticipatory activity (FAA) as well as changes in physiological parameters (e.g. rise in body temperature). Food anticipation is thought to be controlled by a circadian foodentrainable oscillator (FEO), independent of the central clock in the suprachiasmatic nucleus (SCN). Recent studies showed that disruption of some of the well-known circadian clock genes fails to affect the FEO, therefore questioning the hypothesized SCN-like molecular mechanisms responsible for food anticipation. In this context, we focused on the Rev-erbα clock gene. We showed that Rev-erbα -/-mice subjected to RF exhibited reduced FAA compared to wild-type littermates as demonstrated by decreased locomotor activities, regardless of the lighting conditions. In accordance with behavioral data, the anticipatory thermogenesis was nearly absent and the food-anticipatory peak of corticosterone was also diminished in Rev-erbα -/-mice. Conversely, hepatic clock gene expression was shifted in response to daytime feeding in both genotypes, indicating that disrupted FAA is not the consequence of impaired liver synchronization in Rev-erbα -/-mice. On the other hand, PER2 expression was not phase-adjusted to mealtime in the cerebellum while p-ERK expression was not clearly responsive to scheduled RF in several hypothalamic regions. The present results indicate that mice lacking Rev-erb exhibit altered food anticipation, suggesting that this gene may be a component of the FEO. 0.05). ~ for main effect of time (top left corner) or vs. control animals (above data point) (P < 0.05). (A) PER2 and (B) p-ERK immunoreactive (ir) cells in hypothalamic nuclei and cerebellum of mice under AL conditions or exposed to RF schedules. Note that staining intensity was taken into consideration for all hypothalamic nuclei; while for the cerebellum, the number of labeled purkinje cells was counted. Data for ZT0 are double-plotted. ~ for main effect of time (top left corner) (P < 0.05).

Supplemental Figure 1. Food intake and body mass of mice maintained in LD or DD conditions before and during scheduled feeding.

Food intake (left) and body mass (right) determined weekly in (A) LD conditions and (B) DD conditions. The period of restricted feeding is indicated by the hatched bars. Supplemental Figure 2. Food-anticipatory parameters in mice maintained in LD or DD conditions under restricted feeding.

(A) Percent of wheel-running activity and (B) general activity over total daily activity 1 h prior to meal access in three independent series of animals (i.e., LD1, DD and LD2). (C) Note that heterozygous mice (+/-) for Rev-erbα were added to the results. Results of Rev-erbα +/+ and Rev-erbα -/-mice depicted on figure A, B and C for the LD1 experiment are reproduced from (K) 24-h blood glucose profiles from tail bleed in HFD conditions. (L) Mean blood glucose levels over 24 h from chow (data from Figure 1D) and HFD conditions (data from Figure S5K) Rev-erbα+/+ (data from Figure S5K). Rev erbα+/+ (open circles) and -/-mice (closed circles). Note that mice from each feeding conditions were age-matched at the time of sampling.

Values are represented as mean ± SEM. # main effect of feeding condition (P < 0.05); main effect of time (P < 0.05); * main effect of genotype (upper left corner) or significant difference between genotypes for a given time point and feeding condition (above bar graph) (P < 0.05). Supplemental Figure 6. Gene expression in chow-and high-fat fed Rev-erbα+/+ and -/-animals. mRNA expression of glucose and lipid metabolism genes in (A) liver, (B) retroperitoneal white adipose tissue, (C) rectus femoris skeletal muscle. Data are means ± SEM (n = 6 / group). # main effect of feeding condition (upper left corner) or significant difference between feeding conditions for a given genotype and time point (above bar graph) (P < 0.05); ~main effect of time (upper left g g g yp p ( g p ) ( );

( pp corner) or significant difference between time points for a given genotype and feeding condition (above bar graph) (P < 0.05); * main effect of genotype (upper left corner) or significant difference between genotypes for a given time point and feeding condition (above bar graph) (P < 0.05). For abbreviations of the different genes, see table S1. main effect of time (upper left corner) or significant difference between time points for a given genotype and feeding condition (above bar graph) (P < 0.05); * significant difference between genotypes g g yp for a given time point and feeding condition (above bar graph) (P < 0.05).

Supplemental

Supplemental Figure 8. Enhanced adaptive thermoregulation in Rev-erbα-/mice.

Body temperature before (Time 0, ZT2) and during exposure to 4°C (n = 6 / group). Values are expressed as mean ± SEM. ~main effect of time (upper left corner); * main effect of genotype (upper left corner) (P < 0.05).

Appendix 1 -Food anticipation without daytime light

Food-anticipatory activity in a skeleton photoperiod

Below are depicted double-plotted actograms of wheel-running activity from two wild-type and two Rev-erbα -/-mice.

Note that the 12-h light phase or the two 1-h light pulses are indicated by yellow rectangles. A few days of ad libitum (AL) feeding conditions are illustrated. During restricted feeding (RF), food access is indicated by the orange rectangle.

When wild-type mice are challenged with a 6-h restricted feeding schedule, they showed robust food-anticipatory activity, regardless of the lighting conditions. Wheel-running activity levels in anticipation of mealtime, however, were higher during the skeleton photoperiod. This indicates that light can potentially affect the behavioral output of the food-entrainable oscillator. Interestingly, irrespective of the lighting conditions, Rev-erbα -/-mice did not exhibit food-anticipatory activity.

Food-anticipatory activity in constant darkness following mealtime jetlag test

Below are depicted double-plotted actograms of wheel-running activity from two wild-type and two Rev-erbα -/-mice.

Animals are in constant dark conditions (indicated by the above dark bar). A few days in ad libitum (AL) feeding conditions are illustrated. During restricted feeding (RF), food access is indicated by the orange rectangle. After a first period of food restriction at a specific circadian phase, mealtime was 6-h phase-advanced (i.e., jet-lag test).

During the first restricted feeding period, wild-type mice showed foodanticipatory activity, which could be resynchronized after a 6-h phase advance of mealtime. In Rev-erbα -/-mice, food anticipation was not observed at the two feeding times.

Appendix 2 -Behavioral tests

To assess anxiety-or depressive-like behaviors, 32 age-matched mice (including 12 wild-type, 10 Rev-erbα +/-and 10 Rev-erbα -/-mice) were used (sex ratio 1:1).

All mice were exposed to a 12 h/12 h LD schedule. Note that animals were all tested around the onset of the active period.

Tests were separated by several days and conducted in the following order: 1) Elevated plus maze; 2) Light-dark box test; 3) Forced swimming test. Prior to the first test, animals were equally accommodated to being handled by the experimenter, who was blinded to the genotype.

Animals were tested in their housing room, except for the Light-dark test for which mice were moved to an adjacent room.

Elevated plus maze a) Protocol

The elevated plus maze is a method to evaluate anxiety responses in rodents (i.e, conflict between their preference for protected closed areas and their motivation to explore a novel environment). For more information, see Walf and Frye 2007.